Suppressive Effect Of Up-regulation Of P21 And Down-regulation Of MTH 1 On HEPG 2 In Vitro And In Vivo

Objective: RNAa and RNAi technology were used to up-regulate p21 gene expression and down-regulate MTH 1 gene expression in HepG 2 tumor model ofnude mice to study the inhibitory effect on hepatocellular carcinoma and provide new idea for liver cancer treatment.Methods: Liver cancer HepG 2 cells were cultured in vitro.When HepG 2 cells were grown to logarithmic growth phase,HepG 2 tumor cells were injected into the right forelimb of the nude mice to establish an implanted nude mouse model.The nude mice bearing tumors were randomly divided into 4 groups: control group(CT),saRNA-p21 transfection group(P21),siRNA-MTH 1 transfection group(MTH 1)and co-transfection group(P21+MTH 1).Then,6 mice in each group.After the tumor animal model was successfully established,19.8 ?g RNAs per mouse was injected beside the tumor,once three days for 30 days.Some physiological index,tumor volume change and survival time of each group of nude mice were observed during the administration.After the administration,the tumor tissues were taken out for dehydration and embedding to prepare frozen sections.The pathological morphology of tumor cells in each group was observed by HE staining.The relative expression of p21 and MTH 1 mRNA in each group was detected by real-time fluorescent quantitative PCR.The expression of P21 and MTH 1 in tumor tissues of each group was detected by immunofluorescence method.Western blot and ELISA were used to detect the relative expression levels of P21,MTH 1 and apoptosis-related proteins Bak and Caspase-3in tumor tissues of different groups.At the same time,the HepG 2 tumor model of nude mice was established for the efficacy of RNAs and cisplatin.In the comparative study,nude mice with successful tumors were randomly divided into three groups: control group(CT),double transfection group(P21+MTH 1),and cisplatin group,with 6 rats in each group.After the tumor animal model was successfully established,it was administered by the tumor side,once every three days,and RNAs and cisplatin were administered 40 ug per mouse for 30 days.The living conditions,tumor volume changes and survival time of each group of nude mice were observed during the administration.Results: The animal model of HepG 2 tumor in nude mice was successfully established,the tumor formation rate was 100%,and the survival rate of nude mice was 100%.The tumor volume growth curve showed that the tumor growth rate of P21 group and MTH 1 group was lower than that of CT.In the group,the difference between P21+MTH 1 group was more significant;qPCR and WB results showed that after transfection of saRNA targeting p21 gene and siRNA targeting MTH 1 gene in vivo,the transfection group compared with the control group,p21 mRNA level and The protein level was significantly increased,the mRNA level and protein level of MTH 1 were significantly decreased,and the differencewas more significant in the combined transfection group.The ELISA results showed that the expression of Bak protein in the tumor tissue of the combined double transfection group increased,but there was no significant change in the single transfection group.In addition,the expression of Caspase-3 protein was increased in the experimental group transfected with RNAs,and the expression level of double transfection group was significantly higher than that of single transfection group.The tumor volume growth curve of the drug efficacy experiment showed that the cisplatin group and the double transfection group had similar inhibition on tumor volume growth compared with the control group,and the survival rate of the double transfection group was much higher than that of the cisplatin group.Conclusion: Up-regulation of p21 gene and down-regulation of MTH 1 gene expression can effectively inhibit the growth of HepG 2 tumors.The co-regulatory effect of p21 and MTH 1gene expression was more pronounced than when only MTH 1 or p21 gene expression was regulated.In addition,the combination of siRNA targeting p21 gene and saRNA targeting MTH 1 gene can induce apoptosis of HepG 2 cells,which may be related to the increased expression of Bak in Bak/Bcl-xl apoptosis pathway.And the combined regulation of oncogene expression is better than single regulation.The results of the comparison of drug efficacy showed that the therapeutic effect of RNAs compared with cisplatin was similar during the treatment,but the toxic side effects on the body were much smaller.