Dual functions of the ΔNp63-miR-141-3p-YAP1 regulatory axis in cervical cancer progression are dependent on histological subtype

Abstract

Cervical cancer (CC) poses a significant global health challenge, necessitating the development of novel therapeutic strategies. The interplay between the p63 isoform (ΔNp63), microRNA-141-3p (miR-141-3p), and Yes-associated protein 1 (YAP1) has emerged as a potential area of interest in cancer progression. This study aimed to investigate the functional relationship between the between the p63 isoform (ΔNp63), miR-141-3p and YAP1 in modulating migration, invasion, and epithelial-mesenchymal transition (EMT) in two CC cell lines, CaSki, and HeLa, which are human cervical squamous cell carcinoma (SCC) and adenocarcinoma (ADC) cells, respectively. The Gene Expression Omnibus (GEO) datasets, were utilized to assess the expression profiles of TP63 and YAP1 in the cervical SCC and ADC samples by using the GEO2R tool. The prediction software (miRDIP, miRmap, and TargetScan), and RT-qPCR were used to determine the relationship between genes. Different assays were performed for proliferative, migratory and invasive abilities of cells. The results were confirmed by western blot analysis. The ΔNp63-miR-141-3p-YAP1 axis exhibited distinct, cell-line-specific functions. In HeLa cells, this axis promoted a prometastatic phenotype by upregulating YAP1, leading to increased proliferation, migration, invasion, and EMT. Conversely, in CaSki cells, the same axis demonstrated an antimetastatic function by downregulating YAP1. YAP1 expression was significantly higher in HeLa cells compared to CaSki cells. In HeLa cells, YAP1 expression appeared to be regulated not solely by the upstream ΔNp63-miR-141-3p axis, suggesting its role as a key oncogene in HeLa cell progression. MiR-141-3p demonstrated context-dependent effects, exhibiting both pro- and anti-metastatic activities depending on the specific cell line. These findings highlight the complex and subtype-specific functions of the ΔNp63-miR-141-3p-YAP1 regulatory network in cervical cancer progression. In summary, our data highlights the different functions of ΔNp63-miR-141-3p-YAP1 axis in regulating proliferation, migration and invasion as well as EMT of different cervical cancer cells.

Keywords: Cervical cancer; EMT; YAP1; miR-141-3p; ΔNp63.

Fig. 1

A, B In silico analysis of TP63 and YAP1 expression patterns in 19 Squamous cell carcinoma (SCC) and 9 Adenocarcinoma (ADC) tissue samples. Expression levels of TP63 and YAP1 in patients with SCC compared to patients with ADC based on the GSE27388 dataset (****P < 0.0001). C Expression levels of ΔNp63 and YAP1 in CaSki and HeLa cells. ΔNp63 is a major isoform of TP63 and our experimental data specifically refer to ΔNp63 expression, while in silico analysis pertains to TP63 as a whole gene. D Possible interactions between p63-miR141-3p-YAP1 and key regulators of EMT based on in silico analysis. GAPDH was used as an internal control. ** P < 0.01, and *** P < 0.001 indicate significant differences.

Fig. 2

Dual neoplastic features of ΔNp63 knockdown in cervical cancer cell lines A ΔNp63 was silenced in CaSki and HeLa cells by transfection with the specific sh-ΔNp63 construct (qRT‒PCR results). An empty vector was used as a control. B, C After ΔNp63 silencing, proliferation was evaluated through MTT and colony formation assays. D, E Transwell migration and invasion assays revealed that ΔNp63 regulates the migration and invasion of CaSki and HeLa cells in different manners. ** P < 0.01, and *** P < 0.001 represent significant differences; ns = not significant vs. control.

Fig. 3

Suppressive and oncogenic roles of miR-141-3p on CaSki and HeLa cells. A Efficiency of inhibitor/mimic transfection was evaluated by qRT‒PCR. NC-oligos were used as a negative control. B, C Following either miR-141-3p silencing or overexpression, both MTT and colony formation were used to assess cell proliferation. D, E Transwell assays indicated that ΔNp63 regulates the migratory and invasive properties of CaSki and HeLa cells in different manners. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 indicate significant differences.

Fig. 4

sh-ΔNp63/miR-141-3p mimic co-transfection features are comparable to miR-141-3p mimic single transfection. A q-PCR results showed that the expression of miR-141-3p is under the control of ΔNp63. B, C cell proliferation results of sh-ΔNp63/miR-141-3p mimic co-transfection; MTT performed at time points of 0, 24, 48, 72, and 96 h, and colony formation tracked for 11 days D, E Transwell assays were used to identify the influence of single and co-transfections on cell migration and invasion. * P < 0. 1,* * P < 0.01, *** P < 0.001, and **** P < 0.0001 represent significant differences; ns = not significant vs. control.

Fig. 5

The effect of the ΔNp63-miR-141 axis on YAP1 expression. A, B YAP1 expression was evaluated by qRT‒PCR after ΔNp63 knockdown, miR-141 down and upregulation, and sh-∆Np63/miR-141-3p mimic co-transfection in CaSki and HeLa cells. Untreated cells (Cntrl), NC-oligos (Scramble), and mock (sh-NC) were used as control groups. Data for transfected groups were compared to those of the untreated control group. C Western blot results of YAP1, after transfection with sh-∆Np63, miR-141-3p inhibitor and sh-∆Np63/miR-141-3p mimic. Densitometric analysis of western blot bands was performed using ImageJ software, and the results were normalized to GAPDH. Data are presented as mean ± SD from at least three independent experiments. * P < 0. 1, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 indicate a significant difference. Original blots are presented in Supplementary Figs. 1–2.

Fig. 6

Functional roles of ΔNp63-miR-141-3p axis in the EMT process. A Western blot was used to evaluate the expression levels of EMT markers after ΔNp63 silencing and/or miR-141-3p up and downregulation in CaSki and HeLa cells. Densitometric analysis of western blot bands was performed using ImageJ software, and the results were normalized to GAPDH. Data are presented as mean ± SD from at least three independent experiments. B The images show untransfected CaSki cells, transfected with sh-ΔNp63, miR-141-3p inhibitor, miR-141-3p mimic and co-transfected with sh-ΔNp63/ miR-141mimic. Compared to other groups, CaSki cells transfected with sh-ΔNp63 and miR-141-3p inhibitor, exhibited a spindle-like morphology and less cell-cell junctions. Magnification ×10, scale bar 200 μm. Original blots are presented in Supplementary Figs. 3–8.

Fig. 7

Schematic illustration of the ΔNp63–miR-141-3p–YAP1 axis in HeLa (ADC) and CaSki (SCC) cervical cancer cell lines. This regulatory axis promotes EMT, migration, and invasion in HeLa cells but suppresses these processes in CaSki cells through differential YAP1 modulation.