Combined MiRNA With DNA Methylation As A Diagnosis Panel For Esophageal Squamous Cell Carcinoma And The Study Of Methylation Of Candidate Gene TLX2 In Esophageal Squamous Cell Carcinoma

Esophageal cancer(EC)is a common gastrointestinal malignancy.Globally,more than 455,800 new cases of EC are diagnosed in 2002 and 49% of cases are in China.In China,esophageal cancer is the second most common types of cancer,the fourth in the all cancer mortality.There are two main types of esophageal cancer-squamous cell carcinoma(ESCC)and adenocarcinoma(EAC),which have quite distinct etiology andepidemiology.In the United States,more than 80% of esophageal cancer are EAC,but ESCC is the predominante type in Asian and African countries,and about 90% EC are this type in China.Although there are more biomarkers in the diagnosis of ESCC,but because of the lack of enough sensitivity and high specificity,still can't meet the clinical requirements.Therefore,it is important to search for new biomarkers or associated multiple biomarkers to improve the sensitivity and specificity for the diagnosis of ESCC.MicroRNAs(miRNAs)are a class of small non-coding RNA molecules of 18-25 nucleotides that are thought to be involved in the development of cancer.Recent studies have demonstrated that miRNAs can serve as useful biomarkers for cancer.Single miRNA as biomarker has low sensitivity and specificity,and the combination multiple miRNAs can improve the diagnostic efficacy.Based on the microRNA-seq data of EC from TCGA database,we analyzed to obtain each mature miRNA of each sample and then screened using two criteria: abundance at least above 500 reads / millon;P< 0.05(Wilcoxon's rank sum test).The selected mi RNAs were screened again by Random Forest.Finally we selected nine differentially expressed miRNAs as our candidate miRNAs.They were miR-93-5p,miR-21-3p,miR-30d-5p,miR-25-3p,miR-30e-3p,miR-21-5p,miR-181a-5p,miR-29c-3p,and miR-103a-3p.Taking into account the literature,we listed these nine miRNAs as candidate miRNAs.The expression of these nine miRNAs was detected by qRT-PCR in 93 pairs of ESCC cancer and matched paracancerous tissues.MiR-30e-3p,miR-21-3p and miR-21-5p showed significant differences in expression levels compared ESCC tissues with paracancer tissues.The expression of miR-30e-3p in ESCC was significantily lower than that in adjacent tissues.The expression of miR-21-3p and mi R-21-5p in ESCC was significantly higher than that in adjacent tissues.Combining these three miRNAs in the diagnosis of ESCC,the sensitivity was 0.62,the specificity was 0.91 and the area under the ROC curve(AUC)was 0.80,indicating that the diagnostic system has diagnostic effect.Studies have shown that DNA methylation can also be used as a biomarker for the diagnosis of ESCC.On the basis of our analysis of the 3 miRNAs,we analyzed the DNA methylation betweenESCC tissues and adjacent tissues,with the aim of further improving the effect of the diagnosis of ESCC.ELMO1,KCNA3,KCNA6 and TLX2 genes were put on our candidate list with hypermethylation in esophageal cancer by using the TCGA database and Pubmed.The four genes were sequenced in 94 pairs of ESCC cancer and matched paracancerous tissues.The results showed the methylation of KCNA3,KCNA6 and TLX2 gene in ESCC was significantly higher than adjacent tissues.These prompted that three genes could be used as molecular biomarkers to diagnose ESCC.The diagnostic effect was analyzed by logistic regression,the sensitivity was 0.79,the specificity was 0.84 and the area under the ROC curve(AUC)was 0.84.Based on the strategy of improving the diagnostic efficiency by using multiple different types of biomarkers,we combined the miRNA diagnosis system with the gene methylation diagnosis system again.Logistic regression analysis showed that the sensitivity,specificity and AUC of 6 molecular markers of ESCC were 0.86,0.87 and 0.90,respectively,which greatly improving the accuracy of the diagnosis.Among these differential methylation genes,we found that methylation difference of TLX2 gene was the most significant,and its function in ESCC has not been reported.Therefore,we selected the TLX2 gene as a candidate gene.Firstly the expression of TLX2 mRNA was measured by qRT-PCR in 50 pairs of ESCC and adjacent tissues.The results showed that TLX2 expression in ESCC was significantly lower than that in adjacent tissues.We speculated that it is likely the high methylation of the TLX2 gene leads to a decrease expression in its mRNA level.Then Then Ec-109 and CaEs-17 treated with demethylated drugs 5-Aza-2,-deoxycytidine resulted in a significant increase in TLX2 expression,further demonstrating that the TLX2 gene was regulated by methylation.Luciferase assays validated that hypermethylation of the TLX2 promoter region resulted in a decrease in its expression level.Moreover,TLX2 overexpression inhibited ESCC cell proliferation.These results suggested that TLX2 played an important role in development of ESCC.As a result,we identified a combination of three mi RNAs(miR-30e-3p,miR-21-3p,miR-21-5p)and three gene methylation(KCNA3,KCNA6,TLX2)as a method of diagnosis of ESCC,which may assist clinical diagnosis.In addition,we showed that TLX2 gene is regulated by methylation in ESCC and TLX2 can inhibit the proliferation of ESCC cells.AII above will lay a basis for further exploring the molecular biological mechanism of gene methylation in the development of ESCC.