miR-584 inhibits cell proliferation, migration and invasion in vitro and enhances the sensitivity to cisplatin in human cervical cancer by negatively targeting GLI1

Abstract

Cervical cancer is the most lethal malignancy amongst women worldwide. MicroRNAs (miRNAs/miRs) play a critical role in the progression of cervical cancer. Compelling evidence indicates that miR-584 acts as a tumor suppressor in some types of cancers. However, the function of miR-584 in cervical cancer has not been illustrated. In the present study, the effects and mechanism of miR-584 in the process of proliferation, migration and invasion, and drug sensitivity to cisplatin in cervical cancer were determined. miR-584 expression decreased markedly in cervical cancer tissues and cell lines compared with healthy control samples. Dual-luciferase reporter assays confirmed that glioma-associated oncogene 1 (GLI1) is a novel molecular target of miR-584. The overexpression of miR-584 inhibited the expression of GLI1, reduced cell proliferation, migration and invasion, and induced apoptosis in HeLa cells. However, the silencing of miR-584 in CaSki cells produced the opposite effects. In addition, the overexpression of GLI1 in HeLa-cells overexpressing miR-584 markedly reversed the miR-584-induced inhibitory effect. Flow cytometry results showed that miR-584 enhanced cisplatin sensitivity by promoting chemotherapy-induced apoptosis. Therefore, miR-584 acted as a tumor suppressor miRNA and might be a novel target gene for future cervical cancer treatments.

Keywords: cervical cancer; cisplatin; glioma-associated oncogene 1; microRNA-584; migration and invasion; proliferation.

Figure 1.

Expression of miR-584 is downregulated in human cervical cancer tissues and cells. (A) RT-qPCR was used to detect the expression of miR-584 in 30 pairs of human cervical cancer tissues and normal tissues. (B) The expression of miR-584 in cervical cancer cell lines and normal cervical cell line Ect1/E6E7 were explored using RT-qPCR. *P<0.05. RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA.

Figure 2.

miR-584 inhibits cervical cancer cell proliferation, migration and invasion. (A) miR-584 expression in HeLa cells transfected with mimics or miR-NC and CaSki cells transfected with inhibitors or anti-NC was detected by reverse transcription-quantitative PCR. (B) The cell viability of HeLa cells was tested with a CCK-8 assay. (C) A colony formation assay was used to analyze the proliferation rates of HeLa and CaSki cells. (D) The cell viability of CaSki cells was tested with a CCK-8 assay. (E) A Transwell assay was used to analyze the migration and invasion capability of HeLa cells. (Scale bar, 100 µm; magnification, ×100). (F) A Transwell assay was used to analyze the migration and invasion capability of CaSki cells (Scale bar, 100 µm; magnification, ×100). *P<0.05. miR-NC, mimic negative control; CCK-8, Cell Counting Kit-8; miR, micro-RNA; anti-NC, inhibitor negative control.

Figure 3.

GLI1 is a direct target gene of miR-584. (A) The 3′-UTR of GLI1 mRNA includes a highly conserved binding site for miR-584. (B) 293T cells were co-transfected with miR-584 mimics or inhibitors and wt GLI1 mRNA 3′-UTR or mut GLI1 mRNA 3′-UTR. Luciferase activity was analyzed following 24 h of transfection. (C) mRNA expression of GLI1 was analyzed in HeLa and CaSki cells. β-actin was used as an internal control. (D) The protein expression levels of GLI1 were detected in HeLa and CaSki cells. β-actin served as an internal control. (E) Relative GLI1 mRNA expression was analyzed in tumor tissues the corresponding adjacent normal tissues by reverse transcription-quantitative PCR. β-actin served as internal control. (F) The correlation between the mRNA expression of GLI1 and miR-584 in cervical cancer tissue samples analyzed by Pearson's correlation analysis. *P<0.05. 3′UTR, 3′-untranslated region; wt, wild-type; mut, mutant; miR, microRNA; miR-NC, mimic negative control; anti-NC, inhibitor negative control; GLI1, glioma-associated oncogene 1.

Figure 4.

GLI1 is a functional target gene of miR-584. (A) HeLa cells were co-transfected with miR-584 mimics and GLI1 plasmid or empty plasmid. The protein expression levels of GLI1 were then analyzed by western blotting. (B) Cell proliferation of HeLa cells was analyzed using a Cell Counting Kit-8 assay. (C) Cell proliferation of HeLa cells was analyzed using a colony formation assay. (D) A Transwell assay was used to analyze the migration and invasion capability of HeLa cells (Scale bar, 100 µm; magnification, ×100). *P<0.05. miR, microRNA; GLI1, glioma-associated oncogene 1.

Figure 5.

miR-584 promotes drug sensitivity to cisplatin in cervical cancer cells. HeLa and CaSki cells transfected with miR-584 mimics or miR-NC were incubated with or without 10 µM cisplatin for 24 h. Overexpression of miR-584 increased the proportion of apoptotic cells compared with the miR-NC group in HeLa and CaSki cells. The combination of miR-584 and cisplatin significantly enhanced the apoptosis rate of HeLa and CaSki cells compared with cisplatin or miR-584 mimics, respectively. (A) The apoptosis rate of HeLa. (B) The apoptosis rate of CaSki. *P<0.05. miR, microRNA; miR-NC, mimic negative control.