The Clinical Significance And Functional Mechanism Of Human Augmin Complex Submit 3(Haus3)in Hepatocellular Carcinoma

Backgrounds:Hepatocellular carcinoma(HCC)is a kind of the most prevalent neoplasm.A large population of HBV infected patients were reported in China,which accounts for more than 50%newly diagnosed HCC patients in the world.HCC is characterized by highly malignant and fast progressing,and the manifestation were usually found in a late stage of the disease.Liver resection and liver transplantation were recommended as the most efficient option for the curative therapy of HCC.However,HCC was still reported with a high recurrence and a poor survival rate.Efficient prognostic biomarker is still in need,which could also be potential therapeutic targets for HCC.Aim:This research aims to study the differential expressed proteins between HCC tissues and the paired tumor adjacent tissues using isobaric tags for relative and absolute quantitation(iTRAQ)with 2D LC-MS/MS methods.The identified protein Human augmin complex submit 3(Haus3)were explored for expression pattern and the mechanism.We expect to find an effective and efficient prognostic biomarker and a candidate therapeutic target for HCC.Methods:1.15 pairs of HCC and paired tumor adjacent tissues were collected.ITRAQ with 2D LC-MS/MS methods were applied to identify the differently expressed proteins.TCGA-LICH mRNA-seq dataset were set as cohort l,which were used to validate the expression and the prognostic significance of Haus3.137 primary HCC tumor samples and paired adjacent tissues were collected to establish cohort 2.Qualitative Real time PCR(qRT-PCR),western blot and immunochemistry(IHC)were applied to analyze the expression and the clinical significance of Haus3.2.Human HCC cell line SMMC-7721 and Bel-7402 were applied in this study.siRNA and lenti-virus were used for knockdown and overexpression of Haus3.Flow cytometry(FCM)were used to detect cell cycle and cell apoptosis,cell growth were detected by CCK-8 assay and xenograft tumor model.To further exploring the mechanism of Haus3 regulating G2/M phrase transition,correlation analysis were performed on website GEPIA in the TCGA-LIHC mRNA-seq dataset.Western blot were applied to check protein expression and the phosphorylation,Immunofluorescence(IF)assay were applied to detect the relationship of Haus3 with spindle and centrosome related proteins.Results:1.ITRAQ with 2D LC-MS/MS identified 40 differently expressed proteins,including 30 proteins high expressed in tumor tissue and 10 proteins low expressed in tumor tissues.Human augmin complex submit 3(Haus3)was selected for further research.Haus3 was proved high expressed in 50 paired tumor tissues comparing with tumor adjacent tissues,in the mRNA-seq dataset from TCGA-LIHC(p<0.0001).We then explore the expression of Haus3 in 137 paired HCC tumor tissues and tumor adjacent tissues.Western blot,qRT-PCR and IHC found the consistent results.Correlation analysis with clinic pathological parameters showed high Haus3 correlated with larger tumor size(p=0.025)and more tumor lesions(p＝0.004).Kaplan-Meier analysis showed elevated levels of Haus3 in tumor tissues correlated with poor tumor prognosis.High Haus3 and AFP≥20ng/ml are independent prognostic factors for HCC patients underwent liver resection.Additionally,in the subgroup of HCC patients with AFP≥20ng/ml,high Haus3 is still have a significant relationship with poor prognosis(p=0.001).2.Stable over-expression of Haus3 accelerates the HCC cell growth.Haus3 knock-down decreased the HCC cell growth and xenograft growth.Effect of Haus3 disruption on cell apoptand osis was not observed with statistical difference.G2/M phrase cell percentage were significantly increased in Haus3 knock-down HCC cell lines and decreased in Haus3 over-expression cell lines.To further understand the mechanism of Haus3 regulating G2/M phrase transition,we analyzed the correlation of Haus3 and G2/M phrase regulators and found the mRNA level of Haus3 showed positive relationship with PLK1,cdc25c,cdkl,CCNB1(R=0.6,0.5,0.6,0.54).Western blot showed Hans3 knock-down decrease the phosphorylation of PLK1,and the expression of cdc25c,but increase the phosphorylation of cdkl and expression of cyclin B1.Immunofluorescence and western blot assay showed Haus3 knock-down decrease the expression of centrosome related protein(a-tubulin)and spindle related protein(γ-tubulin).Conclusion:1.Applied with proteomics techniques,we found 40 differentially expressed in HCC tissues and tumor adjacent tissues.Comparing with the paired tumor adjacent tissue,Haus3 was found high expressed in HCC tumor tissues,and was significantly related to the tumor size and tumor lesions.High Haus3 is an independent early prognostic risk factor.2.Haus3 regulates HCC cell G2/M phrase transition through the phosphorylation of PLK1 and the activity of cdk1/cylin B1 complex.It also controls cell enter M phrase through regulating the expression of spindle related proteins3.Haus3 is a promising prognostic biomarker for HCC patients undergoing liver resection.