MiRNA199a-3p suppresses tumor growth, migration, invasion and angiogenesis in hepatocellular carcinoma by targeting VEGFA, VEGFR1, VEGFR2, HGF and MMP2

Abstract

Increasing significance of tumor-stromal interaction in development and progression of cancer implies that signaling molecules in the tumor microenvironment (TME) might be the effective therapeutic targets for hepatocellular carcinoma (HCC). Here, the role of microRNA miR-199a-3p in the regulation of TME and development of HCC has been investigated by several in vitro and in vivo assays. Expression of miR-199a-3p was observed significantly low in HCC tissues and its overexpression remarkably inhibited in vivo tumor growth and metastasis to lung in NOD-SCID mice. In vitro restoration of miR-199a-3p expression either in endothelial cells (ECs) or in cancer cells (CACs) significantly diminished migration of ECs in co-culture assay. Again incubation of miR-199a-3p transfected ECs with either conditioned media (CM) of CACs or recombinant VEGF has reduced tube formation, in ECs and it was also dropped upon growth in CM of either anti-VEGF antibody-treated or miR-199a-3p-transfected CACs. In addition, bioinformatics and luciferase-reporter assays revealed that miR-199a-3p inhibited VEGF secretion from CACs and VEGFR1 and VEGFR2 expression on ECs and thus restricted cross talk between CACs and ECs. Again, restoration of miR-199a-3p in hepatic stellate cells (HSCs) reduced migration and invasion of CACs in co-culture assay, while it was enhanced by the overexpression of HGF suggesting miR-199a-3p has hindered HSC-CACs cross talk probably by inhibiting HGF and regulating matrix metalloproteinase MMP2, which were found as targets of miR-199a-3p subsequently by luciferase-reporter assay and gelatin zymography, respectively. Thus, these findings collectively highlight that miR-199a-3p restricts metastasis, invasion and angiogenesis in HCC and hence it may be considered as one of the powerful effective therapeutics for management of HCC patients.

Figure 1

Anti-tumorigenic role of miR-199a-3p. miR-199a-3p expression was compared between (a) HCC tumor and normal liver (NL) tissues, (b) HCC tumor and adjacent non-tumor tissues and (c) HCC cell lines and normal liver (NL). (d) Tumor volume at different time points and (e) tumor weight of subcutaneous HCC in either vector or miR-199a-3p overexpressed cell line injected NOD/SCID mice. (f) Size and number of the nodule formed in the lung after 4 weeks of injection of either vector or miR-199a-3p overexpressed cell line through tail vein in NOD/SCID mice. (g) Hematoxylin and eosin staining of the lung section. Area enclosed by dotted line indicates metastatic growth. *P<0.05, **P<0.01 and ***P<0.005 respectively

Figure 2

miR-199a-3p inhibits migration and angiogenesis in ECs by regulating cross talk between CACs and ECs. Endothelial cell recruitment assay in Boyden chamber: HUVEC cells were co-cultured with HepG2 and SNU449 separately. premiR-199a-3p and control vector plasmids were transfected either in (a) HUVEC or (b) in HCC cell lines. Bar diagram represented percentage of migrated cells though 8μm filter. In vitro tube formation in ECs was performed after similar transfection in either (c) HUVEC or (d) HCC cell lines. HUVECs were cultured on matrigel-coated plate with CM of cancer cell lines. Bars represented percentage of cellular branches. All the experiments were repeated three times. *P<0.05 and **P<0.01, respectively

Figure 3

VEGFA in cancer cells and VEGFR1 and VEGFR2 on endothelial cells are suppressed by miR-199a-3p. (a) Luciferase-reporter assay with VEGFR2 were determined separately in premiR-199a-3p-transfected wild-type and miR-199a-3p binding-site mutant 3′-UTRs of VEGFA, VEGFR1, HepG2 cells. (b) Ectopic expression of miR-199a-3p was confirmed at different time points. (c) Protein expression of VEGFA and VEGFR2 in SNU449 and HUVEC cells, respectively. By ELISA, secreted VEGFA was determined in (d) culture media of control vector, premiR-199a-3p and premiR-199a-3p with anti-miR-199a-3p-transfected SNU449 cells and (e) serum of healthy volunteer (n=8) and HCC patients (n=7). (f) Tube-formation assay was performed with premiR-199a-3p or vector-transfected HUVECs in the presence of 20 ng/ml of recombinant VEGFA and anti-VEGF antibody or control IgG-treated CM of SNU449. (g) VEGFR2, ERK1/2 and phospho-ERK1/2 levels were determined in transfected HUVEC cells cultured in the presence of 20 ng/ml of recombinant VEGFA. α-Tubulin was used as loading control in western blot. *P<0.05, **P<0.01 and ***P<0.005

Figure 4

miR-199a-3p inhibits migration and invasion of cancer cells by regulating the cross talk between cancer cells (HepG2 or SNU449) and hepatic stellate cells (LX2). (a) Migrated cancer cells in transwell plate upon co-culturing with either LX2 cells or mock media. LX2 cells were transfected with vector and premiR-199a-3p, and SNU449 cells were either (b) co-cultured with transfected LX2 or (c) grown on matrigel pre-coated plate with CM of transfected LX2 cells. Average number of SNU449 cells migrated and invaded was counted. *P<0.05 and **P<0.01

Figure 5

miR-199a-3p reduces secretion of HGF from LX2 cell line to inhibit its paracrine effect on cancer cells. (a) 3′-UTR-reporter-luciferase assay of HGF in HepG2 cells transfected with vector and premiR-199a-3p. LX2 cells were transfected with vector, pBABE-puro HGF, HGF-shRNA and premiR-199a-3p. Intracellular HGF expression was determined by (b) western blot and secreted HGF by ELISA in (c) cell culture media and (d) serum of HCC patients. (e) Migration and (f) invasion ability of SNU449 cells in the presence or absence of HGF secretion from LX2 was determined. *P<0.05, **P<0.01 and ***P<0.005

Figure 6

miR-199a-3p regulates extracellular matrix remodeling by targeting MMP2. (a) 3′-UTR-reporter-luciferase assay of MMP2 in HepG2 cells co-transfected with control vector and premiR-199a-3p. (b) MMP2 activity was determined by gelatin zymography in LX2, SNU449 and HepG2. (c) Western blot and (d) gelatin zymography of LX2 cells either transfected with premiR-199a-3p or control vector. **P<0.01