miR-122-5p modulates the radiosensitivity of cervical cancer cells by regulating cell division cycle 25A (CDC25A)

Abstract

Cervical cancer is one of the most common gynecological malignancies globally, Unfortunately, radiotherapy and chemotherapy are not effective at treating some cases of this disease, and the 5-year survival rate is only 40-50%. Cell division cycle 25A (CDC25A) has been shown to induce radioresistance in a variety of tumor cells, but the role of CDC25A in the radioresistance of cervical cancer has not been fully elucidated. Here, we report that CDC25A is highly expressed and miR-122-5p lowly expressed in cervical cancer tissues and cells. The TargetScan database was used to predict CDC25A as a target of miR-122-5p, and the interactions between miR-122-5p and CDC25A were further confirmed by western blot, real-time PCR and dual-luciferase reporter assay. Under X-ray irradiation, up-regulation of CDC25A can promote the radiation resistance of cervical cancer cells, whereas overexpression of miR-122-5p or knockdown of CDC25A inhibits the survival and induces apoptosis of cervical cancer colonies. In conclusion, our data suggest that miR-122-5p enhances the radiosensitivity of cervical cancer cells by targeting CDC25A.

Keywords: CDC25A; cervical cancer; miR-122-5p; radiosensitivity.

Figure 1

CDC25A was significantly up‐regulated and miR‐122‐5p was significantly down‐regulated in cervical cancer tissues and cells. The representative IHC images of the three levels of staining for CDC25A (strongly positive, weakly positive and negative) are shown in (A). (B) The number of strongly positive, weakly positive and negative expressions of CDC25A in 77 cases of cervical cancer and adjacent tissues was counted. (C) The expression levels of CDC25A in cervical cancer and normal tissues were compared using The Cancer Genome Atlas (TCGA) public dataset. (D, E) The expression of CDC25A in normal cervical epithelial cells (END1/E6E7) and six cervical cancer cell lines (HeLa, HeLa229, C‐33A, CaSki, ME180 and SiHa cells) was detected by western blot (n = 3). (F) qRT‐PCR was used to detect the expression of miR‐122‐5p in cervical cancer tissues and adjacent tissues (n = 3). (G) The expression of miR‐122‐5p in six cervical cancer cells and normal cervical epithelial cells was detected by qRT‐PCR (n = 3). Error bars represent SD. Comparisons between groups were analyzed using t‐tests (two‐sided). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50 μm. CESC, cervical squamous cell carcinoma.

Figure 2

CDC25A reduces the radiosensitivity of cervical cancer cells. (A) Cells models with overexpressed CDC25A (left) and underexpressed CDC25A (right) were successfully constructed. (B) Expression levels of HeLa and SiHa cells at different doses of radiation were detected (n = 3). (C) CCK‐8 was used to evaluate the activity of cervical cancer cells when being exposed to irradiation (n = 3). (D) Flow cytometry was used to evaluate the percentage of apoptotic cervical cancer cells under irradiation (n = 3). (E) Colony formation assay was used to evaluate the cell proliferation under irradiation (n = 3). Error bars represent SD. Comparisons between groups were analyzed using t‐tests (two‐sided). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Targeting relationship between miR‐122‐5p and CDC25A. (A) Prediction of the binding site between miR‐122‐5p and CDC25A by TargetScan. (B, C) qRT‐PCR was used to detect the expression of miR‐122‐5p and CDC25A mRNA in cervical cancer cells (n = 3). (D) Western blot was used to detect the expression of CDC25A in cervical cancer cells (n = 3). (E) WT CDC25A + miR‐122‐5p, mutant type CDC25A + miR‐122‐5p and WT CDC25A + NC were transfected into 293T cells, and the three luciferase activities were compared (n = 3). (F) There was a negative correlation between miR‐122‐5p and CDC25A expression in 77 patients with cervical cancer (Spearman's correlation, P < 0.0001, R = −0.6294). Error bars represent SD. Comparisons between groups were analyzed using t‐tests (two‐sided). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

miR‐122‐5p enhances the radiosensitivity of cervical cancer cells by modulating CDC25A. (A) Western blot was used to detect the expression level of CDC25A after transfection (n = 3). (B) After being treated with 4 Gy ionizing radiation, the cell viability of transfected HeLa and SiHa cells was examined (n = 3). (C) After being exposed to irradiation (4 Gy), the survival of cervical cancer cells was detected by colony formation assay (n = 3). (D) After being exposed to irradiation (4 Gy), the percentage of apoptotic cervical cancer cells was detected by flow cytometry (n = 3). Error bars represent SD. Comparisons between groups were analyzed using t‐tests (two‐sided). *P < 0.05, **P < 0.01, ***P < 0.001.