Figure 1.
Elevated FHIT expression inhibits metastasis in human NSCLCs.
(A) Representative BLI plots of lung metastases in mice after injecting 1×106 A549 or H1299 NSCLC cells stably expressing FHIT or a control vector; n = 4. (B) Lungs were extracted at 8 weeks after tail vein injection and the number of nodules was counted. (C) Representative images of FHIT-expressing or control cells that invaded through the filter and were stained with crystal violet. Scale bar, 40 µm. The results are means ± s.d, n = 4 experiments. *P<0.001. (D) Wound healing assay of confluent monolayer of FHIT-expressing or control A549 and H1299 cells at 0 h and 24 h. See Supplementary Fig. S3, that shows representative images of migration. *P<0.001, **P<0.0001.
Figure 2.
FHIT modulates EMT in NSCLCs through the suppression of EMT-related genes.
(A), (B) Phase contrast images of FHIT-expressing or control A549 and H1299 cells, and Immunoblot analysis of EMT markers in both cell lines, respectively. Scale bar, 50 µm. (C), (D) Phase contrast and immunofluorescence images for Vimentin and E-cadherin staining in TGF-β1-induced A549 cells. DAPI staining was used to detect nuclei and merged with Vimentin and E-cadherin stained images. Scale bar, 50 µm (for brightfield images), 20 µm (for immunofluorescence images). (E) Immunoblot and qRT-PCR analyses of expression of EMT markers, Fibronectin (FN1), E-cadherin, N-cadherin, Vimentin and Snail, in response to TGF-β1 treatment of A549 cells with and without FHIT expression. RT-qPCR values were normalized to the housekeeping gene GAPDH. Experiments were performed three times and the data are presented as the mean ± s.d. *P<0.05 and **P<0.01 by Student's t-test.
Figure 3.
FHIT expression leads to activation of miR-30c in lung cancer.
(A) Unsupervised hierarchical clustering of FHIT-regulated miRNAs in H1299/FHIT cells; P<0.05 (B) RT-qPCR analysis showing up-regulation of FHIT and miR-30c in adjacent normal tissues compared to tumor tissues (n = 19). (C) XY scatter plots showing positive correlation between FHIT and miR-30c in primary lung tumors (n = 20). (D) Kaplan-Meier plots of metastasis-free survival of breast cancer patients, stratified by expression of FHIT (2290 patients) or miR-30c (210 patients). Data obtained from the Kaplan-Meier plotter database (kmplot.com/analysis) or microRNA survival analyses in cancer (www.bioprofiling.de).
Figure 4.
miR-30c inhibits TGF-β-induced EMT through direct targeting of Vimentin and Fibronectin.
(A) Phase contrast images of A549/miR-30c cells in response to TGF-β1 treatment. Scale bar, 100 µm. (B), (C) Immunofluorescence images and Western blot analysis of EMT markers, E-cadherin, FN1 and Vimentin, in miR-30c-transfected A549 cells treated with TGF-β1, respectively. Scale bar, 20 µm. (D) RT-qPCR analysis showing induction of E-cadherin and a decline in the expression of Vimentin in TGF-β-induced A549/miR-30c cells. *P<0.05 and **P<0.01 by Student's t-test. (E) RT-qPCR for miR-30c in response to TGF-β1 treatment. *P<0.001 by Student's t-test. (F) miR-30c down-regulates mesenchymal markers, Vimentin and Fibronectin by directly targeting their 3′ UTRs. Luciferase reporters assays using wild type or mutant 3′ UTR were performed after transfection into A549/FHIT or A549/FHIT cells with miR-30c knockdown. Experiments were performed three times and the data are presented as the mean ± s.d.
Figure 5.
miR-30c down-regulates the expression of MTDH and HMGA2 by directly targeting their 3′UTRs.
(A) Luciferase activity assays of luciferase reporters with wild type or mutant 3′UTRs of MTDH or HMGA2 were performed after transfection into A549/FHIT or A549/FHIT cells with miR-30c knockdown. (B), (C) Western blot analysis for HMGA2 or MTDH in miR-30c-transfected cells, H1299 and A549. miR-20a was another negative control. (D) MTDH or HMGA2 protein levels in anti-miR-30c-transfected cells stably expressing FHIT. (E) RT-qPCR for MTDH in presence of miR-30c. (F) Correlation between expression levels of miR-30c and MTDH in NSCLC samples (n = 30). Pearson's correlation was used. y, the relative expression level of miR-30c, U6, as an internal control. x, the relative expression level of MTDH, GAPDH, as an internal control. Experiments were performed three times and the data are presented as the mean ± s.d. *P<0.01 and **P<0.001 by Student's t-test.
Figure 6.
miR-30c inhibits metastasis through the suppression of MTDH and HMGA2 in NSCLCs.
(A) Representative BLI plots of lung metastasis of mice injected (IV) with 1×106 A549 cells stably expressing miR-30c or a control vector; n = 4. (B) Representative H&E stained lung section. The arrows highlight metastatic nodules. *P<0.05 by Student's t-test. (C) Representative BLI plot of metastasis after IV injection with 1×106 A549 cells after knockdown of MTDH, HMGA2, or control; n = 5. (D) Representative H&E stained lung section and the number of lung metastatic nodules. The arrows indicate metastatic nodules. *P<0.001. (E), (F) Invasion and migration assays in control and miR-30c-overexpressing cells, or control, MTDH and HMGA2 knockdown cells, respectively. *P<0.001 **P<0.01 by Student's t-test. (G) Immunohistochemistry assay for FHIT, MTDH and HMGA2 in primary lung tumors and their matched lymph node metastases. Scale bar, 100 µm. (H) The expression pattern of FHIT, miR-30c, MTDH, and HMGA2 in normal (N), non-metastatic (N.M) and metastatic tissues (M). *P<0.001 **P<0.05 by Student's t-test.
Figure 7.
Proposed model for the function of the FHIT and miR-30c in lung metastasis and EMT.