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CircCRIM1 Promotes Hepatocellular Carcinoma Proliferation and Angiogenesis by Sponging miR-378a-3p and Regulating SKP2 Expression.

ABSTRACT: Mounting evidence has demonstrated that circular RNAs have an important function in tumorigenesis and cancer evolvement. CircCRIM1 has been shown to be a poor prognostic element in multiple human malignancies. However, the clinical significance and mechanism of circCRIM1 in hepatocellular carcinoma (HCC) is still unclear. The present study confirmed the expression level of circCRIM1 using quantitative real-time PCR. In addition, circCRIM1 siRNA and overexpression vectors were used for transfection into LM3 or Huh7 cells to down- or up-regulate the expression of circCRIM1. In vitro and in vivo experiments were performed to explore the function of circCRIM1 in HCC. RNA pull-down, RNA immunoprecipitation, fluorescent in situ hybridization, and luciferase reporter assays were conducted to confirm the relationship between miR-378a-3p and circCRIM1 or S-phase kinase-associated protein 2 (SKP2) in HCC. Then, circCRIM1 was up-regulated in HCC and its expression level was significantly associated with poor prognosis and clinicopathologic characteristics. CircCRIM1 enhanced the proliferation and angiogenesis of HCC cells in vitro and promoted xenograft growth in vivo. Moreover, circCRIM1 upregulated the expression of SKP2 by functioning as a sponge for miR-378a-3p. These findings suggest that circCRIM1 boosts the HCC progression via the miR-378-3p/SKP2 axis and may act as a crucial epigenetic therapeutic molecule target in HCC.

SUBMITTER: Ji Y

PROVIDER: S-EPMC8634842 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As a recently discovered noncoding RNA, circular RNAs (circRNAs) have been identified to play key roles in cancer biology; however, the detailed functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) remain largely unclarified. RNA-seq analysis was used to screen the expression profiles of circRNAs in HCC. CircZNF566 expression in HCC tissues and cell lines was detected by qRT-PCR. In vitro CCK-8, colony formation, wound healing, transwell migration, and invasion assays and in vivo tumorigenesis and metastasis assays were conducted to determine the functions of circZNF566. Luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed to confirm the relationship between circZNF566 and miR-4738-3p. Bioinformatics analysis and luciferase reporter assays were employed to determine whether miR-4738-3p regulates tryptophan 2,3-dioxygenase (TDO2) expression. Finally, immunohistochemistry (IHC) was used to detect the level of TDO2 and determine its prognostic value. CircZNF566 was significantly upregulated in HCC tissues and cell lines. High circZNF566 expression in HCC tissues was positively correlated with clinicopathological features and poor prognosis. Functionally, in vitro experiments showed that circZNF566 promoted HCC cell migration, invasion, and proliferation, whereas in vivo experiments showed that circZNF566 promoted tumorigenesis and metastasis. Mechanistically, circZNF566 acted as a miR-4738-3p sponge to relieve the repressive effect of miR-4738-3p on its target TDO2. In addition, miR-4738-3p suppressed HCC cell migration, invasion, and proliferation, while TDO2 was positively correlated with pathological features and poor prognosis and promoted cell migration, invasion, and proliferation in HCC. CircZNF566 is a novel tumor promoter in HCC and functions through the circZNF566/ miR-4738-3p /TDO2 axis; in addition, circZNF566 may serve as a novel diagnostic marker, prognostic indicator, and target for the treatment of HCC.

Project description:Objective:To explore the regulatory effects of circZNF609 on proliferative and migratory capacities of gastric cancer (GC) and its underlying mechanism. Methods:Expression level of circZNF609, CDK6 and miR-483-3p in GC tissues and cells were detected qRT-PCR verification. CCK-8 and transwell assay were conducted the cell viability and migratory capacities of GC cells. Dual luciferase assay was enrolled to confirm the interaction among circZNF609, CDK6 and miR-483-3p. Western blot was used to detect the protein level of CDK6. Results:Expression levels of circZNF609 were higher in GC patients by qRT-PCR.GC patients with higher expression of circZNF609 were expected to have a higher TNM stage and lower 5-year survival than those with lower expression. ROC curves showed a well diagnostic value of circZNF609 in GC. Treatment of RNase R in GC cells downregulated the expression of ZNF609, whereas circZNF609 expression did not change. Furthermore, cytoplasmic expression of circZNF609 was higher than those of nuclear expression. Besides, biological experiments indicated that overexpression of circZNF609 promoted the proliferative and migratory capacities of GC cells. To demonstrate the underlying mechanism of circZNF609, we found that circZNF609 bound to miR-483-3p, which presented a lower expression in GC tissues than that of paracancerous tissues. Both circZNF609 and miR-483-3p could bind to Ago2, suggesting that circZNF609 may act as a sponge of miR-483-3p. In addition, the effect of overexpressed circZNF609 on cellular behaviors of GC cells were partly reversed by overexpression of miR-483-3p. Bioinformatics suggested that CDK6 has a potential binding site with miR-483-3p. The expression of CDK6 markedly increased in GC tissues and cells, which was negatively correlated with miR-483-3p expression. Dual-luciferase reporter gene results indicated that miR-483-3p could bind to the 3'-UTR of CDK6. Moreover, miR-483-3p downregulated CDK6 at both mRNA and protein levels. Overexpression of miR-483-3p inhibited proliferative and migratory capacities of GC cells, which were reversed by CDK6 overexpression. Conclusion:In summary, the expression of circZNF609 is upregulated in GC. CircZNF609 can be used as the sponge of miR-483-3p to regulate the expression level of CDK6, thus participating in the progression of GC by regulating the proliferative and migratory capacities of GC cells.

Project description:Circular RNAs (circRNAs) have been identified from various tissues and species, but their regulatory functions during developmental processes are not well understood. We examined circRNA expression profiles of two developmental stages of bovine skeletal muscle (embryonic and adult musculus longissimus) to provide first insights into their potential involvement in bovine myogenesis. We identified 12 981 circRNAs and annotated them to the Bos taurus reference genome, including 530 circular intronic RNAs (ciRNAs). One parental gene could generate multiple circRNA isoforms, with only one or two isoforms being expressed at higher expression levels. Also, several host genes produced different isoforms when comparing development stages. Most circRNA candidates contained two to seven exons, and genomic distances to back-splicing sites were usually less than 50 kb. The length of upstream or downstream flanking introns was usually less than 105 nt (mean≈11 000 nt). Several circRNAs differed in abundance between developmental stages, and real-time quantitative PCR (qPCR) analysis largely confirmed differential expression of the 17 circRNAs included in this analysis. The second part of our study characterized the role of circLMO7-one of the most down-regulated circRNAs when comparing adult to embryonic muscle tissue-in bovine muscle development. Overexpression of circLMO7 inhibited the differentiation of primary bovine myoblasts, and it appears to function as a competing endogenous RNA for miR-378a-3p, whose involvement in bovine muscle development has been characterized beforehand. Congruent with our interpretation, circLMO7 increased the number of myoblasts in the S-phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted the proliferation of myoblasts and protected them from apoptosis. Our study provides novel insights into the regulatory mechanisms underlying skeletal muscle development and identifies a number of circRNAs whose regulatory potential will need to be explored in the future.

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CircCRIM1 Promotes Hepatocellular Carcinoma Proliferation and Angiogenesis by Sponging miR-378a-3p and Regulating SKP2 Expression.

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