Fig 1: KLF4 inhibits MSI2 to regulate non-small cell lung cancer via the JAK2/STAT3 signalling pathway. (A) Western blotting analysis of the expression of JAK2/STA3 signalling pathway markers in the transfected A549 cell line treated with pKLF4, pMSI2, pKLF4 + pMSI2, and pKLF4 + colivelin. (B) Western blotting analysis of the expression of JAK2/STA3 signalling pathway markers in the transfected H520 cell line treated with pKLF4, pMSI2, pKLF4 + pMSI2, and pKLF4 + colivelin. The results were obtained from four independent experiments and are expressed as the mean ± SD. *P<0.05, ⁎⁎P<0.01, ⁎⁎⁎P<0.001. KLF4, Krüppel-like factor 4; MSI2, Musashi-2; pKLF4, KLF4 overexpression plasmid; pMSI2, MSI2 overexpression plasmid; p-, phosphorylated; WASF3, Wiskott-Aldrich syndrome protein family member 3.
Fig 2: KLF4 inhibits MSI2 to regulate non-small cell lung cancer via the JAK2/STAT3 signalling pathway. (A) MSI2 expression in A549 and H520 cells transfected with pMSI2 was measured via western blotting. (B) Cell proliferation, (C) migration (scale bar, 500 µm) and (D) invasion (scale bar, 200 µm) assays using A549 and H520 cells treated with pKLF4, pMSI2, pKLF4 + pMSI2, and pKLF4 + colivelin. The results were obtained from four independent experiments and are expressed as the mean ± SD. *P<0.05, ⁎⁎P<0.01, ⁎⁎⁎P<0.001. KLF4, Krüppel-like factor 4; MSI2, Musashi-2; pKLF4, KLF4 overexpression plasmid; pMSI2, MSI2 overexpression plasmid; NC, negative control.
Fig 3: Overexpression of KLF4 suppresses NSCLC cell migration and invasion via inhibition of the JAK2/STAT3 signalling pathway. (A) Western blotting assay of KLF4 expression in human NSCLC cell lines (H1975, H460, A549, H520 and NCI-1299) and normal pulmonary epithelium cell (16HBE). (B) Western blotting assay of KLF4 expression in A549 and H520 cells transfected with empty vector or KLF4-overexpression plasmids. (C) Cell proliferation, (D) colony formation, (E) migration (scale bar, 500 µm) and (F) invasion assays (scale bar, 200 µm) were performed with KLF4-transfected A549 and H520 cell lines. (G) Western blotting analysis of the expression of JAK2/STA3 signalling pathway markers in KLF4-transfected A549 and H520 cell lines. The results were obtained from four independent experiments and are expressed as the mean ± SD. *P<0.05, ⁎⁎P<0.01, ⁎⁎⁎P<0.001. KLF4, Krüppel-like factor 4; p-, phosphorylated; WASF3, Wiskott-Aldrich syndrome protein family member 3; NSCLC, non-small cell lung cancer; MSI2, Musashi-2.
Fig 4: KLF4 is downregulated and MSI2 is upregulated in NSCLC specimens. (A) MSI2 and (B) KLF4 expression in normal lung tissues (n=20) and NSCLC specimens (n=30), as determined via reverse transcription-quantitative PCR. (C) Correlation between KLF4 and MSI2 expression in NSCLC specimens (n=30), analysed using Pearson's correlation coefficient analysis. (D) Associations between clinicopathological parameters and MSI2/KLF4 expression in NSCLC specimens according to the World Health Organization classification (I/II vs. III/IV) or tumour size (≥3 vs. <3 cm). The results were expressed as the mean ± SD. ⁎⁎P<0.01, ⁎⁎⁎P<0.001. (E) Kaplan-Meier survival curves comparing the cumulative overall survival probability in patients with low and high MSI2 expression levels. KLF4, Krüppel-like factor 4; MSI2, Musashi-2; NSCLC, non-small cell lung cancer.
Fig 5: KLF4 inhibits the transcription of MSI2 expression in non-small cell lung cancer cell lines. (A) Western blotting assay of KLF4 expression in A549 and H520 cells transfected with siRNA targeting KLF4 (si-KLF4-1, si-KLF4-2, si-KLF4-3 and si-KLF4-4). Reverse transcription-quantitative assays of MSI2 expression in A549 and H520 cells transfected with (B) pKLF4 or (C) si-KLF4-2. (D-E) Fold enrichment of ChIP assay in A549 demonstrated the direct binding of KLF4 to MSI2’s promoter. (D) A549 and H520 cells were co-transfected with MSI2 and pKLF4, si-KLF4-2 or control vector. Promoter activity in the cells was examined using a dual-luciferase reporter assay. The results were obtained from four independent experiments and are expressed as the mean ± SD. *P<0.05, ⁎⁎P<0.01, ⁎⁎⁎P<0.001. KLF4, Krüppel-like factor 4; MSI2, Musashi-2; si-, small interfering RNA; NC, negative control; pKLF4, KLF4 overexpression plasmid.
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