The Role And Molecular Mechanism Of MicroRNA-153-5p Targeting AGO1 In Regulating The Malignant Progression Of Renal Clear Cell Carcinoma

Background:Renal cell carcinoma(RCC)is the most common malignant tumor of the adult kidney in my country,and it is also the urological solid tumor with the worst prognosis.Clear cell RCC is the most common pathological subtype of RCC.Further research on the molecular mechanism of RCC metastasis,screening and identification of ccRCC diagnosis and prognostic molecular biomarkers are urgently needed research topics.MicroRNAs(miRNAs),as a non-coding RNA molecule with a length of 19-25 nucleotides in eukaryotes,specifically bind to the 3’UTR of its target gene mRNA,thereby inhibiting the expression of its target gene to play its biological role.Current studies have shown that miRNAs can affect the occurrence and development of tumors through their biological effects.MicroRNA-153-5p(MiR-153-5p)is a miRNA molecule produced at the 5’ end of its precursor molecule.Recent research results show that miR-153-5p is involved in the biological process of tumorigenesis and development,and is expected to become a potential molecular therapeutic target and a molecular biomarker for tumor prognosis.Previous bioinformatics detection revealed that the expression of miR-153-5p was significantly up-regulated in ccRCC tissues and had a significant correlation with the clinicopathological characteristics of the tumor.However,the function and underlying molecular mechanism of miR-153-5p in the occurrence and progression of ccRCC have not been fully elucidated.Further research is needed to clarify the effect of miR-153-5p and its clinical value as a molecular marker.Objective:To investigate the expression difference of miR-153-5p in clear cell renal cell carcinoma and normal renal tissue and the correlation between its expression and the clinicopathological characteristics of the tumor;to explore the biology of miR-153-5p regulating the malignant progression of clear cell renal cell carcinoma The role and its underlying molecular mechanism;exploring the clinical value of miR-153-5p and its target genes as potential molecular targets and prognostic biomarkers in renal clear cell carcinoma.Methods:(1)Bioinformatics analysis was used to identify miR-153-5p as a miRNA molecule related to ccRCC progression,and RT-qPCR was used for expression verification;(2)Bioinformatics analysis was used to explore the relationship between miR-153-5p and clinical symptoms of ccRCC patients.Correlation between pathological parameters;(3)CCK8 assay,tranwells assay and scratch assay were used to up-regulate or down-regulate miR-153-5p expression through transient transfection to explore the changes in ccRCC proliferation and invasion and migration abilities;(4)Construct a subcutaneous tumorigenesis model of renal cancer in nude mice,and further verify the promoting effect of miR-153-5p on the growth of ccRCC by in vivo experiments:(5)RT-qPCR and Western blot were used to detect the mRNA and protein expression changes of downstream potential target genes after overexpression of miR-153-5p;further apply the dual luciferase reporter plasmid system to explore and confirm the direct relationship between miR-153-5p and downstream target genes binding relationship;(6)Verify the correlation between the downstream target gene(AGO1)and the clinicopathological parameters of ccRCC patients and the effect on the proliferation,invasion and migration phenotype of renal cancer cell lines;(7)Reversion experiments further verify that AGO1 mediates the tumor-promoting effect of miR-153-5p.Results:Part I:The differential expression analysis of ccRCC tissue miRNAs in The Cancer Genome Atlas(TCGA)database was performed using bioinformatics algorithms.These analyzes were based on the three dimensions of metastasis,prognosis and differential expression(relative to normal tissue)of ccRCC.For the differential analysis of the metastasis dimension,we divided ccRCC patients into a metastasis(M1)group and a non-metastasis(M0)group.For the prognostic dimension,patients were divided into<1-year survival(1 year)group and>5-year survival(5 years)group.Volcano plot results showed that miR-153-5p was significantly overexpressed in ccRCC tissues and significantly downregulated in both the M0 group and the 5-year group.Therefore,we identified miR-153-5p as a miRNA molecule that promotes ccRCC progression.Upregulation of miR-153-5p expression was found to be significantly associated with clinical features of ccRCC malignant progression.The molecular expression of miR-153-5p was significantly increased in metastatic ccRCC(M1),and the expression of miR-153-5p was also significantly upregulated in ccRCC patients with higher TNM stage and histological stage.The expression of miR-153-5p in renal cancer cell lines was also significantly higher than that in normal renal tissues.In addition,survival analysis results showed that the overall survival of patients with high expression of miR-153-5p was significantly lower than that of patients with low expression of miR-153-5p.Multivariate regression analysis further confirmed that miR-153-5p still has significant prognostic value after adjusting for gender,age,histological grade and stage,suggesting that miR-153-5p is an independent prognostic factor for ccRCC.In addition,KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis and GO(Gene Ontology)analysis found that miR-153-5p is mainly enriched in DNA replication,proliferation biology functional areas and PI3K/Akt signaling pathway,suggesting that miR-153-5p It may exert its effect through the above pathways.Part II:By transfecting ccRCC cells with miR-153-5p inhibitor and mimics,it was found that downregulation of miR-153-5p significantly inhibited ccRCC cell proliferation,cell invasion and migration;upregulation of miR-153-5p significantly promoted ccRCC cell proliferation,cell invasion and migration phenotypes.At the animal level,down-regulation of miR-153-5p can significantly inhibit the growth of subcutaneous tumors in nude mice.Mechanistically,knockdown of miR-153-5p significantly down-regulated the expression levels of PI3K and p-Akt,suggesting that PI3K/Akt may be involved in the tumor-promoting effect of miR-153-5p.In order to further explore the molecular mechanism of miR-153-5p mediating ccRCC proliferation,invasion and migration,the potential downstream target genes of miR-153-5p were screened by bioinformatics algorithm.RT-qPCR and Western blot detection found that the overexpression of miR-153-5p significantly down-regulated the expression of AGO1 mRNA and protein levels.Subsequently,the results of dual luciferase reporter plasmid experiments confirmed that AGO1 is a direct functional target of miR-153-5p.Part III:Clinical correlation analysis found that downregulation of AGO1 expression was significantly associated with clinical features of ccRCC malignant progression.AGO1 expression was significantly decreased in metastatic ccRCC(M1),and AGO1 expression was also significantly downregulated in ccRCC patients with higher TNM stage and advanced histological stage.In addition,the results of survival analysis showed that the overall survival and disease-free survival of patients with high expression of AGO1 were significantly higher than those of patients with low expression of AGO1.Multivariate regression analysis further confirmed that AGO1 still had significant prognostic value after adjusting for gender,age,histological grade and stage,suggesting that AGO1 was an independent prognostic factor for ccRCC.Further studies found that knocking down the expression of AGO1 significantly promoted the proliferation,invasion and migration of ccRCC cells.After cells were co-transfected with miR-153-5p inhibitor and AGO1 small interfering RNA,AGO1 knockdown significantly restored the inhibition of proliferation,invasion and migration phenotypes induced by miR-153-5p downregulation,and partially reversed miR-153-5p downregulation mediated PI3K/Akt signaling pathway inhibition.Finally,at the clinical level,prognostic analysis shows that miR-153-5p combined with AGO1 can more accurately predict the prognosis of patients,suggesting that miR-153-The combined biomarker of 5p and AGO1 has high clinical value.Conclusions:1.MiR-153-5p promotes the biological behavior of ccRCC proliferation,invasion and migration,and is an independent factor affecting the poor prognosis of ccRCC;2.AGO1 is the direct downstream target of miR-153-5p and participates in mediating miR The tumor-promoting effect of-153-5p;3.AGO1 inhibits the biological behavior of ccRCC proliferation,invasion and migration,and is an independent predictor for the prognosis of ccRCC;4.miR-153-5p combined with AGO1 biomarkers has a higher clinical predictive value.