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Autophagy

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GRSF1-mediated MIR-G-1 promotes malignantbehavior and nuclear autophagy by directlyupregulating TMED5 and LMNB1 in cervical cancercells

Zhen Yang, Qi Sun, Junfei Guo, Shixing Wang, Ge Song, Weiying Liu, Min Liu &Hua Tang

To cite this article: Zhen Yang, Qi Sun, Junfei Guo, Shixing Wang, Ge Song, Weiying Liu, MinLiu & Hua Tang (2018): GRSF1-mediated MIR-G-1 promotes malignant behavior and nuclearautophagy by directly upregulating TMED5 and LMNB1 in cervical cancer cells, Autophagy, DOI:10.1080/15548627.2018.1539590

To link to this article: https://doi.org/10.1080/15548627.2018.1539590

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ARTICLE

GRSF1-mediated MIR-G-1 promotes malignant behavior and nuclear autophagy bydirectly upregulating TMED5 and LMNB1 in cervical cancer cellsZhen Yang*, Qi Sun*, Junfei Guo, Shixing Wang, Ge Song, Weiying Liu , Min Liu, and Hua Tang

Tianjin Life Science Research Center and Department of Pathogen Biology, Collaborative Innovation Center of Tianjin for Medical Epigenetics,School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China

ABSTRACTEmerging evidence has revealed that miRNAs could upregulate the expression levels of target genes.However, the molecular mechanism underlying upregulation of targets mediated by miRNAs remainsunclear. In this study, we found a novel miRNA named MIR-G-1 by GRSF1-RNA immunoprecipitation(RIP)-deep sequencing, which could directly target and upregulate LMNB1 and TMED5 in a GRSF1-dependent manner in cervical cancer cells. In addition, upregulated MIR-G-1 in cervical cancer promoteda malignant phenotype in vitro and in vivo. TMED5 could interact with WNT7B and thus activated thecanonical WNT-CTNNB1/β-catenin signaling pathway. MIR-G-1 mediated the activation of this pathway.Furthermore, MIR-G-1 promoted serum starvation-induced nuclear macroautophagy/autophagy, andaccelerated taxol (TAX)-induced DNA-damage repair in cervical cancer cells. Collectively, these findingsmay provide a new insight into the upregulation mechanism and nuclear autophagy mediated bymiRNAs and provide a potential biomarker for cervical cancer.

Abbreviations: 3ʹUTR: 3′ untranslated region; EMSA: electrophoretic mobility shift assay; EMT: epithelial-mesenchymal transition; GRSF1: G-rich RNA sequence binding factor 1; IF: immunofluorescence; IP:immunoprecipitation; IHC: immunohistochemistry; lnc: long noncoding; miRNA:microRNA; TAX: taxol;TMED5: transmembrane p24 trafficking protein 5

ARTICLE HISTORYReceived 5 January 2018Revised 14 October 2018Accepted 17 October 2018

KEYWORDSCervical cancer; GRSF1;LMNB1; novel miRNA;nuclear autophagy; TMED5

Introduction

MicroRNAs (miRNAs) are a class of naturally occurring shortRNAs (~ 18–22 nucleotides) that regulate the expression oftarget genes by affecting translational repression or mRNAcleavage [1,2], which play an important role in the tumorigen-esis, functioning as oncogenes or tumor suppressors in can-cers [3,4]. Given that miRNAs suppress the expression levelsof target genes by binding with partial complementarity totheir target sites in the 3′ untranslated region (UTR) of spe-cific mRNAs [5,6], emerging evidence has revealed thatmiRNAs could upregulate the expression of target genes bydifferent molecular mechanisms [7,8]. For example, Huanget al. revealed that MIR744 upregulates the expression ofCCNB1 by promoting enrichment of RNA polymerase II(RNAP II) and trimethylation of histone 3 at lysine 4(H3K4me3) at the CCNB1 transcription start site [9]. In addi-tion, MIR122 can enhance hepatitis C virus (HCV) genereplication by targeting 5ʹ-noncoding elements in the HCVgenome [10]. Furthermore, MIR369-3 activates mRNA trans-lation by targeting AU-rich elements in 3ʹUTRs under condi-tions of serum starvation [11]. More importantly, ourprevious study has demonstrated that GRSF1 (G-rich RNAsequence binding factor 1) mediates the MIR346-dependentupregulation of TERT/hTERT by directly binding to the

MIR346 sequences, and then facilitates the recruitment ofTERT mRNA to ribosomes to promote translation in anAGO2-independent manner [12]. However, whether GRSF1mediates the other miRNAs to upregulate the expression oftarget genes remains unknown.

GRSF1 was originally identified as an RNA-binding proteinwith high affinity for G-rich sequences [13], which plays keyroles in all steps of post-transcriptional regulation of RNAs,including RNA transport and localization, RNA stability,RNA splicing, and translation by binding with the uniquemRNAs via RNA-binding domains in a sequence- and struc-ture-specific manner [14–16]. Recently, Noh et al. reportedthat GRSF1 can interact with the long noncoding (lnc)-RMRPand facilitate the localization of lnc-RMRP into the mitochon-drial matrix [17]; lnc-RMRP was well known for being acomponent of the nuclear RNase MRP complex, which parti-cipates in the processing of ribosomal RNA in yeast [18].These data indicate that GRSF1 mediates the function ofnoncoding RNAs to regulate the process of transcriptionand the expression of mRNA and protein.

Autophagy is a highly conserved homeostatic mechanismfrom yeast to human that targets cellular contents to thelysosomal compartment to regulate a wide range of cellularfunctions, which can be selective and nonselective [19,20].

CONTACT Hua Tang htang2002@yahoo.com Tianjin Life Science Research Center and Department of Pathogen Biology, Collaborative Innovation Center ofTianjin for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, 22 Qi-Xiang-Tai Road, Tianjin 300070, China

*These authors contributed equally to this work.Supplemental data for this article can be accessed here.

AUTOPHAGYhttps://doi.org/10.1080/15548627.2018.1539590

© 2018 Informa UK Limited, trading as Taylor & Francis Group

According to the unique substrate delivered, selective autop-hagy is termed, for example, mitophagy [21], reticulophagy[22], lysophagy [23], proteaphagy [24], nucleophagy [25] andxenophagy [26]. However, whether miRNAs play a role in theprocess of nuclear autophagy remains unclear. In addition,some papers reported that autophagy can regulate DNAdamage repair [27]. To investigate the role of MIR-G-1 onDNA repair, we used TAX to induce DNA damage accordingto previous references [28,29].

In the present study, we identified a novel miRNA namedMIR-G-1 by GRSF1-RIP-deep sequencing in HeLa cells. Thelevels of MIR-G-1 in cervical cancer tissues and serum andcervical cancer cell lines were upregulated compared to thecontrol groups. MIR-G-1 overexpression promoted cell pro-liferation, migration and invasion, accelerated cell cycle andEMT progression, inhibited apoptosis and anoikis, andenhanced the resistivity for cis-platinum by upregulatingTMED5 in cervical cancer cells. MIR-G-1 overexpression invivo promoted the tumor growth. In addition, we found thatTMED5 could interact with WNT7B and thus activated theWNT-CTNNB1/β-catenin pathway. MIR-G-1 mediated theactivation of this pathway. MIR-G-1 overexpression promotedthe serum starvation- induced nuclear autophagy by targetingand up-regulating LMNB1, and accelerated the TAX-inducedDNA-damage repair in cervical cancer cells. Furthermore, we

demonstrated that MIR-G-1 upregulates TMED5 and LMNB1in a GRSF1-dependent manner in the silencd-GRSF1-HeLacells by shRNA-GRSF1 (shR-GRSF1-HeLa cells).

Collectively, we revealed that the novel MIR-G-1 pro-moted nuclear autophagy and malignant behavior in cervicalcancer cells by targeting LMNB1 and TMED5 in a GRSF1-dependent manner. These findings may provide a newinsight into the upregulation mechanism and nuclear autop-hagy mediated by miRNAs, and might provide a potentialbiomarker for cervical cancer.

Results

Analysis of mirnas from flag-grsf1-rip deep sequencing incervical cancer cells

To identify whether GRSF1 can mediate the other miRNAs up-regulating their target genes expression in HeLa cells, a Flag-GRSF1-RIP-small RNA library was constructed and sequenced.As shown in Figure S1, 618 known miRNAs and 12 novelmiRNAs were enriched in the complex of Flag-GRSF1-RIP(Figure S1). In addition, the sequencing data showed approxi-mately 400,303 (2.91%) reads of known miRNAs and 823(0.01%) reads of novel miRNAs (Figure 1(a)). Nucleotide biasanalysis indicated that 18 to 25 nucleotide conserved miRNAs

Figure 1. Analysis of miRNAs from Flag-GRSF1-RIP deep sequencing. (a) Venn diagram of differential RNA enrichment in the Flag-GRSF1-RIP complex. (b) Firstnucleotide bias of the novel miRNAs. (c) Nucleotide bias at each position of the novel miRNAs. (d) Statistics of pathway enrichment in the Flag-GRSF1-RIP complex.(e) RT-qPCR showed the levels of G-miRNAs in HeLa cells. Experiments were performed 3 times, and data are presented as means ± SD.*P < 0.05; **P < 0.01;***P < 0.001; ns, not significant.

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prefer G or C at the first position (Figure 1(b)). We firstanalyzed these novel miRNAs, which showed that C wasmost often used (74.2%) as the first nucleotide at the 5′ end(Figure 1(c)). For further functional annotation, all predictedtargets were analyzed by GO terms using the Blast 2 GOprogram with default parameters. There were 8611 predicatedtargets were identified for the top 47 enriched GO categories interms of biological process, cellular component and molecularfunction (Figure S2). Furthermore, KEGG pathway annotationshowed that these target genes were significantly enriched in 20canonical pathways, especially in metabolic pathways andtumorigenesis-related pathways (Figure 1(d)). RT-qPCR wasused to verify the accuracy of the sequencing data, and theresults showed that these novel miRNAs were existent in HeLacells and the levels of MIR-G-1 were comparatively highercompared with the other novel miRNAs (Figure 1(e)).

MIR-G-1, a novel identified mirna, is upregulated incervical cancer cells

According to the sequencing data, we found that MIR-G-1had a standard structure of precursor-MIR-G-1 (chr 22:11629598–11629709: +) (Figure 2(a)) and mature-MIR-G-1(MIR-G-1-5p and MIR-G-1-3p) (Figure 2(b)). RT-qPCRassay indicated that the MIR-G-1-5p was higher than that ofMIR-G-1-3p in HeLa and S12 cells, indicating that MIR-G-1-

3p may degrade during the maturation process of MIR-G-1(Figure 2(c)). Furthermore, northern blot assay also con-firmed the existence of pre-MIR-G-1 and MIR-G-1-5p inHeLa and C33A cells (Figure 2(d)).

To investigate the function of MIR-G-1, we first performedRT-qPCR analysis to examine the expression of MIR-G-1 invarious human cancer cells including cervical cancer. Asshown in Figure 2(e), MIR-G-1 expression was significantlyupregulated in 3 cervical cancer cell lines (HeLa, C33A andSiHa) compared with that in an immortalized normal humancervical epithelial cell line (S12). In addition, MIR-G-1 levelswere also detected in other cancer cell lines (BGC-823, QGY-7703, SW480, SW620 and HepG2) (Figure 2(e)). Next, weexamined the levels of MIR-G-1 in serum and tissues ofpatients with cervical cancer by RT-qPCR. As shown inFigure 2(f,g), MIR-G-1 levels were higher in cervical cancerserum and tissues compared with that in the control groups(Figure 2(f,g)). These data indicated that the novel MIR-G-1 isupregulated and may be an onco-miRNA in cervical cancer.

MIR-G-1 promotes proliferation of cervical cancer cells invitro and tumor growth in vivo

To assess the biological function of MIR-G-1 in cervical cancertumorigenesis, we first verified the efficiency of overexpressionand knockdown plasmids of MIR-G-1 in HeLa and C33A cells

Figure 2. The novel miRNA MIR-G-1 is upregulated in cancer. (a) Prediction for the standard structure of pre-MIR-G-1. (b) The sequence of mature MIR-G-1-3p andMIR-G-1-5p. (C) RT-qPCR showed the level of MIR-G-1-3p and MIR-G-1-5p in HeLa and S12 cells. (d) Northern blot assay indicated that MIR-G-1 is existent in HeLa andC33A cells. (e) RT-qPCR showed the level of MIR-G-1-5p in diverse cancer cells. (f) RT-qPCR showed the level of MIR-G-1-5p in the serum of cervical cancer patients andnormal people. (g) RT-qPCR showed the level of MIR-G-1-5p in tissues. Experiments were performed 3 times, and data are presented as means ± SD.*P < 0.05;**P < 0.01; ***P < 0.001; ns, not significant.

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(Figure S3(a)). MTT assays were then performed to investigate therole ofMIR-G-1 in cell viability. Enforced expression ofMIR-G-1indeed promoted cell viability in HeLa and C33A cells, whereastransfection of anti-MIR-G-1 inhibited cell viability of these cells(Figure 3(a)). Furthermore, colony formation assays and EdUassays also demonstrated that ectopic expression of MIR-G-1increased cell proliferation in HeLa and C33A cells, whereasdownregulation of MIR-G-1 inhibited cell proliferation of thesecells (Figure 3(b,c)) and S3B-C). Then, we hypothesized that thetumorigenic role of MIR-G-1 on HeLa and C33A cells may becorrelated with cell cycle progression.

To test this hypothesis, these treated cells were subjected toFITC-ANXA5/annexin V-propidium iodide/PI staining followedby flow cytometry analysis. Our results showed that pri-MIR-G-1transfection decreased the proportion of HeLa and C33A cells inthe G0/G1 phase and increased the proportion of G2/S phase cells,and then increased the PI index, whereas anti-MIR-G-1 transfec-tion had an opposite effect (Figure 3(d) and S3(d)).

To further explore whether MIR-G-1 could mediate tumorgrowth, HeLa-anti-MIR-G-1 cells or control cells wereinjected into 6 paired nude mice through subcutaneous injec-tion. As shown in Figure 3(e), tumors grew much slower andwith reduced tumor weight (Figure 3(e)), followed by thedownregulation of MKI67/KI67 expression by immunohisto-chemistry (IHC), in the anti-MIR-G-1 group than in thecontrol group (Figure 3(f) and S3(e)). These data demonstratethat MIR-G-1 can promote cell growth in vitro and in vivo byaffecting cell cycle progression.

MIR-G-1 inhibits apoptosis and anoikis, and promotes theresistance to DDP, EMT progression and nuclearautophag

Because MIR-G-1 promotes cell proliferation, we investi-gated whether MIR-G-1 can play an oncogenic role byinhibiting apoptosis in cervical cancer cells. The apoptosis

Figure 3. MIR-G-1 promotes cell proliferation and tumor growth, and inhibits apoptosis and anoikis. (a) MTT assay showed that overexpression of MIR-G-1 promotedcell viability. (b) Colony formation ability was higher after transfection with pri-MIR-G-1. Scale bar: 50 mm. (c) EdU assay was used to detect cell proliferation. Scalebar: 80 μm. (d) Flow cytometric cell cycle analysis showed that MIR-G-1 overexpression results in a significant decrease in the cellular population in the G0/G1 phase.(e) Representative graph of tumor size and the mean tumor weights 30 days after inoculation. (f) MKI67 expression in tissues was shown by IHC. Scale bar: 80 μm. (g)Flow cytometric apoptosis showed that MIR-G-1 overexpression significantly decreased the apoptosis rate in HeLa and C33A cells. (h) BCL2, cleavage of CASP3 andPARP expression level in the indicated plasmid-transduced HeLa cells after TAX (3 µg/ml) for 24 h was shown by western blot assay. (i) External cell morphology wasanalyzed using microscopy. Scale bar: 80 μm. (j) Western blot results for ITGB1, BCL2 and BAX in the indicated HeLa cells. Experiments were performed 3 times, anddata are presented as means ±SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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assay by flow cytometry showed that the percentage ofapoptotic cells was significantly lower in cells transfectedwith pri-MIR-G-1 but was higher in cells transfected withanti-MIR-G-1 (Figure 3(g)). To further validate that MIR-G-1 can suppress apoptosis, we investigated the effects ofMIR-G-1 on several well-characterized biochemical mar-kers [30,31] for apoptosis via western blot assay, includingBCL2, cleaved CASP3 and PARP. As expected, transfec-tion of pri-MIR-G-1 reduced the cleavage of CASP3 andPARP, and increased BCL2 expression in HeLa cells,whereas anti-MIR-G-1 transfection inhibited BCL2 expres-sion and induced the cleavage of CASP3 and PARPexpression in HeLa cells (Figure 3(h) and S3(f)). In addi-tion, as shown in Figure 3(i), HeLa-MIR-G-1 cellsappeared to be mesothelial compared with epithelialHeLa cells (Figure 3(i)), indicating that dysregulation ofMIR-G-1 may be relevant to the tumor metastasis.

Due to anoikis resistance playing a crucial role in thetumor metastasis [32,33], we first investigated whether MIR-G-1 could affect the anoikis of HeLa cells upon pre-treatmentwith the anoikis method [34]. We found that MIR-G-1 over-expression can trigger the anoikis resistance of cervical cancercells at 12, 24, and 48 h, which was confirmed by the upre-gulation of BCL2 and downregulation of BAX and ITGB1[35,36] using western blot assay (Figure 3(j) and S4A). Inaddition, we also found that MIR-G-1 overexpression inhib-ited cell-matrix adhesion, whereas MIR-G-1 knockdownenhanced cell-matrix adhesion in HeLa and C33A cells(Figure 4(a) and S4(b)). Furthermore, ectopic expression ofMIR-G-1 in HeLa cells enhanced the resistance for cis-plati-num in a dose- and time-dependent manner (Figure 4(b)).Transwell assay also showed that MIR-G-1 overexpressionenhanced the migration and invasion of both HeLa andC33A cells by accelerating EMT progression, including upre-gulation of VIM and ICAM1 and downregulation of CDH1(Figure 4(c,d) and S4(c,d)).

Specifically, Mowers et al. reported that autophagy plays animportant role in cell viability and differentiation, resistanceto anoikis, migration and invasion, the EMT process andtumor cell dormancy [37]. In this sense, western blot findingsdemonstrated that MIR-G-1 overexpression decreased theexpression of SQSTM1, and increased the expression ofLC3-II and BECN1 in HeLa cells during starvation (Figure 4(e) and S4(e)). Furthermore, we found by immunofluores-cence (IF) staining a substantial amount of endogenous lipi-dated LC3-II in the nucleus of HeLa cells with pri-MIR-G-1overexpression during starvation (Figure 4(f)). We also foundthat the endogenous and exogenous lipidated LC3-II in thenucleus of nuclear autophagy occurred at 12 h during starva-tion, and disappeared at 48 h during starvation, indicatingthat HeLa cells at 48 h starvation underwent cell death(Figure 4(g,h)). Intrestingly, the lipidated LC3-II in thenucleus of nuclear autophagy did not occur at 12, 24, 36, or48 h during starvation when cells were treated with exogenousEGFP-LC3, based on the EGFP antibody via IF staining(Figure 4(i)), indicating that the EGFP-LC3 may not beresponsible for the whole process of selective autophagy inHeLa cells. However, the molecular mechanism of this phe-nomenon remains unclear.

MIR-G-1 directly targets and upregulates TMED5 andLMNB1

To further explore the molecular mechanism by whichMIR-G-1contributes to cervical cancer progression, TargetScan andRNAhybrid were used to predict potential targets of MIR-G-1.Among the candidates, GAS1, PCDH10, TNKS2, LMNB1 andTMED5 were predicted to be the MIR-G-1 target genes(Figure S5(a-e)). An enhanced green fluorescent protein(EGFP) reporter assay was used to validate whether MIR-G-1directly binds the 3ʹUTRs of these mRNAs. Intriguingly, wefound that MIR-G-1 overexpression only increased the EGFPintensity of the 3ʹUTRs of LMNB1 and TMED5 in cervicalcancer cells; MIR-G-1 knockdown markedly decreased theEGFP intensity of the 3ʹUTRs of LMNB1 and TMED5 in HeLaand C33A cells (Figure 5(a,b)). In addition, western blot assayshowed that the EGFP protein levels of EGFP-fused LMNB1 andTMED5 3ʹUTR were also upregulated by pri-MIR-G-1 transfec-tion, and downregulated by anti-MIR-G-1 transfection in HeLacells (Figure 5(c)). However, the EGFP intensity of the 3′UTRmutant reporters of LMNB1 and TMED5 were not affected bythe alteration ofMIR-G-1 levels (Figure 5(d)). Meanwhile,MIR-G-1 overexpression increased, but MIR-G-1 knockdowndecreased, endogenous LMNB1 and TMED5 mRNAs (RT-qPCR) and protein (IF and western blot) levels compared withthe control groups (Figure 5(e,f) and S6(a,b)).

Moreover, we subsequently examined the expression levelsof LMNB1 and TMED5 in subcutaneous xenotransplantedtumors of nude mice via western blot assay and IHC staining.Lower LMNB1 and TMED5 levels were observed in tumortissues from the anti-MIR-G-1-HeLa cells compared with thatfrom the pSilencer-HeLa group (Figure 5(g,h) and S6(c)). Tofurther confirm LMNB1 and TMED5 are the direct targets ofMIR-G-1, we detected the mRNA levels of LMNB1 andTMED5 in cervical cancer tissues and the adjacent noncan-cerous tissues. RT-qPCR analysis showed a significant upre-gulation of LMNB1 and TMED5 in cervical cancer tissuescompared with the adjacent non-cancerous tissues(Figure S6(d)). Among the 25 pairs of cervical tissues, weobserved a significant correlation between the levels of MIR-G-1, LMNB1 and TMED5 (Figure S6(e)). These results indi-cate that MIR-G-1 can directly target TMED5 and LMNB1 incervical cancer.

Increased TMED5 expression promotes cell proliferation,migration, invasion and EMT progression

Due to the dysregulation of TMED5 in cervical cancer, wenext determined the functional impact of TMED5 on cer-vical cancer cells. First, we constructed a TMED5 overex-pression vector (pTMED5) and a knockdown plasmid (shR-TMED5) (Figure S6(f,g)) and performed the functionalassays. Then, MTT, colony formation and EdU assayswere performed to identify the role of TMED5 on cellproliferation. As shown in Figure 6(a-c) and S5H-I, cellproliferation was increased by TMED5 overexpression anddecreased by TMED5 knockdown in cervical cancer cells(Figure 6(a-c) and S6(h-i)). In addition, cell cycle analysisshowed that TMED5 overexpression decreased the number

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Figure 4. MIR-G-1 inhibits cell adhesion, promotes drug resistance, migration, invasion and nuclear autophagy. (a) Cell-matrix adhesion assay of HeLa and C33A cellsat 90 min with overexpression or knockdown of MIR-G-1. Scale bar: 50 μm. (b) MIR-G-1 enhanced the drug resistance of HeLa cells to cis-platinum at 24 and 48 h. (c)Transwell assays showed that overexpression of MIR-G-1 promoted cell migration and invasion. Scale bar: 50 μm. (d) Western blot assay showed the protein levels ofICAM1, VIM and CDH1 after transfection with the indicated plasmids in HeLa cells. (e) Western blot assay showed the protein levels of LC3, SQSTM1 and BECN1during starvation at 24 h after transfection with the indicated plasmids in HeLa cells. (f) IF showed the distribution of endogenous LC3 in HeLa cells during starvationat 24 h after transfection with the indicated plasmids. Scale bar: 20 μm. (g-h) IF showed the distribution of endogenous and exogenous LC3 in HeLa cells duringstarvation at 0, 12, 24, 36, and 48 h. Scale bar: 20 μm. (i) EGFP intensity showed the distribution of the exogenous EGFP-LC3 in HeLa cells during starvation at 0, 12,24, 36, and 48 h. Scale bar: 20 μm. Experiments were performed 3 times, and data are presented as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, notsignificant.

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of cells in the G0/G1 phase and increased the number in theS/G2 phase, resulting in upregulation of the PI index(Figure 6(d) and S6J). An apoptosis assay showed that thepercentage of apoptotic cells was significantly lower in cellstransfected with pTMED5 but was higher in cells trans-fected with shR-TMED5 (Figure 6(e)). Increased BCL2expression and decreased cleaved CASP3 and PARP expres-sion by western blot assay also demonstrated the inhibition

of apoptosis by TMED5 overexpression in HeLa cells(Figure 6(f) and S7(a)).

Transwell migration and invasion assays showed that over-expression of TMED5 significantly increased cell migrationand invasion, whereas shR-TMED5 decreased cell migrationand invasion in HeLa and C33A cells (Figure 6(g) and S7B).Western blot assay showed that ectopic expression of TMED5decreased CDH1 expression but increased ICAM1 and VIM

Figure 5. MIR-G-1 targets TMED5 and LMNB1 in cervical cancer. (a) EGFP reporter assay showed cells that were cotransfected with the indicated 3ʹUTR and pri-MIR-G-1 or pcDNA3. (b) EGFP reporter assay showed cells that were cotransfected with the indicated 3ʹUTR and anti-MIR-G-1 or pSilencer. (c) Western blot showed the EGFPprotein level for cells transfected with the indicated plasmids. (d) EGFP reporter assay showed cells that were cotransfected with the indicated 3ʹUTR-mut and pri-MIR-G-1 or pcDNA3, anti-MIR-G-1 or pSilencer. (e) IF showed the distribution and expression of TMED5 and LMNB1 in HeLa cells transfected with the indicatedplasmids. Scale bar: 20 μm. (f) Western blot assay showed the expression levels of TMED5 and LMNB1 in HeLa cells transfected with the indicated plasmids. (G)Western blot assay showed the expression levels of TMED5 and LMNB1 in subcutaneous xenotransplanted tumor tissues. (h) IHC showed the expression levels ofTMED5 and LMNB1 in subcutaneous xenotransplanted tumor tissue. Scale bar: 50 μm. Experiments were performed 3 times, and data are presented as means ± SD.*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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expression in HeLa cells (Figure 6(h) and S7(c)). Furthermore,TMED5 overexpression enhanced the resistance for cis-plati-num in a dose- and time-dependent manner in HeLa cells(Figure 6(i)). More importantly, TMED5 expression was sig-nificantly associated with the clinical stage (Figure 6(j)), indi-cating that TMED5 may be a valuable molecular marker forcervical cancer patients.

Interaction of TMED5 and WNT7B activates the wnt-ctnnb1/β-catenin pathway

Bioinformatics software of STRING was used to predict theinteracting proteins for TMED5. According to the predictedscore and functional assay, WNT7B was selected for furtherstudy (Figure 7(a)). As shown in Figure 7(b), IF assays showedthat the distribution of TMED5 or Flag-WNT7B was largely

Figure 6. TMED5 promotes a cervical cancer cell malignant phenotype. (a) MTT assay showed that TMED5 overexpression promoted cell viability. (b) Colonyformation ability was higher after transfection with pTMED5. Scale bar: 50 mm. (c) EdU assay was used to detect cell proliferation. Scale bar: 80 μm. (d) Flowcytometric cell cycle analysis showed that overexpression of TMED5 results in a significant increase in the cellular population in the G2/S phase in HeLa and C33Acells. (e) Flow cytometric apoptosis showed that overexpression of TMED5 significantly decreased the apoptosis rate in HeLa and C33A cells. (f) Western blot assayshowed that the BCL2, cleavage of CASP3 and PARP expression level in the indicated plasmid-transduced HeLa cells after TAX (3 µg/ml) for 24 h. (g) Transwell assaysshowed that overexpression of TMED5 promoted cell migration and invasion. Scale bar: 50 μm. (h) Western blot assay showed the protein levels of ICAM1, VIM andCDH1 after transfection with the indicated plasmids in HeLa cells. (i) TMED5 overexpression enhanced the drug resistance of HeLa cells to cis-platinum at 24 and 48 h.(j) IHC showed the expression level of TMED5 in human tumor tissues in different clinical stages. Scale bar: 50 μm. Experiments were performed 3 times, and data arepresented as means ±SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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overlapping with the distribution of Flag-WNT7B or TMED5,suggesting the colocalization of TMED5 and WNT7B withinHeLa cells (Figure 7(b)). In addition, a specific interactionbetween TMED5 and WNT7B was confirmed by immunopre-cipitation (IP) experiments in which we used either TMED5or WNT7B antibody in HeLa cells (Figure 7(c)).

Next, to determine whether TMED5 regulated the WNT7Bprotein level, we performed western blot assay, and the resultsshowed that TMED5 overexpression enhanced the expressionlevels of WNT7B in HeLa cells (Figure 7(d) and S7(d)).However,WNT7B overexpression had no effect on the expressionlevels of TMED5 (Figure 7(e) and S7(e)). Given thatWNT7B wasessential for theWNT-CTNNB1/β-catenin pathway in pancreaticadenocarcinoma [38], we next investigated the possibility that

MIR-G-1 and TMED5 affect the activation of the WNT-CTNNB1/β-catenin signaling pathway in cervical cancer cells.The localization CTNNB1 in HeLa cells was examined by IFafter transfection with the indicated plasmids. The nuclear dis-tribution of CTNNB1 protein was increased in pri-MIR-G-1- orpTMED5- or pWNT7B-transfected cells and decreased in shR-TMED5- or shR-WNT7B-transfected cells (Figure 7(f)).Moreover, pri-MIR-G-1 or pTMED5 was able to significantlyenhance protein levels of some well known WNT-CTNNB1/β-catenin target genes, such as CCND1, MYC andMT-CO2/COX2,whereas shR-TMED5 or shR-WNT7B was partly able to rescueMIR-G-1-induced upregulation of these genes in HeLa cells(Figure 7(g) and S7(f)). TOP/FOP luciferase reporter assays alsoidentified the activation of WNT-CTNNB1/β-catenin byMIR-G-

Figure 7. Interaction of TMED5 and WNT7B activates the WNT-CTNNB1/β-catenin pathway. (a) STRING showed the predicted interacting proteins of TMED5. (b) IFassay showed the overlapping distribution of TMED5 and WNT7B in HeLa cells. Scale bar: 50 μm. (c) IP assay showed the interaction of TMED5 and WNT7B in HeLacells. (d-e) Western blot assay showed the expression levels of TMED5 and WNT7B in HeLa cells transfected with the indicated plasmids. (f) IF assay showed thedistribution of CTNNB1 in HeLa cells transfected with the indicated plasmids. Scale bar: 80 μm. (g) Western blot assay showed the expression levels of total-CTNNB1,active-CTNNB1, CCND1, MYC and MT-CO2/COX2 in HeLa cells transfected with the indicated plasmids. (h) Top/Fop luciferase reporter assay was performed to detectthe WNT activity. (i) IHC showed the expression levels of CTNNB1 in subcutaneous xenotransplanted tumor tissue. Scale bar: 50 μm. Experiments were performed 3times, and data are presented as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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1 and TMED5 in HeLa cells (Figure 7(h)). In vivo, MIR-G-1 alsopromoted activated CTNNB1 protein expression in nude micetissues by IHC (Figure 7(i)).

Rescue experiments further show that the MIR-G-1-TMED5-WNT7B pathway promotes malignancy in CC

To further confirmwhether the effects ofMIR-G-1 onmalignancyare mediated by TMED5 or WNT7B, rescue experiments wereperformed. Results showed that WNT7B overexpression pro-moted cell proliferation (Figure 8(a-c) and S7(g,h)), cell cycleprogression (Figure 8(d)), migration and invasion (Figure 8(g,h)and S7(j)) and drug resistance (Figure 8(i)) in cervical cancer cells.Knockdown ofWNT7B expression in cells expressing pri-MIR-G-1 blocked the MIR-G-1-induced promotion of proliferation

(Figure 8(a-c) and S7(g,h)), the cell cycle (Figure 8(d)), migrationand invasion (Figure 8(g, h) and S7(j)) and drug resistance(Figure 8(i)). WNT7B overexpression inhibited apoptosis.Knockdown of WNT7B expression in cells expressing pri-MIR-G-1 blocked the MIR-G-1-induced suppression of apoptosis inHeLa and C33A cells (Figure 8(e,f) and S7K). In addition, IHCshowed that WNT7B was downregulated in the anti-MIR-G-1group (Figure 8(j)).

MIR-G-1 accelerates the TAX-induced DNA-damage repairby promoting LMNB1-mediated nuclear autophagy

Recently, starvation-induced nuclear autophagy was discov-ered in yeast [25]. In addition, Dou et al. reported that therole of autophagy in degrading nuclear components depend

Figure 8. MIR-G-1 plays an oncogenic role in cervical cancer by regulating WNT7B expression. (a-c) Overexpression of WNT7B promoted cell proliferation. Scale bar:50 mm. (d) Overexpression of WNT7B promoted cell cycle progression. (e-f) Overexpression of WNT7B inhibited apoptosis. (g-h) Overexpression of WNT7B promotedmigration, invasion and EMT progression. Scale bar: 50 μm. (i) Overexpression of WNT7B promoted drug resistance. (j) IHC showed the expression levels of WNT7B insubcutaneous xenotransplanted tumor tissue. Scale bar: 50 μm. Experiments were performed 3 times, and data are presented as means ±SD.*P < 0.05; **P < 0.01;***P < 0.001; ns, not significant.

10 Z. YANG ET AL.

on LMNB1 [39]. We found that LMNB1 overexpressionduring starvation resulted in a substantial amount of endo-genous lipidated LC3-II in the nucleus (Figure 9(a)). LMNB1knockdown during starvation resulted in a small amount oflipidated LC3-II in the nucleus, indicating that LMNB1 isinvolved in degrading nuclear components during nuclearautophagy (Figure 9(a)). In addition, LMNB1 overexpressionduring starvation resulted in its own enrichment in thenuclear membrane and regulated autophagy-related proteinexpression, including the upregulation of LC3-II and BECN1and downregulation of SQSTM1 (Figure 9(b) and S8A).Earlier findings suggested that autophagy conveys resistanceto DNA damage in epidermal cells [40], and loss of

autophagy strongly impairs the resistance of epidermal ker-atinocytes to extrinsic and intrinsic redox stress, and resultsin severely increased DNA damage and a senescent pheno-type [41]. We reported that LMNB1 regulates the removal ofDNA breaks using comet assays. We observed that LMNB1-overexpressing HeLa cells repair DNA breaks more rapidlythan control vector-treated cells. Knockdown of LMNB1delayed the DNA damage repair in HeLa cells. In addition,overexpression of pri-MIR-G-1 in HeLa cells expressing shR-LMNB1 blocked the shR-LMNB1-induced suppression ofDNA damage repair (Figure 9(c)). In order to probe theeffect of LMNB1 on DNA repair specifically of double-strand breaks, RT-qPCR assay were performed and the

Figure 9. MIR-G-1 plays an oncogenic role in cervical cancer cells by regulating LMNB1expression. (a) IF assay showed the expression levels of LMNB1 and LC3 inHeLa cells during starvation at 24 h treated with the indicated plasmids. Scale bar: 20 μm. (b) Western blot assay showed the expression levels of LC3, SQSTM1,BECN1 and LMNB1 during starvation at 24 h after transfection with the indicated plasmids in HeLa cells. (c) Comet assays showed the degree of DNA breaks in HeLacells transfected with the indicated plasmids treated with 10 µg/ml TAX for 4 h. Scale bar: 50 μm. (d) RT-qPCR showed the mRNA levels of TP53BP1 and H2AFX inHeLa cells transfected with the indicated plasmids under 10 µg/ml TAX treatment at 4 h in HeLa cells. (e) IF assay showed the TP53BP1 and H2AFX foci in HeLa cellstansfected with the indicated plasmids and treated with 10 µg/ml TAX for 4 h. Scale bar: 20 μm. Experiments were performed 3 times, and data are presented asmeans ±SD.*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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results showed that LMNB1 overexpression changed themRNA levels, including downregulation of TP53BP1 andH2AFX (Figure 9(d)), which are the factors required forthe recognition of DNA damage and the formation of repaircomplexes [42,43]. Furthermore, LMNB1 overexpressioninhibited the formation of TP53BP1 or H2AFX foci, andLMNB1 knockdown enhanced the formation of TP53BP1and H2AFX foci in HeLa cells (Figure 9(e)).

MIR-G-1 upregulates TMED5 and LMNB1 in a grsf1-dependent manner

To investigate whether GRSF1 regulates the MIR-G-1mediated upregulation of TMED5 and LMNB1, many experi-ments were performed. As shown in Figure 10(a) and S8B,MIR-G-1 or GRSF1 overexpression could upregulate the pro-tein levels of TMED5 and LMNB1 in HeLa cells, whereasknockdown of GRSF1 in cells expressing pri-MIR-G-1 blockedthe upregulation of TMED5 and LMNB1 (Figure 10(a) and S8(b)). In addition, knockdown of MIR-G-1 and GRSF1decreased the protein levels of TMED5 and LMNB1 in HeLacells (Figure 10(b) and S8(c)). Next, we used shRNA totransiently knock down GRSF1 in HeLa cells, and the shR-GRSF1 stable HeLa cell line (#12 clone) was extremely effec-tive (Figure 10(c) and S8D). We found that the EGFP proteinlevels almost had no change following the indicated transfec-tion in the #12 shR-GRSF1 stable HeLa cell line (Figure 10(d)and S8(e)). Levels of endogenous TMED5 and LMNB1 in the#12 shR-GRSF1 stable HeLa cell line were enhanced whentransfected with pcDNA3 and pGRSF1. Conversely, levels ofendogenous TMED5 and LMNB1 in the #12 shR-GRSF1stable HeLa cell line were reduced in cells transfected withpcDNA3 and shR-GRSF1 or anti-MIR-G-1 and shR-GRSF1.However, the endogenous TMED5 and LMNB1 levels werealmost the same in the #12 shR-GRSF1 stable HeLa cell linetreated with pcDNA3 and pri-MIR-G-1 or pcDNA3 and anti-MIR-G-1 (Figure 10(e) and S8(f)). To confirm the directinteraction of GRSF1 and MIR-G-1, RNA EMSA was per-formed. First, we mapped the 5 GRSF1 fragments (Figure 10(f,g)). Then, the results showed that biotin-conjugated MIR-G-1 obviously shifted complexes in the presence of extractsprepared from the #12 shR-GRSF1 stable HeLa cells expres-sing Flag-GRSF1, and EMSA showed that the fourth fragment(Flag-GRSF1-4) may be responsible for MIR-G-1 binding(Figure 10(h)). These results indicate that GRSF1 binds toMIR-G-1 and mediates its up-regulation of TMED5 andLMNB1 expression.

Discussion

Dysregulation of miRNAs has emerged as promising molecu-lar markers due to their involvement in development andprogression of many cancers [42–45]. For example, MIR214suppresses cell growth, migration and invasion through thenovel target of HMGA1 in human cervical and colorectalcancer cells [46]. In addition, ectopic expression ofMIR130A in human breast cancer cells promotes AKT phos-phorylation, cell survival and tumor growth by targetingPTEN [47]. Furthermore, overexpression of MIR218 decreases

cell proliferation, invasion, colony formation, and tumorsphere formation in vitro and represses tumor growth invivo by negatively regulating IL6 receptor and JAK3 geneexpression in lung cancer [48]. In the present study, werevealed that overexpression of the novel MIR-G-1 enhancedcell proliferation, migration and invasion, accelerated cellcycle and EMT progression, inhibited apoptosis and anoikis,promoted nuclear autophagy, enhanced the resistance for cis-platinum in vitro and promoted tumor growth in vivo.

Although much of the initial work on miRNA pathwaysfocused on its gene silencing mechanisms [49,50], recentevidence has demonstrated that miRNAs could activate targetgene expression via different mechanisms [8]. Vasudevanet al. reported that AU-rich elements in TNF/TNFalpha(tumor necrosis factor) mRNA are transformed into a trans-lation activation signal, recruiting AGO (argonaute, RISCcatalytic component) and FXR1 (FMR1 autosomal homolog1) factors associated with micro-ribonucleoproteins [51].Richard et al. found that activation is regulated by the G0-controlling cAMP-PRKG2 pathway and they identify theendogenous miRNAs in the immature oocyte required toincrease expression of the cell state regulator MYT1 in anAGO2-dependent manner [52]. However, we previouslydemonstrated that GRSF1-mediated MIR346 upregulatesTERT expression in HeLa cells by facilitating the recruitmentof TERT mRNA to ribosomes to promote translation in anAGO2-independent manner [12]. In this study, we appliedGRSF1-IP RNA sequencing and obtained some GRSF1-boundmiRNA. Among them, a novel miRNA-bound GRSF1, MIR-G-1 was characterized. We revealed that MIR-G-1 may bind tothe 3ʹUTR of TMED5 and LMNB1 to enhance their expres-sion in a GRSF1-dependent manner.

Buechlinget al. reported that opm (a TMED5 homolog),which is a highly conserved type 1a transmembrane protein ofthe conserved g-subfamily of p24 proteins, is a conservedregulator of canonical Wnt/wg signalling in Drosophila [53].However, the molecular mechanism of how TMED5 regulatesWNT signaling is not clear in human. In this study, we foundthat overexpression of TMED5 promotes a malignant pheno-type, including cell proliferation, cell cycle and EMT progres-sion, apoptosis, cell migration and invasion, and drugresistance. Moreover, our results demonstrated that TMED5interacts with WNT7B in HeLa cells. WNT7B could mediatethe autocrine WNT-CTNNB1/β-catenin signaling and ancho-rage-independent growth in pancreatic adenocarcinoma [38],which is the same as our results in cervical cancer cells thatWNT7B overexpression promoted cell proliferation, cellmigration and invasion, EMT progression and drug resistanceby activating WNT-CTNNB1/β-catenin signaling. WNT pro-teins are a family of developmentally important signalingmolecules, which play key roles in several cellular processes,including proliferation, differentiation, migration, and pro-grammed cell death [54,55]. These data indicated thatTMED5 could activate WNT-CTNNB1/β-catenin signalingand then promote the development and progression of cervi-cal cancer by interacting with WNT7B and increasing itsexpression in cervical cancer cells.

LMNB1 has been implicated in many cell physiology activ-ities, including DNA replication and transcription, resistance

12 Z. YANG ET AL.

to oxidative stress, chromosome distribution, nuclear andchromatin organization, cell cycle regulation, cell develop-ment and differentiation, nuclear migration and nuclearautophagy [39,56–60]. However, the potential involvementof LMNB1 mediated by miRNAs in nuclear autophagy andDNA repair pathways has not been well characterized. In thispaper, we found that MIR-G-1 promoted nuclear autophagy

and DNA damage repair. Overexpression of MIR-G-1 duringstarvation led to a substantial amount of endogenous lipidatedLC3-II in the nucleus of HeLa cells by targeting and upregu-lating LMNB1, decreased the expression of SQSTM1, andincreased the expression of LC3-II, BECN1 and LMNB1 inHeLa cells. In addition, overexpression of MIR-G-1 or LMNB1following 10 µg/ml TAX treatment at 4 h, downregulated the

Figure 10. MIR-G-1 upregulates TMED5 and LMNB1 in a GRSF1-dependent manner. (a-b) Western blot assay showed the expression levels of TMED5 and LMNB1transfected with the indicated plasmids in HeLa cells. (c) Western blot assay showed the expression levels of GRSF1 after treatment with the shR-GRSF1 technology inHeLa cells. (d) Western blot assay showed the EGFP protein levels in #12 shR-GRSF1 HeLa cells transfected with the indicated plasmids. (e) Western blot assay showedthe expression levels of TMED5 and LMNB1 in #12 shR-GRSF1 HeLa cells transfected with the indicated plasmids. (f-g) GRSF1 mapping fragments were shown andidentified by western blot assay. (h) RNA EMSA assay showed the interaction of GRSF1 or GRSF1-1 or GRSF1-2 or GRSF1-3 or GRSF1-4 and MIR-G-1. (i) The diagram ofMIR-G-1 upregulating TMED5 and LMNB1 to promote malignancy and nuclear autophagy in cervical cancer cells. Experiments were performed 3 times, and data arepresented as means ± SD.*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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levels of TP53BP1 and H2AFX in HeLa cells, suggesting thatMIR-G-1 and LMNB1 may be novel targets for the sensitiza-tion of cancer cells to DNA-damaging therapies.

Taken together, this study highlights that MIR-G-1 upre-gulates TMED5 and LMNB1 in a GRSF1-dependent mannerand promotes malignant behavior and nuclear autophagy inthe cervical cancer cells (Figure 10(i)).

Materials and methods

Materials and cell culture

All clinical samples of human cervical tissues and serum wereobtained from the Department of Oncology and Gynecologyof Taishan Medical University at Xintai Hospital. Writteninformed consent was obtained from all enrolled patients,and all relevant investigations were performed according tothe principles of the Declaration of Helsinki. The specimenswere snap-frozen immediately after collection and were storedat −80°C. Total RNA was extracted from the human samplesand purified using the miRVana miRNA Isolation kit(Thermo Fisher Scientific, AM1561) according to the manu-facturer’s instructions. The study was approved by the ethicsReview Committee of Tianjin Medical University (Ethicsapproval number: TMUaMEC2014004). Cervical cancer cellsand other cancer cells used in this study were obtained fromthe American Type Culture Collection (HeLa, ATCC® CCL-2™. C33A, ATCC® HTB-31™. SiHa, ATCC® HTB-35™. SW480,ATCC® CCL-228™. SW620, ATCC® CCL-227™. HepG2,ATCC® HB-8065™. QGY-7703, ATCC® CRL-7703™) and werecultured according to the indicated conditions (https://www.atcc.org/). S12, an immortalized human cervical keratinocytecell line, was a kind gift from Prof. Wang (Tongji Hospital,Tongji Medical College, Huazhong University of Science andTechnology, Wuhan, Hubei, China).

RNA extraction and quantitative RT-PCR

Total RNA was extracted from human tissues and cultured cellsusing Trizol reagent (Sigma, T9424) according to the manufac-turer’s instructions. The total RNA concentration was determinedusing a NanoDrop Lite (Thermo Fisher Scientific, 2000c). TheRT-qPCR assay was performed according to previously publishedprotocols [61,62]. All the primers used are in Table S1.

Plasmid construction

For the MIR-G-1 expression vector (pri-MIR-G-1), 400 basepairs containing the MIR-G-1-encoded region were amplifiedfrom genomic DNA and cloned into the pcDNA3 vector(Ambion, V86520). The knockdown plasmid of MIR-G-1 wasobtained by annealing double-strand DNA and inserting it intothe pSilencer 2.1-U6-neo vector (Ambion, 113P06). The 3ʹUTRsof the predicted target genes that containMIR-G-1-binding sitesand mutant 3ʹUTR fragments with MIR-G-1-binding sites wereobtained by annealing double-strand DNA or PCR product andinserting it into the pcDNA3-EGFP vector (Tianjin SaierBiotechnology, SRC0062). The shR-TMED5, shR-LMNB1 andshR-WNT7B plasmids expressing siRNA targeting TMED5,

LMNB1 or WNT7B were constructed by annealing double-strand hairpin cDNA and inserting it into the pSilencer 2.1-U6-neo vector (Ambion, 113P06). The full-length sequences ofhuman TMED5 (Tianjin Saier Biotechnology, SRCL13219),LMNB1 (Tianjin Saier Biotechnology, SRCL07384) andWNT7B (Tianjin Saier Biotechnology, SRCL14434) cDNAswere obtained by RT-PCR and cloned into pcDNA3 vector.All primers used are provided in Table S1.

Cell proliferation assay, colony-formation assay, EGFPreporter assay, cell cycle and apoptosis flow cytometricand Transwell assay

These assays were performed as described previously[61,63,64]. For MTT assay, cells were seeded in 96-well platesat 5000 cells per well 1 day prior to transfection and the cellviability at 0, 24, 48 and 72 h post-transfection was deter-mined by MTT assay. Then, 10 µl of 5mg/ml MTT (Sigma,298931) was added and incubated for 6 h. The medium wasaspirated and MTT was dissolved in 100 µl DMSO (Sigma,67685) and absorbance was read at 570 nm using a QuantUniversal Microplate Spectrophotometer.

Colony-formation assay

For colony-formation assay, cells were counted at 24 h post-transfection and seeded into 12-well plates in triplicate at adensity of 300 HeLa or C33A cells per well. and then culturedfor approximately 14 days. Culture medium was replacedevery 3 days, and then cultured for approximately 14 daybefore staining with crystal violet (Sigma, 548629), and colo-nies with more than 50 cells were counted.

EGFP reporter assay

Cells were seeded in 48-well plates 1 day before transfectionand then co-transfected with pri-MIR-G-1 or anti-MIR-G-1and pEGFP-GAS1 3ʹUTR, pEGFP-PCDH10 3ʹUTR, pEGFP-LMNB1 3ʹUTR, pEGFP-TMED5 3ʹUTR, pEGFP-TNKS23ʹUTR, pEGFP-TMED5 3ʹUTR mut, or pEGFP-LMNB13ʹUTR mut. The vector pDsRed2-N1 (Clontech, 632406),which expresses RFP, was included for transfection normal-ization. After transfection for 48 h, the cells were lysed usingradio-immunoprecipitation assay (RIPA) lysis buffer (Sigma,R0278), and the EGFP and RFP intensities were measuredwith a fluorescence spectrophotometer (Hitachi, F4500).

Cell cycle and apoptosis flow cytometry analyses

At 48 h after transfection, transfected HeLa and C33A cells wereharvested by trypsinization and resuspended in cold phosphate-buffered saline (Beyotime, C0221A) for analysis. The rate ofapoptosis among the cells was detected by flow cytometry (BDBiosciences) using an ANXA5/annexinV-FITC-PI apoptosisdetection kit (BD Biosciences, 556463) according to the manu-facturer’s protocol. For the analysis of cell cycle, the resuspendedcells were fixed and stained with PI using a Cycletest™ Plus DNAReagent kit (BD Biosciences, 340242) according to the manu-facturer’s manual, and were detected by flow cytometry. The

14 Z. YANG ET AL.

percentages of cells in G1, S, and G2 phases were ascertainedusing software (FLOWJO, FlowJo7.6).

Transwell migration and invasion assay

Briefly, HeLa and C33A cells were seeded into 8-μm cellculture inserts and placed in 24-well cell culture plates. Inaddition, the upper chamber was coated with 100 ul of dilutedMatrigel (Sigma, 126–2.5) for the invasion assay. The lowerchamber was filled with 600 µl of 20% FBS (Gibco, 10,099–141) medium. HeLa and C33A cells (6 × 104) in 200 μl ofserum-free medium were gently loaded onto each filter insert(upper chamber) and then incubated at 37°C for 48 h. Thefilter inserts were removed from the chambers, fixed withmethanol (Sigma, V900806) for 10 min and stained withHarris’ hematoxylin (Sigma, H9627) for 20 min. The sampleswere subsequently washed, dried and mounted onto slides.The migratory and invasive cells were stained blue, visualizedunder an inverted microscope and then counted in 6 randomfields for statistical analysis.

Antibodies

BCL2 (Wanlei Biotechnology, WL01556; 1:500); cleaved CASP3(Wanlei Biotechnology,WL01992; 1:1000); cleavedPARP (WanleiBiotechnology, WL01932; 1:2000); BECN1(Wanlei Bio-technology; WL02237; 1:1500); LMNB1 (Wanlei Biotechnology,WL01775; 1:1000); CTNNB1 (Wanlei Biotechnology, WL0962a;1:2000); BAX (Wanlei Biotechnology, WL01637; 1:2000); ITGB1(Wanlei Biotechnology, WL02236; 1:500); GAPDH (SaierBiotechnology, SRP00849; 1:3000,); CDH1 (Saier Biotechnology,SRP05266; 1:500); VIM (Saier Biotechnology, SRP01327; 1:3000);ICAM1 (Saier Biotechnology, SRP01174; 1:1500); TMED5 (SaierBiotechnology, SRP08852; 1:500); WNT7B (Saier Biotechnology,SRP07074; 1:1000); MYC (Saier Biotechnology, SRP00871; 1:500);CCND1 (Saier Biotechnology, SRP00456; 1:1000); MT-CO2/COX2 (Saier Biotechnology, SRP00794b; 1: 500); EGFP (SaierBiotechnology, SRP12235; 1:1500); SQSTM1 (SaierBiotechnology, SRP11189; 1:1500); GRSF1 (Saier Biotechnology,SRP01890; 1:1500); LC3 (Saier Biotechnology, SRP01707; 1:200).

TOP/FOP flash reporter assays

To assay the transcriptional activity of the WNT pathway, theindicated plasmids were cotransfected with either the WNTsignaling reporter TopFlash or the negative control FopFlashaccording to the manufacturer’s protocol (Millipore, 17–285).HeLa and C33A cells were transiently transfected with either2 µg pTopFlash (TCF reporter plasmid) or pFopFlash(mutant, inactive TCF binding site) plasmids, and 0.5 µgpSV40-Renilla plasmid (Promega, E6911) as an internal con-trol for 48 h. The Dual-Luciferase reporter assay system(Promega, e1910) was used to assay the firefly and Renillaluciferase activity ratio.

Western blot analysis

Cell extracts were cleaned with 1x PBS buffer (Beyotime,C0221A), prepared with RIPA buffer (Sigma, R0278)

supplemented with cocktail (Roche, 11873580001), and proteinconcentrations were quantified using the BCA protein assay kit(Beyotime, P0010S) according to the manufacturer’s protocols.Antibodies to BCL2 (WL01556), cleaved CASP3 (WL01992) andPARP (WL01932), BECN1 (WL02237), LMNB1 (WL01775),CTNNB1 (WL0962a), ITGB1 (WL02236) and BAX (WL01637)were purchased from Wanlei Biotechnology. Antibodies toGAPDH (SRP00849), CDH1 (SRP05266), VIM (SRP01327),ICAM1 (SRP01174), TMED5 (SRP08852), WNT7B (SRP07074),MYC/C-MYC (SRP00871), CCND1 (SRP00456), MT-CO2/COX2 (SRP00794b), EGFP (SRP12235), SQSTM1 (SRP11189),GRSF1 (SRP01890) and LC3 (SRP01707) were purchased fromTianjin Saier Biotechnology. The secondary goat anti-rabbit anti-body was obtained from Sigma (Sigma, R0881). LabWorks ImageAcquisition andAnalysis Software (UVP, Upland, CA, USA) wereused to quantify band intensities.

IF staining

The subsequent protocol was previously reported by Chen etal. (65). The cells were washed with PBS (Beyotime, C0221A)and then fixed with 4% paraformaldehyde (Sigma, V900894)for 30 min at room temperature. After being washed 3 timeswith PBS (Beyotime, C0221A), the cells were blocked in 10%donkey serum (Solarbio, S9100) for 10 min. The cells weresubsequently incubated with primary antibodies specific forLC3 (Saier Biotechnology, SRP01707), EGFP (SaierBiotechnology, SRP12235), LMNB1 (Wanlei Biotechnology,WL01775), TMED5 (Saier Biotechnology, SRP08852) andCTNNB1 (Wanlei Biotechnology, WL0962a) overnight at 4°C. The next day, the cells were washed with PBS, after whichthey were incubated with fluorescent-conjugated secondaryantibodies (Beyotime, A0562), followed by DAPI (Beyotime,C1002). Images were captured by confocal microscopy.

RNA immunoprecipitation assay

The RIP assay was carried out following the methoddescribed by Ufer et al. [66] with some modifications.HeLa cells transfected with Flag-GRSF1 were trypsinized,resuspended inchilled 1× PBS (Beyotime, C0221A), and cen-trifuged at 5000 g for 5 min at 4°C 24 h post transfection.The pellets were resuspended in lysis buffer (50 mM Tris-HCl [Sigma, T3253], pH 7.5, 1% NP-40 [Beyotime, P0013F],0.5% sodium deoxycholate [Sigma, D6750], 1 mM EDTA,140 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 100 U/mlRNasin [Beyotime, R0102], and proteinase inhibitor cocktail[Roche, 11,873,580,001]), incubated on ice for 10 min withoccasional flicking, and then centrifuged at 16000g at 4°C for5 min. The supernatant was transferred to a new 1.5-ml tube.The samples were pre-cleaned by adding 50 µl of protein G(MBL, MJS002V2) slurry to each tube and rotating thesamples at 4°C for 2 h. After centrifugation at 5000 g for5 min at 4°C, the supernatant was transferred to a new tube.Anti-Flag antibody (Cell Signaling Technology, 5407S;10 µg) was added to the supernatants; the same amount ofmouse IgG (Abcam, ab190475) was added to the nonspecificcontrol sample. After a rotation at 4°C overnight, 50 μl ofprotein G slurry was added to the supernatants and the

AUTOPHAGY 15

samples were rotated at 4°C for 2 h. Following centrifugationat 5000 g for 5 min at 4°C, the supernatants were discardedand the samples were resuspended in 1 ml lysis buffer. Thesamples were centrifuged at 5000 g for 5 min at 4°C and thenthe supernatants were discarded. This step was thenrepeated. The pellet was then washed twice with high saltlysis buffer (the same as the lysis buffer above except thatNaCl is at 700 mM). The RNAs in the pellets were releasedfrom the samples after proteinase K (Abcam, ab64220;100ug/ml) treatment for 30 min at 50°C, extracted withphenol:chloroform:isoamylalcohol (25:24:1), and then preci-pitated in alcohol overnight. The RNAs were then quantifiedby RT-qPCR and used for deep sequencing.

Cell-matrix adhesion assay

Plates (96-well) were coated overnight with 10 mg/ml FN1/fibronectin (Sigma, 10838039001) at 4°C and were blockedwith 1% (w:v) BSA (Sigma, A7030). Cells were seeded on the96-well plates at a density of 5 × 10 4/well in triplicate,allowed to adhere at 37°C for at least 10 min, and werethen washed 3 times with PBS. The cells were fixed with4% (v:v) paraformaldehyde (Sigma, V900894), stained with0.5% (w:v) crystal violet (Solarbio, G1062) for 10 min, andthen lysedwith 30% (v/:v) glacial acetic acid (Aladdin,A116172) for 15 min. Cells on the substrate were imagedafter 90 min using an Olympus IX 71.

In vivo tumor xenograft studies

Animal protocols were approved by Tianjin MedicalUniversity Animal Care and Use Committee. The methodswere carried out in accordance with the approved guidelines.Six-week-old female BALB/c athymic nude mice (Institute ofZoology, Chinese Academy of Sciences, Shanghai, China)were used for the in vivo study. A total number of 1 × 107

stably transfected (pSilencer or anti-MIR-G-1) HeLa cells wereimplanted subcutaneously into the armpit of nude mice. Sixmice were included in each group. Tumor weights were mea-sured using an electronic scale, and the Student’s t-test wasused to compare tumor growth among groups.

Statistical analysis

All analyses were performed using SPSS 19 for Windows(SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 forWindows (GraphPad Software Inc., San Diego, CA, USA).For comparisons of 2 treatment groups, the Student t testwas used. For comparisons of 3 or more groups, one-wayANOVA was used with the Bonferroni post-hoc test forcomparison of 2 selected treatment groups; the Dunnettpost-hoc test was used for comparisons of the other treatmentgroups with the corresponding controls. Data are presented asthe means ± standard deviation (SD) from at least 3 indepen-dent experiments. Statistical analyses were performed usingStudent’s t-tests. P < 0.05 was considered to indicate a statis-tically significant difference.

Acknowledgments

We thank Dr Yang in Xintai Affiliated Hospital of Taishan MedicalUniversity for providing the clinical samples of human cervical tissuesand serum.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported in part by the National Natural ScienceFoundation of China (No: 91629302; 81572790; 81830094; 31270818)and the Natural Science Foundation of Tianjin (No: 12JCZDJC25100).

ORCID

Weiying Liu http://orcid.org/0000-0002-6710-3341

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