In the evolving landscape of cancer research, circRNAs have recently emerged as a novel class of ncRNAs originating from eukaryotic pre-mRNA. These intriguing molecules were comprehensively characterized in 2013, with claims of their association with tumorigenesis in various human organs and their potential role in gene expression regulation(18). Emerging research have shown that a variety of circRNAs played an essential role in HCC oncogenesis, and tumor progression as the tumor promoter or suppressor. Overexpression of the several circRNAs might promote the development of HCC, such as circDLG1, circRBM23, circCFH(19–21), acting as the competent endogenous RNAs by sponging the miRNAs to regulate the gene expression. Conversely, downregulation of other circRNAs in the HCC cells, such as circITCH and circZKSCAN1(22, 23), might suppress the oncogenesis via regulating miRNAs or encoding effective inhibitory proteins directly. Our previous study adds a new dimension to this landscape, highlighting the role of circEPS15 in the invasion and metastasis of HCC by encoding a small polypeptide composed of 150-amino acid protein(24). However, no studies have investigated whether hepatocellular carcinoma patients of different genders exhibit similar expression and functional roles of circRNAs.
Therefore, we comparatively analyzed circRNAs expression profiles of tumors in male and female patients with HCC. To identify target circRNA, we performed a comprehensive and multi-faceted approach: First, we completed circRNA expression profiling, followed by a meticulous intersection analysis. From this analysis, we identified seven parental genes that exhibited pairing with target circRNAs among the 50 up-regulated differentially expressed circRNAs. As the key molecules within the protein-protein interaction(PPI) network, circKIF18A plays pivotal roles in regulating mechanism. These selections were grounded in their inferred regulatory roles within pathways closely linked to liver cancer, as predicted by the KOBAS V3.0 database. To corroborate the expression alterations initially identified through microarray data, we conducted qRT-PCR analyses on the aforementioned seven selected circRNAs using female and male tumor tissues. This analysis revealed significant up-regulation for circKIF18A in male tumor tissues, which is in line with the circRNA profiles. Furthermore, we explored the functional role of circKIF18A by performing flow cytometry, wound healing assay, and transwell assay to assess its impact on cell proliferation, migration, and apoptosis. Remarkably, our results demonstrated that the knockdown of circKIF18A significantly suppressed cell proliferation and migration while promoting apoptosis, earning itself the potential role as the therapeutic target.
After elucidating the functions of circKIF18A, it is imperative to delve deeper into the characteristics and role of its parental gene, kinesin family member 18A (KIF18A), particularly in the context of hepatocellular carcinoma (HCC) oncogenesis. As a member of the kinesin superfamily, KIF18A mainly regulates chromosome aggregation, inhibits centromere movement, participates in chromosome stability, and maintains oscillatory attenuation of mitotic spindle microtubules(25). Several studies have illuminated that overexpression of KIF18A can be found in malignancies, such as lung adenocarcinoma, prostate cancer, clear cell renal carcinoma and breast cancer etc.(26–29). Meanwhile, upregulation of KIF18A promotes tumor cell proliferation and inhibits apoptosis, earning itself becoming the potential monitoring indicator and therapeutic target(30, 31). In our study, in alignment with existing evidence, we consistently observed the overexpression of KIF18A in HCC tissues when compared to adjacent non-tumor tissue. This trend was mirrored in the expression of circKIF18A, which is encoded by the same host genes. Moreover, elevated KIF18A expression levels in HCC patients correlated with an unfavorable prognosis (p = 0.001, p < 0.05). These findings suggest that KIF18A may indeed serve as a potential prognostic biomarker for HCC. Additionally, we made a novel discovery that deserves special mention --knocking down circKIF18A significantly inhibited the invasion and metastasis of hepatocellular carcinoma cells. This observation marks the first instance in which we have identified a similar functional role between circKIF18A and its parent gene, KIF18A.
To delve deeper into the potential molecular mechanisms underlying the cancer-promoting effects of circKIF18A, we embarked on an extensive phase of our study involving comprehensive bioinformatic analyses. We conducted GO analysis on the differentially expressed proteins between the KD and NC groups. In biological processes, our analysis reveals significant enrichment in cellular processes, biological regulation, and metabolic processes, highlighting their pivotal roles in cancer progression. In terms of molecular functions, the enrichment predominantly involves binding activities, consistent with the regulatory functions observed in oncogenic pathways. Within cellular components, enrichment is particularly evident in cell parts and organelle components, reflecting their crucial involvement in cellular homeostasis and signaling cascades. We propose that circKIF18A potentially regulates cytoplasmic metabolism via RNA or protein binding. Furthermore, Western blot results suggest that circKIF18A shows no significant association with cell cycle DNA regulation, consistent with the GO analysis outcomes.
To effectively get the signaling pathway by which circKIF18A regulates the HCC growth, we conducted the KEGG enrichment analysis among mRNA-relative DEGs identified from mRNA-seq libraries. And these DEGs were enriched for genes highly in “ESTROGEN_SIGNALING_PATHWAY”, which may lead to the discrepancy of morbidity between males and females because of different estrogen levels. Next, GSEA analysis indicated that “ESTROGEN_SIGNALING_PATHWAY” was highly positively correlated to the level of circKIF18A. Then, we identified 40 Differentially Expressed Genes (DEGs) situated within the Estrogen signaling pathway and found that KPNA2 is the most significant DE protein. Via utilizing GEPIA analysis, we elucidated that high KPNA2 had a negative impact on OS (p < 0.001) among HCC patients. These findings emphasized the potential significance of KPNA2 and the estrogen signaling pathway as valuable prognostic indicators for HCC.
In the context of our study, the protein KPNA2 emerged as a focal point, distinguished by its most significance and its pronounced association with overall survival. These characteristics collectively suggest a substantial relationship between KPNA2 and circKIF18A, positioning KPNA2 as a potential key regulator, influenced by circKIF18A, in the orchestration of HCC tumorigenesis, progression, and apoptosis. KPNA2, also known as importinα-1, a member of the nuclear transporter family, is involved in the nucleocytoplasmic transport pathway of a variety of tumor-associated proteins. Evidence claimed that the plasma concentration of KPNA2 in HCC patients was lower than in healthy individuals(32). A reduction in KPNA2 could inhibit the proliferation and metastasis of HCC cells significantly, which provides the new theoretical foundation for the treatment of HCC(33). Moreover, served as a direct target of miR-139, KPNA2 was up-regulated in HCC and higher KPNA2 level is associated with poor patient prognosis(34). Exploring the potential interaction between circKIF18A and miR-139 in subsequent investigations would be a valuable direction to pursue.
Based on our study and the observed gender differences in hepatocellular carcinoma incidence and mortality, we hypothesize that estrogen may regulate circRNA expression levels via the estrogen signaling pathway, thereby influencing tumor progression and survival prognosis. Lei Wang et al. found there were 10,287 and 8,592 circRNAs found to be induced or repressed by estrogen treatment respectively in breast cancer cells(35). Yanli Wu et al. pointed out that overexpression or silence of estrogen receptor could lead to significant change of the expression level of circRNA in HCC tumor cells(36). Therefore further experiments are required to verify whether estrogen could regulate the expression of circKIF18A.
In conclusion, a novel circRNA named circKIF18A was found upregulated in male patients with HCC, promoting cell proliferation, and migration, while inhibiting apoptosis, highlighting its potential role as a therapeutic target and prognosis biomarker in HCC. Importantly, bioinformatic analysis illuminated a protein named KPNA2 may participate in the cirKIF18A molecular regulatory mechanism through a common signal pathway known as the Estrogen signaling pathway. These findings provide valuable insights for explaining the observed disparities in incidence and mortality rates of HCC between male and female patients, and guide future research directions aimed at unraveling the regulatory mechanisms mediated by circKIF18A and its interaction with estrogen and KPNA2 in HCC.