serum piwi-interacting rnas pir-020619 and pir-020450 are ... · early diagnostic biomarkers for...

CANCER EPIDEMIOLOGY, BIOMARKERS & PREVENTION | RESEARCH ARTICLE

Serum PIWI-Interacting RNAs piR-020619 andpiR-020450 Are Promising Novel Biomarkers for EarlyDetection of Colorectal CancerZhenfeiWang1, Hao Yang2, DaguangMa3, YongpingMu1, Xiaohui Tan4, Qin Hao5, Li Feng6, Junqing Liang1,Wen Xin7, Yongxia Chen1, Yingcai Wu1, Yongfeng Jia1,8, and Haiping Zhao6

ABSTRACT◥

Background: Early diagnosis can significantly reduce colorectalcancer deaths.We sought to identify serumPIWI-interacting RNAs(piRNAs) that could serve as sensitive and specific noninvasivebiomarkers for early colorectal cancer detection.

Methods:Wescreened the piRNAexpressionprofile in sera from7patientswith colorectal cancer and7normal controls using smallRNAsequencing. Differentially expressed piRNAs were measured in atraining cohort of 140 patients with colorectal cancer and 140 normalcontrols using reverse transcription quantitative PCR. The identifiedpiRNAs were evaluated in two independent validation cohorts of180 patients with colorectal cancer and 180 normal controls. Finally,the diagnostic value of the identified piRNAs for colorectal adenoma(CRA) was assessed, and their expression wasmeasured in 50 patientswith lung cancer, 50 with breast cancer, and 50 with gastric cancer.

Results: The piRNAs piR-020619 and piR-020450 were consis-tently elevated in sera of patients with colorectal cancer as comparedwith controls. A predicative panel based on the two piRNAs wasestablished that displayed high diagnostic accuracy for colorectalcancer detection. The two-piRNA panel could detect small-size andearly-stage colorectal cancer with an area under the ROC curve of0.863 and 0.839, respectively. Combined use of the two piRNAs couldeffectively distinguish CRA from controls. Aberrant elevation of thetwo piRNAswas not observed in sera of patients with lung, breast, andgastric cancer.

Conclusions: Serum piR-020619 and piR-020450 show a strongpotential as colorectal cancer-specific early detection biomarkers.

Impact: The field of circulating piRNAs could allow for noveltumor biomarker development.

IntroductionColorectal cancer is currently the third most common malignancy

worldwide with over 1.4 million new cases and over 680,000 deathsannually (1). The prognosis of colorectal cancer largely depends onthe TNM stage at the time of diagnosis, with the 5-year survival ratedecreasing from about 90% in early-stage disease (stages 0–II) to 12–13% in metastatic disease (stage IV; ref. 2). Unfortunately, colorectalcancer is usually asymptomatic at the early stage. Consequently,

most patients are diagnosed at the advanced stage, when tumorinvasion and dissemination have already occurred and operativecurative resection cannot be performed. Therefore, early detection iscrucial for improving the outcome of patients with colorectal cancer.

Colonoscopy is the gold standard for diagnosing colorectal cancer,but its invasiveness, high cost, and complicated bowel preparationmakes it inappropriate for large-scale screening (3). Fecal occult bloodtests and stool DNA tests are both noninvasive assays, but the formerhas low sensitivity and specificity and the latter is expensive (3).

Blood tumor marker detection has been advocated for cancerscreening because it is simple, economical, and noninvasive. However,commonly used colorectal cancer biomarkers, such as carcinoem-bryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9), areinadequate for early diagnosis because of their poor sensitivities (3).Recent studies have shown abnormal noncoding RNA profiles in theserum/plasma of patients with colorectal cancer compared withhealthy people (4, 5). Some researchers have attempted to developearly diagnostic biomarkers for colorectal cancer from serum/plasmalong noncoding RNAs (lncRNA) and miRNAs. However, serum/plasma lncRNAs are prone to degradation (6), substantially influenc-ing measurement accuracy. Serum/plasma miRNAs are more stablethan serum/plasma lncRNAs, but the use of miRNAs identified astumor biomarkers has some limitations. First, their performance forearly detection of colorectal cancer is still unsatisfactory (7). Second,often times four to five serum/plasma miRNAs are used in combina-tion to perform a diagnosis (8, 9), which can be financially burdensometo patients and requires extensive work from clinical laboratories.Finally, most of the colorectal cancer serum/plasma miRNA biomar-kers are discovered on the basis of a small study population from asingle medical center, and their clinically diagnostic value requiresfurther verification (9). Thus, it is urgently necessary to develop stable,sensitive, specific, and cost-effective circulating biomarker assays for

1The Laboratory for Tumor Molecular Diagnosis, Affiliated People's Hospital ofInnerMongoliaMedical University, Huhhot, China. 2Department ofRadiotherapy,Affiliated People's Hospital of Inner Mongolia Medical University, Huhhot, China.3Department of Thoracic Surgery, Affiliated People's Hospital of Inner MongoliaMedical University, Huhhot, China. 4College of Traditional Chinese Medicine,Inner Mongolia Medical University, Huhhot, China. 5Department of Gastrointes-tinal Surgery, Affiliated Hospital of Inner Mongolia Medical University, Huhhot,China. 6Department of Abdominal Tumor Surgery, Affiliated People's Hospital ofInner Mongolia Medical University, Huhhot, China. 7TransGen Biotech Co. Ltd.,Beijing, China. 8Basic Medicine College, Inner Mongolia Medical University,Huhhot, China.

Note: Supplementary data for this article are available at Cancer Epidemiology,Biomarkers & Prevention Online (http://cebp.aacrjournals.org/).

Z. Wang and H. Yang contributed equally to this article.

Corresponding Authors: Yongfeng Jia, Inner Mongolia Medical University,Huhhot 010021, China. Phone: 13394735216; E-mail: [email protected]; andHaiping Zhao, Affiliated People's Hospital of Inner Mongolia Medical University,42 Zhaowuda Road, Huhhot 010021, China. E-mail: [email protected]

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early detection and screening of colorectal cancer in asymptomaticpopulations.

In addition to lncRNAs and miRNAs, PIWI-interacting RNAs(piRNA), with a length of 26 to 30 nucleotides, are an important classof noncoding RNAs with increasingly demonstrated involvement inhuman malignancies (10). More than 30,000 piRNAs have beenidentified in the human genome, a number far exceeding than thatof miRNAs (�2,000; refs. 10, 11). Dysregulated piRNA expression hasbeen detected in multiple tumor tissue types, contributing to theproliferation, migration, invasion, stemness-maintenance, and drug-resistance of tumor cells (12–14). Tumor cells can release piRNAs intothe extracellular space in the formof free, protein-binding or exosome-enclosed molecules, which can travel to serum, plasma, saliva, andother body fluids (11, 15, 16). The piRNAs in body fluids can be moreresistant to oxidation and degradation than miRNAs because of thedistinctive 20-O-methyl modification at their 30-ends (16, 17). It hasbeen demonstrated that piR-57125 remains extremely stable in serumand plasma samples regardless of repeated freeze–thaw cycles or long-term incubation at room temperature (18). In light of these character-istics, circulating piRNAs should be considered as a novel potentialsource of noninvasive biomarkers for early cancer detection.

In this study, for the first time, we performed a large-scale, bi-centerinvestigation to identify serum piRNA markers that can effectivelydistinguish patients with colorectal cancer, especially early-stage(TNM stages I–II) patients with colorectal cancer, from normalcontrols. Moreover, the expression of piRNA markers was measuredin serum samples of patients with CRA, and lung, breast, and gastriccancers to further evaluate their diagnostic potential.

Materials and MethodsStudy design

The study consisted of four phases.The first phase was a discovery phase in which 14 serum samples

from 7 patients colorectal cancer with TNM I to II stages and 7normal controls were collected and subjected to small RNAsequencing to explore differentially expressed piRNAs. The piRNAswith log (fold change) >3 or <�3, P value <0.01, and Q value<0.001 were identified as differentially expressed piRNAs betweenpatients and controls.

The second phase was a training phase in which the differentiallyexpressed piRNAs identified in phase I was further tested in anindependent cohort of 140 patients with colorectal cancer and 140normal controls using reverse transcription quantitative PCR (RT-qPCR). The piRNAswith cycle threshold (Ct) values ≤ 30 in over 95%samples, fold change >2, and P value <0.01 were selected as potentialmarkers. Then, a predictive piRNA panel was constructed from thepotential markers based on the logistic regression model for differ-entiation of patients and controls.

The third phase was a validation phase in which the diagnosticperformance of the piRNA panel was evaluated in two independentcohorts containing 180 patients with colorectal cancer and 180 normalcontrols from two different medical centers. In this phase, the expres-sion of the piRNAs in the panel was also measured in 12 paired serumsamples from patients with colorectal cancer before and one monthafter surgery.

The fourth phase was an external validation phase in which theexpression of piRNA markers in the serum samples of 40 patientswith CRA was examined, and their performance to distinguish CRAfrom control individuals was assessed. In addition, the expression ofpiRNA markers in the serum samples of 50 patients with lung

cancer, 50 with breast cancer, and 50 with gastric cancer wasassayed.

Study populationThe patients with colorectal cancer and control individuals in the

first and second phases were from the Affiliated Hospital of InnerMongolia Medical University.

In the third phase, the 100 patients with colorectal cancer and 100control individuals in validation cohort 1 were from the AffiliatedHospital of Inner Mongolia Medical University, and the 80 patientswith colorectal cancer and 80 control individuals in validation cohort 2were from the Affiliated People's Hospital of Inner Mongolia MedicalUniversity. The 12 patients with colorectal cancer donating pairedserum samples before and after surgery were from the AffiliatedPeople's Hospital of Inner Mongolia Medical University.

In the fourth phase, the patients with CRA, lung cancer, and breastcancer were from the Affiliated People's Hospital of Inner MongoliaMedical University, and the gastric cancer patients were from theAffiliated Hospital of Inner Mongolia Medical University.

All the lesions were diagnosed by histopathology analysis and thecancers were staged according to the TNM system of the InternationalUnion against Cancer. The involved patients had not received anyanticancer treatments such as surgery, chemotherapy, radiotherapy, orcytotherapy before serum collection. Serum CEA and CA19-9 levelswere measured in the Medical Laboratories of the two hospitals, withthe cut-off values being 5 ng/mL for CEA and 27U/mL for CA19-9. Allthe control individuals were recruited from the physical examinationdepartments of the two hospitals, and were confirmed to have noindication for cancer in the physical examinations, no history of anymalignancy, or no diagnosis of a benign neoplasm in the colorectum.Individuals with pulmonary and gastrointestinal chronic inflamma-tion, as well as abnormal heart rate, blood pressure, and bodytemperature were excluded from the control group. The distributionof age and gender in the control group was similar to that in colorectalcancer group. All the patients and controls were of the same ethnicity.Demographic and clinical features of the patients and controls havebeen described in Supplementary Tables S1 and S2. Written informedconsent was obtained from all participants. This study was conductedin accordance with the Declaration of Helsinki and approved by theMedical Ethics Committee of Inner Mongolia Medical University.

Serum preparation and RNA extractionFive milliliters of fasting venous blood from each individual was

drawn before breakfast and sampled in a coagulating tube. Within1 hour, the tube was centrifuged at 4,000 rpm for 10 minutes at 4�C toobtain serum. Then, 1 mL aliquots of the serum were transferred to1.5 mL RNase-free tubes and centrifuged at 12,000 rpm for 10minutesat 4�C to remove cell debris. The serum samples were then transferredto RNase-free tubes and stored at �80�C until use.

Total RNA was extracted from serum samples using the miRNeasySerum/Plasma Kit (Qiagen) following themanufacturer's instructions.For small RNA sequencing, the quantity and integrity of RNA wereassessed with the Qubit 2.0 (Life Technologies) and Agilent 2100bioanalyzer (Agilent Technologies). For RT-qPCR, the quantity andpurity of RNA was assessed with the SpectraMax QuickDrop spec-trophotometer (Molecular Devices).

Small RNA sequencingFourteen libraries were prepared from the serum RNA of 7 patients

and 7 controls according to the procedure described previously (19),and sequencing was conducted on the BGISEQ-500 platform

Wang et al.

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(BGI-Shenzhen). The raw sequences were filtered to remove the tagswith low quality, with 50 contamination, with poly A, without 30 primeror shorter than 18 nucleotides. The obtained clean reads were mappedagainst piRNABank (http://pirnabank.ibab.ac.in/request.html) withBowtie 2 allowing up to one mismatch per read. Differentiallyexpressed piRNAs were screened using the DEGseq R package.

RT-qPCRComplementary DNA was reverse-transcribed from 100 ng total

RNAwith themiRcute PlusmiRNAFirst-Strand cDNAKit (Tiangen).Real-time PCR was performed on an ABI PRISM 7500 SequenceDetection System (Applied Biosystems) using the miRcute PlusmiRNA qPCR Kit (Tiangen). The PCR reactions were incubated at95�C for 15 minutes, followed by 40 cycles of 94�C for 20 seconds and60�C for 34 seconds, and then ramped from 60 to 95�C to obtainmelting curves. After the cycling, melting curve analyses and electro-phoreses on agarose gels were conducted to evaluate the specificity ofthe PCR products. The Ct values of three candidate internal controlswere analyzed with NormFinder to identify an internal control.Relative expression levels of piRNAs were calculated by the 2�DCt

method, normalizing to the internal control. The piRNA-specificprimers were designed and synthesized by Sangon Biotech ShanghaiCo., Ltd.

Statistical analysisExpression levels of the analyzed serum piRNAs in patients and

normal controls were compared using the nonparametric Mann–Whitney test. The diagnostic performance of the piRNAs for discrim-inating patients from controls was evaluated by receiver-operatingcharacteristic (ROC) curve analysis. The difference of the area underthe ROC curve (AUC) was assessed with Medcalc 18.2.1. The com-parison of gender distribution, sensitivity, and specificity was per-formed using Pearson's chi-squared test. The difference in age betweenpatients with colorectal cancer and controls was analyzed by unpairedunequal variance t test. Paired samples before and after the surgerywere analyzed using two-tailed Wilcoxon test. Logistic regressionanalysis was performed with Statistical Package for the Social Sciencesversion 18.0. All tests of significance were two-tailed, with a P valuelower than 0.05 indicating statistical significance.

ResultsDiscovery of differentially expressed piRNAs

During the discovery phase, 14 small RNA libraries establishedfrom the serum samples of 7 patients with colorectal cancer and 7controls were subjected to small RNA sequencing. The sequencedsamples contained on average 29,480,448 � 2,283,578 raw reads and24,208,284 � 1,517,389 clean reads. More than 98.9% of the cleanreads had a Q value ≥20. In the clean reads of each sample,563,551,725 � 68,838,132 bases passed the filter, of which over98.9% had a Q value ≥20, indicating a high quality of the obtaineddata. Mapping the data to the piRNABank, 179,454 � 58,935 readswere annotated, being a proportion of 0.74 � 0.27% of the cleanreads (Supplementary Table S3). Sequence analysis of the annotatedpiRNAs revealed a strong propensity for a 50 uridine nucleoside, whichis consistent with prior reports of a 50 uridine bias in piRNAs.

According to the criteria described in the study design, nine piRNAswere found aberrantly upregulated and six piRNAs downregulated inthe sera of patients with colorectal cancer compared with normalcontrols (Supplementary Table S4). Because we aimed to developpotential biomarkers which were practical and convenient to identify

colorectal cancer in clinical application, we focused on the nineupregulated piRNAs and further tested their expression in the trainingphase.

Identification of an internal controlThe results of the small RNA sequencing analysis revealed that piR-

001918, piR-017716, and piR-018883 were expressed in all serumsamples, and their fold change between patients with colorectal cancerand normal controls was small (1.000, 1.087, and 1.094, respectively).These three piRNAswere considered as candidate internal controls forquantification of serumpiRNAs, and their expressionwasmeasured inrandomly selected 40 serum samples of controls and 40 serum samplesof patients with colorectal cancer using RT-qPCR. The stability valuecalculated from the qPCR data by NormFinder ranked piR-001918 asthe most suitable internal control.

Evaluation of the upregulated piRNAs during the training phaseThe nine upregulated piRNAs discovered in phase I was further

tested on an independent training cohort of 140 patients with colo-rectal cancer and 140 normal controls. Among the nine piRNAs, piR-000330, piR-022421, piR-017458, and piR-001169 had Ct valuesgreater than 30 in over 5% of samples. Both piR-019544 and piR-017723 exhibited similar expression levels between patients withcolorectal cancer and normal controls. piR-020619, piR-020450, andpiR-020814 were confirmed to be upregulated in patients with colo-rectal cancer compared with normal controls (SupplementaryTable S5; Fig. 1A). The diagnostic performance of piR-020619,piR-020450, and piR-020814 was evaluated by a ROC curve analysis,and the AUCs were 0.871 (sensitivity¼ 84.29%, specificity¼ 76.43%),0.841 (sensitivity ¼ 81.43%, specificity ¼ 75.00%), and 0.680 (sensi-tivity ¼ 58.57%, specificity ¼ 72.86%), respectively (SupplementaryTable S6; Fig. 1B).

Establishment of a predictive colorectal cancer piRNA panelA stepwise logistic regression model to estimate the risk of being

diagnosed with colorectal cancer that included piR-020619, piR-020450, and piR-020814 was applied to the training cohort. piR-020619 and piR-020450 turned out to be significant predictors ofcolorectal cancer risk. The predicted probability of being diagnosedwith colorectal cancer from the logit model based on the two-piRNApanel was calculated using the following equation: logit(P)¼�3.793þ0.592� piR-020619þ 0.498� piR-020450. The calculated probabilitywas used to construct an ROC curve to evaluate the diagnosticperformance of the piRNA panel. The panel yielded an AUC of0.879, which was larger than those for CEA (0.657, P < 0.0001) andCA19-9 (0.557, P < 0.0001). The sensitivity for the panel was 86.43%,whichwas higher than those for CEA (37.86%,P< 0.0001) andCA19-9(18.57%, P < 0.0001). The specificity for the panel was 89.29%, whichwas similar to those for CEA (93.57%, P ¼ 0.0769) and CA19-9(92.86%, P ¼ 0.1821; Supplementary Table S6; Fig. 1C).

Validation of the piRNA panelDuring the validation phase, patients with colorectal cancer also

displayed higher levels of serum piR-020619 and piR-020450 than didnormal controls (Fig. 2). The parameters estimated from the trainingphase were used to predict the probability of being diagnosed withcolorectal cancer in the two independent validation cohorts. Invalidation cohort 1, the piRNA panel demonstrated an AUC of0.883 [larger than those for CEA (0.658, P < 0.0001) and CA19-9(0.596, P < 0.0001)], a sensitivity of 86.00% [higher than those for CEA(40.00%, P < 0.0001) and CA19-9 (27.00%, P < 0.0001)], and a

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specificity of 92.00% [similar to those for CEA (93.00%, P ¼ 1.0000)and CA19-9 (95.00%, P¼ 0.4503; Fig. 2A; Supplementary Table S6)].In validation cohort 2, the piRNA panel demonstrated an AUC of0.913 [larger than those for CEA (0.681, P < 0.0001) and CA19-9(0.625, P < 0.0001)], a sensitivity of 88.75% [higher than those for CEA(42.50%, P < 0.0001) and CA19-9 (28.75%, P < 0.0001)], and aspecificity of 93.75% [similar to those for CEA (93.75%, P ¼0.4795) and CA19-9 (96.25%, P ¼ 0.4795; Fig. 2B; SupplementaryTable S6). When the data from the two cohorts were processed in amixed-up manner, similar results were obtained (Fig. 2C; Supple-mentary Table S6).

In addition, the expression of piR-020619 and piR-020450 wasdetected in 12 paired serum samples from patients with colorectalcancer before and 1 month after surgery. Both the piRNAs showeddecreased expression levels in postoperative samples (Fig. 3).

Logistic regression analysis of the piRNA panelNext, we performed a stepwise binary logistic regression analysis

to evaluate whether the diagnostic usefulness of the piRNA panelwas affected by clinical features of colorectal cancer cases, includingage, gender, tumor location, TNM stage, lymph node metastasis,and distant metastasis. The results showed that the piRNA panel

Figure 1.

Diagnostic performance of piR-020619 and piR-020450 for colorec-tal cancer in the training phase.A, Expression levels of serumpiR-020619 and piR-020450 weresignificantly higher in patients withcolorectal cancer than in normal con-trols (P < 0.0001). B, ROC curvesof piR-020619 (AUC ¼ 0.871) andpiR-020450 (AUC ¼ 0.841) for colo-rectal cancer detection. C, ROCcurves of the two-piRNA panel, CEA,and CA19-9 for colorectal cancerdetection. The two-piRNA panel dis-played significantly higher perfor-mance than CEA and CA19-9 forcolorectal cancer detection (P <0.0001).

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Figure 2.

Diagnostic performance of piR-020619 and piR-020450 for colorectal cancer in the validation phase. A, In validation cohort 1, levels of serum piR-020619 andpiR-020450 were significantly elevated in patients with colorectal cancer compared with normal controls (P < 0.0001); the two-piRNA panel displayedsignificantly higher performance than CEA and CA19-9 for colorectal cancer detection (P < 0.0001). B, In validation cohort 2, levels of serum piR-020619and piR-020450 were also significantly elevated in patients with colorectal cancer compared with normal controls (P < 0.0001); the two-piRNA panelalso displayed significantly higher performance than CEA and CA19-9 for colorectal cancer detection (P < 0.0001). C, When the data from the two cohortswere mixed together, the analyses gave similar results.






remained a strong predictor of colorectal cancer regardless of thesesubgroupings of the patients in the training and validation cohorts(Supplementary Table S7).

Diagnostic performance of the piRNA panel for small colorectaltumors

There were 71 patients with colorectal cancer with tumors≤ 3 cm in the training and validation cohorts of this study. Weassessed the performance of the piRNA panel in differentiatingthe small-tumor patients from normal controls. The piRNA panelpresented an AUC of 0.863, which was larger than those for CEA(0.587, P < 0.0001) and CA19-9 (0.548, P < 0.0001). The sensi-tivity for the panel was 81.69%, higher than those for CEA(23.94%, P < 0.0001) and CA19-9 (15.49%, P < 0.0001). Thespecificity for the panel was 90.94%, similar to those for CEA(93.44%, P ¼ 0.0991) and CA19-9 (94.06%, P ¼ 0.0051; Fig. 4A;Supplementary Table S8). Among the 71 patients with smalltumor lesions, 54 patients were CEA negative and 60 patientswere CA19-9 negative. The piRNA panel was able to distinguishthe CEA-negative and CA19-9-negative patients from normalcontrols, as evidenced by an AUC of 0.844 (sensitivity ¼77.78%, specificity ¼ 90.94%) and 0.855 (sensitivity ¼ 80.00 %,specificity ¼ 90.94), respectively (Fig. 4B and C; SupplementaryTable S8).

Diagnostic performance of the piRNA panel for early-stagecolorectal cancer

There were 173 patients with early-stage (TNM stages I to II)colorectal cancer in the training and validation cohorts of thisstudy. We further assessed the performance of the piRNA panel indifferentiating the patients with early-stage colorectal cancer fromnormal controls. The piRNA panel exhibited an AUC of 0.839,which was larger than those for CEA (0.595, P < 0.0001) andCA19-9 (0.536, P < 0.0001). The piRNA panel had a sensitivityof 76.79%, which was higher than those for CEA (25.60%, P <0.0001) and CA19-9 (13.10%, P < 0.0001), and had a specificity of90.94%, which was similar to those for CEA (93.44%, P ¼ 0.0991)and CA19-9 (94.06%, P ¼ 0.0551; Fig. 4D; SupplementaryTable S8). Among the 173 early-stage patients, 125 were CEAnegative and 146 were CA19-9 negative. The piRNA panel wasalso able to distinguish the CEA-negative and CA19-9-negativepatients from normal controls, with an AUC of 0.839 (sensitivity¼ 76.80%, specificity ¼ 90.94%) and 0.828 (sensitivity ¼ 74.66%,specificity ¼ 90.94%), respectively (Fig. 4E and F; SupplementaryTable S8).

Diagnostic performance of piR-020619 and piR-020450 forCRA

To evaluate the diagnostic performance of piR-020619 andpiR-020450 for precancerous colorectal cancer lesions, we measur-ed the expression of the two piRNAs in the sera of 40 patientswith CRA, and observed an elevated expression compared withcontrol individuals (Fig. 5). ROC curve analyses revealed thatpiR-020619 and piR-020450 could discriminate patients with CRAfrom normal controls, with an AUC of 0.701 (sensitivity ¼ 82.50%,specificity ¼ 55.90%) and 0.689 (sensitivity ¼ 67.50%, specificity ¼72.50%), respectively. Combining the two piRNAs resulted in anincreased AUC of 0.779 with 72.50% sensitivity and 76.60% spec-ificity (Fig. 6).

SerumpiR-020619 and piR-020450 levels in patientswith lung,breast, and gastric cancers

The expression levels of piR-020619 and piR-020450 in the sera ofpatients with lung, breast, and gastric cancer were similar to those ofnormal controls (Fig. 5), suggesting that the twopiRNAs could serve ascolorectal cancer-specific early diagnostic biomarkers.

DiscussionPIWI-interacting RNAs were first discovered in D. melanogaster

testis tissue in 2001, and were initially thought to be expressed solelyin gonadal tissues, where they function to safeguard the germlinegenome against transposon-induced mutations. Recent studiesdemonstrate extensive expression of piRNAs in various humansomatic tissues, where they play an important role in maintainingcellular homeostasis by silencing transposable elements, keepingnormal DNA methylation and histone modification patterns, con-trolling mRNA stability, modulating transcription factor activity, aswell as regulating protein phosphorylation and protein-proteininteractions (20–25).

Dysregulated piRNA expression leads to amplification andmobilization of transposable elements, hypermethylation or hypo-methylation of genomic DNA, and disruption of normal histonemodifications (26–30). These abnormalities further cause a varietyof genomic instability events such as insertion mutations, geneamplifications, and chromosomal rearrangements (31). Becausegenomic instability is a major driving force of carcinogenesis andcancer evolution (32), some piRNAs may be dysregulated at veryearly phases of cancer development. In this study, for the first time,we discovered that expression of serum piR-020619 and piR-020450was significantly elevated in CRA and patients with early-stage

Figure 3.

Changes of serum piR-020619 andpiR-020450 levels in 12 patients withcolorectal cancer before and 1 monthafter surgical removal of the tumors.The levels of serum piR-020619 andpiR-020450 significantly decreasedafter surgical removal of the tumors(P ¼ 0.01 and 0.008, respectively).

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Figure 5.

Expression of piR-020619 and piR-020450 in sera of normal controls, andpatients with CRA, stage I to II CRC,stage III to IV colorectal cancer, lungcancer, breast cancer, and gastric can-cer. The levels of serum piR-020619and piR-020450 in normal controlswere significantly lower than those inpatients with CRA, stage I to II colo-rectal cancer, and stage III to IV colo-rectal cancer (P < 0.01), whereas theywere similar to those in patients withlung, breast, and gastric cancers (P >0.05).

Figure 4.

Diagnostic performance of the two-piRNA panel for small-size and early-stage colorectal cancer in the combined training and validation cohorts. A, The two-piRNApanel displayed significantly higher performance than CEA and CA19-9 for detection of small-size colorectal cancer (P <0.0001).B, The ROC curve of the two-piRNApanel for detection of CEA-negative small colorectal cancer (AUC ¼ 0.844). C, The ROC curve of the two-piRNA panel for detection of CA19-9-negative smallcolorectal cancer (AUC ¼ 0.855). D, The two-piRNA panel displayed significantly higher performance than CEA and CA19-9 for detection of early-stage colorectalcancer (P < 0.0001). E, The ROC curve of the two-piRNA panel for detection of CEA-negative early-stage colorectal cancer (AUC¼ 0.839). F, The ROC curve of thetwo-piRNA panel for detection of CA19-9-negative early-stage colorectal cancer (AUC ¼ 0.828).






colorectal cancer compared with normal controls, suggesting thatthe aberrant expression of the two piRNAs occurs early in theprecancerous stages of colorectal cancer. Activation of the twopiRNAs may contribute to the pathological process of colorectalcancer initiation, and their abnormal upregulation in sera can serveas a sensitive indicator for early colorectal cancer detection.

Most patients with early-stage colorectal cancer do not present anyphenotypic symptoms, and are often missed on routine physicalexamination. The conventional serum tumor markers CEA andCA19-9 are useful in therapeutic evaluation and recurrence surveil-lance, but are still inadequate for colorectal cancer screening mainlybecause of low sensitivity in early-stage patients (3). In our studycohorts, CEA and CA19-9 indeed demonstrated poor performance forearly diagnosis of colorectal cancer, as their sensitivities were only25.60% and 13.10%, respectively. In comparison, the two-piRNApanelestablished in our study generated a greatly improved sensitivity of76.79% in detecting early-stage colorectal cancer. In recent years, theabnormally methylated SEPT9 gene has been tested for utility incolorectal cancer screening. However, the reported sensitivities ofplasma SEPT9 methylation in diagnosing early-stage patients oftenvary between over 30% to less than 70% (33–35), which is lower thanthat for our piRNA panel. Thus, the two-piRNA panel may be a usefulnoninvasive biomarker for early-stage colorectal cancer screening inasymptomatic populations.

Up to 85%of colorectal cancers originate fromadenomatous polyps.Timely recognition and removal of CRA can prevent the benignprecancerous lesions from progressing to malignancy. Thus far, feweffective blood biomarkers for CRAhave been developed. In this study,we found that the expression levels of serum piR-020619 and piR-020450 were higher in patients with CRA than in normal controls, andthat combined use of the two piRNAs had significant diagnostic valuefor CRA with a sensitivity of 72.50%. Therefore, the two piRNAs maybe suitable for the early detection of precancerous lesions of colorectalcancer and help reduce colorectal cancer incidence and mortality.

Circulating tumor biomarkers can be organ-specific or nonspecific.Organ-specific biomarkers provide the advantage of directly identi-fying tumor tissue sites. The serum levels of the tumor markers CEAand CA19-9 can be elevated in multiple malignant conditions, makingit difficult to specify the precise lesion location. In this study, theexpression of serum piR-020619 and piR-020450 was increased in

patients with colorectal cancer but not in patients with lung, breast,and gastric cancers, suggesting a potential of the two piRNAs to serveas colorectal cancer-specific circulating tumor biomarkers. One pub-lished study focusing on several types of human normal and tumortissues discovered that the dysregulation of piRNA expression intumor tissues was organ-specific (36). This specificity in both seraand tissues of patients with cancer suggests significant value of piRNAsas tumor biomarkers.

The expression levels of serum piR-020619 and piR-020450 signif-icantly decreased after the colorectal tumors were surgically removed,suggesting that the excessive circulating piR-020619 and piR-020450in the patients might originate from colorectal cancer tissues. Theycould be overexpressed in colorectal cancer cells and directly promotethe cellular malignant behaviors such as proliferation, migration,invasion, stemness-maintenance, or drug-resistance by disrupting theepigenetic regulation of the transposable elements, protein-codinggenes, or other noncoding RNAs vital for cancer risk. We willinvestigate the roles of the two piRNAs in colorectal cancer initiationand development in future work.

In conclusion, serum piR-020619 and piR-020450 are promisinglynovel colorectal cancer-specific biomarkers that can effectively detectsmall colorectal cancer lesions, early-stage colorectal cancer, and CRA.This study not only provides potential tools for colorectal cancerscreening in asymptomatic populations, but also implies that circu-lating piRNAs are a valuable source for developing new noninvasivetumor biomarkers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors’ ContributionsConception and design: Z. Wang, Y. Jia, H. ZhaoDevelopment of methodology: W. Xin, Y. Chen, Y. WuAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): Z. Wang, H. Yang, D. Ma, Y. Mu, L. Feng, J. LiangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Yang, Y. Mu, X. TanWriting, review, and/or revision of the manuscript: Z. Wang, H. Yang, X. TanAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): Z. Wang, D. Ma, Y. Mu, Q. Hao, Y. JiaStudy supervision: H. Zhao

Figure 6.

ROC curves of piR-020619, piR-020450, and the combination of piR-020619 and piR-020450 for discriminating CRA from normal controls (AUC¼0.701, 0.689, and0.771, respectively).

Wang et al.





AcknowledgmentsThis work was supported by the National Natural Science Foundation of

China 81760552 (to Z. Wang) and 81860534 (to H. Yang), by the Program of theInner Mongolia Natural Science Foundation 2016MS0824 (to Z. Wang), by theInner Mongolia Science & Technology Plan Project 201802152 (to Z. Wang), bythe Supporting Program for Outstanding Youth in Science and Technology ofInner Mongolia Autonomous Region NJYT-17-B30 (to Z. Wang), by the TumorBiotherapy Collaborative Innovation Project (to Y. Jia), by the “Keji BaiwanGongcheng” project of Inner Mongolia Medical University YKD2015KJBW008

(to Z. Wang), and by the TransGen dream fund for life science 2018-04(to Z. Wang).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 19, 2019; revised December 27, 2019; accepted February 7,2020; published first February 17, 2020.

References1. Douaiher J, Ravipati A, Grams B, Chowdhury S, Alatise O, Are C. Colorectal

cancer—global burden, trends, and geographical variations. J Surg Oncol 2017;115:619–30.

2. Hofsli E, SjursenW, PrestvikW, Johansen J, RyeM, TranøG, et al. Identificationof serum microRNA profiles in colon cancer. Br J Cancer 2013;108:1712–9.

3. Vatandoost N, Ghanbari J, Mojaver M, Avan A, Ghayour-Mobarhan M,Nedaeinia R, et al. Early detection of colorectal cancer: from conventionalmethods to novel biomarkers. J Cancer Res Clin Oncol 2016;142:341–51.

4. Chandra Gupta S, Nandan Tripathi Y. Potential of long non-coding RNAs incancer patients: from biomarkers to therapeutic targets. Int J Cancer 2017;140:1955–67.

5. PengH,Wang J, Li J, ZhaoM,Huang S-k, GuY-y, et al. A circulating non-codingRNA panel as an early detection predictor of non-small cell lung cancer. Life Sci2016;151:235–42.

6. Umu SU, Langseth H, Bucher-Johannessen C, FrommB, Keller A,Meese E, et al.A comprehensive profile of circulating RNAs in human serum. RNA Bio 2018;15:242–50.

7. WikbergML, Myte R, Palmqvist R, van Guelpen B, Ljuslinder I. Plasmami RNAcan detect colorectal cancer, but how early? Cancer Med 2018;7:1697–705.

8. Guo S, Zhang J, Wang B, Zhang B, Wang X, Huang L, et al. A 5-serum miRNApanel for the early detection of colorectal cancer. OncoTargets Ther 2018;11:2603–14.

9. Hollis M, Nair K, Vyas A, Chaturvedi LS, Gambhir S, Vyas D. MicroRNAspotential utility in colon cancer: early detection, prognosis, and chemosensitivity.World J Gastroenterol 2015;21:8284–92.

10. Weng W, Li H, Goel A. Piwi-interacting RNAs (piRNAs) and cancer: emergingbiological concepts and potential clinical implications. BBA-Rev Cancer 2019;1871:160–9.

11. Chalbatani GM, Dana H, Memari F, Gharagozlou E, Ashjaei S, Kheirandish P,et al. Biological function and molecular mechanism of piRNA in cancer.Pract Lab Med 2018;13:e00113.

12. Weng W, Liu N, Toiyama Y, Kusunoki M, Nagasaka T, Fujiwara T, et al. Novelevidence for a PIWI-interacting RNA (piRNA) as an oncogenic mediator ofdisease progression, and a potential prognostic biomarker in colorectal cancer.Mol Cancer 2018;17:16.

13. Ai L, Mu S, Sun C, Fan F, Yan H, Qin Y, et al. Myeloid-derived suppressor cellsendow stem-like qualities to multiple myeloma cells by inducing piRNA-823expression and DNMT3B activation. Mol Cancer 2019;18:88.

14. Tan L, Mai D, Zhang B, Jiang X, Zhang J, Bai R, et al. PIWI-interacting RNA-36712 restrains breast cancer progression and chemoresistance by interactionwith SEPW1 pseudogene SEPW1P RNA. Mol Cancer 2019;18:9.

15. Sadik N, Cruz L, Gurtner A, Rodosthenous RS, Dusoswa SA, Ziegler O, et al.Extracellular RNAs: a new awareness of old perspectives. Methods Mol Biol2018; 1740:1–15.

16. HongY,WangC, FuZ, LiangH, Zhang S, LuM, et al. Systematic characterizationof seminal plasma piRNAs as molecular biomarkers for male infertility. Sci Rep2016;6:24229.

17. Kurth HM, Mochizuki K. 20-O-methylation stabilizes Piwi-associated smallRNAs and ensures DNA elimination in tetrahymena. RNA 2009;15:675–85.

18. Yang X, Cheng Y, Lu Q, Wei J, Yang H, Gu M. Detection of stably expressedpiRNAs in human blood. Int J Clin Exp Med 2015;8:13353–8.

19. Fehlmann T, Reinheimer S, Geng C, Su X, Drmanac S, Alexeev A, et al. cPAS-based sequencing on the BGISEQ-500 to explore small non-coding RNAs.Clin Epigenetics 2016;8:123–32.

20. Czech B, Munaf�o M, Ciabrelli F, Eastwood EL, Fabry MH, Kneuss E, et al.piRNA-guided genome defense: from biogenesis to silencing. Annu Rev Genet2018;52:131–57.

21. Fu A, Jacobs DI, HoffmanAE, Zheng T, Zhu Y. PIWI-interacting RNA 021285 isinvolved in breast tumorigenesis possibly by remodeling the cancer epigenome.Carcinogenesis 2015;36:1094–102.

22. Huang XA, Yin H, Sweeney S, Raha D, Snyder M, Lin H. A major epigeneticprogramming mechanism guided by piRNAs. Dev Cell 2013;24:502–16.

23. Peng L, Song L, Liu C, Lv X, Li X, Jie J, et al. piR-55490 inhibits the growth of lungcarcinoma by suppressing mTOR signaling. Tumor Biol 2016;37:2749–56.

24. Yin J, Jiang XY, Qi W, Ji CG, Xie XL, Zhang DX, et al. piR823 contributes tocolorectal tumorigenesis by enhancing the transcriptional activity of HSF 1.Cancer Sci 2017;108:1746–56.

25. Mai D, Ding P, Tan L, Zhang J, Pan Z, Bai R, et al. PIWI-interacting RNA-54265is oncogenic and a potential therapeutic target in colorectal adenocarcinoma.Theranostics 2018;8:5213–30.

26. Yu Y, Xiao J, Hann SS. The emerging roles of PIWI-interacting RNA in humancancers. Cancer Manage Res 2019;11:5895–909.

27. Zhang H, Ren Y, Xu H, Pang D, Duan C, Liu C. The expression of stem cellprotein Piwil2 and piR-932 in breast cancer. Surg Oncol 2013;22:217–23.

28. Yan H, Wu Q, Sun C, Ai L, Deng J, Zhang L, et al. piRNA-823 contributes totumorigenesis by regulating de novo DNA methylation and angiogenesis inmultiple myeloma. Leukemia 2015;29:196–206.

29. Wu D, Fu H, Zhou H, Su J, Zhang F, Shen J. Effects of novel ncRNA molecules,p15piRNAs, on the methylation of DNA and histone H3 of the CDKN2Bpromoter region in U937 cells. J Cell Biochem 2015;116:2744–54.

30. Liu J, Zhang S, Cheng B. Epigenetic roles of PIWI-interacting RNAs (piRNAs) incancer metastasis. Oncol Rep 2018;40:2423–34.

31. Moyano M, Stefani G. piRNA involvement in genome stability and humancancer. J Hematol Oncol 2015;8:38.

32. Negrini S, Gorgoulis VG, Halazonetis TD. Genomic instability—an evolvinghallmark of cancer. Nat Rev Mol Cell Biol 2010;11:220–8.

33. Church TR, Wandell M, Lofton-Day C, Mongin SJ, Burger M, Payne SR, et al. ,et al. Prospective evaluation of methylated SEPT9 in plasma for detection ofasymptomatic colorectal cancer. Gut 2014;63:317–25.

34. Lee HS, Hwang SM, Kim TS, Kim D-W, Park DJ, Kang S-B, et al. Circulatingmethylated septin 9 nucleic acid in the plasma of patients with gastrointestinalcancer in the stomach and colon. Transl Oncol 2013;6:290–6.

35. Wang Y, Chen P-M, Liu R-B. Advance in plasma SEPT9 gene methylationassay for colorectal cancer early detection. World J Gastrointest Oncol 2018;10:15–22.

36. Martinez VD, Vucic EA, Thu KL, Hubaux R, Enfield KS, Pikor LA, et al. Uniquesomatic andmalignant expression patterns implicate PIWI-interacting RNAs incancer-type specific biology. Sci Rep 2015;5:10423.






Published OnlineFirst February 17, 2020.Cancer Epidemiol Biomarkers Prev Zhenfei Wang, Hao Yang, Daguang Ma, et al. Colorectal CancerAre Promising Novel Biomarkers for Early Detection of Serum PIWI-Interacting RNAs piR-020619 and piR-020450

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