Role And Machanism Of Long Non-coding RNA CASC9 In The Proliferation Of Colorectal Cancer

Objective: Colorectal cancer is one of the most common malignancies and the second leading cause of cancer-related death worldwide.Increasing studies have shown that long non-coding RNAs(lnc RNA)are regarded as important regulators in the occurrence and progression of colorectal cancer.As a newly discovered lnc RNA in recent years,CASC9 has been proved to play an oncogenic role in a variety of cancer tissues.However,its role and mechanism in colorectal cancer have rarely been studied.In this study,we aimed to explore the expression level of CASC9 in tumor tissues and cell lines of colorectal cancer,analyze the association between CASC9 and clinicopathological features,and verify the function and mechanism of CASC9 in colorectal cancer cells in vitro,which will provide an important molecular target for the clinical treatment of colorectal cancer.Methods : The tumor tissues and adjacent normal tissues of 50 colorectal cancer patients who had received clinical surgical treatment were collected.The expression level of CASC9 was detected by real-time quantitative PCR,and the relationship between CASC9 and clinicopathological features of colorectal cancer patients was statistically analyzed.Five colorectal cancer cell lines and a normal colon epithelial cell were cultured in vitro,and the expression level of CASC9 was detected.Two cell lines with higher expression level were selected as the research objects.The si RNA targeting CASC9 was designed to transfect colorectal cancer cell lines.Cell proliferation ability was detected by CCK8,colony formation assay and Ed U assay.The relative expression level of CASC9 in the nucleus and cytoplasm of colorectal cancer was detected by subcellular fractionation assay.Bioinformatics website star Base v2.0 were used to predict the binding sites and correlations between mi R-576-5p and CASC9 as well as between mi R-576-5p and AKT3,respectively.Dual luciferase reporting assay and real-time quantitative PCR was used to verify their targeted binding relationships and correlation.In colorectal cancer cell lines,cell proliferation and AKT3 protein expression were detected by down-regulating the expression of CASC9 and/or mi R-576-5p.Results: Real-time quantitative PCR results showed that the expression of CASC9 was dramatically increased in tumor tissues and cell lines of colorectal cancer compared with adjacent normal tissues and normal colon epithelial cell lines(P < 0.05).Moreover,the abnormal expression of CASC9 was correlated with the clinical stage of colorectal cancer(P < 0.05).HT29 and SW620 cells were selected for in vitro experiments.The results of cell functional experiments showed that interfering with CASC9 expression effectively inhibited colorectal cancer cells proliferation(P < 0.05).The results of subcellular fractionation assay showed that CASC9 was mainly enriched in the cytoplasm of colorectal cancer cells.Bioinformatics website prediction and experimental verification showed that mi R-576-5p has a targeted binding site with the 3′-untranslated regions of CASC9 and AKT3,and its expression is negatively correlated with CASC9 and AKT3 in colorectal cancer tissues.Moreover,AKT3 has been proved to be a downstream target gene of mi R-576-5p and is negatively regulated by mi R-576-5p.CCK8 and Western blot assay showed that down-regulation of mi R-576-5p eliminated the inhibitory effect of CASC9 silencing on colorectal cancer cells proliferation,and reversed the result of AKT3 expression reduction induced by CASC9 silencing.Conclusion:CASC9 is an oncogenic lnc RNA in colorectal cancer,which can act as a competitive endogenous RNA of mi R-576-5p to indirectly regulate the expression of AKT3,thereby promoting colorectal cancer cells proliferation.The novel molecular mechanism may provide a new idea for molecular targeted therapy of colorectal cancer.