core role of trpc6 channels in regulating airway re ...jan 16, 2020 · 130 a very efficient...

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Core Role of TRPC6 Channels in Regulating Airway Re-modelling in Chronic 1

Obstructive Pulmonary Disease 2

Qiongyu Hao 1, 2*† Kun Zhao

3* Raoying Xie

4 Jie Wei

5 Wenyue Gu

6† Zhenghua Fei4† 3

1. Department of Internal Medicine, Charles Drew University of Medicine and Science, Los 4

Angeles, CA, 90059, USA 5

2. David Geffen UCLA School of Medicine, Los Angeles, CA, 90095, USA 6

3. Department of Oncology, Huaian Hospital of Huaian City, No.161 Zhenhuailou East Road, 7

Huaian 223200, PR China. 8

4. Department of Oncology, the 1st Affiliated Hospital of Wenzhou Medical University, 9

Wenzhou 325000, PR China. 10

5. Department of Basic Medical Sciences, Medical College, Xiamen University, Xiamen 11

361101, PR China. 12

6. Department of Pathology, Yancheng Hospital Affiliated Southeast University, Yancheng, 13

224000, China. 14

* Qiongyu Hao and Kun Zhao contribute equally to this work as joint first authors. 15

† Corresponding authors: Qiongyu Hao: [email protected]; Wenyue Gu: 16

[email protected]; Zhenghua Fei: [email protected] 17

Key words 18

TRPC6, miR-13a/b-5p, NF-κB, Airway Re-modeling; COPD. 19

Abbreviations list 20

TRPC6, Transient Receptor Potential Channel 6; miR-135a/b-5p, microRNA-135a/b-5p; 21

COPD, Chronic Obstructive Pulmonary Disease; ASMCs, Airway Smooth Muscle Cells; 22

PASMCs, Pulmonary Airway Smooth Muscle Cells; HASMCs, human airway smooth 23

muscle cells; MASMC, Mouse Airway Smooth Muscle Cells; PSS, Physiologic Saline 24

Solution; EMSA, Electrophoretic Mobility Shift Assays; IP, Immunoprecipitation; GSEA, 25

Gene Set Enrichment Analysis. 26

27

28

29

30

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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 16, 2020. ; https://doi.org/10.1101/2020.01.16.908699doi: bioRxiv preprint

https://doi.org/10.1101/2020.01.16.908699

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Abstract 31

Rationale: The mechanistic role of canonical transient receptor potential 6 (TRPC6) channel 32

in chronic obstructive pulmonary disease (COPD) is poorly understood. Objectives: The 33

purpose of this study is to determine the role of TRPC6 channel in COPD and its underlying 34

signaling mechanisms in human airway smooth muscle cells (HASMCs). Methods and 35

Main Results: The present study examined the effects of TRPC6 channel on nicotine and 36

cigarette induced HASMCs proliferation, migration and mouse airway remodeling models. 37

mRNA and protein expression of TRPC6 were increased in cultured HASMCs incubated 38

with nicotine using real-time PCR and western blot analysis. Nicotine treatment significantly 39

increased TRPC6 transcriptional activity through NF-κB in HASMCs with Co-IP and 40

electrophoretic mobility shift assays (EMSA). Nicotine treatment also increased ROS level in 41

HASMCs, this increase was attenuated by Nox inhibitor apocynin. miR-135a/b-5p down-42

regulated mRNA and protein level of TRPC6 in HASMCs, while luciferase reporter assay 43

showed that miR-135a/b-5p targeted at the 3’-UTR of TRPC6 mRNA. microRNA-135a/b-5p 44

(miR-135a/b-5p), with a negative correlation to TRPC6 expression, was low in airway 45

smooth muscle of COPD patients. Cigarette-induced airway remodeling mice model also 46

exhibited a large increase in smooth muscle cell proliferation and smooth muscle layer mass 47

with immunohistochemistry assay, this well-characterized airway remodeling was eliminated 48

by lentivirus of TRPC6 knockdown or miR-135a/b-5p overexpression. Conclusions: 49

Nicotine exposure results in increased HASMCs proliferation and migration through NF-κB 50

signaling. Inhalation of cigarette causes airway smooth muscle layer re-modeling due to 51

altered TRPC6 elicited Ca2+

influx, miR-135a/b-5p abolishes this change both in vitro and in 52

vivo. 53

54

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Introduction 55

Chronic obstructive pulmonary disease (COPD) is a heterogeneous, progressive lung disease 56

characterized by chronic pulmonary inflammation, persistent airways remodeling and airflow 57

limitation. Due to lack of effective treatment, it is a leading cause of death in the United 58

States and worldwide 1. The major risk factor for COPD is exposure to cigarette smoke which 59

contains noxious inflammatory and oxidant agents 2. Ca

2+ signaling has played an essential 60

role in mediating airway smooth muscle cells (ASMCs) proliferation and migration, which 61

has been considered as important characters in the developing COPD 3. TRPC channel has 62

been recognized as non-selective cation channel, which mainly causes Ca2+

and Na+ entry 63

and participates numerous intracellular signaling pathways. This gives them the potential to 64

contribute to the regulation of numerous Ca2+

-dependent cellular functions from cell growth 65

to myocyte contraction. The family of canonical transient receptor channel (TRPC) includes 66

at least seven members (TRPC1-7), which mediates an array of signal transduction pathways 67

4-6. One important member of the TRPC channel family is TRPC6 channel. TRPC6 is widely 68

expressed in a variety of tissues and organs, including the kidney, placenta, lung, heart, brain, 69

skin and vasculature 7-10

. Evidence indicates that TRPC6 plays a key role in normal 70

physiology and disease states of the pulmonary vasculature 11

. Na+ influx through TRPC6 71

channels in pulmonary airway smooth muscle cells (PASMCs) leads to membrane 72

depolarization and activation of voltage-gated L-type Ca2+

channels mediating the bulk of the 73

Ca2+

influx and contraction of smooth muscle cells 12

. Thus, TRPC6 channels may be a 74

potential therapeutic target for the control of pulmonary hemodynamics and gas exchange in 75

physiological and pathological conditions. 76

The miR-135 family is highly conserved among mammals and consists of 2 members, miR-77

135a and miR-135b, which have been reported to play prominent roles as oncogenes in the 78

development of various types of cancer, including the pathogenesis of non-small cell lung 79

cancer, the colorectal cancer 13 14

and breast cancer 15-17

. miR-135a/b-5p are known to directly 80

regulate expression of several genes, including TRPC1 18

, HOXA10 19

and c-myc 20

. Despite 81

these findings, the exact function of the two miR-135 family members remain largely 82

unknown, particularly their function in smoking-associated lung diseases. In this study, 83

applying microRNA research assays, we identified miR-135a/b-5p posttranscriptional 84

regulated TRPC6 expression, which was required to HASMCs proliferation after nicotine 85

exposure. 86

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Airway smooth muscle cells are important regulators of lung function and pathological 87

changes like COPD, asthma and pulmonary hypertension. In this research article, we will 88

discuss the regulating factors of TRPC6 channel in human airway smooth muscle cells 89

(HASMCs) and the functional important role in the development of smoking-induced COPD, 90

which could provide novel therapeutic targets for treating these diseases. 91

Materials and Methods 92

MethodsThe animal protocol was approved by the Institute’s Animal Ethics Committee of 93

Wenzhou Medical University, and the investigation complied with the National Institutes of 94

Health Guide for the Care and Use of Laboratory Animals. All the studies involving human 95

samples were approved by the Ethics Committee of Wenzhou Medical University, and 96

conformed to the principles outlined in the Declaration of Helsinki. An expanded materials 97

and methods section is available in the supplemental data, which includes detailed 98

information on the following aspects: primary culture human ASMCs, cell culture and 99

transient transfection, production of lenti-virus, cell viability assays, migration assays, 100

immunoprecipitation, western blot analysis, nuclear and cytoplasmic protein extracts, 101

electrophoretic mobility shift assays (EMSA), luciferase reporter gene assays, quantitative 102

real-time RT-PCR, measurement of intercellular ROS, generation of SMC-specific TRPC6 103

knockdown mice, measurement of muscle contraction and histological assessments. 104

Statistical Analysis 105

Statistical analyses are performed with the GraphPad Prism software. Data are expressed as 106

means ± SEM. Statistical analysis was performed using paired Student’s t test for 107

comparisons before and after treatment in the same sample, unpaired (independent) Student’s 108

t test for 2-sample comparisons, one-way ANOVA with an appropriate post hoc test for 109

multiple-sample comparisons, and two-way ANOVA for comparisons of the means of 110

populations that were classified in 2 different ways or the mean responses in an experiment 111

with 2 factors 21

. Values of P< 0.05 were considered statistically significant. The Pearson 112

correlation coefficient, a measure of the linear dependence between two variables X and Y, 113

was analyzed in the inverse correlation between miR-135a/b-5p and TRPC6. It has a value 114

between +1 and −1 inclusive, where 1 is total positive linear correlation, 0 is no linear 115

correlation, and −1 is total negative linear correlation. 116

Results 117

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TRPC6 channels mediate the increased proliferation and migration of ASMCs from 118

patients with COPD. 119

To study the effect of proliferation of TRPC6 in HASMCs, normal and COPD HASMCs 120

were freshly isolated and cultured. HASMCs were plated at a low confluence and cultured for 121

continuous days. Cell proliferation was monitored by measuring absorbance at 490 nm after 122

adding MTS solution. HASMCs from COPD patients were more proliferative than normal 123

HASMCs (Fig. 1A). TRPC6 overexpression facilitated the normal HASMCs proliferation 124

(Fig. 1B). Both of knockdown and inhibitor (SAR 7334) of TRPC6 channels inhibited COPD 125

HASMCs growth (Fig. 1C). Expression level of TRPC6 protein in COPD HASMCs was 126

about 3-fold higher than in normal HASMCs (Fig. 1D). COPD HASMCs showed a 2-fold 127

increase in transwell migration. Knockdown of TRPC6 channel protein abolished this 128

increase in COPD HASMCs (Fig. 1G, and Sup. Fig. 1). Western blot analysis demonstrated 129

a very efficient up-regulation (Fig. 1E) and down-regulation (Fig. 1F) of TRPC6 protein. 130

Hsa-miR-135a/b-5p regulate TRPC6 expression and activity to play an important role 131

in the COPD human ASMCs. 132

We next sought to identify specific microRNAs that could modulate TRPC6 expression. We 133

therefore focused on the regulatory mechanisms and biological significance of TRPC6 in 134

HASMCs. miR-135a/b-5p has been predicted targets on the 3’- UTR of TRPC6 channel 135

mRNA (Sup. Tab. 1). Among top 6 microRNAs that were down-regulated in lungs from 136

patients with COPD compared with smokers without COPD 22

and 6 microRNAs that were 137

predicted to bind to TRPC6-3’-UTR based on the “microrna.org” algorithm 138

(http://www.microrna.org/), indeed, miR-135a/b-5p were shown to decrease TRPC6 139

expression in 293T cells compared with other microRNAs by western blot analysis (Fig. 2A). 140

Transfection with miR-135a/b-5p mimics reduced TRPC6 mRNA and protein level, 141

regardless of normal or COPD HASMCs (Fig. 2B and 2C). Nicotine significantly increased 142

the TRPC6 mRNA expression in TRPC family members in normal HASMCs (Fig. 2F). To 143

verify the effect of nicotine and miR-135a/b-5p on TRPC6 expression, HASMCs were 144

treated with nicotine and infected with lentivirus of miR-135a/b-5p. In normal HASMCs, 145

nicotine increased TRPC6 channel expression dramatically, and miR-135a/b-5p 146

overexpression reduced TRPC6 protein expression faintly (Fig. 2D). In COPD HASMCs, 147

nicotine increased TRPC6 channel expression faintly, while miR-135a/b-5p overexpresion 148

reduced TRPC6 protein dramatically (Fig. 2E). Probable explanation is TRPC6 expression 149

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was already high in COPD patient than normal. To identify the regulatory sequence in 3’-150

UTR of TRPC6, full length and additional two reporters with truncated sequence in 3’-UTR 151

of TRPC6 were established. A computational search predicted the site of miR-135a/b-5p 152

binding to the TRPC6- 3’ UTR (Fig. 2G). HASMCs infected with lentivirus of miR-135a/b-153

5p were transfected with pmiR-TRPC6-3’UTR-FL or TRPC6- 3’UTR truncates respectively, 154

luciferase reporter activity of TRPC6- 3’UTR-FL and TRPC6- 3’UTR-1-49 were decreased, 155

the empty vector and TRPC6- 3’UTR-1-43 did not (Fig. 2H), suggesting the sequence from 156

43-49 of 3’UTR of TRPC6 was essential to bind between miR-135a/b-5p and TRPC6 mRNA. 157

In addition, decreased level of the miR-135a/b-5p by nicotine (Fig. 2I) can further increase 158

TRPC6 expression. These lines of evidence collectively demonstrated that miR-135a/b-5p 159

recognized and regulated TRPC6 mRNA through specific binding to its 3’ UTR. If the miR-160

135a/b-5p binding with TRPC6 mRNA degraded TRPC6 mRNA, down-regulation of miR-161

135a/b-5p in COPD might also enhance TRPC6 expression. As determined by real-time PCR 162

analysis of the expression levels of miR-135a/b-5p and TRPC6 mRNA, a significant negative 163

correlation between the miR-135a/b-5p and TRPC6 mRNA was identified in normal and 164

COPD bronchial smooth muscle samples (Fig. 2J), suggesting a protective role of miR-165

135a/b-5p in the occurrence and development of COPD through targeting and degrading 166

TRPC6 mRNA. To study the effect of miR-135a/b-5p and TRPC6 on HASMCs migration, 167

normal and COPD HASMCs were infected with lentivirus of miR-135a/b-5p and treated with 168

SAR 7334. COPD HASMCs migrated faster than normal, miR-135a/b-5p and inhibitor of 169

TRPC6 abolished this increase (Fig. 2K and Sup. Fig. 2). 170

Nicotine promotes NF-κB translocation and increases NF-κB transcriptional activity 171

through α7 nAChR in a ROS-dependent manner. 172

To understand the molecular profiling of HASMCs responding to nicotine stimulation, a 173

genome-wide unbiased approach to analyze gene expression by RNA sequencing was 174

performed. Gene set enrichment analysis (GSEA) analysis of the whole transcriptome using 175

the curated gene set compilation “BIOCARTA” revealed a profound impact on gene 176

expression networks. Enrichment in gene sets corresponding to 177

‘‘BIOCARTA_NFKB_PATHWAY’’ and ‘‘BIOCARTA_NFAT_PATHWAY’’, are the top 178

enriched signatures (Fig. 3A). These results indicate that NF-κB signaling genes and 179

signaling genes were upregulated upon nicotine treatment in HASMCs. To further explore 180

the effect of nicotine on NF-κB activation, normal HASMCs were treated with nicotine in an 181

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increasing concentration manner. Whereas nicotine treatment resulted in nuclear 182

accumulation of NF-κB and reduced cytoplasmic NF-κB (Fig. 3B). Normal HASMCs were 183

exposed to nicotine or PBS for 24hours, then cytosol were extracted and subjected to co-184

immunoprecipitation studies to evaluate the effect of nicotine to the protein-protein 185

interactions between IκBα and NF-κB. The IκBα and NF-κB protein-protein interaction was 186

weakened by nicotine treatment (Fig. 3C). These results supported the notion of promotional 187

effects of nicotine on NF-κB translocation. Previous studies have revealed that the 188

mitochondrial complex I inhibitor rotenone and the complex III preubisemiquinone site 189

inhibitor myxothiazol block the hypoxia-induced increase in [ROS]i and [Ca2+

]i in PASMCs 190

23 24. Thus, we sought to examine the effects of rotenone and myxothiazol on the nicotine 191

stimulus in [ROS]i in HASMCs. Apocynin is thought to be a specific Nox inhibitor by 192

preventing the association of p47phox with the membrane-bound Nox subunits 25

, thus, we 193

examined the effect of apocynin on the nicotine-induced increase in [ROS]i in HASMCs to 194

determine the potential role of Nox in nicotine responses. Nicotine exposures for 24hours 195

caused a significant increase in [ROS]i. However, treatments with apocynin (1μM) for 30 min 196

significantly blocked nicotine-induced increase in [ROS]i (Fig. 3D). Our data indicates that 197

the mean increase in DCF fluorescence after nicotine stimulation was not significantly lower 198

in cells treated with either rotenone (10 μM) or myxothiazol (10 μM) for 30 min compared to 199

cells treated with PBS (Fig. 3D). Whereas, nicotine activates NF-κB activity, however, this 200

increase was abolished by N-acetyl-cysteine (NAC) commonly used to inhibit ROS and MG 201

624 (inhibitor of α7 nAChR) (Fig. 3E and 3F). Taken together, we conclude nicotine 202

activates NF-κB transcriptionally activity through α7 nAChR in an ROS-dependent manner 203

in HASMCs. 204

Nicotine increases TRPC6 channel expression and activity through NF-κB. 205

To determine the possibility of the NF-κB binding site for TRPC6 expression, we employed a 206

luciferase reporter construct driven by the TRPC6 promoter sequence. Plasmids were 207

transfected into the normal HASMCs and luciferase activity assays were performed. 208

Manipulation of the nicotine and NF-κB positively correlates with cellular TRPC6 209

transcriptional activity (Fig. 4A). TRPC6 mRNA expression level was increased by nicotine 210

in HASMCs, however, NF-κB_sh, and BAY 11-7082 abolished nicotine caused TRPC6 211

mRNA expression increase (Fig. 4B). HASMCs infected with lentivirus of p65_sh and 212

p50_sh (NF-κB_sh) were followed by stimulation with nicotine. As expected, TRPC6 protein 213

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level did not significantly change in the knockdown of NF-κB group (Fig. 4C). Similar 214

results were obtained by treating HASMCs with NF-κB inhibitor, BAY 11-7082 (10μM) (Fig. 215

4D). Nicotine increased TRPC6 protein expression in a time-dependent manner. The rate of 216

nicotine-induced TRPC6 expression almost reached to the maximum at 10th hour after 217

nicotine stimulation. These data demonstrated that nicotine can promote TRPC6 mRNA and 218

protein expression in an NF-κB dependent mechanism. We performed EMSA to further 219

verify the putative interaction between NF-κB and NF-κB response element (NRE) of 220

TRPC6 promoter. In the probe-shift assays, increasing exposure hours of nicotine was shown 221

to significantly increase the intensity of the shifted band (Fig. 4E). In the supershift assays 222

(Fig. 4F), incubation of p65 antibody with HASMCs nuclear extract pre-incubated with 223

biotin-labeled wild-type probe led to the formation of a supershifted band with larger 224

molecular weight than the shifted band, suggesting the formation of an NRE-WT/p65/anti-225

p65 antibody complex. In addition, pre-incubation of nuclear extract with a 200-fold excess 226

of unlabeled WT probe completely diminished the shifted band, whereas a 200-fold excess of 227

unlabeled mutant probe failed to do so (Sup. Fig. 3A). Moreover, incubation of nuclear 228

extract with biotin-labeled mutant probe (NRE-MT) did not result in any shifted band (Sup. 229

Fig. 3B). Furthermore, pre-incubation of nuclear extract with biotin-labeled MT probe did 230

not prevent the formation of the shifted band after subsequent incubation with biotin-labeled 231

WT probe. Together, these data clearly demonstrated the interaction of NF-κB with NRE-WT 232

in TRPC6 promoter in HASMCs. 233

Cigarette smoke causes ASM hyperresponsiveness and remodeling in mice model and 234

NFAT signaling activation in Mouse Airway Smooth Muscle Cells (MASMCs). 235

Inhalation of cigarette smoke enhanced methacholine-induced increase of airway resistance 236

and contraction, tail vein injection of lentivirus of SMC specific TRPC6_shRNAs abolished 237

this enhancement in mice with cigarette-evoked airway disease (Fig. 5A and 5B). To assess 238

the downstream pathway responding to TRPC6 altered [Ca2+

]i in the regulation of 239

proliferation, we measured the expression of NFAT and its target genes. Cigarette smoke 240

could result in a significant increase in NFAT and its targets gene we examined, however, 241

TRPC6 knockdown specifically abrogated this increase of up-regulated cell cycle genes (Fig. 242

5C). Cigarette-inhaled mice also exhibited a large increase in ASM mass and cell 243

proliferation, determined by α-SM cell actin and Ki67 immunohistochemistry staining. This 244

well characterized airway re-modelling (increased thickness of ASM layer and the number of 245

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Ki67-positive cells) was eliminated by tail vein injection of lentivirus of SMC specific 246

TRPC6_shRNAs (Fig. 5D and 5E). Taken together, lentivirus of SMC specific 247

TRPC6_shRNAs can specifically knockdown the TRPC6 protein in ASMCs, thereby block 248

cigarette induced airway hyper-responsiveness and re-modelling. Taking these data together, 249

we proposed that cigarette smoke through TRPC6 could determine the selection of NFAT-250

mediated cell cycle. 251

miR-135a/b-5p attenuates airway hyperresponsiveness and remodeling in a murine 252

model of asthma-COPD overlap syndrome. 253

Some patients with COPD appear more similar to those with asthma clinically, suggesting 254

underlying the clinical and pathologic overlap between asthma and COPD 26

. The allergen 255

ovalbumin-induced asthmatic airway disease mouse model was generated according to a 256

modified protocol 27

. Consistent with this knockdown of TRPC6, tail vein injection of 257

lentivirus of miR-135a/b-5p abolished airway hyperresponsiveness in mice with ovalbumin 258

and cigarette evoked airway disease, evidenced by the absence of the enhanced 259

methacholine-induced increase in airway resistance and contraction (Fig. 6A and 6B). 260

Ovalbumin treated mice also exhibited a large increase in ASM mass and HASMCs 261

proliferation, determined by α-SMC actin and Ki67 immunohistochemistry staining. This 262

well-characterized airway re-modelling was eliminated by tail vein injection of lentivirus of 263

miR-135a/b-5p (Fig. 6C and sup. Fig. 5). 264

TRPC6 channel protein expression and airway thickness are in parallel increased in 265

ASM tissues from patients with COPD 266

To study the role of TRPC6 protein expression level in normal and COPD bronchial smooth 267

muscle tissues, western blot analysis of TRPC6 expression was performed. As shown in Fig. 268

7A, it was significantly upregulated by ~2 fold in COPD versus normal bronchial smooth 269

muscle tissues. Consistent with western blot analysis of TRPC6 protein, 270

immunohistochemical staining analysis by α-SMC actin and TRPC6 antibody demonstrated, 271

the percentage of TRPC6 protein positive cells was much higher in COPD than normal 272

samples (Fig. 7B). Correlation study showed TRPC6 protein expression was proportional 273

with the bronchial smooth muscle layer thickness (pink) (Sup. Fig. 6). 274

Discussion 275

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Ca2+

signaling is central to agonist-elicited intrinsic contraction force generation in airway 276

smooth muscle 28

. Moreover, it is also a well-known pathway in mediating airway smooth 277

muscle cells proliferation. However, the signaling and transduction events that link [Ca2+

]i 278

regulation and cigarettes-induced the airway response are less established 29 30

. Cigarette 279

smoking is a major contributing factor in the onset and exacerbation of COPD 31 32

, 280

contributing to airway re-modelling. Nicotine, a well-believed major component of cigarette 281

smoking extract, has been demonstrated plays an essential role in developing enhanced 282

airway contraction and airway smooth muscle cell proliferation. Nicotine elevated [Ca2+

]i in 283

rat airway smooth muscle cells via activating and up-regulating α7 nAChR in ASMCs 33

34

. 284

Therefore, mapping the molecular mechanisms for nicotine induced alterations in bronchial 285

smooth muscle cells may offer clues into COPD pathogenesis and treatment. TRPC6 is 286

highly expressed in smooth muscle as well as in brain and cardiomyocytes. But few 287

physiological roles have been correlated with its expression. In this regard, we found 288

increased expression of TRPC6 and decreased miR-135a/b-5p in ASM tissue of bronchial 289

obtained from humans with COPD. Furthermore, even though there was an inter-individual 290

variation for miR-135a/b-5p and TRPC6 expression across our sample set, we were also able 291

to demonstrate a significantly inverse correlation existed between these two measurements in 292

normal and COPD bronchial smooth muscle tissue, suggesting that TRPC6 channel 293

expression was downregulated by miR-135a/b-5p in vivo as well. To date, however, given the 294

relative lack of research on miR-135a/b-5p in human smooth muscle disease, we wished to 295

investigate its functionality further by investigating how miR-135a/b-5p acts upon other 296

targets might contribute to the disruption of normal molecular pathways in COPD. Our study 297

is the first to show that miR-135a/b-5p expression is downregulated in a panel of bronchial 298

smooth muscle tissue of COPD patients in comparison with normal donor. Our in vitro assays 299

showed that over-expression of miR-135a/b-5p inhibited proliferation and migration of 300

HASMCs. We therefore conclude that miR-135a/b-5p can suppress proliferation in HASMCs. 301

Although several targets of miR-135a/b-5p have been biologically verified 35 36

, none has 302

been proven in HASMCs. This is also the first study to show that TRPC6 is regulated by 303

miR-135a/b-5p in HASMCs. More supporting evidence point out that miR-135a/b-5p 304

expression inversely correlated with TRPC6 levels, which suggested that miR-135a/b-5p 305

could be an important genetic marker for this disease. We propose that stratifying patients 306

into groups which show high or low miR-135a/b-5p levels could be a useful tool to identify 307

low- and high-risk for COPD progression, as others have done for miR-135a/b-5p expression 308

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in bladder cancer 37

, oral carcinoma 38

, lung cancer 39

, colorectal cancer 40

, and acute 309

leukemia 41

. We therefore conclude that miR-135a/b-5p is one of the factors contributing to 310

the regulation of TRPC6 expression in COPD and may explain why altered expression of 311

miR-135a/b-5p has been reported in COPD. This is significant because it means miR-135a/b-312

5p plays an integral role in control of cell proliferation and its aberrant expression is therefore 313

likely to disrupt normal cell growth. However, the effect of miR-135a/b-5p on TRPC6 alone 314

is not likely fully explain the phenotypic effects observed and it is worth remembering that 315

miR-135a/b-5p will undoubtedly exert an effect on several other targets that control cell 316

growth as well. It is important to emphasize that it is the precise balance of the interactions 317

discussed here, which will determine the overall functionality of miR-135a/b-5p in the cell 318

and explains the apparent ability of miR-135a/b-5p to act in contrasting fashions in different 319

disease. 320

Overall, the results presented here describe a novel pathway for HASMCs exposed to 321

nicotine in promoting proliferation through nicotine →α7 nAChR→ROS→NF-322

κB→TRPC6→NFAT signaling which have not been reported previously (Fig. 7C). We 323

propose a model in which nicotine, via NF-κB and miR-135a/b-5p, enhances expression and 324

activation of TRPC6 in ASM, leading to a phenotype switch from a contractile/quiescent to a 325

synthetic/proliferative type. 326

327

Acknowledgement 328

Contract grant sponsor: This study was supported by a grant from the Wenzhou Science and 329

Technology Burea (grant no. 2019Y0355) and the Natural Science Foundation of Zhejiang 330

(grant no. LQ18H070005). 331

Author contributions 332

Conception and design: Q.H., W.G., and Z.F. Experiments carry out: Q.H. and K.Z. 333

performed studies on molecular mechanism and function. J.W. performed the experiments on 334

the smoking mouse model. R.X. performed the human samples analysis. Data interpretation 335

and discussion: Q.H., W.G., and Z.F. 336

Conflicts of interest 337

The authors declare that they have no competing interests. 338

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1. Mannino DM, Buist AS. Global burden of COPD: risk factors, prevalence, and future trends. 340 Lancet 2007;370(9589):765-73. doi: 10.1016/S0140-6736(07)61380-4 341

2. Tannus-Silva DG, Rabahi MF. State of the Art Review of the Right Ventricle in COPD 342 Patients: It is Time to Look Closer. Lung 2016 doi: 10.1007/s00408-016-9961-5 343

3. Ten Broeke R, Blalock JE, Nijkamp FP, et al. Calcium sensors as new therapeutic targets 344 for asthma and chronic obstructive pulmonary disease. Clinical and experimental 345 allergy : journal of the British Society for Allergy and Clinical Immunology 346 2004;34(2):170-6. 347

4. Svobodova B, Groschner K. Reprint of "Mechanisms of lipid regulation and lipid gating in 348 TRPC channels". Cell calcium 2016;60(2):133-41. doi: 10.1016/j.ceca.2016.06.010 349

5. Liao Y, Plummer NW, George MD, et al. A role for Orai in TRPC-mediated Ca2+ entry 350 suggests that a TRPC:Orai complex may mediate store and receptor operated Ca2+ 351 entry. Proceedings of the National Academy of Sciences of the United States of 352 America 2009;106(9):3202-6. doi: 10.1073/pnas.0813346106 353

6. Xu SZ, Sukumar P, Zeng F, et al. TRPC channel activation by extracellular thioredoxin. 354 Nature 2008;451(7174):69-72. doi: 10.1038/nature06414 355

7. Boulay G, Zhu X, Peyton M, et al. Cloning and expression of a novel mammalian homolog 356 of Drosophila transient receptor potential (Trp) involved in calcium entry secondary 357 to activation of receptors coupled by the Gq class of G protein. J Biol Chem 358 1997;272(47):29672-80. 359

8. Garcia RL, Schilling WP. Differential expression of mammalian TRP homologues across 360 tissues and cell lines. Biochem Biophys Res Commun 1997;239(1):279-83. doi: 361 10.1006/bbrc.1997.7458 362

9. Riccio A, Medhurst AD, Mattei C, et al. mRNA distribution analysis of human TRPC family 363 in CNS and peripheral tissues. Brain Res Mol Brain Res 2002;109(1-2):95-104. 364

10. Muller M, Essin K, Hill K, et al. Specific TRPC6 channel activation, a novel approach to 365 stimulate keratinocyte differentiation. J Biol Chem 2008;283(49):33942-54. doi: 366 10.1074/jbc.M801844200 367

11. Weissmann N, Dietrich A, Fuchs B, et al. Classical transient receptor potential channel 6 368 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas 369 exchange. Proc Natl Acad Sci U S A 2006;103(50):19093-8. doi: 370 10.1073/pnas.0606728103 371

12. Gudermann T, Mederos y Schnitzler M, Dietrich A. Receptor-operated cation entry--more 372 than esoteric terminology? Sci STKE 2004;2004(243):pe35. doi: 373 10.1126/stke.2432004pe35 374

13. Holleman A, Chung I, Olsen RR, et al. miR-135a contributes to paclitaxel resistance in 375 tumor cells both in vitro and in vivo. Oncogene 2011;30(43):4386-98. doi: 376 10.1038/onc.2011.148 377

14. Zhou W, Li X, Liu F, et al. MiR-135a promotes growth and invasion of colorectal cancer 378 via metastasis suppressor 1 in vitro. Acta Biochim Biophys Sin (Shanghai) 379 2012;44(10):838-46. doi: 10.1093/abbs/gms071 380

15. Yang Y, Ishak Gabra MB, Hanse EA, et al. MiR-135 suppresses glycolysis and promotes 381 pancreatic cancer cell adaptation to metabolic stress by targeting 382 phosphofructokinase-1. Nat Commun 2019;10(1):809. doi: 10.1038/s41467-019-383 08759-0 384

16. Taipaleenmaki H, Browne G, Akech J, et al. Targeting of Runx2 by miR-135 and miR-203 385 Impairs Progression of Breast Cancer and Metastatic Bone Disease. Cancer Res 386 2015;75(7):1433-44. doi: 10.1158/0008-5472.CAN-14-1026 387

17. Nagel R, le Sage C, Diosdado B, et al. Regulation of the adenomatous polyposis coli 388 gene by the miR-135 family in colorectal cancer. Cancer Res 2008;68(14):5795-802. 389 doi: 10.1158/0008-5472.CAN-08-0951 390

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18. He F, Peng F, Xia X, et al. MiR-135a promotes renal fibrosis in diabetic nephropathy by 391 regulating TRPC1. Diabetologia 2014;57(8):1726-36. doi: 10.1007/s00125-014-3282-392 0 393

19. Tang W, Jiang Y, Mu X, et al. MiR-135a functions as a tumor suppressor in epithelial 394 ovarian cancer and regulates HOXA10 expression. Cell Signal 2014;26(7):1420-6. doi: 395 10.1016/j.cellsig.2014.03.002 396

20. Yamada Y, Hidaka H, Seki N, et al. Tumor-suppressive microRNA-135a inhibits cancer 397 cell proliferation by targeting the c-MYC oncogene in renal cell carcinoma. Cancer Sci 398 2013;104(3):304-12. doi: 10.1111/cas.12072 399

21. Xie W, Kantarcioglu M, Bush WS, et al. SecureMA: protecting participant privacy in 400 genetic association meta-analysis. Bioinformatics 2014;30(23):3334-41. doi: 401 10.1093/bioinformatics/btu561 402

22. Ezzie ME, Crawford M, Cho JH, et al. Gene expression networks in COPD: microRNA and 403 mRNA regulation. Thorax 2012;67(2):122-31. doi: 10.1136/thoraxjnl-2011-200089 404

23. Rathore R, Zheng YM, Li XQ, et al. Mitochondrial ROS-PKCepsilon signaling axis is 405 uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle 406 cells. Biochem Biophys Res Commun 2006;351(3):784-90. doi: 407 10.1016/j.bbrc.2006.10.116 408

24. Wang QS, Zheng YM, Dong L, et al. Role of mitochondrial reactive oxygen species in 409 hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes. 410 Free Radic Biol Med 2007;42(5):642-53. doi: 10.1016/j.freeradbiomed.2006.12.008 411

25. Stolk J, Hiltermann TJ, Dijkman JH, et al. Characteristics of the inhibition of NADPH 412 oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J 413 Respir Cell Mol Biol 1994;11(1):95-102. doi: 10.1165/ajrcmb.11.1.8018341 414

26. Christenson SA, Steiling K, van den Berge M, et al. Asthma-COPD overlap. Clinical 415 relevance of genomic signatures of type 2 inflammation in chronic obstructive 416 pulmonary disease. American journal of respiratory and critical care medicine 417 2015;191(7):758-66. doi: 10.1164/rccm.201408-1458OC 418

27. Xiao JH, Zheng YM, Liao B, et al. Functional role of canonical transient receptor potential 419 1 and canonical transient receptor potential 3 in normal and asthmatic airway 420 smooth muscle cells. American journal of respiratory cell and molecular biology 421 2010;43(1):17-25. doi: 10.1165/rcmb.2009-0091OC 422

28. Wylam ME, Xue A, Sieck GC. Mechanisms of intrinsic force in small human airways. 423 Respir Physiol Neurobiol 2012;181(1):99-108. doi: 10.1016/j.resp.2012.01.011 424

29. Song T, Zheng YM, Vincent PA, et al. Canonical transient receptor potential 3 channels 425 activate NF-kappaB to mediate allergic airway disease via PKC-alpha/IkappaB-alpha 426 and calcineurin/IkappaB-beta pathways. FASEB journal : official publication of the 427 Federation of American Societies for Experimental Biology 2016;30(1):214-29. doi: 428 10.1096/fj.15-274860 429

30. Song T, Hao Q, Zheng YM, et al. Inositol 1,4,5-trisphosphate activates TRPC3 channels 430 to cause extracellular Ca2+ influx in airway smooth muscle cells. American journal of 431 physiology Lung cellular and molecular physiology 2015;309(12):L1455-66. doi: 432 10.1152/ajplung.00148.2015 433

31. Demedts IK, Demoor T, Bracke KR, et al. Role of apoptosis in the pathogenesis of COPD 434 and pulmonary emphysema. Respiratory research 2006;7:53. doi: 10.1186/1465-435 9921-7-53 436

32. Daijo H, Hoshino Y, Kai S, et al. Cigarette smoke reversibly activates hypoxia-inducible 437 factor 1 in a reactive oxygen species-dependent manner. Scientific reports 438 2016;6:34424. doi: 10.1038/srep34424 439

33. Jiang Y, Dai A, Zhou Y, et al. Nicotine elevated intracellular Ca(2)(+) in rat airway 440 smooth muscle cells via activating and up-regulating alpha7-nicotinic acetylcholine 441 receptor. Cellular physiology and biochemistry : international journal of experimental 442

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cellular physiology, biochemistry, and pharmacology 2014;33(2):389-401. doi: 443 10.1159/000356678 444

34. Hong W, Peng G, Hao B, et al. Nicotine-Induced Airway Smooth Muscle Cell Proliferation 445 Involves TRPC6-Dependent Calcium Influx Via alpha7 nAChR. Cell Physiol Biochem 446 2017;43(3):986-1002. doi: 10.1159/000481651 447

35. Goto Y, Kojima S, Nishikawa R, et al. The microRNA-23b/27b/24-1 cluster is a disease 448 progression marker and tumor suppressor in prostate cancer. Oncotarget 449 2014;5(17):7748-59. doi: 10.18632/oncotarget.2294 450

36. Qin W, Shi Y, Zhao B, et al. miR-24 regulates apoptosis by targeting the open reading 451 frame (ORF) region of FAF1 in cancer cells. PLoS One 2010;5(2):e9429. doi: 452 10.1371/journal.pone.0009429 453

37. Inoguchi S, Seki N, Chiyomaru T, et al. Tumour-suppressive microRNA-24-1 inhibits 454 cancer cell proliferation through targeting FOXM1 in bladder cancer. FEBS Lett 455 2014;588(17):3170-9. doi: 10.1016/j.febslet.2014.06.058 456

38. Lin SC, Liu CJ, Lin JA, et al. miR-24 up-regulation in oral carcinoma: positive association 457 from clinical and in vitro analysis. Oral Oncol 2010;46(3):204-8. doi: 458 10.1016/j.oraloncology.2009.12.005 459

39. Zhao G, Liu L, Zhao T, et al. Upregulation of miR-24 promotes cell proliferation by 460 targeting NAIF1 in non-small cell lung cancer. Tumour Biol 2015;36(5):3693-701. doi: 461 10.1007/s13277-014-3008-4 462

40. Gao Y, Liu Y, Du L, et al. Down-regulation of miR-24-3p in colorectal cancer is 463 associated with malignant behavior. Med Oncol 2015;32(1):362. doi: 464 10.1007/s12032-014-0362-4 465

41. Akbari Moqadam F, Boer JM, Lange-Turenhout EA, et al. Altered expression of miR-24, 466 miR-126 and miR-365 does not affect viability of childhood TCF3-rearranged 467 leukemia cells. Leukemia 2014;28(5):1008-14. doi: 10.1038/leu.2013.308 468

469

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Figure 1 TRPC6’s effect on growth and mobility in normal and COPD ASMCs. 470

A. COPD HASMCs proliferated faster than normal HASMCs. HASMCs isolated from 471

normal donors and COPD patients were cultured for 1, 2, 3, 4, 5, 6, 7, 8 days. MTS assay 472

was performed for proliferation analysis. Experiment was repeated in three independent 473

groups. *, p<0.05; **, p<0.01; ***, p<0.005 compared with normal group. 474

B. TRPC6 promoted normal HASMCs proliferation. Normal HASMCs were infected with 475

lentivirus of TRPC6 or control, then cultured for 1, 2, 3, 4, 5, 6, 7, 8 days. MTS assay was 476

performed to proliferation analysis. Experiment was repeated in three independent groups. 477

*, p<0.05; **, p<0.01; ***, p<0.005 compared with control group. 478

C. Knockdown and inhibition of TRPC6 inhibited HASMCs proliferation. COPD HASMCs 479

were infected with lentivirus of TRPC6_SH, control and treated with SAR 7334 (1μM) 480

respectively for 1, 2, 3, 4, 5, 6, 7, 8 days. MTS assay was performed to proliferation 481

analysis. Experiment was repeated in three independent groups. *, p<0.05; **, p<0.01, 482

compared with control group. 483

D. TRPC6 protein expression was increased in cultured COPD HASMCs than normal 484

HASMCs. HASMCs were isolated and cultured from normal bronchia of control subjects 485

and bronchia of patients with COPD. Western blots (left) for TRPC6 expression were 486

followed by densitometric analysis (right), ***, p<0.001. 487

E. TRPC6 was overexpressed and knocked down (F) in HASMCs. The HASMCs were 488

infected with increasing amounts of lenti-viral particles of Human_TRPC6 or 489

Human_TRPC6_SH for 5 days. Cells were harvested and analyzed by western blotting 490

for the TRPC6 expression, GAPDH was used as internal control. Right panel is 491

quantification, *, p<0.05; **, p<0.01; ***, p<0.005. 492

G. COPD promoted migration of HASMCs through TRPC6. Normal and COPD HASMCs 493

were infected with lentivirus of TRPC6_SH and TRPC6 respectively. Subsequent analysis of 494

cell migration was performed by crystal violet dye staining of HASMCs that migrated into 495

transwell. Right panel is quantification. The data are shown as the number of cells migrated 496

in 3 separate experiments. *, p<0.05; **, p<0.01; compared with control. #, p<0.05 compared 497

with normal. 498

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Figure 2 miR-135a/b-5p inhibits migration of ASMCs by binding TRPC6 mRNA 3’-499

UTR and degrading TRPC6 expression. 500

501

A. Hsa-miR-135a/b-5p downregulated TRPC6 protein level in 293T cells. 293T cells 502

transfected with 12 microRNAs expression plasmids predicted to bind TRPC6 mRNA 3’-503

UTR were harvested and analyzed by western blotting for the TRPC6 expression, 504

GAPDH was used as internal control. Right panel is quantification. Experiment was 505

repeated in three independent groups. ***, p<0.005. 506

B. miR-135a/b-5p deceased mRNA (C) and protein level of TRPC6 in both normal (D) and 507

COPD (E) HASMCs. HASMCs infected with lentivirus of miR-135a/b-5p were treated 508

with nicotine for 24hours. mRNA and protein expression were determined by real-time 509

PCR and western blotting, GAPDH as internal control. 510

C. Nicotine increased mRNA and protein level of TRPC6 in both normal (D) and COPD (E) 511

HASMCs. Experiments were repeated in three independent groups. *, p<0.05; **, p<0.01. 512

F. TRPC3 and TRPC6 mRNA expression was up-regulated by nicotine treatment in normal 513

HASMCs. qPCR was performed for the mRNA analysis, GAPDH as internal control. n=3, 514

*, p<0.05; ***, p<0.005. 515

G. Targeting site for hsa-miR-135a/b-5p was identified in TRPC6 3’-UTR. 516

H. TRPC6_3’UTR_576-589 site was indispensable for binding between hsa-miR-135a/b-5p 517

and TRPC6 mRNA. Two truncated and one full length TRPC6_3’UTR were cloned into 518

pmiR-target vector. These constructs and control vector were transfected into HASMCs 519

infected with lentivirus of miR-135a/b-5p respectively. Firefly luciferase expression was 520

normalized to that of renilla luciferase. Experiments were repeated in three independent 521

groups. ***, p<0.005. 522

I. Nicotine decreased miR-135a/b-5p expression in HASMCs. Normal HASMCs were treated 523

with nicotine for 24hours, then harvested for the miR-135a/b-5p expression analysis by real-524

time PCR. Experiments were repeated in three independent groups. *, p<0.05; **, p<0.01; 525

***, p<0.005. 526

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J. Down-regulation of miR-135a/b-5p was associated with up-regulation of TRPC6 in COPD 527

human bronchial smooth muscle tissue. Total RNA were extracted from normal and COPD 528

patient bronchial smooth muscle, the gene expression relationship was analyzed by real-time 529

PCR. n=23 (normal), n=12 (COPD). 530

K. Hsa-miR-135a/b-5p and SAR 7334 affected migration of normal and COPD HASMCs. 531

Hsa-miR-135a/b-5p and SAR 7334 significantly retarded migration of COPD HASMCs. 532

Normal and COPD HASMCs were infected with lentivirus of miR-135a/b-5p and treated 533

with SAR 7334 respectively. Subsequent analysis of cell migration by transwell assay. The 534

data are shown as the number of cells migrated in three separate experiments, right is 535

quantification. *, p<0.05, compared with normal group; #, p<0.05, ##, p<0.005, compared 536

with control group. 537

Figure 3 Nicotine activates activity NF-κB signaling through α7 nAChR in a ROS-538

dependent manner. 539

A. GSEA plot of enrichment in ‘‘BIOCARTA_NFKB_PATHWAY’’ and 540

“BIOCARTA_NFAT_PATHWAY” signatures MSigDB database in nicotine treatment 541

group versus control group (n = 3) using GSEA BIOCARTA biological process. 542

B. Nicotine promoted translocation of p50 and p65 into the nucleus. HASMCs were treated 543

with nicotine at increasing concentration (0, 10nM, 100nM, 1µM). Cytoplasmic and 544

nuclear fractions were prepared, separated by SDS-PAGE, and immunoblotted with the 545

indicated antibodies. GAPDH, Lamin A antibodies were used as internal controls for 546

cytoplasmic and nuclear proteins. 547

C. Nicotine promoted p65 and p50 dissociation from IκBα. HASMCs were treated with 548

nicotine (1µM) and PBS as vehicle. Cytosol was extracted, and IκBα, p65 and p50 were 549

analyzed by immunoprecipitation in combination with immunoblotting as indicated. 550

D. Pharmacological inhibition of NADPH oxidase significantly attenuated the nicotine-551

induced increase in [ROS]i in HASMCs. H2DCFDA (5 mM) fluorescence was recorded 552

with nicotine treatment at increasing concentration (0, 10nM, 100nM, 1µM) in HASMCs 553

pre-treated with Nox inhibitor apocynin (1μM) for 10 min. Inhibition of mitochondrial 554

complex I with rotenone (12.7 μM) or complex III preubisemiquinone site inhibitor 555

myxothiazol (10 μM) (F) for 10 min partly blocks the nicotine-induced increase in [ROS]i 556

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in HASMCs. Data are repeated in three independent experiments. ***, p<0.005 compared 557

with nicotine (0%) treatment. #, p<0.05; ##, p<0.01 compared with control group. 558

E. NAC attenuates nicotine-induced transcription activity of NF-κB in HASMCs. HASMCs 559

were transfected with NF-κB luciferase reporter plasmid and exposed to nicotine (1µM) 560

and different inhibitors as indicated. NF-κB reporter assays were performed after these 561

treatments. Firefly luciferase expression was normalized to renilla luciferase. 562

Experiments were repeated in three independent groups. *, p<0.05; **, p<0.01 compared 563

with nicotine (0%) group. ##, p<0.01 compared with control group. 564

F. MG 624 abolished nicotine-increased transcription activity of NF-κB in HASMCs. MG 565

624 is specific inhibitor of α7 nAChR. HASMCs were transfected with NF-κB luciferase 566

reporter plasmid. NF-κB reporter assays were performed after nicotine treatment at the 567

increasing concentration (0, 10nM, 100nM, 1 µM). Firefly luciferase expression was 568

normalized to renilla luciferase. Experiment was repeated in three independent groups. **, 569

p<0.01 compared with nicotine (0 µM) group. ##, p<0.01 compared with control group. 570

Figure 4 Nicotine regulates TRPC6 expression and activity through NF-κB. 571

A. Nicotine promoted NF-κB binding to TRPC6 promoter. TRPC6 promoter reporter gene 572

plasmids and NF-κB overexpression plasmids were transfected into HASMCs for 573

36hours. HASMCs were treated with nicotine (1µM) and BAY 11-7082 (10 μM) for 24 574

hours as indicated, then TRPC6 promoter reporter luciferase activity was measured after 575

treatment. Results were averages of three experiments. Firefly luciferase expression was 576

normalized to that of renilla luciferase. n=3, **, p<0.01; ***, p<0.005 compared with no 577

NF-κB and nicotine treatment; ##, p<0.01 compared with control group. 578

B. TRPC6 mRNA expression was up-regulated by nicotine treatment in HASMCs, and this 579

increase was abrogated by knockdown and inhibition of NF-κB. HASMCs infected with 580

lentivirus of p65_SH, p50_SH or treated with BAY 11-7082 (10 μM) were followed by 581

stimulation with nicotine (1µM) as indicated hours. qPCR was performed for the mRNA 582

analysis, GAPDH as internal control. n=3; ***, p<0.005 compared with control. ##, 583

p<0.01 compared with no p65_SH, si-p50_SH and BAY 11-2048 treatment group. 584

C. NF-κB knockdown attenuated nicotine-induced TRPC6 protein increase in HASMCs. 585

Normal HASMCs infected with lentivirus of p65_SH, p50_SH or control_SH were 586

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followed by stimulation with nicotine (1µM) as indicated hours. Protein expression of 587

TRPC6, p65, p50 and GAPDH were determined by immunoblotting, experiment was 588

repeated in three independent groups. 589

D. Inhibition of NF-κB abolished nicotine-induced TRPC6 protein increase in HASMCs. 590

Normal HASMCs were pre-treated with the NF-κB inhibitor BAY 11-7082 (10 μM) and 591

PBS as vehicle for 2 hours, followed by stimulation with nicotine (1µM) as indicated 592

hours, TRPC6 and GAPDH were determined by immunoblotting. 593

E. Electrophoretic mobility shift assays demonstrated interaction between NF-κB and the 594

NF-κB binding site of the TRPC6 promoter in HASMCs. HASMCs were grown and 595

treated with nicotine (1µM) for 0, 2, 4, 6, 8 hours, then cells were harvested for gel-shift 596

and supershift (F) assays analyses. 597

Figure 5 TRPC6 knockdown ameliorates cigarette smoke induced ASM 598

hyperresponsiveness and remodeling in mice. 599

A. Inhalation of cigarette smoke strengthened methacholine-induced increase of airway 600

hyperresponseness. Penh to the muscarinic agonist mAch was assessed by a non-invasive 601

unrestricted whole-body plethysmography system. *, p<0.05; **, p<0.01 compared with 602

mice inhaled PBS and infected with lentivirus of control_sh mice. #, p<0.05; ##, p<0.01 603

compared with mice inhaled cigarette smoke and infected with lentivirus of control_sh. 604

B. Inhalation of cigarette smoke strengthened methacholine-induced increase of airway 605

contraction. In vitro airway muscle contractile responses to mAch were recorded in 606

isolated airway (tracheal) rings from mice. *, p<0.05; **, p<0.01 compared with mice 607

inhaled PBS and infected with lentivirus of control_sh mice. #, p<0.05; ##, p<0.01 608

compared with mice inhaled cigarette smoke and infected with lentivirus of control_sh. 609

C. Cigarette smoke and TRPC6 coordinated the selection of NFAT and its target gene 610

expression. The airway smooth muscle was collect from the above mice model. mRNA 611

levels of NFAT and its target genes were determined by quantitative real-time PCR. ***, 612

p<0.005, compared with mice infected with lentivirus of control_sh and inhaled with PBS; 613

##, p<0.01, compared with mice infected with lentivirus of TRPC6_sh and inhaled with 614

cigarette smoke. 615

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D. Immunohistochemistry co-stains of α-smooth muscle actin (pink) and Ki67 (brown) in 616

airways in mice model infected with lentivirus of TRPC6_sh or control_sh, inhaled with 617

cigarette smoke or PBS as vehicle. Arrows indicated co-localization of α-smooth muscle 618

actin and Ki67. Inserted picture was enlarged part of smooth muscle layer. Scale bars, 20 619

μm. 620

E. Quantification of smooth muscle layer area and Ki67 from panel D. **, p<0.01; ***, 621

p<0.005, compared with mice infected with lentivirus of control_sh and inhaled with PBS. 622

#, p<0.05; ##, p<0.01, compared with mice infected with lentivirus of TRPC6_sh and 623

inhaled with cigarette smoke. 624

Figure 6 miR-135a/b-5p’s effect in a murine model of asthma-COPD overlap syndrome. 625

A. Treatment of ovalbumin and cigarette smoke enhanced methacholine-induced increase of 626

airway resistance. Mice were tail vein injected with lentivirus particles of miR-135a/b-5p. 627

After 10 days, the animals were intraperitoneally injected with ovalbumin and/or inhaled 628

with cigarette smoke. *, compared with mice treated with PBS; **, p<0.01; ***, p<0.005. 629

#, compared with mice treated with ovalbumin+ cigarette smoke; #, p<0.05; ##, p<0.01. 630

B. Treatment of ovalbumin and cigarette smoke enhanced methacholine-induced increase of 631

airway contraction. In vitro airway muscle contractile responses to mAch were recorded 632

in isolated airway (tracheal) rings from above mice model. **, p<0.01; ***, p<0.005 633

compared with mice injected and inhaled with PBS. #, p<0.05; ##, p<0.01 compared with 634

mice injected and inhaled with ovalbumin + cigarette smoke. 635

C. Immunohistochemistry co-stains of α-smooth muscle actin (pink) and Ki67 (brown) in 636

airways of above mice model and quantification of smooth muscle layer area (right panel). 637

Figure 7 TRPC6 channel protein expression in human samples. 638

A. TRPC6 protein expression was increased in bronchia tissue isolated from COPD 639

patients compared with normal donors. Western blots for TRPC6 in tissue extracts of 640

normal donors and COPD bronchial smooth muscle followed by densitometric 641

analysis (right), **, p<0.01. 642

B. Immunohistochemistry co-stains of α-smooth muscle actin (pink) and TRPC6 (brown) 643

in bronchia of normal donors and COPD patients. Arrows indicate co-localization of 644

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α-smooth muscle actin and TRPC6. A relative area of smooth muscle layer (pink) was 645

calculated, quantified using ImageJ software. A percentage of TRPC6-positive cells 646

was calculated by dividing the number of cell with TRPC6-positive cell by the total 647

number of cells with TRPC6 and hematoxylin-positive nucleus. Inserted picture was 648

enlarged part of smooth muscle layer. Bar graph indicates the quantification of airway 649

smooth muscle areas and summary of TRPC6-positive cells in airway smooth muscle 650

layers in each groups. Scale bars, 20 μm. Right panel is quantification of smooth 651

muscle layer area and TRPC6. **, p<0.01. 652

C. A schematic model showing the essential role of miR-135a/b-5p- and NF-κB-653

dependent TRPC6 channels in COPD. In HASMCs, nicotine stimulates the indicated 654

signaling cascades. This activation triggers the NF-κB pathway in a ROS-dependent 655

manner through α7 nAChR, leading to translocation of p65 and p50. Sustained 656

transcriptional activation signaling induces to TRPC6 expression increase. Meantime, 657

down-regulation of miR-135a/b-5p by nicotine stimulation releases the inhibition of 658

TRPC6 by miR-135a/b-5p, involving direct influx of Ca2+

and activation of NFAT 659

signaling, which in turn, induces activation of downstream gene leading to the 660

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Control

TRPC6

GAPDH

TRPC6

BA

GAPDH

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miR-9

23-5p

miR-4

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miR-9

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miR-5

76-3p

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83-5p

miR-1

229-3p

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Figure 2

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TRPC6

GAPDH

Control

Nicotine

miR-1

35b-5p

Control

miR-135b-5p

miR-135b-5pmiR-135a-5p

**

miR-135a/b-5p

Normal (n=23)

COPD (n=12)

Pearson r = -0.6432

miR-135a/b-5p

2R = 0.4138-

P value= 0.0090-

Pearson r = -0.72762R = 0.5294 -

P value= 0.0073 -

0 0

TR

PC

6 m

RN

A

2

3

1

4

5

6

7J

1 2 3 4 5

I

miR

s

1.0

1.2

0.6

Relative Luciferase Activity

1.0 1.50.0 0.5

H

TRPC6-3’UTR0.8

1-43 0.4

*** 0.2

***FL 0.01 43 49 1391

Seed Sequence

TRPC6 mRNA

5’-UTR 3’-UTR CDS

II I I I IGGUCUG U AUU UCUG U UUUA A C A

AC CAT TTT C GATT--- T AG A CG TTC5’ --- 3’ATI

TAT

UI

GGUCUG U AUU CCUG U UUUA A C A U

miR-135a-5p

miR-135b-5p

G

TR

PC

6

mR

NA

1 2 3 1 2 3 1 2 3 51 52 53 51 52 53 51 52 53

Normal

COPD

Normal

COPD

Normal

COPD

ControlK

SAR 7334miR-135a/b-5p2.0

Normal*

Nor

mal

COPD1.5

Ce

lls/f

ield

1.0

CO

PD

0.5

0.0Control

miR-135a/b-5pSAR 7334

miR-135a-5p

*

*****

miR-135b-5p

*

*****

Nicotine

ED F

Nicotine

Luc

Luc

Luc

Luc

1-49

####

##

WITHDRAWN



https://doi.org/10.1101/2020.01.16.908699

p65

GAPDH

Lamin A

p50

p65

p50

GAPDH

Lamin A

C

E

B

NF

-κΒ

Rel

ativ

e A

ctiv

ity

IgG IκBα IgG IκBα

Input IP Input IP

Control

F

p65

p50

p65

p50

IκBα

IgG IκBα IgG IκBα

0

2

4

6

NF-

κΒ R

elat

ive

Act

ivity

4

6

0

2

N-acetyl-cysteine

##

##

MyxothiazolApocynin

RotenoneControl

Cyt

oso

lN

ucl

eu

s

## ####

0

200

400

600

RO

S (

RLU

)

MyxothiazolApocynin RotenoneControl

Control MG 624

**

* *

**

****

Figure 3

1

3

5

3

5

1

0.0

0.2

0.4

0.6

0.1

0.3

0.5

0.7

0.8

75,000

50,000

25,000

0

25,0000

0.0

0.2

0.4

0.6

0.1

0.3

0.5

0.7

0.8

75,000

50,000

25,000

0

75,00050,00025,0000 100,000

A

75,00050,000 100,000

Nicotine

Ra

nke

d lis

t me

tric

(P

rera

nke

d)

En

rich

me

nt S

core

(E

S) Enrichment plot: BIOCARTA_NFKB_PATHWAY Enrichment plot: BIOCARTA_NFAT_PATHWAY

Ra

nke

d lis

t me

tric

(P

rera

nke

d)

En

rich

me

nt S

core

(E

S)

Rank in ordered datasetRank in ordered dataset

Nicotine Nicotine***

NicotineControl Nicotine

D

******

##

## ##

##

****WITHDRAWN



https://doi.org/10.1101/2020.01.16.908699

p50

GAPDH

p65

TRPC6

Control NF-κΒ_Sh

0 2 4 6 8 10 0 2 4 6 8 10

Control BAY 11-7082

TRPC6

GAPDH

IgG

Anti-p65

2nM Biotin Probe-WT

Nuclear Extract

Supershifted Probe

Shifted Probe

- + + + +

+ + + +

+ - +

+ + +

+ - +

+ + + 2nM Biotin Probe-WT

Nuclear Extract

(hrs)

Shifted Probe

C

E

NF-κΒ NF-κΒ_Sh

BAY 11-7082

TRP

C6

mR

NA

1

2

3

4

0

- + - + - + - +

A B

0

2

3

4

5

**

***

##

TR

PC

6 R

elat

ive

Act

ivity

BAY 11-7082

####

Free probe

Free probe

+

(hrs)

F

(hrs)

- + - + - + - + - + - + - - - - - + - +

0 4 6 82

D

Control

**

*** Control

1

Figure 4

Nicotine

Nicotine

Nicotine Nicotine

Nicotine

0 2 4 6 8 10 0 2 4 6 8 10

WITHDRAWN



https://doi.org/10.1101/2020.01.16.908699

Cig

arr

ete

Control

PB

S

2

6

10 PBS+Control

Ccnd3

mR

NA

PBS+TRPC6_sh

Cigarrete+TRPC6_shCigarrete+Control

A D

0

0Air

wa

y R

esi

sta

nce

(P

en

h R

ati

o)

10

#

20 ##**

30

***40

Log[mACH] (μM)-3

0

Fo

rce

(mg

/mg

)

200

400 * ###

##

**** **600

mACH (mg/ml) -2 -1 0 1 2 3

Cigarrete+TRPC6_sh (n=9)Cigarrete+Control (n=7)

C

8

Sm

oo

th M

usc

le L

aye

r Are

a

2

4

6

0

Ki6

7 p

osi

tive

(%

)

0

1

3

2

4

#

PBS+Control

PBS+TRPC6_sh Cigarrete+TRPC6_sh

Cigarrete+Control

0

4

8

100

300

500

Figure 5

NFAT Cdk6 Ccnd1

1

5

9

3

7

PBS+TRPC6_sh (n=9)PBS+Control (n=8)

1 3 10 100 30

TRPC6_sh

E

B

***

## ***##

***

##

***

##

***

##

**

WITHDRAWN



https://doi.org/10.1101/2020.01.16.908699

Airw

ay R

esis

tanc

e (

Pen

h R

atio

)

PBS

Cigarrete+Ovalbumin+miR-135a/b-5p

miR-135a/b-5p

Ovalbumin Cigarrete+Ovalbumin

Cigarrete

Fo

rce (

mg

/mg

)

20

30

0

10

40 1000

0

200

400

800

A

C

Sm

oo

th M

usc

le L

aye

r Are

a

0

1

2

3

4

5

PBS+PBS

PBS+Cigarrete

PBS+Ovalbumin

Cigarrete+Ovalbumin

PBS+miR-135a/b-5p

Cigarrete+Ovalbumin+

miR-135a/b-5p

##

**

##

*****

*

###

Log[mACH] (μM) mACH (mg/ml)

600

*****

##

Ovalmunin+Cigarrete (n=9)PBS+Cigarrete (n=9)PBS+Ovalmunin (n=9)PBS+PBS (n=8)

PBS+PBS+miR-135a/b-5p (n=9)Ovalmunin+Cigarrete+miR-135a/b-5p (n=9)

Ovalmunin+Cigarrete (n=9)PBS+Cigarrete (n=9)

PBS+Ovalmunin (n=9)PBS+PBS (n=8)

PBS+PBS+miR-135a/b-5p (n=9)Ovalmunin+Cigarrete+miR-135a/b-5p (n=9)

-3 -2 -1 0 1 2 3 100

Figure 6

0 303 101

B

WITHDRAWN



https://doi.org/10.1101/2020.01.16.908699

**

Normal

TRPC6

GAPDH

Noamal_

4

Noamal_

3

Noamal_

6

COPD_54

COPD_55

COPD_56

Noamal_

1

Noamal_

5

Noamal_

2

COPD_51

COPD_53

COPD_52

COPD

TR

PC

6/G

AP

DH

1.0

1.5

0.0

0.5

2.0

2.5

CO

PD

Normal

A

** COPD

Sm

ooth

Mus

cle

Laye

r Are

a2

3

4

0

1

TR

PC

6 p

osi

tive

ce

lls

2

3

4

0

1

Figure 7

Noamal_3Noamal_1 Noamal_2

COPD_51 COPD_53COPD_52

Normal

B

No

rma

l

COPDNorm

al

COPD

C

****

WITHDRAWN



https://doi.org/10.1101/2020.01.16.908699

core role of trpc6 channels in regulating airway re ...jan 16, 2020 · 130 a very efficient...

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