MiR-9 is involved in TGF-β1-induced lung cancer cell invasion and adhesion by targeting SOX7

Abstract

MicroRNA (miR)-9 plays different roles in different cancer types. Here, we investigated the role of miR-9 in non-small-cell lung cancer (NSCLC) cell invasion and adhesion in vitro and explored whether miR-9 was involved in transforming growth factor-beta 1 (TGF-β1)-induced NSCLC cell invasion and adhesion by targeting SOX7. The expression of miR-9 and SOX7 in human NSCLC tissues and cell lines was examined by reverse transcription-quantitative polymerase chain reaction. Gain-of-function and loss-of-function experiments were performed on A549 and HCC827 cells to investigate the effect of miR-9 and SOX7 on NSCLC cell invasion and adhesion in the presence or absence of TGF-β1. Transwell-Matrigel assay and cell adhesion assay were used to examine cell invasion and adhesion abilities. Luciferase reporter assay was performed to determine whether SOX7 was a direct target of miR-9. We found miR-9 was up-regulated and SOX7 was down-regulated in human NSCLC tissues and cell lines. Moreover, SOX7 expression was negatively correlated with miR-9 expression. miR-9 knockdown or SOX7 overexpression could suppress TGF-β1-induced NSCLC cell invasion and adhesion. miR-9 directly targets the 3' untranslated region of SOX7, and SOX7 protein expression was down-regulated by miR-9. TGF-β1 induced miR-9 expression in NSCLC cells. miR-9 up-regulation led to enhanced NSCLC cell invasion and adhesion; however, these effects could be attenuated by SOX7 overexpression. We concluded that miR-9 expression was negatively correlated with SOX7 expression in human NSCLC. miR-9 was up-regulated by TGF-β1 and contributed to TGF-β1-induced NSCLC cell invasion and adhesion by directly targeting SOX7.

Keywords: SOX7; adhesion; invasion; lung cancer; microRNA-9; transforming growth factor-beta 1.

Figure 1

The correlation analysis of miR‐9 and SOX7 expression in human NSCLC tissues. (A) Relative miR‐9 mRNA level, (B) relative SOX7 mRNA level in human NSCLC and the adjacent normal tissues. (C) The correlation of miR‐9 mRNA expression and SOX7 mRNA expression in human NSCLC tissues. *P < 0.05 and ^# P < 0.01 compared with the normal.

Figure 2

The expression of miR‐9 and SOX7 in human NSCLC cell lines. (A) Relative miR‐9 mRNA level, (B) relative SOX7 mRNA level in human NSCLC cell lines. *P < 0.05 and ^# P < 0.01 compared with the HBE cell line.

Figure 3

miR‐9 knockdown suppressed TGF‐β1‐induced NSCLC cell invasion and adhesion. (A) Relative miR‐9 mRNA level in, (B) invaded cell number of, (C) cell adhesion activity of A549 and HCC827 cells following transfection with the miR‐NC/miR‐9 in the absence or presence of TGF‐β1. *P < 0.05 and ^# P < 0.01 compared with the miR‐NC; ^## P < 0.01 compared with the TGF‐β1+miR‐NC.

Figure 4

SOX7 overexpression suppressed TGF‐β1‐induced NSCLC cell invasion and adhesion. (A) Relative SOX7 protein level in, (B) invaded cell number of, (C) cell adhesion activity of A549 and HCC827 cells following transfection with the pEGFP‐C1/SOX7‐pEGFP‐C1 in the absence or presence of TGF‐β1. Lane 1, pEGFP‐C1; lane 2, SOX7‐pEGFP‐C1; lane 3, TGF‐β1+pEGFP‐C1; lane 4, TGF‐β1+SOX7‐pEGFP‐C1. *P < 0.05 and ^# P < 0.01 compared with the pEGFP‐C1; ^## P < 0.01 compared with the TGF‐β1+ pEGFP‐C1.

Figure 5

TGF‐β1 induced miR‐9 and SOX7 overexpression in NSCLC cells. (A) Relative miR‐9 mRNA level, (B) relative SOX7 protein level in A549 and HCC827 cells in the absence or presence of TGF‐β1. Lane 1, control; lane 2, TGF‐β1. *P < 0.05 and ^# P < 0.01 compared with the control.

Figure 6

SOX7 was a direct target of miR‐9. (A) Bioinformatics predicted the binding site of miR‐9 with SOX7 3′UTR. (B) The relative luciferase activity of SOX7 wild‐type or mutant 3′UTR in HEK293 cells following transfection with the miR‐9 mimic. (C) Relative miR‐9 mRNA level. (D) Relative SOX7 protein level in HEK293 cells following transfection with the miR‐9 mimic or miR‐9 inhibitor. Lane 1, miR‐NC; lane 2, miR‐9 mimic; lane 3, miR‐9 inhibitor. ^# P < 0.01 compared with the miR‐NC.

Figure 7

SOX7 is involved in miR‐9‐mediated NSCLC cell invasion and adhesion. (A) Relative miR‐9 mRNA level in, (B) relative SOX7 protein level in, (C) invaded cell number of, (D) cell adhesion activity of A549 and HCC827 cells following transfection with the miR‐9 mimic and SOX7‐pEGFP‐C1 in the absence or presence of TGF‐β1. Lane 1, CTRL; lane 2, miR‐9 mimic; lane 3, miR‐9 mimic+SOX7‐pEGFP‐C1; lane 4, TGF‐β1+CTRL; lane 5, TGF‐β1+miR‐9 mimic; lane 6, TGF‐β1+miR‐9 mimic+SOX7‐pEGFP‐C1. *P < 0.05 and ^# P < 0.01 compared with the CTRL; ^## P < 0.01 compared with the miR‐9 mimic.