Functional Study Of CCDC132 Gene Regulating Malignant Proliferation Of Osteosarcoma

Objective We aim to use lentivirus(LV)-mediated RNA interference(RNAi)to knock down the expression of CCDC132 in Osteosarcoma(OS)and observe the effect of CCDC132 on proliferation,apoptosis and cell cycle of Osteosarcoma cells.To reveal the function of CCDC132 in the pathogenesis of osteosarcoma.It provides early and reliable experimental evidence for the possibility of CCDC132 as a clinical therapeutic target for osteosarcoma,and further confirms the effect of CCDC132 on the biological behavior of osteosarcoma cells.Methods 1.Firstly,the expression of CCDC132 in each cell line saos-2,u-2os,mg-63 and HOS was determined by real-time fluorescence PCR;Then,the designed CCDC132 knockdown group lentivirus and the negative control group lentivirus infected cells u-2os were used to detect the protein expression of CCDC132 gene in u-2os cell line by quantitative real-time PCR(q PCR)and calculate the knockdown efficiency.After the infection of 293 T cells,Western blot(WB)was used to verify the effectiveness of the gene target and select the best target.2.The effect of CCDC132 expression on the growth and proliferation of u-2os cells in osteosarcoma cell line was analyzed by celigo cell count after infection of u-2os cells with lentivirus in CCDC132 knockdown group and lentivirus in negative control group.The effect of CCDC132 on u-2os cell cycle was detected by pi-facs cell cycle assay.The effect of CCDC132 gene expression on apoptosis was determined by Annexin v-apc single staining.The effect of CCDC132 expression on cell viability was detected by MTT assay in vitro.Results1.It can be seen from the real-time quantitative PCR results that Sadoc-2,U-2OS,MG-63,HOS and other 4 human osteosarcoma cells express the abundance of CCDC132 gene in high abundance,and the expression of target gene in U-2OS is abundant Degree is highest.2.After successfully infecting the target cell U-2OS,it can be seen from the qPCR results that after sh RNA lentivirus infection,the CCDC132 gene expression level in the U-2OS cells of the experimental group was inhibited(p <0.05),and knocked down The efficiency reached 77.9%.3.After the lentivirus of the gene knockdown group and the lentivirus of the negative control group successfully infected 293 T cells,it can be seen from the Western Blot results that the target sequence has a knock-down effect on the foreign expression of the target gene,and is therefore an effective target..4.Celigo cell counting experiment showed that U-2OS cell proliferation rate in the experimental group was significantly inhibited.It can be seen that CCDC132 played a role in promoting the proliferation of osteosarcoma cells.5.PI-FACS cell cycle test showed that compared with the control group,the number of cells in the sh CCDC132 group in the G1 phase was reduced(P <0.05),and the number of cells in the S phase was reduced(P <0.05).The number of cells increased(P <0.05);CCDC132 gene was significantly correlated with the cycle distribution of U-2OS cells.6.Annexin V-APC single staining cell counting experiment results showed that compared with the control group,the number of apoptotic cells in sh CCDC132 group increased(P <0.05).It is suggested that CCDC132 gene is significantly related to the apoptosis of U-2OS cells.7.MTT test results showed that compared with sh Ctrl group,cell proliferation in sh CCDC132 group was significantly slowed,and cell viability was significantly decreased.It is suggested that CDCC132 gene is significantly related to the proliferation ability of U-2OS cells.Conclusions1.CCDC132 was highly expressed in OS,especially in u-2os,the expression abundance of CCDC132 was significantly higher than that of other cell lines;2.CCDC132 is an effective target,and the expression of osteosarcoma cells is significantly inhibited by lentivirus interference.It may provide a direction for the targeted treatment of osteosarcoma in the future.3.Through the cytological function measurement,we can know that the expression of CCDC132 can promote cell proliferation,enhance the efficiency of cell proliferation,cause cell cycle stagnation and promote apoptosis.