Long Noncoding RNA GAS5 Promotes Proliferation, Migration, and Invasion by Regulation of miR-301a in Esophageal Cancer

Abstract

Long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) has been revealed to be associated with the progression of various cancers. However, the biological roles of GAS5 in esophageal cancer (EC) remain unclear. We aimed to thoroughly explore the functions of GAS5 in EC. The results showed that GAS5 expression was increased in EC cells (ECA109, TE-1, TE-3, and EC9706) compared to SHEE cells. Knockdown of GAS5 decreased cell viability, migration, and invasion and induced apoptosis in EC9706 cells. Moreover, miR-301a appeared to be directly sponged by GAS5, and miR-301a suppression obviously alleviated the protumor effects of GAS5. Furthermore, miR-301a positively regulated CXCR4 expression, and overexpression of CXCR4 induced apoptosis and abolished the promoting effect of miR-301a inhibition on cell viability, migration, and invasion. Besides, miR-301a blocked Wnt/β-catenin and NF-κB signaling pathways by regulation of CXCR4. Our results indicated that GAS5 promoted proliferation and metastasis and inhibited apoptosis by regulation of miR-301a in EC. These data contributed to our understanding of the mechanisms of miRNA-lncRNA interaction and provides a novel therapeutic strategy for EC.

Figure 1

Long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) was upregulated in esophageal cancer (EC) cells. Relative expressions of GAS5 in EC cell lines (ECA109, TE-1, TE-3, and EC9706) and in an esophageal epithelia cell line (SHEE) were detected by quantitative real-time reverse transcriptase (qRT)-PCR. All values are mean ± standard deviation (SD). Ns, no significance. *p < 0.05; **p < 0.01.

Figure 2

lncRNA GAS5 promoted proliferation and metastasis and inhibited apoptosis in EC cells. EC9706 cells were transfected with short hairpin (sh)-GAS5 #1 and sh-GAS5 #2 or negative control (sh-NC). (A) Relative expression of GAS5 was examined by qRT-PCR. (B) Cell viability, (C) migration, (D) invasion, and (E) apoptosis were determined by trypan blue exclusion, Transwell, and flow cytometry assays. (F, G) Expression of the main apoptosis factors was examined by Western blot. All values are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

lncRNA GAS5 acted as a molecular sponge to regulate microRNA-301a (miR-301a) in EC. EC9706 cells were transfected with sh-GAS5 #2. (A) The combination of GAS5 and miR-301a was predicated by TargetScan, microRNA, and NCBI databases. (B) Relative expression of miR-301a was measured by qRT-PCR. (C) Relationship between miR-301a and GAS5 was detected by dual-luciferase reporter activity assay. All values are mean ± SD. **p < 0.01.

Figure 4

miR-301a mediated protumor effects of lncRNA GAS5 in EC cells. EC9706 cells were transfected with sh-GAS5, sh-GAS5 + miR-301a inhibitor, and corresponding controls. (A) Cell viability, (B) migration, (C) invasion, and (D) apoptosis were determined by trypan blue exclusion, Transwell, and flow cytometry assays. (E, F) Expression of the main apoptosis factors was examined by Western blot. All values are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

miR-301a positively regulated chemokine C-X-C motif receptor 4 (CXCR4) expression. EC9706 cells were transfected with miR-301a mimic, inhibitor, and corresponding controls. (A) Relative mRNA expression of CXCR4 was detected by qRT-PCR. (B, C) Relative protein level of CXCR4 was detected by Western blot assay. All values are mean ± SD. **p < 0.01; ***p < 0.001.

Figure 6

miR-301a suppression promoted cell proliferation and metastasis and inhibited apoptosis by regulating CXCR4. EC9706 cells were transfected with miR-301a mimic, miR-301a inhibitor, pEX-CXCR4, sh-CXCR4, and corresponding controls. (A) The protein level of CXCR4 was detected by Western blot. (B, C) Relative expression of CXCR4 was examined by qRT-PCR and Western blot. (D) Cell viability, (E) migration, (F) invasion, and (G) apoptosis were determined by trypan blue exclusion, Transwell, and flow cytometry assays. (H, I) The expression of the main apoptosis factors was examined by Western blot. All values are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 7

miR-301a blocked the Wnt/β-catenin and nuclear factor κ B (NF-κB) signaling pathways by regulation of CXCR4. EC9706 cells were transfected with miR-301a mimic, miR-301a inhibitor, miR-301a inhibitor + sh-CXCR4, and corresponding controls. Relative protein levels of the (A, B) Wnt/β-catenin signal pathway and (C, D) NF-κB signal pathway were examined by Western blot. All values are mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.