miR‑302c‑3p and miR‑520a‑3p suppress the proliferation of cervical carcinoma cells by targeting CXCL8

Abstract

The aim of the present study was to identify the differentially expressed microRNAs (miRs) in cervical carcinoma (CC) tissues and cells and to explore the function of miR‑302c‑3p and miR‑520a‑3p in the proliferation of CC cells. Potential dysregulated miRNAs in CC tissues and tumour‑adjacent tissues were detected. Reverse transcription‑quantitative PCR (RT‑qPCR) was performed to determine the expression of miR‑302c‑3p, miR‑520a‑3p and CXCL8 in CC tissues and cell lines. The target genes of the miRNAs were predicted using miRTarBase and verified by luciferase reporter assays. RT‑qPCR and western blotting were performed to measure the expression of C‑X‑C motif ligand (CXCL)8 after transfection. The effect on proliferation was verified by Cell Counting Kit assay and ethynyl‑2‑deoxyuridine staining. Flow cytometry was utilised to assess the effect on apoptosis. In the present study, miR‑302c‑3p and miR‑520a‑3p were markedly downregulated in CC cell lines compared to the normal cervical cell line H8. Functionally, overexpression of miR‑302c‑3p and/or miR‑520a‑3p inhibited proliferation and promoted the apoptosis of CC cell lines in vitro, while the knockdown of miR‑302c‑3p and/or miR‑520a‑3p had the opposite effect. Furthermore, miR‑302c‑3p and miR‑520a‑3p could both bind to CXCL8. Inhibition of CXCL8 in combination with miR‑302c‑3p and/or miR‑520a‑3p overexpression exerted proliferation‑suppressive and apoptosis‑stimulating effects on CC cells, whereas restoring CXCL8 attenuated the miR‑302c‑3p‑ and miR‑520a‑3p‑induced anti‑proliferative and pro‑apoptotic effects. miR‑302c‑3p and miR‑520a‑3p suppress the proliferation of CC cells by downregulating the expression of CXCL8, which may provide a novel target for the treatment of CC.

Keywords: cervical carcinoma; miR‑302c‑3p; miR‑520a‑3p; C‑X‑C motif ligand 8; proliferation; apoptosis.

Figure 1.

miR-302c-3p and miR-520a-3p are decreased in CC cell lines and clinical CC tissues. (A) Expression profile of 25 mature miRNA sequences in CC tissues and adjacent tissues. (B and C) The relative expression levels of miR-302c-3p and miR-520a-3p in adjacent normal tissues and CC tissues. (D and E) The relation of the expression of miR-302c-3p and miR-520a-3p between CC and tumour-adjacent tissues displayed with correlational analyses. (F and G) The expression of miR-302c-3p and miR-520a-3p in CC cell lines (HeLa-S3 and C-33A) and the normal cervical epithelial cell line H8. Compared with H8 group. n=3; **P<0.01; ***P<0.001. CC, cervical carcinoma; miR, microRNA.

Figure 2.

Downregulation of miR-302c-3p and miR-520a-3p increases proliferation and decreases apoptosis in CC cells. (A) Reverse transcription-quantitative polymerase chain reaction assays to detect the transfection efficiencies of miR-302c-3p and miR-520a-3p mimics, and mimic NC represents mimic control. Compared with blank group. (B) Cell Counting Kit-8 assays of HeLa-S3 (left) and C-33A (right) cell proliferation after transfection with miR-302c-3p/miR-520a-3p mimics. (C) Changes in CC cell proliferation were also determined by EdU staining (EdU/DAPI). Decreased cell proliferation by upregulating miR-302c-3p or/and miR-520a-3p was observed in HeLa-S3 (top) and C-33A (bottom) cells. Compared with mimic NC group. ‘miR-302c-3p mimic’ vs. ‘miR-302c-3p and miR-520a-3p’: P<0.05 (HeLa-S3), P<0.01 (C-33A); ‘miR-520a-3p mimic’ vs. ‘miR-302c-3p and miR-520a-3p’: P<0.01 (HeLa-S3), P<0.001 (C-33A). (D) The changes in apoptosis of CC cells were determined by flow cytometry. ‘miR-302c-3p mimic’ or ‘miR-520a-3p’ vs. ‘miR-302c-3p and miR-520a-3p’: P<0.001. *P<0.05; **P<0.01; ***P<0.001. CC, cervical carcinoma; miR, microRNA.

Figure 3.

CXCL8 is a common target gene of miR-302c-3p and miR-520a-3p. (A) Expression levels of CXCL8 in tumour-adjacent and CC tissues were detected by reverse transcription-quantitative polymerase chain reaction. (B) Association between expression levels of CXCL8 and miR-302c-3p or miR-520a-3p was displayed with correlational analyses. (C) mRNA (top) and protein (bottom) expression levels of CXCL8 in CC cell lines (HeLa-S3 and C-33A cells) and normal cervical epithelial cells. The mRNA (D) and protein (E) expression level of CXCL8 in HeLa-S3 and C-33A cells after transfection with miR-302c-3p/miR-520a-3p mimics compared to blank cells. ‘miR-302c-3p mimic’ vs. ‘miR-302c-3p and miR-520a-3p’: P<0.05 (HeLa-S3), P<0.05 (C-33A); ‘miR-520a-3p mimic’ vs. ‘miR-302c-3p and miR-520a-3p’: P<0.05 (HeLa-S3), P<0.05 (C-33A), the values represent the gray values of the western blotting bands. (F) Diagram of the CXCL8 3′-UTR-containing reporter construct. Representative luciferase activity in 293T cells co-transfected with wild-type or mutated reporter plasmids and miR-302c-3p or miR-520a-3p mimics. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. CC, cervical carcinoma; miR, microRNA; CXCL8, C-X-C motif ligand 8.

Figure 4.

CXCL8 promotes proliferation and suppresses apoptosis in CC cells. (A) mRNA and protein expression levels of CXCL8 in HeLa-S3 cells and C-33A cells after transfection with si-NC and CXCL8 siRNA (si-CXCL8), pcDNA3.1 and pcDNA3.1-CXCL8, which were compared with si-NC group or pcDNA3.1 group, respectively. (B) Proliferation of HeLa-S3 and C-33A cells in response to transfection of CXCL8 siRNA or pcDNA3.1-CXCL8 or negative control plasmid. (C) EdU assay of HeLa-S3 and C-33A cells transfected with CXCL8 siRNA. Graph shows the proportion of EdU positive cells (EdU/DAPI). (D) Flow cytometry was used to determine the apoptosis of HeLa-S3 and C-33A cells transfected with CXCL8 siRNA. CXCL8 siRNA was compared with si-NC. *P<0.05; **P<0.01; ***P<0.001. CC, cervical carcinoma; CXCL8, C-X-C motif ligand 8; si-NC, siRNA control.

Figure 5.

CXCL8 blocks the proliferation-suppressive effects of miR-302c-3p and miR-520a-3p in CC cell lines. (A) Cell counting kit-8 assay of HeLa-S3 and C-33A cells co-transfected with miR-302c-3p/miR-520a-3p mimics and/or CXCL8 plasmid. (B) EdU assays of HeLa-S3 (top) and C-33A (bottom) cells co-transfected with miR-302c-3p/miR-520a-3p mimics or CXCL8 plasmid. Graph shows the proportion of EdU positive cells. *P<0.05; **P<0.01; ***P<0.001. CC, cervical carcinoma; miR, microRNA; CXCL8, C-X-C motif ligand 8.

Figure 6.

CXCL8 blocks the apoptosis-promoting effects of miR-302c-3p and miR-520a-3p in CC cell lines. Flow cytometry shows the efficacy of pcDNA3.1-CXCL8 in restoring the proliferation-promoting and apoptosis-suppressive function in HeLa-S3 and C-33A cells. CXCL8 siRNA and pcDNA3.1-CXCL8 were compared with si-NC and pcDNA3.1, respectively. Cells co-transfected with miR-302c-3p mimic, miR-520a-3p mimic and pcDNA3.1-CXCL8 were compared with miR-302c-3p mimic and pcDNA3.1 as well as miR-520a-3p mimic and pcDNA3.1. *P<0.05; **P<0.01. CC, cervical carcinoma; miR, microRNA; si-NC, siRNA control; CXCL8, C-X-C motif ligand 8.