hsa-miR-21-5p/ZNF367分子轴通过PI3K/Akt通路影响胃癌细胞的增殖和迁移

The hsa-miR-21-5p/ZNF367 molecular axis affects the proliferation and migration of gastric cancer cells through the PI3K/Akt pathway

目的探讨ZNF367通过PI3K/Akt通路抑制胃癌(GC)细胞的增殖和迁移,并初步探讨其作用分子机制。方法收集2015年8月–2018年10月上海市第八人民医院普外科手术切除的GC组织(非坏死部分)和相邻癌旁组织(距肿瘤组织>3 cm)标本46例,同时选取正常人胃黏膜上皮细胞GES1及GC细胞系MGC-803和MKN-45。采用qRT-PCR、Western blot实验检测ZNF367和hsa-miR-21-5p(mi R-21-5p)在组织和细胞系中的表达情况,进行Kaplan-Meier生存分析得出总生存期;利用脂质体LipofectamineTM 2000转染寡核苷酸片段mi RNA-NC、miR-21-5pmimic和miR-21-5pinhibitor至MGC-803细胞,通过CCK8法和划痕实验检测细胞的增殖和迁移能力;采用双荧光素酶报告载体系统验证ZNF367和miR-21-5p的作用靶点,采用qRT-PCR、 Westernblot实验检测miR-21-5pmimic和miR-21-5pinhibitor转染MGC-803细胞中ZNF367、E-cadherin、N-cadherin、Vimentin以及PI3K/Akt通路中p-PI3K和p-Akt的表达情况。结果 ZNF367在胃癌组织和胃癌细胞系中的表达水平分别显著低于癌旁组织和GES-1细胞(P<0.01),且在MGC-803细胞中表达水平最低,而miR-21-5p的表达正相反;在MGC-803细胞中,OE-ZNF367和miR-21-5p inhibitor转染组的细胞增殖和迁移能力显著低于对照组,而si RNA-ZNF367和miR-21-5pmimic转染组的细胞增殖和迁移能力显著高于对照组;生物信息学和荧光素酶活性实验结果表明ZNF367与miR-21-5p具有直接靶向关系;qRT-PCR、Western blot实验结果表明在miR-21-5p inhibitor转染组中,E-cadherin的表达是显著提高的(P <0.01),而N-cadherin、Vimentin、p-PI3K和p-Akt的表达是显著下降的(P<0.01);在miR-21-5pmimic转染组中,结果相反(P <0.01)。结论 hsa-miR-21-5p/ZNF367分子轴通过调控PI3K/Akt通路抑制EMT过程,从而影响胃癌细胞MGC-803的增殖和迁移。

Objective To investigate effect of miR-21-5p/ZNF367 axis on the proliferation and migration of gastric cancer(GC) cells. Methods GC and corresponding para-cancer normal tissues were obtained from 46 gastric cancer patients undergoing surgery in General Surgery Department of Shanghai Eighth People’s Hospital. Meanwhile, GES-1 and GC cell lines MGC-803 and MKN-45 were selected to perform the assays. We used qRT-PCR and Western blot(WB) to detect the expression level of ZNF367 and hsa-miR-21-5 p(miR-21-5p) in the tissues and cell lines, and kaplan-meier survival analysis was performed to obtain total survival period. The LipofectamineTM 2000 was used to transfect oligonucleotide fragments, including miRNA-NC, miR-21-5p mimic and miR-21-5p inhibitor, into MGC-803 cells. The proliferation and migration of the cells were detected by CCK8 and wound healing assay, respectively. Dual luciferase reporter gene assays were used to validate the relationship between ZNF367 and miR-21-5p. The expression level of ZNF367, E-cadherin, N-cadherin, Vimentin, p-PI3K and p-Akt in MGC-803 cells transfected with miR-21-5p mimic and miR-21-5p inhibitor were detected by qRT-PCR and WB experiments. Results The expression level of ZNF367 in GC tissues and cell lines was significantly lower than that in para-cancer tissues and GES-1 cells(P < 0.01), and the expression level was the lowest in MGC-803 cells, while the expression level of miR-21-5p was found in an opposite way(P < 0.01). In MGC-803 cells, the proliferation and migration abilities of cells transfected with over-expressed ZNF367 and miR-21-5 p inhibitor were significantly lower than those of the control group, while the proliferation and migration abilities of cells transfected with siRNA-ZNF367 and miR-21-5p mimic were significantly higher than those of the control group. Bioinformatics and luciferase activity assays showed that ZNF367 had a direct targeting relationship with miR-21-5p. The results of qRT-PCR and WB experiments demonstrated that the expression of E-cadherin was significantly increased in the miR-21-5p inhibitor transfection group(P < 0.01), while the expression of N-cadherin, Vimentin, p-PI3K and p-Akt significantly was decreased. In the transfection group of miR-21-5p mimic, opposite results were observed(P < 0.01). Conclusions ZNF367 was a negative regulator in the proliferation and migration of GC cell MGC-803. The miR-21-5p/ZNF367 molecular axis affects EMT process by regulating PI3K/Akt pathway.