Experimental Study Of The Mechanism Of CDK4/6 Inhibitor SHR6390 Inhibiting The Proliferation Of Cervical Cancer Cells

Purpose:Abnormal regulation of the cell cycle is commonly observed in the development and progression of multiple malignancies.CDK4/6(cyclin-dependent kinase 4/6)is one of the key regulatory genes during cell cycle operation.SHR6390(dalpiciclib,Darsil)is a novel domestic small molecule selective CDK4/6 inhibitor that mechanistically induces G1 cell cycle arrest in tumor cells to lead to tumor cell growth inhibition.In other tumors,such as breast cancer,studies have found that CDK4/6 inhibitors have anti-tumor activity.Cervical cancer usually also has abnormal cyclin D-CDK-Rb signaling pathway.However,there are no relevant studies reporting the application of CDK4/6 inhibitors in the research field of cervical cancer.In this study,we hope to explore the inhibitory effect of SHR6390 on cervical cancer cells at the level of in vitro cell lines and animal models,and to investigate the relevant mechanisms.Method:1.In this study,clinical data and RNA expression data of two cervical cancer data sets(GSE63514 and TCGA)were collected.Compare the expression levels of CDK4/6 messenger RNA in different cervical tissues to determine the the prognostic value of CDK4/6 in cervical cancer.2.The effects of SHR6390 on proliferation activity were analyzed by performing Cell Counting Kit-8 and colony formation,apoptosis and cell cycle were investigated by flow cytometry.3.At the same time,RNA-sequencing(RNA-seq)was performed to find the mainly changed signalling pathway.key protein expression of Cyclin D1/CDK4/6/Rb signalling pathway in cervical cancer cell lines MS751 and Si Ha were explored by western blotting assays.4.Subcutaneous tumor experiment in nude mice:healthy nude mice were subcutaneously vaccinated with human cervical cancer cells Si Ha,waiting for the natural tumor growth to 50-100mm~3,and the tumor-bearing mice were randomly divided into three groups and treated with the corresponding dose concentration of SHR6390.The three groups were control(SHR6390 0mg/kg),low dose(SHR6390 100mg/kg)and high dose(SHR6390 200mg/kg)to record tumor volume changes to verify the effect of SHR6390 in inhibiting cervical cancer cell growth in vivo.Results:1.The expression levels of CDK4 and CDK6 were significantly higher in tumor tissues than normal tissues(P<0.01).High m RNA expression of CDK6 was associated with unfavorable outcomes(P=0.0353).2.The CCK-8 test and colony formation experiments showed that SHR6390 inhibited the proliferation of cervical cancer cell lines MS751and Si Ha cells in vitro with IC50 values of 4.24μM and 12.82μM,respectively.The rates of apoptosis in both MS751 and Si Ha cells enhanced significantly when treated with 5 or 10μMμmol/l of SHR6390(P<0.05).Treated MS751 and Si Ha cells by 5 or 10μM SHR6390respectively,it was found that the rate of apoptosis in the two cervical cancer cells was significantly higher than that of the vehicle control group,and there was an obvious dose-dependent effect.The ratio rate of apoptosis in MS751 cells was 10.24%(vehicle control group),17.05%(5μM SHR6390),32.9%(10μM SHR6390),and the ratio rate of apoptosis in Si Ha cells was 10.88%(vehicle control group),13.48%(5μM SHR6390),32.6%(10μM SHR6390),P<0.05.MS751 cells treated with SHR6390 of 5μM showed G1 phase arrest significantly,while Si Ha cells treated with 10μM SHR6390 also showed remarkable G1 phase cell cycle arrest.3.The RNA-seq revealed that cell cycle pathway was the most influenced signalling pathway after SHR6390 administering.Further,SHR6390 induced d G1 cell cycle arrest in both cell lines significantly.Western blotting showed that 10μM SHR6390 significantly downregulated the expression levels of these three proteins(Rb,p-Rb,and p16).4.Finally,Subcutaneous tumors in nude mice confirmed that SHR6390 showed a dose-dependent inhibition of Si Ha growth in cervical cancer cells.Conclusion:The novel selective CDK4/6 inhibitor SHR6390inhibited the growth of cervical cancer cells via inducing G1 phase cell cycle arrest,inducing cell apoptosis,which suggests that interventions of the cyclin D/CDK/Rb signaling pathway may benefit cervical cancer patients.