Long non-coding RNA 1308 promotes cell invasion by regulating the miR-124/ADAM 15 axis in non-small-cell lung cancer cells

Figures & data

Table 1 Top 10 upregulated lncRNAs in NSCLC tissues

Probe name	Gene symbol	P-value	FC	Regulation
C_15547	lncRNA NONHSAG038390	2.03E–06	28.39	Up
C_79834	lncRNA 1308	7.35E–09	23.60	Up
C_1346	lncRNA SLC32A1	8.15E–05	11.10	Up
C_64647	lncRNA ELTD1-2	2.55E–05	8.69	Up
C_16443	lncRNA PVT1-108	2.10E–08	6.37	Up
C_44036	lncRNA PIK3C2G-2	1.59E–06	5.90	Up
C_87304	lncRNA NONHSAG047350	3.60E–06	5.68	Up
C_2609	lncRNA TSSK2-2	1.92E–05	5.62	Up
C_89598	lncRNA OR4Q3-3	0.001056	5.05	Up
C_33744	lncRNA EFR3A-5	1.12E–07	4.98	Up

Abbreviations: FC, fold change; lncRNA, long non-coding RNA; NSCLC, non-small-cell lung cancer.

Figure 1 lncRNAs are systemically dysregulated in NSCLC tissues.

Notes: Heatmap of expression profiles for the 3,166 lncRNAs showed significant expression changes (913 upregulated and 2,253 downregulated).

Abbreviations: lncRNA, long non-coding RNA; NSCLC, non-small-cell lung cancer.

Figure 2 lncRNA 1308 is upregulated in lung cancer samples and cell lines.

Notes: (A) Expression of lncRNA 1308 was measured in 40 lung cancer samples and matched to adjacent normal tissues by qRT-PCR, and the expression levels of lncRNA 1308 were normalized to actin RNA expression for subsequent analyses. (B) The expression levels of lncRNA 1308 were measured in human BESA-2B cell lines and 5 lung cancer cell lines (A549, H1299, H1975, XWLC, and GLC) by qRT-PCR, and the expression levels of lncRNA 1308 were normalized to actin RNA expression for subsequent analyses. Results are presented as mean ± SD. *P<0.05, **P<0.01, and ***P<0.001.

Abbreviations: GLC, Gejiu lung cancer; lncRNA 1308, long non-coding RNA 1308; qRT-PCR, quantitative reverse transcription PCR; XWLC, Xuanwei lung cancer.

Figure 3 Downregulation of lncRNA 1308 suppresses cell invasion in lung cancer cells.

Notes: (A) lncRNA 1308 expression was quantified by qRT-PCR analysis. lncRNA 1308 expression was interfered in both XWLC and GLC cells. (B) The invasive ability of XWLC and GLC cells was evaluated by in vitro invasion assays after transfection of NC and siRNA of lncRNA 1308. Results are presented as mean ± SD. **P<0.01. Magnification 100×.

Abbreviations: GLC, Gejiu lung cancer; lncRNA 1308, long non-coding RNA 1308; qRT-PCR, quantitative reverse transcription PCR; NC, negative control oligonucleotides; XWLC, Xuanwei lung cancer.

Table 2 Relative miRIP isolation of miRNAs in XWLC cells using lncRNA 1308 probes (lncRNA1308/control >10)

miRNA	lncRNA 1308/control
miR-124	170.1
miR-134	61.4
miR-191	40.8
miR-106a	28.4
miR-107	22.3
miR-22	15.5
miR-23a	15.2
miR-302b	13.8
miR-182	13.0
miR-27b	12.6
let-7b	11.9
miR-296	10.9
miR-16	10.1

Abbreviations: lncRNA, long non-coding RNA; miRIP, microRNA in vivo precipitation; miRNA, microRNA.

Figure 4 Affinity purification of lncRNA mRNA by miRIP in XWLC cells.

Notes: (A) lncRNA 1308 mRNA, probe binding sites, and region analyzed by qRT-PCR. (B) RT-PCR analysis of lncRNA 1308 mRNAs, from lncRNA 1308 probe affinity purification, in XWLC cells. (C) qRT-PCR of multiplex miRNA data of lncRNA 1308 probe affinity purification, in XWLC cells. Data shown are normalized values for the enriched miRNAs (>10 fold).

Abbreviations: Ctl, control probe; lncRNA 1308, long non-coding RNA 1308; miRIP, microRNA in vivo precipitation; miRNA, microRNA; qRT-PCR, quantitative reverse transcription PCR; XWLC, Xuanwei lung cancer.

Figure 5 lncRNA 1308 is a target of miR-124 and miRNA that represses protein levels of ADAM 15

Notes: (A and B) Schematic representation of pMIR firefly luciferase reporter construction. (A) A 525-bp lncRNA 1308 sequence was cloned and constructed in pMIR firefly luciferase reporter. (B) 274 bp of ADAM 15 3′-UTR sequence was cloned and constructed in pMIR firefly luciferase reporter. (C and D) Analysis of luciferase activity. HEK 293T cells were co-transfected with pMIR firefly luciferase reporter plasmids and pTK-Renilla luciferase plasmids, together with negative control and miR-124 mimics. After 36 h, firefly luciferase activity was measured and normalized by Renilla luciferase activity. (E) Effects of miR-124 mimics on the endogenous ADAM 15 protein levels. XWLC and GLC cells were cotransfected with miR-124 mimics or NC. Forty-eight hours after transfection, the cells were isolated, and the expression of ADAM 15 was analyzed by Western blotting. Results were represented as mean ± SD. **P<0.01.

Abbreviations: ADAM15, a disintegrin and a metalloproteinase 15; GLC, Gejiu lung cancer; lncRNA 1308, long non-coding RNA 1308; miRNA, microRNA; NC, negative control oligonucleotides; ns, not significant; XWLC, Xuanwei lung cancer.

Figure 6 lncRNA 1308 acts as a ceRNA to regulate ADAM 15 levels and suppresses cell invasion in lung cancer cells.

Notes: (A) lncRNA 1308 acts as a ceRNA to inhibit ADAM 15 expression in lung cancer cells. XWLC and GLC cells were transfected with lncRNA 1308 siRNA and/or infected with miR-124 inhibitors, and the expression of ADAM 15 was analyzed by Western blotting. (B) lncRNA 1308 acts as a ceRNA to inhibit cell invasion. XWLC and GLC cells were transfected with lncRNA 1308 siRNA and/or infected with miR-124 inhibitors, and the invasive ability of cells was evaluated by in vitro invasion assays. Results are presented as mean ± SD. **P<0.01. Anti-124, miR-124 inhibitor. Magnification 100×.

Abbreviations: ADAM15, a disintegrin and a metalloproteinase 15; ceRNA, competing endogenous RNA; GLC, Gejiu lung cancer; lncRNA 1308, long non-coding RNA 1308; XWLC, Xuanwei lung cancer.