Fig 2: Immunohistochemical antibody detection. The percentage of (a) SmActin, TGF-β, HIF1A, LOX, LOXL2 and TN-C positive area and (b) the percentage of MMP-2, MMP-9 and Fibronectin positive area after immunohistochemical (IHC) detection. Data are presented as mean ± SD (n = 10). Values connected by lines differ significantly from each other (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0005).

Fig 3: Silencing LINC00963 inhibited colon cancer cell invasion and migration-related protein expression, and overexpression of LINC00963 had the opposite effect. a–d The effect of overexpression or silencing of LINC00963 on colon cancer cell invasion was examined by Transwell. e–h LINC00963 overexpression promoted the expressions of MMP-3 and MMP-9, and silencing LINC00963 inhibited the expression of MMP-3 and MMP-9. The experiment was repeated three times independently. GAPDH served as a control. ***P < 0.001 vs siNC; ^^^P < 0.001vs NC

Fig 4: miR-4500 suppresses non-small cell lung cancer cell migration and invasion by downregulating STAT3 in vitro. (A) A Wound healing assay and (B) a Transwell invasion assay of A549 and H1975 cells subsequent to transfection with si-STAT3 or NC or a combination of si-STAT3 and miR-4500 inhibitors. (C) Western blotting determined the protein expression levels of MMP-2 and MMP-9 in A549 and H1975 cells transfected with with si-STAT3 or NC or a combination of si-STAT3 and miR-4500 inhibitors. All data were presented as the mean ± standard deviation and each was performed in triplicate. *P<0.05 vs. the control group. #P<0.05 vs. the si-STAT3 group. miR, microRNA; STAT3, signal transducer and activator of transcription 3; si-, small interfering RNA; MMP, matrix metalloproteinase.

Fig 5: TGF-β signaling was involved in the regulation of LHX9 and metastasis-related proteins. a U2OS or Saos-2 cells were cultured without or with 1, 5, 10 ng/ml TGF-β1 for 24 h (left two panels), or cultured without or with 5 ng/ml TGF-β1 for 24, 48 and 72 h, respectively (right two panels). The expression of FRS2 protein was measured by Western blot, β-actin was used as the loading control. The normalized protein expression levels against β-actin were shown in the below histograms. b U2OS or Saos-2 cells were cultured without or with 1, 5, 10 ng/ml TGF-β1 for 24 h, then the relative expression of LHX9 was assessed by RT-qPCR. The mRNAs were normalized to GAPDH, experiments were performed in triple. c U2OS or Saos-2 cells were cultured without or with 5 ng/ml TGF-β1 for 24, 48 and 72 h, respectively, and the relative expression of LHX9 was measured by Western blot. GAPDH was used as the loading control. The normalized expression of LHX9 against GAPDH was shown in the right histograms. d U2OS or Saos-2 cells were transfected with shNC or shFRS2, then they were cultured for 48 h. The relative expression of TGF-beta and TGF-beta R1 was assessed by RT-qPCR. The mRNAs were normalized to GAPDH mRNA (n = 3). e U2OS or Saos-2 cells were transfected with shNC or shFRS2, then they were cultured in the absence or presence of TGFβR1 inhibitor (SB431542) for 48 h. The expression of LHX9, COL1A1, MMP-9, and MMP-1 was measured by Western blot. GAPDH was used as the loading control. The normalized expression of these proteins against GAPDH was shown in the right histograms. a–e The result was a representative of three independent experiments. Error bars represented mean ± SD. p values were determined by one-way analysis of variance (ANOVA) followed by Tukey post hoc test (a–c, e) or unpaired two-tail Students’ t-test (d). **p < 0.01, *p < 0.05. NS not significant