MiR-145 Target SENP1 To Promote Apoptosis Of Hepatocellular Carcinoma Cells By Increasing SUMOylation Of HK2

Objective: Liver cancer is the sixth most frequently diagnosed cancer in the world.The most common type of liver cancer is hepatocellular carcinoma(HCC),also known as primary liver cancer.It is one of the most serious malignant tumors in the world,accounting for about 90% of all liver cancer cases and the third leading cause of cancer death in the world.According to e Medicine-Health data,due to late diagnosis and poor prognosis,the 5-year survival rate of early HCC is 33%,while the late survival rate is only 2%-11%.Therefore,it is particularly important to develop advanced diagnostic and treatment solutions.In the research,it was found that miRNA participates in the energy metabolism process of many diseases and plays an important role in regulating cell proliferation,aging,apoptosis,etc,so it has received widespread attention in the past few years.Most cancer cells rely on aerobic glycolysis rather than oxidative phosphorylation,and require high levels of nutrients,especially glucose,to meet the nutritional needs of rapid growth and proliferation.As the first speed-limiting enzyme of glycolysis,the binding of HK2 to the outer membrane mitochondria is crucial to its oncogenic activity.However,the regulatory mechanism of the binding of HK 2 to the outer membrane mitochondria is still unclear.SUMOylation is one of the key modifications after translation.Extensive research has linked the various regulation of SUMOylation to the target protein,such as the structure,activity,function,stability,location and interaction with other proteins,while SENPs family participates in the reversible process of SUMOylation.Based on the above analysis and research basis,this study aims to explore miR-145/SENP1 to regulate the SUMOylation level of HK2 and the mechanism of participating during hepatocellular carcinoma,so as to provide new ideas for the treatment of hepatocellular carcinomaMethods: In vitro experiments: HepG2 cells transfected with miR-145 were used to construct cell therapy models.The expression of SENP1,HK2,SUMO1,VDAC1,Cyt-c,C-Caspase3,and Caspase3 at the cellular level was detected by Western blotting and/or q RT-PCR.Immunofluorescence double-staining was performed to detect the co-localization of HK2 with VDAC1.COIP was performed to detect the interaction of HK2 with SUMO1 and VDAC1,respectively;RNA immunoprecipitation was performed to probe the relationship between miR-145 and SENP1;mitochondrial membrane potential and flow cytometry were used to measure the level of apoptosis.In vivo experiment: Sixteen BALB/c-nu nude mice(male,3 weeks old,weight 10g-14g)were purchased and rando m Ly divided into two groups of 8 mice each after one week of adapting under 12 h light/12 h dark laboratory conditions.Group 1 was injected with negative control transfected HepG2 cells;Group 2 was injected with HepG2 cells stably transfected with miR-145 to establish a nude mouse hepatoma transplantation tumor model;Pathological histological changes of transplantation tumors were observed by transplantation tumor volume,weight,tumor weight ratio,HE,TUNEL,and immunohistochemical staining;SENP1,HK2,Cyt-c,and C-Caspase were detected by Western blotting and/or q RT-PCR.Results:1.The GEPIA2 database showed that the expression of SENP1 in liver cancer tissues was higher than that in adjacent tissues,and it was closely related to the overall survival of patients.Western blotting and q RT-PCR results showed that SENP1 was highly expressed in hepatoma cell lines Huh7,Hep3 B and HepG2 and liver cancer tissues.2.HK2 can be SUMOylated in liver cancer cells and high expression of SENP1 reduced the SUMOylation level of HK2 and promoted its localization in the outer mitochondrial membrane.Knockdown of SENP1 by si SENP1 increased the level of SUMOylation of HK2 and inhibited its mitochondrial localization,resulting in reduced lactic acid production,reduced extracellular acidification rate and increased oxygen consumption rate;The mitochondrial membrane potential decreased,and the apoptosis proteins C-Ccaspase3 and Cytc were regulated.3.Transfection of miR-145 mimic into HepG2 cells inhibited SENP1 expression,increased the level of HK2 SUMOylation and reduced binding to VDAC1,decreased both extracellular acidification rate and mitochondrial membrane potential,upregulated C-Ccaspase3 and Cyt-c expression,and significantly increased apoptosis in hepatocellular carcinoma cells.It is suggested that miR-145 may promote apoptosis of hepatocellular carcinoma cells by targeting SENP1 to inhibit HK2 desuccination and mitochondrial localization.4.The vehicle and miR-145 transfected HepG2 cell suspensions were injected into the right anterior axilla of nude mice,and the samples were taken on day 21,which showed that the transplanted tumors in the miR-145-stabilized group were smaller and lighter compared with the vehicle group.HE staining showed that the transplanted tumor cells in the miR-145 stable group were relatively disordered,showing deep nuclear staining,nuclear fragmentation,and nuclear lysis;TUNEL staining showed that the transplanted tumors in the miR-145 stable group had more obvious apoptosis,suggesting that miR-145 inhibited the growth of HepG2 transplanted tumors.Conclusion: miR-145 target SENP1 to promote apoptosis of hepatocellular carcinoma cells by increasing SUMOylation of HK2...