| Background and ObjectiveClock genes are related to the occurrence of many diseases.Recent studies have found that clock genes are closely related to the occurrence and progeression of tumors.However,the regulation mechanisms of clock gene ARNTL2 in colon carcinoma still remain unclear.The aim of this study was to analyze the effects of ARNTL2 knockdown by siRNA on the proliferation and invasion of colon carcinoma and clarify the molecular mechanisms of ARNTL2 in the progression of colon carcinoma.MethodsThe study firstly screened and predicted the expression of the clock gene ARNTL2 in tumor and its relationship with survival and prognosis of colon cancer through bioinformatics analysis.The immunohistochemistry(IHC)analysis were carried out for detecting the expression of ARNTL2 in colon carcinoma and adjacent tissues and analyzing the relationship between ARNTL2 expression and clinicopathological parameters.Meanwhile,Western blot was used to detect its expression in colon carcinoma cell lines,and cell immunofluorescence technique was performed to analyze its intracellular location and distribution.Then RNA interference was used to inhibit the expression of ARNTL2 in colon cancer cells.The effect of ARNTL2 on the growth,invasion and migration of colon cancer cells in vivo and in vitro were analyzed using CCK-8 assay,plate clone formation,wound healing assay,transwell migration and invasion and nude mouse subcutaneous tumor model.Finally,the regulation effect of clock gene change on tumor-related signal pathways were analyzed by ARNTL2 RNA interference and western blot assay in colon cancer cells,respectively.ResultsBioinformatics analysis found that the clock gene ARNTL2 is highly expressed in colon carcinoma tissues,which is closely related to the survival and prognosis of colon cancer.ARNTL2 was highly expressed in tissues and colon carcinoma cells compared with that in the adjacent tissues.The expression of ARNTL2 was statistically associated with T and M stage of colon carcinoma,which was found to be mainly located in the nucleus of colon carcinoma cells by cell immunofluorescence technique simultaneously.Knockdown of ARNTL2 inhibited colon carcinoma cells viability,colony-formation,and migration activity in vitro and reduced the size of tumors in the nude mice in vivo.Moreover,knockdown of ARNTL2 suppressed the expression of SMOC2 targeting ARNTL2 and blocked the expression of EMT and PI3K/AKT/mTOR signaling pathway-related factors simultaneously,further inhibiting the growth and migration of colon carcinoma.ConclusionAccording to the bioinformatics,expression and function studies of ARNTL2 in colon cancer tissues and cells,it is the first time to reveal the mechanism of ARNTL2 gene to regulate the occurrence,invasion and migration of colon cancer.The results clearly demonstrated that knockout of ARNTL2 gene can inhibit the proliferation and invasion of colon cancer cells.At the same time,it is also proved that down-regulation of ARNTL2 gene expression can inhibit PI3K/AKT/mTOR signaling pathway and SMOC2-EMT activity,thereby inhibiting proliferation and migration of cancer cells.These findings revealed the mechanism of ARNTL2 gene knockout on inhibiting the growth of colon cancer cells,and provided a reliable theoretical basis for ARNTL2 gene knockout to exert a tumor suppressor effect in colon cancer. |
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