LncRNA AFAP1-AS1/miR-27b-3p/VEGF-C Axis Modulates Stemness Characteristics in Cervical Cancer Cells
Cervical cancer (CC) remains a significant global health concern, particularly in developing countries, where it accounts for a high mortality rate among women. Despite advancements in early detection and treatment, the prognosis for patients with metastatic CC remains poor. The complexity of CC carcinogenesis, including its occurrence, metastasis, and treatment resistance, necessitates a deeper understanding of the underlying molecular mechanisms. Recent research has highlighted the role of cancer stem cells (CSCs) in tumor progression, drug resistance, and recurrence. CSCs exhibit stem-like properties, including self-renewal, tumorigenesis, and differentiation, making them a critical focus in cancer research. This study explores the regulatory role of the long non-coding RNA (lncRNA) AFAP1-AS1 in cervical cancer stem cells (CCSCs) and its interaction with microRNA-27b-3p (miR-27b-3p) and vascular endothelial growth factor-C (VEGF-C), providing insights into potential therapeutic targets for CC.
The Role of AFAP1-AS1 in Cervical Cancer
AFAP1-AS1, a lncRNA derived from the antisense strand of the AFAP1 gene locus, has been implicated in various cancers as an oncogenic factor. Its upregulation has been associated with poor prognosis in breast cancer, non-small cell lung cancer (NSCLC), gastric cancer (GC), and nasopharyngeal carcinoma. In CC, AFAP1-AS1 has been linked to tumor invasion and metastasis, but its role in CCSCs remained unexplored. This study demonstrates that AFAP1-AS1 is significantly upregulated in CC cell lines, particularly in CD44v6(+) cells, which are known to exhibit stem-like properties. CD44v6, a variant of the hyaluronic acid receptor CD44, is highly expressed in various cancers and is associated with enhanced tumorigenicity and metastasis.
Isolation and Characterization of CD44v6(+) CC Cells
To investigate the role of AFAP1-AS1 in CCSCs, CD44v6(+) cells were isolated from SiHa and Hela CC cell lines using flow cytometry. These cells exhibited remarkable stemness characteristics, including enhanced self-renewal ability, as evidenced by sphere formation assays. Furthermore, CD44v6(+) cells expressed higher levels of stemness markers such as octamer-binding transcription factor 4 (OCT4), osteopontin (OPN), and cluster of differentiation 133 (CD133) compared to CD44v6(-) cells. These findings confirm that CD44v6(+) cells possess CSC-like properties, making them a suitable model for studying CCSC regulation.
AFAP1-AS1 Knockdown Suppresses Stemness and EMT in CCSCs
To elucidate the functional role of AFAP1-AS1, small interfering RNAs (siRNAs) targeting AFAP1-AS1 (siAFAP1-AS1) were transfected into CD44v6(+) cells. AFAP1-AS1 knockdown significantly inhibited sphere formation, indicating a reduction in self-renewal capacity. Additionally, the expression of stemness markers OCT4, OPN, and CD133 was downregulated in siAFAP1-AS1-treated cells. These results suggest that AFAP1-AS1 plays a crucial role in maintaining the stemness of CCSCs.
Epithelial-mesenchymal transition (EMT) is a key process in cancer metastasis, characterized by the loss of epithelial features and the acquisition of mesenchymal properties. AFAP1-AS1 knockdown also suppressed the expression of EMT-related proteins, including Twist1, matrix metalloproteinase-9 (MMP-9), and VEGF-C. Twist1 is a transcription factor that promotes EMT, while MMP-9 and VEGF-C are involved in extracellular matrix remodeling and angiogenesis, respectively. The downregulation of these proteins suggests that AFAP1-AS1 contributes to the metastatic potential of CCSCs.
AFAP1-AS1 Acts as a Competing Endogenous RNA for miR-27b-3p
LncRNAs often function as competing endogenous RNAs (ceRNAs) by sponging microRNAs (miRNAs), thereby regulating the expression of target genes. This study identified miR-27b-3p as a target of AFAP1-AS1. Bioinformatics analysis predicted binding sites between AFAP1-AS1 and miR-27b-3p, which were confirmed through luciferase reporter assays. RNA pull-down assays further validated the interaction, demonstrating that AFAP1-AS1 directly binds to miR-27b-3p. AFAP1-AS1 knockdown increased miR-27b-3p levels, indicating that AFAP1-AS1 negatively regulates miR-27b-3p by acting as a molecular sponge.
miR-27b-3p Inhibits VEGF-C Expression
miR-27b-3p has been reported as a tumor suppressor in various cancers. This study identified VEGF-C as a downstream target of miR-27b-3p. VEGF-C, a member of the VEGF family, is a potent promoter of lymphatic vessel formation and plays a critical role in tumorigenesis and metastasis. Luciferase reporter assays confirmed that miR-27b-3p binds to the 3′ untranslated region (3’UTR) of VEGF-C, leading to its downregulation. Overexpression of miR-27b-3p significantly reduced VEGF-C protein levels, while inhibition of miR-27b-3p increased VEGF-C expression. These findings establish VEGF-C as a functional target of miR-27b-3p in CCSCs.
The AFAP1-AS1/miR-27b-3p/VEGF-C Axis Regulates CCSC Stemness and EMT
The study further investigated the functional relationship between AFAP1-AS1, miR-27b-3p, and VEGF-C. AFAP1-AS1 knockdown increased miR-27b-3p levels, leading to the suppression of VEGF-C. This cascade inhibited the stemness and EMT of CCSCs, as evidenced by reduced sphere formation, downregulation of stemness markers, and decreased expression of EMT-related proteins. To confirm the role of VEGF-C, CD44v6(+) cells were co-transfected with siAFAP1-AS1 and a VEGF-C overexpression vector. VEGF-C overexpression attenuated the inhibitory effects of AFAP1-AS1 knockdown on stemness and EMT, confirming that VEGF-C is a functional downstream target of the AFAP1-AS1/miR-27b-3p axis.
Implications for Cervical Cancer Therapy
The findings of this study highlight the AFAP1-AS1/miR-27b-3p/VEGF-C axis as a critical regulator of CCSC stemness and EMT. AFAP1-AS1 promotes CCSC self-renewal, tumorigenicity, and metastasis by sponging miR-27b-3p and upregulating VEGF-C. Targeting this axis could provide a novel therapeutic strategy for CC, particularly for patients with advanced or metastatic disease. Inhibition of AFAP1-AS1 or upregulation of miR-27b-3p may suppress CCSC stemness and EMT, thereby reducing tumor progression and improving patient outcomes.
Limitations and Future Directions
While this study provides valuable insights into the role of AFAP1-AS1 in CCSCs, further research is needed to validate these findings in vivo. Animal models of CC could help elucidate the therapeutic potential of targeting the AFAP1-AS1/miR-27b-3p/VEGF-C axis. Additionally, clinical studies are required to assess the correlation between AFAP1-AS1 expression and CC patient prognosis. Investigating the role of this axis in other cancers may also broaden its therapeutic applicability.
Conclusion
In summary, this study demonstrates that AFAP1-AS1 plays a pivotal role in regulating the stemness and EMT of CCSCs through the miR-27b-3p/VEGF-C axis. AFAP1-AS1 knockdown inhibits CCSC self-renewal, tumorigenicity, and metastasis by upregulating miR-27b-3p and suppressing VEGF-C. These findings underscore the potential of targeting the AFAP1-AS1/miR-27b-3p/VEGF-C axis as a novel therapeutic approach for cervical cancer, particularly in cases with advanced or metastatic disease.
doi.org/10.1097/CM9.0000000000001665
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