Altern Ther Health Med. 2022 Sep;28(6):132-137.
To detect the expression level of the Mfn2 gene in hepatocellular carcinoma (HCC) and adjacent normal liver tissues and further analyze its anticancer effects.
The expression levels of Mfn2, GLS1 and the autophagy-related proteins lc3b and Beclin1 in liver cancer and adjacent tissues in patients with liver cancer were detected by real-time-quantitative polymerase chain reaction (RT-qPCR). The HepG2 human HCC cell line was cultured in vitro, and the Mfn2 protein was stably expressed through transfection of a high Mfn2 expression plasmid. The Cell-Counting Kit-8 (CCK-8) method was used to observe the effect of Mfn2 overexpression on the activity of HepG2 cells. Furthermore, RT-qPCR and Western blotting were performed to detect the effects of Mfn2 overexpression on the protein expression of GLS1, Beclin1 and lc3b.
Compared with tissues adjacent to cancer tissues, the mRNA levels of Mfn2, GLS1, Beclin1 and lc3b in liver cancer tissues were lower. Compared with normal hepatocytes, the expression of Mfn2, Beclin1 and lc3b in HCC cells was decreased, but the expression of GLS1 was increased. Compared with the control group (NC) transfected with empty plasmid, Mfn2 overexpression led to significant time-dependent inhibition of HepG2 cell activity and GLS1 protein expression (P < .05). In addition, Mfn2 overexpression induced autophagy by triggering the expression of autophagy-related proteins Beclin-1 and lc3b in HCC cells (all P < .05). The effect of transfection with a high-dose Mfn2 plasmid was more obvious than that of transfection with a low-dose Mfn2 plasmid (all P < .05).
The expression of Mfn2, GLS1, Beclin1 and lc3b in HCC was lower than in normal liver tissue. The expression of Mfn2, Beclin1 and lc3b in HCC cells was decreased, but the expression of GLS1 was increased. Overexpression of Mfn2 inhibited GLS1 gene expression by inhibiting the activity of HCC cells and promoted the expression of Beclin1 and lc3b to induce autophagy, thereby exerting an anticancer effect. Further research is needed to clarify the mechanism of Mfn2 activity.
检测长链线粒体融合蛋白 2（Mfn2）基因在肝癌（HCC）及癌旁正常组织中的表达水平，并进一步分析其抗癌作用。
采用实时定量聚合酶链反应（RT-qPCR）检测肝癌患者肝癌组织及癌旁组织中 Mfn2、谷氨酰胺酶 1（GLS1）及自噬相关蛋白 LC3B、Beclin1 的表达水平。体外培养 HepG2 人 HCC 细胞系，通过转染高 Mfn2 表达质粒稳定表达 Mfn2 蛋白。采用细胞计数试剂盒（CCK-8）法观察 Mfn2 过表达对 HepG2 细胞活性的影响。进一步采用 RT-qPCR 和 Western blot 法检测 Mfn2 过表达对 GLS1、Beclin1 和 LC3B 蛋白表达的影响。
与癌旁组织相比，肝癌组织中 Mfn2、GLS1、Beclin1 和 LC3B 的 mRNA 水平均降低。与正常肝细胞相比，HCC 细胞中 Mfn2、Beclin1 和 LC3B 的表达降低，而 GLS1 的表达增加。与空质粒转染的对照组（NC）相比，Mfn2 过表达可显著抑制 HepG2 细胞活性和 GLS1 蛋白表达，呈时间依赖性（均 P<.05）。此外，Mfn2 过表达通过触发自噬相关蛋白 Beclin1 和 LC3B 的表达诱导 HCC 细胞发生自噬（均 P<.05）。高剂量 Mfn2 质粒转染的效果比低剂量 Mfn2 质粒转染的效果更明显（均 P<.05）。
HCC 中 Mfn2、GLS1、Beclin1 和 LC3B 的表达均低于正常肝组织。HCC 细胞中 Mfn2、Beclin1 和 LC3B 的表达降低，而 GLS1 的表达增加。Mfn2 过表达通过抑制 HCC 细胞活性抑制 GLS1 基因表达，并通过促进 Beclin1 和 LC3B 的表达诱导自噬，从而发挥抗癌作用。需要进一步研究以阐明 Mfn2 活性的机制。
