抑制miR-218的表达对人去势抵抗型前列腺癌C4-2细胞增殖、侵袭及迁移能力的影响及其机制

doi:10.3760/cma.j.issn.2095-7041.2019.05.011

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摘要目的探讨抑制miR-218的表达对人去势抵抗型前列腺癌C4-2细胞增殖、侵袭及迁移的影响及其机制。方法培养人去势抵抗型前列腺癌C4-2细胞,分为空白组、阴性对照组(NC组)、miR-218组;空白组未予以任何处理,NC组转染阴性对照序列,miR-218组转染miR-218抑制剂。各组细胞处理后继续培养5 d,然后收集细胞,采用实时荧光定量PCR(qPCR)检测转染后各组细胞miR-218的表达情况,采用WST-1细胞增殖实验和细胞平板克隆形成实验检测转染后各组C4-2细胞增殖、活性情况,采用划痕实验检测各组C4-2细胞迁移距离,采用Transwell小室检测细胞侵袭数量和迁移数量,采用半定量逆转录PCR(RT-PCR)检测TOB1基因mRNA表达情况,采用Western blot检测TOB1基因蛋白表达情况。结果 qPCR结果显示:转染后NC组、miR-218组人去势抵抗型前列腺癌C4-2细胞miR-218的相对表达量分别为1.02±0.06、0.38±0.04,miR-218组中miR-218的表达明显低于NC组,差异有统计学意义(t=15.372, P＜0.05)。WST-1细胞增殖实验结果显示:(1)转染后空白组、NC组随着时间的延长,光密度(OD)值呈上升趋势,而miR-218组OD值在24、48 h时呈上升趋势,72 h时OD 值开始明显下降;(2)空白组、NC组、miR-218组组间比较,OD 值在0、24 h时3组间差异均无统计学意义(P值均＞0.05),48、72 h时miR-218组OD 值均小于空白组、NC组,差异均有统计学意义(F=12.615、24.523,P值均＜0.05)。细胞平板克隆形成实验结果显示:转染后空白组、NC组和miR-218组克隆形成数分别为(42.2±1.2)个、(41.3±2.4)个、(20.6±1.2)个,miR-218组明显少于空白组、NC组,差异均有统计学意义(F=155.530,P＜0.05)。划痕实验和Transwell小室细胞侵袭、迁移实验结果显示:转染后,miR-218组细胞迁移距离、侵袭细胞数和迁移细胞数均低于空白组和NC组,差异均有统计学意义(F=17.625、48.625、38.352,P值均＜0.05);而空白组和NC组间细胞迁移距离、侵袭细胞数和迁移细胞数比较,差异均无明显统计学意义(P值均＞0.05)。RT-PCR和Western blot检测结果显示:转染后空白组、NC组、miR-218组TOB1基因mRNA的相对表达量分别为 0.20±0.02、0.19±0.03、0.35±0.02,TOB1基因蛋白的相对表达量分别为0.22±0.01、0.23±0.02、0.68±0.02,miR-218组细胞中TOB1 mRNA和蛋白水平均明显高于空白组和NC组,差异均有统计学意义(F=18.615、22.523,P值<0.05);而空白组细胞中TOB1 mRNA和蛋白水平与NC组比较,差异均无统计学意义(P值>0.05)。结论抑制人去势抵抗型前列腺癌C4-2细胞miR-218的表达,可抑制该细胞的增殖和活性,并抑制其侵袭和迁移能力,其机制可能与上调TOB1基因mRNA和蛋白表达有关。

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	王苏贵
	皇立媛
	吴自余
	李强
	张先云
	阳东荣
	姜福金

关键词 ：前列腺肿瘤, 微小核糖核酸-218, C4-2细胞株, 细胞增殖, 细胞迁移

Abstract：Objective To investigate the effect of inhibiting the expression of microRNA-218(miR-218) on the proliferation, invasion and migration of human castrated resistant prostate cancer cell line C4-2 and its mechanism.Methods Human castration-resistant prostate cancer C4-2 cells were cultured and divided into three groups: blank control group, negative control group (NC group transfected negative control reagent) and miR-218 group (transfected with inhibitor of miR-218). Cells in each group were cultured for 5 days after treatment. The expression of miR-218 in each group was detected by quantitative real-time PCR (qPCR). WST-1 cell proliferation assay and plate cloning assay were used to detect proliferation and activity of C4-2 cells in each group after transfection. Scratch assay was used to detect the migration distance of C4-2 cells in each group. Transwell assay was used to detect the number of cell invasion and migration. Reverse transcription PCR was used to detect the expression of TOB1 gene. Western blot was used to detect the expression of TOB1 gene.Results The results of qPCR showed that the relative expression of miR-218 in castrated-resistant prostate cancer C4-2 cells in NC group and miR-218 group was 1.02±0.06 and 0.38±0.04 after transfection, respectively. The expression of miR-218 in microRNA-218 group was significantly lower than that in NC group (t=15.372, P＜0.05). The results of WST-1 cell proliferation experiment showed that: (1) OD value of blank group and NC group increased with time, while that of miR-218 group increased at 24 and 48 hours, and began to decrease significantly at 72 hours. (2)Compared with the blank group, NC group and miR-218 group, there was no significant difference in OD value between the three groups at 0 and 24 hours (all P values＞0.05). At 48 and 72 hours, OD value of the miR-218 group was significantly lower than that of the blank group and NC group, and the difference was statistically significant (F=12.615, 24.523, all P values＜0.05). The results of cell plate clone formation experiment showed that the number of clone formation in the blank group, NC group and miR-218 group after transfection was 42.2±1.2, 41.3±2.4 and 20.6±1.2. The number of clone formation in the miR-218 group was significantly less than that in the blank group and NC group (F=155.530, P＜0.05). Scratch assay and Transwell cell invasion and migration assay showed that the migration distance, the number of invasive cells and the number of migrating cells in the miR-218 group were significantly lower than those in the blank group and NC group after transfection (F=17.625, 48.625, 38.352, all P values＜0.05). There was no significant difference in cell migration distance, number of invasive cells and number of migrating cells between blank group and NC group (all P values＞0.05). The results of RT-PCR and Western blot showed that the relative expression levels of TOB1 gene in blank group, NC group and miR-218 group were 0.20±0.02, 0.19±0.03, 0.35±0.02, and the relative expression levels of TOB1 gene protein were 0.22±0.01, 0.23±0.02, 0.68 ±0.02 after transfection, respectively. The levels of TOB1 mRNA and protein in the cells of miR-218 group were significantly higher than those in the blank group and NC group, with statistical significance (F=18.615, 22.523, all P values＜ 0.05), while there was no significant difference in the levels of TOB1 mRNA and protein between the blank group and NC group (all P values＞0.05).Conclusions Inhibiting the expression of miR-218 in human castration-resistant prostate cancer cell line C4-2 can inhibit the proliferation and activity of C4-2 cells and inhibit the invasion and migration of C4-2 cells. The mechanism may be related to the up-regulation of TOB1 gene expression and protein expression.

Key words： Prostatic neoplasms MicroRNA-218 C4-2 cell line Cell proliferation Cell migration

收稿日期: 2018-11-21

基金资助:国家自然科学基金(81472776);江苏省六大人才高峰科研项目(2019-WSW-218);淮安市自然科学基金(HAB201730);徐州医科大学附属淮安医院科研基金(YK201506)

通讯作者: 皇立媛,Email:397052884@qq.com

引用本文:

王苏贵, 皇立媛, 吴自余, 李强, 张先云, 阳东荣, 姜福金. 抑制miR-218的表达对人去势抵抗型前列腺癌C4-2细胞增殖、侵袭及迁移能力的影响及其机制[J]. 中华解剖与临床杂志, 2019, 24(5): 483-489.
Wang Sugui, Huang Liyuan, Wu Ziyu, Li Qiang, Zhang Xianyun, Yang Dongrong, Jang Fujin. Effect of inhibiting the expression of microRNA-218 on proliferation, invasion and migration of human castration-resistant prostate cancer cell line C4-2 and its mechanism. Chinese Journal of Anatomy and Clinics, 2019, 24(5): 483-489.

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http://www.cjac.com.cn/CN/10.3760/cma.j.issn.2095-7041.2019.05.011 或 http://www.cjac.com.cn/CN/Y2019/V24/I5/483

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