LncRNA GATA6-AS1 Inhibits the Progression of Non-Small Cell Lung Cancer via Repressing microRNA-543 to Up-Regulating RKIP

Figures & data

Table 1 The Association Between GATA6-AS1 Expression and the Clinicopathological Characteristics of NSCLC Patients

Characteristics	Number (n=50)	GATA6-AS1 Expression		χ^2	P value
		Low	High
Gender
Male	29	14	15	0.0821	0.7745
Female	21	11	10
Age (years)
<55	28	13	15	0.3247	0.5688
≥55	22	12	10
Lymphatic metastasis
Negative	17	5	12	4.3672	0.0366
Positive	33	20	13
Histology
Squamous cell carcinoma	20	11	9	0.3333	0.5637
Adenocarcinoma	30	14	16
Tumor size
<3cm	13	3	10	5.0936	0.0240
≥3cm	37	22	15

Figure 1 GATA6-AS1 was down-regulated in NSCLC tissues and cells. (A) The expression of GATA6-AS1 in NSCLC tissues and normal lung tissues was analyzed with TCGA data using GEPIA. (B) The expression of GATA6-AS1 in 50 cases of NSCLC and adjacent normal tissues was detected by qRT-PCR. (C) The expression of GATA6-AS1 in normal lung epithelial cell lines (BEAS2B) and NSCLC cell lines (A549, H1299, H292, 95-D, and H460 cells) was detected by qRT-PCR. *P<0.05, **P < 0.01, ***P < 0.001.

Abbreviations: GATA6-AS1, lncRNA GATA6-AS1; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time PCR.

Figure 2 GATA6-AS1 inhibited NSCLC cell proliferation, migration, invasion and EMT. (A) qRT-PCR was used to detect GATA6-AS1 expression in H1299 cells transfected with GATA6-AS1 overexpression plasmid and H460 cells transfected with GATA6-AS1 shRNA. (B) CCK-8 method was used to detect the proliferation of NSCLC cells after overexpression or knockdown of GATA6-AS1. (C and D) Transwell assay was used to detect the migration and invasion of NSCLC cells. (E) Western blot was employed to detect the expressions of EMT indicators (E-cadherin and N-cadherin).*P < 0.05, **P < 0.01, ***P < 0.001.

Abbreviations: GATA6-AS1, lncRNA GATA6-AS1; NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time PCR; EMT, epithelial–mesenchymal transformation; CCK-8, cell counting kit-8.

Figure 3 GATA6-AS1 targeted miR-543. (A) The bioinformatics database LncBase Predicted v2 predicted that GATA6-AS1 contained potential binding site for miR-543. (B) qRT-PCR was adopted to detect the correlation between GATA6-AS1 and miR-543 expression in NSCLC tissues. (C) The targeting relationship between GATA6-AS1 and miR-543 was confirmed by dual-luciferase reporter gene experiments. (D and E) The expression of miR-543 in NSCLC tissues and cells was detected by qRT-PCR. (F) qRT-PCR was used to detect the expression of miR-543 in NSCLC cells with GATA6-AS1 overexpression or knockdown. **P < 0.01, ***P < 0.001, NS: P > 0.05.

Abbreviations: GATA6-AS1, lncRNA GATA6-AS1; NSCLC, non-small cell lung cancer; miR543, microRNA-543; qRT-PCR, quantitative real-time PCR.

Figure 4 MiR-543 partially reversed the inhibitory effect of GATA6-AS1 on NSCLC cells. (A) MiR-543 mimics was transfected into H1299 cells with GATA6-AS1 overexpression; miR-543 inhibitors was used to transfect H460 cells with GATA6-AS1 knockdown. The expression of miR-543 in NSCLC cells was detected by qRT-PCR. (B–E) Up-regulation of miR-543 attenuated the inhibitory effects of GATA6-AS1 overexpression on H1299 cell proliferation, migration, invasion and EMT; inhibiting miR-543 reversed the effects of knockdown of GATA6-AS1 on cell proliferation, migration, invasion, and EMT in H460 cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Abbreviations: GATA6-AS1, lncRNA GATA6-AS1; NSCLC, non-small cell lung cancer; miR543, microRNA-543; qRT-PCR, quantitative real-time PCR; EMT, epithelial–mesenchymal transformation.

Figure 5 GATA6-AS1/miR-543 axis was involved in the regulation of RKIP expression. (A) qRT-PCR was used to detect the expression of RKIP mRNA in NSCLC cells overexpressing or knocking down GATA6-AS1. (B) Western blot was used to detect the expression of RKIP protein in NSCLC cells with GATA6-AS1 overexpression or knockdown. (C and D) Overexpression of miR-543 inhibited the role of GATA6-AS1 overexpression in promoting RKIP mRNA and protein; miR-543 inhibitors reversed the inhibitory effects of knockdown GATA6-AS1 on RKIP mRNA and protein. (E and F) qRT-PCR was adopted to detect the correlation between RKIP mRNA and GATA6-AS1 or miR-543 expression in NSCLC tissues. **P < 0.01, ***P < 0.001.

Abbreviations: GATA6-AS1, lncRNA GATA6-AS1; NSCLC, non-small cell lung cancer; miR-543, microRNA-543; qRT-PCR, quantitative real-time PCR; RKIP, Raf kinase inhibitor protein.

Figure 6 The recovery of RKIP expression in NSCLC cells partially reversed the tumor-suppressive effect of GATA6-AS1. (A–D) RKIP knockdown reversed the inhibitory effects of GATA6-AS1 overexpression on H1299 cell proliferation, migration, invasion, and EMT; overexpression of RKIP reversed the promoting effect of knocking down GATA6-AS1 on the malignant biological behaviors of H460 cells. **P < 0.01, ***P < 0.001.

Abbreviations: GATA6-AS1, lncRNA GATA6-AS1; NSCLC, non-small cell lung cancer; EMT, epithelial–mesenchymal transformation; RKIP, Raf kinase inhibitor protein.