Effects Of MicroRNA-182-5p On Cell Proliferation And Invasion In Esophageal Squamous Cell Carcinoma And Related Molecular Mechanisms

Background and ObjectiveEsophageal cancer mainly includes esophageal adenocarcinoma and esophageal squamous cell carcinoma(ESCC).Global cancer statistics report that there were 572000 new cases and 509000 deaths in 2018.The majority of esophageal cancer are ESCC in world,especially more than 95%of esophageal cancer are ESCC in China.Esophageal cancer is a kind of malignant tumor that has high level of heterogeneity,in addition,most patients are in advanced stage when they are initially diagnosed,therefor,the therapeutic effect of ESCC is not ideal.The present method of treatment are mainly surgery,chemotherapy and radiation,and the immunotherapy strategy is also in clinical trials for excepting to improve patient’s quality of life and five-year survival rates.Relevant research has shown that ESCC is the consequence of the interaction of genes and environmental factors,such as dietary habits of individuals,nutritional status,smoking,drinking,Porphyromonas gingivalis(P.gingivalis)infection,gene amplification or gene mutation,However,pathogenesis of esophageal cancer cannot be fully made clear by these factors.MicroRNAs(miRNAs,miRs)which are widely expressed in tissues are small non-coding single-stranded RNA and they can regulate multiple biological processes.Abnormal expression of miRNAs may cause to various diseases and functions as oncogenes or tumor suppressor genes in tumor intiation.The microRNA-182-5P(miR-182-5p)is a member of miRNA family and previous studies demonstrated that miR-182-5p plays an important role in carcinogenesis including oral squamous cell carcinoma,renal carcinoma,liver cancer,etc.MiR-182-5p have different levels of expression and regulatory mechanism in different types of cancer.Increasing studies remind us that miR-182-5p may be promising diagnostic and prognostic molecular biomarker as well as a potential therapeutic target in malignant tumor,but it has been rarely reported that the functional role of miR-182-5p in ESCC.Therefor,we examined the expression level of miR-182-5p in ESCC tissues,ESCC cells,normal esophageal epithelial tissues and normal esophageal epithelial cells.The expression level of miR-182-5p was analyzed and compared in these specimens.Finally,we investigated the effects of miRNA-182-5p on cell proliferation and invasion in ESCC and explored its possible related molecular mechanisms.Materials and methodsA total of 52 pairs of ESCC tissues and normal esophageal epithelial tissues were collected in the First Affiliated Hospital of Zhengzhou University from June 2013 to May 2018.ESCC cells preserved in liquid nitrogen come from State Key laboratory of esophageal cancer prevention and treatment,The First Affiliated Hospital of Zhengzhou University.The clinicopathological parameters of the patients were obtained from the medical records.RT-qPCR was employed to detect the miR-182-5p expression in ESCC tissues and cells.MiR-182-5p inhibitor,miR-182-5p mimic and negative control(NC)were transfected into ESCC Eca109 and TE1 cells,and miR-182-5p expression was examined after transfection using RT-qPCR.Cell counting kit-8(CCK-8)was utilized to investigate cell proliferation and Transwell chamber was used to detect cell invasion ability.Dual-Luciferase Reporter assay was used to detect the direct interaction of miR-182-5p with cell adhesion molecule 2(CADM2)mRNA,RT-qPCR was employed to CADM2 mRNA expression in ESCC tissues,and the correlation between CADM2 mRNA and miR-182-5p was examined.Finally,Western blot was used to detect the expressions of CADM2,FAK,p-AKT and AKT proteins after transfection.Data were analyzed using the GraphPad Prism 8.0 software.Results1.The miR-182-5p level in ESCC tissues and was significantly higher than that in normal esophageal epithelial tissues,and the difference was statistical significance(P<0.0001).2.RT-qPCR was employed to detect the level of miR-182-5p expression in ESCC tissues.We constructed the media to determine the cut-off values for miR-182-5p level,the five-year survival ratio of ESCC patients with high miR-182-5p level was evidently lower than that of ESCC patients with low miR-182-5p level,and the difference was statistical significance(P<0.05).3.MiR-182-5p expression was tightly associated with TNM staging and lymph node metastasis(P<0.05),but was not correlated with gender,age,smoking,drinking and degree of differentiation(P>0.05).4.The expression of miR-182-5p in ESCC cells was all higher than that in normal esophageal epithelial cell Het-1A(P<0.01),with obviously higher in Ecal 09 and TE1.5.MiR-182-5p inhibitor significantly downregulated the miR-182-5p expression in Eca109 and TE1(P<0.0001),and its downregulation suppressed cell proliferation and invasion ability(P<0.05),conversely,miR-182-5p mimic significantly upregulated the miR-182-5p expression in Eca109 and TE1(P<0.0001),and its upregulation promoted cell proliferation and invasion ability(P<0.05).6.CADM2 mRNA was the direct target of miR-182-5p,and the miR-182-5p level exhibited negative correlation with CADM2 mRNA level in ESCC tissues(r=-0.5004,P<0.001).7.CADM2 mRNA expression was tightly associated with TNM staging and lymph node metastasis(P<0.05),but was not correlated with gender,age,smoking,drinking and degree of differentiation(P>0.05).8.MiR-182-5p inhibitor significantly upregulated the expression of CADM2 protein,but suppressed the expressions of FAK,p-AKT and AKT proteins,whereas miR-182-5p mimic markedly downregulated the expression of CADM2 protein,but promoted the expressions of FAK,p-AKT and AKT proteins.Conclusion1.The miR-182-5p level in ESCC tissues and cells was significantly higher than that in normal esophageal epithelial tissues,and its upregulation promoted cell proliferation and invasion ability.2.MiR-182-5p was potential molecular biomarkers of the TNM staging,lymph node metastasis and patient prognosis.3.CADM2 mRNA was the direct target of miR-182-5p,and the miR-182-5p level exhibited negative correlation with CADM2 mRNA level in ESCC tissues.The miRNA-182-5p/CADM2/FAK/AKT signaling way could regulate the development of ESCC.4.MiR-182-5p is implicated in the occurrence and development of ESCC,and the drugs designed to target the miR-182-5p may provide a new therapeutic direction for ESCC patients.