Abstract
Clear cell renal cell carcinoma (ccRCC) is a prevalent urological carcinoma with high metastatic risk. Circular RNAs (circRNAs) have been identified as effective diagnostic and therapeutic biomarkers for ccRCC. This research aims to disclose the effect and regulatory mechanism of circRNA ribosomal protein L23a (circ_RPL23A) in ccRCC. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to examine circ_RPL23A, microRNA-1233 (miR-1233) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). Cell cycle progression, apoptosis, cell viability, invasion and migration, which were respectively conducted by using flow cytometry, 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT), transwell assays. The levels of ACAT2 protein and cell cycle proteins, proliferation-associated protein, and epithelial-mesenchymal transition (EMT) associated proteins were measured by western blot. Target relationship was analyzed via dual-luciferase reporter assay and RNA pull down assay. The animal model was used to study how circ_RPL23A affects in vivo. Circ_RPL23A was lower expressed in ccRCC tissues and cells. The elevated circ_RPL23A suppressed cell cycle progression, proliferation, migration and invasion but promoted apoptosis in ccRCC cells. MiR-1233 was a target of circ_RPL23A and direct targeted to ACAT2. Besides, circ_RPL23A exerted its anti-tumor effect by sponging miR-1233, and then relieved the inhibition effect of miR-1233 on ACAT2. Overexpression of circ_RPL23A also curbed ccRCC tumor growth in vivo. Circ_RPL23A inhibited ccRCC progression by upregulating ACAT2 expression by competitively binding miR-1233, which might provide an in-depth cognition for ccRCC pathogenesis and circ_RPL23A might be a promising biomarker in ccRCC diagnosis and treatment.
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Abbreviations
- ccRCC:
-
Clear cell renal cell carcinoma
- ACAT2 :
-
acetyl-coenzyme A acetyltransferase 2
- circ_RPL23A :
-
circRNA ribosomal protein L23a
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The data that support the findings of this study are available on request from the corresponding author.
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This work was supported by Provincial Department of Education Fundamental Research Operating Expenses of Basic Research Projects (Grant No. 2018-KYYWF-0968).
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Supplementary Information
Supplementary Fig. 1
Linear RPL23A was downregulated in ccRCC samples. The qRT-PCR was performed to determine the expression of RPL23A in 60 ccRCC and normal samples. Data are reported as mean ± SD.*P < 0.05 vs normal para-carcinoma tissues. (PNG 2780 kb)
Supplementary Fig. 2
Circ_RPL23A depletion accelerated tumor progression in ccRCC cells. (A-B) The transfection efficiency was assessed by qRT-PCR after 786-O and 769-P cells were transfected with si-circ_RPL23A or si-NC. (C-D) Cell cycle progression in transfected cells was detected by PI-flow cytometry assay. (E-F) Western blot assay was administrated for determining cell cycle proteins CyclinD1 and CDK4 upon transfection. (G-H) Cell viability upon transfection was examined by MTT assay. (I-J) The protein levels of cell proliferation marker (Ki67, PCNA) upon transfection were detected by western blot. (K) The apoptosis rate of transfected cells was examined using Annexin V/PI-flow cytometry. (L-M) Transwell assay was used to measure the migration and invasion of transfected cells. (N-O) The determination of snail and E-cadherin proteins was carried out via western blot. Data are reported as mean ± SD, n = 3.*P < 0.05 vs si-NC group (ANOVA). (PNG 28450 kb)
Supplementary Fig. 3
The direct interaction between circ_RPL23A and miR-1233. RNA pull down assay was employed to analyze the enrichment of miR-1233 in 786-O and 769-P cells with biotin-labeled circ_RPL23A-WT or circ_RPL23A-MUT probe. Data are reported as mean ± SD, n = 3. *P < 0.05 vs bio-NC group (ANOVA). (PNG 1379 kb)
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Cheng, L., Cao, H., Xu, J. et al. Circ_RPL23A acts as a miR-1233 sponge to suppress the progression of clear cell renal cell carcinoma by promoting ACAT2. J Bioenerg Biomembr 53, 415–428 (2021). https://doi.org/10.1007/s10863-021-09901-8
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DOI: https://doi.org/10.1007/s10863-021-09901-8