Based On Transcriptomics To Study The Molecular Mechanism Of Ginsenoside Rh2 Inhibiting Cervical Cancer Cell Energy Metabolism-induced Apoptosis

Objective:This study aims to explore the effects of ginsenoside Rh2 on the apoptosis and energy metabolism of cervical cancer-derived cell lines as well as its targets and underlying molecular mechanism via transcriptome sequencing technology,providing a theoretical basis for the development and clinical treatment of new drugs related to cervical cancer.Methods:1.CCK-8 method was used to detect the effects of different concentrations of20（S）ginsenoside Rh2（20（S）-GRh2）and 20（R）ginsenoside Rh2（20（R）-GRh2）on the viability of cervical cancer cells,Hoechst33342 staining method to observe the influence on the morphology of cell apoptosis,Annexin V-FITC/PI to detect the degree of cell apoptosis.2.Flow cytometry and chemiluminescence technology were employed to detect the effect of20（S）-GRh2 on the mitochondrial membrane potential,ATP level and reactive oxygen levels of cervical cancer cells.An extracellular energy metabolism analyzer was used to detect the effect of 20（S）-GRh2 pair on energy metabolism in cervical cancer cells.Western blot was used to detect changes in the protein expression of mitochondrial electron transport chain complex,and spectrophotometry was used to detect the enzyme activity of mitochondrial electron transport chain complex.3.Establish a He La cell transcriptome database before and after administration of 20（S）-GRh2 based on the Illumina Hiseq platform,and apply bioinformatics analysis methods for statistical screening.4.UV spectrophotometry was used to detect the activity of Caspase-3/8/9.Western blot was used to detect the changes of Bax,Bcl-2,cytochrome C,VDAC1 and HK2 protein expression levels,knock down efficiency of VDAC1.Flow cytometry was used to detect apoptosis Death change.Results:1.Compared with the control group,20（S）-GRh2 showed a time-dependent and concentration-dependent inhibitory effect on the cell viability of He La and C33A cell lines,with IC₅₀ of 45μM（He La）and 55μM（C33A）,respectively.20（R）-GRh2 had almost no effect.Annexin V-FITC/PI staining showed that with the increment of drug concentration resulted in significantly increased apoptosis-like cells.20（S）-GRh2 induced the cell apoptosis of He La and C33A cell lines(^***p<0.001,^*p<0.05).2.20（S）-GRh2 can significantly reduce the mitochondrial membrane potential of He La and C33A cells(^***p<0.001,^*p<0.05),and inhibit ATP production(^**p<0.01,^*p<0.05).This inhibitory effect was more significant on HPV-positive cells（He La）;He La cell line was selected for further study.20（S）-GRh2 can i increase the level of reactive oxygen species(^**p<0.01),and inhibit mitochondrial oxidative phosphorylation and glycolysis of Hela cell line the inhibition of oxidative phosphorylation process is more significant;20（S）-GRh2significantly inhibited the activity of mitochondrial electron transport chain complex I,III and V(^**p<0.01),and had no effect on the expression level of chain complex proteins.3.High-throughput transcriptome sequencing was performed on He La cells before and after 45μM 20（S）-GRh2 treatment.Three biological replicates were performed and above6.5G data was obtained.The sequence length was 150bp and the base content distribution was stable.The quality of sequencing was good,with a Q20 value of over 98%and a Q30 value of over 96%.By comparing with the reference genome,more than 94%of Reads were located on the genome,and the utilization of transcriptome data was high.The reads covered on the m RNA were more uniform and random.4.Screen the genes with statistical differences.Compared with the control group,there were 414 differential genes in the 20（S）-GRh2 treatment group,of which 361 were up-regulated and 53 were down-regulated.Gene ontology（GO）enrichment analysis suggests that differential genes were mainly enriched in biological processes such as apoptosis regulation,metabolism,and cellular oxidative stress.5.20（S）-GRh2 induced He La cell apoptosis through the mitochondrial apoptotic pathway,which can significantly reduce the activity of Caspase-3 and Caspase-9(^***p<0.001,^*p<0.05),and promote the expression of Bax protein,the translocation of Bax mitochondria,and the release of cytochrome C into the cytoplasm and reduce the expression of Bcl-2 protein.6.Combined with the results of transcriptome differential gene analysis,Western blot confirmed that 20（S）-GRh2 can up-regulate the expression of VDAC1 protein(^***p<0.001)and inhibit the expression of HK2 protein（^*p<0.05）.The expression trends of VDAC1 and HK2 were negatively correlated;20（S）-GRh2 apoptosis-inducing ability and the expression of pro-apoptotic protein Bax were reduced after VDAC1 knockdown(^##p<0.01).Conclusion:Based on the establishment of transcriptome database and bioinformatics analysis methods,combined with molecular biology research methods,this article finally clarifies:20（S）-GRh2 can up-regulate VDAC1 protein expression,inhibit HK2 protein expression,and promote Bax transfer from cytoplasm to mitochondria,thereby damaging mitochondrial function,inhibiting mitochondrial oxidative phosphorylation and glycolysis pathways,and ultimately promoting the mitochondrial-dependent endogenous apoptotic pathway to induce apoptosis of cervical cancer cells.