Figure 1.
p53 induces miR-182 expression.
(A) p53 expression levels were induced after treatment with doxorubicin (DXR, 1 µg/mL) in M23 and SP6.5 uveal melanoma cell lines. GAPDH was used as an internal control. (B) Induction of miR-182 expression after treatment with doxorubicin (1 µg/mL) in both cell lines. (C) M23 and SP6.5 cells transfected with a siRNA targeting p53 or a scrambled negative control siRNA were treated with 1 µg/mL of doxorubicin for 24 or 48 hours. miR-182 expression levels were indicated, as determined by real-time RT-PCR relative to the level of U6 snRNA expression. Representative data from three independent experiments are shown.
Figure 2.
Ectopic miR-182 inhibits cell proliferation.
(A) MTS assay was performed on days 1 to 5 as indicated after lipofectamine transfection of uveal melanoma cells M23 and SP6.5 with either miR-182 (50 nM) or a negative control scrambled oligonucleotide (NC). The data at each time point are expressed as the mean value ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three independent experiments. (B) M23 and SP6.5 cells were collected 48 hours after transfection with miR-182 or NC, stained with propidium iodide, and analyzed by flow cytometry. Ten thousand cells were evaluated in each sample. The most representative results in three independent experiments are depicted. (C) M23 and SP6.5 cells transfected with miR-182 or NC were seeded at low density. After 7 days, colony formation was assessed by staining with crystal violet. Typical results from three independent experiments are shown.
Figure 3.
Ectopic miR-182 inhibits cell migration and invasion.
Uveal melanoma cells M23 and SP6.5 were transfected with miR-182 or a negative control (NC) for 24 hours and plated on either culture or Matrigel inserts. The number of cells that had migrated through the culture insert pores (A) or Matrigel insert pores (B) was quantified by counting five independent visual fields using a 20X microscope objective. Results represent those obtained in three experiments. *: Differences in cell migration or invasion between miR-182 and negative control transfected cells were significant, p<0.01.
Figure 4.
(A) Caspase 3/7 activity assay was performed on M23 and SP6.5 cells transfected with either miR-182 or a negative control (NC) after doxorubicin (DXR, 1 µg/mL) treatment. Relative caspase 3/7 activity is indicated in comparison to negative control. Results are expressed as the mean value ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three experiments. (B) M23 and SP6.5 cells (miR-182 and NC transfected) were treated with doxorubicin for 48 hours. Cells were then stained with Hoechst 33342 and examined using a 20X fluorescent microscope objective. (C) M23 and SP6.5 cells with both condensed and fragmental nuclei were counted manually following treatment with doxorubicin as described above. Results represent those obtained in three experiments. *: Differences in apoptotic cell number between miR-182 and negative control transfected cells were significant, p<0.01.
Figure 5.
MITF, BCL2 and cyclin D2 (CCND2) are targets of miR-182.
(A) Specific locations of the binding sites were marked with red color and MITF, BCL2 and cyclin D2 3′ UTR were marked with blue color. Alignment between the predicted miR-182 target sites and miR-182, the conserved 7–8 bp “seed” sequence for miR-182:mRNA pairing is indicated. (B) Diagram depicting the pMIR luciferase reporter constructs, containing a CMV promoter, which was utilized to verify the putative miR-182 binding sites. (C) HEK293 cells were co-transfected with miR-182, pLuc-target 3′ UTR, or a mutant 3′ UTR, along with a pRL-SV40 reporter plasmid. After 24 hours, the luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. As shown, only in the presence of both miR-182 and the normal pLuc-MITF 3′ UTR (or equivalent) was there suppression of luciferase activity. Results represent those obtained in three separate experiments. *: Differences in luciferase activity between miR-182 and negative control transfected cells were significant, p<0.01.
Figure 6.
miR-182 downregulates the expression of MITF and c-Met.
(A) MITF expression in M23 and SP6.5 cells after transfection with miR-182 was dramatically reduced by Western blot analysis. GAPDH was used as an internal control. (B) c-Met expression in M23 and SP6.5 cells after transfection with miR-182 was dramatically reduced by Western blot analysis. GAPDH was used as an internal control. (C) miR-182 downregulated expression of p-Akt and p-ERK1/2, but not total Akt or ERK1/2. M23 and SP6.5 cells were either mock, transfected with a negative control or miR-182, lysed, and then used for Western blot analysis.
Figure 7.
Downregulation of c-Met suppresses uveal melanoma cell proliferation, migration and invasion.
(A) Western blot analysis was performed to detect c-Met expression after lipofectamine transfection of uveal melanoma cells M23 and SP6.5 with either c-Met siRNA (50 nM) or a negative control (NC). (B) MTS cell proliferation assay after 3 days is shown. The data are expressed as the mean value ± SEM of the results obtained from triplicates in one experiment. Results represent those obtained in three experiments. (C&D) Uveal melanoma cells M23 and SP6.5, transfected with c-Met siRNA or NC, were quantified for migratory (C) or invasive (D) studies using culture or Matrigel inserts. Results represent those obtained in three experiments. *: Differences in cell migration or invasion between c-Met siRNA and negative control transfected cells were significant, p<0.01.
Figure 8.
miR-182 downregulates multiple cell signal pathways.
Transfected M23 and SP6.5 cells were prepared and used for Western blot analysis with multiple antibodies. GAPDH was used as an internal control. (A) miR-182 downregulated expression of BCL2 and cyclin D2. (B) miR-182 downregulated cell cycle related proteins cyclin E2, CDK2, CDK4, and phosphorylated-Rb (p-Rb). Effect on CDK6 was not pronounced in M23 cells.
Figure 9.
miR-182 suppresses uveal melanoma cell growth in vivo.
(A) Representative photographs of nude mice 4 weeks after inoculation with miR-182 or control lentivirus infected uveal melanoma cells. a: Inoculation with M23 cells; b: Inoculation with SP6.5 cells. (B) Average volume of tumors derived from M23 or SP6.5 cells infected with miR-182 or control lentivirus in nude mice. *: Differences in tumor volume between miR-182 and control infected cells were significant, n = 4 each, p<0.05 for M23 cells inoculation, p<0.01 for SP6.5 cells inoculation.