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微小RNA-365通过靶向Bcl-2诱导肝癌细胞凋亡。

miR-365 induces hepatocellular carcinoma cell apoptosis through targeting Bcl-2.

作者信息

Li Mingfei, Yang Yuan, Kuang Yu, Gan Xianfeng, Zeng Wei, Liu Yuping, Guan Hua

机构信息

Department of Hepatobiliary Surgery, Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, P.R. China.

Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610072, P.R. China.

出版信息

Exp Ther Med. 2017 May;13(5):2279-2285. doi: 10.3892/etm.2017.4244. Epub 2017 Mar 20.

Abstract

Hepatocellular carcinoma (HCC) is currently ranked as the third leading cause of cancer-related mortality worldwide. microRNAs (miRs) serve important roles in the development and progression of HCC. miR-365 has been demonstrated to function as a tumor suppressor in several types of cancer, including HCC; however, the mechanisms by which miR-365 regulates HCC apoptosis remains to be elucidated. In the present study, reverse transcription-quantitative polymerase chain reaction was performed to determine miR-365 expression levels in HCC and normal liver (LO2) cells. miR-365 overexpression was induced in SMC7721 cells using a plasmid-based system, and Cell Counting Kit-8 and TUNEL assays were performed to detect cell activity and apoptosis following miR-365 transfection. A luciferase assay was performed to determine the direct target of miR-365 in apoptosis regulation. Furthermore, a subcutaneously transplanted tumor model was established to evaluate the effects of miR-365 on tumor growth . The tumor tissue was used for further proliferation and apoptosis detection. The results of the present study indicated that miR-365 expression was significantly lower in HCC cells compared with LO2 cells (P<0.01). Transfection of SMC7721 cells with miR-365 plasmid significantly inhibited cell activity by inducing apoptosis (P<0.01). Luciferase assay indicated that miR-365 targets B-cell lymphoma 2 (Bcl-2) directly and therefore induces the downstream expression of pro-apoptotic proteins. The SMC7721 primary tumor growth was significantly reduced by miR-365 transfection (P<0.01). Further investigation demonstrated that the miR-365 group contained significantly fewer cells that were positive for proliferating cell nuclear antigen (P<0.01) and significantly more apoptotic cells (P<0.01). In conclusion, the results of the present study demonstrated that miR-365 may serve a role in inducing HCC apoptosis via directly targeting Bcl-2. This may provide a novel diagnosis and therapy target for the treatment of patients with HCC.

摘要

肝细胞癌(HCC)目前是全球癌症相关死亡的第三大主要原因。微小RNA(miR)在HCC的发生和发展中发挥重要作用。miR-365已被证明在包括HCC在内的多种癌症类型中起肿瘤抑制作用;然而,miR-365调节HCC细胞凋亡的机制仍有待阐明。在本研究中,进行逆转录-定量聚合酶链反应以确定HCC细胞和正常肝(LO2)细胞中miR-365的表达水平。使用基于质粒的系统在SMC7721细胞中诱导miR-365过表达,并进行细胞计数试剂盒-8和TUNEL检测以检测miR-365转染后的细胞活性和凋亡情况。进行荧光素酶检测以确定miR-365在凋亡调节中的直接靶点。此外,建立皮下移植肿瘤模型以评估miR-365对肿瘤生长的影响。肿瘤组织用于进一步的增殖和凋亡检测。本研究结果表明,与LO2细胞相比,HCC细胞中miR-365的表达明显降低(P<0.01)。用miR-365质粒转染SMC7721细胞可通过诱导凋亡显著抑制细胞活性(P<0.01)。荧光素酶检测表明,miR-365直接靶向B细胞淋巴瘤2(Bcl-2),从而诱导促凋亡蛋白的下游表达。miR-365转染显著降低了SMC7721原发性肿瘤的生长(P<0.01)。进一步研究表明,miR-365组中增殖细胞核抗原阳性的细胞明显减少(P<0.01),凋亡细胞明显增多(P<0.01)。总之,本研究结果表明,miR-365可能通过直接靶向Bcl-2在诱导HCC细胞凋亡中发挥作用。这可能为HCC患者的治疗提供新的诊断和治疗靶点。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2383/5443224/de0c684d15ab/etm-13-05-2279-g00.jpg

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