lncINS-IGF2 Promotes Cell Proliferation and Migration by Promoting G1/S Transition in Lung Cancer

Abstract

Long noncoding RNAs are capable of regulating gene expression at multiple levels. These RNA molecules are also involved in a variety of physiological and pathological processes. Emerging data demonstrate that a series of differentially expressed long noncoding RNAs are implicated in tumorigenesis. In the present study, we used microarray analysis to identify long noncoding RNAs that are dysregulated in non-small-cell lung cancer when compared to normal lung tissues. Accordingly, we performed quantitative real-time polymerase chain reaction to analyze the levels of long noncoding RNA and the cis target gene. We further found the oncogene property of long noncoding RNA that long noncoding RNA downexpression inhibits non-small-cell lung cancer cells proliferation and migration based on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and colony formation assays and wound healing as well as transwell assays. The influence of long noncoding RNA on cell cycle of non-small-cell lung cancer cells is also analyzed by flow cytometry. Among the dysregulated long noncoding RNAs, we identified INS-IGF2 readthrough, transcript variant 1, noncoding RNA (NR_003512.3) is upregulated in non-small-cell lung cancer tissues, the cis gene of which is insulin-like growth factor 2 gene hinted by bioinformatics analysis. We also observed that downregulation of INS-IGF2 readthrough, transcript variant 1, noncoding RNA reduces insulin-like growth factor 2 messenger RNA expression. Furthermore, INS-IGF2 readthrough, transcript variant 1, noncoding RNA downregulation suppresses non-small-cell lung cancer cell proliferation and migration. This downregulation results in a concomitant inhibition of the G1/S transition in non-small-cell lung cancer cells. Our findings suggest that INS-IGF2 readthrough, transcript variant 1, noncoding RNA may be an oncogene involved in the development of lung cancer. Therefore, we speculate that INS-IGF2 readthrough, transcript variant 1, noncoding RNA represents a potential therapeutic target for lung cancer.

Keywords: lncINS-IGF2; lncRNA; lung cancer; migration; proliferation.

Figure 1.

Relative expression levels of lncINS-IGF2 in lung cancer tissues and cell lines. A, lncINS-IGF2 expression was assessed following microarray analysis of lung cancer tumor specimens (n = 3) and normal adjacent lung tissue (n = 3). B, The expression of lncINS-IGF2 was measured by qRT-PCR in A549, H1299, and H441 lung cancer cell lines, HBE cells, 293T cells, and HeLa cells. *P < .05 versus normal group. Results are expressed as the mean (SD) of the samples and each analysis was performed in triplicate. qRT-PCR indicates real-time quantitative polymerase chain reaction; SD, standard deviation.

Figure 2.

lncINS-IGF2 knockdown results in downregulation of IGF2 in NSCLC cells. A549 and H1299 cells were transfected with control siRNA or lncINS-IGF2 siRNA for 48 hours. The cells were subsequently harvested to assess knockdown effects (A and B). A549 and H1299 cells were transfected with NC and siRNA of lncINS-IGF2. IGF2 mRNA levels were measured by qRT-PCR assay (C and D). *P < .05 versus NC. Data are expressed as the mean (SD) and each experiment was performed in triplicate. IGF2, insulin-like growth factor 2; NSCLC, non-small-cell lung cancer; siRNA, small interfering RNA; qRT-PCR, real-time quantitative polymerase chain reaction; NC, normal control; SD, standard deviation.

Figure 3.

lncINS-IGF2 knockdown inhibits cell proliferation and colony formation in NSCLC cells. A549 and H1299 cells transfected with control siRNA or lncINS-IGF2 siRNA were trypsinized and plated into 96-well plates for the MTT assay. The asterisk shows the significant differences on the proliferation rate between 2 groups (A and B). Colony formation of A549 and H1299 cells at 14 days after transfection with the indicated oligonucleotides (C and D). *P < .05 versus NC. Data are expressed as the mean (SD) and each experiment was performed in triplicate. NSCLC indicates non-small-cell lung cancer; siRNA, small interfering RNA; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; NC, normal control; SD, standard deviation.

Figure 4.

Downregulation of lncINS-IGF2 retards G1/S transition in NSCLC cells. A549 and H1299 cells were transfected with control siRNA or lncINS-IGF2 siRNA for 48 hours. Cell populations at the G1, S, and G2/M phases were detected by flow cytometry. *P < .05 versus NC. Data are expressed as the mean (SD) and the experiments were performed in triplicate. NSCLC indicates non-small-cell lung cancer; siRNA, small interfering RNA; NC, normal control; SD, standard deviation.

Figure 5.

lncINS-IGF2 knockdown induces inhibition of cell migration in NSCLC cells. A, B, The migration capacity of A549 and H1299 cells was determined using a wound-healing assay. Photographs were taken at ×100 magnification at 0, 24, 48, and 72 hours after wounding; the photographs were taken at the same location in each well. C, D, Representative images of the migration transwell assay; the number of migrated cells was randomly measured by photograph at ×200 magnification in 5 different fields for each chamber. *P < .05 versus NC. Data are defined as mean (SD) following 3 independent experiments. NSCLC indicates non-small-cell lung cancer; NC, normal control; SD, standard deviation.