miR-103a-3p/CHI3L1在卵巢癌细胞增殖和血管拟生中的作用机制

doi:10.3760/cma.j.cn371439-20191224-00031

摘要/Abstract

摘要：

目的探究微小RNA(miR)-103a-3p/壳多糖酶3样蛋白1(CHI3L1)在卵巢癌细胞增殖和血管拟生中的作用机制及对转化生长因子-β(TGF-β)通路的影响。方法通过生物信息学分析miR-103a-3p的表达水平与卵巢癌患者总生存率间的关系。将人卵巢腺癌细胞株SKOV3细胞分为4组:对照组、miR-103a-3p mimic组、miR-103a-3p mimic+CHI3L1组和CHI3L1组。采用定量聚合酶链反应(qPCR)和Western blotting分别检测miR-103a-3p、CHI3L1 mRNA和CHI3L1蛋白的表达水平。采用酶联免疫吸附法检测细胞培养液中YKL-40的表达水平。采用CCK-8法、克隆形成实验和血管生成实验检测4组细胞活力、增殖能力和血管生成能力。通过双荧光素酶报告验证miR-130a-3p靶向CHI3L1。结果 miR-103a-3p高表达组卵巢癌患者总体生存率高于miR-103a-3p低表达组(χ ²=6.187,P=0.048)。对照组、miR-103a-3p mimic组、miR-103a-3p mimic+CHI3L1组和CHI3L1组4组之间miR-103a-3p和CHI3L1 mRNA的水平差异均具有统计学意义(F=198.254,P<0.001;F=60.214,P<0.001),miR-103a-3p mimic组和miR-103a-3p mimic+CHI3L1组的miR-103a-3p水平高于对照组(均P<0.001),CHI3L1组的CHI3L1 mRNA水平显著高于对照组(P<0.001)。4组CHI3L1蛋白的表达水平分别为2.25±0.23、1.19±0.12、2.29±0.28、4.31±0.37,差异具有统计学意义(F=18.675,P<0.001)。4组细胞培养液中YKL-40的表达水平分别为(1.84±0.20)ng/ml、(0.95±0.08)ng/ml、(2.64±0.25)ng/ml、(6.27±0.79)ng/ml,差异具有统计学意义(F=35.297,P<0.001),CHI3L1组的YKL-40水平显著高于对照组(P<0.001),miR-103a-3p mimic组的YKL-40水平低于对照组(P<0.001),miR-103a-3p mimic+CHI3L1组的YKL-40水平高于miR-103a-3p mimic组(P<0.001)。4组的细胞活力分别为100%±2.54%、76.23%±2.13%、104.89%±3.56%、137.42%±2.80%,差异具有统计学意义(F=23.584,P<0.001),miR-103a-3p mimic组的细胞活力显著低于对照组(P<0.001),CHI3L1组显著高于对照组(P<0.001),miR-103a-3p mimic+CHI3L1组显著高于miR-103a-3p mimic组(P<0.001)。4组细胞的克隆形成数分别为(76.85±4.67)个、(21.56±2.85)个、(72.06±5.07)个、(169.63±9.21)个,差异具有统计学意义(F=31.541,P<0.001),miR-103a-3p mimic组细胞的增殖能力显著低于对照组(P<0.001),CHI3L1组显著高于对照组(P<0.001),miR-103a-3p mimic+CHI3L1组显著高于miR-103a-3p mimic组(P<0.001)。4组细胞的相对管长度和管分支差异均具有统计学意义(F=24.254,P<0.001; F=27.564, P<0.001)。4组细胞的TGF-β和Smad水平比较差异均具有统计学意义(F=30.254,P<0.001;F=34.187,P<0.001)。双荧光素酶实验结果显示,与转染NC组相比较,共转染miR-103a-3p与CHI3L1-wt后细胞中荧光素酶活性显著降低。分别使用NC和miR-103a-3p与CHI3L1-mut共转染后细胞中荧光素酶活性变化不大。结论 miR-103a-3p通过直接靶向抑制CHI3L1的表达,从而抑制卵巢癌SKOV3细胞的增殖和血管生成能力,抑制卵巢癌淋巴转移和远端转移,这可能与TGF-β通路有关。

关键词: 卵巢肿瘤, 壳多糖酶3样蛋白质1, 微RNAs, 转化生长因子β, 细胞增殖

Abstract:

Objective To investigate the mechanisms of microRNA (miR)-103a-3p/chitinase-3-like protein 1 (CHI3L1) in the proliferation and vascular mimicry of ovarian cancer cells and its effect on transforming growth factor-β (TGF-β) pathway. Methods The relationship between the expression level of miR-103a-3p and the overall survival rate of ovarian cancer patients was analyzed by bioinformatics. The human ovarian adenocarcinoma SKOV3 cells were divided into 4 groups: control group, miR-103a-3p mimic group, miR-103a-3p mimic+CHI3L1 group and CHI3L1 group. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the expression levels of miR-103a-3p, CHI3L1 mRNA and CHI3L1 protein respectively. The expression level of YKL-40 in cell culture fluid was detected by enzyme-linked immunosorbent assay. The cell viability, proliferation ability and angiogenesis ability of the 4 groups were detected by CCK-8 method, clone formation experiment and angiogenesis experiment. The dual luciferase report verified that miR-130a-3p targeted CHI3L1. Results The overall survival rate of ovarian cancer patients with high expression of miR-103a-3p was higher than that of patients with low expression of miR-103a-3p (χ ²=6.187, P=0.048). The differences in miR-103a-3p and CHI3L1 mRNA levels among the control group, miR-103a-3p mimic group, miR-103a-3p mimic+CHI3L1 group and CHI3L1 group were statistically significant (F=198.254, P<0.001; F=60.214, P<0.001), miR-103a-3p mimic group and miR-103a-3p mimic+CHI3L1 group had higher miR-103a-3p levels than the control group (all P<0.001), CHI3L1 group had higher CHI3L1 mRNA level than the control group (P<0.001). The expression levels of CHI3L1 protein in the 4 groups were 2.25±0.23, 1.19±0.12, 2.29±0.28 and 4.31±0.37, and the difference was statistically significant (F=18.675, P<0.001). The expression levels of YKL-40 in the cell culture fluids of the 4 groups were (1.84±0.20) ng/ml, (0.95±0.08) ng/ml, (2.64±0.25) ng/ml, (6.27±0.79) ng/ml, and the difference was statistically significant (F=35.297, P<0.001). The YKL-40 level of the CHI3L1 group was significantly higher than that of the control group (P<0.001), the miR-103a-3p mimic group was lower than the control group (P<0.001), and the miR-103a-3p mimic+CHI3L1 group was higher than the miR-103a-3p mimic group (P<0.001). The cell viabilities of the 4 groups were 100%±2.54%, 76.23%±2.13%, 104.89%±3.56% and 137.42%±2.80%, and the difference was statistically significant (F=23.584, P<0.001). The cell viability of the miR-103a-3p mimic group was significantly lower than that of the control group (P<0.001), the CHI3L1 group was higher than the control group (P<0.001), and the miR-103a-3p mimic+CHI3L1 group was higher than the miR-103a-3p mimic group (P<0.001). The number of clones formed in the 4 groups were 76.85±4.67, 21.56±2.85, 72.06±5.07 and 169.63±9.21, and the difference was statistically significant (F=31.541, P<0.001). The proliferation capacity of the miR-103a-3p mimic group was significantly lower than that of the control group (P<0.001), the CHI3L1 group was higher than the control group (P<0.001), and the miR-103a-3p mimic+CHI3L1 group was significantly higher than the miR-103a-3p mimic group (P<0.001). The differences in the relative tube lengths and the tube bramches of the 4 groups were both statistically significant (F=24.254, P<0.001; F=27.564, P<0.001). The differences in TGF-β and Smad levels of the 4 groups were both statistically significant (F=30.254, P<0.001; F=34.187, P<0.001). The results of dual luciferase experiments showed that compared with the NC group, the luciferase activity in cells co-transfected of miR-103a-3p and CHI3L1-wt was significantly reduced. The difference of luciferase activity between the cells transfected with NC and co-transfected with miR-103a-3p and CHI3L1-mut was not significant. Conclusion MiR-103a-3p can directly inhibit the expression of CHI3L1 and inhibit the proliferation and angiogenesis of ovarian cancer SKOV3 cells to inhibit ovarian lymphatic metastasis and distant metastasis, which may be related to the TGF-β pathway.

Key words: Ovarian neoplasms, Chitinase-3-like protein 1, MicroRNAs, Transforming growth factor beta, Cell proliferation

杨立芬, 宋伟, 许大伟, 武军, 高然. miR-103a-3p/CHI3L1在卵巢癌细胞增殖和血管拟生中的作用机制[J]. 国际肿瘤学杂志, 2020, 47(6): 333-339.

Yang Lifen, Song Wei, Xu Dawei, Wu Jun, Gao Ran. Mechanisms of miR-103a-3p/CHI3L1 in proliferation and vascular mimicry of ovarian cancer cells[J]. Journal of International Oncology, 2020, 47(6): 333-339.

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组别	miR-103a-3p	CHI3L1
对照组	0.86±0.07	3.26±0.29
miR-103a-3p mimic组	19.83±1.72^a	1.21±0.12^a
miR-103a-3p mimic+CHI3L1组	18.72±2.04^a	3.95±0.40^b
CHI3L1组	0.85±0.09	24.95±2.17^a
F值	198.254	60.214
P值	<0.001	<0.001

组别	TGF-β	Smad
对照组	1.56±0.13	2.42±0.22
miR-103a-3p mimic组	0.92±0.08^a	1.10±0.10^a
miR-103a-3p mimic+CHI3L1组	1.60±0.15^b	2.73±0.24^b
CHI3L1组	2.48±0.21^a	4.18±0.32^a
F值	30.254	34.187
P值	<0.001	<0.001