| Objective:(1)To explore the inhibitory effect of Platycodin D on the growth of nonsmall cell lung cancer in vitro and in vivo.(2)The core targets of Platycodin D on nonsmall cell lung cancer were predicted and screened based on genome-wide CRISPR/Cas9 technology,TCGA database and PPI network.To provide positive theoretical support for the late application of Platycodin D in the clinical treatment of non-small cell lung cancer.Methods:(1)MTS assay was used to detect the effect of Platycodin D on non-small cell lung cancer A549 cells in vitro to explore the proliferation and inhibition of cells.Flow cytometry was used to detect the effect of Platycodin D on cell cycle and apoptosis of A549 cells.(2)Platycodin D was used to inhibit the growth of non-small cell lung cancer cell line A549 in vivo,and the tumor model of A549 tumor-bearing mice was established in Balb/c nude mice.They were randomly divided into three groups(nasty 10)tumor model group,Platycodin D group and cisplatin group.According to the relevant literature reports and the body surface area conversion formula of mice,the dose of Platycodin D in mice was 4.9mg/kg and the dose of cisplatin was 2mg/kg,the frequency of administration is intraperitoneal injection every other day.The tumor volume,tumor weight and body weight of mice were measured according to the standard flow,and the administration period was 21 days.The experimental mice were treated,the blood was taken,the tumor tissue was separated,the lung,liver and kidney were taken,and the tumor proliferation,inhibition rate as well as the change of body weight of mice were calculated.(3)The potential target genes of NSCLC affected by Platycodin D were screened based on CRISPR/Cas9 combined with high-throughput screening technique.(4)Clinical gene expression and prognosis of potential targets were analyzed based on TCGA database and PPI network.(5)The A549 cells in control group and Platycodin D group were collected Total RNA was extracted and reverse transcribed,and the core target sites were verified by qRT-PCR.Results:(1)Platycodin D had a good and stable effect on A549 cells in vitro.Flow cytometry showed that Platycodin D induced G2/M phase and S phase cell cycle arrest in A549 cells,and induced apoptosis in A549 cells in a concentration-dependent manner.(2)Platycodin D inhibi ted the growth of A549 tumor-bearing mice in vivo.Platycodin D significantly inhibited the growth of non-small cell lung cancer cells and had less effect on the quality of life of mice compared with cisplatin group.(3)Screening of potential target genes of Platycodin D on A549 cells by CRISPR/Cas9 combined with high-throughput screening technique,a total of 18 potential target genes:HSF2BP,FAM110 A,CIPC,DPPA2,CYP2A6,PRKRIP1,PDZRN4,TMEM79,CSTB,HBA1,TTCC36,FAM43 A,BRINP2,RBPMS,LMX1 B,CMAS,ZFP28 and KLC4;Based on TCGA database and PPI network prediction analysis,nine potentially important target genes were screened,which were HSF2 BP 、 FAM110A、 CMAS、 CSTB、 LMX1B、 PRKRIP1、TMEM79、DPPA2 and RBPMS.(4)The results of qRT-PCR showed that there were two significant differences in the expression of 18 core targets in A549 cells treated with PD at 24 h and 48 h as well as 12 h and 24 h,respectively,which were HSF2 BP and FAM110A.There were six significant differences in the expression of 18 targets at 24 h,which were PDZRN4 、 PRKRIP1、CYP2A6、 TMEM79、 DPPA2 and CIPC.Conclusion: In vitro cell experiments showed that Platycodin D could effectively inhibit the growth of non-small cell lung cancer A549 cells,and the effect was significant.Platycodin D induced G2 / M phase and S phase cell cycle arrest as well as induced apoptosis in A549 cells in a concentration-dependent manner.In vivo animal experiments showed that Platycodin D could inhibit the tumor growth of A549 tumor-bearing mi ce,and had less effect on the quality of life of A549 tumor-bearing mice compared with cisplatin group.Based on CRISPR/Cas9 library screening and related bioinformatics analysis,Platycodin D may act on non-small cell lung cancer A549 cells through 18 potentially important target genes: HSF2 BP,FAM110A,CIPC,DPPA2,CYP2A6,PRKRIP1,PDZRN4,TMEM79,CSTB,HBA1,TTCC36,FAM43 A,BRINP2,RBPM S,LMX1 B,CMAS,ZFP28 and KLC4.The results of predictive analysis combined with TCGA database and PPI network construction showed that the expression of nine target genes HSF2BP、FAM110A、CMAS、CSTB、LMX1B、PRKRIP1、TMEM79、DPPA2and RBPMS were significantly different in patients with non-small cell lung cancer,and had a great impact on the prognosis.The results of molecular biological verification showed that Platycodin D may exert its anti-tumor effect through eight potentially importan t target genes: HSF2 BP,FAM110A,PDZRN4,PRKRIP1,CYP2A6,TMEM79,DPPA2 and CIPC. |
