Expression Level And Role Of Long Non-coding RNA LINC01137 In Esophageal Squamous Cell Carcinoma

Objective: Esophageal squamous cell carcinoma(ESCC)is a common aggressive malignancy with a high mortality rate.Exploring the molecular mechanism of its development and finding new diagnostic and therapeutic targets are great significance for the treatment of ESCC.Studies have found that long non-coding RNA(lncRNA)is involved in the development of many diseases,and the relationship between lncRNA and tumors has attracted more and more attention.A large number of reports have shown that lncRNA is closely related to the occurrence,development,migration,invasion and survival time of a variety of tumors.In recent years,studies have found that the occurrence of ESCC is also regulated by a variety of lncRNAs.Our study investigated the expression level of lncRNA LINC01137 in ESCC and its role in the growth,migration and invasion of ESCC cells.The results showed that lncRNA LINC01137 was significantly up-regulated in ESCC tissues and cell lines,which suggesting that LINC01137 may play a role in the development of ESCC.Small interfering RNA(siRNA)was used to knocked down LINC01137 expression in ESCC cells.The proliferative and invasive effects of LINC01137 downregulation on ESCC cells were measured.Our study may provide new insight into the mechanisms underlying ESCC tumorigenesis.Methods:1.Firstly,we detected the expression level of LINC01137 in 10 paired ESCC and adjacent normal tissues by RT-qPCR.We also detected the expression level of LINC01137 in a normal esophageal squamous cell line(Het-1a)and four ESCC cell lines(Eca109,Kyse130,Kyse150,Kysete1).2.Then we designed two siRNAs target LINC01137 and transfectd them into ESCC cells to knock down the expression of the LINC01137.3.We used CCK-8 assay,Colony Formation assay to analyze the effect of downregulation LINC01137 on the proliferation ability of ESCC cells.4.We used Transwell assay to investigate the effect of knockdown LINC01137 on the migration and invasion ability of ESCC cells.5.Western Blot was used to detect the effect of LINC01137 knockdown on proliferation and EMT-related proteins.6.The localization of LINC01137 in ESCC cells was detected by cytoplasmic assay.Results:1.The expression of LINC01137 in ESCC tissuesLINC01137 expression in 10 paired ESCC tissues and adjacent normal esophageal tissues was detected by RT-qPCR,and the results showed that LINC01137 expression was significantly higher in ESCC than that in adjacent normal esophageal tissues,with statistical significance(P<0.05).2.The expression of LINC01137 in ESCC cellsThe expression of LINC01137 was detected in 1 normal esophageal squamous epithelial cell line(Het-1a)and 4 ESCC cell lines(Eca109,Kyse130,Kyse150,Kysete1)by RT-qPCR.The results showed that LINC01137 expression in ESCC cells was significantly higher than that in normal esophageal squamous epithelial cells,with statistical significance(P<0.01).Meanwhile,the expression of LINC01137 in Eca109 and Kyse130 cells was relatively higher than that in the other two ESCC cell lines.Therefore,Eca109 and Kyse130 cells were used for subsequent experiments.3.SiRNA was constructed to knock down the expression of LINC01137We designed two pairs of siRNA targeting LINC01137(siLINC01137-1 and siLINC01137-2)to down-regulate the expression of LINC01137 in ESCC cells.Eca109 and Kyse130 cells were transfected with siLINC01137-1 and siLINC01137-2,and total RNA was extracted 48 hours after transfection.The results of RT-qPCR showed that compared with the control group,the expression of LINC01137 in siLINC01137 s transfected groups was reduced by more than 50%(P<0.01),which indicating that both siRNA targeting LINC01137 could significantly knockdown the expression of LINC01137.4.The effect of LINC01137 knockdown on proliferation,migration and invasion of ESCC cellsEca109 and Kyse130 cells were transfected with siLINC01137-1 and siLINC01137-2,and the proliferative and invasive effects of LINC01137 downregulation in ESCC cells were measured by CCK-8,Colony Formation and Transwell assays.The results showed that,compared with the control group,knockdown of LINC01137 expression significantly inhibited proliferation,migration and invasion of Eca109 and Kyse130 cells,with significant statistical difference(P<0.05).5.Western Blot assay was used to detect the changes of proliferation and EMT related proteins after knocked down of LINC01137SiLINC01137-1 and siLINC01137-2 were transfected into Eca109 and Kyse130 cells.Western Blot assay was used to detect the expression of proliferation and EMT related proteins after LINC01137 expression was down regulated.The results showed that the expressions of proliferation related proteins,CyclinD1 and Survivin,as well as EMT-related proteins,Snail,Vimentin and Mmp9,was significantly decreased in the experimental group comparing with the control group(P<0.05).6.Localization of LINC01137 in ESCC cells.Cytoplasmic and nuclear components of Eca109 and Kyse130 cells were separated and total RNA in nuclear and cytoplasmic fractions was extracted respectively.The expression of LINC01137 in cytoplasm and nucleus was detected by RT-qPCR.The results showed that the expression of LINC01137 in the cytoplasm of both cells was significantly higher than that in the nucleus,indicating that LINC01137 was mainly located in the cytoplasm(P<0.05).Conclusion:The expression of LINC01137 in ESCC tissues was significantly higher than that in normal esophageal tissues,and the expression of LINC01137 in ESCC cell lines was also significantly higher than that in normal esophageal squamous cell lines.Knock down of LINC01137 expression significantly inhibited the proliferative,migratory and invasive ability of ESCC cells,and obviously reduced the expression of proliferative and EMT-related proteins.