The Expression And Regulation Mechanism Of Long Non-coding RNA LINC01021 In Esophageal Squamous Cell Carcinoma And Its Effect On Biological Characteristics Of Esophageal Cancer Cells

Part one Expression of long non-coding RNA LINC01021 in esophageal cancer cell lines and esophageal squamous cell carcinoma tissuesObjectives: To detect the expression of long non-coding RNA LINC01021 in human esophageal cancer cell lines and esophageal squamous cell carcinoma(ESCC)tissues,and to discuss clinicopathologic and prognostic significance of it in ESCC.Methods: RT-qPCR method was applied to detect the mRNA expression levels of LINC01021 in esophageal cancer cell lines(TE1,TE13,Eca109,Kyse150,Kyse170)and 52 esophageal squamous cell carcinomas(ESCC)tissues and corresponding noncancerous tissues.Result:1.The mRNA expression of LINC01021 in esophageal cancer cell lines.The LINC01021 gene was overexpressed in all five esophageal cancer cell lines,especially in Eca109 cells.2.The mRNA expression of LINC01021 in ESCC tissues and corresponding noncancerous tissues.The expression of LINC01021 in ESCC tissues was obviously higher than that in corresponding noncancerous tissues(P<0.05).The expression of LINC01021 was closely correlated with TNM stages,pathological differentiation and lymph node metastasis(P<0.05),but not with gender and age(P>0.05).Part two Effect of long non-coding RNA LINC01021 on the biological characteristics of esophageal cancer cellsObjectives: To detect the effect of knock down lncRNA LINC01021 on proliferation,migration,invasion ability and cell cycle of human esophageal cancer cell lines and its related mechanism.Methods:1 Design and synthesis LINC01021 shRNA vector.LINC01021 shRNA was transfected into human esophageal cancer cell line Eca109.Transfection efficiency was evaluated by RT-qPCR.2 MTS and clone formation experiment were respectively applied to detect the proliferation ability of esophageal cancer cell lines with LINC01021 knocked down.3 Scratch test was applied to detect the migration ability of esophageal cancer cell lines with LINC01021 knocked down.4 Transwell chamber invasion assay was applied to detect the invasion ability of esophageal cancer cell lines with LINC01021 knocked down.5 Flow cytometry was applied to detect the cell cycle of esophageal cancer cell lines with LINC01021 knocked down.6 RT-q PCR method was applied to detect the mRNA expression levels of EMT-related genes(E-cadherin,Vimentin,Snai1,Twist1,and ZEB1)in esophageal cancer cell lines with LINC01021 knocked down.Result:1.LINC01021 shRNA plasmid screening and quantification of knockdown Efficiency: Eca109 cells knocked down by LINC01021 gene were successfully constructed.The results of RT-q PCR analysis showed that the relative expression level of LINC01021 gene in Eca109 transfection group was significantly lower than that in control group,and the relative expression level of LINC01021 gene in LINC01021sh4 transfection group was the lowest(P<0.05).The transfection efficiency was sufficient to continue the further experiment.2.The effect of LINC01021 gene knocked down on cell proliferation abilities of esophageal cancer cell lines.MTS and clone formation experiment demonstrated that the knock down of LINC01021 gene could inhibit the proliferation ability of Eca109 cells in vitro(P<0.05).3.The effect of LINC01021 gene knocked down on cell migration abilities of esophageal cancer cell lines.Scratch test demonstrated that the knock down of LINC01021 gene could inhibit the migration ability of Eca109 cells in vitro(P<0.05).4.The effect of LINC01021 gene knocked down on cell invasion abilities of esophageal cancer cell lines.Transwell chamber invasion assay demonstrated that the knock down of LINC01021 gene could inhibit the invasion ability of Eca109 cells in vitro(P>0.05).5.The effect of LINC01021 gene knocked down on cell cycle of esophageal cancer cell lines.Flow cytometry assay demonstrated that the knock down of LINC01021 increased relative proportion in S and G2/M phase.In addition,knock down of LINC01021 inhibited esophageal cancer cells proliferation probably through the induction of S and G2/M phase cell cycle arrest.6.The effect of LINC01021 gene knocked down on the expression of EMT-related gene.Knock down of LINC01021 gene significantly up-regulated E-cadherin mRNA expression(P<0.05)and down-regulated the mRNA expression of Vimentin,Snail,Twist1,and ZEB1 in Eca109 cells(P<0.05).Conclusion:1.The high expression of LINC01021 in ESCC tissues and esophageal cancer cell lines maybe related to the occurrence and development of esophageal squamous cell carcinoma.2.LINC01021 can affect the proliferation,invasion and migration of esophageal cancer cells in vitro,and regulate the proliferation of esophageal cancer cells through affecting cell cycle.The effect of LINC01021 gene on the biological behavior of esophageal cancer cells may be related to EMT.