MicroRNA-216b regulated proliferation and invasion of non-small cell lung cancer by targeting SOX9

Abstract

Micro (mi)RNAs are small, evolutionarily conserved and endogenous noncoding RNA molecules between 19 and 24 nucleotides in length. The potential roles of miRNAs in the carcinogenesis and progression of non-small cell lung cancer (NSCLC) have been studied previously. In the present study, it was revealed that miRNA-216b (miR-216b) expression was lower in NSCLC tissue and cell lines compared with that in adjacent healthy lung tissue samples and the normal bronchial epithelial 16HBE cell line, respectively. The ectopic expression of miR-216b inhibited the proliferation and invasion of NSCLC cells in vitro. SRY-Box 9 (SOX9) was identified as a direct target of miR-216b in NSCLC. In addition, SOX9 small interfering RNA was able to mimic the effects of miR-216b overexpression on cell proliferation and invasion in NSCLC. Therefore, the data reported in the present study demonstrate that miR-216b is an important tumor suppressor in NSCLC. These data may contribute to the understanding of the molecular mechanism underlying the carcinogenesis and progression of NSCLC, and provide novel therapies for patients with NSCLC.

Keywords: SRY-Box 9; invasion; microRNA-216b; non-small-cell lung cancer; proliferation.

Figure 1.

Expression levels of miR-216b were downregulated in NSCLC tissues and cell lines. (A) miR-216b was significantly downregulated in NSCLC tissues compared with the adjacent non-tumor lung tissues. (B) Quantitative analysis of the miR-216b by qPCR in four NSCLC cell lines. Data is presented as the mean ± standard deviation. Experiments were performed in triplicate and repeated three times. *P<0.05. miR, microRNA; NSCLC, non-small cell lung cancer.

Figure 2.

miR-216b inhibited NSCLC cell proliferation. (A) H23 and A549 cells were transfected with miR-216b mimics or NC. Reverse transcription quantitative polymerase chain reaction was adopted to determine the miR-216b expression subsequent to transfection. (B) Effect of miR-216b overexpression on the proliferation of H23 and A549 cells was assessed using cell proliferation assay. *P<0.05. miR, microRNA; NC, negative control.

Figure 3.

Effect of miR-216b overexpression on the invasion capacity of H23 and A549 cells was evaluated using cell invasion assay. Upregulation of miR-216b expression decreased the invasion capacity of H23 and A549 cells. *P<0.05. miR, microRNA; NC, negative control.

Figure 4.

miR-216b directly targeted SOX9 and decreased its expression. (A) Predicted miR-216b binding sequence in the SOX9 3′UTR. The corresponding mutant counterpart was generated in the SOX9 3′UTR sequence in the complementary site for the seed region of miR-216b. (B) The pGL3-SOX9-3′UTR Wt or pGL3-SOX9-3′UTR Mut luciferase reporter vector was co-transfected into HEK293T cells with miR-216b mimics or NC. (C) SOX9 expression at mRNA level measured using qPCR. (D) SOX9 expression at protein levels subsequent to transfection with miR-216b mimics or NC measured using western blot analysis. *P<0.05. has-miR, human microRNA; NC, negative control; SOX9, sex determining region Y-Box 9; UTR, untranslated region; wt, wild type; Mut, mutant.

Figure 5.

SOX9 downregulation was the mechanism of miR-216b inhibiting the proliferation and invasion of NSCLC cells. (A) H23 and A549 cells were transfected with SOX9 siRNA or siRNA-NC. Western blot analysis was used to measure the SOX9 expression subsequent to transfection. (B) Effect of SOX9 downregulation on the proliferation of H23 and A549 cells was assessed using a cell proliferation assay. (C) Effect of SOX9 downregulation on the invasion capacity of H23 and A549 cells was evaluated using a cell invasion assay. *P<0.05. SOX9, sex determining region Y-Box 9; siRNA, small interfering RNA; NC, negative control.