LINC00963通过miR-214-5p/SEMA4C调控结直肠癌细胞的增殖、迁移和侵袭

LINC00963 regulates the proliferation,migration and invasion of colorectal cancer cells via miR-214-5p/SEMA4C

目的:探讨基因间长链非编码RNA00963(LINC00963)对结直肠癌细胞增殖、迁移和侵袭的影响及分子机制。方法:通过LncBase Predicted v.2预测LINC00963和miR-214-5p的调控关系并通过双荧光素酶实验进行验证,StarBase v3.0预测LINC00963和SEMA4C相关性。常规培养结直肠癌细胞系HCT116、SW480,将结直肠癌细胞分为si-nc组、si-LINC00963组、miR-nc组、miR-214-5p mimic组。采用实时荧光定量聚合酶链反应检测LINC00963、miR-214-5p和SEMA4C的表达水平,细胞计数试剂盒8(CCK-8)检测细胞增殖,划痕和Transwell实验检测细胞迁移和侵袭。Western blot法检测细胞中SEMA4C蛋白表达水平。结果:通过LncBase Predicted v.2预测发现LINC00963和miR-214-5p存在结合位点,StarBase v3.0显示LINC00963和SEMA4C呈正相关,双荧光素酶报告实验显示LINC00963与miR-214-5p存在相互作用。在HCT116、SW480细胞转染24 h后,与si-nc组比较,si-LINC00963组结直肠癌细胞增殖活力、划痕愈合力和侵袭细胞的数量均减弱,细胞中miR-214-5p表达增高,SEMA4C的mRNA和蛋白表达水平显著下降,差异有统计学意义(P<0.05);将miR-214-5p mimic同样转染至HCT116、SW480细胞24 h后,与miR-nc组比较,miR-214-5p mimic组miR-214-5p表达增高,SEMA4C的mRNA和蛋白表达水平显著下降,结直肠癌细胞增殖活力、划痕愈合力和侵袭细胞的数量均减弱,差异有统计学意义(P<0.05)。结论:LINC00963在结直肠癌中呈现高表达,下调其表达可增加miR-214-5p的表达,降低SEMA4C的表达,从而抑制结直肠癌细胞增殖、迁移和侵袭。

Objective:To investigate the effect of long intergenic non-coding RNA 00963(LINC00963)on the proliferation,migration and invasion of colorectal cancer cells and its molecular mechanism.Methods:The relationship between LINC00963and miR-214-5p was predicted by LncBase Predicted v.2 and verified by Dual Luciferase Assay.StarBase v3.0 was used to predict the relationship between LINC00963 and SEMA4C.The colorectal cancer cell lines HCT116 and SW480 were all cultured in a standard medium.Colorectal cancer cells were divided into si-nc group,si-LINC00963 group,miR-nc group and miR-214-5p group.The real-time qPCR was used to detect the expression levels of LINC00963,miR-214-5p and SEMA4C.Cell counting kit-8(CCK-8)was used to detect cell proliferation.Scratch and Transwell assays were performed to detect cell migration and invasion.The expression of SEMA4C protein was detected by Western blot.Results:The prediction of LncBase Predicted v.2 showed that there was a binding site between LINC00963 and miR-214-5p.StarBase v3.0 showed a positive correlation between LINC00963 and SEMA4C.Dual Luciferase Assay showed that LINC00963 interacted with miR-214-5p.After transfection of HCT116 and SW480 cells for 24 h,compared with si-nc group,the proliferation activity,scratch healing ability and the number of invasive cells decreased,the expression of miR-214-5p increased,and the expression of SEMA4C mRNA and protein significantly decreased in si-LINC00963 group(P<0.05).After transfection of miR-214-5p mimic into HCT116and SW480 cells for 24 h,compared with miR-nc group,the expression of miR-214-5p increased,the expression of SEMA4C mRNA and protein significantly decreased,and the proliferation activity,scratch healing ability and the number of invasive cells decreased in the miR-214-5p mimic group(P<0.05).Conclusions:LINC00963 is overexpressed in colorectal cancer.Down-regulating the expression of LINC00963 may increase the expression of miR-214-5p and decrease the expression of SEMA4C,thus inhibiting the proliferation,migration and invasion of colorectal cancer cells.