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hep26373-sup-0001-suppfig1.tif3910KSupporting Fig. 1. miR-195 represses tumor angiogenesis in vitro. (A) An endothelial recruitment assay revealed the suppressive effect of miR-195 on the HCC cell-promoted migration of endothelial cells (ECs). HUVECs were seeded in the upper compartments of transwell chambers, and MHCC-97H cells that were non-transfected (panel 2) or transfected with NC (panel 3) or miR-195 (panel 4) were seeded in the lower compartments. Co-cultures were conducted for 12 h in Serum-Free Medium for endothelial cells (SFM). SFM (panel 1), in which no tumor cells were placed in the bottom chamber, was used as a control. (B) Restoration of miR-195 inhibited the HCC cell-promoted tube formation of ECs. HUVECs were cultured for 6 h in the presence of SFM (panel 1) or 100% tumor-cell conditioned medium (TCM) from MHCC-97H cells, which were non-transfected (panel 2) or transfected with NC (panel 3) or miR-195 duplex (panel 4). Scale bar, 100 µm. *, P< 0.05; **, P< 0.01; ***, P< 0.001.
hep26373-sup-0002-suppfig2.tif4634KSupporting Fig. 2. miR-195 suppresses tumor migration and invasion in vitro. (A, B) Restoration of miR-195 inhibited the migration and invasion of HCC cells. MHCC-97H cells that were non-transfected (panel 1) or transfected with NC (panel 2) or miR-195 duplex (panel 3) were added to transwell chambers without (A) or with (B) Matrigel coatings and were incubated for 20 h, followed by staining with crystal violet. Scale bar, 100 µm. **, P< 0.01.
hep26373-sup-0003-suppfig3.tif604KSupporting Fig. 3. Analysis of miR-195 expression in QGY-miR-195-LUC and the control line QGY-control-LUC by qPCR. QGY-miR-195-LUC cells were cultured with (+) or without (-) Dox for 48 h; QGY-control-LUC cells were cultured without Dox. The miR-195 level was normalized to RNU6 expression. No error bar is shown for the control because the normalized miR-195 level was set to 1. *, P< 0.05.
hep26373-sup-0004-suppfig4.tif2264KSupporting Fig. 4. miR-195 suppresses tumor growth and angiogenesis in vivo. (A, B) miR-195 suppressed tumor growth in vivo. Orthotopic xenograft tumor growth (A) was monitored by in vivo bioluminescent luciferase detection at the indicated days after tumor implantation. Tumor volumes (B) were measured on the 50th day after tumor implantation. Tumors developed in the implanted sites in 6/8 mice in the miR-195-off group and 5/8 in the miR-195-on group. The dissected tumors were measured for length (L) and width (W) with calipers, and the tumor volumes (V) were calculated according to the formula: V = (L×W2) ×0.5. (C) Orthotopic xenograft tumors from miR-195-on mice displayed lower microvessel densities (MVD). On the 50th day after implantation, tumor tissues from the miR-195-off or -on groups were stained for CD146. Scale bar, 50 µm. (D, E) Restoration of miR-195 expression inhibited tumor metastasis in the orthotopic xenograft model. The intrahepatic (D) and pulmonary metastases (E) were detected by ex vivo bioluminescent imaging on the 50th day after implantation. For the imaging of intrahepatic metastases (D), the left hepatic lobe with primary tumors was removed before imaging. For (A-E), QGY-miR-195-LUC cells were inoculated under the capsule of the left hepatic lobe of BALB/c nude mice. All mice were fed with Dox for the first 10 days prior to division into the miR-195-on and -off groups, followed by Dox withdrawal in the miR-195-on group and continued Dox treatment in the miR-195-off mice for another 40 days. For (A), (D) and (E), luciferase signals were quantified by measuring the photons/second. *, P< 0.05; **, P< 0.01.
hep26373-sup-0005-suppfig5.tif600KSupporting Fig. 5. In vivo bioluminescent detection of primary xenograft tumors on the 40th day after implantation. Tumors developed in the implanted sites in 9/15 mice in the miR-195-off group and 8/14 in the miR-195-on group. Luciferase signals were quantified by measuring the photons/second. The central horizontal line represents the mean value; the error bars represent the SEM.
hep26373-sup-0006-suppfig6.tif2732KSupporting Fig. 6. Restoration of miR-195 expression inhibits tumor metastasis in an orthotopic xenograft model. (A, B) miR-195 expression suppressed intrahepatic metastasis. (C, D) miR-195 expression repressed pulmonary metastasis. The intrahepatic and pulmonary metastases were detected by ex vivo bioluminescent imaging (A, C) and histopathological analysis (B, D). On the 40th day after implantation, mice were sacrificed 5 min after the injection of luciferin, and the organs were collected and imaged ex vivo. For imaging of the intrahepatic metastases, the left hepatic lobe with primary tumors was removed before imaging. After ex vivo imaging, the livers and lungs were serially sectioned and subjected to Hematoxylin-eosin-staining and histopathological analysis. The central horizontal line, mean value; error bar, SEM.
hep26373-sup-0007-suppfig7.tif4252KSupporting Fig. 7. VEGF is a direct target of miR-195. (A) miR-195 and its putative binding sequence in the 3'UTR of VEGF. Mutations were generated in the complementary site that binds to the seed region of miR-195. (B) Restoration of miR-195 decreased the level of secreted VEGF. TCM from MHCC-97H cells that were non-transfected or transfected with NC or miR-195 duplex were analyzed by ELISA. (C) Knockdown of endogenous miR-195 enhanced the cellular VEGF level. QGY-7703 cells that were non-transfected (lane 1) or transfected with anti-NC (lane 2) or anti-miR-195 (lane 3) for 48 h were analyzed by immunoblotting. GAPDH was used as an internal control. The results were reproduced in three independent experiments and representative immunoblots are shown. (D) Restoration of miR-195 expression reduced VEGF levels in orthotopic xenograft tumors. (E) The level of miR-195 correlated inversely with VEGF expression in human HCC tissues. For (D) and (E), a brown signal was considered to be a positive VEGF stain. Images were captured at a magnification of 400X. Scale bar, 50 µm. *, P < 0.05; ***, P< 0.001.
hep26373-sup-0008-suppfig8.tif1172KSupporting Fig. 8. miR-195 exerts its anti-angiogenic function by inhibiting VEGFR2-signaling in ECs. (A) Restoration of miR-195 in HCC cells attenuated VEGFR2 signaling in ECs. Twenty-four hours after seeding, HUVECs were grown in SFM for 12 h and were then cultured for 15 min in the presence of SFM (lane 1) or 100% TCM from MHCC-97H cells that were non-transfected (lane 2) or transfected with NC (lane 3) or miR-195 (lane 4), followed by immunoblotting for phosphor-Tyr1175-VEGFR2, VEGFR2, phosphor-T202/Y204-ERK1/2 and ERK1/2 expression. β-actin was used as an internal control. (B) Antagonism of miR-195 in HCC cells enhanced VEGFR2 signaling in ECs. Twenty-four hours after seeding, HUVECs were grown in SFM for 12 h, then were cultured for 15 min in the presence of SFM (lane 1) or 100% TCM from QGY-7703 cells that were non-transfected (lane 2) or transfected with anti-NC (lane 3) or anti-miR-195 (lane 4), followed by immunoblotting as in (A). The results in both (A) and (B) were reproduced in three independent experiments, and representative immunoblots are shown.
hep26373-sup-0009-suppfig9.tif250KSupporting Fig. 9. Knockdown of VEGF inhibits HCC cell-promoted angiogenesis in vitro. (A) Cellular levels of VEGF were reduced by si-VEGF transfection. QGY-7703 cells that were non-transfected (lane 1) or transfected with NC (lane 2), miR-195 (lane 3) or si-VEGF (lane 4) were analyzed by RT-PCR. Hypoxanthine-guanosine phosphoribosyl transferase (hPRT) was amplified in the same reactions as an internal control. The results were reproduced in three independent experiments and a representative image is shown. (B) Knockdown of VEGF inhibited the HCC cell-promoted migration of ECs. HUVECs were seeded in the upper compartments of transwell chambers, and QGY-7703 cells transfected with NC (bar 1) or si-VEGF (bar 2) were seeded in the lower compartments. Co-cultures were conducted for 12 h in SFM. (C) Knockdown of VEGF repressed the HCC cell-promoted tube formation of ECs. HUVECs were cultured for 6 h in the presence of 100% TCM from QGY-7703 cells, which were transfected with NC (bar 1) or si-VEGF (bar 2). The capillary tube formation of HUVECs was assessed subsequently. *, P< 0.05; **, P< 0.01.
hep26373-sup-0010-suppfig10.tif593KSupporting Fig. 10. Over-expression of VEGF attenuates the anti-angiogenic effect of miR-195. (A) Immunoblotting showed the efficiency of VEGF over-expression. Forty-eight hours after transfections with pc3-Gab or pc3-Gab-VEGF, QGY-7703 cells were analyzed by immunoblotting. GAPDH was used as an internal control. (B) Introduction of VEGF antagonized the anti-angiogenic effect of miR-195. HUVECs were cultured in TCM derived from QGY-7703 cells that were co-transfected with the following RNA duplex/expression plasmid combinations: NC/empty vector (panel 1), miR-195/empty vector (panel 2), NC/VEGF (panel 3) or miR-195/VEGF (panel 4). The capillary tube formation of HUVECs was assessed subsequently. The results in both (A) and (B) were reproduced in three independent experiments, and representative images are shown. Scale bar, 100 µm.
hep26373-sup-0011-suppfig11.tif6004KSupporting Fig. 11. VAV2 and CDC42 are direct targets of miR-195. (A) miR-195 and its putative binding sequences in the 3'UTR of VAV2 and CDC42. Mutations were generated in the complementary sites that bind to the seed region of miR-195. (B) Expression of miR-195 reduced the protein levels of cellular VAV2 and CDC42. MHCC-97H cells that were non-transfected (lane 1) or transfected with NC (lane 2) or miR-195 (lane 3) for 48 h were analyzed by immunoblotting. (C) Knockdown of endogenous miR-195 enhanced the expression of VAV2 and CDC42. HCC cells that were non-transfected (lane 1) or transfected with anti-NC (lane 2) or anti-miR-195 (lane 3) for 48 h were analyzed by immunoblotting. For (B) and (C), GAPDH was used as an internal control. The results were reproduced in three independent experiments and representative immunoblots are shown. (D) Restoration of miR-195 expression reduced VAV2 and CDC42 expression in orthotopic xenograft tumors. (E) miR-195 expression correlated inversely with VAV2 or CDC42 expression in human HCC tissues. For (D) and (E), a brown signal was considered to be a positive VAV2 or CDC42 stain. The images were captured at a magnification of 400X. Scale bar, 50 µm. ***, P< 0.001.
hep26373-sup-0012-suppfig12.tif255KSupporting Fig. 12. Knockdown of VAV2 and CDC42 inhibits tumor migration in vitro. (A) Cellular mRNA levels of VAV2 and CDC42 were reduced by si-VAV2 and si-CDC42 transfection. QGY-7703 cells that were non-transfected (lane 1) or transfected with NC (lane 2), miR-195 (lane 3), si-VAV2 (lane 4) or si-CDC42 (lane 5) were analyzed by RT-PCR. GAPDH was used as an internal control. The results were reproduced in three independent experiments and a representative image is shown. (A) Knockdown of VAV2 and CDC42 inhibited the migration of HCC cells. QGY-7703 cells transfected with NC (bar 1), si-VAV2 (bar 2) or siCDC42 (bar 3) were added to transwell chambers and incubated for 12 h, followed by staining with crystal violet. ***, P< 0.001.
hep26373-sup-0013-suppfig13.tif179KSupporting Fig. 13. Over-expression of VAV2 or CDC42 attenuated the anti-migration effect of miR-195. (A) Immunoblotting showed the efficiencies of VAV2 and CDC42 over-expression. Forty-eight hours after transfections with pc3-Gab, pc3-Gab-VAV2 or pc3-Gab-CDC42, QGY-7703 cells were analyzed by immunoblotting. GAPDH was used as an internal control. The results were reproduced in three independent experiments and a representative immunoblot is shown. (B) Introduction of VAV2 or CDC42 antagonized the anti-migration effect of miR-195. QGY-7703 cells that were co-transfected with the following RNA duplex/expression plasmid combinations: NC/empty vector (bar 1), miR-195/empty vector (bar 2), NC/VAV2 (bar 3), miR-195/VAV2 (bar 4), NC/CDC42 (bar 5), miR-195/CDC42 (bar 6) were added to transwell chambers and incubated for 12 h, followed by staining with crystal violet. **, P< 0.01; ***, P< 0.001.
hep26373-sup-0014-suppfig14.tif1891KSupporting Fig. 14. Overexpression of miR-195 inhibited lamellipodia formation in QGY-7703 cells. QGY-7703 cells that were non-transfected or transfected with NC, miR-195, si-VAV2 or si-CDC42 for 48 h were added to Matrigel-coated plates and incubated for 1 h, followed by staining for filamentous actin. Representative images of the lamellipodia (arrows) in cells are shown. Arrows indicate the mature lamellipodias. Scale bar, 10 µm.
hep26373-sup-0015-suppfig15.tif593KSupporting Fig. 15. The level of miR-195 correlated inversely with the proliferation rate of tumor cells in human HCC tissues. This correlation was analyzed in the 90 HCC tissues described in Fig. 5C. The median value of the proportion of Ki-67-positive tumor cells in 90 cases was chosen as the cut-off point for separating the low- (n = 45) from the high-proliferation-rate (n = 45) tumors. **, P< 0.01.
hep26373-sup-0016-suppfig16.tif98KSupporting Fig. 16. Effect of miR-195 over-expression on the growth of QGY-7703 (A) and MHCC-97H cells (B). Cells that were non-transfected or transfected with NC or miR-195 duplex were grown in 24-well plates, and the cell numbers were counted every 24 h for 5 days after the transfections. *, P< 0.05; ***, P< 0.001.
hep26373-sup-0017-suppfig17.tif293KSupporting Fig. 17. Effect of miR-195 over-expression on the growth and migration of MHCC-97L (A), Huh-7 (B), SMMC-7721 (C) and HCT-116 cells (D). Cells that were non-transfected or transfected with NC or miR-195 duplex were analyzed for the growth (panel 1) and migration (panel 2). *, P< 0.05; **, P< 0.01; ***, P< 0.001.
hep26373-sup-0018-suppfig18.tif143KSupporting Fig. 18. Effect of miR-195 over-expression on Rb signaling in MHCC97L and QGY-7703 cells. Forty-eight hours after transfections with the indicated RNA duplexes, cells were subjected to immunoblotting or RT-PCR analysis. pRb, total Rb protein; ppRb, phosphorylated pRb. β-actin, internal control.
hep26373-sup-0019-suppfig19.tif52KSupporting Fig. 19. miR-195 affects various phenotypes of cancer cells by regulating different signaling pathways.
hep26373-sup-0020-suppinfo.doc108KSupporting Information
hep26373-sup-0021-supptables.doc51K

Supporting Table 1. Univariate and Multivariate Analysis of Factors Associated with RFS a

Supporting Table 2. Sequences of RNA and DNA Oligonucleotides

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