miR-150-5p在卵巢癌中的表达及其生物学功能研究

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摘要目的探讨微小RNA-150-5p(miR-150-5p)在卵巢癌组织中的表达水平及其通过磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路对卵巢癌细胞增殖和凋亡能力的影响。方法选取2016年4月—2018年12月于武警上海总队医院妇科就诊的82例卵巢癌患者作为研究对象,患者年龄22~65周岁,平均(43.5±6.8)岁。采用qRT-PCR检测病变组织及其相应癌旁组织中miR-150-5p的表达水平。以人卵巢癌SKOV3细胞株作为空白对照组,转染miR-150-5p抑制剂的人卵巢癌SKOV3细胞株作为miR-150-5p抑制剂组,转染阴性对照质粒的人卵巢癌SKOV3细胞株作为阴性对照组,MTT法检测细胞增殖能力,流式细胞术检测细胞凋亡,生物信息学网站Targetscan 预测miRNA-150-5p与PI3K的靶向结合情况,荧光素酶试验验证miR-150-5p与PI3K的靶向关系,Western blotting检测Cleaved caspase-3、PI3K、Akt、磷酸化蛋白激酶B(Phosphorylated protein kinase,p-Akt)p-Akt蛋白的表达。结果 82例卵巢癌患者病变组织及相应癌旁组织中miR-150-5p的相对表达水平分别为(1.64±0.10)和(0.99±0.08),差异有统计学意义;空白对照组和阴性对照组中miR-150-5p的表达差异无统计学意义,miR-150-5p抑制剂组中miR-150-5p的相对表达量为(0.11±0.03),明显低于空白对照组和阴性对照组,差异有统计学意义。空白对照组和阴性对照组24 h、48 h和72 h细胞增殖率的差异无统计学意义,而miR-150-5p抑制剂组24 h、48 h 和72 h细胞增殖率的差异分别为(9.61±1.32)%、(24.54±3.61)%和(33.78±3.72)%,同一时间点均明显低于空白对照组和阴性对照组,差异有统计学意义。空白对照组和阴性对照组48 h细胞凋亡率的差异无统计学意义,而miR-150-5p 抑制剂组的细胞凋亡率为(16.65±2.93)%,明显高于空白对照组[(0.74±0.10)%]和阴性对照组[(1.79±0.21)%],差异有统计学意义。荧光素酶报告基因分析证实miR-150-5p与PI3K存在靶向结合位点。miR-150-5p抑制剂组PI3K、p-Akt蛋白表达低于空白对照组和阴性对照组,差异有统计学意义;Cleaved caspase3蛋白表达高于空白对照组和阴性对照组(P＜0.01);结论 miR-150-5p在卵巢癌中作促癌基因,其调控作用可能与PI3K/Akt信号通路有关。

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	蒋小平
	樊华
	严建耀
	朱红玲

关键词 ：卵巢癌, 微小RNA-150(miR-150-5p), PI3K/Akt信号通路, 增殖, 凋亡

Abstract：Objective To investigate the expression of microRNA-150-5p (miR-150-5p) in ovarian cancer tissue and explore the effect on proliferation and apoptosis of thyroid cancer cells through phosphatidylinositol 3- kinase(PI3K)/protein kinase B(Akt) signaling pathway.Methods Eighty-two ovarian cancer patients were enrolled in the gynecology department, Shanghai Armed Police Corps Hospital from April 2016 to December 2018. The patients were 22~65 years old, with an average of (43.5±6.8) years old. The expression of miR-150-5p was detected by qRT-PCR. Taking human ovarian cancer SKOV3 cell line as blank control group, human ovarian cancer SKOV3 cell line transfected with miR-150-5p inhibitor as miR-150-5p inhibitor group, human ovarian cancer SKOV3 cell line transfected with negative control plasmid as negative control group. Cell proliferation ability was determined by MTT, cell apoptosis ability was determined by flow cytometry.Targeted binding of miR-150-5p to PI3K was predicted by Targetscan of bioinformatics website, and the targeting relationship between miR-150-5p and PI3K was verified by luciferase assay. Cleaved caspase-3, PI3K, Akt, phosphorylated protein kinase(p-Akt) protein expression was determined by western blotting.Results The relative expression levels of miR-150-5p in the lesion tissues and adjacent tissues of 68 patients with Ovarian Cancer were 1.64±0.10 and 0.99±0.08 respectively. The cell proliferation rates of miR-150-5p inhibitors group at 24, 48 and 72 h did not show statistically significant difference, while those in miR-150-5p inhibitors group was (9.61±1.32)%、(24.54±3.61)% and (33.78±3.72)% respectively, which were significantly lower than blank control group and negative control group. The apoptosis rates of blank control group and negative control group did not show statistically significant difference, while the apoptosis rate of miR-150-5p inhibitors group was (16.65±2.93) %, significantly higher than those of blank control group and negative control group, which was (0.74±0.10)% and (1.79±0.21)% respectively. Luciferase reporter gene analysis confirmed the existence of targeted binding sites between microrna-150-5p and PI3K. There was no statistical significance in the expression of PI3K, p-Akt and Cleaved caspase-3 protein between blank control group and negative control group. The protein expression of PI3K and p-AKT in miR-150-5p inhibitor group was significantly lower than those of blank control group and negative control group, and Cleaved caspase-3 protein was significantly higher than that of blank control group and negative control group.Conclusion MiR-150-5p acts as an oncogene in ovarian cancer, and its regulatory effect may be related to PI3K/Akt signaling pathway.

Key words： ovarian cancer mcrioRNA-150(miR-150-5p) PI3K/Akt signaling pathway proliferation apoptosis

收稿日期: 2019-06-17

引用本文:

蒋小平, 樊华, 严建耀, 朱红玲. miR-150-5p在卵巢癌中的表达及其生物学功能研究[J]. 中国生育健康杂志, 2020, 31(4): 335-340.
JIANG Xiaoping, FAN Hua, YAN Jianyao, ZHU Hongling. Expression of miR-150-5p in ovarian cancer and its biological function. Chinese Journal of Reproductive Health, 2020, 31(4): 335-340.

链接本文:

http://cjrh.bjmu.edu.cn/CN/ 或 http://cjrh.bjmu.edu.cn/CN/Y2020/V31/I4/335

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