茯苓多糖通过LncRNA HCG11/miR-539-3p调控肝癌细胞自噬、化疗耐药的机制

陈永芳; 李艳珂; 张淑静

doi:10.13386/j.issn1002-0306.2023110291

茯苓多糖通过LncRNA HCG11/miR-539-3p调控肝癌细胞自噬、化疗耐药的机制

Mechanism of Poria cocos Polysaccharide Regulating Autophagy and Chemotherapy Resistance of Hepatocellular Carcinoma Cells through LncRNAHCG11/miR-539-3p 539-3p

摘要

摘要: 目的：探讨茯苓多糖（Poria cocos polysaccharide，PCP）对肝癌细胞增殖、自噬和化疗耐药的影响及机制。方法：对数期HepG-2/DDP细胞随机分组：对照组、PCP组（7.5、15、30 µg/mL PCP）、shNC 组、HCG11 siRNA组（转染50 nmol/L HCG1干扰序列）、pcDNA组（转染2 µg pcDNA）、pcDNA3.1-HCG11组（转染2 µg pcDNA3.1-HCG11）、PCP组（30 µg/mL）和PCP+pcDNA3.1-HCG11组（转染2 µg pcDNA3.1-HCG11+30 µg/mL PCP）。MTT检测肝癌细胞耐药情况和抑制率，Western blot检测Beclin-1、LC3 I和LC3 II水平，实时荧光定量PCR（Real time quantitative PCR，qRT-PCR）检测细胞株中LncRNA HCG11和miR-539-3p的水平，双荧光素酶报告实验检测LncRNA HCG11和miR-539-3p的靶向关系，裸鼠成瘤实验验证体内瘤体的瘤重、瘤体积及自噬相关蛋白水平。结果：4~128 µg/mL PCP抑制HepG-2/DDP细胞增殖，7.5、15、30 µg/mL的PCP能显著降低HepG-2/DDP细胞中MRP1、P-gp、Beclin-1、LC3II的水平，下调HCG11水平，增加miR-539-3p的表达水平（P<0.01）；与对照组比较，HCG11 siRNA 组HepG-2/DDP细胞HCG11相对表达水平、IC₅₀（顺铂）、Beclin-1、LC3 II/ LC3 I比值显著降低，miR-539-3p水平和抑制率（顺铂）显著增加（P<0.01）；与PCP组比较，PCP+pcDNA3.1-HCG11处理组HCG11相对表达水平显著升高，Belin-1、LC3 II/LC3 I比值显著增加（P<0.01）；在不同浓度的顺铂处理下，与PCP组比较，PCP+pcDNA3.1-HCG11 组抑制率显著降低（P<0.01）；与PCP组比较，PCP+HCG11 shRNA 组肿瘤体积和肿瘤重量显著降低，Belin-1、LC3 II/LC3 I水平显著下调（P<0.05）。结论：PCP能通过LncRNA HCG11/miR-539-3p调控耐药和自噬，并影响肝癌细胞的增殖。

Abstract: Objective: To explore the effects and mechanism of Poria cocos polysaccharide (PCP) on proliferation, autophagy and chemotherapy resistance of hepatocellular carcinoma cells. Methods: HepG-2/DDP cells in logarithmic phase were randomly divided into control group, PCP group (7.5, 15, 30 μg/mL PCP), shNC group, HCG11 siRNA group (transfection of 50 nmol/L HCG1 interference sequence), pcDNA group (transfection of 2 μg pcDNA), pcDNA3.1-HCG11 group (transfection of 2 μg pcDNA3.1-HCG11), PCP group (30 μg/mL PCP) and PCP+pcDNA3.1-HCG11 group (transfection of 2 μg pcDNA3.1-HCG11+30 μg/mL PCP). MTT assay was used to detect the drug resistance and inhibition rate of hepatocellular carcinoma cells, western blot was used to detect the levels of Beclin-1, LC3, and quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the levels of LncRNA HCG11 and miR-539-3p in the cell lines. Double luciferase report experiment was used to detect the targeting relationship between LncRNA HCG11 and miR-539-3p, and nude mice tumorigenesis experiment was used to verify the influence of tumor weight, tumor volume and autophagy-related protein level in nude mice. Results: 4~128 µg/mL PCP had a certain inhibitory effect on HepG-2/DDP cells. PCP of 7.5, 15, 30 μg/mL could significantly decrease the levels of MRP1, P-gp, Beclin-1 and LC3II, decrease the level of HCG11 and increase the expression of miR-539-3p in HepG-2/DDP cells (P<0.01). Compared with the control group, the relative expression level of HCG11, IC₅₀ (cisplatin), Beclin-1 and LC3II/LC3I in HCG11siRNA group decreased significantly, while the level of miR-539-3p and inhibition rate (cisplatin) increased significantly (P<0.01). Compared with PCP group, the relative expression level of HCG11 in PCP+pcDNA3.1-HCG11 group increased significantly, while the ratio of Belin-1 and LC3 II/LC3 I increased significantly (P<0.01). Compared with PCP group, the inhibition rate of PCP+pcDNA3.1+HCG11 group was significantly decreased under different concentrations of cisplatin (P<0.01). Compared with PCP group, tumor volume and tumor weight in PCP+HCG11shRNA group were significantly decreased, and the levels of Belin-1 and LC3II/LC3I were significantly decreased (P<0.05). Conclusion: PCP can affect the proliferation of hepatocellular carcinoma cells by regulating drug resistance and autophagy through LncRNA HCG11/miR-539-3p.