The Research Of The Effect And Mechanism Of PCT3 In Inhibiting Proliferation And Invasion Of Prostate Cancer Cells

Objective:Prostate cancer is the most common malignancy of the urogenital system in older men.Worldwide,prostate cancer is the second most common malignancy in men.In recent years,the incidence of prostate cancer in China is increasing rapidly,which poses a great threat to the health of elderly men in China.At the same time,due to the relative lack of early prostate cancer screening system in China,about 60%of the first diagnosed prostate cancer patients are advanced and metastatic advanced prostate cancer with poor prognosis,which is significantly higher than the 16%in European and American countries.For patients who have lost the opportunity of local treatment in the middle and late stage,castration combined with anti-androgenic block is a clinically recommended first-line treatment,effective for about 80-90%of patients.However,after 18-24 months of treatment,most patients will develop drug resistance and PSA will rise again,and eventually develop castration resistant prostate cancer(CRPC).In recent years,great progress has been made in basic research and clinical diagnosis and treatment of prostate cancer.New endocrine dr?gs such as enzaluramide and abiraterone have improved the prognosis of patients with CRPC to some extent,but the effect of these treatments is still limited and can only extend the survival of patients for a few months.Therefore,the new treatment method is still an important research direction in the academic circle.Extracting natural compounds from traditional Chinese medicine is one of the important sources of medicine.Plants of the genus pygmy have long pillars and heavy floors P.Forrestii(Takht.)h.Li has the pharmacological effects of anti-tumor,anti-inflammatory,hemostasis,anti-oxidation,and protection of vascular endothelial cells.As its main active component,PCT3 has been found to have different inhibitory effects on brain tumor cells,leukemia cells and malignant lymphoma cells.However,the inhibitory effect of PCT3 on prostate cancer and its molecular mechanism are still unclear.In this study,molecular biology experiments and transcriptome sequencing were used to study the anticancer effect and molecular mechanism of PCT3 on prostate cancer.Methods:In the first part,the project identified the effect of PCT3 on the biological behavior of prostate cancer cells in vitro.Including comparing the effects of PCT3 and heavy floor saponins on the proliferation of normal epithelial cells RWPE,prostate cancer cells LNCAP,PC3 and DU145.CCK8,EdU,clonal assay was used to detect the inhibitory effect of the dr?g on cell proliferation.Scratch experiment,transwell invasion experiment to explore the inhibitory effect of dr?gs on the ability of cell invasion and metastasis;Flow cytometry and western blot were used to study the apoptosis of prostate cancer cells induced by PCT3.In the second part,the expression of lncRNAs and mRNAs in prostate cancer cells before and after PCT3 acting on LNCAP and PC3 was detected by high-thro?ghput expression profile microarray,and lncRNAs and mRNAs with different expressions were screened.By comparing KEGG and GO databases,the enrichment analysis of cell signaling pathway and cell function of differential genes was conducted.Finally,rt-pcr was used to verify IncRNAs and mRNAs with significant differences in expression after PCT3 treatment.In the third part,based on the previous transcriptome results,in vivo and in vitro experiments were conducted to explore the correlation between the down-regulation of IQGAP3 protein expression of PCT3 in prostate cancer cells and its inhibitory effect on prostate cancer.Lncap-iqgap3 and pc3-iqgap3 were transfected with lentivirus and treated with PCT3.CCK8,clone molding to detect the inhibition of cell proliferation;The scratch experiment and transwell invasion experiment were used to detect the inhibition of cell invasion and metastasis.PC3 and pc3-iqgap3 were used to construct a subcutaneous implantation model of prostate cancer in nude mice,and the changes of tumor growth after intratumoral injection of PCT3 were observed.Finally,western blot was used to detect the ERK signaling pathway and emt-related proteins in prostate cells after the effect of PCT3,to explore the specific molecular mechanism of PCT3 down-regulating IQGAP3 and inhibiting prostate cancer cells.Results:In the first part,PCT3 showed similar anticancer effect to other anti-tumor saponins in prostate cancer,and its toxicity to normal prostate cancer cells was weaker than that of other heavy building saponins.The results of cell biological function experiments showed that 4?g/mL PCT3 could significantly inhibit the proliferation of LNCAP and pc-3 of prostate cancer cells,2?g/mL PGT3 could significantly inhibit the migration and invasion of cells,and 4?g/mL PCT3 could induce apoptosis of cells.In the second part,thro?gh high-thro?ghput expression profile microarray detection,172 mRNA and 98 lncRNA expressions were significantly changed in LNCAP cells after PCT3 action.In PC3 cells,1321 mRNA and 251 lncRNA expressions were significantly changed.Thro?gh bioinformatics analysis,the genes with significant changes in expression after PCT3 were mostly enriched in apoptosis,dr?g response,negative regulation of cell proliferation,cell cycle,and cell functions related to DNA replication.Finally,the differentially expressed genes of the two cells were intersected,and we determined that 41 mRNA and 5 lncmas were significantly altered after PCT3 treatment.Thro?gh rt-pcr verification,we speculated that the regulation of PCT3 on the expression of NEAT1,MALAT1,TIPIN,LYAR,IQGAP3,GINS2,ZGRF1 and other genes in prostate cancer cells might be related to the molecular mechanism of prostate cancer inhibition.In the third part,in vivo and in vitro experimental results showed that the down-regulation of IQGAP3 expression in prostate cancer cells by PCT3 might be related to its inhibitory effect on the proliferation and invasion and metastasis of prostate cancer cells,but not to the inhibitory effect on hormone-sensitive prostate cancer.Western blot results of signaling protein showed that PCT3 inhibited hormone resistance to proliferation of prostate cancer cells by down-regulating IQGAP3,which may be related to regulation of the ERK/AKT signaling pathway.By down-regulating IQGAP3,PCT3 inhibits the hormone to resist the migration and invasion of prostate cancer cells,which may be related to the regulation of EMT.Conclusion:In this study,it was determined that low concentration PCT3 had significant inhibitory effect on prostate cancer proliferation and metastasis.Its regulation of prostate cancer transcriptome was found and its potential dr?g targets were screened.Finally,it was proved that PCT3 could inhibit the effect of hormone on prostate cancer resistance by regulating the expression of IQGAP3 and the effect was related to the regulatory signaling pathway.This study provides a theoretical basis for PCT3 as the treatment of prostate cancer and explores a new therapeutic target for prostate cancer.