Autophagy Upregulated HERC3 Expression To Activate TGF-β Pathway And Promote EMT And Chemoradiotherapy Resistance In Glioblastoma

Glioma is the most common primary central nervous system(CNS)tumor,accounts for about 70%of the CNS primary tumors.According to the histological classification of WHO(2007),glioma can be divided into 4 grades,of which grade Ⅰ/Ⅱ are low-grade gliomas,and grade Ⅲ/Ⅳ are high-grade gliomas,also known as malignant gliomas.Glioblastoma multiform(GBM)as the grade IV glioma,that is highly malignant and diffcult to treat,and its prognosis is poor.Despite the National Comprehensive Cancer Network recommended postoperative radiotherapy in combination with temozolomide(TMZ)chemotherapy(Stupp regimen)as a standard treatment for GBM,it can only improve the overall survival time of patients in 2 months.TMZ was reported to play the role of GBM chemotherapy by inducing autophagy.Our previous studies have confirmed that after treating with TMZ,some autophagic cells survive for a short time,and induced the occurrence of EMT to promote the migration and invasion of surviving cells,and mediated chemoradiotherapy resistance.In this study,we use TMZ to induce autophagy of glioblastoma and found that autophagy can significant upregulate the expression of ubiquitin ligase HERC3,which promotes the ubiquitination degradation of SMAD7(I-SMAD)through the autolysosome pathway and activates the TGF-β pathway finally induces the EMT in GBM cells,which provides insights into a better understanding of the mechanism of chemoradiation resistance of GBM.Chapter 1.Related proteomic analysis in autophagic glioblastoma cellsMethods:U87MG cells was treated with 200nM Rapamycin for 6h,8h,12h,24h and 48h,and then these GBM cell lysates was followed by Isobaric Tags for Relative and Absolute Quantification(iTRAQ)proteomic analysis.Results:The iTRAQ proteomics analysis showed that ubiquitin ligase HERC3,PAI1 and SMAD3(receptor-regulated SMAD,R-SMAD,A important role in TGF-βpathway)were significantly overexpressed,but the downregulation of SMAD7(the inhibitory proteins of TGF-β pathway).Chapter 2.Related proteomic analysis in autophagic glioblastoma cellsMethods:Immunohistochemistry and Western blot were used to detect the expression of HERC3 of the GBM tissue microarray,tumor specimens and 5 pairs of GBM primary cell lines.Survival analysis of the data from The Cancer Genome Atlas(TCGA)and follow-up data of 110 patients with GBM in the department of neurosurgery of Nanfang hospital to study the correlation between the prognosis of GBM patients and HERC3.Results:The expression of HERC3 is positively associated with the malignancy and t he invasion of GBM.Chapter 3.The biological behavior of HERC3 in glioblastoma cells.Methods:After the GBM cells were treated with TMZ,RAPA and LG for different time periods,the expression of HERC3 was verified by Western blot and immunofluorescence.Based on the above results,the expression of HERC3 and morphological changes of GBM cells were studied by immunocytochemistry at the peak of HERC3 expression.After inhibiting the autophagy of GBM cells,the expression of HERC3 and TGF-β pathway-related protein expression was verified byWestern blot.Chosing two cells with different HERC3 expression background(HERC3 was highly expressed in LN229,and T98G with low expression of HERC3),respectively,interference and overexpression of HERC3 by lentivirus,The characteristic variations and TGF-β pathway activation of LN229 and T98G were assessed by Western blot,wound healing assay and Transwell chamber invasion assay.Results:Western results verified the upregulation of HERC3 expression by TMZ,Rapa and LG treatment in GBM cells,and the expression peak was at 12h(TMZ),16h(Rapa)and 12h(LG)after treatment,which was consistent with the result of iTRAQ analysis.Immunocytochemistry and immunofluorescence at 12h showed that the expression of HERC3 was significantly upregulated and distributes in both cytoplasm and nuclei,and with the longest cellular pseudopod under microscopy.Western blot analysis showed 3-MA,CQ and si-ATG7 significant suppressed the expression of HERC3 induced by autophagy,Meanwhile,upregulated SMAD7,and inhibited the TGF-β signaling pathway.After HERC3 was knocked down,the expression of SMAD7 was upregulated,TGF-β signaling pathway and EMT were inhibited in LN229 cells.In contrast,HERC3 overexpression downregulated the expression of SMAD7,upregulated the EMT and activated the TGF-β signaling pathway in T98G cells.Chapter 4.HERC3 mediated TGF-β pathway activationMethods:SBE luciferase reporter plasmids were constructed for Luciferase reporter assay,and PAII and NOTCHI primers were synthesized for QRT-PCR to detect the effect of HERC3 on TGF-β pathway.The effect of HERC3 on the TGF-β pathway at the protein level was examined by Western blot.Results:Luciferase reporter gene assay,QRT-PCR,Western blot confirmed that HERC3 can mediate the upregulation of TGF-β pathway downstream target genes,and induce GBM cells EMT.Chapter 5.HERC3 induces SMAD7 ubiquitination and degradation byautophagy lysosomesMethods:Myc-HERC3,Flag-SMAD7 and HA-Ub plasmids were constructed and transfected into HEK293T cells,cycloheximide(CHX)was added to inhibit the synthesis of protein,the expression of Flag-SMAD7 was detected by Western blot.Gradient expression of Myc-HERC3 in HEK293T,and Flag-SMAD7 was detected by Western blot.HERC3 was overexpressed in T98G cells,the ubiquitination of endogenous SMAD7 was detected by Co-immunoprecipitation.And HEK293T cells with Myc-HERC3,Flag-SMAD7 and HA-Ub Co-transfection was used to confirm the ubiquitination of exogenous SMAD7 by Co-immunoprecipitation.LN229 cells were treated with autophagy inhibitor 3-MA,chloroquine(CQ)and proteasome inhibitor MG 132,and the expression of SMAD7 was detected by Western blot.Result:HERC3 downregulated the expression of SMAD7 in the time-dependent manner,and the decreased level of SMAD7 was also dependent on the dose of HERC3.The Co-immunoprecipitation and Western blot analysis indicated that HERC3 significantly increased the ubiquitination of endogenous and exogenous SMAD7.MG 132 slightly increased the expression of SMAD7 with Rapamycin treatment,while autophagic inhibitor,3-MA and CQ significantly changeover the SMAD7 level inhibited by Rapamycin.In summary,HERC3 induces the degradation of SMAD7 ubiquitination and degrades by autophagic lysosomes rather than the proteasome.