| Objective: Renal cell carcinoma(RCC)is a type of malignant tumor originating from renal tubular epithelial cells,accounting for more than 90% of all kidney cancer cases,referred to as renal cancer.Renal cell carcinoma includes more than 10 histological and molecular subtypes,among which clear cell renal cell carcinoma(cc RCC)is the most common pathological subtype,accounting for about 70% of all renal cancer patients.Smoking,obesity and hypertension are thought to be closely linked to kidney cancer.Localized renal cell carcinoma can be treated by surgical resection,and the 5-year survival rate is 95%.However,metastatic kidney cancer is not sensitive to either radiotherapy or chemotherapy,and the 5-year survival rate drops to 20%.In the past two decades,several targeted drugs have been developed for the treatment of advanced RCC,including tyrosine kinase inhibitors(TKIs)and immune checkpoint inhibitors(ICIs).However,the clinical application of these drugs is limited due to the intolerability of drug toxicity,low drug response rate,acquired drug resistance,and economic burden.Therefore,the research on the molecular mechanism of the occurrence and development of RCC and the precise treatment of advanced RCC are still the challenges we are facing at present.Genomic studies have shown that RCC has a wide range of genetic mutations and significant intratumoral heterogeneity,which are closely related to disease diagnosis,prognosis,and treatment response.It has been known that gene mutations associated with RCC,such as VHL,FLCN,TFE3,FH,and SDHB,can alter tumor responses to oxygen,iron,nutrition,and energy metabolism.As driving genes of RCC,they are closely related to the progression and prognosis of this disease.In-depth study of these RCC driver genes has increased our understanding of the biology of this cancer and will contribute to the early diagnosis of RCC and the development of new targeted therapeutics.Therefore,this study focused on discovering new differentially expressed genes in RCC,and clarifying their molecular functions and mechanisms in tumor genesis and development.RAD52 Motif Containing 1(RDM1)is a protein-coding gene located on chromosome 17q11.2.Human RDM1 protein is composed of 284 amino acids,andRDM1 expression is particularly abundant in testicular tissue.RDM1 protein contains two motifs: one is found in RAD52,which plays a role in DNA double-strand break repair and homologous recombination repair;The other is RNA recognition motif(RRM).Previous studies have found that RDM1 has RNA-DNA binding ability and may be involved in DNA homologous recombination repair and DNA double-strand damage repair.Recently,the relationship between RDM1 and tumor has aroused the interest of researchers.For example,RDM1 plays a carcinogenic role in human lung adenocarcinoma,ovarian cancer,and breast cancer.However,whether RDM1 plays a role in the development and progression of cc RCC remains unknown.This project will further study the molecular mechanism of RDM1 driving and regulating the occurrence and development of cc RCC,further enrich and improve the genetic molecular map of renal clear cell carcinoma,and the results will contribute to the early diagnosis and prognosis assessment of cc RCC,and provide a theoretical basis for precise and individualized targeted therapy.Methods: Firstly,whole-gene m RNA expression of 69 pairs of cc RCC and paracancer tissue samples in TCGA database was analyzed.According to its high expression level in tumor samples and research significance reported in literature,we found that 22 genes may be related to cc RCC.The cc RCC cells 786-O were transfected with short haircard RNA(sh RNA)to knock down the above target genes.Cell viability was detected by Celigo analysis.Knockdown of ERCC6 L,PLEKHO1,GLYATL2,OIP5,and RDM1 significantly inhibited the proliferation of cancer cells.Among them,related studies on ERCC6 L,PLEKHO1,and OIP5 in RCC have been reported.The expression of RDM1 m RNA in cc RCC tissues and its relationship with disease progression and prognosis were preliminarily determined by analyzing TCGA database.By collecting surgical specimens of cc RCC and obtaining clinicopathological data of patients,RDM1 m RNA expression differences in cc RCC tissues and peritumoral tissues were analyzed by RTq PCR.Immunohistochemistry(IHC)technique was used to detect the expression of RDM1,and the survival curve was plotted.In functional experiments,the expression of RDM1 was knocked down in RCC cell lines 786-O and caki-1 by lentivirus transfection technology,and the effect on cell proliferation and colony formation were detected.Theeffect of RDM1 knockdown on cell cycle distribution and apoptosis was observed by flow cytometric analysis.CCK8 and colony formation assay were used to detect the effect of overexpression of RDM1 on the proliferation of cancer cells,and CCK8 assay was also used to detect the response of cancer cells to platinum chemotherapy after overexpression of RDM1.Xenograft tumor model was used to observe the effect of RDM1 knockdown on tumor proliferation in vivo.In terms of molecular mechanism,gene microarray and bioinformatics technology were used to analyze the changes of downstream gene transcription level after RDM1 knockdown,and the downstream target protein of RDM1 was identified as MCM2.PCR and western blot were used to verify the changes of the above key genes and proteins.Co-immunoprecipitation(Co-IP)and immunofluorescence analysis(IF)were used to observe whether there was direct interaction between RDM1 and MCM2 and its intracellular localization.Flow cytometric analysis and colony formation assay were used to determine whether MCM2 supplementation could rescue the cell phenotypic changes caused by RDM1 knockdown.Finally,TMA data were used to analyze the expression and localization of RDM1 and MCM2 in cc RCC tissues,and to conduct a joint analysis of the prognosis of patients with cc RCC.Results: 1.In this study,by analyzing TCGA database,it was found that RDM1 m RNA was up-regulated in cc RCC tissues,and was associated with disease progression and poor prognosis.RDM1 m RNA level in cc RCC tissues was significantly higher than that in normal adjacent renal tissues(P < 0.001).The expression level of RDM1 in cc RCC tissue microarray(TMA)was significantly higher than that in normal kidney tissue(P < 0.05),and the overall survival rate of patients with high expression of RDM1 was significantly lower than that of patients with low expression of RDM1.2.Knockdown RDM1 significantly inhibited cell proliferation and colony formation ability in cc RCC 786-O and caki-1 cell lines.Flow cytometric analysis demonstrated that lowering the m RNA expression of RDM1 induce cell cycle arrest in G1 phase.RDM1-knockdown significantly induced apoptosis of cc RCC cells as compared with controls.Knockdown RDM1 significantly inhibited tumor growth in these cc RCC xenograft tumor models.In addition,overexpression of RDM1 did not affect cell proliferation and the sensitivity ofRCC cells to platinum chemotherapy drugs.3.The downstream target gene of RDM1 was predicted to be MCM2 by gene chip and bioinformatics technology.Differential expression of RDM1 could lead to corresponding linkage changes of MCM2 m RNA and protein levels.The co-localization of RDM1 and MCM2 was observed by Co-IP analysis,but there was no direct interaction between RDM1 and MCM2 at the protein level.4.Through the analysis of clinical cc RCC tissue samples,it was found that the m RNA expression levels of RDM1 and MCM2 were positively correlated.Based on TMA data,we found co-localization of RDM1 and MCM2 in cc RCC tissues,and patients with high expression of both RDM1 and MCM2 had a worse overall prognosis.Conclusion: In this study,we found that RDM1 is a potential oncogene of cc RCC,and screening for RDM1 expression in patients with cc RCC may be helpful to evaluate the prognosis of patients.It was revealed that RDM1-MCM2 axis may regulate the occurrence and development of cc RCC,providing a theoretical basis for the treatment of a new target,and targeting RDM1 may become a new choice for the treatment of advanced cc RCC. |
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