MicroRNA-425 induces apoptosis and suppresses migration and invasion of human cervical cancer cells by targeting RAB2B

Abstract

Dysregulation of microRNA-425 (miR-425) has been reported in several human cancers. However, the role of miR-425 in human cervical cancer via modulation of RAB2B expression is still unclear. This study was therefore designed to examine the expression and decipher the role of miR-425 in cervical cancer. The qRT-PCR was used for expression analysis. MTT and EdU assays were used for the determination of cell viability and proliferation, respectively. Annexin V/PI staining was used to detect apoptosis. Wound healing and transwell assays were used to monitor cell migration and invasion. Western blotting was used for protein expression analysis. The in vivo study was performed in xenografted mice model. The results of the present study revealed miR-425 to be significantly (P = 0.032) down-regulated in cervical cancer tissues and cell lines. Additionally, low expression of miR-425 was associated with significantly (P = 0.035) lower survival rate of the cervical cancer patients. Overexpression of miR-425 resulted in significant (P = 0.024) decline of cervical cancer cell proliferation via induction of apoptosis. The induction of apoptosis was associated with up-regulation of Bax and down-regulation of Bcl-2. Besides, the migration and invasion of cancer cells significantly (P < 0.01) decreased under miR-425 overexpression. Additionally, miR-425 could inhibit the growth of xenografted tumors in vivo. In silico analysis and dual luciferase assay revealed RAB2B as the direct target of miR-425 in cervical cancer. RAB2B was found to be significantly (P < 0.05) up-regulated in cervical cancer tissues and cell lines and miR-425 overexpression suppressed the expression of RAB2B. Additionally, silencing of RAB2B could suppress the growth of cervical cancer cells but its overexpression could rescue the tumor-suppressive effects of miR-425. Taken together, the results revealed the tumor-suppressive roe of miR-425 and point towards its therapeutic potential in the management of cervical cancer.

Keywords: RAB2B; apoptosis; cervical cancer; metastasis; micro-RNA; xenograft mice.

Figure 1.

miR-425 has considerable down-regulation in cervical cancer which predicts the disease survival. (a) qRT-PCR based expression analysis of miR-425 in cervical cancer and normal adjacent tissues. (b) Kaplan-Meier survival analysis of cervical cancer with reference to miR-425 expression. (c) qRT-PCR based expression analysis of miR-425 in cervical cancer cell lines (HeLa, siHa, DoTc2 and C-33 A) and normal cervical epithelial cell line, NCE. The experiments were performed in triplicate and expressed as mean ± SD (*P < 0.05).

Figure 2.

miR-425 over-expression restricted the cancer cell growth and declined proliferative viability of cervical cancer cells. (a) qRT-PCR based expression analysis of miR-425 in DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC. (b) MTT assay for the analysis of viability of DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC. (c) EdU assay for assessment of proliferative viability of DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC. The experiments were performed in triplicate and expressed as mean ± SD (*P < 0.05).

Figure 3.

miR-425 over-expression induced apoptosis in vitro in cervical cancer cells. (a) Analysis of nuclear morphology of DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC using AO/EB staining (green colour depicts normal, yellow color early apoptotic and red color late apoptotic cells). (b) Analysis of nuclear morphology of DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC using DAPI staining. (c) Analysis of apoptosis of DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC using flow cytometry. (d) Western blotting of Bax and Bcl-2 from DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC. The experiments were performed in triplicate and expressed as mean ± SD (*P < 0.05).

Figure 4.

miR-425 over-expression minimized the cancer cell migration and invasion. (a) Analysis of migration of DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC using wound-healing method. (b) Analysis of invasion of DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC using transwell-chamber assay. (c) Western blotting of MMP-2 and MMP-9 from DoTc2 cervical cancer cells transfected with miR-425 mimics or miR-NC. The experiments were performed in triplicate and expressed as mean ± SD (*P < 0.05).

Figure 5.

miR-425 restricted in vivo xenograft tumor growth and proliferation. (a) Analysis of tumor size under administration of miR-425 mimics or miR-NC. (b) Analysis of tumor volume under administration of miR-425 mimics or miR-NC. (c) Average tumor weight under administration of miR-425 mimics or miR-NC. (d) Immune-histochemical fluorescence staining of caspase-3 (cleaved) and ki67 from mice tumors under administration of miR-425 mimics or miR-NC. The experiments were performed in triplicate and expressed as mean ± SD (*P < 0.05).

Figure 6.

RAB2B is the functional molecular target of miR-425 in cervical cancer. (a) TargetScan analysis for prediction of molecular target of miR-425. (b) Dual luciferase reporter assay for the interaction analysis of miR-425 with 3’-UTR of RAB2B. (c) qRT-PCR based expression analysis of miR-425 in cervical cancer and normal adjacent tissues. (d) qRT-PCR based expression analysis of miR-425 in cervical cancer cell lines (HeLa, siHa, DoTc2 and C-33 A) and normal cervical epithelial cell line, NCE. (e) Western blotting of RAB2B form DoTc2 cancer cells transfected with miR-425 mimics or miR-NC. (f) MTT assay for the analysis of viability of DoTc2 cervical cancer cells transfected with si-RAB2B or si-NC. (g) MTT assay for the analysis of viability of DoTc2 cervical cancer cells transfected with miR-425 mimics, miR-NC or miR-425 mimics plus pcDNA-RAB2B. The experiments were performed in triplicate and expressed as mean ± SD (*P < 0.05).