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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 1  |  Page : 395-400

Abberent expression of oncogenic and tumor-suppressive microRNAs and their target genes in human adenocarcinoma alveolar basal epithelial cells


1 Department of Molecular Genetics, Science and Research Branch, Islamic Azad University, Fars; Department of Molecular Medicine, Biotechnology Research Center, Pasture Institute of Iran, Tehran, Iran
2 Department of Molecular Genetics, Science and Research Branch, Islamic Azad University, Fars, Tehran, Iran
3 Department of Medical Science, Tracheal Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Department of Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong
5 Department of Molecular Medicine, Biotechnology Research Center, Pasture Institute of Iran, Tehran, Iran

Date of Web Publication13-Apr-2016

Correspondence Address:
Morteza Karimipoor
Department of Molecular Medicine, Biotechnology Research Center, Pasture Institute of Iran, 1316943551 Pasteur Institute of Iran, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.148673

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 > Abstract 


Context: Lung cancer is one of the most serious types of cancer that often diagnosed at advanced stage. MicroRNAs (miRNAs) are small non-coding molecules which silence gene expression of target gene (s) at posttranscriptional level. They are key regulators of cell cycle, apoptosis, anti-cancer drug responsiveness and metastasis.
Aims: Identification of the differential expression level of miR-15a/16, miR-21, miR-34a, miR-126, miR-128 and miR-210 in A549 cell line versus normal tissues and their correlation with selected corresponding target genes.
Materials and Methods: A549 cell line was cultured in F-12K medium and miRNA was extracted from normal tissues (2-3 cm adjacent to tumor tissue) and A549 cell line. cDNA was synthesized with specific stem-loop primers for each miRNA, while OligodT primer was used for target genes cDNA synthesis. Real-time quantitative polymerase chain reaction. (RT-qPCR) was used to analyze the expression pattern of miRNAs and target genes in A549 and normal non-small cell lung carcinoma. (NSCLC) tissues.
Results: miR-15a/16, miR-34a, miR-126 and miR-128 were down-regulated significantly. (>2-fold change), while miR-21 and miR.210 were up-regulated in A549. Bcl-2 as miR-34a target gene was down-regulated while Hif-1α and Akt-3 were up-regulated that might be miR-210 and miR-34a target genes, respectively.
Conclusion: The significant differential expression level of these miRNAs made them as candidate biomarkers in NSCLC tumor tissues of patients. Perhaps Bcl-2 down-regulation and Akt-3 up-regulation can be linked with survival signals in A549 cell line. We can conclude that Bcl-2 and Akt-3 might be therapeutic targets to inhibit cell proliferation in NSCLC.

Keywords: A549, Akt-3, Bcl-2, Hif-1α, miRNA, NSCLC, RT-qPCR


How to cite this article:
Tafsiri E, Darbouy M, Shadmehr MB, Cho WC, Karimipoor M. Abberent expression of oncogenic and tumor-suppressive microRNAs and their target genes in human adenocarcinoma alveolar basal epithelial cells. J Can Res Ther 2016;12:395-400

How to cite this URL:
Tafsiri E, Darbouy M, Shadmehr MB, Cho WC, Karimipoor M. Abberent expression of oncogenic and tumor-suppressive microRNAs and their target genes in human adenocarcinoma alveolar basal epithelial cells. J Can Res Ther [serial online] 2016 [cited 2019 Aug 23];12:395-400. Available from: http://www.cancerjournal.net/text.asp?2016/12/1/395/148673




 > Introduction Top


Lung cancer is the first death-related cause of cancer in the world. It is categorized histologically to two main classes, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC consists 80% of all lung cancer cases which divide to three main subtypes including, adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma.[1] Lung cancer is caused by several genetic modifications on various oncogenes and tumor suppressor genes which are involved in cell cycle control, DNA repair and apoptosis.[2] The most common genetic alterations of lung cancer are upregulation of CCND1 and Bcl-2, mutations of KRAS or members of ERBB family, Rb inactivation, p16INK4a, TP53 mutation or gene inactivation and anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML-4) fusion gene.[3]

A549 cell line is an adenocarcinoma human alveolar basal epithelial cell which was first cultured by D.J. Giard, et al. in 1972 from lung cancerous tissue explants of a 58-year-old Caucasian male.[4] A549 cells are squamous, grow as monolayer cells and adherent to the culture flask (http://a549.com/).

MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression at posttranscriptional level. They function in different biological and physiological processes including proliferation, apoptosis, invasion, metastasis and differentiation.[5],[6] They are double-stranded molecules which have stem-loop structure in nucleus and called pri-miRNA. RNaseIII endonuclease, Drosha, cleaves pri-miRNA to a ~70 nucleotide structure which is called pre-miRNA.[7],[8] The cleaved pre-miRNA is exported to cytoplasm through exportin-5.[9] and in the cytoplasm Dicer, another RNase III endonuclease, cleave pre-miRNA further to a 17-26 nucleotide structure and load it on a microprocessor complex which is called RNA-induced silencing complex (RISC). One strand of miRNA in this complex target 3'-untranslated region (UTR) of mRNA (s) and degrade it or block its translation.[10],[11]

miRNAs can have oncogenic (oncomiRs) or tumor suppressor (TSmiRs) role in different types of cancers. Upregulation of oncomiRs usually target tumor suppressor genes leading to cancer development. Whereas, downregulation of TSmiRs facilitate the activity of target oncogenes.[12],[13] The first microRNA which was studied in cancer was miR-15a/16 that is located on 13q14 and are frequently deleted or down-regulated in different types of cancer, including leukemia, lung and prostate cancer.[14] miR-15a/miR-16 are involved in apoptosis through targeting Bcl-2 as an anti-apoptotic gene.[15]

The careful selection of endogenous control gene has a key role in quantification of miRNAs expression level. Reference gene should be constitutively expressed at the same level in nearly all tissues and not affected by external or internal factors.[16] According to the tests which were done by Applied Biosystems they found that among common housekeeping genes including, ACTB and GAPDH, there are great variation in gene expression among different tissues and cell lines.[17] It has been suggested that SnRNAs and SnoRNAs, including RNU6B, RNU44 and RNU48, had higher abundance and were considered as more appropriate endogenous control for miRNAs, as they are closer in length to miRNAs and are constitutively expressed in different types of tissues and cell lines (http://www3.appliedbiosystems.com).

In this study we evaluated the expression level of some TSmiRs such as miR-15a/miR-16, miR-34a, miR-126 and miR-128 and in parallel oncomiRs like miR-21 and miR-210 in A549 in comparison with normal tissue.


 > Materials and Methods Top


Normal tissue collection and cell culture

A549 human broncho-alveolar lung carcinoma was obtained from Cell Bank of Pasteur Institute (Tehran, Iran). Cells were cultured in Kaighn's modified F-12K medium (Gibco, Life Technologies) with 10% fetal bovine serum (FBS) (Gibco, Life Technologies) at 37°C in 5% CO2. Cells were cultured in the presence of 100 unit/ml penicillin and 100 µg/ml streptomycin. Briefly, 1 ml aliquot of cells were thawed as quickly as possible in water bath at 37°C then the cells were transferred to 9 ml warm media and mixed gently. After that the cells were spinned down at 14xg for 5 minutes. Next, the media was discarded and resuspended in 10 ml warm medium. The cells were placed in incubator when the confluency reached to 70-90%, 0.25% (W/V) trypsin and 0.53 mM EDTA (Gibco, Invitrogen) solution were added then cells were incubated at 37°C for 5 minutes. The cells were observed under an inverted microscope until cell layer is dispersed. Finally, 3X to 5X complete growth medium was added to inactivate trypsin and RNA extraction procedure on A549 cells was followed.

All the patients underwent lung resection (either lobectomy or pneumonectomy) based on the oncologic principles. The normal tissue was taken from the normal (non-tumoral) part of the resected lung as far as possible from the tumor. Ten normal tissues were chosen which were in stage I, non-smoker and adenocarcinoma. The informed consent was taken from all patients before surgical resection and approval was obtained from the ethic committee of Masih Daneshvari Hospital.

RNA isolation and cDNA synthesis

Total RNA of A549 cell line and normal tissues were extracted by miRNeasy kit according to manufacturer's instruction (Qiagen, USA). The purity of extracted RNA was checked by Nanodrop (the ratio of absorbance at 260 and 280nm ≥ 1.8). Besides, quality assessment of total RNA was checked by running the extracted RNA on denaturing agarose gel electrophoresis followed by ethidium bromide staining. Total RNA of 10 normal tissues were pooled in a way to reach to the final concentration of 1000 ng/µl. Then cDNA synthesis was done based using two procedures, one for target genes which cDNA was synthesized through 2-step RT-PCR kit (Vivantis, USA) with OligodT primers and another for miRNAs that the same protocol were used except that stem-loop specific microRNA primer was used instead of oligodT primers [Table 1].[18],[19] Ten pmol stem-loop specific primer was incubated at 95°C for 8 minutes to linearize the loop structure. Then it was placed at room temperature for 10 minutes followed by addition of 10 mM dNTP, RNA (1000 ng) and H20 to the stem-loop primer. The mix was incubated at 65°C for 5 minutes and immediately chilled on ice for 2 minutes. Finally, 10X reaction buffer, Reverse Transcriptase enzyme and RNase-free water were added to final volume of 20 µl. The whole volume was incubated at 42°C for 60 minutes. At the end, in order to inactivate the enzyme, the reaction was incubated at 85°C for 5 minutes.
Table 1: The list of specific miRNAs and RNU6 stem-loop primer sequences

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miRNA target prediction

To predict miRNA target genes, various microRNA-target interactions databases and web-based tools such as picTar, Tarmir, miRecord, mirGen, miRmap, mirDIP, miRanda, Tarbase, DIANAmicroT, StarBase, TargetScan (version 5.1), mirWalk and mirTarBase, were used for each selected miRNA [Table 2]. To exclude the over-prediction, several target prediction software and databases were used.
Table 2: List of the miRNA target prediction databases

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Real time RT-PCR (qRT-PCR)

Specific TaqMan probes, specific forward primers and universal reverse primer were designed with our team by using Allele ID6 and Oligo7 software [Table 3]. The TaqMan RT-PCR reactions were performed in MicroAmp Optical 96-well plate using SDS v. 1.0.1 software (ABI System 7300, Applied Biosystems, USA). Briefly, each reaction well contained 5 µl TaqMan master mix, 1 µl specific forward and reverse primers (10 pmol), 0.1 µl specific probe (1 pmol) and 2 µl (800 ng) of cDNA, for a total reaction mixture of 12 µl. Non-Template Control (NTC) was included in each assay. The thermal-cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for one minute. Serial dilutions of target cDNA were made to determine the dynamic range and amplification efficiency of each target by plotting corresponding Ct values versus log cDNA concentrations. The Bcl-2, Akt-3 and Hif-1 α genes were analyzed by SYBR Green based q-PCR (Applied Biosystems) using SDS v. 1.0.1 software (ABI System 7300, Applied Biosystems) [Table 4]. Each reaction for SYBR Green based RT-PCR (total volume 20 μl) contained 10 µl SYBR Green mix, 1 µl mixture of specific forward and reverse primers (10 pmol), and 2 µl of cDNA (800 ng). Non-Template Control (NTC) was included in each assay. The thermal-cycling conditions were as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 1 minute. Serial dilutions of target cDNA were made to determine the dynamic range and amplification efficiency of each target by plotting corresponding Ct values versus log cDNA concentrations. Prism 5.0b (GraphPad; La Jolla, CA, USA) was used for graphing.
Table 3: The list of specific TaqMan primers for studied miRNAs

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Table 4: List of SYBR Green q-PCR of Bcl-2, Akt-3, Hif-1α and GAPDH

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 > Results Top


In order to consider the differences in miR-15a/16, miR-21, miR-34a, miR-126, miR-128 and miR-210 expression level in A549 cell line, TaqMan RT-PCR was performed. To evaluate the fold change of these miRNAs, ΔΔCt of the pooled normal tissue of 10 patients (2-3 cm adjacent to tumor tissue) was compared with A549 cell line for each miRNA separately and the relative expression level of miRNAs was calculated by comparative ΔΔCT method.[20]

The optimization process on miRNAs was performed by drawing the standard curve for miRNAs and RNU6b in serial dilutions of cDNA from 800-50ng in duplicate. The standard curve for miR-16 was shown in [Figure 1]a. The graph provides information about Ct of miR-16 based on log concentration of cDNA. As regards the serial dilutions 200 ng which equals to log concentration of 2.3-25ng with log concentration of 1.4 were almost on linear standard curve. It is noticeable that 800 ng and 400 ng were too far from standard curve. Therefore, 200 ng was selected for all miRNAs and endogenous control. Consequently, the expression level of miR-15a/16, miR-21, miR-34a, miR-126, miR-128 and miR-210 were evaluated in A549 which some of the miRNAs and RNU6 expression pattern is shown in [Figure 1]b. The comparison of ΔCt of specified miRNAs in A549 and pooled normal tissues demonstrated that two miRNAs (miR-21 and miR-210) were up-regulated while miR-15a, miR-16, miR-34a, miR-126 and miR-128 were down-regulated. Among down-regulated miRNAs the fold change of miR-34a, miR-126, miR-16, miR-15a and miR-128 were as follows: Negative 25, 7, 4, 1.6, and 1.6. While the fold change of miR-210 and miR-21 were positive 39 and 1.6. As these results show miR-15a, miR-16, miR-21, miR-34a, miR-126, miR-128 and miR-210 expression level were significantly changed between A549 cell line and pooled normal tissues. It is noticeable that the fold change of miR-34 and miR-210 were remarkable.
Figure 1: Real time qRT-PCR of miRNAs. (a) Optimizing TaqMan qRT-PCR of miR-16 and standard curve from 800-25 ng of A549 cDNA. The serial dilution was made in 800 ng, 400 ng, 200 ng, 100 ng, 50 ng and 25 ng. Eight hundred and 400 ng were crossed out as they were so far from linear graph. (b) The fold change of miR-15a/miR-16, miR-21, miR-34a, miR-126, miR-128 and miR-210 in A549. miR-15a, miR-16, miR-34a, miR-126 and miR-128 were down-regulated in the order of 1.6, 4, 25, 7 and 1.6 fold in A549 cell line when compared with pooled normal tissues. miR-21 and miR-210 were up-regulated in 1.6 and 39 fold, respectively. (c) The fold change of Bcl-2, Akt-3 and Hif-1α as target genes in comparison with miR-34a and miR-210

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According to bioinformatics analysis which predicts targets for miRNAs three target genes were selected based on miRNAs' complementary match between 3'-UTR of target gene and miR-15a/16 and miR-34a. Bcl-2 was the candidate target gene for miR-15a/16 and miR-34a in target prediction databases with high score. The 3'-UTR of Akt-3 was in complementary match with miR-15a/16 in databases and Hif-1 α was the candidate gene for miR-126 with not much high score. The expression level of Bcl-2, Akt-3 and Hif-1 α were evaluated by SYBR Green based qPCR in A549 cell line and compared with pooled normal tissue. As shown in [Figure 1]c. Bcl-2 was down-regulated significantly (>-fold), but Akt-3 and Hif-1 α were up-regulated. The data shows a positive correlation between miR-210 with Hif-1 α and negative correlation between miR-15a/miR-16 and miR-34a with Akt-3 but we could not find the same link between miR-34a and Bcl-2.


 > Discussion Top


It is the first study which focuses mainly on expression level of miR-15a/miR-16, miR-21, miR-34a, miR-126, miR-128 and miR-210 together in A549 cell line. Dysregulation of apoptosis and proliferation are the mechanisms which have a key role in tumorgenesis and might lead to survival of cells that are genetically instable.[21] miR-15a/16 were down-regulated in A549 that can regulate apoptosis by targeting Bcl-2.[22] This study demonstrated that Bcl-2 gene is down-regulated significantly while miR-15a/16 and miR-34a had a decrease in their expression level. According to EMBL-EML database on gene expression level it has been documented that Bcl-2 gene is down-regulated in most of the NSCLC cell lines except that NCI-H1155 and NCI-H1770 that are driven from metastatic sites, therefore there might be a kind of correlation between Bcl-2 expression level and late stages of NSCLC. Druz et al., has demonstrated that miR-15a-3p had a pro-apoptotic role in A549 by activating caspase 3/7.[23] Furthermore, miR-15a/16 and miR-34 are capable of apoptosis induction and cell cycle arrest through Rb gene inactivation or down-regulation of miR-15a/16 and miR-34 in NSCLC.[24] However, there might be other mechanisms including, reduced expression of Dicer which is frequently observed in many lung cancer patients.[25] In B-cell neoplasm it is documented that PAX5 is able to reduce promoter activity of miR-15a/16 that it might be similar in lung cancer.[26] Moreover, point mutation in miR-16 primary transcript is associated with down-regulation of mature miR-16.[27] miR-15a/16 are also downregulated in CLL and pituitary adenoma,[28] while they are frequently up-regulated in cervical cancer.[29] Tumorgenecity in cervical cancer might be independent of miR-15a/16 but it seems more likely due to inactive form of Rb. Bandi, et al., have shown the same mechanism in NSCLC, as in some cases miR-15a/16 are increased while expression level of Rb is decreased. Therefore, miR-15a/16 can have dual function as tumor suppressive or oncogenic function.[24]

miR-34a, which was down-regulated significantly in A549, showed a remarkable rise in Akt-3 gene, therefore it can be concluded that miR-34a might regulate expression level of Akt-3 gene negatively. According to Expression  Atlas More Details website (http://www.ebi.ac.uk/gxa/) Calu-1, NCI-H1155, NCI-H1651 and NCI-H1703 cell lines, which all belong to non-small-cell lung cancer, showed an increase in Akt-3 gene expression level.

miR-21 is up-regulated in A549 cell line with positive 1.6 fold change, it is mainly considered as an oncomiR which can reduce apoptosis and increase proliferation through targeting tumor suppressor genes, BIM.[30] and PDCD4.[31] The overexpression of miR-21 is frequent in different types of cancers including, lung, breast, stomach, colon and pancreas.[32],[33],[34] Hatley, et al., have shown that in a k-Ras G12D NSCLC mouse model, the incidence of lung cancer is related to upregulation of miR-21.[35] Liyun et al., have shown that downregulation of miR-21 through anti-mir-21 could increase sensitivity of A549 cell line to cisplatin.[36] Chemotherapeutic response might be associated with miR-21, since downregulation of miR-21 can cause an increasing sensitivity of MCF-7 cell line to tropotecan.[37]

miR-126 is down-regulated in A549 cell line which target EGFL7 and might inhibit proliferation of lung cancer cells.[38] miR-126 is a metastasis inhibitor in relapsing breast cancer, leukemia and cervical cancer.[29],[39],[40]

miR-128 involves in apoptosis inhibition and increased resistance to chemotherapeutic agents including, cisplatin, doxorubicin and 5-fluorouracil].[41] miR-128 targets a repressive transcription factor E2F5. miR-128 overexpression leads to elimination of repressive effect of E2F5 on p21wf1 The constitutive expression of p21wf1 in cytoplasm will recruit its anti-apoptotic activity against anticancer drugs.[41] miR-128 downregulation is documented in lung cancer,[42] as there is a link between miR-128 and p53. They showed that the overexpression of miR-128 is associated with increased p53 level and induction of apoptosis in HEK293T cell line.[43]

miR-210 is one of miRNAs which is regulated in hypoxia condition and its expression level is related to the advanced stage of NSCLC.[44],[45] This study showed that miR-210 has a significant increase in A549 cell line, about positive 39 fold, which can induce G0 to G1 entry in cell cycle [46] and inhibit apoptosis through caspase-3/7 activity.[47] The transcription of miR-210 is regulated by Hif-1 α in a way that this transcription factor binds to the promoter of miR-210 and increases its expression level.[48] However, miR-210 might negatively target Hif-1 α and can inhibit its expression. Our data suggests that in A549 by overexpression of miR-210 the expression level of Hif-1 α is raised either, therefore upregulation of miR-210 can be partly due to overexpression of Hif-1 α gene which is activated in hypoxia.

In summary we found that miR-15a/16, miR-34a, miR-126 and miR-128 were down-regulated in A549 cell line in comparison with normal tissue in adenocarcinoma lung cancer. While miR-21 and miR-210 were up-regulated which suggests their oncogenic role in adenocarcinoma. Our finding was in agreement with previous studies on miR-15a/16, miR-21, miR-34a, miR-126, miR-128 and miR-210 expression level in A549 cell line. The future perspective is the evaluation of these miRNAs in NSCLC tumor tissues with different histological subtypes prospective in a study and consider any correlation between miRNAs expression level with smoking and other clinic pathological features of the patients, including lymph node, tumor size, stage and histological subtype. Furthermore, there might be some correlations between expression level of miR-15a/16, miR-21 and miR-126 with platinum-based chemotherapy. As there is a negative correlation between miR-15a/16, miR-34a with Akt-3 so these miRNAs can be considered as therapeutic targets for inducing cell death and apoptosis.


 > Acknowledgment Top


We would like to acknowledge the support by grants from the Molecular Medical Genetics Department of Pasteur Institute of Iran and Tracheal Diseases Research Center, NRITLD of Shahid Beheshti University of Medical Sciences

 
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