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Silencing circ-USP1 protects the renal tubular from kidney injury induced by hypoxia via modulating miR-194-5p/DNMT3A axis in acute renal allografts.

ABSTRACT: Recent studies indicate that circular RNAs are involved in dysregulation of kidney injury. Nevertheless, the underlying mechanisms remain largely unclear. Therefore, this study sought to investigate the role of circ-USP1 in the pathogenesis of early renal allografts. Thirty-two male C57BL/6J mice aged between 6 and 8 weeks were divided into the sham and allograft groups. Thereafter, the association between miR-194-5p, circ-USP1 and DNMT3A was confirmed using a combination of bioinformatics and the luciferase reporter gene assay. Additionally, the expression of circ-USP1, miR-194-5p and DNMT3A mRNA was detected through qPCR. Afterwards, the Western blot assay was performed to examine the expression of DNMT3A protein. Finally, the TUNEL assay was conducted to determine the rate of apoptosis in DNMT3A cells. The expression of circ-USP1 increased, while that of miR-194-5p decreased in renal allografts. Additionally, silencing circ-USP1 reduced kidney injuries caused by renal allografts in mice. Moreover, miR-194-5p was a target for circ-USP1, and DNMT3A was a target of miR-194-5p. Finally, it was shown that silencing circ-USP1 reduced DNMT3A expression in the kidney of mice that received renal allografts. Circ-USP1 functions as a competing endogenous RNA for miR-194-5p. This occurs in order to regulate DNMT3A expression in kidney injury induced by hypoxia in acute renal allografts.

SUBMITTER: Yao T

PROVIDER: S-EPMC8256350 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Objectives: Circular RNAs (circRNAs) are involved in carcinogenesis, though their expression profile in renal cell carcinoma (RCC) is uncharacterized. The tumor suppressor gene miR-145-5p is expressed in RCC tissues, but its relationship with circRNAs is unknown. Thus, we aimed to identify differentially expressed circRNAs in RCC tissues and to explore the interaction between these circRNAs and miR-145 in the development of RCC. Methods: We performed high-throughput sequencing and bioinformatics analyses to examine the expression pattern of circRNAs in RCC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to functionally annotate differentially expressed circRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for sequence verification. Small interfering RNAs were employed to investigate the function and mechanism of circRNAs in RCC. The relationship between miR-145-5p and circRNAs was confirmed using luciferase, RNA immunoprecipitation (RIP), and biotin-coupled probe RNA pull-down assays. Results: Fifty-three circRNAs were significantly and differentially expressed in RCC compared to normal control tissue. Bioinformatic analyses indicated that two significantly upregulated circRNAs, circ-AFF2 and circ-ASAP1, had sequences corresponding to miR-145 response elements. Consistently, the luciferase reporter, RIP, and biotin-coupled probe RNA pull-down assays showed that circ-AFF2 and circ-ASAP1 may repress miR-145 by acting as sponges. circ-AFF2 and circ-ASAP1 were highly expressed in RCC patient-derived tumor samples; their overexpression correlated with poor prognosis and low miR-145 levels. Knockdown of circ-AFF2 or circ-ASAP1 in RCC cell lines inhibited proliferation, underscoring their oncogenic function. A circRNA-miRNA network was constructed for RCC using the differentially expressed circRNAs and projected miRNAs. Candidate genes were verified by RT-qPCR and western blot, indicating that circ-AFF2 and circ-ASAP1 may be connected to RCC proliferation and metastasis. Conclusion: circ-AFF2 and circ-ASAP1 were upregulated in RCC and likely promote tumor progression by sponging miR-145. Therefore, both circRNAs should be investigated further as potential diagnostic and therapeutic targets for RCC.

Project description:BackgroundNumerous studies proved that long non-coding RNA (lncRNA) is involved in the progression of multifarious diseases, especially in some carcinomas. As a potential tumor biomarker, plasmacytoma variant translocation 1 gene (PVT1) is involved in the development and progression of multifarious cancers. Nevertheless, the intrinsic and concrete molecular mechanism of PVT1 in bladder cancer still remained unclear, which is also the dilemma faced in many non-coding RNA studies.ResultsOur research revealed that PVT1 was significantly higher expression in bladder carcinoma specimens and cell lines. Further experiments indicated that knockdown or overexpression of PVT1 restrained or promoted the malignant phenotype and WNT/β-catenin signaling in bladder cancer cells. Meanwhile miR-194-5p was in contrast and miR-194-5p could partially reverse the function of PVT1 in malignant bladder tumor cells. As a microRNA sponge, PVT1 actively promotes the expression of b-cells lymphoma-2-associated transcription factor 1 (BCLAF1) to sponge miR-194-5p and subsequently increases malignant phenotypes of bladder cancer cells. Therefore, it performs a carcinogenic effect and miR-194-5p as the opposite function, and serves as an antioncogene in the bladder carcinomas pathogenesis.ConclusionPVT1-miR-194-5p-BCLAF1 axis is involved in the malignant progression and development of bladder carcinomas. Experiments revealed that PVT1 has a significant regulatory effect on bladder cancer (BC) and can be used as a clinical diagnostic marker and a therapeutic molecular marker for patients suffering from BC.MethodsIn urothelial bladder carcinoma specimens and cell lines, the relative expression levels of PVT1 and miR-194-5p were detected by quantitative reverse transcription PCR (RT-qPCR). Through experiments such as loss-function and over-expression, the biological effects of PVT1 and miR-194-5p on the proliferation, migration, apoptosis and tumorigenicity were explored in bladder cancer cells. Co-immunoprecipitation, proteomics experiments, dual luciferase reporter gene analysis, western blot and other methods were adopted to investigate the PVT1 potential mechanism in bladder carcinomas.

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Silencing circ-USP1 protects the renal tubular from kidney injury induced by hypoxia via modulating miR-194-5p/DNMT3A axis in acute renal allografts.

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