Abstract
Circular RNAs have been reported to play significant roles in regulating pathophysiological processes while also guiding clinical diagnosis and treatment of hepatocellular carcinoma (HCC). However, only a few circRNAs have been identified thus far. Herein, we investigated the role of a specific closed-loop structure of hsa_circ_101555 that was generated by back-splicing of the host gene casein kinase 1 gamma 1 (CSNK1G1) in the development and proliferation of HCC. We investigated the expression of Hsa_circ_101555 in HCC and normal tissues using bioinformatics. The expression level of hsa_circ_101555 was further detected by fluorescence in situ hybridization and qRT-PCR in ten HCC patients. Transwell, migration, WST-1 assays, and colony formation assays were used to evaluate the role of hsa_circ_101555 in HCC development and proliferation. The regulatory mechanisms of hsa_circ_101555 in miR-145-5p and CDCA3 were determined by dual luciferase reporter assay. A mouse xenograft model was also used to determine the effect of hsa_circ_101555 on HCC growth in vivo. hsa_circ_101555 showed greater stability than the linear RNA; while in vitro and in vivo results demonstrated that hsa_circ_101555 silencing significantly suppressed cell proliferation, migration, and invasion of HCC cells. Rescue experiments further demonstrated that suppression of miR-145-5p significantly attenuated the biological effects of hsa_circ_101555 knockdown in HCC cells. We also identified a putative oncogene CDCA3 as a potential miR-145-5p target. Thus, our results demonstrated that hsa_circ_101555 might function as a competing endogenous RNA of miR-145-5p to upregulate CDCA3 expression in HCC. These findings suggest that hsa_circ_101555 may be a potential therapeutic target for patients with HCC.
Highlights
Hepatocellular carcinoma (HCC), the most prevalent form of primary liver cancer, represents one of the most common malignant tumors globally[1]
We found that the proliferation and colony-forming ability of HCC cells co-transfected with si-hsa_circ_101555_003 and miR-145-5p inhibitor were higher than those of HCC cells transfected with only siRNA-hsa_circ_101555, suggesting that the downregulation of miR-145-5p could partially reverse the proliferation-inhibitory effect of siRNAhsa_circ_101555 (Fig. 5I–L)
We found that: (1) hsa_circ_101555 is highly expressed in HCC tissues and HCC cell lines (HepG2 and HCCLM3); (2) silencing hsa_circ_101555 in a murine xenograft model significantly reduces the growth of HCC; (3) hsa_circ_101555 is likely required to sustain proliferation, migration, and invasion in HCC cell lines; (4) hsa_circ_101555 acts as an miR-145-5p sponge, while silencing hsa_circ_101555 significantly inhibits cell growth; (5) hsa_circ_101555 sponges miR-145-5p to promote CDCA3 expression; and (6) eIF4A3 induces hsa_circ_101555 cyclization and increases hsa_circ_101555 expression
Summary
Hepatocellular carcinoma (HCC), the most prevalent form of primary liver cancer, represents one of the most common malignant tumors globally[1]. Gu et al Cell Death and Disease (2021)12:356 lack 5′ caps and 3′ polyadenylated tails, making them more stable than linear RNAs6. CircRNAs reportedly function as molecular sponges for microRNAs (miRNAs)[7,8] and RNA-binding proteins[9], while serving as vital regulators of gene transcription and expression[10,11]. CircRNAs are highly conserved across multiple species and exhibit tissue-specific and development stage-dependent expression patterns[12,13]. These features imply that circRNAs possess significant functions in biological and pathological processes. Compared with other ncRNAs, such as miRNAs and long noncoding RNAs, the research on circRNA in HCC remains in its infancy. Only a few functional circRNAs have been discovered and characterized in HCC21, while a large number remain to be explored or identified
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