TMUB1 Negatively Regulates Hepatocellular Carcinoma Cells Proliferation Via Regulating STAT1

BackgroundHepatocellular carcinoma?Hepatocellular carcinoma,HCC?is a common malignant tumor.There are a variety of treatment methods for HCC,such as surgical resection,liver transplantation,radiotherapy and chemotherapy,radiofrequency ablation,molecular targeted therapy and so on.However,the 5-year survival rate of HCC is still less than 12%1.About 748,300 new cases of HCC and 695,900 deaths are reported worldwide every year,half of which are in China.In recent years,the morbidity and mortality of HCC are on the rise in China.Due to the high incidence and mortality of HCC,the research on the prevention and treatment of HCC has been widely carried out all over the world.Currently,the pathogenesis of HCC remained unclear,so finding a definitive and effective treatment is difficult.Finding effective treatments through the in-depth study of the pathogenesis of HCC thus remains the focus of current HCC research.TMUB1 was first reported in 2005,and its expression in hepatocytes was significantly increased after hepatectomy.The TMUB1 gene is located at the 4q11 position on chromosome 4 in rats?located on chromosome 7 in humans?and has a total length of 1381bp.The TMUB1 protein contains 245 amino acids and includes a nuclear export signal?NES?at the amino terminus and a region containing a similar ubiquitin structure?UBL;121-175 aa?.TMUB1 is a transmembrane protein,acts as a ubiquitin-like protein participated in the ERAD?endoplasmic reticulum-associated protein degradation?pathway.As a nuclear-cytoplasmic shuttle protein,TMUB1 is released from the membrane and shuttles between the cytoplasm and the nucleus to exert its biological functions.Early studies indicated that TMUB1 has an inhibitory effect on the proliferation of normal hepatocytes.IL-6 and miR-27a/b can regulate the expression of TMUB1,as well as its posttranscriptional modification,thereby promoting its inhibition of hepatocyte proliferation.TMUB1 regulates hepatocyte regeneration by inhibiting STAT3phosphorylation and interfering with CAML binding to cyclophilin B.TMUB1 also acts as an important factor in the p14^arf-p53 pathway and participates in inhibiting tumorigenesis by repairing DNA.However,whether TMUB1 plays a role in hepatocellular carcinoma is not clear.Both STAT3 and STAT1 are important members of the JAK/STAT pathway,and they antagonize each other.The possible mechanisms include:1)inhition of expression of each other;2)phosphorylation competition,affecting downstream gene expression;3)interfering with the formation of functional dimers.Previous studies have found that TMUB1 have effect on STST3 function,so we conclude that TMUB1 may have an effect on the biological characteristics of HCC by regulating STAT1 fuction.Methods1.Approved by the Ethics Committee of the Army Medical University and Army Medical Center,we gathered 132 paraffin specimens from HCC patients who have undergone partial hepatectomy at Army Medical Center between January 2013 and December 2014.Clinical pathological including gender,age,tumor size,tumor cells differentiation,TNM stage,extrahepatic metastasis,vascular invasion,lymph node metastasis and other patient-related information.We followed the patients until January 1,2019,and recorded relapse and deaths.2.Immunohistochemical staining was used to detect the expression of STAT1 in liver cancer tissue specimens,and we analyzed the correlation between the expression of STAT1and TMUB1,and the relationship between the co-expression of TMUB1/STAT1 and the pathological characteristics of HCC tissues and the prognosis of patients.3.We cultured HCC cell lines Hep G2,Huh7,MHCC97h,LM3,SMMC-7721 and normal liver cell lines LO2.Western-blot and qPCR were used to detect TMUB1 expression in different cell lines,and then we selected Huh7 cells which had higher expression of TMUB1 to transfect with interference plasmid,and MCHH97h cells which had lower expression of TMUB1 to infected with overexpression plasmid,CCK-8 was used to detect cell proliferation ability,and Transwell test was used to detect cell migration and invasion ability.4.We changed the expression of TMUB1 with interfered and overexpression plasmid,western-blot and qPRC detected the expression of STAT1 and CCND1.Huh7 cells were transfected with TMUB1 interference plasmid and STAT1 overexpression plasmid simultaneously,MHCC97 cells were transfected with TMUB1 overexpression plasmid and STAT1 interference plasmid simultaneously,western-blot was used to detect TMUB1,STAT1,CCND1 expression,and EdU was used to detect cell proliferation ability.5.The bioinformatics method was used to screen TMUB1 and STAT1 related miRNAs networks through various online analysis software and database.The miRNAs were used as the bridge nodes to screen mRNAs and lnc RNAs.TMUB1,TMUB1+STAT1 ce RNAs regulatory networks were constructed to predict the mechanism of TMUB1 in HCC and the regulatory mechanism of TMUB1 on STAT1.Result1.TMUB1 is negatively correlated with HCC pathological malignancy and low expression of TMUB1 indicates poor prognosis.The tumor sizes were significantly larger in TMUB1 low expression group?6.8±4.0 cm?than those in TMUB1 high expression group?5.2±2.5 cm??P<0.001?.The HCC differentiation in TMUB1 high expression group was better than those in TMUB1 low expression group?P=0.021?.The proportion of multiple tumors in TMUB1 low expression group?14.4%,n=19?was significantly higher than that in TMUB1 high expression group?9.1%,n=12??P=0.011?.Regarding TNM stage,there were significantly more patients with stage III?6.8%?and stage IV?2.3%?in TMUB1 low expression group than patients with stage III?1.5%?and stage IV?0.8%?in TMUB1 high expression groups?P=0.004?.But the vascular invasion or metastasis between the two groups had no statistically significant differences.In addition,the results of postoperative follow-up showed that the tumor-free survival time was obviously lower in TMUB1 low expression group than in TMUB1 high expression group?P=0.014?,and the overall survival time was also obviously lower in TMUB1 low expression group than in TMUB1 high expression group?P=0.001?.The mean postoperative survival time was lower in TMUB1low expression group?828±102 days?than in TMUB1 high expression group?1180±150days?.2.STAT1 expression and TMUB1 expression were positively correlated,P<0.0001,pearson r=0.47.The tumor size of the TMUB1^lowSTAT1^low group?7.8±4.4?cm??was significantly larger than that of the TMUB1^highSTAT1^low?6.7±3.0?cm??,TMUB1^LowSTAT1^high?5.3±2.7?cm??,and TMUB1^highSTAT1^high?4.7±2.0?cm??groups?p<0.001?.The proportion of multiple tumors in the TMUB1^lowSTAT1^low group?8.3%,n=11?was significantly higher than that in the TMUB1^high/STAT1^high?5.3%,n=7?,TMUB1^highSTAT1^low?3.0%,n=4?and TMUB1^lowSTAT1^high groups?6.1%,n=8?.Regarding TNM stage and HCC differentiation,the TMUB1^highSTAT1^highigh group was better than those of TMUB1^highSTAT1^low,TMUB1^LowSTAT1^high and TMUB1^lowSTAT1^low groups?P=0.016?.But there were no statistically significant differences in the four groups for vascular invasion or metastasis.The overall survival of the TMUB1^highSTAT1^high group were longer than those of TMUB1^highSTAT1^low,TMUB1^LowSTAT1^high and TMUB1^lowSTAT1^low groups?P=0.004?.3.TMUB1 suppressed HCC proliferation but had no significant effect on HCC metastasis.Proliferation was suppressed in TMUB1-overexpressing MHCC97h cells s,while proliferation was significantly enhanced in TMUB1-knockdown Huh7 cells.The migration and invasion had no significantly changes in two groups.4.TMUB1 regulates the expression of STAT1.When TMUB1 expression was upregulated,STAT1 m RNA and protein levels were both upregulated compared with control MHCC97h cells.Cyclin D1 m RNA and protein levels were both downregulated compared with control MHCC97h cells.When TMUB1 expression was downregulated,STAT1 m RNA and protein expression levels were both downregulated compared with control Huh7 cells.Cyclin D1 mRNA and protein levels were upregulated compared with control Huh7 cells.5.MHCC97h cells proliferation were significantly suppressed by overexpression of TMUB1 and were rescued by STAT1 interference plasmid.In contrast,the promotive effect of TMUB1 knockdown on proliferation in Huh7 cells was abo lished by STAT1overexpression plasmid.These data demonstrate that TMUB1 suppresses HCC proliferation vis regulating expression of STAT1 at least partially.6.We found that TMUB1 mRNA was not significantly down-regulated in the HCC tissue through bioinformatics analysis,which indicated that TMUB1 mRNA might be inhibited by post-transcriptional regulators.We screened miR-615-2p,miR-423-5p,miR-222-3p and miR-25-3p which were up-regulated in HCC,and constructed the TMUB1ceRNA regulatory networks.Conclusion:1.TMUB1 is negatively correlated with HCC pathological malignancy and low expression of TMUB1 indicates poor prognosis.2.TMUB1 suppressed HCC proliferation but had no significant effect on HCC metastasis.3.TMUB1 suppresses HCC proliferation via regulating STAT1 signaling.4.TMUB1 might be inhibited in HC by post-transcriptional inhibition of miRNA.