Department of Neurology, the First Hospital of Changsha, Changsha, Hunan 410011, P.R. China.
Department of Gastroenterology, the First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, Guangdong 518028, P.R. China.
Acta Biochim Pol. 2023 Apr 17;70(2):239-246. doi: 10.18388/abp.2020_5657.
Sorafenib is the first-line treatment for hepatocellular carcinoma (HCC), but its efficacy is limited by the drug resistance of HCC cells. MiR-375 has been shown to be an inhibitor of autophagy that contributes to sorafenib resistance of HCC cells. In this context, this study probed into the unaddressed molecular target of miR-375 in inhibiting the autophagy of HCC cells under sorafenib treatment.
Western blotting and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) have been applied to measure the expressions of miR-375 and SIRT5 in parental HCC cells (HepG2 and Huh7) and sorafenib-resistant HCC cells (HepG2/so and Huh7/so). HepG2/so cells were accordingly transfected with miR-375 mimic, miR-375 inhibitor, sh-SIRT5, pcDNA3.1-SIRT5 or negative control. Expressions of p62, LC3I and LC3II in HCC cells have been measured by Western blotting. Viability and apoptosis of HCC cells have been assessed by CCK-8 (cell counting kit 8) and flow cytometry respectively. Bioinformatics techniques and dual-luciferase reporter assay have been used to predict and verify the targeting relationship between miR-375 and SIRT5.
MiR-375 was under-expressed and SIRT5 was over-expressed in HCC cells. An autophagy inhibitor impaired the survival of HepG2/so cells transfected with miR-375 inhibitor. An autophagy activator enhanced the drug resistance of HepG2/so cells transfected with miR-375 mimic. MiR-375 suppressed the drug resistance of HepG2/so cells by inhibiting autophagy. SIRT5 enhanced the drug resistance of HepG2/so cells by promoting autophagy and it could be targeted by miR-375.
MiR-375 suppresses autophagy to attenuate the sorafenib resistance of HCC cells by regulating SIRT5. The findings of this study may provide new therapeutic targets for treating HCC.
索拉非尼是肝细胞癌（HCC）的一线治疗药物，但它的疗效受到 HCC 细胞耐药性的限制。miR-375 已被证明是一种自噬抑制剂，可导致 HCC 细胞对索拉非尼产生耐药性。在这种情况下，本研究探讨了 miR-375 在索拉非尼治疗下抑制 HCC 细胞自噬的未解决的分子靶点。
Western blot 和 qRT-PCR（定量逆转录-聚合酶链反应）用于测量亲本 HCC 细胞（HepG2 和 Huh7）和索拉非尼耐药 HCC 细胞（HepG2/so 和 Huh7/so）中 miR-375 和 SIRT5 的表达。HepG2/so 细胞分别用 miR-375 模拟物、miR-375 抑制剂、sh-SIRT5、pcDNA3.1-SIRT5 或阴性对照转染。Western blot 法检测 HCC 细胞中 p62、LC3I 和 LC3II 的表达。CCK-8（细胞计数试剂盒 8）和流式细胞术分别评估 HCC 细胞的活力和凋亡。生物信息学技术和双荧光素酶报告基因检测用于预测和验证 miR-375 和 SIRT5 之间的靶向关系。
miR-375 在 HCC 细胞中表达下调，SIRT5 表达上调。自噬抑制剂可损害转染 miR-375 抑制剂的 HepG2/so 细胞的存活。自噬激活剂增强了转染 miR-375 模拟物的 HepG2/so 细胞的耐药性。miR-375 通过抑制自噬来抑制 HepG2/so 细胞的耐药性。SIRT5 通过促进自噬增强 HepG2/so 细胞的耐药性，并且可以被 miR-375 靶向。
miR-375 通过调节 SIRT5 抑制自噬，从而减轻 HCC 细胞对索拉非尼的耐药性。本研究的结果可能为治疗 HCC 提供新的治疗靶点。
