The Function Of LncRNA CASC9/miR-505/ERCC1 Pathway On The Growth And Migration Of Gastric Cancer

Backgroud:Gastric cancer is a malignant tumor that originates from the gastric mucosa epithelium,and has the highest incidence among various malignant tumors in China.However,the mechanism of gastric cancer occurrence and development is not clear.Recent studies have shown that long non-coding RNA(LncRNA)is widely involved in the occurrence and development of gastric cancer and server as cancer biomarkers and therapeutic target for gastric cancer.Studies have shown that CASC9 plays an important role in a variety of tumors.Nevertheless,the function and its molecular mechanism of CASC9 in gastric cancer need to be further studied.Purpose:Measure the expression of the CASC9/miR505/ERCC1 pathway in cancer tissue samples from gastric cancer patients and related adjacent healthy tissues;Detect the expression of CASC9/miR505/ERCC1 pathway in gastric cancer cells and normal human gastric mucosal epithelial cell line;Investigate the function of CASC9 in the viability,proliferation and migration ability of gastric cancer cells;Elucidate the underlying molecular mechanism of the role of CASC9 in gastric cancer;Clarify the effect of CASC9/miR-505/ERCC1 pathway on the viability,proliferation and migration ability of gastric cancer cells.Methods:1.Collect samples of cancer tissues from gastric cancer patients and adjacent healthy tissues,grind the tissue samples using liquid nitrogen,extract the total RNA using Trizol reagents,transcribed them into cDNA,and detect the expression of CASC9/miR505/ERCC1 pathway by real-time quantitative PCR.At the same time,extract total proteins by RIPA lysis,and apply western blot assay to detect the expression of ERCC1 in cancer tissue samples and adjacent healthy tissues of gastric cancer patients.2.Culture gastric cancer cell lines SGC-7901,MKN-45,HGC-27,MGC-803,and normal human gastric mucosal epithelial cell lines GES-1.Trizol method was used to extract RNA and transcribed them into cDNA.Finally,real-time quantitative PCR was performed to detect the expression of CASC9/miR-505/ERCC1 pathway;3.Synthesize small interfering RNA of CASC9,transfect them into gastric cancer cell lines MGC-803 and MKN-45 using Lipo-2000 regents,then collect cells and detect the expression of CASC9 by qPCR experiment;measure the cell viability by CCK8 experiment;test cell proliferation by the EdU experiment;detect the migration potential by migration chamber experiment;4.Perform the following experiments separately:1)Synthesize small interfering RNA of CASC9,transfect them into gastric cancer cell lines MGC-803 and MKN-45 with Lipo-2000 regents,then collect cells and detect miR-505 expression by qPCR experiment;2)Dual luciferase reporter gene detects the binding between CASC9 and miR-505;3)Synthetic miR-505 mimics and inhibitors and transfect them into gastric cancer cell lines MGC-803 and MKN-45 with Lipo-2000 regents,then the cells were collected,and miR-505 and ERCC1 expression were detected by qPCR experiments,furthermore,the protein level of ERCC1 were measured by WB experiment;4)Double luciferase reporter gene detects the binding between miR-505 and ERCC1;5)Gastric cancer cell lines MGC-803 and MKN-45 were divided into three groups:control small interfering RNA,CASC9 small interfering RNA,CASC9 small interfering RNA+miR-505 inhibitor and the expression of ERCC1 were measured by qPCR and WB experiments;5.Gastric cancer cell lines MGC-803 and MKN-45 were divided into four groups:control small interfering RNA,CASC9 small interfering RNA,CASC9 small interfering RNA+miR-505 inhibitor,CASC9 small interfering RNA+miR-505 inhibitor+ERCC small interfering RNA.Then,cell viability were detected by CCK8 experiment;cell proliferation were tested by EdU experiment;cell migration potential were measured by migration chamber experiment.Results:1.(1)The expression level of CASC9 and ERCC1 in cancer tissue samples of gastric cancer patients is significantly higher than that in adjacent healthy tissue samples;the expression level of miR-505 in cancer tissue samples of gastric cancer patients is significantly lower than corresponding healthy tissue adjacent;(2)17 out of the 20 patients,CASC9 and ERCC1 RNA expression levels in cancer tissues are higher than those in adjacent healthy tissues;18 out of the 20 patients miR-505 expression level in cancer tissues is higher expression levels than that in adjacent healthy tissues;(3)17 out of the 20 patients,the protein level of ERCC 1 in the cancer tissue of patients is higher than corresponding healthy tissue adjacent;2.Compared with normal human gastric mucosal epithelial cell line GES-1,the expression of CASC9 and ERCC1 are up-regulated in gastric cancer cells SGC7901,MKN-45,HGC-27 and MGC-803;compared with normal human gastric mucosal epithelial cell line GES-1,the expression of miR-505 is down-regulated in gastric cancer cells SGC-7901,MKN-45,HGC-27 and MGC-803;compared with normal human gastric mucosal epithelial cell line GES-1,the protein level of ERCC1 is up-regulated in gastric cancer cells SGC-7901,MKN-45,HGC-27 and MGC-803;3.Small interfering RNA of CASC9 can effectively reduce the expression of CASC9 in gastric cancer cell lines MKN45 and MGC-803;inhibiting the expression of CASC9 can significantly reduce the cell viability,proliferation ability and migration of gastric cancer cell lines MKN-45 and MGC803 ability;4.1)Inhibiting the expression of CASC9 does not regulate the expression level of miR-505;2)The double luciferase reporter gene shows that CASC9 can effectively bind to miR-505;3)miR-505 mimics can significantly increase the amount of miR-505 and inhibit the expression of ERCC1,while miR-505 inhibitors can significantly reduce the amount of miR-505 and up-regulate the expression of ERCC1;4)The double luciferase reporter gene showed that ERCC1 can effectively bind to miR-505;5)Inhibiting the expression of CASC9 can significantly inhibit the expression of ERCC1,while miR-505 inhibitor can reduce that;5.Regulating the CASC9/miR-505/ERCC1 pathway can affect the viability,proliferation and migration of gastric cancer cells.Conclusion:This study demonstrats that CASC9/miR-505/ERCC1 pathway is abnormally expressed in gastric cancer tissues,and further clarifies that CASC9 up-regulates ERCC1 expression by competitively binding the miR-505,thereby promoting the growth and migration of gastric cancer cells.