MiR-34b-3p Represses The Chemoresistance Of Bladder Cancer Cells By Regulating The CCND2 And P2RY1 Genes

Chapter 1.The correlation between the expression of CCND2 and P2RY1 genes and the expression of miR-34b-3p genes in bladder cancer chemotherapy-sensitive and tolerant cellsPurpose:To investigate the correlation between the expression level of CCND2 and P2RY1genes and the expression level of miR-34b-3p in bladder cancer chemotherapeutic drug sensitive cell line?5637?and bladder cancer chemotherapeutic drug tolerant cell line?EJ?,and whether CCND2 and P2RY1 are the target genes of miR-34b-3p.Method:1.The expression of miR-34b-3p gene in drug-sensitive cell line?5637?and drug-tolerant cell line?EJ?of bladder cancer was detected by qRT-PCR.2.Western and qRT-PCR techniques were used to detect the expression of CCND2 and P2RY1 genes in drug-sensitive cell line?5637?and drug-tolerant cell line?EJ?of bladder cancer.3.Mimic and antagomir of miR-34b-3p were transfected into bladder cancer chemosensitive cell line?5637?and chemoresistance cell line?EJ?by cell transfection technology.The expression of microRNA-34b-3p was detected by qRT-PCR.The expression of CCND2 and P2RY1 was detected by Western and qRT-PCR.4.Detection of CCND2 and P2RY1 genes in bladder cancer chemotherapeutic drug sensitive cell line?5637?and bladder cancer chemotherapeutic drug tolerant cell line?EJ?by double luciferase reporter gene is regulated by miR-34b-3p.Result:1.The expression level of miR-34b-3p gene in bladder cancer chemosensitive cell line?5637?was lower than that in tolerant cell line?EJ?.2.The expression level of CCND2 and P2RY1 genes in bladder cancer chemosensitive cell line?5637?was higher than that in chemotherapy tolerant cell line?EJ?.3.The expression of miR-34b-3p was increased and CCND2 and P2RY1 were decreased after transfection of mimic of miR-34b-3p in bladder cancer chemosensitive cell line?5637?,and the expression of miR-34b-3p was decreased after antagomir in tolerant cell line?EJ?,while the expression of CCND2 and P2RY1 was increased.4.The double luciferase reporter gene indicates that CCND2 and P2RY1 are the target genes of miR-34b-3p in bladder cancer cell lines.Conclusion:qRT-PCR,Western and double luciferase reporter gene assay showed that the expression levels of CCND2 and P2RY1 genes were negatively correlated with the expression levels of miR-34b-3p,and miR-34b-3p was involved in regulating the expression of CCND2 and P2RY1 genes.Chapter 2:miR-34b-3p Represses the Chemoresistance of Bladder Cancer Cells by Regulating the CCND2 and P2RY1 GenesPurpose:To investigate the effects of miR-34b-3p,CCND2 and P2RY1 genes on chemotherapy tolerance,apoptosis and cell cycle of bladder cancer cells.To investigate the effect of miR-34b-3p/CCND2/P2RY1 on chemotherapy tolerance of bladder cancer cells in nude mice subcutaneous transplantation experiment.Method:1.By cell transfection technology,mimic and antagomir of miR-34b-3p were transfected into bladder cancer chemosensitive cell line?5637?and chemosensitivity tolerant cell line?EJ?respectovety.And the expression of miR-34b-3p was detected by qRT-PCR.CCK8 assay was used to determine the cell viability of five clinical chemotherapeutics on bladder cancer cells:paclitaxel?PA?,pirarubicin?PI?,epirubicin hydrochloride?EH?,adriamycin?AD?and cisplatin?CI?.2.By cell transfection technology,the si-CCND2 and si-P2RY1 gene was transfected into bladder cancer chemosensitive cell line?5637?.The changes of CCND2 and P2RY1 gene expression were detected by qRT-PCR and Western assay.CCK8 assay was used to determine the cell viability of five clinical chemotherapeutics on bladder cancer cells:paclitaxel?PA?,pirarubicin?PI?,epirubicin hydrochloride?EH?,adriamycin?AD?and cisplatin?CI?.3.CCND2 and P2RY1 lentiviruses were infected in bladder cancer chemotherapeutic tolerant cell line?EJ?.The changes of CCND2 and P2RY1 gene expression were detected by qRT-PCR.CCK8 assay was used to determine the cell viability of five clinical chemotherapeutics on bladder cancer cells:paclitaxel?PA?,pirarubicin?PI?,epirubicin hydrochloride?EH?,adriamycin?AD?and cisplatin?CI?.4.Mimic and antagomir of miR-34b-3p were transfected into bladder cancer chemosensitive cell line?5637?and chemotherapy tolerant cell line?EJ?,respectively.interfering RNA of CCND2 and P2RY1 genes were transfected into bladder cancer chemosensitive cell line?5637?respectively,and cell apoptosis was detected by flow cytometry.5.Mimic and antagomir of miR-34b-3p were transfected into bladder cancer chemosensitive cell line?5637?and chemosensitive cell line?EJ?respectively.Interfering RNA of CCND 2 and P2RY1 genes were transfected into bladder cancer chemosensitive cell line?5637?.Cell cycle changes were detected by flow cytometry.6.After transfection of mimic of miR-34b-3p into bladder cancer chemosensitive cell line?5637?,nude mice were subcutaneously tumorigenic.Paclitaxel?PA?and PBS were injected intraperitoneally.The growth of tumorigenic volume was measured regularly.After death,the tumors were weighed and the expression of CCND2 and P2RY1 genes was detected.Result:1.Mimic and antagomir of miR-34b-3p were transfected into bladder cancer chemosensitive cell line?5637?and chemotherapy tolerant cell line?EJ?,respectively,and the expression level of miR-34b-3p was increased and decreased,respectively.CCK8 assay showed that cell viability decreased after transfection of mimic,but increased after antagomir.2.The expression levels of CCND2 and P2RY1 genes in chemosensitive bladder cancer cells?5637?transfected with si-CCND2 and P2RY1 were lower than those of control cells?NC?detected by qRT-PCR and Western technique.CCK8 assay showed that the cell viability of transfected si-CCND2 and si-P2RY1 decreased in varying degrees.3.CCND2 and P2RY1 over-expressed lentiviruses were constructed in tolerant cell lines?EJ?.qRT-PCR assay showed that the expression levels of CCND2 and P2RY1genes were increased.CCK8 assay showed that the survival rate of cells infected with CCND2 and P2RY1 over-expressed lentiviruses increased in varying degrees.4.Mimic and antagomir of miR-34b-3p were transfected into sensitive cell line?5637?and chemotherapeutic tolerant cell line?EJ?,respectively,and the apoptotic rate increased and decreased,while interfering RNA of CCND2 and P2RY1 gene was transfected into sensitive cell line?5637?,respectively,and the apoptotic rate increased.5.Mimic and antagomir of miR-34b-3p were transfected into sensitive cell lines?5637?and tolerant cell lines?EJ?,respectively.The proportion of G2/M in cell cycle increased and decreased.Interfering RNA of CCND2 and P2RY1 genes was transfected in sensitive cell lines,and the G2/M ratio in the cell cycle was changed.6.In the nude mice subcutaneous transplantation tumor experiment,the tumor formation of nude mice showed that the tumor volume of the 3PM+PBS group was significantly smaller than that of the NC+PBS group,while the tumor volume of the3PM+PA group was significantly smaller than that of the NC+PA group.The volume of paclitaxel treatment group is again smaller than the PBS treatment group.The expression of CCND2 and P2RY1 protein in the 3PM+PBS group was lower than that in the NC+PBS group,while the CCND2 and P2RY1 protein expression levels in the3PM+PA group were significantly lower than those in the NC+PA group.Conclusion:CCK8 experiments showed that miR-34b-3p inhibited the chemotherapeutic tolerance of bladder cancer cells,while CCND2 and P2RY1 to promote the chemotherapeutic tolerance of bladder cancer cells.Apoptosis,cell cycle and subcutaneous transplantation of bladder cancer cells in nude mice showed that miR-34b-3p,CCND2 and P2RY1 genes affected chemotherapy tolerance of bladder cancer cells.Chapter 3:miR-34b-3p/CCND2/P2RY1 participates in chemotherapy tolerance of bladder cancer Cells through Notch and PKC/Ca⁺⁺signaling pathwaysPurpose:To investigate the effect of miR-34b-3p/CCND2/P2RY1 on signal pathways related to bladder cancer cells and cancer.Method:1.Seven cancer-related signal transduction reporter gene plasmids were transfected into bladder cancer chemosensitive cell line?5637?and chemotherapy tolerant cell line?EJ?,respectively,to establish the difference spectrum of signal transduction pathway activity between the two cell lines.2.Mimic and antagomir of miR-34b-3p were transfected into bladder cancer chemosensitive cell line?5637?and chemotherapy tolerant cell line?EJ?,respectively,to identify the changes of pathway activity with significant differences.3.The transfection of si-CCND2 and si-P2RY1 into bladder cancer chemosensitivity?5637?was performed to identify the changes of the pathway activity with significant differences.4.qRT-PCR was used to detect the expression of key transcription factors in seven cancer-related signaling pathways in bladder cancer sensitive cells and tolerant cells,si-CCND2 and si-P2RY1 were transfected into sensitive cells,and qRT-PCR was used to detect the expression of key transcription factors in seven cancer-related signaling pathways in bladder cancer chemosensitive cells.Result:1.Seven common cancer-related signal transduction pathway reporter gene detection systems showed that Notch,NF-kB and PKC/Ca⁺⁺had significant differences in signal transduction pathway activity in Bladder Cancer Chemosensitivity and tolerance cells.2.The signal activities of Notch and PKC/Ca++were consistent with the mimic and antagomir transfection of miR-34b-3p in chemotherapeutic sensitive and tolerant cells of bladder cancer,respectively.3.Transfection of si-CCND2 and si-P2RY1 in sensitive cells,the signal activity of Notch and PKC/Ca++changed in accordance with the law.4.The key transcription factors in p53/DNA dam age,TGF?,NF-kB,MAPK/ERK and PKC/Ca⁺⁺cancer-related signaling pathways are higher in bladder cancer-resistant cells than in sensitive cells;The expression levels of Notch and Hedgehog transcription factors in bladder cancer sensitive cells were lower than those in resistant cells.The expressions of RBP-J?and NFAT were increased after mimic,si-CCND2 and si-P2RY1 transfected into miR-34b-3p in bladder cancer sensitive cells 5637,respectively.Conclusion:miR-34b-3p/CCND2/P2RY1 participates in chemotherapy tolerance of bladder cancer through Notch and PKC/Ca⁺⁺signaling pathways.