LncRNA NNT-AS1调节miR-130a-5p/CCNT2信号轴对膀胱癌细胞增殖及肿瘤干细胞干性的影响

doi:10.3969/j.issn.1006-6233.2026.02.08

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摘要 目的: 探讨LncRNA NNT-AS1调节miR-130a-5p/细胞周期蛋白T2(CCNT2)信号轴对膀胱癌细胞增殖及肿瘤干细胞干性的影响。方法: RT-qPCR检测人正常膀胱上皮细胞SV-HUC-1及人膀胱癌细胞系BIU-87、HT-1376、T24中LncRNA NNT-AS1、miR-130a-5p、CCNT2表达。将T24细胞和膀胱癌干细胞均随机分为正常组、pcDNA组、pcDNA-NNT-AS1组、si-NC组、si-NNT-AS1组,分组转染48h,RT-qPCR检测LncRNA NNT-AS1d对miR-130a-5p/CCNT2轴的调控作用,并以双荧光素酶报告基因实验验证它们之间靶向关系;CCK-8法检测各组细胞增殖;细胞成球实验检测各组膀胱癌干细胞干性;Western blot检测各组膀胱癌干细胞干性标志蛋白表达。回复实验:将T24细胞及膀胱癌干细胞随机分为si-NNT-AS1组、si-NNT-AS1+NC-miR-130a-5p组、si-NNT-AS1+anti-miR-130a-5p组、si-NNT-AS1+pcDNA组、si-NNT-AS1+pcDNA-CCNT2组,分组转染后以同法检测各组T24细胞及膀胱癌干细胞干性;将T24细胞及膀胱癌干细胞随机分为正常组、miR-130a-5p mimic组、miR-130a-5p mimic+pcDNA-CCNT2组,分组转染后以同法检测各组T24细胞及膀胱癌干细胞干性。结果: 人膀胱癌细胞系中LncRNA NNT-AS1与CCNT2表达升高(P<0.05),miR-130a-5p表达降低(P<0.05)。LncRNA NNT-AS1可靶向调控miR-130a-5p/CCNT2信号轴。相比正常组,si-NNT-AS1组24,48和72h时T24细胞活力、膀胱癌干细胞成球数与分化抗原簇44(CD44)、八聚体结合转录因子(Oct4)、乙醛脱氢酶1A1(ALDH1A1)、Nanog蛋白相对表达降低(P<0.05);pcDNA-NNT-AS1组24,48和72h时T24细胞活力、膀胱癌干细胞成球数与CD44、Oct4、ALDH1A1、Nanog蛋白相对表达升高(P<0.05);si-NC组、pcDNA组细胞以上指标均无明显差异(P>0.05)。下调miR-130a-5p、过表达CCNT2均可逆转敲低LncRNA NNT-AS1对T24细胞和膀胱癌干细胞干性的抑制作用。过表达CCNT2可逆转miR-130a-5p对T24细胞增殖和膀胱癌干细胞干性的抑制作用。结论: LncRNA NNT-AS1可通过靶向调控miR-130a-5p/CCNT2信号轴而促进膀胱癌细胞增殖并增强膀胱癌干细胞干性,敲低LncRNA NNT-AS1可抑制膀胱癌细胞增殖及膀胱癌干细胞干性。

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关键词 ：膀胱癌细胞, LncRNA NNT-AS1, miR-130a-5p/CCNT2, 增殖, 肿瘤干细胞, 干性

Abstract：Objective: To discuss the effects of lncRNA NNT-AS1 on the proliferation of bladder cancer cells and the stemness of tumor stem cells by regulating the miR-130a-5p/cyclin T2 (CCNT2) signal axis. Methods: The expression of lncRNAs NNT-AS1, miR-130a-5p and CCNT2 in normal bladder epithelial cells SV-HUC-1 and human bladder cancer cell lines BIU-87, HT-1376 and T24 were detected by RT-qPCR. T24 cells and bladder cancer stem cells were randomly assigned into normal group, pcDNA group, pcDNA-NNT-AS1 group, si-NC group and si-NNT-AS1 group. After grouping and 48 hours of transfection, RT-qPCR was used to detect the regulatory effect of lncRNA NNT-AS1d on the miR-130a-5p/CNT2 axis, and dual-luciferase reporter gene experiments were used to verify their targeting relationship. CCK-8 method was used to detect cell proliferation in each group. Cell spherulation test was used to detect the stemness of bladder cancer stem cells in each group. Western blot was used to detect the stemness marker protein of bladder cancer stem cells in each group. Reply experiment: T24 cells and bladder cancer stem cells were randomly assigned into si-NNT-AS1 group, si-NNT-AS1+NC-miR-130a-5p group, si-NNT-AS1+anti-miR-130a-5p group, si-NNT-AS1+pcDNA group and si-NNT-AS1+pcDNA-CCNT2 group. After grouping and transfection, the stemness of T24 cells and bladder cancer stem cells in each group was detected by the same method. T24 cells and bladder cancer stem cells were randomly grouped into normal group, miR-130a-5p mimic group and miR-130a-5p mimic+pcDNA-CCNT2 group. After grouping and transfection, the stemness of T24 cells and bladder cancer stem cells in each group were detected by the same method. Results: The lncRNA NNT-AS1 and CCNT2 in human bladder cancer cell line increased (P<0.05), and miR-130a-5p decreased (P<0.05). LncRNA NNT-AS1 could target and regulate the miR-130a-5p/CNT2 signaling axis. Compared with the normal group,in the si-NNT-AS1 group,the T24 cell activity,bladder cancer stem cell spherulation,and the relative expression of cluster of differentiation 44 (CD44), octamer binding transcription factor 4 (Oct4), aldehyde dehydrogenase 1A1 (ALDH1A1) and Nanog proteins decreased at 24, 48 and 72 hours (P<0.05); while the T24 cell activity, bladder cancer stem cell spherulation, and the relative expression of CD44, Oct4, ALDH1A1 and Nanog proteins in the pcDNA-NNT-AS1 group increased at 24, 48 and 72 hours(P<0.05); in addition, there was no conspicuous difference in the above cellular indicators in the si-NC group and the pcDNA group (P>0.05).Both downregulation of miR-130a-5p and overexpression of CCNT2 could reverse the inhibitory effects of lncRNA NNT-AS1 knockdown on the stemness of T24 cells and bladder cancer stem cells. Overexpression of CCNT2 could reverse the inhibitory effect of miR-130a-5p on the proliferation of T24 cells and the stemness of bladder cancer stem cells. Conclusion: LncRNA NNT-AS1 can promote the proliferation of bladder cancer cells and enhance the stemness of bladder cancer stem cells by targeting the miR-130a-5p/CCNT2 signal axis. Knocking down lncRNA NNT-AS1 can inhibit the proliferation of bladder cancer cells and the stemness of bladder cancer stem cells.

Key words： Bladder cancer cells LncRNA NNT-AS1 miR-130a-5p/CCNT2 Proliferation Tumor stem cells Stemness

基金资助:新疆维吾尔自治区自然科学基金资助项目,(编号:2021D01C189)

通讯作者: 倪泽称

引用本文:

艾合买提·卡德尔, 郭峰, 雷鹏, 张小安, 梁泽兰, 史振峰, 倪泽称. LncRNA NNT-AS1调节miR-130a-5p/CCNT2信号轴对膀胱癌细胞增殖及肿瘤干细胞干性的影响[J]. 河北医学, 2026, 32(2): 225-234.
AIHEMAITI Kadeer, GUO Feng, LEI Peng, et al. Effects of LncRNA NNT-AS1 on Proliferation of Bladder Cancer Cells and Stemness of Tumor Stem Cells by Regulating miR-130a-5p/CCNT2 Signal Axis. HeBei Med, 2026, 32(2): 225-234.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2026.02.08 或 http://www.hbyxzzs.cn/CN/Y2026/V32/I2/225

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