| Objective:Renal cell carcinoma(RCC)is one of the malignant cancers with an increasing morbidity and mortality in recent years.For example,the global incidence of RCC is over than 72,000 by 2023.The incidence rate of clear cell renal cell carcinoma(ccRCC)accounts for 75-80%of RCC.Although the targeted therapies such as targeting of VEGFR/PDGFR tyrosine kinases and mTOR pathway,and the immunotherapies significantly improved the prognosis of metastatic ccRCC,30%of RCC patients with metastasis showed a primary resistance to the molecular targeted drugs.Unfortunately,those patients who were initially sensitive to the treatments displayed "secondary drug resistance" around one year after the treatments,which greatly reduces the efficacy of the molecular targeted drugs.To overcome this serious issue,it is urgent to clarify the precise molecular mechanisms behind the progression of RCC and develop a promising therapeutic strategy to treat RCC patients.NOTCH1-mediated signaling pathway plays a vital role in the regulation of the cellular communication during organ development.Upon the ligand binding,NOTCH1 is subjected to its cleavage,and then its intracellular domain(NICD)forms an active complex with various transcription factors within cell nucleus,thereby transactivating its downstream target genes such as HES1(hairy and enhancer of split 1),c-MYC and VCAM1.Among them,HES1 has been shown to be a transcriptional regulator directly regulated by NOTCH1 and play a pivotal role in down-regulation of NOTCH1-mediated downstream signaling.Of note,accumulating evidence suggests that HES1 has oncogenic role in numerous cancers including breast cancer,lung cancer,prostate cancer and melanoma.In support of this notion,it has been also described that NOTCH1 requires HES1 to exert its oncogenic function.To date,it still remains unclear how HES1 could participate in NOTCH1-induced development of RCC.MicroRNA(miRNA)is a small non-coding RNA(18-22 nucleotides in length)which binds to the 3’-untranslated region(3’-UTR)of its target mRNA to prohibit its translation.Its uncontrolled expression is frequently observed in a variety of cancers such as RCC,indicating that miRNA might play a crucial role in the regulation of proliferation,differentiation,apoptosis and stress response of cancers.Indeed,it has been described that a variety of miRNAs including let-7c,miR-15a and miR-34a are involved in the regulation of NOTCH1 signaling pathway.Since miRNA might have an ability to attenuate its target gene expression implicated in cancer development,it is suggestive that miRNA is an attractive natural product used for an alternative therapy.Thus,the identification of the candidate miRNA(s)participated in NOTCH1-HES1 regulatory complex contributes to the better understanding of NOTCH1-HES1 induced RCC development.Methods:1.The transcriptome data of renal clear cell carcinoma in the TCGA database was used to analyze the association between the NOTCH pathway related factors and malignant phenotypes of renal clear cell carcinoma.Subsequently,the collected clinical samples were used to detect the protein expression level and RNA level of HES1 in the cancer tissue and its adjacent tissues by western blot and qPCR,so as to analyze whether HES1 has specificity in renal clear cell carcinoma.At the same time,the expression of HES1 in renal clear cell carcinoma and its effect on the prognosis of patients were analyzed in conjunction with transcription data from TCGA.Then,by knocking down HES1 gene expression,EdU,Transwell and CCK8 methods were used to verify the effects of HES1 on the proliferation,migration and viability of renal cell carcinoma.2.GEO multi-data combined analysis was used to screen out the miRNAs that were heterologous to renal cell carcinoma,and the correlation between miR-13 8-2 and NOTCH1-HES1 pathway was analyzed by combining TCGA.In addition,TCGA was used to analyze the correlation between the mature miR-13 8-5p and miR-13 8-2-3 of miR-13 8-2 and NOTCH1 and HES1,respectively,and the transcription expression of the two mature miR-138-5p in renal clear cell carcinoma,as well as the influence of miR-13 8-2 on the prognosis of patients with renal cell carcinoma.In addition,the mRNA expression of mature miR-138-5p and miR138-2-3p in clinical samples was detected by qPCR to determine whether they were specific in cancer tissues.According to the collected patient information,the influence of two miRNA mature bodies on the prognosis of patients was analyzed.In addition,the effects of mature miR-138-5p and miR-138-2-3p mimics and inhibitors on the proliferation,migration and viability of renal cell carcinoma were verified by EdU,Transwell and CCK8 methods,respectively.Subsequently,we analyzed the HES1-related ChIP-seq data in Encode database to determine whether HES1 was enriched near the promoter of miR-1382.Subsequently,knockdown and overexpression of HES1 were used to detect its regulatory effects on miR-138-2 and its mature miR-138-5p and miR-138-2-3p.And through ChIP experiment,it was verified that HES1 was enriched near the promoter of miR-138-2.In addition,a mutant plasmid was designed to verify the binding site of HES1 on the miR-138-2 gene by double luciferase assay.The function of HES1 in vivo and its regulatory effect on miR-138-2 were detected by knocking down HES1 in vivo in the tumor-forming laboratory of nude mice.3.By analyzing Targetscan database,it was determined that miR-138-2 might regulate mRNA levels of MAML1,APH1A and NOTCH1.By transfecting the mimics and inhibitors of mature miR-13 8-5p and miR-13 82-3p,mRNA levels of MAML1,APH1A and NOTCH1 were detected by qPCR and western blot to determine whether miR-38-2 had a regulatory relationship with them.Subsequently,the mutant plasmid was constructed by double luciferase assay to detect the specific binding sites of mature miR-138-5p and miR-138-2-3p on the mRNA of MAML1,APH1A and NOTCH1.In vivo experiments of tumor formation in nude mice,the gene expression of miR-138-2 was knocked down to observe the in vivo effects of miR-138-2 on tumors and the in vivo regulatory mechanism of NOTCH1-HES1 axis.Subsequently,dibenzazepine,a classic inhibitor of NOTCH1 pathway,was used to detect the degree of inhibition on cell viability by CCK8 method,the inhibition effect on NOTCH1-HES1 axis was detected by western blot,and the activation effect on miR-138-2 was detected by qPCR.In addition,CCK8 method was used to detect the inhibitory effect of dibenzazepine combined with miR-138-2 overexpression on renal cell carcinoma activity.In addition,after stabilizing the overexpression of HES1 or NOTCH1,the overexpression plasmid of miR-138-2 was transfected.In the response experiment,CCK8 and Transwell were used to detect the cell viability and migration ability,respectively,to determine the regulatory relationship between them.Results:1.Through transcriptomic data analysis in TCGA,only a small number of mutations exist in the entire NOTCH pathway in renal clear cell carcinoma,but it is closely associated with various malignant phenotypes and multi-drug resistance in renal clear cell carcinoma.In the detection of clinical tumor samples,both the protein level and mRNA level of HES1 are highly expressed in tumor tissues.In addition,TCGA analysis showed that HES1 was highly expressed in renal clear cell carcinoma,and its high expression was associated with poor prognosis of patients.After the knockdown of HES1 in renal cancer cells,we found that the proliferation ability of renal cancer cells was significantly decreased,and the migration ability and cell vitality of the cells were significantly inhibited.2.According to the results of multi-transcriptomic joint analysis in GEO,the expression of miR-138-2 has significant specificity.Subsequently,in the GESA analysis of TCGA,there was a significant negative correlation between miR-138-2 and NOTCH pathway.Moreover,the mature miR-138-5p and miR-138-2-3p were negatively correlated with NOTCH1 and HES1.Moreover,low expression of miR-138-2 was associated with poor prognosis of patients.Analysis of clinical samples showed that the mature miR-138-5p and miR-138-2-3p of miR-138-2 were significantly down-regulated in tumor tissues.In addition,after exogenous overexpression of miR-138-5p and miR-138-2-3p,the proliferation,migration and cell viability of renal cancer cells were significantly inhibited.However,knockdown of endogenous miR-138-5p and miR-138-2-3p significantly enhanced the proliferation,migration and cell viability of renal cancer cells.Through the analysis of HES1 ChIP-seq data in Encode database,HES1 may be enriched in the vicinity of miR-138-2 promoter.The content changes of miR-138-2 and its mature miR-13 8-5p and miR-138-2-3p were opposite to the changes of HES1 in subsequent knockdown or overexpression.This proves that HES1 has negative regulation on miR-138-2.In ChIP experiments,HES1 was highly enriched near the promoter of miR-138-2,and the binding site of HES1 on the miR-138-2 gene was finally determined by constructing double luciferase mutant plasmids.In vivo experiments of tumor formation in nude mice,knocking down HES1 gene inhibited tumor growth,and miR-138-2 was activated in tumor tissues.3.Through miRNA target database prediction,we found that MAML1,APH1A and NOTCH1 may be potential downstream of miR-138-2.mRNA levels of MAML1,APH1A and NOTCH1 were reversed by exogenous overexpression or endogenous knockdown of miR-138-5p and miR-138-2-3p.By constructing double luciferase mutant plasmids,the binding sites of miR-138-5p and miR-138-2-3p on MAML1,APH1A and NOTCH1 mRNA were identified.In addition,overexpression of miR-138-2 significantly inhibited tumor growth in vivo nude mice,and NOTCH1-HES1 axis-related proteins were significantly inhibited in tumor tissue.Subsequently,dibenzazepine,a classic inhibitor of the NOTCH1 pathway,was used to significantly inhibit the activity of renal cancer cells and inhibit the expression of proteins in the Notch1-HES1 axis.When used in combination with exogenous miR-138-2,dibenzazepine enhances its ability to inhibit the activity of kidney cancer cells.In the response experiment,after HES1 or NOTCH1 was overexpressed at the stable point,miR-138-2 overexpressed plasmid was transfected,which could restore the activity and migration ability of kidney cancer cells,and further established the HES 1-Mir-138-2-Notch1 axis.Conclusion:HES1 is specifically highly expressed in renal cell carcinoma,and can promote a variety of malignant phenotypes of renal cell carcinoma,and is an important epigenetic regulator.In renal cell carcinoma,HES1 can inhibit the gene expression of miR138-2 through transcription and reduce the contents of miR-138-5p and miR-138-3p in the mature body of miR-138-2.However,miR-138-5p and miR-138-3p could inhibit the mRNA of MAML1,APH1A and NOTCH1.In renal cell carcinoma,tumor proliferation and migration can be uncontrolled due to the presence of HES1-miR-138-2-NOTCH1 positive feedback axis.When NOTCH pathway inhibitor is combined with miR-138-2 overexpression,it can effectively inhibit the activity of kidney cancer cells. |
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