Enhancement Of PI3K/AKT/HIF1α-VEGF Pathway Via Transcriptional Activation Of SCARB1 By SREBP2 Promotes Malignant Biological Behavior Of Hepatocellular Carcinoma

Background and Objective:Hepatocellular carcinoma(HCC)is characterized by high incidence and mortality rates,and most patients have already developed into the middle or late stages once diagnosed,losing the chance for curative treatment such as transplantation,resection,or ablation.HCC also has a high recurrence rate and poor long-term prognosis.Therefore,identifying molecular markers for early diagnosis,recurrence monitoring,and survival prediction of HCC is crucial for patient stratification and optimizing medical intervention.The low early diagnosis rate,high recurrence rate,and poor prognosis of HCC are mainly attributed to the high proliferative activity of tumor cells,local infiltration,and high rates of distant metastasis.Hypoxia is a intrinsic feature of solid tumors.Previous studies have recognized the pivotal role of hypoxia in tumor angiogenesis,cell proliferation,differentiation,and apoptosis.Further research has shown that hypoxia affects cancer’s potential mechanisms,including DNA damage repair,cell metabolism,and tumor immunity,leading to transcriptional heterogeneity in cancer stem cell phenotype,invasiveness,and chemotherapy and radiation resistance.Investigating the molecular regulatory mechanism of HCC hypoxia-related genes will aid in understanding HCC’s malignant biological behavior.Therefore,we established a prognostic model of hypoxia-related genes in HCC patients using bioinformatics methods and selected scavenger receptor class B type I(SCARB1)gene that ranked first in the model for further research.SCARB1 is a protein-encoding gene that is mainly involved in cholesterol metabolism.Research has shown that SCARB1 is highly expressed in various malignant tumors and has a close relationship with tumors’ malignant biological behavior,making it a potential target for cancer treatment.In the preliminary experiment,we found that the expression of SCARB1 in HCC cancer tissue was higher than in adjacent tissue,and that SCARB1 and HIF-1α expression are positively correlated.However,how the upstream molecular regulates SCARB1 and how its downstream regulates HIF-1α are still unclear.Therefore,the purpose of this study is to explore whether SCARB1 enhances the PI3K/AKT/HIF-1α-VEGF pathway through regulating the transcription factor Sterol-regulatory element binding protein 2(SREBP2),promoting HCC’s malignant biological behavior.Methods:1.We established a prognostic model in hepatocellular carcinoma(HCC)patients based on the hypoxia-related genes using online bioinformatics methods and select the target gene SCARB1 for next studying.2.We used immunohistochemical staining to detect the expression level of SCARB1 in cancer tissues and adjacent tissues.Subsequently,we performed a analysis(Chisquare)to determine the association between SCARB1 expression and the clinicopathological characteristics of patients.Cox regression analysis was conducted to analyze the factors affecting the prognosis of HCC patients and to determine whether the expression level of SCARB1 could serve as an independent prognostic factor for HCC patients.3.We used colony formation,CCK8,Ed U,transwell test,scratch test,and flow cytometry to detect changes in the proliferation,invasion,migration,cell cycle,and apoptosis of liver cancer cells following SCARB1 inhibition and overexpression.4.We established subcutaneous tumor-bearing models of NOD-SCID mice and divided them into the control group,SCARB1 knockout group,and inhibitor(BLT-1)group to further explore the effect of the SCARB1 gene on tumor growth in mice.We also conducted immunohistochemical staining of tumor tissues in both groups to detect Ki-67,HIF-1α,and VEGF expression.5.For mechanism studying,we predicted through GESA enrichment analysis and PPI protein network analysis that SCARB1 may activate the PI3K/AKT signal pathway while promoting cholesterol uptake through HDL-mediated ways to promote the malignant biological behavior of HCC.We analyzed the cholesterol content in HCC patients with high SCARB1 expression using MRI.Western blot was used to analyze the expression changes of PPI3 K,P-AKT HIF-1α,and Ki-67 after SCARB1 inhibition and overexpression.6.We used Western blot to detect changes in SCARB1,PPI3 K,P-AKT,HIF-1α,and Ki-67 expression after SREBP2 inhibition and overexpression.The dual luciferase assay was performed to verify whether SREBP2 could activate the binding site of the SCARB1 promoter region.7.SCARB1 was overexpressed in the SREBP2-knockdown Huh7 cell line.Subsequently,the expression levels of SCARB1,PPI3 K,P-AKT,HIF-1α,and Ki-67 were detected,respectively.Results:1.Analysis of the HCC sample immunohistochemical staining,q RT-PCR,and Western blot revealed that the expression of SCARB1 was higher in cancer tissues/cells than in adjacent tissues/cells.Furthermore,the expression of SCARB1 was found to be negatively correlated with overall survival.In addition to SCARB1 expression,the pathological grade and clinical TNM stage were identified as independent risk factors for poor prognosis in HCC patients.2.The proliferation assay demonstrated that knockdown of SCARB1 inhibited cell proliferation,while overexpression of SCARB1 promoted cell proliferation.Transwell and scratch assays revealed that the ability of tumor cells to invade and migrate decreased after SCARB1 knockdown,while overexpression of SCARB1 increased these abilities.Cell cycle analyses showed that the proportion of cells in the S phase decreased after SCARB1 knockdown.Conversely,after overexpression of SCARB1,the proportion of cells in the S phase increased.The apoptosis assay showed that the apoptosis rate increased after SCARB1 knockdown,but decreased after overexpression.Moreover,in vivo studies showed that the weight and volume of tumor tissue significantly decreased,and the expression levels of Ki-67,HIF-1α,and VEGF decreased in immunohistochemical analyses after knockdown of SCARB1 or adding an inhibitor.3.Mechanistically,MRI revealed a significant increase in cholesterol content in HCC tumor patients with high SCARB1 expression.Western blot analyses demonstrated that,compared with the control group,PPI3 K,P-AKT,Ki-67,and HIF-1αdecreased after SCARB1 knockdown and increased after overexpression.These results suggested that SCARB1 activates the PI3K/AKT/HIF-1α-VEGF signal pathway while promoting cholesterol uptake through HDL-mediated ways in HCC cells.4.JASPAR and PROMO databases were used to jointly screen the transcription factor SREBP2,which was found to regulate SCARB1 expression.Knockdown of SREBP2 inhibited cell proliferation,invasion,and migration,and increased cell apoptosis,while overexpression of SREBP2 had the opposite effect.Furthermore,the expression levels of SCARB1,PPI3 K,P-AKT,HIF-1α,and Ki-67 were consistently decreased after knocking down SREBP2 and increased after its overexpression.Fluorescein assays confirmed that SREBP2 can bind to the upstream CTCAGGTGAT sequence of SCARB1 and transcriptionally activate SCARB1Conclusion:1.SCARB1 promotes the proliferation,invasion,migration,and inhibits cell apoptosis.2.The high expression of SCARB1 is an independent risk factor for poor prognosis in HCC.3.SCARB1 activates the PI3K/AKT/HIF-1α-VEGF signal pathway while promoting cholesterol uptake in HCC cells via HDL-mediated ways,thereby promoting the proliferation,invasion,and migration of HCC cells,and inhibiting tumor cell apoptosis.4.SREBP2 induces upregulation of SCARB1 expression in HCC through transcriptional activation.Ultimately,SREBP2 promotes the proliferation,invasion,and migration of HCC cells,and inhibits tumor cell apoptosis in a SCARB1-dependent manner.