Mechanisms Of PLK1 Regulating Proliferation And Apoptosis Of Renal Cell Carcinoma Cells Through Phosphorylation Of MCM3

Objective: Renal cell carcinoma(RCC)is one of the most common cancers in the urinary system.Its clinical manifestations are relatively insidious at the onset,and there are no obvious clinical symptoms at the early stage of the disease.As a result,when a large number of patients are diagnosed with renal cell carcinoma,the cancer cells in the patients have metastasized far away.Patients with advanced renal cell carcinoma are insensitive to radiotherapy and chemical therapy.Immunotargeting therapy is prone to drug resistance.Therefore,the best management of renal cell carcinoma is early diagnosis and early treatment.PLK1 has the function of activating or inhibiting the substrate regulated by PLK1 and regulating the process of cell proliferation cycle.Upregulation of PLK1 is associated with many cancers,and PLK1 has been identified as a prognostic marker for many cancers.Studies have shown that microchromosome maintenance protein 3(MCM3)plays an important role in many processes,including the proliferation cycle of DNA replication cells.However,it is not clear whether MCM3 has the same effect in renal clear cell carcinoma.Based on the biological functions of PLK1 and MCM3,we further explored the interaction between them.PLK1 regulates the functional sites of MCM3.The effect of phosphorylation of MCM3 protein by PLK1 on the proliferation and apoptosis of renal cell carcinoma,that is,the detailed mechanism of the role of MCM3 and PLK1 in the occurrence and development of renal cell carcinoma,in order to determine the key molecular markers.To lay a scientific experimental and theoretical foundation for the diagnosis and treatment of renal cell carcinoma.Methods: 1.The expression of PLK1 and MCM3 in renal cell carcinoma and adjacent tissues were detected by immunohistochemistry and Western blotting.The expression and localization of these two proteins in renal cell carcinoma cell lines were also studied to explore the relationship between the expression of PLK1 and clinical prognosis.2.The effects of PLK1 and MCM3 on the proliferation and apoptosis of renal cell lines 786-O and ACHN were investigated by cloning,CCK-8 and Ed U cell proliferation experiments,respectively.Secondly,the effects of PLK1 and MCM3 on cell growth,proliferation and apoptosis were verified by tumorigenic experiments in nude mice.3.Predictive analysis of bioinformatics website,immunoprecipitation,Phos-tag labeling,immunofluorescence and immunoblotting were used to verify and confirm the action mode and site of PLK1 on MCM 3.4.By establishing stable transfected cell lines,co-transfected cell lines,cloning and selection techniques,flow cytometry and immunoblotting techniques,the effects of PLK1 on the function of MCM3 cells and the downstream pathways involved were explored.5.The phosphorylation of PLK1 on MCM3 and the function of MCM3s112 locus were detected by tumorigenesis experiment and immunohistochemical technique in nude mice.The regulatory relationship and mechanism between PLK1 and MCM3 were further validated,as well as their effects on cell proliferation and apoptosis.Results: 1.The expression of PLK1 in tumor tissues of 90 RCC patients was higher than that in adjacent tissues and renal cell lines than that in normal epithelial cells.It was also found that the expression of MCM3 in the two groups was similar to that of PLK1.Immunohistochemical results showed that the expression level of PLK1 protein in renal cancer tissues was significantly higher than that in adjacent tissues,and with the increase of tumor grade,the expression level of PLK1 protein increased.The expression level of PLK1 was significantly correlated with the prognosis of renal cell carcinoma patients.The tumor diameter,recurrence rate and stage were positively correlated with the high expression of PLK1.2.We constructed 786-O and ACHN stable transfection cell lines that overexpressed and knocked down the expression of PLK1 and MCM3.CCK8 assay showed that the proliferation efficiency of 786-O and ACHN stable transfected cell lines overexpressing PLK1 or MCM3 was significantly higher than that of blank group,while the proliferation efficiency of cells decreased significantly after PLK1 or MCM3 were knocked out respectively(P < 0.05).The results of clone formation showed that the number of cell clusters in 786-O and ACHN stable transfected cell lines overexpressing PLK1 or MCM3 was significantly higher than that in blank group,while the number of cell clusters decreased significantly after knocking out PLK1 or MCM3 respectively(P < 0.05).The results of Ed U cell proliferation experiment showed the same trend as CK8 and clone formation.3.In 786-O cell lines overexpressing PLK1,immunoprecipitation assay was carried out.The results showed that PLK1 and MCM3 had obvious binding phenomenon.Further immunofluorescence assay confirmed that PLK1 and MCM3 were mainly bound to the nucleus.Phos-tag assay showed that there was phosphorylation between PLK1 and MCM3.In 786-O cells with high expression of PLK1,MCM3 showed a high level of phosphorylation.Based on the above results,the relationship between PLK1 and MCM3 was further predicted on the bioinformatics website.It was found that the phosphorylation site of PLK1 to MCM3 might be in Ser112.The mutant plasmid of MCM3Ser112 locus was constructed and co-transfected with MCM3S112 A plasmid in cells overexpressing PLK1 by Phos-tag assay.Compared with the control group,the phosphorylation of MCM3 was obvious.The phosphorylation modification of MCM3 by PLK1 was further confirmed at Ser112 locus.4.In 786-O and ACHN cells over-expressing PLK1,clone formation experiments confirmed that the cell growth ability was significantly enhanced compared with the blank control group(P < 0.05),while in the group co-transfected with MCM3s112 A plasmid,this trend was significantly reversed.The same trend was also found in the cell cycle experiment.In the M CM3s112 A group,PLK1 reversed the effect of PLK1 on cell cycle and increased the G2/M phase(P < 0.05).Flow cytometry showed that the number of apoptotic cells in the co-transfected MCM3s112 A cell group was significantly higher than that in the over-expressed PLK1 cell group,and P-MCM3 also promoted proliferation and inhibited apoptosis in renal cell carcinoma cells.After over-expression of PLK1,mutation of MCM3Ser112 site could reverse the effect of PLK1 on cells,which further confirmed that PLK1 affected the kidney by regulating the phosphorylation of MCM3.Proliferation and apoptosis of cancer cells;5.The tumorigenicity of nude mice in vivo was consistent with that in vitro.The tumour volume of mice injected with ACHN cells overexpressing PLK1 was significantly larger than that of the control group,which could be reversed in ACHN cells co-transfected with PLK1 and MCM3s112 A.Immunohistochemical experiments confirmed that the expression of Ki-67 protein in PLK1 overexpression group was significantly higher than that in co-transfection MCM3s112 A group,which further confirmed the role of PLK1-MCM3 regulatory axis in proliferation and apoptosis of renal cell carcinoma.Conclusion: 1.The expression levels of PLK1 protein in renal cell carcinoma cell lines(ACHN,786-O,CAKI and 769-P)and renal cell carcinoma tissues were significantly higher than those in immortalized renal tubular epithelial cell lines(HKC)and adjacent tissues,respectively.2.High expression of PLK1 protein can promote the proliferation rate of RCC cells,and low expression of PLK1 protein can reduce the proliferation rate of RCC cells.3.The number and level of PLK1-expressing cells in renal cancer tissues are closely related to the severity of disease.The growth of tumors in patients with high expression of PLK1 protein is accelerated,easy to metastasis and recurrence,and the average survival time is lower than that in patients with low expression of PLK1 protein.4.The expression of MCM3 was high in renal cell carcinoma.PLK1 and MCM3 can bind to each other in vivo,and PLK1 induces MCM3 phosphorylation.5.The phosphorylation of MCM3 Ser112 point mutant plasmid(MCM3 S112A)co-transfected PLK1 with high expression of 786-O and ACHN cell lines MCM3 was significantly inhibited;PLK1 phosphorylated the Ser112 site of MCM3,promoted the proliferation of renal cell carcinoma and inhibited apoptosis;MCM3 Ser112 point mutation affected the phosphorylation of MCM3 by PLK1,inhibited the proliferation of renal cell carcinoma and promoted the apoptosis of cancer cells.6.MCM3 Ser112 locus may become a new target for clinical treatment of renal cell carcinoma,and be used to develop new targeted therapeutic drugs.