Long Non Coding RNA OIP5-AS1 Inhibits The Proliferation Of Esophageal Squamous Cell Carcinoma Cells By Targeting MiR-30a-5p Via FOXD1/ERK1/2 Pathway

Objectives:In recent years,it has been widely reported in tumor studies that long non-codingRNA(LncRNA OIP5-AS1)plays an important role in the occurrence and development of a variety of tumors,and there are more and more researches on targeted therapeutic drugs for LncRNA.This study aims to reveal the following points: the molecular mechanism of LncRNA OIP5-AS1 by regulating miR-30a-5 to inhibit proliferation of esophageal squamous cell carcinoma cells through down-regulation of the expression of FOXD1 and ERK1/2,the binding and regulation relationship between LncRNA OIP5-AS1 and miRNA-30a-5p,the negative regulation of miRNA-30a-5p on the expression of FOXD1,the regulation relationship of FOXD1 on the expression of ERK1/2,and the effect of the expression of ERK1/2 on the phenotype of esophageal cancer cells.Methods:Firstly,the expression levels ofRNA-OIP-AS1 and miRNA-30a-5p in esophageal cancer tissues and normal tissues were detected by q-PCR(gene amplification),WB(Western blot),IHC(immunohistochemistry),to study their relationship and correlation with esophageal cancer,and these expressions and relationships were also verified in esophageal cancer cells and normal cells.Secondly,the expression of FOXD1 in esophageal cancer cells and normal cells was detected by WB and q PCR.The expression level of miRNA-30a-5p was detected by inhibiting LncRNA-OIP-AS1 in esophageal cancer cells,and their regulatory relationship was analyzed.The expression level of FOXD1 was detected by up-regulation of miRNA-30a-5p.Predicted in Targetscan database and detected by dual luciferase reporter gene assay,the interaction between miRNA-30a-5p and FOXD1 was analyzed.The expression of FOXD1 was detected by overexpression and inhibition of miRNA-30a-5p,and the regulatory relationship between miRNA-30a-5p and FOXD1 was determined.Then,FOXD1 was inhibited,the expression of ERK1/2 was detected,and the regulatory relationship between FOXD1 and ERK1/2 was analyzed.Inhibiting the expression of ERK1/2,the proliferation,the number of cell clones,the early apoptosis and invasion rate of esophageal cancer cells were detected by MTT,crystal violet staining,flow cytometry and small chamber invasion,and the effects of ERK1/2expression on the phenotype of esophageal cancer cells were analyzed.Thirdly,by inhibiting the expression ofRNA-OIP-AS1 we detected the protein expression of miRNA-30a-5p,FOXD1,ERK1/2 and the changes of proliferation,apoptosis and invasion ability of esophageal cancer cells.Finally,we injected subcutaneous tumor-forming cells with inhibition of LncRNA-OIP-AS1 into nude mice,detected the growth rate and size of the tumor body,collected the tumor tissue,and detected the expression of FOXD1 and ERK1/2 in the tumor tissue by WB and IHC,to verify the conclusion at the level of esophageal cancer cells.Results:The expression of LncRNA OIP5-AS1 in esophageal cancer tissues and cells was significantly higher than that in normal tissues and cells,whereas the expression of miRNA-30a-5p in esophageal cancer tissues and cells was significantly lower than that in normal tissues and cells.The expression of FOXD1 in esophageal cancer tissues and cells was significantly higher than that in normal tissues and cells.Target Scan predicted that miRNA-30a-5p and FOXD1 mRNA had a binding region of 3′-UTR.The results of dual luciferase reporter gene assay showed that miRNA-30a-5p and FOXD1 had an interaction and a negative regulation relationship.FOXD1 expression can promote the expression of ERK1/2,and ERK1/2 expression can promote the proliferation,cell clone formation and invasion ability of esophageal cancer cells,and inhibit the early apoptosis rate of esophageal cancer cells.In vivo experiments showed that inhibition of LncRNA OIP5-AS1 reduced the tumor formation of esophageal cancer cells.The expression of FOXD1 and ERK1/2decreased significantly.Conclusions:1.The expression of LncRNA OIP5-AS1 in esophageal cancer tissues and cells was significantly higher than that in normal tissues and cells,while the expression of miRNA-30a-5p was on the contrary.2.LncRNA OIP5-AS1 negatively regulated miRNA-30a-5p in esophageal cancer cells.MiRNA-30a-5p targets FOXD1 in the 3′-UTR region of esophageal cancer cells and negatively regulates FOXD1 expression.3.Both FOXD1 and ERK1/2 were significantly higher in esophageal cancer tissues and cells than in normal tissues and cells.FOXD1 can positively regulate the expression of ERK1/2,and the expression of ERK1/2 can promote the proliferation,cell clone formation and invasion ability of esophageal cancer cells,and inhibit the early apoptosis rate of esophageal cancer cells.In vivo experiments of esophageal carcinoma cells in nude mice showed the same results as at the cellular level.4.In other words,by inhibiting the up-regulation of miRNA-30a-5p expression by LncRNA OIP5-AS1,the expression of FOXD1 and ERK1/2 can be inhibited so as to inhibit the proliferation,invasion and promote apoptosis of esophageal cancer cells.This conclusion may provide some new insights for the development of targeted therapies for esophageal cancer.