Journal of Reports in Pharmaceutical Sciences

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 8  |  Issue : 1  |  Page : 18--23

The effect of ibuprofen on expression of Cox-1/2-related miRNAs in MKN- 45 -derived cancer stem-like cells


Hassan Akrami, Behnam Karimi, Zohreh Salehi, Sajjad Sisakhtnezhad 
 Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran

Correspondence Address:
Hassan Akrami
Department of Biology, Faculty of Science, Razi University, Kermanshah
Iran

Abstract

Context: Ibuprofen is an anti-inflammatory drug that non-selectively blocks cyclooxygenases-1/2 (COX-1/2) enzymes and thus reduces the risk tumorigenesis. This study was designed to detect microRNAs (miRNAs) that target Cox-1/2 mRNA and to investigate the effect of ibuprofen on the expression of the miRNAs in MKN-45-derived gastric cancer stem-like cells (CSLCs). We were also aimed to find signaling pathways modulated by the miRNAs. Subjects and Methods: The miRWalk database was used to recognize miRNAs that targeted Cox-1/2 genes. CSLCs were derived from MKN-45 cell line and were then treated with ibuprofen. Consequently, the effect of ibuprofen was evaluated on the expression of the miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, DIANA tools were used to identify signaling pathways that modulated by the miRNAs. Results: Our bioinformatic investigation showed that hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669 targeted both Cox-1 and Cox-2 mRNAs. The qRT-PCR results indicated that hsa-mir-16-5p and hsa-mir-4669 were overexpressed 2.34 and 9.47 folds, respectively, while hsa-mir-483-5p under-expressed (2.08 folds) in ibuprofen-treated CSLCs relative to untreated cells. Moreover, it found that these miRNAs are involved in PI3K-Akt, P53, transforming growth factor-beta, phosphatidylinositol and insulin signaling pathways, cell cycle, extracellular matrix receptor interaction, gap junction, small cell lung cancer, prostate cancer, and chronic myeloid leukemia. Conclusions: We suggest that ibuprofen may reduce the risk of gastric cancer by affecting the expression of miRNAs that target Cox-1/2. however, further research is necessary to unravel its exact effects.



How to cite this article:
Akrami H, Karimi B, Salehi Z, Sisakhtnezhad S. The effect of ibuprofen on expression of Cox-1/2-related miRNAs in MKN- 45 -derived cancer stem-like cells.J Rep Pharma Sci 2019;8:18-23


How to cite this URL:
Akrami H, Karimi B, Salehi Z, Sisakhtnezhad S. The effect of ibuprofen on expression of Cox-1/2-related miRNAs in MKN- 45 -derived cancer stem-like cells. J Rep Pharma Sci [serial online] 2019 [cited 2019 May 23 ];8:18-23
Available from: http://www.jrpsjournal.com/text.asp?2019/8/1/18/255058


Full Text

 Introduction



MicroRNAs (miRNAs) are a class of small endogenous noncoding RNAs that play a crucial role in the posttranscriptional regulation of genes.[1] Some miRNAs regulate cell proliferation and apoptosis processes, which are important in cancer formation and progression, by targeting oncogenes and tumor suppressor genes. These miRNAs function as oncogenes or tumor suppressors and thus named Oncomir and tumor suppressor miRNA, respectively.[2] Recent studies have been shown that some miRNAs play important roles as tumor suppressors or oncogenes in different cancers such as gastric cancer.[3] Liu et al. found that the expression profile of miRNAs was changed in MKN-45-derived cancer stem-like cells (CSLCs) relative to the MKN-45 cells.[4] Cancer stem cells (CSCs) are defined as the unique subpopulation in the tumors that can initiate tumor formation. They have self-renew potency and can also differentiate into various kinds of cells.[5] CSCs also induce the growth, metastasis, and chemo- and radio-resistant of tumors.[6] In this regard, it has also been found that miRNAs have a crucial role in the function of CSCs through regulating the expression of different genes involved in the CSC characteristics and functions.[7]

Ibuprofen is a member of nonsteroidal anti-inflammatory drugs derived from propionic acid and is a nonselective inhibitor of COX, an enzyme involved in prostaglandin synthesis through the arachidonic acid pathway.[8] Indeed, ibuprofen inhibits hydrophobic channel isozyme COX through direct interaction with the channel.[9],[10] COX is a key enzyme for conversion of arachidonic acid to prostaglandins and has two isozymes COX-1 and COX-2.[11] It has been found that overexpression of COX-2 in cancer cell lines promotes their ability to invade surrounding tissues as well as increases cell invasion in gastric cancer.[12] Ibuprofen inactivates COX-2 and thereby inhibits cancer cell proliferation.[13] It is also able to induce apoptosis in cancerous cells and to inhibit angiogenesis in gastric cancer.[14],[15] Although the exact mechanism of action of ibuprofen is remained to be clarified, it found that oncomir and tumor suppressor miRNA expression was changed in the ibuprofen-treated CSCs. For example, ibuprofen delays the development of tactile allodynia and suppresses spinal miR-155 in a rat model.[16] It showed that some miRNAs downregulated the expression of Cox-1 and Cox-2 genes and thereby inhibited cell invasion.[17] For example, COX-2 is a target of hsa-mir-16 in human hepatoma.[18] Given the importance of ibuprofen in regulating the expression of miRNAs in cells, this study was aimed to find miRNAs that target Cox-1/ 2 mRNA and to evaluate the effect of ibuprofen on the expression of the miRNAs in MKN-45-derived CSLCs. As well as, it was attempted to identify the signaling pathways that modulated by the miRNAs. In the current study, bioinformatic analysis was performed to find miRNAs that target Cox-1/ 2 mRNA. Consequently, we selected hsa-mir-16-5p, hsa-mir-4669, and hsa-mir-483-5p and then studied the effect of ibuprofen on the expression of these miRNAs in MKN-45-derived CSLCs. Finally, bioinformatic investigation was used to identify the signaling pathways that affected by these miRNAs.

 Materials and Methods



Bioinformatic analysis to find microRNAs and their affected signaling pathways

In the current study, different online databases were used to recognize miRNAs that target Cox-1/ 2 mRNAs and to find the signaling pathways that affected by the identified miRNAs. We used miRWalk, microT4, miRmap, PITA, RNA 22, TargetScan, and RNA hybrid in the miRWalk database (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) to identify miRNAs that target Cox-1/ 2 mRNAs. The results were filtered and miRNAs with P ≤ 0.05 were selected from the list. After miRNA selection, the signaling pathways that affected by the selected miRNAs were obtained using DIANA tools (http://diana.imis.athena-innovation.gr/DianaTools).

Isolation and culture of MKN-45-derived cancer stem-like cells

The MKN-45 cell line was obtained from Pastor Institute (Tehran, Iran) and cultured in Dulbecco's modified Eagle medium/F12 supplemented with 10% fetal bovine serum (Gibco, Germany), penicillin (100 U/mL), and streptomycin 100 μg/mL (Sigma, Germany). To isolate CSLCs, the MKN-45 cells were cultured on the nonadhesive surface of 10 cm 2 culture plastic dishes coated with agarose (1.5%) (Sigma, Germany) and incubated at 37°C and 5% CO2 for 2 weeks. The culture medium was changed every 3 days until the sphere formation, which was the indication for the presence of CSLCs in culture.[19]

Treatment of cancer stem-like cells with ibuprofen

To investigate the effect of ibuprofen on the expression of miRNAs in the MKN-45-derived CSLCs, they were treated with 700 μM of the compound. The ibuprofen-treated CSLCs were then incubated at 37°C and 5% CO2 for 48 h.

Gene expression analysis

Total RNA was extracted from ibuprofen-treated and untreated CSLCs using a Qiagen RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. Reverse transcriptase reactions were performed by specific stem-loop RT primer, including 5'-GTCGTATCCAGTGCAGGGTCCGA GGTATTCGCACTGGATACGACCGCCAA-3', 5'-GTCGTATCCAGTGCAGGGTCCGAGGT ATTCGCACTGGATACGACCTCCCT-3', and 5'-G TCGTATCCAGTGCAGGGTCCGAGGTATTCGC ACTGGATACGACCCTCCT-3' for hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669, respectively. The effect of ibuprofen on the expression of miRNAs was evaluated using specific primers in quantitative real-time polymerase chain reaction (qRT-PCR). The nucleotide sequences of the forward primers were 5'-CGCGCTAGCAGCACGTAAAT-3', 5'-GTGCGTAAGACGGGAGGAAAGA-3', and 5'-C GCATAGTCCGGGAAGTGGAG-3' for hsa-mir- 16-5p, hsa-mir-483-5p, and hsa-mir-4669, respectively. In addition, a reverse universal primer contained 5'-GTGCAGGGTCCGAGGT-3' nucleotide sequence was used for all miRNAs. In this study, glyceraldehyde-3-phosphate dehydrogenase (forward primer: 5'-ACTCTGGTAAAGTGGATATTGTTGC-3' and reverse primer: 5'-GGAAGATGGTGATGGGATTTC-3') was also used as an endogenous control for relative quantification of gene expression by RT-PCR [Table 1]. In this study, the expression level of miRNAs relative to the internal control was assessed using Livak et al. method.[20]{Table 1}

Statistical analysis

In the present study, statistical analysis was performed using SPSS software (version 16.0) through a Student's t-test. Data were presented as mean ± standard deviation and P value cutoffs <0.05 were used to identify statistically significant data.

 Results



Cancer stem-like cell formation and ibuprofen treatment

CSLCs were derived from gastric cell line MKN-45 during 2 weeks after cells seeded on nonadhesive agarose-coated plastic dishes. This culture condition was prevented MKN-45 from adhering to the dish surface and led to producing spheroid body-forming cells, which also known as CSLCs [Figure 1]. Consequently, the produced CSLCs were treated with ibuprofen.{Figure 1}

Bioinformatic analysis to identify microRNAs that target Cox-1/2

Our bioinformatic analysis showed that hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669 targeted both Cox-1 and Cox-2 mRNAs (P ≤ 0.05). Results were shown in [Table 2]. Finally, hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669 were selected to investigate the effect of ibuprofen on the expression of miRNAs in MKN-45-derived CSLCs.{Table 2}

MicroRNA expression analysis in ibuprofen-treated cancer stem-like cells in comparison to untreated samples

qRT-PCR was performed to evaluate the effect of ibuprofen on the expression of hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669 in CSLCs [Figure 2]. The qRT-PCR results indicated that hsa-mir-16-5p and hsa-mir-4669 were upregulated 2.34 and 9.47 folds, respectively, whereas hsa-mir-483-5p (2.08 folds) underexpressed in ibuprofen-treated CSLCs relative to untreated cells [Figure 3].{Figure 2}{Figure 3}

Signaling pathway analysis of hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669

We used DIANA tools to identify signaling pathways influencing by hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669. Results indicated that hsa-mir-16-5p was associated with PI3K-Akt, p53, mammalian target of rapamycin (mTOR), transforming growth factor-beta (TGF-β) and ErbB signaling pathways, cell cycle, small-cell lung cancer, and prostate cancer. Moreover, we found that hsa-mir-483-5p is related to PI3K-Akt signaling pathway, focal adhesion, extracellular matrix (ECM)-receptor interaction, gap junction, and prostate cancer. In addition, bioinformatic investigation showed that hsa-mir-4669 probably modulates phosphatidylinositol signaling system, insulin signal transduction, and chronic myeloid leukemia.

 Discussion



miRNAs are a class of ~22-nt endogenous noncoding small RNAs that are associated with the development and progression of a variety of human cancers such as gastric cancer.[3] It has been found that the expression profile of miRNAs in tumor initiation cells, CSCs, has differed from that in noncancerous cells.[4] In addition, Chemical compounds, such as Ibuprofen, by targeting of effectors-mediated signaling pathways, influence on proliferation, differentiation and apoptosis of CSCs, and thus act as anti-cancer drug.[15] Ibuprofen is a member of nonsteroidal anti-inflammatory drugs that used as a potential antitumor drug.[8] The effect of ibuprofen was investigated on cell proliferation, apoptosis, angiogenesis, and expression of stemness marker genes.[14] Mechanistically, it showed that ibuprofen works through inhibiting the COX enzymes.[13] Overexpression of COX-2 in cancer cell lines increased their ability to invade surrounding tissues. In addition, it promoted cell invasion in gastric cancer.[12] Despite these studies, the exact mechanism of action of ibuprofen is unknown, and therefore, this study was designed to detect miRNAs that target Cox-1/ 2 transcript and to find the signaling pathways associated with the identified miRNAs. In this regard, our bioinformatic investigation indicated that hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669 posttranscriptionally modulate the expression of Cox-1/ 2 genes. Therefore, we evaluated the effect of ibuprofen on the expression of hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669 in MKN-45-derived CSLCs using qRT-PCR. The results of the quantitative gene expression analysis indicated that ibuprofen induced the expression of hsa-mir-16-5p and hsa-mir-4669, while it reduced the expression of hsa-mir-483-5p in CSLCs. Interestingly, the highest level of upregulation was seen for hsa-mir-4669 in ibuprofen-treated CSLCs (9.47 folds) as compared with untreated controls. A previous study by Liu et al. demonstrated that miR-4669 is one of the miRNAs that upregulated in the spheroid-forming cells as compared to the parental cell line MKN-45.[4] Therefore, it is proposed that hsa-mir-4669 acts as a tumor suppressor miRNA. Collectively, our bioinformatic data and gene expression analysis indicated that ibuprofen may inhibit cancer initiation and progression through upregulation of hsa-mir-4669 and consequently downregulating the Cox-1/ 2 transcript. Moreover, bioinformatic investigation demonstrated that hsa-mir-4669 is related to regulation of the phosphatidylinositol and insulin signal transduction, and therefore, it proposes that hsa-mir-4669 may affect the CSLCs characteristics and functions through regulating these signaling pathways.

hsa-mir-16-5p is a tumor suppressor miRNA and it has been reported that hsa-mir-16-5p was downregulated in non-small cell lung cancer.[21] Our bioinformatic evaluation also showed that hsa-mir-16-5p is a potential regulator of Cox-1/ 2 expression, and thus, the effect of ibuprofen was assessed on the expression of mir-16-5p in the MKN-45-derived CSLCs. Results revealed that the expression of hsa-mir-16-5p was also upregulated 2.34 folds in ibuprofen-treated CSLCs as compared to untreated cells. Therefore, these data propose that ibuprofen probably affects CSLC biology through overexpression of hsa-mir-16-5p and downregulating the Cox-1/ 2 transcript. In addition, DIANA tools indicated that hsa-mir-16-5p modulates PI3K-Akt, p53, mTOR, TGF-β, and ErbB signaling pathways, and therefore, it can suggest that hsa-mir-16-5p probably influences on CSLC characteristics through regulating these signaling pathways.

In the present study, we also found that hsa-mir-483-5p posttranscriptionally regulated the expression of Cox-1/ 2 genes. Functionally, a previous study indicated that hsa-mir-483-5p acts as tumor suppressor miRNAs and reduced cell proliferation in adrenocortical carcinoma cell lines.[22] Hsa-mir-483-5p also suppresses cell proliferation through induction of G0/G1 arrest in glioma cells.[23] Moreover, upregulation of hsa-mir-483-5p is associated with poorer disease-specific survival.[24] According to the results of these studies, we expected that ibuprofen treatment upregulates hsa-mir-483-5p expression in CSLCs, but our findings revealed that the expression of hsa-mir-483-5p was decreased 2.08 folds in ibuprofen-treated CSLCs as compared to untreated samples. In addition, bioinformatic investigation showed that hsa-mir-483-5p is associated with PI3K-Akt signaling pathway, focal adhesion, gap junction, and ECM-receptor interaction. Therefore, we propose that ibuprofen may influence on the CSLC communications and interactions through downregulation of hsa-mir-483-5p.

 Conclusions



We deduce that ibuprofen may reduce the risk of gastric cancer through modulating the expression of hsa-mir-16-5p, hsa-mir-483-5p, and hsa-mir-4669 and thus by regulating Cox-1/ 2 expression and signaling pathways that are involved in cancer initiation and progression. However, the exact mechanism and the role of these regulations are needed to be elucidated in further study.

Acknowledgments

We would like to acknowledge and extend our gratitude to Diba Farahani for her contribution in completing this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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