Table 1.
Correlation between miR-34a expression and clinicopathologic parameters in 75 TSCC patients.
Figure 1.
miR-34a correlated with lymph node metastases of TSCC patients and inhibited migration and invasion of TSCC cell lines.
(A), Relative levels of miR-34a in 75 surgical specimens of TSCC and matched adjacent nonmalignant tissues was quantified by qRT-PCR. Data were presented as log2 fold change(△△Ct values,TSCC/Nonmalignant, T/N). (B), Means of miR-34a relative levels for 75 surgical specimens of TSCC and the matched adjacent nonmalignant tissues. Data were presented as 2−△Ct (miR-34a-U6) values (*P<0.05). (C), Means of miR-34a relative levels in SCC-15, CAL27 cell lines and primary normal human oral keratinocyte (HOK) cells. Data were presented as 2−△Ct (miR-34a-U6) values (**P<0.01). (D), Means of miR-34a relative levels from formalin-fixed paraffin-embedded tissues including a group of 15 TSCC patients with positive lymph node metastases compared with another group of 15 TSCC patients with negative lymph node metastases matched for age, gender, pathologic differentiation, and TNM stage. Data were presented as 2−△Ct (miR-34a-U6) values (**P<0.01). (E), Cell viability was measured using CCK-8 assays, the data were presented as means ±SD as indicated (*P<0.05, **P<0.01). (F) and (G) Representative photomicrographs of wound healing and transwell assays results for SCC-15 and CAL27 cells transfected with pcDNA3.0-miR-34a (miR-34a) or pcDNA3.0 (NC) (×200 magnification, **P<0.01).
Figure 2.
miR-34a targets MMP9 and MMP14.
(A), The sequence of miR-34a (middle) matches the coding sequence (CD) of MMP9 and 3′untranslated region (UTR) of MMP14 (top). Bottom, mutations of the CD of MMP9 and 3′UTR of MMP14. (B), miR-34a inhibited wild-type, but not mutated MMP9 CD and MMP14 3′UTR luciferase reporter activity. CAL27 cells were co-transfected with firefly luciferase reporter plasmids containing wild type or mutant MMP9 CD and MMP14 3′UTR, and pRL-TK plasmid (a plasmid expressing rellina luciferase) and pcDNA3.0-miR-34a (miR-34a) or pcDNA3.0 (NC) as indicated. After 24 h, firefly luciferase activities were measured and normalized by use of renilla luciferase activities. Data were presented as mean ±SD (*P<0.05). (C), The relative MMP9 and MMP14 mRNA levels determined by qRT-PCR in miR-34a or NC transfected CAL27 cells (*P<0.01). (D), Inhibition of the expression of MMP9 and MMP14 proteins. Representative Western blotting image (top) and the quantification (bottom) of MMP9 and MMP14 proteins in miR-34a transfected SCC-15 and CAL27 cells. β-actin was used as internal control and was also detected by Western blot (**P<0.01).
Figure 3.
Inhibition of MMP9 and MMP14 was responsible for the TSCC cells migration and invasion effects of miR-34a.
(A) and (B), Inhibition of cell migration and invasion by knockdown of MMP9 and MMP14. CAL27 cells were transfected with siRNA control, siMMP9-1, siMMP9-2, siMMP14-1, and siMMP14-2 as indicated. (C) and (D), MMP9 and MMP14 overexpression partially rescues miR-34a-reduced cell migration and invasion. Representative photomicrographs of wound healing and transwell assays results for stable expression miR-34a CAL27 cells transfected with pcDNA3.0-MMP9 (MMP9) or pcDNA3.0-MMP14 (MMP14) (×200 magnification, **P<0.01).
Figure 4.
Inverse correlation between miR-34a and MMP9 or MMP14 protein levels in TSCC.
The expression of MMP9 or MMP14 proteins was examined by immunohistochemistry (IHC) and miR-34a expression was detected by qRT-PCR and in situ hybridization (ISH). (A), Statistical analysis of the expression of miR-34a in tumor vs. nonmalignant tissue. Spearman's rank correlation analysis was performed, with r and P values as indicated. (B), The concurrence of high miR-34a expression and corresponding low expression of MMP9 or MMP14 were confirmed in TSCC and nonmalignant specimens by ISH with miR-34a detection probe or Scramble-miR and IHC, Primary antibodies were omitted in negative control for IHC (200× magnification). (C), The concurrence of low miR-34a expression and corresponding high expression of MMP9 or MMP14 was confirmed in TSCC and high miR-34a expression and corresponding low expression of MMP9 or MMP14 were confirmed in the matched nonmalignant specimens by ISH with miR-34a detection probe or Scramble-miR and IHC. Primary antibodies were omitted in negative control for IHC (200× magnification).