DNAJC10 Promotes Glioblastoma Proliferation And Invasion Through TGFβ1 Signaling Pathway

Malignant glioma is the most common malignant tumor in the central nervous system(CNS),accounting for more than 70% of primary CNS malignant tumors,and is characterized by high recurrence rate,resistance to chemotherapy and radiotherapy,and high mortality after tumor resection.In recent years,the worldwide medical technology of diagnosis and treatment is rapidly growing,and the treatment of intracranial glioma patients gradually tends individualized and precise,but for most patients with surgical resection combined with radiation and chemotherapy treatment is still the mainstream.However,the clinical effect of treatment is still not very satisfied.Therefore,the exploration and development of novel and effective therapeutic targets for glioma has become an important and urgent research topic.ERdj5 encoded by Dna J Heat Shock Protein Family(Hsp40)C10(DNAJC10)gene is an endoplasmic co-chaperone protein in eukaryotic cells.It is an important component of the endoplasmic reticulum associated degradation complex and is involved in the recognition and degradation of misfolded proteins within cells,reducing incorrect disulfide bond binding in misfolded glycoproteins.In recent years,many tumor studies have shown that HSPs play an important role in the occurrence and development of tumors,and abnormal expression of HSPs has been found in a variety of cancers,including lung cancer,gastric cancer,breast cancer,prostate cancer and melanoma.As stress response molecules,heat shock protein genes are involved in a variety of intracellular physiological processes and pathways,which are very important for the occurrence,development and clinical progress of cancers.It has been found that knockdown of DNAJC10 expression can weaken the resistance of glioma cells and melanoma cells to endoplasmic reticulum stress inducer toxic carrolactone and anti-tumor drug trefenamide.However,the role of DNAJC10 in glioma malignant progression and its underlying mechanism remains unclear.It has been reported in the early relevant literature that DNAJC10 plays an important role in exocrine cytokines and protein degradation complexes,and the secretion of cytokines and the degradation of abnormal proteins are the most important function of tumor cells to resist to abominable environment.According to the result of mass spectrometry in the related literature shows that TGF beta is one of the important substrates of DNAJC10 protein.However,whether DNAJC10 promotes the malignant progression of glioma in vivo and in vitro by regulating TGFβ signaling pathway and its detailed molecular mechanism is not yet clear.In this study,transcriptome data from large glioma databases,such as TCGA and CGGA,were mined to determine the transcriptome expression level of DNAJC10 in glioma samples of all grades and its correlation with the overall prognosis of glioma patients.The expression levels of DNAJC10 protein and m RNA in WHO grade II-IV glioma and normal control brain tissues were detected by real-time fluorescence quantitative PCR and western blot.In order to study the effect of DNAJC10 on malignant progression of glioma cells,we constructed DNAJC10knock-down lentivirus(sh-DNAJC10),as well as three site mutation(3*-CXXA-)lentivirus(OE-DNAJC10-4*-CXXA-flag)in DNAJC10 molecule,and transfected them into glioma U87 and LN229 cell lines.The stably expressed sh-DNAJC10 glioma cell line was constructed and the corresponding expression level was verified.The effects of DNAJC10 on proliferation of U87 and LN229 glioma cells were detected by CCK-8 assay,monoclonal cell colony formation and Ed U assays.And the effects of DNAJC10 on migration and invasion ability of U87 and LN229 glioma cells were detected by wound healing and Transwell invasion assays.Protein immunoprecipitation was used to detect the binding of DNAJC10 to endogenous TGFβ1,and the expression and secretion changes of TGFβ1 after knockdown DNAJC10 were detected using western blot and ELISA assays.DNAJC10 knockdown was confirmed to affect the proliferation,migration and invasion ability of glioma cell lines by down-regulating TGFβ1 pathway.In addition,intracranial experiments in nude mice were performed to verify the carcinogenic effect of DNAJC10 in xenograft tumor experiment and the value of DNAJC10 in survival and prognosis evaluation of nude mice with tumor.In this study,we revealed that DNAJC10 was overexpressed in glioma specimens and that high expression of DNAJC10 was significantly associated with poor prognosis in glioma patients.We found that knockdown of DNAJC10 in glioma cell lines would significantly inhibite the proliferation,migration,and invasion of glioma cells.Reports indicated that TGFβ1 might be the substrate of DNAJC10,so the interaction between DNAJC10 and TGFβ1 was confirmed by immunoprecipitation,and the protein expression and secretion of TGFβ1 were inhibited by the knockdown of DNAJC10.We hypothesized that DNAJC10 may affect the proliferation,migration and invasion of glioma cells through the TGFβ1pathway.Recovery experiments showed that the proliferation,migration and invasion of DNAJC10 knockdown group were restored to original levels after the addition of exogenous TGFβ1,which was consistent with our expected results.In addition,intracranial tumor formation and immunohistochemistry experiments in nude mice showed that the survival time was significantly prolonged in DNAJC10 knockdown group,and the expression of tumor proliferation biomarker Ki-67 was significantly decreased,as well as the expression of TGFβ1.The results of this study suggest that DNAJC10 can enhance the proliferation,migration,invasion and growth of glioma cells by regulating TGFβ1 pathway.Therefore,we believe that DNAJC10 may be a potential target for clinical treatment of glioma.