The Oncogenetic Function And Diagnosistic Value Of MiR-31and MiR-1322in Esophageal Squamous Cell Carcinoma

Oesophageal cancer ranks seventh and sixth in cancer incidence and mortality rate worldwide, respectively. A total of50%of all oesophageal cancer worldwide occurs in China. Esophageal squamous cell carcinoma (ESCC) accounts for approx.90%of all oesophageal carcinomas diagnosed at an advanced stage. The difficulty in managing ESCC is due to its aggressive invasion and early metastasis to lymph nodes, adjacent tissue and organs. The majority of cases are diagnosed at a relatively late stage of the disease, when the chances of surgical intervention are lost. Even with surgery, the median survival rate of ESCC patients after RO resection (the complete removal of all tumour with microscopic examination of margins showing no tumour cells) is less than2years. Additionally, ESCC is relatively resistant to both chemotherapy and radiotherapy. Many studies have shown that the development and metastasis of ESCC relates to the dysregulation of several oncogenes and tumour suppressor genes in multiple pathways. Understanding the molecular pathogenesis of ESCC, and especially the mechanisms of tumorigenesis and metastasis, is extremely important for developing novel biomarkers and treatment strategies.microRNAs (miRNAs), which are endogenous small single-stranded non-coding RNAs ranging from19to25nt, play an important role in epigenetic and post-transcriptional regulation networks. Their roles in cancer development and metastasis lead to an extensive exploration of oncogenetic or tumour-suppressivemiRs inmultiple cancers, ofwhich some were potential diagnostic and prognostic markers for certain cancers. Since miRs have been shown to be stable in serum and an increasing number reports have revealed that circulating miRs could serve as a diagnostic marker for various cancers.The expression changes of miR-31are frequently reported in multiple cancers. Interesting, miR-31is widely up-regulated inmost SCCs. In the first part the expression of miR-31was detected in45paired ESCC tissues and523serum samples using real-time RT (reverse transcription)-PCR. The result showed that miR-31was up-regulated in77.8%of the ESCC tissues. Serum miR-31levels in ESCC patients were significantly higher than in normal controls (P<0.001). Patients with high-levels of serum miR-31also had a poorer prognosis in relapse-free survival (P=0.001) and tumour-specific survival (P=0.005). ROC analysis suggested that miR-31can serve as a potential diagnostic and prognostic biomarker for ESCC. In vitro studies showed that miR-31promoted ESCC colony formation, migration and invasion. Luciferase reporter and Western blot assays confirmed that three tumour suppressor genes, namely EMP1(epithelial membrane protein1), KSR2(kinase suppressor of ras2) and RGS4(regulator of G-protein signalling4), were targeted by miR-31. The oncogene role of miR-31in ESCC mainly is dued to the down-regulation of several tumor suppresion genes especially EMP1.Esophageal cancer-related gene2(ECRG2), also known as SPINK7) is regarded as a tumor suppressor gene in esophageal cancer. It was first cloned and identified by our laboratory (Genbank Accession Number AF268198). There is a triplet TCA short tandem repeat (STR) polymorphism located in the3'-UTR of ECRG2with two specific alleles, TCA3(TCATCATCA) and TCA4(TCATCATCATCA). The TCA3allele is proved to be a risk factor for ESCC by that subjects who carried the TCA3/TCA3genotype were at an increased risk of ESCC and a significantly poorer prognosis. In the second part we investigated all the microRNAs predicted to bind at the ECRG2STR region and regulation the function of ECRG2. miR-580,miR-1182,miR-1272,miR-1322were predicted to bind at the STR region. The expression of TCA4is higher than TCA3because that miR-1322could significantly down-regulate the with TCA3allele (<0.01), but it could not down-regulate the with TCA4allele significantly (>0.05). MiR-1322was also expressed significantly higher in ESCC tissue and serum samples than in controls (both<0.01). ROC analysis suggested that miR-1322can serve as a potential diagnostic biomarker for ESCC. MiR-1322can regulate in an allele-specific manner and that serum levels of miR-1322can serve as a potential diagnostic biomarker for patients with ESCC.In conlusion, we verified the oncogene function and potential diagnosis biomarker of miR-31and miR-1322in ESCC, which shed light on the early detection and target therapy for ESCC. [Abstract] MicroRNA-31(miR-31) is frequently altered in numerous cancers. The aim of the present study was is to investigate the role of miR-31in oesophageal squamous cell carcinoma (ESCC). Method We measured miR-31in45paired ESCC tissues and523serum samples using real-time RT (reverse transcription)-PCR. The serum samples were divided into a discovery group (120ESCCs and121normal controls), a validation group (81ESCCs and81controls), and a final group comprising six other common tumours (colorectal, liver, cervical, breast, gastric and lung cancers; total n=120). A Mann-Whitney U test and Wilcoxon matched-pairs test were used for the statistics. Result miR-31was up-regulated in77.8%of the ESCC tissues. Serum miR-31levels in ESCC patients were significantly higher than in normal controls (P<0.001). It yielded an ROC (receiver operating characteristic) AUC (area under the curve) of0.902[95%CI (confidence interval),0.857-0.936] in the discovery group and a similar result in the validation group [ROC AUC,0.888(95%CI,0.819-0.939)]. Patients with high-levels of serum miR-31also had a poorer prognosis in relapse-free survival (P=0.001) and tumour-specific survival (P=0.005). In vitro studies showed that miR-31promoted ESCC colony formation, migration and invasion. Luciferase reporter and Western blot assays confirmed that three tumour suppressor genes, namely EMP1(epithelial membrane protein1), KSR2(kinase suppressor of ras2) and RGS4(regulator of G-protein signalling4), were targeted by miR-31. Conclusion We conclude that miR-31plays oncogenetic functions and can serve as a potential diagnostic and prognostic biomarker for ESCC. [Abstract] Aims A short tandem repeat (STR) polymorphism in the3'UTR region of esophageal cancer-related gene2(ECRG2, also known as) has been widely reported to be associated with the incidence and the prognosis of esophageal squamous cell carcinoma (ESCC). This study explores how the microRNA binding to the STR. region affects expression in ESCC. Method Dual-luciferase reporter assays were used to verify the effects of the four microRNAs (miR-580, miR-1182, miR-1272, and miR-1322) predicted to bind the STR region of the3'untranslated region (UTR). The expression of identified effective microRNA was then analyzed in44paired ESCC and adjacent normal tissues and402case-controlled serum samples (divided into a discovery group and an independent validation group) by real-time RT-PCR assay. Result We found that only miR-1322could significantly down-regulate the with TCA3allele (<0.01), but it could not down-regulate the with TCA4allele significantly (>0.05). MiR-1322was also expressed significantly higher in ESCC tissue and serum samples than in controls (both<0.01). Additionally, serum levels of miR-1322yielded an under receiver operating characteristic (ROC) curve area of0.847(95%CI,0.795-0.890) for discriminating ESCCs from healthy controls in the discovery group and a similar result was obtained in the validation group (under ROC area is0.845;95%CI,0.780-0.897). Conclusion We conclude that miR-1322can regulate in an allele-specific manner and that serum levels of miR-1322can serve as a potential diagnostic biomarker for patients with ESCC.