The Mechanism Of SSRP1 Affecting Apoptosis Of HCC Cells By Regulating Mitochondrial Function Through TRAP1

Background:Hepatocellular carcinoma(HCC)is a primary malignant tumor occurred in liver.According to WHO statistics,the overall survival rate of patients with liver cancer is18%,and the survival rate of patients with advanced liver cancer is as low as 2.5%.Early liver cancer can be treated by surgery,but it is easy to recur.Patients with advanced liver cancer usually use first-line drugs such as sorafenib and tyrosine kinase inhibitor for systemic treatment.However,their ability to increase the survival period of patients is limited,and drug resistance usually occurs within half a year.According to reports,epigenetics plays an important role in the occurrence of liver cancer and drug tolerance.Abnormal epigenetic regulation can lead to gene expression imbalance and induce liver cancer.Therefore,aiming at epigenetics,restoring its normal modification and correct gene expression is an effective approach for the treatment of liver cancer.Structure specific recognition protein-1(SSRP1),a subunit of the histone chaperone chromatin transcription(FACT)complex,is involved in the disassembly and assembly of nucleosome in the epigenetic regulation process.It regulates the expression of target genes by participating in chromatin related processes,including chromatin remodeling,transcription initiation,and transcription extension.SSRP1 is highly expressed in various tumor tissues including HCC,and is associated with cell cycle,proliferation,and apoptosis.This study found that SSRP1 may be associated with mitochondrial relative apoptosis in HCC cells,but its potential mechanism is unclear.Although some studies have shown that epigenetic regulation is closely related to mitochondrial function,such as histone deacetylase inhibitors that can inhibit mitochondrial dysfunction in mice,inducing oxidative stress and cell damage;However,most research on histone chaperones,including SSRP1,aiming at upstream m RNA regulation or DNA repair mechanisms rather than the effects on cellular biological functions such as mitochondrial homeostasis.Therefore,this study aims to discuss the regulatory mechanism of SSRP1 on mitochondrial function and apoptosis in HCC cells through mitochondrial function.TNF receptor associated protein 1(TRAP1)is a member of the heat shock protein 90(HSP90)family.It is also the only HSP90 family menmber that mainly distributes in mitochondria and directly interacts with the oxidative phosphorylation complex.TRAP1 is highly expressed in multiple tumors,and it can promote tumorigenesis by regulating mitochondrial related proteins to participate in oxidative phosphorylation and maintain mitochondrial function.However,there has not been systematic study on TRAP1 in HCC.This study found that downregulation of SSRP1 leads to mitochondrial damage and apoptosis in HCC cells;Downregulation of SSRP1 inhibited the expression of TRAP1.Therefore,our work explored the mechanism of SSRP1 in epigenetic regulation and mitochondrial function of HCC through TRAP1,and provides a new insight and direction for the clinical treatment of HCC through mitochondrial homeostasis,especially oxidative phosphorylation.Objective:Taking HCC cells as the research object,small interfering RNA was used to observe the effects of SSRP1 inhibition on apoptosis,mitochondrial function and oxidative phosphorylation of HCC cells in vitro and in vivo,and explore the relevant mechanism of regulating mitochondrial function in liver cancer cells through TRAP1.Method and Results:(1)SSRP1 is highly expressed in HCC,which has clinical diagnostic significance and is related to survival.Using TCGA database for analysis,the results showed that the level of SSRP1 in HCC was significantly higher than that in normal liver tissue and negatively correlated with overall survival and disease-free survival;The ROC curve analysis results show that SSRP1 has a strong clinical diagnostic correlation.(2)The effect of si-SSRP1 on the proliferation and apoptosis of HCC cells was observed in vitro.Experimental cell lines: human HCC cell lines Hep G2 and HCCLM3.Experimental groups: si-NC group,si-SSRP1-1 and si-SSRP1-2 group.CCK-8 was used to detect the effect of SSRP1 on the viability of HCC cells and the results showed that inhibiting SSRP1 expression can inhibit cell viability;Using Annexin V-FITC/PI staining and DAPI staining to detect the effect of SSRP1 on apoptosis of HCC cells,the results showed that downregulating SSRP1 increased the proportion of apoptosis,and apoptotic phenotypes such as nuclear pyknosis and fragmentation appeared in the nucleus;Western Blot assay was used to detect the expression of apoptosis related proteins,and it was found that inhibition of SSRP1 resulted in a downregulation of the apoptosis related protein Bcl-2/Bax ratio,while the expression of Cleared Caspase3 was upregulated.The above results suggest that targeting SSRP1 can inhibit the vitality of liver cancer cells and induce apoptosis.(3)The effect of SSRP1 inhibition on mitochondrial function and oxidative phosphorylation of HCC cells in vitro.JC-1 detection showed that the mitochondrial membrane potential of HCC was decreased after down-regulation of SSRP1;ATP detection,DCFH-DA and Mito SOX probe results showed that after SSRP1 inhibition,the ATP production was decreased and the production of reactive oxygen species was increased significantly,indicating that the mitochondrial function was damaged.These results suggest that downregulating the expression of SSRP1 can induce mitochondrial damage in liver cancer cells.R01 probe and Seahorse XF-24 mitochondrial oxygen consumption rate(OCR)showed that the oxidative phosphorylation was damaged;Western Blot showed that si-SSRP1 could down-regulate the expression of oxidative respiratory chain related protein NDUFV1,SDHB,ATP5F1,COX7 C,and COXIV in HCC cells.It is suggested that the change of oxidative respiratory chain related protein caused by SSRP1 may be the mechanism of maintaining mitochondrial function and apoptosis inhibition.(4)Explore the possible mechanism of SSRP1 in HCC cells on apoptosis and oxidative phosphorylation.1)The mechanism of SSRP1 on mitochondrial related proteins was preliminarily explored.The q PCR results showed that SSRP1 did not affect the m RNA expression of oxidative phosphorylation related proteins,and MG132 addition could inhibit the decrease in mitochondrial phosphorylation protein levels caused by SSRP1 downregulation.These results suggest that SSRP1 regulates the protein expression level through the protein quality control pathway.TCGA database was used and the downstream target genes of SSRP1 were predicted and screened.It was found that TRAP1 was positively correlated with SSRP1 expression,highly expressed in HCC and negatively correlated with the survival period;QPCR,Western Blot and Ch IP-q PCR were used and showed that SSRP1 was enriched at the TRAP1 transcription start site,and the expression of TRAP1 was subsequently downregulated after SSRP1 was downregulated.The above results demonstrate that TRAP1 is a key target gene for SSRP1 to function.2)Targeted si-TRAP1 was designed and synthesized,and use CCK-8 kit,flow cytometry,ATP chemiluminescence detection kit,DCFH-DA,and Mito SOX probes to detect the effects of TRAP1 on apoptosis and mitochondrial homeostasis;The effects of TRAP1 on oxidative phosphorylation and related proteins in liver cancer cells were observed using R01 fluorescent probe kit,Seahorse XF-24 energy metabolism analyzer,and Western Blot.The results showed that,consistent with the downregulation of SSRP1 expression,downregulation of TRAP1 expression can induce apoptosis and mitochondrial damage in HCC cells,and inhibit oxidative phosphorylation and the expression of related proteins NDUFV1,SDHB,ATP5F1,COX7 C,and COXIV.These results suggest that SSRP1 may maintainin the mitochondrial function and inhibit apoptosis through TRAP1.3)Overexpression TRAP1 plasmid was synthesized and Western Blot showed that si-SSRP1 can reverse the effect of TRAP1 overexpression plasmid.Prove that SSRP1 is an upstream regulatory factor of TRAP1.CCK-8 kit,flow cytometry,ATP chemiluminescence detection kit,DCFH-DA,and Mito SOX probes were used to detect the effects of TRAP1 c DNA on apoptosis and mitochondrial damage in HCC cells under SSRP1 inhibition.The results showed that overexpression of TRAP1 can restore the apoptosis and mitochondrial damage induced by SSRP1 inhibition in liver cancer cells;R01 fluorescent probe kit,Seahorse XF-24 energy metabolism analyzer,and Western blot showed that overexpression of TRAP1 restored the oxidative phosphorylation damage induced by SSRP1 inhibition,and restored the expression of oxidative phosphorylation related proteins NDUFV1,SDHB,ATP5F1,COX7 C,and COXIV.The above results indicate that SSRP1 maintains the stability of mitochondrial oxidative phosphorylation complexes through TRAP1 mediated protein quality control,thereby maintaining mitochondrial function and inhibiting liver cancer cell apoptosis.(5)The therapeutic effect and mechanism of targeted SSRP1 on HCC were verified in vivo.HCCLM3 cells were used to construct a nude mouse subcutaneous transplant tumor model;Nude mice were divided into two groups and injected with si-NC and si-SSRP1 around the tumor,respectively;Measure the tumor volume and draw a curve every three days,and observe the mental state of the mice during this period;After 21 days of administration,euthanize the mice and remove the tumor.The results showed that compared with the control group(si-NC group),the tumor volume of si-SSRP1 group was significantly reduced.Immunohistochemical results showed that si-SSRP1 could reduce the expression of Ki-67,Bcl-2,and Cleared Caspase-3 and enhance the expression of Bax.At the same time,si-RNA with 2-O-Me modification did not present biological toxicity.Western Blot showed that TRAP1 and mitochondria-related protein were down-regulated in si-SSRP1 treatment group,which confirmed the mechanism of targeted SSRP1 treatment for liver cancer.Conclusion:(1)SSRP1 is highly expressed in HCC with good clinical diagnostic relevance for HCC,and is negatively correlated with survival;(2)SSRP1 regulates the expression of mitochondrial oxidative phosphorylation protein through protein quality control,maintains the mitochondrial function of HCC cells,and inhibits cell apoptosis;(3)There is a positive correlation between TRAP1 and SSRP1 expression;TRAP1 is highly expressed in liver cancer cells and negatively correlated with the survival period of liver cancer patients(4)SSRP1 maintains the stability of oxidative phosphorylation related proteins in HCC cells and inhibits their degradation,stabilizing mitochondrial function and inhibiting apoptosis of HCC cells through transcriptional regulation of TRAP1 expression.