MicroRNA-1/206 targets c-Met and inhibits rhabdomyosarcoma development

Abstract

MicroRNAs (miRNAs) are endogenous short (approximately 22) nucleotide RNAs that regulate gene function by modification of target mRNAs. miRNA-1 (miR-1) and miRNA-206 (miR-206) are highly expressed in skeletal muscle. Due to the tissue-specific nature of miR-1/206 for skeletal muscles, we investigated the role of miR-1/206 in the development of rhabdomyosarcoma. Initially, we demonstrated that miR-1/206 expression was suppressed in rhabdomyosarcomas and found at very low levels in a rhabdomyosarcoma RD cell line. Transient transfection of miR-1/206 into cultured RD cells led to a significant decrease in cell growth and migration. Using bioinformatics, we identified two putative miR-1/206 binding sites within the 3'-untranslated region of the human c-Met mRNA. miR-1/206 was then shown to have activity on mRNA expression by targeting the c-Met 3'-untranslated region. The expression of c-Met protein was shown to be down-regulated by subsequent Western blot analysis. Conversely, up-regulation of c-Met was confirmed in tissue samples of human rhabdomyosarcoma, with its level inversely correlated with miR-1/206 expression. In vivo, miR-1/206-expressing tumor cells showed growth delay in comparison with negative control. Our results demonstrated that miR-1/206 suppressed c-Met expression in rhabdomyosarcoma and could function as a potent tumor suppressor in c-Met-overexpressing tumors. Inhibition of miR-1/206 function could contribute to aberrant cell proliferation and migration, leading to rhabdomyosarcoma development.

FIGURE 1.

miR-1/206 expression is down-regulated in human RD rhabdomyosarcoma cell line and human rhabdomyosarcoma specimens. A, Northern blot analysis was performed to detect the expression of miR-1 and miR-206 in the RD cell line (RD), in comparison with human skeletal muscle (SKM). miR-1 and miR-206 were highly expressed in human skeletal muscle tissues but were down-regulated in RD cells. U6 small nuclear RNA (U6) was used as an internal control. B, miR-1 and miR-206 were expressed at low levels in eight human rhabdomyosarcoma specimens. C, quantitative representation of the data from panel B by measuring the intensities of each band and then normalizing to the intensity of the internal control U6 small nuclear RNA.

FIGURE 2.

Exogenous miR-1/206 inhibits RD cell proliferation through induction of apoptosis and G₁ cell cycle arrest. A, RD cell growth was assessed after transfection with miR-1 or miR-206. The negative control (NC) scrambled oligonucleotide does not encode for any known miRNA. The number of cells was dramatically reduced after transfection with miR-1 (panel c) or miR-206 (panel d) as compared with mock (panel a) and NC (panel b) transfection. Photos were taken 72 h after transfection. B, the MTS cell proliferation assay was carried out and plotted from days 1 to 5 as indicated after Lipofectamine transfection of RD cells with miR-1 (50 nm), miR-206 (50 nm), or NC. Cells transfected with miR-1/206 were less metabolically active than cells transfected with NC. The data are expressed as the mean value ± S.E. (error bars) of the results obtained from triplicates in one experiment. Results are representative of three separate experiments. C, transfection with miR-1/206 induced DNA fragmentation in RD cells as demonstrated by Hoechst staining. Photographs of cells following transfection are shown as indicated: panel a, mock; panel b, negative control; panel c, miR-1; and panel d, miR-206. Red arrows denote cells with fragmented nuclei. D, caspase 3/7 activity assay was performed on RD cells transfected with either NC or miR-1/206. Relative caspase 3/7 activity is indicated in comparison with negative control. Results are expressed as the mean value ± S.E. (error bars) of the results obtained from triplicates in one experiment. Results are representative of three separate experiments. *, differences between miR-1/206 and negative control transfected cells were significant, p < 0.05. E, cell cycle arrest was evaluated using flow cytometry. RD cells, collected 48 h after transfection, were stained with propidium iodide and analyzed by flow cytometry. Cells transfected with miR-1 and miR-206 had a higher proportion of cell cycle arrest at the G₁ phase in comparison with mock or NC. 10,000 cells were evaluated in each sample. Results depicted are representative of three independent experiments. F, miR-1/206 regulated cell cycle-related proteins that are important for cell cycle G₁ phase progression and G₁/S transition. RD cells were transfected with miR-1/206, mock, or a negative control. Cell lysates were prepared and used for Western blot analysis with CDK2, CDK4, CDK6, E2F1, E2F3, p21, and phosphorylated Rb (p-Rb) antibodies. GAPDH was used as a loading control.

FIGURE 3.

Transfection of miR-1/206 reduces the migration of RD cells. RD cells were transfected with miR-1/206 or NC for 24 h and plated on culture inserts in DMEM containing 20 ng/ml HGF to assess the number of migrating cells. The number of cells that had migrated through the pores was quantified by counting 10 independent visual fields using a ×20 microscope objective. Results are expressed as the mean ± S.E. (error bars) for the data obtained from three independent assays. *, differences in cell migration between miR-1/206 and negative control transfected cells were significant, p < 0.01.

FIGURE 4.

c-Met is a direct target of miR-1/206. A, predicted miR-1/206 binding sites in c-Met 3′-UTR. Specific locations of the binding sites were marked with red color, and c-Met 3′-UTR was marked with blue color. B, alignment between the predicted miR-1/206 target sites and miR-1/206 is shown. The conserved, 7-bp seed sequence for miR-1/206:mRNA pairing is also indicated. C, diagram depicting the pMIR luciferase reporter constructs, containing a cytomegalovirus (CMV) promoter, which was utilized to verify the putative miR-1/206 binding sites (see “Experimental Procedures”). Luc, luciferase; poly A, poly(A) tail. D, HEK293 cells were co-transfected with miR-1 or miR-206, pLuc-MET 3′-UTR, along with a pRL-SV40 reporter plasmid. After 24 h, the luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. The data are expressed as the mean value ± S.E. (error bars) of the results obtained from three independent experiments. *, differences in luciferase activity between miR-1/206 and negative control transfected cells were significant, p < 0.01. E, c-Met expression levels in RD cells after transfection with miR-1/206 were determined by Western blot analysis. As compared with the NC miRNA, miR-1/206 expression dramatically reduced the levels of c-Met in RD cells. GAPDH was used as an internal control.

FIGURE 5.

Introduction of miR-1/206 down-regulates targets of c-Met. RD cells were transfected with miR-1, miR-206, or NC. Cell lysates were prepared and used for Western blot analysis with antibodies specific for phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated Akt (p-Akt), total Akt, phosphorylated FAK (p-FAK), and total FAK. miR-1/206 down-regulated expression levels of p-ERK1/2, p-Akt, and p-FAK, but not total ERK1/2, Akt, or FAK.

FIGURE 6.

The expression levels of c-Met in human rhabdomyosarcoma specimens are up-regulated. A, tissue samples were prepared and used for Western blot analysis. SKM, skeletal muscle. B, the majority of samples examined (samples 2–8) showed an up-regulation of c-Met. Expression of c-Met was shown after normalization to GAPDH.

FIGURE 7.

Introduction of miR-1/206 in RD cells suppresses tumor growth in nude mice. A, mice were inoculated with lentivirus-infected RD cells expressing miR-1, miR-206, or NC. Representative photographs of nude mice 8 weeks after inoculation are shown. B, average volume of tumors derived from RD cells infected with NC or miR-1/206 in nude mice. *, differences in tumor volume between miR-1/206 and NC infected cells were significant, n = 6 each, p < 0.01. Error bars indicate S.E.