| 柳月霞,魏菊红,刘小丽.长链非编码 RNA膀胱癌相关转录本 1靶向调控微小 RNA-20b-5p对宫颈癌细胞自噬及顺铂敏感性的影响[J].安徽医药,2021,25(4):789-793. |
| 长链非编码 RNA膀胱癌相关转录本 1靶向调控微小 RNA-20b-5p对宫颈癌细胞自噬及顺铂敏感性的影响 |
| Effected of LncRNA BLACAT1 targeted regulation of miR-20b-5p onautophagy and cisplatin sensitivity of cervical cancer cells |
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| DOI：10.3969/j.issn.1009-6469.2021.04.038 |
| 中文关键词: 宫颈肿瘤 LncRNA BLACAT1 miR-20b-5p 自噬 顺铂敏感性 |
| 英文关键词: Uterine cervical neoplasms LncRNA BLACAT1 miR-20b-5p Autophagy Cisplatin sensitivity |
| 基金项目: |
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| 中文摘要: |
| 目的探讨长链非编码 RNA膀胱癌相关转录本 1(LncRNA BLACAT1)对宫颈癌细胞自噬及顺铂敏感性的影响及其分子机制。方法本研究起止时间 2018年 6月至 2019年 10月，体外培养人正常宫颈上皮细胞 HUCEC、宫颈癌细胞株 ME180、C33A、Hela与顺铂耐药宫颈癌细胞株 Hela/DDP，实时荧光定量聚合酶链反应(qRT-PCR)检测细胞中 BLACAT1、微小 RNA-20b5p(miR-20b-5p)的表达量。以 Hela/DDP细胞为研究对象，分别将 BLACAT1小分子干扰 RNA(si-BLACAT1)及其阴性对照(siNC)、 si-BLACAT1与 miR-20b-5p特异性寡核苷酸抑制剂的阴性对照(anti-miR-NC)、 si-BLACAT1与 miR-20b-5p特异性寡核苷酸抑制剂(anti-miR-20b-5p)转染至 Hela/DDP细胞。甲基噻唑基四唑(MTT)检测细胞抑制率为 50%时的顺铂浓度(顺铂 IC50值)以此评估顺铂敏感性；蛋白质印迹法(Western blotting)检测 p62、微管相关蛋白轻链 3I(LCB3-Ⅰ)、微管相关蛋白轻链 3II(LCB3-Ⅱ)表达量。双荧光素酶报告实验验证 BLACAT1与 miR-20b-5p的靶向关系。结果与 HUCEC相比，宫颈癌细胞株 ME180、C-33A、Hela中 BLACAT1的表达水平升高［(0.80±0.16)比(3.41±0.14)、(3.72±0.14)、(2.52±0.15)］(P＜0.05)miR-20b5p的表达水平降低［(1.00±0.16)比(0.42±0.16)、(0.33±0.14)、(0.38±0.15)］(P＜0.05)，Hela细胞与 Hela/DDP细胞相比差，异有统计学意义［(2.52±0.15)比(5.23±0.14)；(0.38±0.15)比(0.21±0.11)］(P＜0.05)；与 si-NC组相比， si-BLACAT1组 p62蛋白水平降低［(0.82±0.13)比(0.24±0.11)］(P＜0.05)LCB3-Ⅱ蛋白水平升高［(0.64±0.15)比(0.95±0.13)］(P＜0.05)，顺铂 IC50值降低［(16.82±3.24)比(6.21±1.25)］(P＜0.05)；双荧，光素酶报告实验证实 BLACAT1能够靶向结合 miR-20b-5p；与 si-BLACAT1+antimiR-NC组相比， si-BLACAT1+anti-miR-20b-5p组 p62蛋白水平升高［(0.27±0.13)比(1.66±0.15)］(P＜0.05)LCB3-Ⅱ蛋白水平降低［(2.60±0.15)比(0.85±0.12)］(P＜0.05)顺铂 IC50值升高［(6.23±1.20)比(15.67±2.16)］(P＜0.05)。结论，LncRNA BLACAT1靶向抑制 miR-20b-5p表达而调控宫颈癌细，胞自噬从而降低顺铂敏感性。 |
| 英文摘要: |
| Objective To investigate the effect of LncRNA BLACAT1 on autophagy and cisplatin sensitivity of cervical cancer celland its molecular mechanism.Methods The start and end time of this research was from June 2018 to October 2019. Human normal cervical epithelial cells HUCEC, cervical cancer cell lines ME180, C-33A, Hela, and cisplatin-resistant cervical cancer cell line Hela/ DDP were cultured in vitro. The expression levels of BLACAT1 and miR-20b-5p were detected by qRT-PCR. With Hela/DDP cells as research objects, si-NC, si-BLACAT1, si-BLACAT1 and anti-miR-NC, si-BLACAT1 and anti-miR-20b-5p were transfected into Hela/DDP cells, respectively. The cisplatin concentration (IC50 value of cisplatin) at 50% cytostatic rate was measured by MTT to evaluatethe sensitivity of cisplatin. Western blotting was used to detect the expression of p62, LCB3-Ⅰ and LCB3-Ⅱ. The dual luciferase report experiment verified the targeting relationship between BLACAT1 and miR-20b-5p.Results Compared with HUCEC, the expression levels of BLACAT1 in cervical cancer cell lines ME180, C-33A, and Hela were significantly increased [(0.80±0.16) vs. (3.41± 0.14)/(3.72±0.14)/(2.52±0.15)] (P<0.05), and the expression levels of miR-20b-5p were significantly reduced [(1.00±0.16) vs. (0.42± 0.16)/(0.33±0.14)/(0.38±0.15)] (P<0.05), Hela/ DDP cells[(2.52±0.15) vs. (5.23±0.14)] and Hela cells [(0.38±0.15) vs. (0.21±0.11)] had a statistically significant difference (P<0.05). Compared with the si-NC group, the protein level of p62 in the si-BLACAT1 group was significantly reduced [(0.82±0.13) vs. (0.24±0.11)] (P<0.05), the protein level of LCB3-Ⅱ was significantly increased [(0.64±0.15) vs. (0.95±0.13)] (P<0.05), the IC50 value of cisplatin decreased significantly [(16.82±3.24) vs. (6.21±1.25)] (P<0.05). Double luciferase reporting experiments confirmed that BLACAT1 was able to target miR-20b-5p. Compared with the si-BLACAT1 + anti-miR-NC group, the protein level of p62 in the si-BLACAT1 + anti-miR-20b-5p group was significantly increased [(0.27±0.13) vs. (1.66±0.15)] (P<0.05), and the protein level of LCB3-Ⅱ was significantly reduced [(2.60±0.15) vs. (0.85±0.12)] (P<0.05), the IC50 value of cisplatin increased significantly [(6.23±1.20) vs. (15.67±2.16)] (P<0.05).Conclusion LncRNA BLACAT1 regulates autophagy of cervical cancer cells and reduces cisplatin sensitivity through targeted inhibition of miR-20b-5p expression. |
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