[Vincristine inhibits the proliferation of ovarian cancer cells by regulating the demethylation of RASSF2A]

Zhonghua Zhong Liu Za Zhi. 2021 Sep 23;43(9):932-938. doi: 10.3760/cma.j.cn112152-20200229-00148.
[Article in Chinese]

Abstract

Objective: To investigate the effect of vincristine on the proliferation of ovarian cancer cells by regulating RASSF2A demethylation. Methods: SKOV3 cells were infected with control (LV-NC) and RASSF2A lentivirus (LV-RASSF2A) and treated with or without vincristine. Cell counting kit-8 (CCK-8) was used to detect the activity of ovarian cancer cells (SKOV3) treated with different doses of vincristine. Colony formation assay was used to detect the proliferation of SKOV3 cells. Flow cytometry was used to detect the apoptosis of SKOV3 cells. Real time polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of RASSF2A in IOSE-29 and SKOV3 cells. Western blot was used to examine the protein expression of RASSF2A in IOSE-29 and SKOV3 cells. Methylation-specific PCR was used to detect methylation and demethylation levels of RASSF2A gene in IOSE-29 and SKOV3 cells. Results: The cell viabilities of SKOV3 cell treated with 6.25 nmol/L, 12.5 nmol/L, 25 nmol/L, 50 nmol/L and 100 nmol/L vincristine were (87.19±4.49)%, (73.67±8.62)%, (66.35±6.04)%, (50.32±6.00)% and (34.92±6.11)%, respectively, lower than (100.46±4.69)% of control group (P<0.05). The half maximal inhibitory concentration of vincristine at 48 hours was 50.02 nmol/L. The proliferation abilities of SKOV3 cells in vincristine 12.5 nmol/L group, 25 nmol/L group and 50 nmol/L group were (41.70±2.21)%, (32.15±1.80)% and (23.00±2.01)%, respectively, significantly lower than (100.78±5.66)% in the control group (all P<0.05). The apoptotic rates of SKOV3 cells in vincristine 12.5 nmol/L group, 25 nmol/L group and 50 nmol/L group were (3.65±0.27)%, (5.21±0.76)% and (10.46±1.00)%, respectively, significantly higher than (2.12±0.23)% in the control group (all P<0.05). Compared with the IOSE-29 group (1.00±0.07 and 0.68±0.04), the mRNA expression (0.32±0.04) and protein expression (0.24±0.02) of RASSF2A were down-regulated in SKOV3 cells (P<0.05). Compared with the LV-NC group [(101.60±4.39)%, (100.73±3.29)%, (4.06±0.30)%], over-expression of RASSF2A down-requlated cell viability (68.92±3.94)%, inhibited proliferation (16.38±2.16)%, and promoted apoptosis (8.65±0.56)%, (P<0.05). Conclusion: Vincristine can increase RASSF2A expression and inhibit ovarian cancer cell proliferation by promoting the demethylation of RASSF2A promoter.

目的: 探讨长春新碱调控RAS相关结构域蛋白2A(RASSF2A)去甲基化对卵巢癌细胞增殖的影响。 方法: 将卵巢癌SKOV3细胞分为不同浓度长春新碱组,将慢病毒转染至LV-NC组(转染LV-NC慢病毒液,感染复数为70)和LV-RASSF2A组(转染LV-RASSF2A慢病毒液,感染复数为70)SKOV3细胞,空白组加入等量磷酸盐缓冲液。采用CCK-8法检测6.25 nmol/L长春新碱组、12.5 nmol/L长春新碱组、25 nmol/L长春新碱组、50 nmol/L长春新碱组和100 nmol/L长春新碱组SKOV3细胞的活力,集落形成实验检测SKOV3细胞的增殖能力,流式细胞术检测SKOV3细胞的凋亡情况,逆转录聚合酶链反应检测人正常卵巢上皮细胞IOSE-29和SKOV3细胞中RASSF2A mRNA的表达水平,Western blot检测IOSE-29和SKOV3细胞中RASSF2A蛋白的表达水平,甲基化特异性聚合酶链反应检测IOSE-29和SKOV3细胞中RASSF2A甲基化和去甲基化水平。 结果: 6.25 nmol/L长春新碱组、12.5 nmol/L长春新碱组、25 nmol/L长春新碱组、50 nmol/L长春新碱组和100 nmol/L长春新碱组SKOV3细胞的存活率分别为(87.19±4.49)%、(73.67±8.62)%、(66.35±6.04)%、(50.32±6.00)%和(34.92±6.11)%,均低于对照组[(100.46±4.69)%,均P<0.05]。培养48 h时,长春新碱半数抑制浓度为50.02 nmol/L。12.5 nmol/L长春新碱组、25 nmol/L长春新碱组和50 nmol/L长春新碱组SKOV3细胞的增殖率分别为(41.70±2.21)%、(32.15±1.80)%和(23.00±2.01)%,均低于对照组[(100.78±5.66)%,均P<0.05)];SKOV3细胞的凋亡率分别为(3.65±0.27)%、(5.21±0.76)%和(10.46±1.00)%,均高于对照组[(2.12±0.23)%,均P<0.05)]。SKOV3细胞中RASSF2A mRNA和蛋白的表达水平分别为0.32±0.04和0.24±0.02,均低于IOSE-29细胞(分别为1.00±0.07和0.68±0.04,均P<0.05)。LV-RASSF2A组SKOV3细胞的存活率、增殖率和凋亡率分别为(68.92±3.94)%、(16.38±2.16)%和(8.65±0.56)%,与LV-NC组[分别为(101.60±4.39)%、(100.73±3.29)%和(4.06±0.30)%]比较,差异均有统计学意义(均P<0.05)。 结论: 长春新碱通过上调RASSF2A的表达,促进RASSF2A启动子去甲基化,以抑制卵巢癌细胞的增殖能力。.

Keywords: Demethylation; Ovarian neoplasms; Proliferation; RAS association domain family; Vincristine.

MeSH terms

  • Apoptosis
  • Cell Line, Tumor
  • Cell Proliferation
  • Demethylation
  • Female
  • Humans
  • Ovarian Neoplasms* / genetics
  • Vincristine / pharmacology

Substances

  • Vincristine