微小RNA-10a-5p促进紫草素抗卵巢癌细胞的作用

杲飞莹; 武强; 潘相羽; 卢丹

doi:10.7619/jcmp.20222460

摘要:

目的初步探究微小RNA-10a-5p(miR-10a-5p)在紫草素抗卵巢癌中的作用。

方法基于数据库分析miR-10a-5p在肿瘤中的表达水平; 瞬时转染miR-10a-5p mimic及NC, 通过逆转录实时定量聚合酶链反应(RT-qPCR)检测miR-10a-5p的表达水平; 使用CCK-8实验检测紫草素对SKOV3细胞的半数抑制浓度(IC₅₀); 通过流式细胞术检测细胞凋亡和细胞周期; 活死细胞染色比较细胞死亡情况。

结果 miR-10a-5p在多种肿瘤中异常表达, 与对照组相比，转染miR-10a-5p mimic后可上调SKOV3细胞中miR-10a-5p的表达水平约80倍，差异有统计学意义(P < 0.000 1); miR-10a-5p可促进卵巢癌细胞的凋亡，提高SKOV3细胞对紫草素的敏感性，其IC₅₀由2.45 μmol/L降低至1.40 μmol/L, 降幅达42.9%。与NC相比，转染mimic可促进SKOV3细胞的凋亡，差异有统计学意义(6.73%与17.71%, P=0.007 5)。与无紫草素相比，紫草素可显著促进卵巢癌细胞的凋亡，差异有统计学意义(6.73%与82.48%, P < 0.000 1); 且miR-10a-5p可协同紫草素促进SKOV3的凋亡，差异有统计学意义(82.48%与96.80%, P < 0.000 1)。活死细胞染色结果显示, miR-10a-5p联合紫草素使更多细胞显著发生死亡(P < 0.000 1); miR-10a-5p联合紫草素使S期细胞占比显著增高(P < 0.000 1), 从而抑制细胞增殖。通过Targetscan7.2预测发现, GATA6可能是miRNA-10a-5p的一个作用靶点。Kaplan-Meier Plotter数据库分析提示, GATA6的高表达与卵巢癌的不良预后相关，瞬时转染mimic后, GATA6 mRNA和其蛋白表达水平明显著下调(P < 0.000 1)。

结论 miR-10a-5p和紫草素均可促进SKOV3凋亡，有协同抗卵巢癌的作用，且miR-10a-5p可显著提高SKOV3细胞对紫草素的药物敏感性，其可能是通过靶向作用于GATA6而产生抑癌作用。

Abstract:

Objective To preliminarily explore the role of microRNA-10a-5p (miR-10a-5p) in the anti-ovarian cancer effect of shikonin.

Methods The expression level of miR-10a-5p in tumors was analyzed based on database; the expression of miR-10a-5p was detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) after transient transfection of miR-10a-5p mimic and NC; the CCK-8 assay was used to detect half inhibitory concentration (IC₅₀) of shikonin in SKOV3 cells; the cell apoptosis and cell cycle were detected by flow cytometry; the cell death of each group was compared with the staining of live and dead cells.

Results The miR-10a-5p was abnormally expressed in a variety of tumors. Compared with the control group, the expression level of the miR-10a-5p in SKOV3 cells could be significantly up-regulated by about 80 times after transfecting miR-10a-5p mimic (P < 0.000 1); miR-10a-5p can promote the apoptosis of ovarian cancer cells and improve the sensitivity of SKOV3 cells to shikonin. The IC₅₀ of mir-10A-5P decreased by 42.9% from 2.45 μmol/L to 1.40 μmol/L. Compared with NC group, transfection mimic could significantly promote the apoptosis of SKOV3 cells (6.73% versus 17.71%, P=0.007 5). Compared with the non-shikonin group, shikonin significantly promoted the apoptosis of ovarian cancer cells (6.73% versus 82.48%, P < 0.000 1); in addition, miR-10a-5p could significantly promote the apoptosis of SKOV3 with shikoin (82.48% versus 96.80%, P < 0.000 1). The staining results of live and dead cells showed that miR-10a-5p combined with shiverin caused significant death of more cells (P < 0.000 1); the miR-10a-5p combined with shiverin significantly increased the proportion of S-phase cells (P < 0.000 1), thus inhibiting cell proliferation. GATA6 was predicted to be a target of miRNA-10a-5p by Targetscan7.2. Kaplan-Meier Plotter database analysis suggested that high expression of GATA6 was associated with poor prognosis of ovarian cancer, and the mRNA and protein expression levels of GATA6 were significantly down-regulated after transient transfection with mimic (P < 0.000 1).

Conclusion Both miR-10a-5p and shikonin can promote the apoptosis of SKOV3 cell, and have the synergistic anti-ovarian cancer effect. And miR-10a-5p can significantly improve the drug sensitivity of SKOV3 cell to shikonin, which may be through targeting GATA6 to produce cancer inhibitory effect.

微小RNA-10a-5p促进紫草素抗卵巢癌细胞的作用

MicroRNA-10a-5p promotes the anti-ovarian cancer effect of shikonin