Nicotine promotes the development of non-small cell lung cancer through activating LINC00460 and PI3K/Akt signaling

Abstract

Objective: Nicotine, the main ingredient in tobacco, is identified to facilitate tumorigenesis and accelerate metastasis in tumor. Studies in recent years have reported that long intergenic non-protein coding RNA 460 (LINC00460) is strongly associated with lung cancer poor prognosis and nicotine dependence. Nonetheless, it is unclear whether nicotine promotes the development of lung cancer through activation of LINC00460. Methods: We determined that LINC00460 expression in lung cancer tissues and the prognosis in patients with non-small cell lung carcinoma (NSCLC) using Gene Expression Profiling Interactive Analysis (GEPIA) website and The Cancer Genome Atlas (TCGA) database. Through in vitro experiments, we studied the effects of nicotine on LINC00460 in NSCLC cells lines using Cell Counting Kit-8 (CCK-8), transwell test, flow cytometry, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot assays. Results: We identified the significant up-regulated expression level of LINC00460 in NSCLC tissues and cell lines, especially, the negative correlation of LINC00460 expression level with overall survival (OS). In in vitro experiments, LINC00460 was overexpressed in NSCLC cell lines under nicotine stimulation. Nicotine could relieve the effect of LINC00460 knockdown on NSCLC cell proliferation, migration and apoptosis. The same influence was observed on PI3K/Akt signaling pathway. Conclusions: In summary, this is the first time to examine the potential roles of LINC00460 in lung cancer cell proliferation, migration and apoptosis induced by nicotine. This may help to develop novel therapeutic strategies for the prevention and treatment of metastatic tumors from cigarette smoke-caused lung cancer by blocking the nicotine-activated LINC00460 pathway.

Keywords: LINC00460; apoptosis; cell migration; nicotine; non-small cell lung cancer; proliferation.

Figure 1. Expression of LINC00460 in NSCLC tissues based on GEPIA data

(A) Expression levels of LINC00460 in NSCLC tissues (n=483) and normal tissue (n=347) from the TCGA database were analyzed. *P<0.05. (B) OS after 20 years of patients with low or high expression levels of LINC00460 was analyzed by Kaplan–Meier method and log rank test. P=0.0037.

Figure 2. Expression of LINC00460 in NSCLC cell lines under nicotine induced

(A,B) Expression levels of LINC00460 in NSCLC cell lines (A549 and H1299) under nicotine stimulation were measured by qRT-PCR. *P<0.05, **P<0.01 vs. 0 μg/ml group. (C,D) The time-dependent analysis of LINC00460 expression levels in NSCLC cell lines (A549 and H1299). **P<0.01 vs. NC group. (E) The expression levels of LINC00460 knockdown mRNA were assessed using qRT-PCR, *P<0.05 vs. NC group. LINC00460-KD, small interfering LINC00460 RNA.

Figure 3. Effect of LINC00460 knockdown on NSCLC cells proliferation

(A) OD values in A549 cell were detected by CCK-8 assay in three groups for 0, 24, 48, 72 h. *P<0.05 vs. NC group. (B) OD values in H1299 cell were detected by CCK-8 assay in three groups for 0, 24, 48, 72 h. *P<0.05 vs. NC group.

Figure 4. Effect of LINC00460 knockdown on NSCLC cell migration

(A) Microscopic images of A549 cells passing though the microwells of the transwell chamber were detected by transwell assay. The number of migrated cells was counted. *P<0.05 vs. NC group, bar = 200 μm. (B) Microscopic images of H1299 cells passing though the microwells of the transwell chamber were detected by transwell assay. The number of migrated cells was counted. *P<0.05 vs. NC group, bar = 200 μm.

Figure 5. Effect of LINC00460 knockdown on apoptosis in NSCLC A549 cells

(A) Flow cytometric plots show specific cell populations in A549 cells. Q2 were late apoptotic cells, Q3 were live cells, and Q4 were early apoptotic cells. (B) Expression of activated Bcl-2, Bax and Cleaved Caspase-3 were determined by Western blot and shown by bar graphs in (C). *P<0.05 vs. NC group. Data were expressed as mean ± SD.

Figure 6. Effect of LINC00460 knockdown on apoptosis in NSCLC H1299 cells

(A) Flow cytometric plots show specific cell populations in H1299 cells. Q2 were late apoptotic cells, Q3 were live cells, and Q4 were early apoptotic cells. (B) Expression of activated Bcl-2, Bax and Caspase3 were determined by Western blot and showed by bar graphs in (C). *P<0.05 vs. NC group.

Figure 7. Effect of LINC00460 knockdown on activation of PI3K pathway in NSCLC cell lines

(A,B) Represented bands of PI3K/Akt signaling associated proteins were assessed by Western blot in A549 and H1299 cells. (C,D) Relative protein expression levels of proteins were showed by column diagram. *P<0.05 vs. NC group.