HBX-modulated MYH9/GSK3β/β-catenin-c-Jun Positive Feedback Loop Promotes Stemness And Chemoresistance Of Hepatocellular Carcinoma

BackgroundsHepatocellular carcinoma(HCC)is currently one of the most common causes of cancer-related death and one of the most commonly diagnosed cancers.Cancer stem cells(CSCs)are responsible for cancer recurrence and metastasis,and are related to poor prognosis in patients with HCC.MYH9 is thought to play a critical role in cancer progression.However,the role of MYH9 in HCC is yet to be investigated,and the association between MYH9 and HCC stemness remains unclear.Contents and methods1.To identify the proteins interacting with Hepatitis B viral X(HBX).(1)Coimmunoprecipitation(Co-IP)assay combined with mass spectrometry-based quantitative proteomics were adopted to identify the interactive proteins of HBX.(2)The colocalizion of the interactive proteins in HCC cells were detected by immunofluorescence and confocal microscopy.2.To explore biological function of MYH9 in HCC and the molecular mechanism involved.(1)The effect of MYH9 on stemness,migration and invasion,proliferation and chemoresistance of HCC cell was analyzed by hepatosphere formation assay,flow cytometry analysis,migration,invasion and wound healing assay,MTT assay,colony-formation assay,EdU incorporation assay and tumor xenograft study in vitro and in vivo.(2)Western blot analysis was adopted to determine the expression of Sox2,Oct4,Nanog,CD44,ABCG2,ABCB1,E-Ca,N-Ca,Vimentin,Slug,c-Jun,c-Myc,CCND1,β-catenin and GSK3β in HCC cells.3.To study the relationship between MYH9 and GSK3β,and the molecular mechanism that HBX and MYH9 activate the Wnt/β-catenin signaling.(1)Co-IP was applied to test the interaction between GSK3β,APC,Axin1,β-catenin and MYH9 in HCC cells.(2)Cycloheximide(CHX)chase assay was performed to investigate the effect of HBX and MYH9 on the the stability of GSK3β,APC,Axinl and β-catenin in HCC cells.(3)Co-IP was applied to observe the effect of HBX and MYH9 on the interaction between β-catenin,Ubiquitin,TRAF6 and GSK3β in HCC cells,and figure out the ubiquitination site in GSK3β.(4)The effect of GSK3β on stemness,migration and invasion,proliferation and chemoresistance of HCC cells was analyzed by flow cytometry analysis,migration and invasion assay,and EdU incorporation assay in vitro.Western blot analysis was adopted to determine the expression of Sox2,Oct4,Nanog,CD44,ABCG2,ABCB1,E-Ca,N-Ca,Vimentin,Slug,c-Jun,c-Myc,CCND1,β-catenin and GSK3β in HCC cells.4.To investigate how HBX promotes MYH9 expression and how MYH9 enhances Ubiquitin expression.(1)MYH9 and Ubiquitin might be transcriptionally regulated by c-Jun based on the ChIP-seq binding peaks in conjunction with UCSC,PROMO and JASPAR bioinformatics software.(2)Realtime-PCR and western-blot were used to reveal the effect of c-Jun on MYH9 and Ubiquitin expression in HCC cells.(3)Electrophoretic mobility shift assay(EMS A)was performed to identify whether the binding sites of c-Jun was functional.(4)Chromatin immunoprecipitation(ChIP)assay was performed to confirm the binding of c-Jun in the transcription regulatory region of MYH9 and Ubiquitin.(5)Luciferase reporter assay was performed to verify the acceleration of MYH9 and Ubiquitin transcription by c-Jun.5.To analyze the relationship between MYH9 and GSK3β expression,and the influence on patients prognosis in HCC.(1)Bioinformatic analysis was performed to analyze MYH9 and GSK3βexpression in HCC tissues and paracarcinoma tissues,and their relationship was further explored.(2)Immunohistochemistry(IHC)was performed to detect the expression of MYH9 and GSK3β expression in HCC tissues and paracarcinoma tissues,and their relationship was further explored.(3)Univariate and multivariate survival analysis were conducted to detect the influence of MYH9 on patients prognosis in HCC.Results(1)HBX interacts with MYH9 to activate Wnt/β-catenin signaling and promotes stemness,migration and invasion,proliferation and chemoresistance of HCC cells.(2)MYH9 interacts with GSK3β and degrades its expression by ubiquitination,resulting in the activation of Wnt/β-catenin signaling.(3)HBX enhances stemness,migration and invasion,proliferation and chemoresistance of HCC cells through the MYH9/c-Jun positive feedback loop.(4)HBX enhances stemness,migration and invasion,proliferation and chemoresistance of HCC cells through the MYH9/c-Jun/Ubiquitin-mediated ubiquitination of GSK3β.(5)MYH9 expression was elevated in HBV-positive HCC compared to that in HBV-negative HCC,and MYH9 expression was negatively associated with GSK3β expression.High MYH9 expression predicts poor prognosis for HCC patients.ConclusionsMYH9 plays a oncogenetic role in HCC.HBX activates Wnt signaling through MYH9/GSK3 β/β-catenin-c-Jun positive feedback loop to promote stemness,migration and invasion,proliferation and chemoresistance of HCC cells.