miR-130a 通过 CYLD 调控膀胱癌细胞自噬的机制研究。

Department of Urology, the first People's Hospital of Jingzhou (The First Affiliated Hospital of Yangtze University), Jingzhou, Hubei 434000, P.R. China.

J BUON. 2020 May-Jun;25(3):1636-1642.

PURPOSE

The study aimed to explore the regulatory mechanism of micro ribonucleic acid (miR)-130a in the autophagy of bladder cancer cells through cylindromatosis (CYLD).

METHODS

Human bladder cancer T24 cell line was used as the objects of the study. After miR-130a was knocked down using small-interfering RNA (siRNA) in T24 cell line, the changes in expressions of miR-130a and CYLD in each group were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The cell proliferation in each group was detected using cell counting kit-8 (CCK8) assay and flow cytometry. The changes in mRNA and protein levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin1 were determined using qRT-PCR and Western blotting. The autolysosomes were detected through acridine orange (AO)/ethidium staining bromide (ER) staining. Moreover, CYLD was knocked down using siRNA, and then the changes in mRNA expressions of miR-130a, LC3 and Beclin1 in each group were detected through qRT-PCR.

RESULTS

After interference in miR-130a with siRNA, miR-130a-siRNA group had a significantly lower mRNA expression of miR-130a compared with NC-siRNA group and a significantly higher mRNA expression of CYLD (p<0.05), obviously inhibited cell proliferation (p<0.05), and decreased significantly mRNA and protein expressions of LC3 showing Beclin1 (p<0.05), and an evidently smaller number of autolysosomes. After knockdown of CYLD using siRNA, the mRNA expression of miR-130a had no significant changes (p>0.05), while the mRNA expressions of LC3 and Beclin1 declined significantly in CYLD-siRNA group compared with those in NC-siRNA group (p<0.05).

CONCLUSION

MiR-130 can promote the autophagy of bladder cancer cells through regulating CYLD, thus facilitating the proliferation of tumor cells.

目的

本研究旨在通过胱抑素（CYLD）探讨微小 RNA（miR）-130a 对膀胱癌细胞自噬的调控机制。

方法

以人膀胱癌 T24 细胞系为研究对象，采用小干扰 RNA（siRNA）敲低 T24 细胞系中的 miR-130a，通过实时定量逆转录聚合酶链反应（qRT-PCR）检测各组 miR-130a 和 CYLD 的表达变化。采用细胞计数试剂盒-8（CCK8）检测各组细胞增殖，流式细胞术检测细胞凋亡。采用 qRT-PCR 和 Western blot 检测微管相关蛋白 1 轻链 3（LC3）和 Beclin1 的 mRNA 和蛋白水平变化。通过吖啶橙（AO）/溴乙锭（ER）染色检测自噬体。此外，采用 siRNA 敲低 CYLD，通过 qRT-PCR 检测各组 miR-130a、LC3 和 Beclin1 的 mRNA 表达变化。

结果

用 siRNA 干扰 miR-130a 后，miR-130a-siRNA 组的 miR-130a 表达水平明显低于 NC-siRNA 组，CYLD 表达水平明显高于 NC-siRNA 组（p<0.05），细胞增殖明显受到抑制（p<0.05），LC3 和 Beclin1 的 mRNA 和蛋白表达明显降低（p<0.05），自噬体数量明显减少。用 siRNA 敲低 CYLD 后，miR-130a 的 mRNA 表达无明显变化（p>0.05），而 CYLD-siRNA 组的 LC3 和 Beclin1 的 mRNA 表达明显低于 NC-siRNA 组（p<0.05）。