miR‑218 functions as a tumor suppressor gene in cervical cancer

Abstract

Previous microRNA (miR) microarray analysis revealed that miR‑218 is downregulated in cervical cancer tissues. The present study aimed to further evaluate the expression of miR‑218 in cervical cancer specimens, determine the association between its expression with disease progression, and investigate the roles of miR‑218 in cervical cancer cells. Tissue specimens were obtained from 80 patients with cervical squamous cell carcinoma, 30 patients with high‑grade cervical intraepithelial neoplasia [(CIN) II/III] and 15 patients with low‑grade CIN (CINI); in addition, 60 plasma samples were obtained from patients with cervical cancer, and 15 normal cervical tissue specimens and 30 plasma samples were obtained from healthy women. These samples were used for analysis of miR‑218 expression via reverse transcription‑-quantitative PCR. In addition, tumor cells were transfected with miR‑218 mimics, human papillomavirus (HPV)16 E6/E7 small interfering RNA, or their respective negative controls to determine the viability, colony formation, migration and invasion of cells using MTT, colony formation, wound healing and Transwell assays, respectively. Target genes of miR‑218 were bioinformatically predicted and analyzed using Gene Ontology (GO) terms. The results revealed that miR‑218 was downregulated in the tumor tissues and plasma of patients with cervical cancer, with expression associated with the advanced clinicopathological characteristics of patients, including HPV positivity, tumor size, blood vessel invasion and lymph node metastasis. Furthermore, miR‑218 overexpression reduced tumor cell viability and xenograft growth, and suppressed tumor cell migration and invasion. HPV was detected in 75% of the 80 patients with cervical cancer, and HPV positivity was inversely associated with miR‑218 expression. In addition, bioinformatics analysis predicted that roundabout guidance receptor 1 (ROBO1) was a target gene of miR‑218; miR‑218 overexpression significantly reduced ROBO1 levels. Furthermore, GO analysis revealed that ROBO1 was involved in regulating cell proliferation, adhesion and migration, and the cell cycle. In conclusion, the findings of the present study suggested that miR‑218 may possess antitumor activities in cervical cancer.

Keywords: cervical cancer; microrna; mir-218; biomarker.

Figure 1.

Differential expression of miR-218 in LSIL, HSIL and cervical cancer tissues. Tissue specimens obtained from 80 cervical squamous cell carcinoma lesions, 30 HSILs, 15 LSILs and 15 normal cervical tissue specimens were analyzed for miR-218 expression via reverse transcription-quantitative PCR. **P<0.01 and ***P<0.001. HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; miR-218, microRNA-218.

Figure 2.

Downregulation of miR-218 in plasma samples from patients with cervical cancer. miR-218 expression in plasma samples from patients with cervical cancer and healthy controls, as determined via reverse transcription-quantitative PCR analysis. *P<0.01. miR-218, microRNA-218.

Figure 3.

Effects of miR-218 expression on cervical cancer cell viability and xenograft growth. (A) Cervical cancer cell lines SiHa, HeLa and C-33A were transfected with miR-218 mimics or NC, and harvested to analyze miR-218 expression via reverse transcription-quantitative PCR. (B) Viability of stable miR-218-expressing SiHa and C-33A cells as determined by MTT assays (*P<0.05). (C) Colony formation of stable miR-218-expressing SiHa and C-33A cells. (D) Stably miR-218-overexpressing SiHa cells were injected into nude mice. These mice were then sacrificed 4 weeks later. (E) Tumor xenograft volume (n=6). (F) Tumor xenograft weight (n=6). **P<0.01. C-218/-NC, C-33A cells transfected with miR-218 mimics/NC; H-218/-NC, HeLa cells transfected with miR-218 mimics/NC; S-218/-NC, SiHa cells transfected with miR-218 mimics/NC; miR-218, microRNA-218; NC, negative control.

Figure 4.

Effects of miR-218 overexpression on tumor cell migration and invasion. (Aa) Stable miR-218-expressing SiHa, HeLa and C-33A cells were grown and subjected to wound-healing assays. Images were acquired under a fluorescence microscope to demonstrate the efficiency of gene infection (the lentiviruses contained the GFP gene). (Ab) Quantification of the wound healing assay. Transwell (Ba) migration and (Ca) invasion assays of S-218, H-218 and C-218 cells. (Bb and Cb) Quantification of the Transwell assays. **P<0.01. C-218/-NC, C-33A cells transfected with miR-218 mimics/NC; H-218/-NC, HeLa cells transfected with miR-218 mimics/NC; S-218/-NC, SiHa cells transfected with miR-218 mimics/NC; miR-218, microRNA-218; NC, negative control.

Figure 5.

HPV16 E6/E7 regulates miR-218 expression in cervical cancer. (A) HPV status was determined in 80 cases of cervical cancer tissues via PCR analysis, whereas miR-218 levels were evaluated via RT-qPCR analysis. It was revealed that miR-218 expression was low in HPV-positive cervical cancer tissues compared within HPV-negative cervical cancer tissues. (B) Relative miR-218 levels in SiHa (HPV16+), HeLa (HPV18+) and C-33A (HPV-) cells. (C) SiHa cells were transfected with HPV16 E6/E7 siRNA or respective NC siRNA for 48 h and then subjected to RT-qPCR analysis. Expression of HPV16 E6 or E7 was significantly knocked down in HPV16 E6/E7 siRNA-infected SiHa cells. (D) Relative miR-218 levels in HPV16 E6/E7 siRNA-transfected cervical cancer cells compared with NC siRNA-transfected cells. **P<0.01. HPV, human papillomavirus; miR-218, microRNA-218; RT-qPCR, reverse transcription-quantitative PCR; siRNA, small interfering RNA.

Figure 6.

Relative expression levels of ROBO1 in miR-218-overexpressing cervical cancer cells. (A) SiHa, (B) HeLa and (C) C-33A cells were transfected with miR-218 mimics or NC, and ROBO1 expression was evaluated via reverse transcription-quantitative PCR. **P<0.01. C-218/-NC, C-33A cells transfected with miR-218 mimics/NC; H-218/-NC, HeLa cells transfected with miR-218 mimics/NC; S-218/-NC, SiHa cells transfected with miR-218 mimics/NC; miR-218, microRNA-218; NC, negative control; ROBO1, roundabout guidance receptor1.