[1]代 晶,张 勇,潘长清,等.RMP 通过AMPK 通路调节线粒体稳态和氧化应激诱导卵巢癌细胞增殖与凋亡的机制研究[J].现代检验医学杂志,2023,38(02):57-62.[doi:10.3969/j.issn.1671-7414.2023.02.011 ]
　DAI Jing,ZHANG Yong,PAN Chang-qing,et al.Mechanism of RMP Regulating Mitochondrial Homeostasis and Oxidative Stress-induced Ovarian Cancer Cell Proliferation and Apoptosis Through the AMPK Pathway[J].Journal of Modern Laboratory Medicine,2023,38(02):57-62.[doi:10.3969/j.issn.1671-7414.2023.02.011 ]
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RMP 通过AMPK 通路调节线粒体稳态和氧化应激诱导卵巢癌细胞增殖与凋亡的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年02期
页码:
57-62
栏目:
论著
出版日期:
2023-03-15

文章信息/Info

Title:
Mechanism of RMP Regulating Mitochondrial Homeostasis and Oxidative Stress-induced Ovarian Cancer Cell Proliferation and Apoptosis Through the AMPK Pathway
文章编号:
1671-7414（2023）02-057-06
作者:
代 晶; 张 勇; 潘长清; 王 丹; 王 君; 杨 芳; 王 亮
（电子科技大学医学院附属绵阳医院 /绵阳市中心医院妇产科，四川绵阳 621000）
Author(s):
DAI Jing;  ZHANG Yong;  PAN Chang-qing;  WANG Dan;  WANG Jun;  YANG Fang;  WANG Liang
（Department of Obstetrics and Gynecology, Mianyang Hospital Affiliated to the School of Medicine of University of Electronic Science and Technology/Mianyang Central Hospital, Sichuan Mianyang 621000, China）
关键词:
RPB5调节蛋白; AMPK通路; 线粒体稳态; 氧化应激; 卵巢癌
分类号:
R737.31；R730.43
DOI:
10.3969/j.issn.1671-7414.2023.02.011
文献标志码:
A
摘要:
目的 探讨 RPB5调节蛋白（RPB5-mediating protein，RMP）对卵巢癌细胞增殖与凋亡的影响以及通过腺苷酸激活蛋白激酶（adenosine 5’-monophosphate (AMP)-activated protein kinase，AMPK）通路调节线粒体稳态和氧化应激的作用机制。方法 实时荧光定量 PCR(qRT-PCR)法检测人输卵管上皮永生化细胞 FTE-187与人卵巢癌细胞系 SKOV3， A2780和 HO8910中 RMP基因的表达。以 SKOV3细胞作为研究对象，利用 siRNA技术敲低 SKOV3细胞中 RMP的表达，再通过 qRT-PCR法验证 RNAi效率。通过平板克隆形成实验和流式细胞技术观察敲低 RMP表达后对 SKOV3细胞增殖能力、周期分布及凋亡能力的影响。通过激光共聚焦显微镜观察敲低 RMP表达后 SKOV3细胞中线粒体形态的变化。 Western blot进一步证实 RMP调控线粒体稳态相关蛋白 AMPK和 p-AMPK以及凋亡蛋白 Bcl-2和 Bax的表达。采用 ROS荧光法检测敲低 RMP表达后对 SKOV3细胞内 ROS水平的影响。结果 RMP在 FTE-187细胞中的表达水平为 1.00±0.13，在 A2780，HO8910和 SKOV3细胞中的表达水平分别为 1.58±0.19，1.88±0.17，2.15±0.10，较 FTE-187中的表达显著上升，差异有统计学意义（F=72.035，P＜ 0.001）。SKOV3细胞转染后， RMP-siR组 RMP表达水平为 0.86±0.20，较 control组（2.06±0.11）和 NC-siR组（1.92±0.23）显著下降，差异有统计学意义（F=90.220， P＜ 0.001）。平板克隆形成实验和流式细胞检测结果表明，敲低 RMP可以通过引起 G2/M期阻滞导致卵巢癌细胞增殖障碍，促进卵巢癌细胞凋亡。通过激光共聚焦显微镜观察到敲低 RMP表达后， SKOV3细胞中点状的线粒体明显增多，线粒体的碎片化也增多，导致线粒体稳态失衡。 Western blot结果表明， control组和 NC -siR组 p-AMPK表达水平分别为 0.75±0.12，0.77±0.17，敲低 RMP表达后， p-AMPK表达水平为 1.39±0.33，较 control组和 NC-siR组升高，差异有统计学意义（F=46.550，P＜ 0.001）。control组和 NC-siR组凋亡相关蛋白 Bax/Bcl-2比值分别为 0.55±0.11和 0.56±0.08，敲低 RMP表达后， Bax/Bcl-2比值增加为 1.57±0.22，差异亦有统计学意义（F=62.027，P＜ 0.001）。荧光显微镜观察到， control组和 NC-siR组荧光强度分别为 100.24%±8.76%和 103.07%±7.93%，敲低 RMP后其荧光强度为 295.14%±12.10%，显著高于 control组和 NC-siR组，差异有统计学意义（F=392.708，P＜ 0.001）。结论 RMP在卵巢癌细胞中呈高表达，敲低 RMP表达后可以通过激活磷酸化 AMPK通路导致卵巢癌细胞线粒体稳态失衡和氧化应激，诱导卵巢癌细胞在 G2/M期发生阻滞，进而抑制卵巢癌细胞增殖，促进其凋亡。
Abstract:
Objective To investigate the effect of the RPB5-mediating protein (RMP) on the proliferation and apoptosis of ovarian cancer cells and the mechanism of regulating mitochondrial homeostasis and oxidative stress through the AMPK pathway. Methods The expression of RMP gene in immortalized human fallopian tube epithelial cells FTE-187 and human ovarian cancer cell lines SKOV3, A2780 and HO8910 was detected by qRT-PCR. Taking SKOV3 cells as the research object, the expression of RMP in SKOV3 cells was knocked down by specific siRNA, and the RNAi efficiency was verified by qRT-PCR method. The effects of knockdown of RMP expression on the proliferation, cycle distribution and apoptosis of SKOV3 cells were observed by plate clone formation assay and flow cytometry. Laser confocal microscopy was used to observe the change in mitochondrial morphology in SKOV3 cells after knockdown of RMP expression. Western blot further confirmed that RMP regulates the expression of mitochondrial homeostasis-related proteins AMPK and p-AMPK as well as apoptosis proteins Bcl-2 and Bax. Results The expression level of RMP in FTE-187 cells was 1.00±0.13, and the expression levels in A2780, HO8910 and SKOV3 cells were 1.58±0.19, 1.88±0.17 and 2.15±0.10, respectively, which was significantly higher than that in FTE-187, the difference was statistically significant (F=72.035, P＜ 0.001). After transfection of SKOV3 cells, the expression level of RMP in RMP-siR group was 0.86±0.20, which was significantly lower than that in control group (2.06±0.11) and NC-siR group (1.92±0.23), and the difference was statistically significant (F=90.220, P＜ 0.001). The results of plate clone formation assay and flow cytometry showed that knockdown of RMP could induce ovarian cancer cell proliferation disorder and promote ovarian cancer cell apoptosis by causing G2/M phase arrest. It was observed by laser confocal microscopy that after knockdown of RMP expression, the number of punctate mitochondria in SKOV3 cells increased significantly, and the fragmentation of mitochondria also increased, resulting in the imbalance of mitochondrial homeostasis. Western blot results showed that the expression levels of p-AMPK in the control group and NC group were 0.75±0.12 and 0.77±0.17, respectively. After knocking down the expression of RMP, the expression level of p-AMPK was 1.59±0.33, which was higher than that in the control group and NC-siR group, the difference was statistically significant (F=46.550, P＜ 0.001). The ratio of apoptosis-related protein Bax/Bcl-2 in the control group and NC-siR was 0.55±0.11 and 0.56±0.08, respectively. After knocking down the expression of RMP, the ratio of Bax/ Bcl-2 was significantly increased to (1.57±0.22), and the difference was also statistically significant (F=62.027, P＜ 0.001). fluorescence microscope observed that the fluorescence intensity of control group and NC-siR group were 100.24%±8.76% and 103.07%±7.93 %, respectively. After knockdown of RMP, the fluorescence intensity was 295.14%±12.10%, which was significantly higher than that of control group and NC-siR group (F=392.708, P＜ 0.001). Conclusion RMP was highly expressed in ovarian cancer cells. Knocking down the expression of RMP can lead to the imbalance of mitochondrial homeostasis and oxidative stress in ovarian cancer cells by activating the phosphorylated AMPK pathway, and induce ovarian cancer cells to block in the G2/M phase, thereby inhibiting ovarian cancer cell proliferation and promoting its apoptosis.

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备注/Memo

备注/Memo:
收稿日期：2022-07-22修回日期：2022-11-28
基金项目：医学工程国家重点实验室开放课题，项目编号：2020KFKT017。
作者简介：代晶（1987-），女，硕士研究生，主治医师，研究方向：妇科肿瘤， E-mail：ohh5101@163.com。
通讯作者：王亮 (1976-)，女，硕士研究生，副主任医师。

更新日期/Last Update: 2023-03-15