HSP90β Promotes Growth Of Hepatoma Cells Via Inhibiting Ubiquitination And Degradation Of YTHDF2

BackgroundLiver cancer ranks the sixth among common malignancies and its mortality rate ranks the third worldwide.Hepatocellular carcinoma(HCC)is one of the most malignant types among all liver cancers.Surgical resection,liver transplantation or local ablation are beneficial to the cure of early HCC.However,due to the insidious onset,rapid progression,high proliferation,invasion and metastasis of liver cancer,patients have progressed to the middle and advanced stages at the time of diagnosis and cannot be treated surgically.The prognosis of these patients is very poor,with palliative therapy including chemotherapy and radiotherapy can generally be performed,with five years survival rate of less than 6%.Therefore,there is an urgent need to elucidate the molecular mechanisms of HCC development and progression and to develop effective druggable targets for the treatment of advanced liver cancer in order to achieve the purpose of therapeutic intervention.It is well-known that tumor development and progression are influenced by epigenetic modifications.Post-transcriptional modifications have attracted much attention in biomedicine.In eukaryotic cells,m~6A is one of the most common as well as popular prevalent modifications of m RNA,and Writer、Eraser、Reader in m~6A are aberrantly expressed in a variety of cancers.Among them,Reader is a selective RNA-binding protein that plays a crucial role in tumor development,progression,metastasis,treatment resistance and recurrence.YTHDF2 is the firstly characterized functional protein and identified as a primary regulator of m RNA stability.An increasing number of studies had demonstrated that the YTHDF2 gene plays an oncogenic role in various cancers,such as HCC,prostate cancer,pancreatic cancer,etc.But so far,the research on YTHDF2 have primarily focused on the function of YTHDF2,while the research on the regulatory mechanisms of YTHDF2 itself is still unclear.ObjectiveThis study aims to investigate whether YTHDF2 is modified by ubiquitination and its related regulatory mechanism in HCC,and to further develop novel strategies to target YTHDF2 degradation and ultimately achieve the purpose of inhibiting tumor growth.This project intends to demonstrate that the ubiquitination regulation of YTHDF2 protein plays a key role in the occurrence and development of HCC by means of biological mass spectrometry,co-immunoprecipitation,western blot and immunofluorescence analysis and other means,elucidate the molecular regulation mechanism of YTHDF2 ubiquitination and degradation,and ultimately provide potential therapeutic strategies for the clinical treatment of liver cancer.Methods1.HCC cells were cultured and passaged.2.Detection of tumor cell viability:According to the experimental purpose,the liver cancer cells need to be processed,the MTS assay was used to determine the rate of cell survival.3.Cell colony formation assay:The tumor cells were treated according to the experimental requirements.The formation of cell colonies was observed using an inverted microscope.The numbers of colonies were also calculated.4.Co-immunoprecipitation and western blot techniques:According to experimental requirements,after corresponding treatment of liver cancer cells,the proteins were collected to detect the expression of the target protein in the tumor cells.5.Real-time fluorescence quantitative nucleic acid amplification detection technology(RT-q PCR):According to the operation guide of kit,and then use c DNA as template for amplification to detect the target gene and obtain the CT value.6.Immunofluorescence test:According to the purpose and requirements of the experiment,the tumor cells were treated,and immunofluorescence experiments were performed to detect the localization,interaction,and fluorescence of the target genes.7.Annexin V-FITC/PI staining assays:After treating the cells according to the experimental purpose,the cells were stained and the kinetic imaging of the cells was observed by an inverted fluorescence microscope.8.Transwell cell migration test:After the cells were treated according to the test purpose,the cells in the chamber were stained,and the cell migration was observed by an inverted microscope,and the migration rate of the tumor cells was calculated.9.Nude mouse transplantation experiment:BALB/C mice were inoculated with liver cancer cells on the back or under the armpit to construct a xenograft model,and randomly divided into two groups(the treatment group and the control group),according to the experimental requirements,regular drug treatment,observation the growth status of the transplanted tumor in nude mice,and the size and weight of the transplanted tumor and the weight was change of the nude mice was compared.Results1.YTHDF2 is regulated by the ubiquitin proteasome systemIn this study,we demonstrated that the proteasome inhibitor MG132 could lead to a remarkably increase in the ubiquitination level of YTHDF2 on two cell lines,Hep G2and Hep3B,by co-immunoprecipitation.The YTHDF2 interacting protein was then identified using Co-IP combined with biological mass spectrometry,and the interaction between YTHDF2 and HSP90βchaperones was found,indicating that HSP90βmay be a chaperone controlling the amount of YTHDF2 protein.2.YTHDF2,HSP90β,STUB1 interact in hepatocellular carcinomaWe identified YTHDF2 interacting proteins using Co-IP in combination with biomass spectroscopy and found that YTHDF2 interacts with HSP90βmolecular chaperones.Co-IP combined with western blot was also used to further to verify that HSP90β,YTHDF2,STUB1 could directly bind to each other in Hep G2 and Hep3B cells.By immunofluorescence combined with confocal laser scanning microscopy,we found that most of the binding of YTHDF2 to STUB1 occurred in the cytosol,and HSP90βalso mainly bound to STUB1 in the cytoplasm.In addition,after treating Hep G2 and Hep3B cells with RNAase,it was found that the binding of YTHDF2 to HSP90βand STUB1 was not significantly inhibited,indicating that its binding was not regulated by RNA.3.HSP90βmaintains the stability of YTHDF2 in hepatocellular carcinomaAfter treating Hep G2 and Hep3B cells with NVP-AUY922,an inhibitor of HSP90β,it was found that inhibiting the function of HSP90βcould down-regulate the protein expression of YTHDF2.Then,RT-q PCR analysis was performed,and it was found that the m RNA level of YTHDF2 did not change significantly after inhibiting the function of HSP90β.Subsequently,the protein levels of YTHDF2 were measured by analysis using CHX tracing experiments and western blot,and it was found that inhibition of HSP90βshortened the half-life of YTHDF2.Moreover,the reduction of YTHDF2 protein after inhibition of HSP90βwas significantly reduced by bortezomib.Inhibition of HSP90βincreased the total ubiquitination of YTHDF2 and K48-linked ubiquitination level of YTHDF2 by Co-IP combined with western blot.4.STUB1 mediates ubiquitination degradation of YTHDF2After treatment with STUB1 si RNA in Hep G2 cells,western blot showed the knockdown of STUB1 expression could up-regulate YTHDF2 protein level,while STUB1 overexpression could down-regulate YTHDF2 protein expression;then RT-q PCR analysis showed that there was no significant change in YTHDF2 m RNA level after knockdown of STUB1 expression.Subsequently,CHX tracking assay analysis to detect the protein levels of YTHDF2 using western blot and found that inhibition of STUB1 prolonged the half-life of YTHDF2.In addition,the use of bortezomib,a proteasome inhibitor,was found to reverse the down-regulation of YTHDF2 protein induced by STUB1 overexpression.Immunoprecipitation combined with western blot was used to examine the effect of STUB1 on the ubiquitination level of YTHDF2 in Hep G2 cells,and it was found that knockdown of STUB1 expression significantly reduced the total ubiquitination of YTHDF2 and K48-linked ubiquitination level of YTHDF2.5.HSP90β,STUB1,and YTHDF2 regulate proliferation and metastasis of HCCFirstly,MTS was used to detect the proliferation inhibition of Hep G2 and Hep3B cells by the knockdown of YTHDF2 and HSP90β,or inhibition of HSP90βfunction.In addition,colony formation assay showed that inhibition of HSP90βsignificantly inhibited the long-term proliferative ability of Hep G2 and Hep3B cells.Subsequently,transwell cell migration assay showed that the deletion of YTHDF2 and HSP90βimpaired the migration ability of Hep G2 and Hep3B cells,while the knockdown of STUB1 significantly increased the migration rate of Hep G2 and Hep3B cells.6.HSP90βinhibitors promoted the sensitivity of HCC to sorafenibHep G2 and Hep3B cells were treated with NVP-AUY922 in combination with sorafenib.Cell viability,apoptosis,and colony formation assays were performed.The results showed that HCC cell growth was significantly inhibited after the combination.Hep G2 cells were inoculated on nude mice to establish the in vivo models and treated with NVP-AUY922,sorafenib,and their combination intraperitoneally.We found that the HSP90βinhibitor in combination with sorafenib could significantly inhibit the growth of ectopic xenografts of HCC in vivo.The results of immunohistochemical assays made from some of harvested tumor tissues further showed that the level of Ki67was reduced after the combination.Conclusion1.HSP90βinteracts with STUB1 to positively and negatively regulate the ubiquitination modification and stability of YTHDF2,and ultimately regulate the growth,invasion and metastasis of HCC cells.2.HSP90βinhibitor can promote the sensitivity of hepatocellular carcinoma cells to sorafenib in vivo and in vitro.