LncRNA LINC01512 Promotes the Progression and Enhances Oncogenic Ability of Lung Adenocarcinoma

Abstract

Previously, a significantly upregulated lncRNA, LINC01512, in lung adenocarcinoma (LAD) was obtained, while its biological function and molecular mechanisms were unclear. The expression level of LINC01512 was estimated by qPCR from 100 pairs of LAD and NT samples. The correlation of LINC01512 to clinical data of LAD patients was analyzed. LINC01512 was knocked down and overexpressed in SPCA-1 and A549 cell lines by lentivirus-mediated technology, and the oncological behavioral changes of SPCA-1 and A549 cells were observed, as well as, tumorigenicity in experimental nude mice. Compared to the adjacent tissues, LINC01512 was obviously upregulated in LAD. The expression level of LINC01512 was closely related to lymph node metastasis and tumor node metastasis (TNM) stage. Survival analysis showed that the survival time of high expression LINC01512 group was significantly shorter than the low-expression group in LAD. Knockdown or overexpression test unanimously confirmed that LINC01512 can increase the ability of cell migration, invasion, proliferation, colony formation, adhesion, and S phase and G2/M phase cells, whereas decrease the apoptosis and G0/G1 phase cells. Nude mice experiments confirmed that LINC01512 significantly enhanced the speed and weight of tumorigenicity. LINC01512 is an oncogenic lncRNA gene that promotes the progression and distinctly enhances the oncogenic ability in lung adenocarcinoma. J. Cell. Biochem. 118: 3102-3110, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.

Keywords: BIOLOGICAL FUNCTIONS; LINC01512; LONG NONCODING RNA; LUNG ADENOCARCINOMA.

Figure 1

Expression level of LINC01512 in LAD tissue, cells, three A549 cell groups, three SPCA‐1 cell groups. (A) Comparison of the relative expression levels of LINC01512 in LAD and adjacent tissues. LINC01512 expression level of LAD was significantly higher than the adjacent tissue. **P < 0.01. (B) The correlation of LINC01512 expression with the survival of LAD prognosis. The survival time of LINC01512 high‐expression group was significantly shorter than that of the low‐expression group. (C) Comparison of LINC01512 expression levels in six LAD cell lines. The expression levels of LINC01512 from NCI‐H1975, SPCA‐1, and NCI‐H441 cells were highly expressed. The expression level of SPCA‐1 was the highest, and that of NCI‐H1299 and LETP‐a2 was moderate, while that of A549 cells was the lowest. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Comparison of interference efficiency of PCR amplification of three LINC01512 interference sequences. siRNA2 group exhibited the highest interference efficiency. (E) Overexpression of LINC01512 in three A549 cell groups. The overexpression level of LINC01512 in A549 cell line was significantly higher than that in the control and NC groups. *P < 0.05, **P < 0.01, ***P < 0.001. The relative expression level of LINC01512 was used to calculate the relative lncRNA concentrations (ΔCt = Ct median lncRNA − Ct median β‐actin), and 2^−ΔΔCt was calculated as the relative expression.

Figure 2

LINC01512 is relative to ability of cell migration and invasion in LAD. (A) Results of cell migration of SPCA‐1 cells. Compared to the NC and control groups, the migration distance of siRNA2 group decreased significantly after LINC01512 was knocked down. (B) Results of cell migration of A549 Cells. Compared to the NC and control groups, the migration distance of LINC01512 group increased significantly after LINC01512 was overexpressed. (C) Comparison of the results of invasion test in different treatment SPCA‐1 cells groups. The OD570 nm of siRNA2 group decreased significantly after LINC01512 was knocked down. (D) Comparison of the results of cell invasion test in different treatment A549 cells groups. The OD570 nm of LINC01512 group increased significantly after LINC01512 was overexpressed. *P < 0.05.

Figure 3

Comparison of cell proliferation in three different treatment groups on different days of the CCK‐8 assay. (A) The curve of three SPCA‐1 groups of different days. (B) Illustration of three SPCA‐1 groups of different days. (C) The curve of three A549 groups of different days. (D) The illustration of three A549 groups of different days. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01.

Figure 4

Results of cell apoptosis and cell cycles. (A) Activity of caspase‐3 changes in the different SPCA‐1 group. (B) Activity of caspase‐9 changes in different SPCA‐1 groups. (C) Activity of caspase‐3 changes in different A549 groups. (D) Activity of caspase‐9 changes in different A549 groups. (E) The result of cell cycle in the control of the SPCA‐1 group. (F) Result of cell cycles in control of A549 group. *P < 0.05, **P < 0.01.

Figure 5

Comparison of the results of adhesion. (A) Adhesion of three SPCA‐1 cell groups; compared to the CON group of siRNA2 groups, the OD550 of Matrigel and FN groups of siRNA2 groups significantly increased after Matrigel and FN treatment. The OD550 in the siRNA2 groups was lower than that in the control and NC groups (P = 0.0044, 0.0038, 0.0032) after CON, Matrigel, and FN treatment, indicating that LINC01512 siRNA resulted in significantly reduced cell adhesion. Compared to the Matrigel and CON groups, NC and siRNA2 groups were significantly higher than the control groups after FN treatment. (B) Adhesion of three A549 cell groups. Compared to the CON group, OD550 in LINC01512 groups was significantly higher than that in the NC and control groups (P = 0.0012, 0.0041, P = 0.0059, 0.0093) after Matrigel and FN treatment. The OD550 of control, NC, and LINC01512 groups was significantly higher than the Matrigel and CON groups (P = 0.033, 0.026, 0.011 and P = 0.0076, 0.0042, 0.0018) after FN treatment. *P < 0.05, **P < 0.01, ***P < 0.001, #P <0.05, ##P < 0.01, △P < 0.05, △△P < 0.01.

Figure 6

Results of cell clone formation were compared. (A) The clone formation rate of siRNA2 group (5.00 ± 0.81%) was significantly lower than that of the control (14.97 ± 1.34%, P = 0.0018) and NC groups (15.13 ± 2.31%, P = 0.0011). (B) Clone formation rate in the control (25.01 ± 1.37%, P < 0.001) and NC groups (26.15 ± 2.48%, P < 0.001) was significantly lower than that in the LINC01512 group (42.57 ± 2.71%) after LINC01512 overexpression. **P < 0.01, ***P < 0.001.

Figure 7

Experimental results of nude mice. (A) Tumor volume of LINC01512 and NC groups of A549 cells. The rate of tumor growth of the LINC01512 group was significantly faster than that of the NC group (P < 0.05). (B) Tumor volume of siRNA2 and NC of SPCA‐1 cells. The rate of tumor growth of the siRNA2 group was significantly slower than that of the NC group (P < 0.05). (C) The growth curve of the tumor from LINC01512 and NC groups of A549 cells. The tumor weight in the LINC01512 group was significantly higher than that in the NC group (t = 4.831, P < 0.001). (D) The growth curve of the tumor from siRNA2 and NC groups of SPCA‐1 cells. The tumor weight in the siRNA2 group was significantly lower than that in the NC group (t = 6.327, P < 0.001).