MiR-613:A Novel Diagnostic And Prognostic Biomarker For Patients With Esophageal Squamous Cell Carcinoma

Purpose:Esophageal carcinoma is a serious malignancy with regards to mortality and prognosis,and is expected to increase in incidence over the next 10 years.The outcome is far from satisfactory with 15-25%overall 5-year survival rate.Tumor markers of esophageal cancer are an advancing research area that could potentially lead to earlier diagnosis as well as playing a part in assessing tumor response to therapy.Recently,platelet count tumor length and nutrition proved to be associated with prognosis of esophageal cancer patients.Therefore,the significance of detecting novel biomarkers for ESCC should be emphasized.The expression profiling of miRNAs has already entered into cancer clinics as diagnostic and prognostic biomarkers to assess tumorigenesis,progression and response to treatment in cancer patients.For instance,serum miR-25,miR-223 and miR-375 have been identified as diagnostic factors for ESCC.Serum miR-24 and miR-200c also have the potential to serve as a noninvasive biomarker for predicting treatment responses to chemotherapy or concurrent chemo-radiation therapy.The miR-613 and miR-1297 function as an oncogene or tumor suppressor in different cancer types.MiR-613 was down-regulated in papillary thyroid cancer(PTC),while it was up-regulated in gastric cancer.It could target FN1 gene and Wnt pathway.However,we still do not understand the level of miR-613 and its mechanism is still not clear in esophageal carcinoma.Our aims are to investigate the expression level of miR-613 in esophageal cancer tissues and serum,and to further explore the value of diagnosis and prognostic in clinical practise.Methods:Patients and samplesWe enrolled serum samples from 75 ESCC patients before surgery,and 75 age-and sex-matched healthy volunteers in Qilu Hospital of Shandong University between October 2014 and March 2015.At the same time,60 pairs of ESCC and paracancerous tissue of the previous patients who underwent surgeries were collected(15 cases did not receive surgery due to their bad condition and the refusal on surgery).Cancerous and paracancerous specimens were taken post-operationally for each patient within 30 min.Meanwhile,109 formalin-fixed paraffin-embedded(FFPE)cancerous tissue samples of 2009 were collected from Qilu Hospital.All patients were pathologically diagnosed as ESCC using surgical specimens or biopsies.None of the ESCC patients had received any anticancer treatment prior to sampling.All data,including age,sex,smoking and drinking habit,tumor length,tumor site,invasion depth(T stage),lymph node metastasis(N stage),distant metastasis(M stage)and differentiation degree were obtained from clinical or pathologic records.RNA isolationTissue samples were preserved at-80℃ prior to RNA isolation.Total-RNA was extracted from fresh tissue,serum and FFPE tissue samples using miRNeasy Mini Kit,miRNeasy Serum/Plasma Kit and miRNeasy FFPE Kit(QIAGEN,Duesseldorf,North lane,German)in accordance with the manufacturer’s protocols.RNA yield was determined from the A260/A280 absorbance ratio using a DeNovix DS-11 spectrophotometer(DeNovix,Wilmington,DE,USA).qRT-PCRAll-in-OneTMmiRNAqRT-PCR Detection Kit(GeneCopoeia,Rockville,USA)was used in reverse transcription(RT).The RT reaction system was 25μl,The 20ul reaction system for qPCR was 20ul.The expression levels of tissue miRNAs were normalized to RNU6,whereas miR-16 was used as an internal control for serum samples.Relative quantification of miRNA expression was calculated with 2-△△Ct method in fresh tissue and serum samples[13]while 2-△Ct method was used for FFPE cancerous tissue samples.Results:The expression level of miR-613,in ESCC tissues compared to paired paracancerous tissuesThe expression levels of miR-613 in 60 pairs of ESCC cancerous and paracancerous tissue samples were analyzed by qRT-PCR.MiR-613 showed lower expression level in cancerous tissues(0.34 ±0.56 vs 1.08 ±0.07,P<0.001).We also analyzed the relationship between miR-613 expression and clinicopathological features in ESCC patients in order to better understand their potential roles in the development and progression of ESCC,We found that the expression level of miR-613 was significantly reduced with increased T stage of ESCC(P = 0.008).The expression level and diagnostic value of serummiR-613 We hypothesized that the expression levels of miRNAs in ESCC tissues would influence serum levels of ESCC patients.Statistically significant differences between ESCC patients and healthy controls in expression levels of miR-613(0.89±0.73 vs 1.71 ±1.03,P<0.001).Reduced expression of serum miR-613 was significantly associated with N stage(P＝0.038).No other significant differences were found between the expression levels of serum miR-613 and clinicopathological characteristics(all P>0.05).We also conducted the Pearson correlation coefficient analysis to value the association of miR-613 expression level in frozen cancerous tissues with its level in serums.It revealed that the Pearson correlation coefficient is 0.812(P<0.001).We further analyzed ROC curve of these two miRNAs to assess their diagnostic value.It showed that the AUC based on serum miR-613 was 0.767±0.040.At the cut-off point(Yuden Index)of 0.875,the sensitivity and specificity were 81.3%and 62.7%for serum miR-613.Further analysis on early stage patients were performed and revealed that the AUC was 0.728 ±0.052 for miR-613(P<0.001).The association of miR-613 with clinical outcomes of ESCC patients We selected 109 patients to assess the expression level of miR-613 in FFPE tissues.The mean level of 0.0016 were regarded as the cut-off value for miR-613.No significant relationship was found between miR-613 expression level and patients’ clinicopathological features.The median survival time of all patients was 39 months(range 6-81).35 patients(32.11%)were alive and 74 patients(67.89%)died during our follow-up period.Kaplan-Meier curve revealed that the down-regulation level of miR-613 was related to worse overall survival(OS)and progression-free survival(PFS)(P = 0.018 and P = 0.035).Furthermore,the multivariate analysis identified miR-613to be independent prognostic factors for OS(P = 0.030)and PFS(P = 0.036).Conclusions:(1)MiR-613 showed lower expression level in cancerous tissues(2)The expression level of miR-613 was significantly reduced with increased T stage of ESCC.(3)MiR-613 expression level in serums is associated with the level infrozen cancerous tissues.Reduced expression of serum miR-613 was significantly associated with N stage and diagnostic value is great significance.(4)the down-regulation level of miR-613 was related to worse overall survival(OS)and progression-free survival(PFS)...