High Glucose Regulates The Proliferation And Metastasis Of HepG2 Cells Through The E2F1/RRBP1 Pathway

Objective Hepatocellular carcinoma(HCC)is the most common primary liver cancer,its morbidity and mortality have been increasing in recent years.And the incidence of diabetes is also increasing year by year.Studies have shown that diabetes is an independent risk factor of HCC.Although there are many common risk factors between diabetes and HCC,its internal mechanism is still unclear.Another student in our research group extracted endoplasmic reticulum from HepG2 cells with different glucose concentration intervention in the early stage,and then performed mass spectrometry analysis on the isolated endoplasmic reticulum,thus obtaining 39 differentially expressed endoplasmic reticulum proteins.Through bioinformatics analysis and cell experiments,we intended to explore the mechanism of poor prognosis in hepatocellular carcinoma patients complicated with diabetes.From the perspective of endoplasmic reticulum(ER),we studied the ways in which high glucose promoted the progression of hepatocellular carcinoma,and screened a new protein that played an important role in this process,so as to provide possible intervention targets for the treatment of such patients.Methods:1.By searching the GEPIA database and reading the literature,candidate proteins(FKBP3,FAM98 A and RRBP1)were screened out from the differentially expressed proteins obtained by mass spectrometry analysis.2.RT-q PCR was used to detect mRNA expression of candidate proteins in HepG2 cells cultured with different glucose concentrations(5.5mmol/L or 25mmol/L),so as to further select those with significant differences in expression as research objects.The target protein screened by the above methods was RRBP1.3.The TCGA database was retrieved and bioinformatics analysis was carried out to identify the expression differences of RRBP1 in liver cancer tissues and adjacent tissues,as well as the signaling pathways that might be activated by RRBP1 differential expression.4.HepG2 cells were divided into Low glucose group(5.5mmol/L)and High glucose group(25mmol/L).The proliferation,migration and invasion of cells were measured by CCK8,Wound-healing experiment and Transwell experiment;and the expression of RRBP1 protein was detected by Western blot.5.HepG2 cells were divided into Low glucose group,High glucose group,High glucose+NC-siRNA group and High glucose+RRBP1-siRNA group.The proliferation,migration and invasion of cells were measured by CCK8,Wound-healing experiment and Transwell experiment.6.Potential upstream transcription factors of RRBP1 were predicted by PROMO and Jaspar websites,and the candidate transcription factors were screened by GEPIA database retrieval and literature review.7.RT-q PCR and Western blot were used to detect the mRNA and protein expression of candidate proteins in HepG2 cells cultured with different glucose concentrations,so as to further select those with significant differences in expression as research objects.The target transcription factor screened by the above methods was E2F1.8.Ch IP and dual-luciferase reporter assay were used to detect whether E2F1 could regulate the transcription of RRBP1.9.HepG2 cells were divided into Low glucose group,High glucose group,High glucose+NC-siRNA group and High glucose+E2F1-siRNA group.The expression of RRBP1 was detected by RT-PCR and Western blot.The proliferation,migration and invasion of cells were measured by CCK8,Wound-healing experiment and Transwell experiment.10.The role of E2F1/RRBP1 pathway in the proliferation and metastasis of HepG2 cells was verified by Rescue assay.Results:1.The mRNA and protein expression of RRBP1 in HepG2 cells cultured with high glucose was significantly higher than that in cells cultured with low glucose.2.According to the bioinformatics analysis results,the expression of RRBP1 in liver cancer tissues was significantly higher than that in adjacent tissues;There were 502 differentially expressed genes in RRBP1 high-expression group compared with RRBP1 low-expression group;These differentially expressed genes could be significantly enriched in the pathways closely related to diabetes and tumorigenesis;In addition,GSEA gene enrichment analysis showed that cancer-related pathways such as cell cycle pathway and DNA repair pathway were significantly activated in the RRBP1 high-expression group.3.The proliferation,migration and invasion abilities of HepG2 cells in high glucose group were significantly higher than that in low glucose group,while the proliferation,migration and invasion abilities of tumor cells were significantly decreased after the expression of RRBP1 was inhibited.4.Through PROMO and Jaspar websites prediction,GEPIA database retrieval,literature review,RT-q PCR and Western blot experiment verification,E2F1 was selected as a possible transcription factor of RRBP1 for the next experiment.5.The mRNA and protein expression of E2F1 in HepG2 cells cultured with high glucose was significantly higher than that in cells cultured with low glucose.6.The results of Ch IP and dual-luciferase reporter assay showed that E2F1 could bind to the promoter of RRBP1 gene and promote the transcription of RRBP1 gene.7.After inhibiting the expression of E2F1 in HepG2 cells,the mRNA and protein expression of RRBP1 decreased significantly.8.After inhibiting the expression of E2F1 in HepG2 cells cultured with high glucose,the proliferation,migration and invasion of tumor cells were significantly decreased.9.The results of Rescue assay showed that E2F1 could further promote the proliferation and metastasis of HepG2 cells by up-regulating the expression of RRBP1.Conclusion:High glucose may promote the expression of RRBP1 in HepG2 cells by upregulating the level of transcription factor E2F1,and then enhance the proliferation,migration and invasion ability of HepG2 cells by regulating cell cycle and promoting DNA repair.In this study,we found that the E2F1/RRBP1 pathway may be one of the mechanisms by which high glucose promotes the development of HCC,and is expected to be a new target for the treatment of HCC complicated with diabetes.