Abstract
Studies showed that long chain non-coding RNAs (lncRNAs) involved in the development and progression of lung cancer. However, the mechanisms of EGFR exon 19 deletion in lung adenocarcinoma were unclear. Lung adenocarcinoma was divided into EGFR exon 19 deletion group and EGFR wild-type group. We studied the differential expression profiles of lncRNAs in EGFR exon 19 deletion in lung adenocarcinoma by high-throughput microarray. Using abundant and varied probes, we were able to assess 30,586 lncRNAs and 26,109 mRNAs in our microarray. Compared with the wild-type EGFR, we found that 1,533 lncRNAs and 1,406 mRNAs were differentially expressed (≥twofold change) in EGFR exon 19 deletion in lung adenocarcinoma, indicating that many lncRNAs were significantly upregulated or downregulated in EGFR exon 19 deletion in lung adenocarcinoma. The 10 lncRNAs were aberrantly expressed in EGFR exon 19 deletion in lung adenocarcinoma compared with wild-type EGFR group validated by real-time RT-PCR. Among these, RP11-325I22.2 and LOC440905 were the most aberrantly expressed in 20 cases of EGFR exon 19 deletion in lung adenocarcinoma samples by real-time RT-PCR. Our study showed lncRNAs expression pattern in EGFR exon 19 deletion in lung adenocarcinoma by microarray. RP11-325I22.2 and LOC440905 might play an important role in the mechanism of EGFR exon 19 deletion in lung adenocarcinoma. The study may provide a new mechanism of EGFR exon 19 deletion in lung adenocarcinoma.
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Introduction
Lung cancer mortality is the highest in all cancers, and its incidence is gradually growing [1]. Lung adenocarcinoma is an important type of non-small cell lung cancer (NSCLC). The epidermal growth factor receptor (EGFR) is a member of EGF-family of extracellular protein ligands. Gefitinib and erlotinib are reversible EGFR tyrosine kinase inhibitors (EGFR-TKIs) combining with EGFR to inhibit EGFR and its downstream pathways. EGFR overexpression has been observed in 40–80 % of NSCLC patients [2]. It was shown that mutations of EGFR kinase domain were more highly sensitive to TKI than the wild-type EGFR in lung adenocarcinoma patients [3–5]. However, these lung adenocarcinoma patients eventually acquired resistance to EGFR-TKI and progressed after 5–9 months of EGFR-TKI therapy. The mechanisms of EGFR-TKI resistance include primary and acquired drug resistance [6]. Mechanisms of primary resistance to EGFR-TKI therapy include K-ras mutations [7, 8], Serine/threonine-protein kinase B-raf mutations [9], Phosphatase, and tensin homolog inactivation [10, 11]. The reasons of acquired resistance involved EGFR secondary mutations [12–14], MET gene amplification [15, 16], hepatocyte growth factor over-expression [17], insulin-like growth factor 1 receptor precursor overexpression [18], and so on. But the mechanisms of EGFR-TKI resistance are complex and have not been fully elucidated. It was important to understand the mechanisms of EGFR-TKI resistance for the treatment of lung cancer, wherein the TK domains of 19 and 21 of exon usually account for about 90 % of EGFR mutations and to these two sites of mutation observed in the clinical treatment with the EGFR-TKIs most closely related to the efficacy.
Long chain non-coding RNAs (long non-coding RNAs, lncRNAs) are non-coding RNAs longer than 200 nucleotides [19]. Many studies have shown that the lncRNAs were associated with some diseases, including cancer [20–22]. The disorders of lncRNAs are also a feature of many cancers and promote the development, invasion, and metastasis by a variety of mechanisms [20, 23]. Studies have shown lncRNAs involved in the development and progression of lung cancer [24–28]. Nowadays, there is no relevant literature of lncRNAs relative to the mechanisms of EGFR exon 19 deletion in lung adenocarcinoma.
In this study, we presented the lncRNAs expression pattern in lung adenocarcinoma with EGFR exon 19 deletion samples compared with the wild-type EGFR gene samples, several of which were evaluated by SYBR Green I real-time RT-PCR in a total of 90 lung adenocarcinoma tissues. Our results showed that lncRNAs expression pattern may provide a new mechanism and preliminary data for the study of EGFR exon 19 deletion in lung adenocarcinoma.
Materials and methods
Patient samples
The lung adenocarcinoma with 5 exon 19 deletion patients and 5 wild-type EGFR patients was prospectively collected from the First Affiliated Hospital of Wenzhou Medical University, China, from April 2012 to August 2013. Otherwise, 90 cases of lung adenocarcinoma tissues (including 20 EGFR exon 19 deletion samples and 70 wild-type EGFR gene samples) were collected for SYBR Green I real-time RT-PCR, see Table 1. EGFR mutation was analyzed by ARMS method, conducted by a D×S EGFR mutation Test Kit, according to the manufacturer’s recommendations (Amoy Diagnostics Co., LTD, China). The diagnosis of lung adenocarcinoma was confirmed by histopathology result. The Institutional Ethics Review Board of the First Affiliated Hospital of Wenzhou Medical University approved this study, and all patients provided informed consent to this study.
RNA extraction
Lung adenocarcinoma cells were obtained by Laser capture microdissection; we combined the five lung adenocarcinoma with exon 19 deletion samples cells and five samples with the wild-type EGFR cells; and the two groups were subjected to RNA extraction. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The integrity of the RNA was assessed by electrophoresis on a denaturing agarose gel. A NanoDrop ND-1000 spectrophotometer was used for the accurate measurement of RNA concentration (OD260), protein contamination (ratio OD260/OD280), and organic compound contamination (ratio OD260/OD230).
Microarray and computational analysis
The Agilent array platform was used for microarray experiments. The sample preparation and microarray hybridization were performed based on the manufacturer’s standard protocols. Briefly, mRNA was purified from total RNA after removal of rRNA using an mRNA-ONLY™ Eukaryotic mRNA Isolation Kit (Epicentre Biotechnologies.USA). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias using a random priming method. The labeled cRNAs were hybridized onto a Human lncRNA Array v3.0 (8 × 60 K, Arraystar), designed for 30,586 lncRNAs and 26,109 mRNAs. The lncRNAs were carefully constructed using the most highly respected public transcriptome databases (Refseq, UCSC Known Genes, GENCODE, etc.), as well as landmark publications (Nature, Cell, Science, etc.). Each transcript was accurately identified by a specific exon or splice junction probe. Positive probes for housekeeping genes and negative probes were also printed onto the array for hybridization quality control. After washing the slides, the arrays were scanned using the Agilent Scanner G2505C and the acquired array images were analyzed with Agilent feature extraction software (version 11.0.1.1). Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.0 software package (Agilent Technologies). The microarray work was performed by KangChen Bio-tech, Shanghai, and People’s Republic of China.
Functional group analysis
GO categories were derived from Gene Ontology (www.geneontology.org), which provides three structured networks of defined terms that describe gene product attributes. The P value denotes the significance of GO term enrichment in the differentially expressed mRNA list (P ≤ 0.05 was considered statistically significant). We also performed pathway analysis for the differentially expressed mRNAs based on the latest Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This analysis allowed us to determine the biological pathway for which a significant enrichment of differentially expressed mRNAs existed (P ≤ 0.05 was considered statistically significant).
SYBR green I real-time RT-PCR
Total RNA was extracted from frozen lung adenocarcinoma tissues with TRIzol reagent (Invitrogen Life Technologies, USA) and then reverse transcribe using Thermo Scientific RT reagent Kit (Thermo Scientific)according to the manufacturer’s instructions. lncRNAs expression in lung adenocarcinoma tissues was measured by SYBR Green I real-time RT-PCR using SYBR Premixes Ex Taq on ABI 7000 instrument. Two lncRNAs that significantly expressed (RP11-325I22.2 and LOC440905) were evaluated in all of the patients included in this study. 2 mg of total RNA was transcribed to cDNA. PCR was performed in a total reaction volume of 20 ul, including 10ul SYBR Premix (2×), 2 ul of cDNA template, 1 ul of PCR Forward Primer (10 mM), 1 ul of PCR Reverse Primer (10 mM), and 6 ul of double-distilled water. The SYBR Green I real-time RT-PCR was set at an initial denaturation step of 10 min at 95 °C, and 95 °C (5 s), 60 °C (30 s) in a total of 40 cycles with a final extension step at 72 °C for 5 min. All experiments were done in triplicate, and all samples normalized to GAPDH. The median in each triplicate was used to calculate relative lncRNAs concentrations (ΔCt = Ct median lncRNAs − Ct median GAPDH). Expression fold changes were calculated.
Statistical methods
A comparison between the two groups was performed by Mann–Whitney U test. P < 0.05 was considered to be statistically significant. The fold change and the Student’s t test were analyzed for statistical significance of the microarray results. The false discovery rate (FDR) was calculated to correct the P value. The threshold value we used to designate differentially expressed lncRNAs and mRNAs was a fold change of ≥2.0 or ≤0.5.
Results
Overview of lncRNAs profiles
To study the potential biological functions of lncRNAs in lung adenocarcinoma with EGFR exon 19 deletion samples, we examined the lncRNA and mRNA expression profiles in human lung adenocarcinoma through microarray analysis (Fig. 1a, b). Among these, 539 lncRNAs were found to be upregulated more than twofold in the lung adenocarcinoma with EGFR exon 19 deletion group compared with wild-type EGFR group, while 994 lncRNAs were downregulated more than twofold (P < 0.05; Table 2; Fig. 1c, d).
LncRNA classification and subgroup analysis
The expression profiles of 426 intergenic lncRNAs indicated that they were differentially expressed (fold change ≥ 2.0, P < 0.05) between EGFR exon 19 deletion group and wild-type EGFR group. Among these, 166 were upregulated and 260 were downregulated. We also identified some nearby coding genes (distance < 300 kb) that may be regulated by these lncRNAs (Table 3). LncRNAs with enhancer-like function (lncRNA-a) were identified using GENCODE annotation. The expression profiles of 46 enhancer-like lncRNAs indicated that they were differentially expressed (fold change ≥ 2.0, P < 0.05) between EGFR exon 19 deletion group and wild-type EGFR group. Among these, 15 were upregulated and 31 were downregulated. We also identified some nearby coding genes that may be regulated by these enhancer-like lncRNAs (Table 4). HoxlncRNAs (lncRNA transcribed from Hox loci LncRNAs) profiles: This data table contains 83 HoxlncRNA clusters (Table 5).
Overview of mRNAs profiles
In total, 1,406 mRNAs were found to be differentially expressed between EGFR exon 19 deletion group and wild-type EGFR group, including 483 upregulated mRNAs and 923 downregulated mRNAs (Table 6; Figs. 1, 2).
Go analysis
The genes corresponding to the downregulated mRNAs included 160 genes involved in biological processes, 59 genes involved in cellular components, and 51 genes involved in molecular functions. The genes corresponding to the upregulated mRNAs included 296 genes involved in biological processes, 21 genes involved in cellular components, and 69 genes involved in molecular functions. These results imply that biological processes relevant to mRNA might play a more important role in the mechanism of EGFR exon 19 deletion in lung adenocarcinoma than these of cellular components and molecular functions.
Pathway analysis
The 27 upregulated pathways were identified, including metabolism of xenobiotics by cytochrome p450, drug metabolism-cytochrome p450, chemical carcinogenesis, and so on. 27 downregulated pathways were identified, including alcoholism, systemic lupus erythematosus, viral carcinogenesis, transcriptional misregulation in cancer, and so on (Fig. 3; Tables 7, 8).
SYBR green I real-time RT-PCR validation
According to fold difference, gene locus, and so on, we initially identified a number of candidated lncRNAs (including LOC440905, AC018865.8, RP11-325I22.2, AFAP1-AS1, CYP4F43P, RP11-133F8.2, AC073135.3, XLOC_003318, BLNK, XLOC_005621) and verified the expression of lncRNAs by SYBR Green I real-time RT-PCR with GAPDH as reference gene, by calculating the 2−ΔΔCT. We found that multiple lncRNAs of gene microarray are consistent with results of SYBR Green I real-time RT-PCR, see Fig. 4. The RP11-133F8.2 and LOC440905 were the most significantly changed lncRNAs of these candidated lncRNAs from 20 cases EGFR exon 19 deletions in lung adenocarcinoma patients and 70 cases wild-type EGFR gene patients. According to Fig. 5, RP11-133F8.2 expression of EGFR exon 19 deletions in lung adenocarcinoma was significantly higher than wild-type EGFR tissues (Mann–Whitney U = 107.00, P = 0.01), while LOC440905 expression of EGFR exon 19 deletions in lung adenocarcinoma was significantly lower than wild-type EGFR tissues (Mann–Whitney U = 189.21, P = 0.001).
Discussion
The most frequent mutations of EGFR gene, as delE746-A750, delL747-p753inss, delL747-T75linss, delL747-S752ins, T790 M mu, L858R mu, occurred in NSCLC patients. These mutations are detected mainly in adenocarcinoma tissue [29–32], wherein the TK domains of 19 and 21 of exon usually accounts for about 90 % of EGFR mutations and were most closely related to the efficacy.
Nowadays, there was no literature about the relation of lncRNAs to EGFR gene mutation in lung adenocarcinoma. In this study, we obtained 1,533 differentially expressed lncRNAs in EGFR exon 19 deletions in lung adenocarcinoma, and our data showed that these lncRNAs may participate in EGFR exon 19 deletions in lung adenocarcinoma.
We fatherly studied the classification and subgroup of lncRNAs and acquired the expression profiles of linRNAs, enhancer-like lncRNAs, and Hox lncRNA cluster. Systematic comparison of the expression pattern of every lncRNA with its immediate 5′ and 3′ Hox gene neighbor showed that the vast majority of lncRNAs (90 %) are coordinately induced with their 3′ Hox genes, while only 10 % of instances are lncRNA expression anticorrelated with 3′ Hox gene expression [33]. LncRNAs in the Hox loci became systematically dysregulated during some cancer progression [20]. LncRNAs are known to function via a variety of mechanisms; however, a common and important function of lncRNAs is to alter the expression of nearby coding genes by affecting the process of transcription [34–36] or directly playing an enhancer-like role [37, 38]. We also identified some nearby coding genes(<300 kb) that may be regulated by lincRNAs and enhancer-like lncRNAs. We studied also the relationship of lincRNA and nearby coding genes.
We found that 10 candidated lncRNAs of microarray are consistent with results of SYBR Green I real-time RT-PCR; it indicated that lncRNAs expressed unusually during EGFR exon 19 deletions in lung adenocarcinoma. The RP11-325I22.2 was most significantly upregulated, and LOC440905 were most downregulated of these candidated lncRNAs, and these of EGFR exon 19 deletions in lung adenocarcinoma was significantly higher or lower than wild-type EGFR by RT-PCR. This result suggested that RP11-325I22.2 and LOC440905 might play in EGFR exon 19 deletions in lung adenocarcinoma; we would further study the biological function of RP11-325I22.2 and LOC440905.
In order to obtain new sight into the function of targets of lncRNAs, GO analysis and KEGG pathway annotation were applied to this target gene pool. GO analysis revealed that the number of genes corresponding to downregulated mRNA was larger than that corresponding to upregulated mRNA. KEGG annotation showed that there are 27 upregulated pathways (metabolism of xenobiotics by cytochrome p450, drug metabolism-cytochrome p450, chemical carcinogenesis, and so on.) and 27 downregulated pathways (including alcoholism, systemic lupus erythematosus, viral carcinogenesis, transcriptional misregulation in cancer, and so on). Our data denoted that these pathways might play important roles in EGFR exon 19 deletions in lung adenocarcinoma.
Conclusions
Our study revealed that a set of lncRNAs are differentially expressed EGFR exon 19 deletions in lung adenocarcinoma. The differentially expressed lncRNAs may be closely related to EGFR exon 19 deletions in lung adenocarcinoma, and we would further study these lncRNAs. This result suggested that RP11-325I22.2 and LOC440905 might play an important role in the mechanism of EGFR exon 19 deletions in lung adenocarcinoma.
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Acknowledgments
This study was supported by the Wenzhou Municipal Science and Technology Bureau of China (Y20110041, Y20130170, Y20140032, Y20140024) and the Zhejiang Provincial Health Department (No. 2014KYA133), the Scientific Research Programs of Department of Education of Zhejiang Province (Y201430425). We thank all of the donors who donated to the Microarray Service at KangChen Bio-technology Company in Shanghai.
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The authors declare that they have no competing interests.
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Yumin Wang and Jie Chen have contributed equally to this study, and all should be considered first author.
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Wang, Y., Chen, W., Chen, J. et al. LncRNA expression profiles of EGFR exon 19 deletions in lung adenocarcinoma ascertained by using microarray analysis. Med Oncol 31, 137 (2014). https://doi.org/10.1007/s12032-014-0137-y
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DOI: https://doi.org/10.1007/s12032-014-0137-y