| Backgrounds and purposesRenal cell carcinoma(RCC)is a common malignant tumor,accounting for 2-3% of adult malignancies.It is one of the few tumors with an increasing incidence worldwide.However,RCC faces the difficulty of early diagnosis and poor treatment.Because RCC is insidious and there are no obvious typical clinical symptoms in the early stage,about 20-30% of patients have already metastasized when diagnosed.In addition to surgery,other treatments such as IL2 and INF? had no significant clinical effect,and quality of life was greatly compromised due to their severe side effects.Therefore,the targeted therapy came into being.The receptor tyrosine kinase inhibitors(TKI)represented by sunitinib were the first-line treatment for advanced RCC.Sunitinib was a multi-target tyrosine kinase inhibitor with both inhibition of neovascularization and tumor cells.Although the targeted drugs have greatly improved the therapeutic prospects of advanced RCC,drug resistance has gradually reduced the clinical effects of targeted drugs.About 20% of patients developed innate resistance when they first took sunitinib,and most patients would develop secondary resistance after 6-11 months of treatment with sunitinib.The molecular and biological mechanisms involved in sunitinib resistance were still unclear,and there was no effective biomarker to predict the resistance and effective treatment to reverse or delay the drug resistance was lacking.Therefore,it is necessary to explore the mechanism and molecular markers of sunitinib resistance in RCC.DNA methylation is a common epigenetic modification by the action of DNA methyltransferase(DNMT).It often occurrs on CpG islands and CpG islands are generally located in the promoter region of genes and contain many binding sites for transcription factors.Methylation in this region is often closely related to gene transcription and is closely related to many diseases such as cancer,aging,and Alzheimer's disease.Therefore,the purpose of our study is to explore whether DNA methylation changing plays a role in RCC sunitinib resistance and whether methylation of DNA can be used as a diagnosis marker for sunitinib resistance,thus finding new targets for the treatment of sunitinib resistance and improving rhe quality of life for RCC patients.Methods1.Use Illumina Human 850 k methylation chip in sunitinib-resistant and sensitive RCC tissues(4:4),to screen for different methylation sites,and use Sequenom Methylation mass spectrometry to verify the methylation chip results in sunitinib-resistant and sensitive RCC tissues(10:10).2.By qPCR,Western blot,immunohistochemistry and Elisa,QPCT was found differently expressed in the tissues and plasma of sunitinib-resistant and sensitive RCC patients.The tissue microarray constructed using tissues of 156 RCC patients was used to detect the expression of QPCT,and the relationship between QPCT and patient prognosis and the response to sunitinib were analyzed based on the follow-up data.3.In vitro,CCK-8 Proliferation experiments,colony formation experiments,and flow cytometry were used to determine whether sunitinib sensitivity of RCC cells was changed when QPCT was knocked down or overexpressed.4.In vivo,786-O cells stably overexpressing QPCT and control cells were injected subcutaneously in nude mice,then sunitinib 40mg/kg/day(experimental group)and saline(control Group)were treated with gavage,the size of the transplanted tumor was measured every five days,data was recorded,and the tumor growth curve was drawn.5.5-Aza-2'-deoxycytidine(Decitabine)was used to inhibit DNA methylation in the QPCT promoter region and then to dectect whether DNA methylation could affect its expression;ChIP assay was used to verify whether NF-kB(p65)could bind to and QPCT promoter region,and whether the degree of methylation in QPCT promoter region affected NF-kB(p65)binding.6.Human proteomic chips was used to detect proteins that might bind to QPCT,by using protein co-immunoprecipitation(Co-IP)and laser confocal in RCC cells,HRAS was found binding to QPCT.In RCC tissue microarray and RCC cells,the relationship between HRAS and sunitinib resistance were verified.Using the "Rescue method",it was further clarified that QPCT affects the sensitivity of RCC cells to sunitinib through HRAS.Results1.Through the methylation chip,9 genes were screened out: IRS1,SKI,PTK2 B,QPCT,C1orf86,B3GNT7,AP4K2,PRKCZ,ACP52.Sequenom Methylation found that IRS1,SKI,PTK2 B,QPCT methylation levels changed significantly.3.QPCT and IRS1 were significantly different expressed in RCC sunitinib-resistant and sensitive tissues at mRNA level;but only QPCT was different expressed in RCC sunitinib-resistant and sensitive tissues at protein level.Immunohistochemistry was used to determine the expression of QPCT in RCC sunitinib-resistant and sensitive tissues.Elisa showed that QPCT was significantly different in plasma of sunitinib-resistant and sensitive patients.It was confirmed by tissue microarray and follow-up data that QPCT expression was associated with RCC prognosis,and high expression of QPCT predicted a poor prognosis and poor response to sunitinib.4.In vitro and in vivo,knockdown of QPCT could promote sunitinib sensitivity and overexpression of QPCT could promote sunitinib resistance of RCC cells.5.QPCT was up-regulated when the methylation of QPCT promoter region was inhibited by Decitabine.6.NF-kB(p65)was highly expressed in sunitatinib-resistant RCC tissues,and it was proved by ChIP assay that NF-kB(p65)could bind to QPCT promoter region and inhibition of methylation in QPCT promoter region could promote the binding of NF-kB(p65),and NF-kB(p65)could regulate the expression of QPCT.7.A total of 355 proteins were detected by human proteome chips to bind to QPCT.Seven target proteins were screened: PTK2,HRAS,CBL,GAB1,NAF1,MAPK8,MAPK10.It was confirmed by protein co-immunoprecipitation(Co-IP)and laser confocal experiments that QPCT and HRAS were combined.Overexpression of HRAS could promote sunitinib resistance in RCC cells and knockdown of HRAS could promote sunitinib sensitivity in RCC cells.It was found that the combination of QPCT could increase the stability of HRAS and reduce its ubiquitination degradation.8.It was demonstrated by "Rescue method" that QPCT affected sunitinib sensitivity of RCC cells through HRAS,and HRAS induced sunitinib resistance in RCC by activating the ERK signaling pathway.ConclusionsThe methylation level of the QPCT promoter region was lower in sunitinib-resistant tissues than sunitinib-sensitive tissues in RCC and QPCT was highly expressed in sunitinib-resistant tissues,as well as the plasma of sunitinib-resistant patients.In vitro and in vivo,knockdown of QPCT could promote sunitinib sensitivity and overexpression of QPCT could promote sunitinib resistance in RCC cells.QPCT expression was increased following inhibition of methylation in QPCT promoter region by Decitabine.ChIP assay demonstrated that NF-kB(p65)which was highly expressed in sunitinib-resistant RCC tissues bound to QPCT promoter region,and inhibition of methylation could promote NF-kB(p65)binding.NF-kB(p65)could regulate the expression of QPCT.QPCT and HRAS combinations were found by human proteomic microarray.QPCT could increase the stability of HRAS and reduce its ubiquitination degradation.HRAS promoted ERK phosphorylation and activated ERK signaling pathway leading to sunitinib resistance in RCC. |
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