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The long non-coding RNA, GAS5, enhances gefitinib-induced cell death in innate EGFR tyrosine kinase inhibitor-resistant lung adenocarcinoma cells with wide-type EGFR via downregulation of the IGF-1R expression.

Dong S, Qu X, Li W, Zhong X, Li P, Yang S, Chen X, Shao M, Zhang L - J Hematol Oncol (2015)

Bottom Line: Our results showed that GAS5 was significantly downregulated in lung adenocarcinoma tissues compared with the paired adjacent non-tumorous tissue samples.However, GAS5 was almost equally expressed between benign tumors compared with the adjacent normal tissues.GAS5 was also overexpressed in EGFR-TKI sensitive cell lines compared with the resistant cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. dsycmu@163.com.

ABSTRACT

Background: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are approved for patients with recurrent non-small cell lung cancer (NSCLC). However, the efficacy of EGFR-TKIs in NSCLC therapy is limited by primary and acquired resistance. Recent studies have revealed that long non-coding RNAs (LncRNA) may be involved in EGFR-TKI resistance. Therefore, a better understanding of the interactive mechanisms underlying LncRNA-mediated EGFR-TKIs resistance may help us to improve clinical response rates.

Method: To investigate the expression of growth arrest-specific 5 (GAS5) in lung adenocarcinoma, we performed real-time reverse-transcriptase polymerase chain reaction. The correlation between GAS5 expression levels and the samples' clinicopathological features was also analyzed. Primary resistance to EGFR-TKIs was identified in the human lung adenocarcinoma cell line A549. Plasmid vectors were used to overexpress GAS5 in A549 cells. MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) colony formation assays and EdU (5-ethynyl-2'-deoxyuridine) assays were used to assess cell proliferation, and flow-cytometric analysis was used to evaluate the apoptosis rate. The expression levels of our target proteins, namely, EGFR, p-EGFR, ERK, p-ERK, Akt, p-Akt, IGF-1R (insulin-like growth factor 1 receptor), and p-IGF-1R, were analyzed by western blotting. A549 cells transfected with pcDNA-GAS5 were injected into nude mice. The transplanted mice were treated with gefitinib to study the effect of GAS5 on the resistance to EGFR-TKIs in vivo.

Results: Our results showed that GAS5 was significantly downregulated in lung adenocarcinoma tissues compared with the paired adjacent non-tumorous tissue samples. Furthermore, lower GAS5 expression levels were associated with larger tumor sizes, poor tumor differentiation, and advanced pathological stages. However, GAS5 was almost equally expressed between benign tumors compared with the adjacent normal tissues. GAS5 was also overexpressed in EGFR-TKI sensitive cell lines compared with the resistant cell line. Using MTT, EdU incorporation, and colony formation assays, we showed that GAS5-expressing A549 cells displayed an elevated level of cell death. In addition to its pro-apoptotic effect in the A549 cell line, GAS5 overexpression also suppressed the growth of A549-derived tumors in nude mice treated with gefitinib. GAS5 overexpression was inversely correlated with the expression of the EGFR pathway and IGF-1R proteins.

Conclusions: Collectively, our results indicated that GAS5 LncRNA may represent a potential biomarker for the diagnosis of lung adenocarcinoma and that GAS5 might play a novel role in the development of the resistance to gefitinib, which could be reversed by overexpressing GAS5.

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qRT-PCR analysis of LncRNA GAS5 in NSCLC tissues and its clinical significance. GAS5 expression was normalized to GAPDH expression. (A) GAS5 was measured in 72 pair NSCLC and paired adjacent normal tissues by qRT-PCR. The data are presented as a fold-change in the tumor tissue relative to the normal tissue. (B) GAS5 expression was significantly lower in the tumors ≥3 cm. (C) GAS5 expression was significantly lower in moderate-poor differentiation tumors. (D) GAS5 expression was significantly lower in patients with a higher pathological stage. *P < 0.05; **P < 0.01.
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Fig1: qRT-PCR analysis of LncRNA GAS5 in NSCLC tissues and its clinical significance. GAS5 expression was normalized to GAPDH expression. (A) GAS5 was measured in 72 pair NSCLC and paired adjacent normal tissues by qRT-PCR. The data are presented as a fold-change in the tumor tissue relative to the normal tissue. (B) GAS5 expression was significantly lower in the tumors ≥3 cm. (C) GAS5 expression was significantly lower in moderate-poor differentiation tumors. (D) GAS5 expression was significantly lower in patients with a higher pathological stage. *P < 0.05; **P < 0.01.

Mentions: The quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) technique was used to assess GAS5 expression in tumor samples and their corresponding adjacent normal tissues in a total of 99 paired samples. In cancerous tissues, GAS5 expression was lower than the average level observed in normal specimens, with an average expression level of 0.76 compared with normal tissue (Figure 1A). In eight metastatic lung cancer tissues, GAS5 expression was also lower than that in the normal specimens, but not significantly (P = 0.087). In the 19 benign lung tumors, GAS5 expression levels were slightly lower than in the adjacent normal tissues, with an average expression level of 0.936 compared with normal tissues (P = 0.342).Figure 1


The long non-coding RNA, GAS5, enhances gefitinib-induced cell death in innate EGFR tyrosine kinase inhibitor-resistant lung adenocarcinoma cells with wide-type EGFR via downregulation of the IGF-1R expression.

Dong S, Qu X, Li W, Zhong X, Li P, Yang S, Chen X, Shao M, Zhang L - J Hematol Oncol (2015)

qRT-PCR analysis of LncRNA GAS5 in NSCLC tissues and its clinical significance. GAS5 expression was normalized to GAPDH expression. (A) GAS5 was measured in 72 pair NSCLC and paired adjacent normal tissues by qRT-PCR. The data are presented as a fold-change in the tumor tissue relative to the normal tissue. (B) GAS5 expression was significantly lower in the tumors ≥3 cm. (C) GAS5 expression was significantly lower in moderate-poor differentiation tumors. (D) GAS5 expression was significantly lower in patients with a higher pathological stage. *P < 0.05; **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419450&req=5

Fig1: qRT-PCR analysis of LncRNA GAS5 in NSCLC tissues and its clinical significance. GAS5 expression was normalized to GAPDH expression. (A) GAS5 was measured in 72 pair NSCLC and paired adjacent normal tissues by qRT-PCR. The data are presented as a fold-change in the tumor tissue relative to the normal tissue. (B) GAS5 expression was significantly lower in the tumors ≥3 cm. (C) GAS5 expression was significantly lower in moderate-poor differentiation tumors. (D) GAS5 expression was significantly lower in patients with a higher pathological stage. *P < 0.05; **P < 0.01.
Mentions: The quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) technique was used to assess GAS5 expression in tumor samples and their corresponding adjacent normal tissues in a total of 99 paired samples. In cancerous tissues, GAS5 expression was lower than the average level observed in normal specimens, with an average expression level of 0.76 compared with normal tissue (Figure 1A). In eight metastatic lung cancer tissues, GAS5 expression was also lower than that in the normal specimens, but not significantly (P = 0.087). In the 19 benign lung tumors, GAS5 expression levels were slightly lower than in the adjacent normal tissues, with an average expression level of 0.936 compared with normal tissues (P = 0.342).Figure 1

Bottom Line: Our results showed that GAS5 was significantly downregulated in lung adenocarcinoma tissues compared with the paired adjacent non-tumorous tissue samples.However, GAS5 was almost equally expressed between benign tumors compared with the adjacent normal tissues.GAS5 was also overexpressed in EGFR-TKI sensitive cell lines compared with the resistant cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, First Hospital of China Medical University, Shenyang, Liaoning Province, 110001, People's Republic of China. dsycmu@163.com.

ABSTRACT

Background: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are approved for patients with recurrent non-small cell lung cancer (NSCLC). However, the efficacy of EGFR-TKIs in NSCLC therapy is limited by primary and acquired resistance. Recent studies have revealed that long non-coding RNAs (LncRNA) may be involved in EGFR-TKI resistance. Therefore, a better understanding of the interactive mechanisms underlying LncRNA-mediated EGFR-TKIs resistance may help us to improve clinical response rates.

Method: To investigate the expression of growth arrest-specific 5 (GAS5) in lung adenocarcinoma, we performed real-time reverse-transcriptase polymerase chain reaction. The correlation between GAS5 expression levels and the samples' clinicopathological features was also analyzed. Primary resistance to EGFR-TKIs was identified in the human lung adenocarcinoma cell line A549. Plasmid vectors were used to overexpress GAS5 in A549 cells. MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) colony formation assays and EdU (5-ethynyl-2'-deoxyuridine) assays were used to assess cell proliferation, and flow-cytometric analysis was used to evaluate the apoptosis rate. The expression levels of our target proteins, namely, EGFR, p-EGFR, ERK, p-ERK, Akt, p-Akt, IGF-1R (insulin-like growth factor 1 receptor), and p-IGF-1R, were analyzed by western blotting. A549 cells transfected with pcDNA-GAS5 were injected into nude mice. The transplanted mice were treated with gefitinib to study the effect of GAS5 on the resistance to EGFR-TKIs in vivo.

Results: Our results showed that GAS5 was significantly downregulated in lung adenocarcinoma tissues compared with the paired adjacent non-tumorous tissue samples. Furthermore, lower GAS5 expression levels were associated with larger tumor sizes, poor tumor differentiation, and advanced pathological stages. However, GAS5 was almost equally expressed between benign tumors compared with the adjacent normal tissues. GAS5 was also overexpressed in EGFR-TKI sensitive cell lines compared with the resistant cell line. Using MTT, EdU incorporation, and colony formation assays, we showed that GAS5-expressing A549 cells displayed an elevated level of cell death. In addition to its pro-apoptotic effect in the A549 cell line, GAS5 overexpression also suppressed the growth of A549-derived tumors in nude mice treated with gefitinib. GAS5 overexpression was inversely correlated with the expression of the EGFR pathway and IGF-1R proteins.

Conclusions: Collectively, our results indicated that GAS5 LncRNA may represent a potential biomarker for the diagnosis of lung adenocarcinoma and that GAS5 might play a novel role in the development of the resistance to gefitinib, which could be reversed by overexpressing GAS5.

Show MeSH
Related in: MedlinePlus