MicroRNA-125a-5p modulates human cervical carcinoma proliferation and migration by targeting ABL2

Abstract

Background: In this study, we intended to understand the regulatory mechanisms of microRNA-125a-5p (miR-125a-5p) in human cervical carcinoma.

Methods: The gene expressions of miR-125a-5p in seven cervical carcinoma cell lines and 12 human cervical carcinoma samples were evaluated by quantitative real-time reverse transcription polymerase chain reaction. Ca-Ski and HeLa cells were transduced with lentivirus carrying miR-125a-5p mimics, and the effects of lentivirus-induced miR-125a-5p upregulation on cervical carcinoma proliferation and migration were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and transwell assays, respectively. In additional, HeLa cells were inoculated into null mice to evaluate the effect of miR-125a-5p upregulation on in vivo cervical carcinoma growth. The direct regulation of miR-125a-5p on its target gene, ABL proto-oncogene 2 (ABL2), in cervical carcinoma was evaluated by quantitative real-time reverse transcription polymerase chain reaction, Western blotting and luciferase reporter assays, respectively. ABL2 was then downregulated by small interfering RNA to examine its effect on cervical carcinoma proliferation and migration.

Results: miR-125a-5p was downregulated in both cervical carcinoma cell lines and human cervical carcinomas. In Ca-Ski and HeLa cells, lentivirus-mediated miR-125a-5p upregulation inhibited cancer proliferation and migration in vitro and cervical carcinoma transplantation in vivo. ABL2 was shown to be directly targeted by miR-125a-5p. In cervical carcinoma, ABL2 gene and protein levels were both downregulated by miR-125a-5p. Small interfering RNA-mediated ABL2 downregulation also had tumor-suppressive effects on cervical carcinoma proliferation and migration.

Conclusion: The molecular pathway of miR-125a-5p/ABL2 plays an important role in human cervical carcinoma. Targeting miR-125a-5p/ABL2 pathway may provide a new treatment strategy for patients with cervical carcinoma.

Keywords: ABL2; cancer migration; cancer proliferation; cervical carcinoma; miR-125a-5p.

Figure 1

Expression of miR-125a-5p in cervical carcinoma. Notes: (A) The gene expression levels of miR-125a-5p in seven cervical carcinoma cells, HeLa, SiHa, Ca-Ski, C-33-A, DoTc24510, HT-3, and C-4I, were measured by qRT-PCR and compared with the gene expression level of miR-125a-5p in two non-carcinoma human cervix epithelial cell lines, GH329 and Ect1/E6E7 cells (^*P<0.05). (B) In human cervical carcinomas, the gene expression level of miR-125a-5p was compared with carcinoma and adjacent non-carcinoma tissues in 12 samples (^*P<0.05). Abbreviation: qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.

Figure 2

miR-125a-5p overexpression reduced in vitro proliferation and migration in cervical carcinoma. Notes: Ca-Ski and HeLa cells were transduced with lentivirus miR-125-mimic to overexpress miR-125a-5p for 6 hours. The control cells were transduced with a negative control miRNA lentivirus (miR-C) for 6 hours. (A) Twenty-four hours after lentiviral transduction, gene expressions of miR-125a-5p in Ca-Ski and HeLa cells were examined by qRT-PCR, ^*P<0.05. (B and C) Twenty-four hours after lentiviral transduction, an MTT assay was performed for 5 days to compare cervical carcinoma proliferation between miR-125a-5p overexpressed cells and control cells; (B): Ca-Ski, (C): HeLa, ^*P<0.05. (D and E) Twenty-four hours after lentiviral transduction, a transwell assay was performed. The representative crystal violet images were shown for miR-125a-5p overexpressed cells and control cells (left panel). The migration capabilities were evaluated by calculating the average migrated cells in 96-well plates and normalized to control (right panel); (D): Ca-Ski, (E): HeLa, ^*P<0.05. Abbreviations: qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 3

miR-125a-5p overexpression reduced in vivo growth of cervical carcinoma. Notes: HeLa cells were transduced with lentivirus of miR-125-mimic to overexpress miR-125a-5p for 6 hours. The control cells were transduced with a negative control miRNA lentivirus (miR-C) for 6 hours. Twenty-four hours after transduction, cells were re-suspended and 1 million healthy cells were then subcutaneously transplanted into the left side of 8-week-old female null mice. (A) The weekly in vivo carcinoma growth was measured by the equation of length × width²/2 (^*P<0.05). (B) Five weeks after in vivo transplantation, mice were sacrificed and tumors were extracted for Ki-67 immunostaining.

Figure 4

miR-125a-5p regulates ABL2 in cervical carcinoma. Notes: Ca-Ski and HeLa cells were transduced with lentivirus miR-125-mimic to overexpress miR-125a-5p for 6 hours. The control cells were transduced with a negative control miRNA lentivirus (miR-C) for 6 hours. Twenty-four hours after lentiviral transduction, the gene expression levels of ABL2 were examined by qRT-PCR in Ca-Ski (A) and HeLa (B) (^*P<0.05). The protein expression levels of ABL2 were examined by Western blotting in Ca-Ski (C) and HeLa (D). The targeting search result was shown for the binding between miR-125a-5p and ABL2 3′-UTR. A mutated ABL2 3′-UTR with nullified miR-125a-5p binding site was also created (E). HEK293T cells were co-transfected with miR-125-mimic, an empty luciferase vector (vector), a luciferase vector inserted with 3′-UTR of wild-type ABL2 (ABL2 WT 3′-UTR), or a luciferase vector inserted with mutated ABL2 3′-UTR (ABL2 Mu 3′-UTR). Forty-eight hours after co-transfection, a dual-luciferase reporter assay was performed. The luciferase activities were measured for all three luciferase vectors and then normalized to the luciferase activity of vector (^*P<0.05) (F). Abbreviations: qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; ABL2, ABL proto-oncogene 2; 3′-UTR, 3′-untranslated regions.

Figure 5

ABL2 downregulation reduced in vitro proliferation and migration in cervical carcinoma. Notes: Ca-Ski and HeLa cells were transfected with siRNA-ABL2 or siRNA-C for 12 hours. (A) Twenty-four hours after siRNA transfection, gene expressions of ABL2 in Ca-Ski and HeLa cells were examined by qRT-PCR, ^*P<0.05. (B and C) Twenty-four hours after siRNA transfection, an MTT assay was performed for 5 days to compare cervical carcinoma proliferation between ABL2 downregulated cells and control cells; (B): Ca-Ski, (C): HeLa, ^*P<0.05. (D and E) Twenty-four hours after siRNA transfection, a transwell assay was performed. The representative crystal violet images were shown for ABL2 downregulated cells and control cells (left panel). The migration capabilities were evaluated by calculating the average migrated cells in 96-well plates and normalized to control (right panel); (D): Ca-Ski, (E): HeLa, ^*P<0.05. Abbreviations: ABL2, ABL proto-oncogene 2; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; siRNA, small interfering RNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.