Abstract:

Objective　To investigate the effect of long chain non-coding RNA (lncRNA) GAS6-AS1 on the malignant biological behaviors of cervical cancer cells regulating the miR-330-5p/polypyrimidine string binding protein 1 (PTBP1) axis. Methods　The experiment was conducted from February to December 2023. The cancer tissue and adjacent tissue of 26 patients with cervical cancer treated at Affiliated Hospital of Shaanxi University of Chinese Medicine from July 2021 to June 2023 were collected. The cervical cancer MS751 cells were divided into a control group, an sh-NC group, an sh-GAS6-AS1 group, an sh-GAS6-AS1+inhibitor NC, group, an sh-GAS6-AS1+miR-330-5p inhibitor group, an sh-GAS6-AS1+oe-NC group, and an sh-GAS6-AS1+oe-PTBP1 group. The expression levels of GAS6-AS1, miR-330-5p, and PTBP1 mRNA in the cells and tissues were detected by the real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The cell proliferation was detected by the cell counting kit 8 (CCK-8 method). The cell migration and invasion were detected by the transwell experiment. The cell apoptosis rate was detected by the flow cytometry. Western blot was applied to detect the expression levels of tumor proliferation antigen (Ki67), Bcl-2 associated X protein (Bax), B lymphoblastoma-2 gene (Bcl-2), matrix metalloproteinase 2 (MMP-2) and PTBP1 proteins in the cells. The targeted relationship of miR-330-5p with GAS6-AS1 and PTBP1 was verified. t test, one-way ANOVA, and SNK-q test were used for the statistical analysis. Results　There were statistical differences in the expression levels of GAS6-AS1, miR-330-5p, and PTBP1 mRNA between the cervical cancer tissue and adjacent tissue (1.84±0.17 vs. 1.03±0.09, 0.34±0.03 vs. 0.98±0.07, 2.13±0.22 vs. 1.06±0.10; all P<0.05). The PTBP1 positive expression rate in the cancer tissue was higher than that in the adjacent tissue [(68.46±7.82)% vs. (21.83±2.79)%; P<0.05]. Compared with those in the normal human cervical epithelial cells H8, the expressions of GAS6-AS1 and PTBP1 mRNA were increased, and the expression of miR-330-5p was decreased in the cervical cancer cells (all P<0.05). The expressions of GAS6-AS1 and PTBP1 mRNA, the value of A₄₅₀ (24 and 48 h), the numbers of cell migration and invasion, and the expressions of Ki67, Bcl-2, MMP-2, and PTBP1 protein in the MS751 cells in the sh-GAS6-AS1 group were lower than those in the sh-NC group. The expression of miR-330-5p, apoptosis rate, and Bax protein expression in the sh-GAS6-AS1 group were higher than those in the sh-NC group (all P<0.05). Knocking down miR-330-5p expression or upregulating ANXA2 expression while downregulating GAS6-AS1 was able to weaken the inhibitory effect of downregulating GAS6-AS1 on the behaviors of the MS751 cells (both P<0.05). Conclusion　Interference with GAS6-AS1 expression can regulate the miR-330-5p/PTBP1 axis, thereby inhibiting the proliferation, migration, and invasion of cervical cancer cells and promoting apoptosis.

Key words:

Cervical cancer, LncRNA GAS6-AS1, MiR-330-5p/PTBP1 axis, Biological behaviors

摘要：

目的　探讨长链非编码RNA（lncRNA）生长停滞特异基因转录的反义RNA（GAS6-AS1）调节miR-330-5p/多聚嘧啶串结合蛋白1（PTBP1）轴对宫颈癌细胞恶性生物学行为的影响。方法　本实验于2023年2月至12月实施。收集2021年7月至2023年6月期间在陕西中医药大学附属医院接受手术治疗的26例宫颈癌患者癌组织和癌旁组织，将宫颈癌MS751细胞分为control组、sh-NC组、sh-GAS6-AS1组、sh-GAS6-AS1+inhibitor NC组、sh-GAS6-AS1+miR-330-5p inhibitor组、sh-GAS6-AS1+oe-NC组、sh-GAS6-AS1+oe-PTBP1组；采用实时荧光定量聚合酶链式反应（qRT-PCR）检测细胞和组织中的GAS6-AS1、miR-330-5p和PTBP1 mRNA表达水平；细胞计数试剂盒8（CCK-8）法检测细胞增殖；Transwell实验检测细胞迁移和侵袭；流式细胞术检测细胞凋亡率；Western blot检测细胞中肿瘤增殖抗原（Ki67）、Bcl-2关联X蛋白（Bax）、B淋巴细胞瘤-2基因（Bcl-2）、基质金属蛋白酶2（MMP-2）、PTBP1蛋白表达量。验证miR-330-5p与GAS6-AS1和PTBP1的靶向关系。采用t检验、单因素方差分析和SNK-q检验进行统计分析。结果　宫颈癌组织GAS6-AS1、miR-330-5p和PTBP1 mRNA表达水平与癌旁组织比较（1.84±0.17比1.03±0.09、0.34±0.03比0.98±0.07、2.13±0.22比1.06±0.10），差异均有统计学意义（均P<0.05）。癌组织中PTBP1阳性表达率为（68.46±7.82）%，高于癌旁组织的（21.83±2.79）%（P<0.05）。宫颈癌细胞中GAS6-AS1和PTBP1 mRNA表达高于正常人宫颈上皮细胞H8，miR-330-5p表达低于正常人宫颈上皮细胞H8（均P<0.05）；sh-GAS6-AS1组MS751细胞中GAS6-AS1和PTBP1 mRNA表达、A₄₅₀（24 h、48 h）值、细胞迁移和侵袭数量、Ki67、Bcl-2、MMP-2、PTBP1蛋白表达均低于sh-NC组，miR-330-5p表达、凋亡率和Bax蛋白表达均高于sh-NC组（均P<0.05）；在下调GAS6-AS1的同时敲低miR-330-5p表达或上调ANXA2表达，均可减弱下调GAS6-AS1对MS751细胞行为的抑制作用（均P<0.05）。结论　干扰GAS6-AS1表达可调控miR-330-5p/PTBP1轴，进而抑制宫颈癌细胞增殖、迁移和侵袭，并促进凋亡。

关键词:

宫颈癌, 长链非编码RNA生长停滞特异基因转录的反义RNA, miR-330-5p/多聚嘧啶串结合蛋白1轴, 生物学行为