Research On Mechanism Of STAMBPL1-mediated Lipid Metabolism In Hepatocellular Carcinoma Regulated By SREBP1

Objective: 1.To observe the expression of STAMBPL1 in Hepatocellular carcinoma(HCC).2.To study the effects of STAMBPL1 on the biological function of hepatocellular carcinoma cells and Wnt/β-catenin signaling pathway.3.To study the effect of SREBP1 activation of STAMBPL1 promoter on lipid metabolism of hepatocellular carcinoma cells.Methods: 1.Tissue samples were collected at Shengjing Hospital Affiliated to China Medical University,Shenyang,Liaoning Province,from September 2020 to August 2022.The expression of STAMBPL1 in 43 pairs of HCC tissues and adjacent tissues was analyzed by real-time quantitative PCR,and the expression of STAMBPL1 in the central position of cancer tissues was detected by immunohistochemistry in 31 groups of HCC tissues,and the correlation between STAMBPL1 expression and pathological indexes of patients was analyzed.The 6 pairs of samples with the most significant differences in Real-time PCR detection were selected,and the STAMBPL1 expression in cancer and adjacent tissues was detected by Western blot.2.Five hepatocellular cancer cell lines Huh-7,Hep 3B2.1-7,PLC/PRF/5,SNU-182,Li-7 were cultured in vitro.The expression level of STAMBPL1 in the cells was detected by Real-time PCR,and the expression level of STAMBPL1 was detected by Western blot.Cells with relatively high STAMBPL1 expression level were screened out,which was defined as H,and cells with relatively low STAMBPL1 expression level,which was defined as L,for subsequent experiments.3.Design and synthesize two STAMBPL1 interference sequences and their control to construct lentivirus vector to infect H cells.STAMBPL1 overexpression vector and its control lentivirus vector were constructed to infect L cells.After screening stable cell lines,the expression of STAMBPL1 was detected by Real-time PCR and Western blot.The proliferation of cells in each group was verified by CCK-8 at 0 h,24 h,48 h and 72 h.Cell proliferation was detected by clonal formation assay.The cell cycle of each group was detected by flow cytometry.Cell migration ability was detected by scratch test.Transwell assay was used to detect cell invasion ability.Lipid accumulation was verified by oil red O staining.The content of triglyceride in cells was detected by the kit.The expressions of FASN and SCD1 proteins were detected by Western blot.The cells in each group were transfected with TOPflash/FOPflash and p RL-TK reporter plasmid.48 h later,the activity of β-catenin was detected by double luciferase assay.The mRNA expressions of c-Myc,Cyclin D1 and Survivin were detected by Real-time PCR.4.In animal experiments,Stamb PL1-interfered or overexpressed stable cells were injected into the right subcutaneous area of 22-24 g nude mice(about 1×106 cells/piece).Tumor volume was measured every 3 days,and the mice were killed 30 days later,and tumor tissues were collected.H cells were divided into:(1)LV-NC-sh RNA(n=6),(2)LV-sh STAMBPL1-1(n=6),and(3)LV-sh STAMBPL1-2(n=6).L cells:(1)LV-vector(n=6),(2)LV-STAMBPL1 OE(n=6).The expressions of STAMBPL1 and Ki-67 in tumor tissues were detected by immunohistochemistry.Lipid accumulation was detected by oil red O staining.The expressions of Cyclin D1 and Survivin in tumor tissues were detected by Real-time PCR.5.A SREBP1 interfering plasmid and its negative control,SREBP1 overexpression plasmid and its negative control were constructed and transfected into H cells.The expression levels of SREBP1 and STAMBPL1 were detected by Real-time PCR and Western blot.A luciferase reporter vector containing STAMBPL1 promoter sequence was constructed and co-transferred into H cells with SREBP1 overexpression plasmid.Luciferase activity was detected 48 h later.In H cells,CHIP verified SREBP1 binding to the STAMBPL1 promoter.Results: 1.The expression of STAMBPL1 was obvious in HCC clinical samples.Patients with high STAMBPL1 expression had tumor diameter ≥10cm and high TNM stage more than patients with low STAMBPL1 expression level.2.The mRNA and protein expressions of STAMBPL1 were the highest in SNU-182 cells,and the lowest in Hep 3B2.1-7 cells.STAMBPL1 silencing inhibited the proliferation of SNU-182 cells,while STAMBPL1 overexpression promoted the proliferation of Hep 3B2.1-7 cells.STAMBPL1 silencing inhibited cell clonogenesis,while STAMBPL1 overexpression enhanced cell clonogenesis.STAMBPL1 silencing increased the percentage of G1 phase cells and decreased the percentage of S phase cells,while STAMBPL1 overexpression decreased the percentage of G1 phase cells and increased the percentage of S phase cells.STAMBPL1 silencing decreased cell mobility and the number of cell invasions,while STAMBPL1 overexpression increased cell mobility and the number of cell invasions.3.STAMBPL1 silencing decreased TOP/FOP transcriptional activity,while STAMBPL1 overexpression increased TOP/FOP transcriptional activity.STAMBPL1 silencing inhibited the expression of c-Myc,Cyclin D1,Survivin mRNA of downstream genes in Wnt/β-catenin signaling pathway,while STAMBPL1 overexpression promoted the expression of c-Myc,Cyclin D1,Survivin mRNA.4.In vivo experiments showed that STAMBPL1 silting inhibited tumor growth and Ki-67 expression in hepatocellular carcinoma,Cyclin D1 and Survivin mRNA expressions were significantly down-regulated,while STAMBPL1 overexpression promoted tumor growth and Ki-67 expression in hepatocellular carcinoma.Cyclin D1 and Survivin mRNA expressions were significantly increased.5.STAMBPL1 silencing inhibited lipid accumulation in tumor tissues,while STAMBPL1 overexpression promoted lipid accumulation in tumor tissues.Overexpression of STAMBPL1 promoted lipid generation in Hep 3B2.1-7 cells.The content of triglyceride in Hep 3B2.1-7 cells overexpressed by STAMBPL1 was significantly increased.Overexpression of STAMBPL1 promoted the expressions of key enzymes FASN and SCD1 for de facto fatty acid synthesis in Hep 3B2.1-7 cells.6.In hepatocellular carcinoma,SREBP1 binds to STAMBPL1 promoter and promotes STAMBPL1 expression by enhancing STAMBPL1 transcriptional activity.Conclusion: In this study,GEO database and clinical hepatocellular carcinoma tissues were used to find that the up-regulation of STAMBPL1 was closely related to the gene’s special involvement in cell cycle,homologous recombination,micro RNAs in cancer,pyrimidine metabolism and other phenotypes.The protein and mRNA expressions of STAMBPL1 in hepatocellular carcinoma tissues were significantly higher than those in adjacent tissues,and STAMBPL1 expression was significantly correlated with tumor diameter and TNM staging indexes,which had clinical significance.Further studies using nude mice and hepatocellular carcinoma cell lines have shown that STAMBPL1 is involved in hepatocellular cancer cell cycle,cell proliferation,cell migration,and invasion ability.These phenotypes are regulated with the knockdown or overexpression of STAMBPL1 via the Wnt/β-catenin signaling pathway.After further investigation,it was found that the up-regulation of STAMBPL1 promoted lipid accumulation in hepatocellular carcinoma tumor tissues,and thus up-regulated the expression levels of FASN and SCD1 proteins.This is mainly through SREBP1 binding to STAMBPL1 promoter,which further activates STAMBPL1 transcriptional activity.