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

Figures & data

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.