N6-methyladenosine modification of CENPK mRNA by ZC3H13 promotes cervical cancer stemness and chemoresistance

Abstract

Background: Stemness and chemoresistance contribute to cervical cancer recurrence and metastasis. In the current study, we determined the relevant players and role of N⁶-methyladenine (m⁶A) RNA methylation in cervical cancer progression.

Methods: The roles of m⁶A RNA methylation and centromere protein K (CENPK) in cervical cancer were analyzed using bioinformatics analysis. Methylated RNA immunoprecipitation was adopted to detect m⁶A modification of CENPK mRNA. Human cervical cancer clinical samples, cell lines, and xenografts were used for analyzing gene expression and function. Immunofluorescence staining and the tumorsphere formation, clonogenic, MTT, and EdU assays were performed to determine cell stemness, chemoresistance, migration, invasion, and proliferation in HeLa and SiHa cells, respectively. Western blot analysis, co-immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter, cycloheximide chase, and cell fractionation assays were performed to elucidate the underlying mechanism.

Results: Bioinformatics analysis of public cancer datasets revealed firm links between m⁶A modification patterns and cervical cancer prognosis, especially through ZC3H13-mediated m⁶A modification of CENPK mRNA. CENPK expression was elevated in cervical cancer, associated with cancer recurrence, and independently predicts poor patient prognosis [hazard ratio = 1.413, 95% confidence interval = 1.078 - 1.853, P = 0.012]. Silencing of CENPK prolonged the overall survival time of cervical cancer-bearing mice and improved the response of cervical cancer tumors to chemotherapy in vivo (P < 0.001). We also showed that CENPK was directly bound to SOX6 and disrupted the interactions of CENPK with β-catenin, which promoted β-catenin expression and nuclear translocation, facilitated p53 ubiquitination, and led to activation of Wnt/β-catenin signaling, but suppression of the p53 pathway. This dysregulation ultimately enhanced the tumorigenic pathways required for cell stemness, DNA damage repair pathways necessary for cisplatin/carboplatin resistance, epithelial-mesenchymal transition involved in metastasis, and DNA replication that drove tumor cell proliferation.

Conclusions: CENPK was shown to have an oncogenic role in cervical cancer and can thus serve as a prognostic indicator and novel target for cervical cancer treatment.

Keywords: Centromere protein K; Cervical cancer; Chemoresistance; N 6-methyladenosine; Stemness.

Fig. 1

ZC3H13 participates in regulating m⁶A modification of CENPK mRNA. a Correlogram displaying the link between CENPK and 21 m⁶A regulators expression as per the TCGA CESC dataset. The red frame emphasizes the correlation between CENPK and 21 m⁶A regulators expression. b Relationship between CENPK and ZC3H13 expression as per the TCGA CESC dataset. c qPCR and Western blotting determining the effect of ZC3H13 knockdown on CENPK expression. d MeRIP assays were adopted to measure the m⁶A modification of CENPK mRNA. The enriched RNAs were subjected to reverse transcription and further analyzed by qPCR and PCR. e Bioinformatics analysis predicting m⁶A sequences within the CENPK 3′-UTR. f Luciferase reporter assays were carried out for validating the effect of ZC3H13 knockdown on the post-transcriptional repression of CENPK in HeLa and SiHa cells. Data are represented as the mean ± SD. ^**P < 0.01; ^***P < 0.001; ns non-significant

Fig. 2

CENPK expression is elevated in cervical cancer and confers poor patient prognosis. a Representative images showing the differential CENPK expression between cervical cancer and adjacent normal tissues. b Representative images showing the relationship between CENPK and Ki67 expression in cervical cancer. c Kaplan–Meier survival analysis disclosing the overall survival of cervical cancer patients according to CENPK expression. d Kaplan–Meier survival analysis displaying the recurrence-free survival of cervical cancer patients according to CENPK expression. e Univariate and multivariate analyses were adopted for correlating CENPK expression, clinicopathological characteristics, and overall survival of cervical cancer patients. f Univariate and multivariate analyses were performed to correlate CENPK expression, clinicopathological characteristics, and recurrence-free survival of cervical cancer patients. g Development of a nomogram for predicting cancer recurrence of cervical cancer patients. HR hazard ratio

Fig. 3

CENPK promotes cervical cancer stemness, chemoresistance, metastasis, and proliferation. Tumorsphere formation (a) and immunofluorescence assays (b) were adopted to measure stemness of CENPK-depleted HeLa and SiHa cells and the control cells. c Clonogenic assays were adopted to estimate chemoresistance of CENPK-depleted HeLa and SiHa cells and the control cells treated with cisplatin and carboplatin. d Transwell assays were performed to elucidate the migration and invasion of CENPK-silenced HeLa and SiHa cells and the control cells. MTT assays (e), and colony-formation assays (f) were applied to evaluate the proliferation of CENPK-silenced HeLa and SiHa cells and the control cells. g Immunofluorescence was adopted for detecting the expression of γ-H2AX (Ser139) in CENPK-silenced HeLa treated with cisplatin (10 μmol/L for 24 h), CENPK-silenced SiHa cells treated with carboplatin (100 μmol/L for 24 h), and control cells. h EdU incorporation assays were used for elucidating DNA replication of CENPK-depleted HeLa and SiHa cells and the control cells. i Western blotting analysis of the expression of proteins associated with stemness (c-Myc), DNA damage repair (p53), epithelial-mesenchymal transition (Vimentin), and DNA replication (p21, CCND1 and c-Jun) in CENPK-depleted HeLa and SiHa cells and control cells. Data are represented as the mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 4

CENPK enhances tumorigenic functions of cervical cancer cells in vivo. a qPCR analyses of CENPK mRNA levels in HeLa and SiHa-derived xenografts with CENPK silencing and controls. b-d Impact of CENPK on cervical cancer proliferation (n = 5/group), metastasis (n = 5/group), and tumor formation ability (n = 6/group) was detected by establishing the xenograft mouse models. Tumor volume (e) and tumor weight (f) were calculated and recorded. g Xenograft tumors were subjected to detection of Ki67 expression by immunohistochemistry (IHC). h Survival analyses were performed to compare the overall survival time of the mice in the CENPK-silenced group (n = 10), the cisplatin/carboplatin-treated group (n = 10), the CENPK knockdown plus cisplatin/carboplatin-treated group (n = 10), and the controls (n = 10). Data are represented as the mean ± SD. ^**P < 0.01, ^***P < 0.001

Fig. 5

CENPK interacts with SOX6 to activate Wnt signaling and inactivate p53 signaling in cervical cancer. a. TOP/FOP luciferase reporter assays were conducted for measuring the impact of CENPK and SOX6 on Wnt signaling activity. b. Co-immunoprecipitation analyses displaying the interaction between CENPK and SOX6. c. Immunofluorescence co-staining showing the colocalization of CENPK and SOX6. The red bars indicated by the arrows represent the colocalization of CENPK and SOX6. d. Co-immunoprecipitation analyses displaying the effect of CENPK knockdown on the interaction between CENPK and SOX6, and the impact of CENPK knockdown on the interplay between SOX6 and β-catenin. e. Immunofluorescence co-staining and cell fractionation assays showing the effect of CENPK on the expression and nuclear translocation of β-catenin and SOX6 in HeLa and SiHa cells incubated with lithium (30 nmol/L). f. Luciferase reporter assays were performed for elucidating the effect of CENPK and SOX6 on p53 signaling activity. g. Chromatin immunoprecipitation analyses were applied to estimate the impact of CENPK on SOX6-mediated c-Myc transcription. h. Cycloheximide chase assays assessing the impact of CENPK and SOX6 on p53 stability in HeLa cells. i. Co-immunoprecipitation analyses verifying the impact of CENPK and SOX6 on p53 ubiquitination in HeLa cells treated with MG132. j. Cell fractionation assays validating the impact of CENPK and SOX6 on p53 nuclear export in HeLa cells. Data are represented as the mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; ns non-significant; IP immunoprecipitation; LiCl lithium

Fig. 6

Working model of the ZC3H13-CENPK-SOX6 regulatory axis in controlling stemness, DNA damage repair, cell cycle, DNA replication, and EMT through Wnt and p53 signaling