Porcupine Inhibitor LGK974 Downregulates The Wnt Signaling Pathway And Inhibits Clear Cell Renal Cell Carcinoma

Background:Renal cell carcinoma(RCC)accounts for 2-3%of all cancers,and the highest incidence is in Western countries[1].According to the Cancer Statistics of the USA in 2019,it is estimated that 73,820 new cases of kidney cancer will be diagnosed and 14,770 patients will die of kidney cancer[2].Compared with the data of 201 8(65340 new cases and 14970 deaths)[3],the number of new cases is increasing and the number of deaths has not decreased significantly though the diagnosis and treatment techniques of kidney cancer have been improving.According to the China Cancer Statistics 2015,it is expected that there will be 66,800 new cases of kidney cancer,and 23,400 patients will die of kidney cancer in 2015[4].Therefore,whether in Western countries or China,the impact of kidney cancer on human health cannot be underestimated.Clear cell renal cell carcinoma(ccRCC)accounts for 75-80%of renal cell carcinoma(RCC)cases and is the most common subtype of RCC[5].It is reported that 20-30%of RCC patients have local or distant metastases at diagnosis,and most patients with advanced RCC do not respond well to chemotherapy or radiotherapy[6].In the clinic,many kidney cancer patients respond well to targeted drugs.Although the targeted drugs for the treatment of RCC have acertain therapeutic effect on advanced RCC,due to the problem of drug resistance,they are still unsatisfactory.Therefore,exploring and applying new therapeutic targets will benefit more patients.As a palmitoyltransferase,Porcupine(PORCN)plays an important role in the activation and secretion of Wnt protein.PORCN exists in the endoplasmic reticulum.Palmitoyl coenzyme A is added to the Wnt protein to activate wnt protein and affect the activity of Wnt signal pathway,but the role of PORCN in clear cell renal cell carcinoma(ccRCC)is little knownIn this study,we used real-time quantitative PCR to explore the difference of PORCN expression between renal carcinoma cell lines and normal renal epithelial cell lines,and then used immunohistochemical technique to explore the difference of PORCN expression between renal clear cell carcinoma and adjacent tissues,and further used bioinformatics analysis technology to explore the effect of high and low expression of PORCN on the prognosis of patients with renal cell carcinoma.In vitro experiments verified the effects of PORCN inhibitor LGK974 on the proliferation,apoptosis,migration,invasion and cycle of renal cell carcinoma cells,as well as the pathway through which LGK974 played a role.Objective:1.To determine the difference of PORCN expression between renal carcinoma cell lines 786-0,A498,ACHN,CAKI and normal renal epithelial cell line HK-2,and the difference of PORCN expression between renal clear cell carcinoma and paracancerous tissues.2.Bioinformatics analysis was used to explore the effect of high and low expression of PORCN on the prognosis of patients with renal cell carcinoma,and to find the 10 genes most related to PORCN,and then further analyze their pathway and co-expression analysis.3.To determine the effect of PORCN inhibitor LGK974 on proliferation,apoptosis,migration and invasion of clear cell renal cell carcinoma.4.To further verify the pathway through which PORCN plays a role in the analysis of Bioinformation.Methods:1.The difference of PORCN expression between renal carcinoma cell line and normal renal epithelial cell line was detected by qRT-PCR.The clinical specimens were processed by paraffin-embedded section technique,and the difference of PORCN expression in cancer tissue and adjacent tissue was detected by immunohistochemical technique2.The Human Protein Atlas website was used to analyze the effect of high and low expression of PORCN on prognosis3.The 10 genes most related to PORCN were found by STRING website,followed by GO analysis and KEGG analysis by WebGestalt website,mutation analysis and coexpression analysis by cbioportal website4.The effect of PORCN inhibitor LGK974 on ccRCC cell proliferation was demonstrated by CCK8 cell proliferation assay5.The effect of PORCN inhibitor LGK974 on the proliferation and colony formation ability of ccRCC cells was proved by colony formation assay.6.The effect of PORCN inhibitor LGK974 on apoptosis and cell cycle of ccRCC cells was proved by flow cytometry,and the effect of PORCN inhibitor LGK974 on the expression of apoptosis-related proteins and cycle-related proteins was detected by Western Blot7.The effects of PORCN inhibitor LGK974 on the migration and invasion of ccRCC cells were confirmed by Wound Healing Assay and Transwell assay,and the effect of PORCN inhibitor LGK974 on the expression of EMT-related proteins was detected by Western Blot technique.8.The effects of PORCN inhibitor LGK974 on the expression of β-catenin and the target genes of Wnt signal pathway cyclinD1,c-Myc,MMP9 and MMP2 in ccRCC cells were detected by Western Blot and qRT-PCR9.Te data are expressed as the mean±SEM.Statistical analysis was performed using Prism Graphpad software.Statistical significance was determined using a t test or one-way ANOVA.*p<0.05;**p<0.01;**p<0.001Result:1.PORCN is highly expressed in renal cell line and renal cell carcinoma and is closely related to poor prognosis2.Through bioinformatics analysis,it was found that the genes most related to PORCN were Wnt signaling pathway related genes.Pathway analysis showed that PORCN and its most related genes were enriched in Wnt pathway,and PORCN and Wnt pathway related genes had varying degrees of mutation and coexpression in renal cell carcinoma.3.CCK8 assay showed that LGK974 killed renal cancer cells in a time-dependent and concentration-dependent manner,and clone formation assay showed that LGK974 could inhibit the clone formation ability of renal cancer cells4.Flow cytometry showed that LGK974 could induce apoptosis of renal cancer cells.WesternBlot assay showed that LGK974 could up-regulate Bax,and down-regulate Bcl2,to induce apoptosis of ccRCC cells.5.Wound Healing Assay showed that LGK974 could inhibit the migration ability of renal cancer cells,and Transwell test once again showed that LGK974 could inhibit the migration and invasion of renal cancer cells.Western Blot experiment showed that LGK974 could reduce the expression level of interstitial markers6.Western Blot experiment showed that LGK974 could reduce the expression level of β-catenin in cytoplasm and target genes cyclinD1 and c-Myc of Wnt signal pathway.qRT-PCR showed that LGK974 could reduce the expression level of MMP9 and MMP2Conclusion:Taken together,we demonstrated that the expression of PORCN was upregulated in some renal cell lines and renal cell carcinoma tissues.PORCN inhibitor LGK974 can inhibit cell proliferation,migration,and invasion,induce apoptosis,and block cell cycle.Further analysis showed that LGK974 could downregulate the activity of the Wnt/β-catenin signaling pathway.These data also provide evidence for the feasibility of targeting PORCN to downregulate the Wnt/β-catenin signaling pathway in renal cell carcinoma and reveal the possibility of PORCN inhibitors as potential targets for the treatment of renal cell carcinoma.