基于miR-34b-3p/PINK1/Bcl-2信号通路探讨蓝萼甲素调控肝癌高转移细胞系HCCLM3自噬与凋亡的机制OA
Mechanism of glaucocalyxin A regulating autophagy and apoptosis in highly metastatic hepatocellular carcinoma cell line HCCLM3 based on miR-34b-3p/PINK1/Bcl-2 signaling pathway
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 2.5 μg/mL 组、GLA 5 μg/mL 组、GLA 10 μg/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、10 μg/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 2.5 μg/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 5 μg/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 10 μg/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 5 μg/mL 组相比,GLA 10 μg/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 apop-tosis 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-immu-noprecipitation.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 expres-sion 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/LC3Ⅰratio was elevated,Bax and Cleaved Caspase-3 expression increased,Bcl-2 expression decreased,and the apoptosis rate in-creased(all P<0.05),and transmission electron microscopy revealed mitochondrial swelling and an increased number of au-tophagosomes;in the GLA 10 μg/mL group,miR-34b-3p expression significantly increased,PINK1 expression decreased a-long 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 ob-served under transmission electron microscopy.Compared with the GLA 5 μg/mL group,the binding level of PINK1 and Par-kin 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.
朱琳琳;张秀
河南医药大学医学技术学院 河南省免疫与靶向药物重点实验室,河南 新乡 453003河南医药大学医学技术学院 河南省免疫与靶向药物重点实验室,河南 新乡 453003
医药卫生
蓝萼甲素肝癌miR-34b-3p/PINK1/Bcl-2信号通路自噬凋亡
glaucocalyxin Ahepatic carcinomamiR-34b-3p/PINK1/Bcl-2 signaling pathwayautophagyapoptosis
《山东医药》 2026 (4)
21-25,5
河南省科技攻关项目(252102310079)河南省科技研发计划联合基金项目(252103810345).
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