Data were all measurement data and expressed as mean standard deviation; ANOVA was used for comparisons among multiple groups, followed by Tukeys post hoc test; * the oe-NC group, # the oe-IGF1R group. Discussion Malignant melanoma has an increasing tendency of poor survival rates, which is an aggressive skin cancer [20]. could pave a new way for the treatment of malignant melanoma. < 0.05; Physique1(a)), indicating that there was differential expression of miR-139-5p in malignant melanoma. Additionally, the miR-139-5p expression of the primary normal skin melanocytes PIGI and five malignant melanoma cell lines (A375, SK-MEL-1, SK-MEL-2, SK-MEL-5, and SK-MEL-28) were evaluated by RT-qPCR, and the results implied that in comparison to PIGI cells, miR-139-5p was poorly expressed in the five malignant melanoma cell lines (all PIGI cell line. Overexpressed mir-139-5p suppresses the proliferation, migration and invasion, and enhances the apoptosis of malignant melanoma cells MiR-139-5p was overexpressed (Physique 2(a)) to further understand whether miR-139-5p could take effects on malignant melanoma. MTT assay (Physique 2(b)), BrdU staining (Physique 2(c)) and colony formation assay (Physique 2(d)) were adopted to measure the development of malignant melanoma cells, and the results revealed that in A375 cells, the cell proliferation reduced in miR-139-5p mimic group (< 0.05), which was relative to the miR-139-5p mimic-NC group. Flow cytometry (Physique 2(e,f)) and Hoechst 33258 staining (Physique 2(g)) suggested that this proportion of cells in G0/G1 phase was enhanced, the proportion of cells in S phase was significantly declined and the apoptosis was obviously increased (all < 0.05) after miR-139-5p has been overexpressed. Besides, Transwell assay (Physique 2(h,i)) was adopted to detect the invasion and migration of cells, the results of which showed that invasion and migration of cells were suppressed in the miR-139-5p mimic group versus those in the miR-139-5p mimic-NC group (< 0.05). Results above expressed for that the overexpressed miR-139-5p led to the suppressed proliferation, migration and invasion and induced apoptosis of malignant melanoma cells. Open in a separate window Physique 2. MiR-139-5p affected the biological functions of malignant melanoma cells. (a), RT-qPCR was used to evaluate the efficiency of miR-139-5p transfection (b-d), the cell proliferation was measured by MTT assay, BrdU staining and colony formation assay, Rabbit polyclonal to PDGF C respectively; (e), cell cycle distribution was detected by PI single staining; (f), cell apoptosis was evaluated by Annexin V/PI double staining; (g), morphological changes of cell apoptosis were observed by Hoechst 33,258 staining; Carbasalate Calcium (h-i), the changes of migration and invasion of cells were, respectively, detected by Transwell assay; data were all measurement data and expressed as mean standard deviation Carbasalate Calcium deviation, the impartial t-test was adopted for statistical analysis. Mir-139-5p targets and down-regulates the expression of IGF1R We have found from the bioinformatics site (http://www.targetscan.org/vert_71/) that there were binding sites between miR-139-5p and IGF1R (Physique 3(a)). Meanwhile, dual-luciferase reporter gene assay was carried out to test whether IGF1R was the target gene of miR-139-5p (Physique 3(b)). The results unraveled that this luciferase activity of IGF1R-Wt was apparently inhibited in the miR-139-5p mimic group relative Carbasalate Calcium to the miR-139-5p mimic-NC group (< 0.05), while there was no significant effect on luciferase activity of IGF1R-Mut (> 0.05). Besides, RT-qPCR and Western blot analysis were used to measure the IGF1R expression in tissues, the results reflected that this expression level of IGF1R in malignant melanoma tissues was markedly enhanced (Physique 3(c,d)), which was relative to adjacent normal skin tissues. Results above implied that miR-139-5p could target and down-regulate the expression of IGF1R. The outcomes of immunohistochemical staining unraveled that IGF1R was mainly expressed in the cytoplasm of the malignant melanoma cells, and positive cells were in red.