microRNA-133b represses the progression of lung cancer through inhibiting SOX9/β-catenin signaling pathway

Abstract

MicroRNA-133b (miR-133b) has been shown to be down-regulated in lung cancer and functions as a tumor repressor. However, the underlying mechanisms of miR-133b in lung cancer are not clear. SOX9, a member of SOX family, serves as an oncogene in lung cancer by activating b-catenin signaling and was identified to be a direct target of miR-133b in breast cancer. Based on these data, the current study was performed to explore whether SOX9/b-catenin signaling is implicated in miR-133b-meditaed lung cancer repression. MiR-133b expression in lung cancer tissues and cells were detected by RT-PCR. CCK-8, colony formation, flow cytometry, transwell chamber and in vivo assays were carried out to determine cell proliferation, colony formation, apoptosis, cell cycle, invasion, and tumorigenesis. We found that miR-133b expression was decreased in lung cancer tissues and cells. Up-regulation of miR-133b reduced cell proliferation and colony formation, induced cell apoptosis and G0/G1 phase arrest, and decreased cell invasion. Besides, miR-133b up-regulation decreased the expression of b-catenin and SOX9. Cell viability inhibition and apoptosis promotion induced by miR-133b up-regulation were all impaired when SOX9 was up-regulated. Furthermore, miR-133b over-expression repressed the tumorigenesis of lung cancer cells with smaller tumor size and lower Ki-67 expression. Taken together, this study clarifies that miR-133b represses lung cancer progression by inhibiting SOX9/b-catenin signaling.

Keywords: MiR-133b; SOX9; lung cancer; β-catenin.

Figure 1

Expression of miR-133b was decreased in lung cancer tissues and cell lines. A. QPCR analysis of the levels of miR-133b in 20 paired lung cancer tissues and adjacent normal lung tissues. B. QPCR analysis of the levels of miR-133b in normal lung cell line BEAS-2B and lung cancer cell lines A549, H1299, and SPC-A1. (**P<0.01).

Figure 2

miR-133b inhibited the malignant phenotypic switching of lung cancer cells. H1299 cells were infected with mimic-miR-133b or mimic-NC, then: A. The expression of miR-133b was detected by QPCR. B. Cell colony formation ability was determined by colony formation assay. C. Cell viability was determined by CCK-8 assay. D, E. Cell cycle and apoptosis were evaluated by flow cytometry with PI staining and Annexin V/PI double staining, respectively. F. Cell invasion was assessed by transwell chamber with Matrigel coated. (n=3, *P<0.05, **P<0.01).

Figure 3

miR-133b repressed the progression of lung cancer through down-regulating β-catenin and SOX9 expression in H1299 cells. A. Western blot analysis of the protein expression of β-catenin and SOX9 after 48 h of H1299 cells infection with mimic-NC or mimic-miR-133b (n=3, *P<0.05, **P<0.01). B. Western blot analysis of the transfection efficiency of OE-SOX9 (n=3, **P<0.01). C. Cell colony formation ability was determined by colony formation assay after H1299 cells were treated with mimic-miR-133b or mimic-miR-133b + OE-SOX9. D. Cell apoptosis was evaluated by flow cytometry after H1299 cells were treated with mimic-miR-133b or mimic-miR-133b + OE-SOX9. (n=3, **P<0.01, compared with control group; #P<0.05, compared with mimic-miR-133b group).

Figure 4

miR-133b reduced the tumor formation ability of lung cancer cells in vivo. H1299 cells were stably transfected with mimic-miR-133b or mimic-NC, then these stably transfected cell lines were injected into nude mice to test their tumorigenesis. A. Tumor weights were measured. B. The expression of Ki-67 in tumor tissues was evaluated by immunohistochemistry (n=10 **P<0.01).