Hsa-miR-411-3P对胃癌细胞作用功能及相关分子机制的研究 <sup>*</sup>

doi:10.13523/j.cb.1909002

中国生物工程杂志

2020, Vol. 40

Issue (4): 1-9 DOI: 10.13523/j.cb.1909002

研究报告

Hsa-miR-411-3P对胃癌细胞作用功能及相关分子机制的研究 ^*

陈雪艳¹,张娜²,陈娟²,杨艳红³,张巨峰^2,^**(

)

1 广东药科大学药学院广州 510006
2 广东药科大学生命科学与生物制药学院广州 510006
3 广东药科大学附属第一医院(临床医学院) 广州 510080

Effect of Hsa-miR-411-3P on Gastric Cancer Cells and Related Mechanisms

CHEN Xue-yan¹,ZHANG Na²,CHEN Juan²,YANG Yan-hong³,ZHANG Ju-feng^2,^**(

)

1 School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
2 School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China
3 The First Affiliated Hospital, School of Clinical Medicine, Guangdong Pharmaceutical University, Guangzhou 510080, China

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摘要：

目的：研究Hsa-miR-411-3P对胃癌细胞功能作用及分子机制。方法：MTT检测Hsa-miR-411-3P对胃癌细胞SGC-7901、AGS增殖的影响;流式细胞术检测Hsa-miR-411-3P对胃癌细胞SGC-7901、AGS周期和凋亡的影响;RNAhybrid2.1.2、Miranda3.3a、TargetScan7.0 预测Hsa-miR-411-3P靶基因,与mRNA测序结果联合分析,取交集基因认为是比较可靠的靶基因,进行GO、KEGG分析;Real-time PCR检测Hsa-miR-411-3P靶基因VAV3、ROCK2、PLD1、PTCH1的表达。结果：过表达Hsa-miR-411-3P抑制SGC-7901、AGS细胞增殖,促进凋亡,使SGC-7901细胞周期阻滞在S期,AGS细胞周期阻滞在G1期;软件预测和测序结果联合分析获得235个交集靶基因;Hsa-miR-411-3P靶基因分子功能集中在酶结合、蛋白质结合、转移酶活性等;生物学过程集中在代谢过程、细胞组件组装、解剖结构发展、细胞成分生物发生等(P<0.05);KEGG信号通路集中在胰岛素信号通路、cAMP信号通路、AMPK信号通路、FOXO信号通路等(P<0.05);VAV3、ROCK2、PLD1、PTCH1共同参与cAMP信号通路;过表达Hsa-miR-411-3P的SGC-7901、AGS细胞中,VAV3、ROCK2 mRNA表达量均减少,PLD1 mRNA表达量在SGC-7901细胞中增加,在AGS细胞中减少;PTCH1 mRNA表达量在SGC-7901细胞中减少,在AGS细胞中增加。结论：过表达Hsa-miR-411-3P将下调靶基因VAV3、ROCK2的表达,从而影响cAMP信号通路,抑制SGC-7901、AGS细胞增殖,促进凋亡,使SGC-7901细胞周期阻滞在S期,AGS细胞周期阻滞在G1期。

关键词： 胃癌; miRNA; Hsa-miR-411-3P

Abstract:

Objective: To study the functional effects of Hsa-miR-411-3P on gastric cancer cells and related molecular mechanisms. Method: The effect of Hsa-miR-411-3P on the proliferation of gastric cancer cells SGC-7901 and AGS was detected by MTT; The effect of Hsa-miR-411-3P on the cycle and apoptosis of gastric cancer cells SGC-7901、 AGS was detected by flow cytometry; Three online tools: RNAhybrid2.1.2, Miranda3.3a, TargetScan7.0 predicted Hsa-miR-411-3P target genes, and combined with mRNA sequencing results, the intersection genes were considered reliable target genes, and then performed GO, KEGG analysis; Real-time PCR was used to detect the expression of Hsa-miR-411-3P target genes VAV3, ROCK2, PLD1, and PTCH1. Result: Overexpression of Hsa-miR-411-3P inhibited the proliferation of gastric cancer cells SGC-7901 and AGS, promoted the apoptosis of gastric cancer cells SGC-7901, AGS, arrested the cell cycle of SGC-7901 in S phase, and arrested AGS cell cycle in G1 phase. Three online tools: RNAhybrid2.1.2, Miranda3.3a, TargetScan7.0, predicted Hsa-miR-411-3P target genes, and combined with mRNA sequencing results, the intersection genes were considered to be reliable target genes, and a total of 235 intersection targets were obtained. Take 235 intersection target genes as target gene sets for subsequent analysis. GO and KEGG analysis of 235 cross-target genes found that their molecular function was concentrated in enzyme binding, protein binding, transferase activity, etc. Biological processes was concentrated in metabolic processes, assembly of cellular components, development of anatomical structures, and biogenesis of cellular components, etc.(P<0.05). KEGG signaling pathway was concentrated in insulin signaling pathway, cAMP signaling pathway, AMPK signaling pathway, FOXO signaling pathway, etc. (P<0.05). Insulin signaling pathway, AMPK signaling pathway, FOXO signaling pathway, and cAMP signaling pathway are all closely related to gastric cancer. The Hsa-miR-411-3P target genes VAV3、 ROCK2、 PLD1 and PTCH1, which are highly correlated with gastric cancer, all participate in the cAMP signaling pathway.VAV3、 ROCK2、 PLD1 and PTCH1 may have a negative regulatory relationship with Hsa-miR-411-3P, because in the sequencing results, the expression of Hsa-miR-411-3P was up-regulated after berberine treatment of SGC-7901 cells, but the expression of VAV3、 ROCK2、 PLD1 and PTCH1 was down-regulated. For this, verification was performed by using Real-time PCR. It was found that overexpression of Hsa-miR-411-3P would decrease the expression of VAV3 and ROCK2 mRNA in SGC-7901、 AGS cells; overexpression of Hsa-miR-411-3P would increase the expression of PLD1 mRNA in SGC-7901 cells and decrease the expression of PLD1 mRNA in AGS cells;overexpression of Hsa-miR-411-3P would decrease the expression of PTCH1 mRNA in SGC-7901 cells and increase the expression of PTCH1 mRNA in AGS cells. It was indicated that overexpression of Hsa-miR-411-3P could down-regulate the expression of target genes VAV3 and ROCK2, because the expression of PLD1 and PTCH1 was differently in two gastric cancer cells, which was controversial and need to be further studied by other gastric cancer cells. Conclusion: Overexpression of Hsa-miR-411-3P will decrease the expression of target genes VAV3、 ROCK2 to affect cAMP signaling pathway, inhibit the proliferation of SGC-7901, AGS cells, promote apoptosis, and arrest SGC-7901 cell cycle in S phase, AGS cell cycle in G1 phase.

Key words: Gastric cancer miRNA Hsa-miR-411-3P

收稿日期: 2019-09-02 出版日期: 2020-05-18

ZTFLH:

R737.9

基金资助: * 广东省科技计划项目(2014A020212303);广东省中医药局科研项目(20182079);广州市越秀区科技计划项目(2018-WS-011)

通讯作者: 张巨峰 E-mail: jfzhang111@163.com

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引用本文:

陈雪艳,张娜,陈娟,杨艳红,张巨峰. Hsa-miR-411-3P对胃癌细胞作用功能及相关分子机制的研究 ^*[J]. 中国生物工程杂志, 2020, 40(4): 1-9.

CHEN Xue-yan,ZHANG Na,CHEN Juan,YANG Yan-hong,ZHANG Ju-feng. Effect of Hsa-miR-411-3P on Gastric Cancer Cells and Related Mechanisms. China Biotechnology, 2020, 40(4): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.1909002 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I4/1

表1 引物序列

表2 Hsa-miR-411-3P对胃癌细胞增殖的影响

图1 Hsa-miR-411-3P对胃癌细胞增殖的影响

图2 Hsa-miR-411-3P对胃癌细胞周期的影响

图3 Hsa-miR-411-3P对胃癌细胞凋亡的影响

表3 Hsa-miR-411-3P靶基因的GO分子功能分析

表4 Hsa-miR-411-3P靶基因的GO生物学过程分析

表5 Hsa-miR-411-3P 靶基因KEGG分析

图4 测序结果中Hsa-miR-411-3P及其靶基因VAV3、ROCK2、PLD1、PTCH1表达量

图5 H sa-miR-411-3P 对靶基因表达的影响

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