Figure 1.
Overexpression of miR-127 inhibits HCC cell migration and tumor growth.
(A) Wound healing assay. MHCC97H cells were transfected with either an empty vector pTarget (-) or a miR-127 expression vector in pTarget (+) (2 µg). Wound closure was photographed 48 hr later (left) and the residual gap between the migrating cells was quantified (right), which is expressed as a percentage of the initial scraped area. (B) Cell migration assay. MHCC97H cells were transfected with pTarget or pTarget-miR127 (2 µg), a negative (neg) control or miR-127 inhibitor (anti-miR-127) (20 nM). Cells that had migrated through the membrane of Transwell inserts were fixed, stained with crystal violet, and visualized by microscopy. (C) Cell invasion assay. MHCC97H cells were transfected with pTarget or pTarget-miR127 (2 µg), and seeded onto pre-coated inserts with BME solution and incubated for 16 hr. The invaded cells were dissolved in Cell Dissociation Solution/Calcein-AM and absorbance was measured. (D) Tumorigenesis assay. Control 97HLuc-Sico or 97HLuc-miR-127 cells were grafted subcutaneously in the dorsa of athymic mice, and tumor growth was monitored using the Xenogen bioluminescent imaging system by day 9. The number represents tumor images detected (photons/s/cm2/steridian). *p<0.01, miR-127 vs. pTarget control; ¥p<0.01, anti-miR-127 vs. negative control.
Figure 2.
Overexpression of miR-127 inhibits MMP13 3′UTR activity and gene expression.
(A) Sequence alignment between miR-127 and the 3′UTR of MMP13 in human, mouse, and rat. Solid line: seed match region. (B) Transient transfection assay. Hela cells were co-transfected with the control pTarget (0), pTarget-miR-127, and MMP13 (left), PPP1R9B (middle), or MMP13 mutant (mut) 3′UTRs (right). Luciferase (Luc) activity was normalized to Renilla activity (act). PPP1R9B served as a negative control. (C) Transient transfection assay to determine the effect of pTarget-miR-127 (100 ng) and miR-127 inhibitor (20 nM) on MMP13 3′UTR reporter activity. (D) Western blots (WB) of MMP13 protein expression in MHCC97H cells that were transfected with pTarget-miR127 (2 µg) or treated with a miR-127 inhibitor (anti-miR-127) (20 nM). (E) qPCR analysis of miR-127 expression under the same experimental conditions as (D). (B-E): Data are expressed as means ± SD of triplicate assays. *p<0.01, miR-127 vs. pTarget control; ¥p<0.01, anti-miR-127 vs. negative control.
Figure 3.
Overexpression of miR-127 inhibits MMP13 and TGFβ-mediated induction of HCC cell migration.
(A) Cell migration assay. MHCC97H cells were transfected with MMP13 siRNA (siMMP13) (20 nM) in the absence or presence of pTarget or pTarget-miR-127 (2 µg). After cells were seeded onto the inserts, lower chamber media were treated with MMP13 peptide (50 ng/ml) as indicated. Cells that had migrated through the membrane were fixed and stained with crystal violet (left). Quantitative results on the right. (B-C) qPCR analysis of MMP13 (B) and miR-127 (C) expression under the same experimental conditions as (A). (D) Cell migration assay. MHCC97H and Hepa-1 cells were transfected with pTarget or pTarget-miR-127 (2 µg), in the absence or presence of TGFβ (5 ng/ml). Migrated cells were stained with crystal violet and visualized by microscopy (left). Quantitative results on the right. (A-D): *p<0.01, miR-127 vs. pTarget in control group; ¥p<0.01, pTarget in MMP13 group vs. pTarget in control group; ‡p<0.01, miR-127 vs. pTarget in MMP13 group; §p<0.01, miR-127 vs. pTarget in siMMP13 group.
Figure 4.
TGFβ represses miR-127 expression by enhancing c-Jun activity.
(A) qPCR analysis of MMP13 (left), c-Jun (middle), and miR-127 (right) expression in MHCC97H cells treated with TGFβ. *p<0.01, TGFβ (+) vs. TGFβ (-) group. (B) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter (pro) luciferase (luc) reporter, and c-Jun or c-Fos expression plasmids. *p<0.01, c-Jun (+) vs. c-Jun (-) group. (C) Transient transfection assay. Hela cells were co-transfected with miR-127 or AP1 promoter reporter along with c-Jun/c-Fos plasmid (100 ng) in the absence or presence of TGFβ (5 ng/ml). *p<0.01, c-Jun/Fos vs. control without (-) or with (+) TGFβ; ¥p<0.01, control with (+) TGFβ vs. control without (-) TGFβ. (D) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter reporter, c-Jun (100 ng) and/or p53 plasmids (100 ng). *p<0.01, c-Jun or p53 vs. control; ¥p<0.01, c-Jun/p53 vs. p53. (B-D) Luciferase (Luc) activity was normalized by Renilla activity (act). (E) qPCR analysis of miR-127 and p53 expression in HepG2 cells transfected with control (con) or p53 siRNAs (si-p53) (20 nM), or in HCT116p53+/+ (+) and HCT116p53−/− (-) cells. *p<0.01, si-p53 vs. control. (F) qPCR analysis of miR-127 expression in MHCC97H cells that were overexpressed with c-Jun and/or p53. *p<0.01, p53 vs. control (-); ¥p<0.01, c-Jun/p53 vs. p53.
Figure 5.
TGFβ enhances c-Jun-mediated repression of miR-127 via ERK and JNK pathways.
(A-C) qPCR analysis of c-Jun (A), miR-127 (B), and MMP13 (C) mRNA expression in MHCC97H cells treated with TGFβR inhibitor (SB 431542, 10 µM), NFκB inhibitor (BAY-11-7082, 5 µM, ERK inhibitor (PD 98059, 50 µM), p38 inhibitor (SB 203580, 10 µM) and JNK inhibitor (SP 600125, 50 µM),) in the absence (−) or presence of TGFβ (+) (5 ng/ml). Statistical results represent the mean ± SD. *p<0.01, TGFβ (+) vs. TGFβ (−) group.
Figure 6.
miR-127 is downregulated in a subset of HCC specimens.
(A-B) qPCR analysis of miR-127 expression (A) and MMP13 mRNA (B, left), and Western blot (WB) analysis of MMP13 protein (B, right) in 5 pairs of surrounding controls and HCC specimens. *p<0.01, tumor vs. non-tumor.
Figure 7.
Model of negative-feedback regulation of miR-127 expression by TGFβ/c-Jun that controls MMP13 expression and stability in HCC.
TGFβ activates the oncogene c-Jun through ERK and JNK pathways. The activation of c-Jun serves a dual function, which involves induction of MMP13 gene expression but repression of miR-127 gene transcription by inhibiting miR-127 promoter activity. c-Jun also antagonizes p53 activation of the miR-127 promoter and gene transcription. On the other hand, overexpression of miR-127 decreases MMP13 protein levels by binding to its 3′UTR and causing MMP13 degradation, thus diminishing TGFβ-mediated HCC migration.