Study On The Mechanism Of TRIP13 Promoting Tumor Proliferation And Drug Resistance And Its New Small Molecular Targeting Compounds

Objective:To explore the mechanism of proliferation and drug resistance of TRIP 13 in bladder cancer and the mechanism of drug targeting TRIP13.Methods:1)Through TCGA and GEP databases,the relationship between TRIP 13 and tumors was explored by analyzing tissue expression and survival curve in clinical data samples of cancer patients and normal control group.2)We constructed overexpression and knockdown of TRIP 13 T24 and J82 cell lines which were dectected by Western blot;MTT assay and clonogenic assay were used to detect cell proliferation;Cell cycle analysis was used to detect the effect of cell growth;For flow cytometry,cells were double-stained with APC-labelled AnnexinV and PI to detect apoptosis;Immunofluorescence was used to detect the effect of TRIP 13 on DNA damage;Western blot was used to detect the expression of MAD2,RAD50 and gamma H2AX proteins,and to elucidate the mechanism of TRIP 13 promoting tumor proliferation and drug resistance.3)MTT assay was used to detect the effect of VCP17 and VCP20 on the activity of tumor cells;Annexin-V APC/PI double staining was used to detect apoptotic cells,and Western blot was used to detect the expression of apoptotic proteins such as PARP and Casepase 3;Cell cycle distribution was detected by cell cycle assay,and cell cycle-related proteins such as Cyclin B1 were detected.The effects of VCP17 and VCP20 on osteoclast differentiation were investigated by TRAP staining,and the expression of various cytokines was detected by real-time fluorescent quantitative PCR.The effect of NF-κB signaling pathway was detected by Western blot;MTT assay was used to explore the possibility of combination of VCP17 and VCP20 with bortezomib.4)We constructed a mouse model of subcutaneous xenotransplantation and a mouse model of myeloma to detect the anti-tumor activity of VCP 17 and VCP20 in vivo.Results:1)The expression of TRIP13 in tumor tissue was significantly higher than that in normal control group(P<0.05).Survival curve analysis showed that the survival time of patients with high expression of TRIP 13 was significantly shortened(P=0.0118).Overexpression of TRIP 13 was associated with poor prognosis of bladder cancer.2)MTT assay showed that the proliferation rate of T24 and J82 cells in TRIP 13 knockdown group was slower than that in control group(P<0.05).The proliferation rate of T24 and J82 cells in TRIP 13 overexpression group was faster than that in control group(P<0.01).At the same time,the IC50 of TRIP 13 cells overexpressed by chemotherapy drugs(cisplatin and adriamycin)was higher than that in control group;The results of cloning experiment showed that the number of clones formed by over-expressed TRIP 13 cells was significantly higher than that of wild-type cells(P<0.05);the proportion of G2/M cells in over-expressed TRIP 13 cell lines increased significantly(P<0.05);flow cytometry showed that over-expressed TRIP 13 cell lines produced fewer apoptotic signals than the control cells;Western blot results showed that over-expressed TRIP 13 cells produced fewer apoptotic signals than control cells;Western blot results showed that the expression of MAD2 was decreased when TRIP 13 was overexpressed and increased when TRIP 13 was knocked down.At the same time,wild-type and overexpressed TRIP 13 cells treated with same drugs showed that the expression of yH2AX was decreased and RAD50 was increased in overexpressed TRIP13 cell lines;Immunofluorescence results showed that the fluorescence intensity of yH2AX was higher when TRIP 13 was knocked down than control group.These results suggest that TRIP13 promotes the proliferation and drug resistance of cancer cells by inhibiting SAC signal and DNA damage.3)MTT assay showed that VCP17 and VCP20 had good anti-tumor activity;Cell cycle assay showed that VCP17 and VCP20 treated cells blocked at G2/M phase(P<0.05),and Western blot showed that Cyclin B1 expression decreased;Flow cytometry showed that apoptotic signals were produced after drug treatment,and cleaved PARP and Caspase 3 were detected by Western blot;Western blot showed that the expression of TRIP 13 decreased,and the expression of NF-κB signaling pathway-related proteins p65 and IκBαdecreased,while the expression of p-p65 increased;TRAP staining showed that VCP17 and VCP20 could inhibit the differentiation of osteoclasts;Real-time fluorescence quantitative PCR showed that VCP17 inhibited the expression of IL-6 and TNF-a and increased the expression of VEGF,while VCP20 increased the expression of IL-6 and TNF-α;MTT results showed that the combination of VCP17 and VCP20 with bortezomib could increase the killing ability to tumors.4)In the mouse model of xenogeneic subcutaneous tumor transplantation,the volume of transplanted tumors in mice was significantly smaller than that in the control group(P<0.05),VCP17 and VCP20 could inhibit the growth of transplanted tumors in mice;In the mouse model of myeloma,we found that there was no significant difference in survival curve between VCP17 group and VCP20 group(Pvcp17-0.3071、Pvcp20=0.5846).Conclusions:1.TRIP 13 is overexpressed in bladder cancer patients and negatively correlated with prognosis.TRIP 13 plays a carcinogenic role in tumors by inhibiting spindle assembly checkpoint signals and DNA damage,activating DNA repair and promoting the proliferation and drug resistance of cancer cells.2.In vitro,VCP17 and VCP20 have good anti-tumor activity,induce apoptosis and block cell growth,and effectively inhibit TRIP 13 expression and NF-κB signaling pathway;In vivo,VCP17 and VCP20 also have some anti-tumor effects.