山东医药

目的 以miR-34b-3p/PINK1/Bcl-2信号通路为靶向，探讨蓝萼甲素(GLA)调节肝癌细胞线粒体自噬与凋亡的作用机制。方法 以肝癌高转移细胞系HCCLM3为对象，设对照组、GLA 2.5μg/mL组、GLA 5μg/mL组、GLA 10μg/mL组。对照组常规培养，其余三组加入相应终浓度的GLA(2.5、5、10μg/mL)。采用实时荧光定量PCR法检测细胞miR-34b-3p的表达；Western blotting法检测细胞PINK1、Parkin、LC3、Bax、Bcl-2、Cleaved Caspase3的表达；流式细胞仪检测细胞凋亡率；荧光显微镜观察PINK1蛋白在细胞内的定位分布；免疫共沉淀法检测细胞中PINK1与Parkin的结合状态；透射电镜拍摄观察细胞自噬与凋亡形态。结果 与对照组相比，GLA 2.5μg/mL组细胞miR-34b-3p表达、线粒体自噬与凋亡通路相关蛋白(PINK1、Parkin、LC3Ⅱ/LC3Ⅰ、Bax、Bcl-2、Cleaved Caspase3)相对表达量及细胞凋亡率差异无统计学意义(P均>0.05);GLA 5μg/mL组细胞miR-34b-3p表达差异无统计学意义(P>0.05),但PINK1、Parkin表达升高，LC3Ⅱ/LC3Ⅰ升高，Bax、Cleaved Caspase3表达升高，Bcl-2表达降低，细胞凋亡率升高(P均<0.05),透射电镜下可见线粒体肿胀、自噬小体增多；GLA 10μg/mL组细胞miR-34b-3p表达显著升高，PINK1表达降低且胞内荧光强度减弱，Parkin、Bcl-2表达降低，Bax、Cleaved Caspase3表达升高，细胞凋亡率升高(P均<0.05),透射电镜下可见典型凋亡形态。与GLA 5μg/mL组相比，GLA 10μg/mL组细胞中PINK1与Parkin结合水平降低。结论 GLA可能通过靶向miR-34b-3p/PINK1/Bcl-2信号通路调节HCCLM3细胞自噬与凋亡，且作用效果与剂量密切相关。

Objective To investigate the mechanism by which glaucocalyxin A(GLA) regulates mitophagy and apoptosis in hepatocellular carcinoma cells with a focus on the miR-34b-3p/PINK1/Bcl-2 signaling pathway. Methods The highly metastatic hepatocellular carcinoma cell line HCCLM3 was used. Cells were divided into the control group and GLA-treated groups(2.5, 5, and 10 μg/mL). Cells in the control group were cultured under routine conditions, while cells in the GLA groups were treated with 2.5, 5, and 10 μg/mL GLA, respectively. The expression of miR-34b-3p was detected by quantitative real-time PCR(qPCR). The protein expression levels of PINK1, Parkin, LC3, Bax, Bcl-2, and Cleaved Caspase-3 were determined by Western blotting. The apoptosis was analyzed by flow cytometry. The intracellular localization of PINK1 was observed by fluorescence microscopy. The interaction between PINK1 and Parkin was assessed by co-immunoprecipitation. Cellular autophagy and apoptosis were observed by transmission electron microscopy. Results Compared with the control group, no statistically significant differences were found in the expression of miR-34b-3p, the relative expression levels of mitophagy-and apoptosis-related proteins(PINK1, Parkin, LC3Ⅱ/LC3Ⅰ, Bax, Bcl-2, Cleaved Caspase-3), or the apoptosis rate in the GLA 2.5 μg/mL group(all P>0.05); in the GLA 5 μg/mL group, miR-34b-3p expression showed no statistically significant difference(P>0.05), whereas the expression levels of PINK1 and Parkin increased, the LC3Ⅱ/LC3Ⅰ ratio was elevated, Bax and Cleaved Caspase-3 expression increased, Bcl-2 expression decreased, and the apoptosis rate increased(all P<0.05), and transmission electron microscopy revealed mitochondrial swelling and an increased number of autophagosomes; in the GLA 10 μg/mL group, miR-34b-3p expression significantly increased, PINK1 expression decreased along with reduced intracellular fluorescence intensity, Parkin and Bcl-2 expression decreased, while Bax and Cleaved Caspase-3 expression increased, and the apoptosis rate increased(all P<0.05), and typical apoptotic morphology was observed under transmission electron microscopy. Compared with the GLA 5 μg/mL group, the binding level of PINK1 and Parkin was reduced in the GLA 10 μg/mL group.Conclusion GLA may regulate autophagy and apoptosis in HCCLM3 cells by modulating the miR-34b-3p/PINK1/Bcl-2 signaling pathway, and its effects are dose-dependent.