MicroRNA-378 enhances migration and invasion in cervical cancer by directly targeting autophagy-related protein 12

Abstract

Cervical cancer is the second most common type of cancer among women worldwide and a leading cause of mortality in women. Metastases reduce the overall survival rate in patients with cervical cancer. Thus, it is clinically urgent to investigate the molecular mechanism of cervical cancer metastasis. The aim of the present study was to investigate the mechanism of microRNA (miR)‑378 in the metastasis of cervical cancer. In the present study, miR‑378 expression levels were significantly upregulated in cervical cancer tissues and cervical intraepithelial neoplasia III tissues when compared with normal cervix tissues. Re‑expression of miR‑378 significantly promoted tumor migration and invasion in vitro, and metastasis in vivo, while downregulation of miR‑378 suppressed the effect in vitro. Luciferase reporter assay revealed that autophagy‑related protein 12 (ATG12) was a direct target of miR‑378 and its expression was downregulated by miR‑378. In cervical cancer tissues with lymph node metastasis, miR‑378 was upregulated while ATG12 was downregulated when compared with lymph node negative cases. To the best of our knowledge, the present study is the first to provide evidence that miR‑378 may be associated with ATG12. Collectively, the data of the present study suggested that miR‑378 may function as an oncogene by promoting metastasis in cervical cancer. The finding that miR‑378 targets ATG12 indicated that miR‑378 may have a potential role in autophagy. These findings may provide novel insights into the mechanism of metastasis in cervical cancer and a novel therapeutic target for the treatment of cervical cancer.

Keywords: microRNA-378; cervical cancer; migration; invasion; autophagy-related protein 12.

Figure 1.

miR-378 was frequently upregulated in CIN III and LN positive CA tissues. (A) miR-378 was significantly upregulated in CA tissues (n=55) and CIN III tissues (n=70) when compared with N tissues (n=60). (B) In patients with cervical cancer, the positive group (n=17) with LN metastasis exhibited significantly higher expression levels of miR-378 than the negative group (n=38). Relative expression was normalized to U6. *P<0.01 and **P<0.05 as indicated. CA, cervical cancer; CIN III, cervical intraepithelial neoplasia III; LN, lymph node; miR, microRNA; N, normal cervix.

Figure 2.

Overexpression of miR-378 promoted cell migration and invasion of cervical cancer. (A) Relative expression of miR-378 was determined by reverse transcription-quantitative polymerase chain reaction. (B) Overexpression in C-33A cells promoted cell migration and knockdown of miR-378 in HeLa cells inhibited cell migration (magnification, ×100). (C) Overexpression in C-33A cells promoted cell invasion and knockdown of miR-378 in HeLa cells inhibited cell invasion (magnification, ×100). (D) Effects of miR-378 on the protein expression of ATG12 were determined by western blot analysis in C-33A cells transfected with GIPZ-miR-378 and HeLa cells transfected with miR-378 inhibitor. (E) Quantitative analysis of the western blot results in Fig. 2D. Each bar represents the grey area of each band and the statistical significance was analyzed from three independent replicates. The data are presented as the mean ± standard deviation. *P<0.05. ATG12, autophagy-related protein 12; CCND1, cyclin D1; miR, microRNA; NC, negative control; pRb, retinoblastoma.

Figure 3.

ATG12 was identified as a direct target of miR-378. (A) Putative binding site of miR-378 in the 3′-UTR of ATG12. (B) Luciferase reporter assay in C-33A and HeLa cells. Cells were co-transfected with miR-378 mimics or control plus WT or MUT ATG12 3′-UTR. Firefly luciferase activity was normalized to Renilla luciferase activity. **P<0.01 vs. NC. (C) Representative images of ATG12 stain in cervical cancer tissues (magnification, ×100). (D) immunohistochemistry staining of the cervical cancer tissues revealed that ATG12 expression was upregulated in LN negative group (52.9% positive) when compared with the LN positive group (20% positive). *P<0.05. (E) Inhibition of ATG12 in C-33A cells promoted cell migration and invasion (magnification, ×100). ATG12, autophagy-related protein 12; LN, lymph node; miR, microRNA; MUT, mutant; si, small interfering; UTR, untranslated region; WT, wild-type.

Figure 4.

miR-378 promoted cervical cancer metastasis in vivo. (A) Representative hematoxylin and eosin staining sections of distant metastatic nodules in lungs. The upper image is the NC group and the lower image is the GIPZ-miR-378 group. (B) Representative images of lungs at the 8th week post-transfection; the left and right panels present the NC and the GIPZ-miR-378 mouse groups, respectively. (C) The number of distant metastatic nodules in the lung were counted and compared between the control and GIPZ-miR-378 groups. **P<0.01. miR, microRNA; NC, negative control.