Abstract：Objective: To investigate the regulation and mechanism of microRNA-135a/b (miR-135a/b) in the migration of uveal melanoma (UM) cells. Methods: Experimental study. Quantitative RT-PCR was performed to determine the level of miR-135a/b in UM species and the paraneoplastic tissue. The microRNAmimics were transfected in UM cell line (M23 and SP6.5) to overexpress miR-135a/b, transfected nonsense sequence as control group (NC). Small interference RNAtransfection and lentiviral infection were taken to downregulated and upregulated the protein level of SIRT1, respectively. Nonsense small interference RNA (siNC) and lentiviral vector with nonsense sequence (Lv-NC) were used as control groups. The cell migratory behavior was measured by scratch assay and transwell assay. The dual-luciferase reporter assay was taken to confrm the target of miR-135a/b. The protein level of SIRT1 was determined by Western blot. Data analysis using independent samples t-test. Results: Compared with paraneoplastic tissue, miR-135a/b was downregulated signifcantly in UM species (miR-135a: t=6.38, P=0.003, miR-135b: t=23.26, P<0.001). The scratch assay and transwell assay showed that the migratory distance was reduced (miR-135a: t=36.01, 8.98, P<0.01; miR-135b: t=27.53, 10.51, P<0.01) and the number of migratory cells was decreased (miR-135a: t=7.04, 7.02, P<0.01; miR-135b: t=5.01, 7.32, P<0.01) in miR-135a/b overexpression M23 and SP6.5 cells compared with NC group. The dual-luciferase reporter assay revealed that miR-135a/b could signifcantly inhibit the fuorescence value of luciferase in SIRT1 wild type group (miR-135a: t=2.88, P=0.028; miR-135b: t=4.99, P=0.003). The protein level of SIRT1 in M23 and SP6.5 cells was downregulated in miR-135a/b transfected compared to the NC group (miR-135a: t=9.93, 4.13, P<0.01; miR-135b: t=4.66, 6.20, P<0.01). The scratch assay and transwell assay showed that the migratory distance was reduced (siSIRT1-I: t=13.88, 11.78, P<0.01; siSIRT1-II: t=31.20, 6.27, P<0.01) and the number of migratory cells was decreased (siSIRT1-I: t=7.57, 4.66; P<0.01; siSIRT1-II: t=5.58, 3.25; P<0.05) in M23 and SP6.5 cells with downregulated the protein level of SIRT1, compared to the siNC groups. The transwell assay showed that the number of migratory cells was decreased (miR-135a+Lv-SIRT1: t=4.10, 2.75, P<0.05; miR-135b+Lv-SIRT1: t=2.99, 2.49, P<0.05) in M23 and SP6.5 cells with upregulated the protein level of SIRT1 and over expression of miR-135a/b, compared with UM cells only over expression of miR-135a/b. Conclusions: The expression level of miR-135a/b was significantly downregulated in UM and miR-135a/b inhibited the migration of UM cells targeting SIRT1 mRNA3' untranslated region, supporting miR-135a/b-SIRT1 axis plays critical roles in UM development.
浙公网安备 33030202001892号