LncRNA Linc00152靶向miR-193a-3p对胃癌细胞增殖、侵袭和迁移的影响

Lnc RNA Linc00152 Targeting miR-193a-3p on Proliferation,Invasion and Migration of Gastric Cancer Cells

关键词:  Linc00152  胃癌  miR-193a-3p  细胞周期素D1  上皮细胞间充质转化

Key words:  Linc00152  gastric cancer  miR-193a-3p  cyclin D1  epithelial-mesenchymal transition

摘要:

目的 探讨LncRNA Linc00152靶向调控miR-193a-3p对胃癌细胞增殖、侵袭和迁移的影响。方法 采用qRT-PCR法检测正常胃黏膜细胞GES-1及4种胃癌细胞(MGC803、BGC803、SGC7901、AGS)中Linc00152的表达水平。MGC-803细胞分为pcDNA3.1-GFP-Linc00152组、pcDNA3.1-GFP组;AGS细胞分为si-Linc00152组、si-NC组,分别转染相应的Linc00152过表达和抑制载体。qRT-PCR法检测Linc00152、细胞周期素D1(cyclin D1,CCND1)和miR-193a-3p基因的表达水平,CCK-8检测细胞的活性,细胞克隆试验检测细胞的克隆能力,Annexin VFITC/PI染色后采用流式细胞术检测转染细胞凋亡率,Transwell法检测细胞侵袭,荧光素酶报告试验检测Linc00152和miR-193a-3p靶向关系,Western blotting检测Bcl-2、Bax、CCND1、E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的蛋白表达水平。并将si-Linc00152组、si-NC组细胞接种裸鼠,观察裸鼠体质量和一般状态,处死后称重记录肿瘤体积、质量,qRT-PCR法测定肿瘤组织Linc00152、CCND1和miR-193a-3p的表达水平。结果 与GES-1细胞相比,胃癌细胞MGC803、BGC803、SGC7901、AGS中Linc00152的表达升高,其中Linc00152在胃癌MGC803细胞中表达较低,而在胃癌AGS细胞中的表达较高,选用胃癌MGC803、AGS细胞进行后续试验。与pcDNA3.1-GFP组相比,pcDNA3.1-GFP-Linc00152组细胞的活性显著升高,克隆能力增强,细胞凋亡减少,侵袭能力显著上升,CCND1、Bcl-2、波形蛋白的表达显著升高,miR-193a-3p和E-钙黏蛋白的表达显著降低。双荧光素酶报告基因系统检测结果显示,Linc00152可直接调控miR-193a-3p的转录活性,且Linc00152可显著上调MGC-803细胞CCND1蛋白表达。与si-NC组相比,si-Linc00152组的裸鼠体内的异种移植瘤体积和体质量显著下降,CCND1和Linc00152的表达显著下降,miR-193a-3p的表达显著上升。结论 Linc00152可靶向作用于miR-193a-3p促进胃癌细胞增殖、侵袭和迁移,其作用机制与细胞周期、上皮细胞间充质转化以及细胞凋亡有关。

Abstract:

OBJECTIVE To explore the effect of LncRNA Linc00152 on the proliferation, invasion and migration of gastric cancer cells by miR-193a-3p sponging.METHODS The expression level of Linc00152 in normal gastric mucosal cells(GES-1) and four gastric cancer cells(MGC803, BGC803, SGC7901, AGS) was detected by qRT-PCR. MGC-803 cells were divided into pcDNA3.1-GFP-Linc00152 group, pcDNA3.1-GFP group, while AGS cells were divided into si-Linc00152 group and si-NC group. Each group was treated with corresponding Linc00152 overexpression and knockdown vector, gene levels of Linc00152. Cyclin D1(CCND1) and miR-193a-3p were measured by qRT-PCR, cell proliferative ability was tested by CCK-8 and colony formation assay, apoptosis of transfected cells was detected by flow cytometry after Annexin VFITC/PI staining, invasion ability was observed by Transwell, luciferase reporter assay was performed or measuring the relationship between Linc00152 and miR-193a-3p. Western blotting was used to determine the protein levels of Bcl-2, Bax, CCND1, E-cadherin and Vimentin. The cells of si-Linc00152 and si-NC group were inoculated into nude mice to observe the body weight and general state of nude mice, and the tumor volume and weight were recorded after death. The expression levels of Linc00152, CCND1 and miR-193a-3p in tumor tissue were measured by qRT-PCR.RESULTS The expression of Linc00152 in gastric cancer cells MGC-803, BGC-803, SGC-7901 and AGS was higher than that in GES-1 cells, in which Linc00152 was lower in gastric cancer MGC-803 cells and higher in gastric cancer AGS cells. The MGC-803 cells and AGS cells were used for further experiment. Compared with pcDNA3.1-GFP group, cell proliferative ability, cell invasion and migration ability, and the gene and protein levels of CCND1, protein levels of Bcl-2 and vimentin were increased notably, and apoptosis, the gene level of miR-193a-3p, protein level of E-cadherin were decreased significantly in pcDNA3.1-GFP-Linc00152 group. In the Luciferase reporter assay, miR-193a-3p overexpression resulted in a significant decrease in luciferase activity of the Linc00152-WT reporter, but no effect on Linc00152-MUT luciferase activity. Compared with si-NC group, the volume and weight of tumour, gene levels of CCND1 and Linc00152 in vivo were decreased markedly, and the gene level of miR-193a-3p increased notably in si-Linc00152 group.CONCLUSION Linc00152 targeting miR-193a-3p on the proliferation, invasion and migration of gastric cancer cells, and the mechanism related to cell cycle, epithelial-mesenchymal transition and apoptosis.