Introduction: Hepatocellular carcinoma (HCC) carries a poor prognosis, and circular RNAs (circRNAs) are pivotal in its biology. This study aimed to investigate the role of a novel circRNA, circ₀001387, in HCC pathophysiology. Methods: A circRNA-miRNA-mRNA axis was identified via bioinformatics analysis of GEO databases. Functional assays included qRT-PCR, CCK-8, wound healing, transwell, flow cytometry for apoptosis/cell cycle, and western blot for protein detection (CDK4, c-Myc, CyclinD1, GSK-3β, p-GSK-3β, E-cadherin, N-cadherin, MCM6). in vivo tumor growth was assessed in nude mouse xenografts. Interactions were validated by dual-luciferase reporter assays. results: 3. Results 3. 1 Construction of competing endogenous RNA (ceRNA) regulatory network and screening of hub genes We analyzed gene expression datasets GSE155949, GSE108724, and GSE60502 using R software. Through this analysis, we identified 36 downregulated circRNAs (DECs), 63 differentially expressed miRNAs (DEMs), and 344 differentially expressed genes (DEGs), as shown in Figure 1A-C. Utilizing the circBase database, we identified a total of 1225 target genes potentially regulated by DECs. After intersecting these with DEMs, we obtained 21 miRNAs for the construction of a ceRNA network, as depicted in Additional Figure A. Additionally, we identified 6238 target genes that may be regulated by DEMs using TargetScan and miRDB. Intersection with DEGs yielded 109 mRNAs for the subsequent construction of the ceRNA network, detailed in Additional Figure B. The ceRNA regulatory network was constructed and presented in Figure 1D. Subsequently, the 109 genes within the ceRNA regulatory network were subjected to KEGG pathway enrichment analysis using the David database, as shown in Additional Figure C. Furthermore, survival analyses through the TCGA database indicated that 19 genes were significantly associated with poor outcomes in HCC, illustrated in Additional Figure D. To establish the comprehensive circRNA-miRNA-mRNA network, we employed Cytoscape software, as visualized in Figure 1E. The CytoHubba plugin within Cytoscape was utilized to perform degree-based algorithms to analyze gene relationships. The results highlighted MCM6 as a hub gene with a central role in the network, as demonstrated in Figure 1F-G. Based on these findings, we decided to further investigate the regulatory axis of circ₀001387, miR-4324, and MCM6 both in vivo and in vitro. 3. 2 Cicr₀001387 or MCM6 increases and miR-4324 decreases in HCC tissues and cell lines The expression levels of circ₀001387, miR-4324, and MCM6 in HCC tissues and cell lines were evaluated using RT-qPCR. The results revealed that miR-4324 was significantly downregulated, whereas circ₀001387 and MCM6 were upregulated in HCC tissues and cell lines compared to adjacent normal tissues or normal cell lines, as depicted in Figure 1H-M. Among the cell lines tested, HepG2 exhibited notably aberrant expression patterns, which led to its selection for further experimental investigation. 3. 3 Circ₀001387 Exacerbates the Malignant Phenotype of HCC In vitro Firstly, RT-qPCR analysis was conducted to assess the transfection and expression efficiency of si-circ₀001387 and OE-circ₀001387 in HepG2 cells (Fig. 2A). Subsequent assays, including CCK-8, Wound healing, and Transwell, demonstrated that overexpression of circ₀001387 significantly enhanced the growth, migration, and invasion of HepG2 cells, while its knockdown had the opposite effect (Fig. 2C-E). Flow cytometry results indicated that overexpression of circ₀001387 led to fewer cells in the G1 phase, more cells in the S phase, and a decreased apoptosis rate compared to other groups (Fig. 2F-G). Western blot assays further confirmed that overexpression of circ₀001387 promoted epithelial-mesenchymal transition (EMT), whereas its knockdown showed the opposite effect (Fig. 2B). These results suggest that circ₀001387 plays an oncogenic role in the development of HCC. 3. 4 Circ₀001387 acts as a sponge for miR-4324, and its effect is reversed by miR-4324 in HCC cells Previous bioinformatics analysis identified a potential binding region between circ₀001387 and miR-4324. Dual-luciferase reporter assays were performed to validate this regulatory relationship. The results showed that the effect of the WT circ₀001387 vector was attenuated by the miR-4324 mimic in HepG2 cells, whereas no effect was observed with the MUT vector (Fig. 3A-B). Additionally, miR-4324 levels were found to increase upon circ₀001387 knockdown and decrease upon its overremovedFig. 3C). Conversely, the level of circ₀001387 was also found to be regulated by miR-4324 expression levels (Additional Fig F). These findings indicate that circ₀001387 and miR-4324 may mutually modulate each other’s expression. Further in vitro studies revealed that the oncogenic effects mediated by circ₀001387 were partially reversed by miR-4324 inhibition (Fig. 3D-M). In summary, circ₀001387 appears to promote HCC progression by acting as a sponge for miR-4324. 3. 5 MCM6 is a target gene of miR-4324 and can reverse the effect of miR-4324 in HCC A binding region between miR-4324 and MCM6 was identified using the TargetScan database. Dual-luciferase reporter analysis confirmed the regulatory relationship, with suppressed luciferase activity observed in the presence of the miR-4324 mimic for the MCM6-WT but not the MCM6-MUT (Fig. 4A-B). RT-qPCR analysis of HCC cell lines with miR-4324-mimic or inhibitor revealed significant downregulation of MCM6 in the miR-4324-mimic cell line and upregulation in the miR-4324-inhibitor cell line (Fig. 4C). Furthermore, the level of miR-4324 was found to be inversely related to MCM6 expression levels in HepG2 cells (Fig. 4D). In vitro studies showed that the effects of miR-4324 knockdown on HCC cell development, invasion, migration, and apoptosis were partially reversed by MCM6 knockdown (Fig. 4E-M). Collectively, these results suggest that MCM6 is a target gene of miR-4324 and can reverse the effects of miR-4324 in HCC. 3. 6 Circ₀001387 positively regulates the expression of MCM6, and its effect on HCC can be partially reversed by MCM6 Bioinformatics analysis suggested that circ₀001387 may regulate downstream target gene expression and exert oncogenic effects through the miR-4324/MCM6 axis. To verify this, RT-qPCR and Western blotting were used to assess MCM6 expression levels, which were found to be significantly reduced in circ₀001387-knockdown HepG2 cells and enhanced in circ₀001387-overexpressing cells (Fig. 5A-B). Functional rescue experiments using MCM6 overexpression demonstrated that the effects of circ₀001387 knockdown on HCC progression were mitigated by MCM6 overremovedFig. 5C-L). These results indicate that MCM6 is an important target gene of circ₀001387, and the effects of circ₀001387 in HCC can be partially reversed by MCM6. 3. 7 Overexpression of Circ₀001387 inhibits tumor growth in vivo To investigate the effect of circ₀001387 on tumor growth in vivo, HepG2 cells transfected with OE-circ₀001387, OE-NC, and OE+Phenoxodiol were injected into nude mice. Weekly measurements of tumor volume indicated that tumors in the OE-circ₀001387 group were larger and heavier than those in the other groups (Fig. 6A-D). RT-qPCR and Western blot analysis of collected tumor samples showed significantly increased MCM6 mRNA expression in the circ₀001387 overexpression group compared to the OE-NC group (Fig. 6E-F). 3. 8 Activation of the cell cycle pathway is involved in the oncogenic functions of the Circ₀001387/miR-4324/MCM6 regulatory axis KEGG analysis indicated a relationship between MCM6 and cell cycle signaling pathways. Western blot analysis both in vitro and in vivo assessed the activation of the Cell Cycle pathway. Expression levels of CDK4, c-Myc, CyclinD1, and p-GSK-3β, which are associated with cell cycle pathways, were found to change in response to circ₀001387 modulation and MCM6 interference (Fig. 6G-H). Furthermore, treatment with a Cell Cycle inhibitor (Phenoxodiol) in vivo partially reversed the changes in these proteins induced by circ₀001387 (Fig. 6I). Overall, these results suggest that the circ₀001387/miR-4324/MCM6 regulatory axis contributes to HCC tumorigenesis, at least in part, by activating the Cell Cycle pathway. Results: Circ₀001387 and MCM6 were upregulated in HCC tissues and cells, while miR-4324 was downregulated. Overexpression of circ₀001387 promoted HCC cell proliferation, migration, invasion, and tumor growth in vivo, while inhibiting apoptosis and cell cycle arrest. Knockdown produced opposite effects. Mechanistically, circ₀001387 directly sponged miR-4324, which targeted MCM6. Rescue experiments confirmed that miR-4324 inhibition or MCM6 overexpression reversed the effects of circ₀001387 knockdown, whereas MCM6 silencing offset miR-4324 knockdown. Discussion: Our findings delineate a novel oncogenic axis in which circ₀001387 drives HCC progression by sequestering miR-4324 to elevate MCM6 expression. This pathway underscores the significant regulatory role of circRNAs in HCC and aligns with emerging evidence of competing endogenous RNA networks in cancer. Study limitations include the need for further clinical validation of this axis as a biomarker.
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