The Effect of miR-34a-5p and miR-145-5p Ectopic Expression on Cell Proliferation and Target Gene Expression in the MDA-MB-231 Cell Line

İlknur SUER; Murat KAYA; Emre ÖZGÜR

doi:10.4274/nkmj.galenos.2021.770512

ABSTRACT

Conclusion:

miR-34a-5p and miR-145-5p may have an impact on the breast cancer cell line MDA-MB-231 proliferation and EMT mechanism. At the same time, according to our study results, it was revealed that E2F1, MEK1 and CDK4 genes, whose expression level decreased after transfection of mimic miR-34a-5p, could be targeted by miR-34a-5p in breast cancer, and that the expression level of KLF4, which decreased as a result of mimic miR-145-5p transfection, could be the target.

Results:

The proliferation of the miR-34a-5p or miR-145-5p transfected cells decreased compared to the control groups. The expression of E2F transcription factor 1 (E2F1) (p=0.009), mitogen activated protein kinase 1 (MEK1) (p=0.001) and cyclin dependent kinase 4 (CDK4) (p=0.005) genes, which were among the genes targeted by miR-34a-5p, were significantly reduced. EMT markers were significantly changed in miR-34a-5p transfected cells (E-Cad increase p=0.01; Vimentin decrease p=0.008). Kruppel-like factor 4 (KLF4) (p=0.007) targeted miR-145-5p were significantly reduced and EMT markers were significantly changed in miR-145-5p transfected cells (E-Cad increase p=0.0005; Vimentin decrease p=0.006).

Materials and Methods:

Firstly, literature search and in silico analysis were performed to detect possible target genes of miR-34a-5p and miR-145-5p, which are known to be tumor suppressors. Mimic miR-34a-5p and miR-145-5p were transfected to the breast cancer cell line MDA-MB-231. Deregulated genes were investigated by the quantitative real-time polymerase chain reaction compared to control cells. Also, the effect of these miRNAs on proliferation was determined using the Water Soluble Tetrazolium Salt-8 method. Finally, the expressions of epithelial mesenchymal transition (EMT) markers, which are known to be important in the metastatic process, are examined.

Aim:

It was aimed to investigate the effect of miR-34a-5p and miR-145-5p on breast cancer cell line MDA-MB-231 and to determine the expression of target genes of these microRNAs (miRNAs).

Keywords:

MDA-MB-231, breast cancer, miRNA, miR-34a-5p, miR-145-5p

INTRODUCTION

Breast cancer is the most common type of cancer in women, and it occupies a large place among cancer-related deaths in women all over the world, especially among women in underdeveloped countries¹. MicroRNAs (miRNAs) are single-stranded, non-protein-coding RNAs containing approximately 19-24 nucleotides and repress their target mRNAs. Depending on the functions of target genes, miRNAs are biomolecules that play a vital role in various cellular processes such as differentiation, proliferation and apoptosis, and their deregulation is effective in the development of many types of cancer². The miR-34 family, which was characterized in 2007, has three members, miR-34a-5p, miR-34b and miR-34c. The miR-34 family of tumor suppressor miRNAs is known to target many cancer-related genes, including p53. It has been reported that these miRNA family members are deregulated in a large number of cancer types³. One of the other most important tumor suppressor miRNA is miR-145-5p. The expression level of miR-145-5p has been shown to be frequently decreased in many cancers, including colorectal cancer, hematological cancers, prostate cancer, ovarian cancer, B-cell tumor, and breast cancer⁴. Undoubtedly, it is extremely important in terms of the molecular mechanism of cancer to learn that this decrease in the expression levels of miR-34a-5p and miR-145-5p, whose expression levels are found to be decreased in many cancers, contribute to the cancerization process by increasing their expression of target genes^4,5. However, studies on miR-34a-5p and miR-145-5p miRNAs targeting which genes in breast cancer and through which pathways are effective in the cancer process are limited.

Within the scope of the study, it was aimed to investigate the effects of related miRNAs on cell proliferation by transfection of mimic miR-145-5p and miR-34a-5p to MDA-MB-231 cells, which are triple negative breast cancer cell line.

Afterwards, the expression levels of one of the target genes of miR-34a-5p, Mouse double minute 4 (MDM4), E2F1, Insulin-like growth factor 1 receptor (IGF1R), MEK1, CDK4, cluster of differentiation 44 (CD44), eukaryotic translation initiation factor 4E (EIF4E) targeted by neurogenic locus notch homolog protein 2 (NOTCH2) and target genes of miR-145-5p, CDK4, Cyclin-dependent kinase 6 (CDK6), Epidermal growth factor receptor (EGFR), SRY-Box Transcription Factor (SOX2), KLF4, jagged canonical notch ligand 1 (JAG1) genes were investigated and it was aimed to investigate whether the expression levels of related genes are correlated with miR-34a-5p and miR-145-5p. Finally, it was aimed to investigate the relationship of miR-34a-5p and miR-145-5p with metastasis by looking at the expression of epithelial mesenchymal transition (EMT) markers that are known to contribute to the metastatic process.

GİRİŞ

Meme kanseri kadınlarda en sık görülen kanser türüdür ve özellikle az gelişmiş ülkelerdeki kadınlar başta olmak üzere tüm dünyada kanserle ilişkili kadın ölümleri arasında büyük bir yer tutmaktadır¹. MikroRNA’lar (miRNA) yaklaşık 19-24 nükleotid içeren, tek zincirli, protein kodlamayan ve hedef mRNA’larını baskılayan RNA’lardır. Hedef genlerin fonksiyonlarına bağlı olarak miRNA’lar hücrede farklılaşma, proliferasyon ve apoptoz gibi hayati derecede öneme sahip olan çeşitli hücresel süreçte rol oynayan biyomoleküllerdir ve deregülasyonları birçok kanser çeşidinin gelişmesinde etkili olmaktadır². 2007 yılında karakterize edilen miR-34 ailesinin miR-34a-5p, miR-34b ve miR-34c olmak üzere üç tane üyesi bulunmaktadır. Tümör baskılayıcı miRNA’lardan olan miR-34 ailesinin p53 dahil çok sayıda kanser ile ilişkili geni hedeflediği bilinmektedir. Oldukça fazla sayıda kanser türünde bu miRNA aile üyelerinin deregüle olduğu bildirilmiştir³. Diğer en önemli tümör baskılayıcı özellik sergileyen miRNA’lardan birisi de miR-145-5p’dir. Kolorektal kanser, hematolojik kanserler, prostat kanseri, over kanseri, B-hücreli tümör ve meme kanserinin de dahil olduğu çok sayıdaki kanserde miR-145-5p’nin ifade seviyesinin sıklıkla azaldığı gösterilmiştir⁴. Birçok kanserde ifade seviyeleri azalmış olarak tespit edilen miR-34a-5p ve miR-145-5p’nin ifadelerindeki bu azalmanın hangi hedef genlerin ekspresyonlarının artmasıyla kanserleşme sürecine katkı sağladığını öğrenmek şüphesiz kanserin moleküler mekanizması açısından son derece önemlidir^4,5. Buna rağmen miR-34a-5p ve miR-145-5p miRNA’larının, meme kanserinde hangi genleri hedefleyerek hangi yolaklar üzerinden kanser sürecinde etkili olduğu ile ilgili çalışmalar sınırlı sayıdadır.

Çalışma kapsamında üçlü negatif meme kanseri hücre hattı olan MDA-MB-231 hücrelerine mimik miR-145-5p ve miR-34a-5p transfeksiyonu yapılarak ilgili miRNA’ların hücre proliferasyonuna etkilerinin araştırılması hedeflenmiştir. Sonrasında ise miR-34a-5p’nin hedef genlerinden mouse double minute 4 (MDM4), E2F transkripsiyon faktör 1 (E2F1), insülin benzeri büyüme faktörü 1 reseptör (IGF1R), mitojen aktive protein kinaz 1 (MEK1), CDK4, cluster of differentiation 44 (CD44), nörogenik lokus notch homolog protein 2’nin (NOTCH2) ve miR-145-5p’nin hedeflediği ökaryotik translasyon başlatma faktörü 4E (EIF4E), CDK4, siklin bağımlı kinaz 6 (CDK6), epidermal büyüme faktörü reseptörü (EGFR), SRY-Box transkripsiyon faktörü (SOX2), KLF4, jagged canonical notch ligand 1 (JAG1) genlerinin ekspresyon seviyeleri araştırılarak ilgili genlerin ifade düzeylerinin miR-34a-5p ve miR-145-5p ile korele olup olmadığının araştırılması hedeflenmiştir. Son olarak metastatik sürece katkı sağladığı bilinen epitelyal mezenkimal geçiş (EMT) belirteçlerinin ekspresyonuna bakılarak miR-34a-5p ve miR-145-5p’nin metastaz ile ilişkisinin araştırılması hedeflenmiştir.

MATERIALS AND METHODS

Cell Culture and miRNA Transfection

For the functional study of miR-34a-5p and miR-145-5p non-coding RNAs in vitro, MDA-MB-231 breast cancer cell line 1% antibiotic (penicillin/streptomycin) and 10% fetal bovine serum (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) was cultured and replicated in RPMI-1640 (Roswell Park Memorial Institute-1640) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium at 37 °C in a 5% CO₂ incubator.

In our study, in which an in vitro functional experiment of miR-34a-5p and miR-145-5p synthetic mimic (Thermo Fisher Scientific Inc.) was planned separately in replicated cells, an oligonucleotide sequence control miRNA (non-targeting control miRNA, control nt miRNA) (Thermo Fisher Scientific Inc.) that did not target any gene was used (miR-145-5p 5’-GUCCAGUUUUCCCAGGAAUCCCU-3’), non-targeting control miRNA (5’-UCACAACCUCCUAGAAAGAGUAGA-3’), miR-34a-5p (5’-UGGCAGUGUCUUAGCUGGUUGU-3’).

24 hours after seeding the appropriate number of cells (well/300,000 cells) on 6-well plates, transfection of miRNA mimic was performed in the cells. Mimics were transported into the cell with Lipofectamine 2000 reagent (Thermo Fisher Scientific Inc.) by applying the manufacturer’s protocol in transfection. Then, the cells were incubated at 37 °C and 24 hours after transfection, cells transfected with miR-145-5p, miR-34a-5p and cells transfected with non-targeting control miRNA were taken separately in 1000 µL Trizol (MRC Inc.) in eppendorfs and RNA was then brought to -80 °C for isolation.

Isolation of RNA from Cells After Transfection

Total RNA was isolated from cells taken in trizol (MRC Inc.) in accordance with the manufacturer’s protocol, and RNA concentration and purity were measured with the help of NanoDrop ND-2000c (Thermo Fisher Scientific Inc.) spectrophotometer.

Confirmation of miRNA cDNA Synthesis and Transfection by miRNA qRT-PCR

Using TaqMan probes, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of miR-34a-5p and miR-145-5p in cell lines and to check whether the transfection processes were effective. MiRNA cDNA synthesis using total 30 ng RNA was performed using TaqMan MicroRNA reverse transcriptase kit (Applied Bio., Foster City, CA, United States) and miRNA RT primers according to the manufacturer’s protocol. TaqMan assays were used for qRT-PCR, and qRT-PCR was performed on LightCycler^® 480 (Roche) instrument using TaqMan Universal Master Mix (Thermo Fisher Scientific Inc.) kit with TaqMan miRNA probes (Thermo Fisher Scientific Inc.) and RNU43 (control miRNA) probes (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. Experiments were performed in duplicate and 2^-^ΔΔ^Ctmethod was used for relative quantitation analysis.

In Silico Prediction of Target Genes for Expression of miR-34a-5p and miR-145-5p After Transfection

Target prediction tools for prediction of related genes which are the tools such as miRBase (http://www.mirbase.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and miRTargetLink (https://ccb-web.cs.uni-saarland.de/mirtargetlink/) and databases such as Genecards, NCBI, PubMed were used. That the relationship of the gene selected to be investigated selectively in the prediction phase of possible target genes and the related miRNA has not been shown in the MDA-MB-231 breast cancer cell line before, that the expression level of the selected gene in breast cancer has been shown to increase in breast cancer previously in the literature, and that the selected gene is targeted by the relevant miRNA at least in two different cancers were taken into account. The miRTargetLink database was also used to schematize genes with strong interactions of miR-34a-5p and miR-145-5p⁶.

cDNA Synthesis and qRT-PCR Procedures for the Target Genes of miR-34a-5p and miR-145-5p

The cDNA extraction with SCRIPT cDNA synthesis kit (Jena Bioscience) and qPCR SybrMaster (Jena Bioscience) and qRT-PCR processes were performed in accordance with the manufacturer’s protocol, using a total of 1000 ng of RNA obtained from the study group and control group cells.

While the expression levels of MDM4, E2F1, IGF1R, MEK1, CDK4, CD44 and NOTCH2 genes were determined in miR-34a-5p transfected cells, the expression levels of EIF4E, EGFR, SOX2, CDK4, CDK6, JAG1 and KLF4 genes were examined in miR-145-5p transfected cells. The primer sequences of the examined genes are shown in Table 1. qRT-PCR processes were performed on a LightCycler^® 480 (Roche) device.

Investigation of the Effect of miR-34a-5p and miR-145-5p on Cell Proliferation

After cells were transfected with miR-34a-5p and miR-145-5p synthetic mimic and control miRNA (non-targeting control miRNA), whether there was a change in the proliferation of cells were examined using the Cell Proliferation Reagent Water Soluble Tetrazolium Salt-8 (WST-8) (EcoTech Biotechnology) according to the manufacturer’s protocol.

Cells seeded in triplicate at 5000 cells per well on 96-well plates were cultured at 37 °C in a 5% CO₂ incubator. Proliferation changes in cells transfected after 24 hours were measured at the following 24^th, 48^th and 72^nd hours. For measurement, 10 µL of WST-8 (EcoTech Biotechnology) solution was added to each well and cells were incubated at 37 °C for three hours in the dark. Cell viability was evaluated using a plate reader (Thermo Varioskan flash) device capable of measuring absorbance at 450 nm.

Investigation of the Effect of miR-34a-5p and miR-145-5p on Cell Migration with EMT Marker

In order to examine whether the transfected miRNAs contribute to the migration and invasion potential of cells, the expression levels of E-cadherin, N-cadherin, Vimentin genes, which are important EMT markers in epithelial mesenchymal transition, were evaluated by qRT-PCR technique. qRT-PCR processes were performed using qPCR SybrMaster (Jena Bioscience) in accordance with the manufacturer’s protocol.

Statistical Analysis

For normalization of gene expression, β-actin primers were used as internal control and experiments were performed in duplicate. The 2^-^ΔΔ^Ct method was used for the relative quantitation analysis. Statistical analyzes were performed using Student’s t-test and data were presented as mean±standard deviation. Data with a p value less than 0.05 were considered statistically significant. GraphPad Prism 5 and Statistical Package for the Social Sciences 21 programs were used for graphic drawings.

GEREÇ VE YÖNTEMLER

Hücre Kültürü ve miRNA Transfeksiyonu

miR-34a-5p ve miR-145-5p kodlamayan RNA’ların in vitro ortamda fonksiyonel olarak çalışılması için MDA-MB-231 meme kanseri hücre hattı %1 antibiyotik (penisilin/streptomisin) ve %10 fetal bovin serum (Gibco, Thermo Fisher Scientific Inc, Waltham, MA, ABD) ilave edilen RPMI-1640 (Roswell Park Memorial Institute-1640) (Gibco, Thermo Fisher Scientific, Waltham, MA, ABD) besiyerinde 37 °C’de %5 CO₂’li inkübatörde kültür edilerek çoğaltıldı.

Çoğaltılan hücrelerde ayrı ayrı olacak şekilde miR-34a-5p ve miR-145-5p sentetik mimiğinin (Thermo Fisher Scientific Inc.) in vitro fonksiyonel deneyi planlanan çalışmamızda herhangi bir geni hedeflemeyen bir oligonükleotid dizisi kontrol miRNA (non-targeting kontrol miRNA, kontrol nt miRNA) (Thermo Fisher Scientific Inc.) olarak kullanıldı (miR-145-5p 5’-GUCCAGUUUUCCCAGGAAUCCCU-3’), non-targeting kontrol miRNA (5’-UCACAACCUCCUAGAAAGAGUAGA-3’), miR-34a-5p (5’-UGGCAGUGUCUUAGCUGGUUGU-3’).

6-kuyucuklu platelere uygun sayıda hücre (kuyucuk/300.000 hücre) ekimi yapıldıktan 24 saat sonra, hücrelerde miRNA mimiğinin transfeksiyon işlemleri gerçekleştirildi. Transfeksiyonda üretici firmanın protokolü uygulanarak lipofektamin 2000 reagent (Thermo Fisher Scientific Inc.) ile mimiklerin hücre içerisine taşınması sağlandı. Ardından hücreler 37 °C’de inkübasyona bırakılarak transfeksiyondan 24 saat sonra miR-145-5p, miR-34a-5p transfekte edilen hücreler ile non-targeting kontrol miRNA transfekte edilen hücreler ayrı ayrı ependorflarda 1000 µL trizol (MRC Inc.) içerisine alınarak daha sonra RNA izole edilmek üzere -80 °C’ye kaldırıldı.

Transfeksiyon Sonrası Hücrelerden RNA İzole Edilmesi

Trizol (MRC Inc.) içerisine alınan hücrelerden üretici firmanın protokolüne uygun şekilde total RNA izolasyonu gerçekleştirilerek, RNA konsantrasyon ve saflıkları NanoDrop ND-2000c (Thermo Fisher Scientific Inc.) spektrofotometre yardımıyla ölçüldü.

miRNA cDNA Sentezi ve miRNA qRT-PZR ile Transfeksiyon Doğrulanması

TaqMan probları kullanılarak miR-34a-5p ile miR-145-5p’nin hücre hatlarındaki ifade düzeylerinin tespiti ve transfeksiyon işlemlerinin etkili bir şekilde olup olmadığının kontrol edilmesi amacıyla kantitatif gerçek zamanlı polimeraz zincir reaksiyonu (qRT-PZR) yapıldı. Total 30 ng RNA kullanılarak miRNA cDNA sentezi, TaqMan MicroRNA reverse transcriptase kit (Applied Bio., Foster City, CA, İngiltere) ve miRNA RT primerleri kullanılarak üretici firmanın protokolüne uygun olarak yapıldı. qRT-PZR için TaqMan assayleri kullanılmış olup, qRT-PZR işlemleri LightCycler^® 480 (Roche) cihazında ve TaqMan Universal Master Mix (Thermo Fisher Scientific Inc.) kitiyle TaqMan miRNA probları (Thermo Fisher Scientific Inc.) ve RNU43 (kontrol miRNA) probları (Thermo Fisher Scientific Inc.) kullanılarak üretici firmanın protokolüne göre gerçekleştirildi. Deneyler iki tekrarlı olacak şekilde yapılmış olup rölatif kantitasyon analizi için 2^-^ΔΔ^Ctmetodu kullanıldı.

miR-34a-5p ve miR-145-5p Transfeksiyonu Sonrası İfadesi İncelenecek Hedef Genlerin İn Silico Tahminle Belirlenmesi

İlgili genlerin tahmininde hedef tahmin araçları olan miRBase (http://www.mirbase.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/), miRTargetLink (https://ccb-web.cs.uni-saarland.de/mirtargetlink/) gibi araçların kullanımının yanı sıra Genecards, NCBI, PubMed gibi veritabanlarından da yararlanıldı. Olası hedef genlerin tahmini aşamasında seçici davranılarak araştırılmak üzere seçilen genin ilgili miRNA ile ilişkisinin MDA-MB-231 meme kanseri hücre hattında daha önceden gösterilmemiş olması, seçilen genin meme kanserinde ekspresyon seviyesinin arttığının daha önce literatürde gösterilmiş olması ve seçilen genin ilgili miRNA tarafından hedeflendiğinin en az iki farklı kanserde gösterilmiş olması gibi çeşitli kriterler dikkate alındı. Ayrıca miRTargetLink veritabanı miR-34a-5p ve miR-145-5p’nin güçlü etkileşim gösterdiği genlerin şematize edilmesinde de kullanıldı⁶.

miR-34a-5p ve miR-145-5p’nin Hedef Genleri için cDNA Sentezi ve qRT-PZR İşlemleri

Çalışma grubu ile kontrol grubu hücrelerden elde edilen RNA’dan total 1000 ng kullanılacak şekilde, SCRIPT cDNA synthesis kit (Jena Bioscience) ile cDNA eldesi ve qPZR SybrMaster (Jena Bioscience) ile qRT-PZR işlemleri üretici firmanın protokolüne uygun olarak gerçekleştirildi. miR-34a-5p transfekte edilen hücrelerde MDM4, E2F1, IGF1R, MEK1, CDK4, CD44 ve NOTCH2 genlerinin ifade düzeyleri belirlenirken, miR-145-5p transfekte edilen hücrelerde ise EIF4E, EGFR, SOX2, CDK4, CDK6, JAG1 ve KLF4 genlerinin ifade düzeyleri incelendi. İncelenen genlere ait primer dizileri Tablo 1’de gösterilmiştir. qRT-PZR işlemleri LightCycler^® 480 (Roche) cihazında gerçekleştirildi.

Hücrelerin Proliferasyonuna miR-34a-5p ve miR-145-5p’nin Etkisinin Araştırılması

Hücreler miR-34a-5p ve miR-145-5p sentetik mimiği ve kontrol miRNA (non-targeting kontrol miRNA) ile transfekte edildikten sonra hücrelerin proliferasyonunda değişim olup olmadığı Cell Proliferation Reagent WST-8 (Water Soluble Tetrazolium Salt-8) (EcoTech Biotechnology) ile üretici firmanın protokolü kullanılarak incelendi. 96-kuyucuklu platelere kuyucuk başına 5000 hücre düşecek şekilde üç tekrarlı olarak ekilen hücreler 37 °C’de %5 CO₂’li inkübatörde kültür edildi. 24 saat sonra transfeksiyon uygulaması yapılan hücrelerdeki proliferasyon değişimi, takip eden 24., 48. ve 72. saatlerde ölçüldü. Ölçüm için WST-8 (EcoTech Biotechnology) solüsyonundan her kuyucuğa 10 µL eklendi ve hücreler 37 °C’de üç saat karanlık ortamda inkübe edildi. Hücre canlılığı 450 nm’de absorbans ölçümü yapabilen plak okuyucu (Thermo Varioskan flash) cihaz kullanılarak değerlendirildi.

Hücrelerin Göçüne miR-34a-5p ve miR-145-5p Etkisinin EMT Marker ile Araştırılması

Transfeksiyonu yapılan miRNA’ların hücrelerin migrasyon ve invazyon potansiyeline katkıları olup olmadığını incelemek üzere EMT’de önemli bazı EMT belirteçleri olan E-cadherin, N-cadherin, Vimentin genlerinin ekspresyon seviyeleri qRT-PZR tekniğiyle değerlendirildi. qRT-PZR işlemleri qPZR SybrMaster (Jena Bioscience) kullanılarak üretici firmanın protokolüne uygun olarak gerçekleştirildi.

İstatistiksel Analiz

Gen ekspresyonunda normalizasyon için internal kontrol olarak β-aktin primerleri kullanılmış olup deneyler iki tekrarlı olacak şekilde yapıldı. Rölatif kantitasyon analizi için 2^-^ΔΔ^Ct metodu kullanıldı. İstatistiksel analizler Student’s t-test kullanılarak yapıldı ve veriler ortalama±standart sapma olarak gösterildi. P değeri 0,05’ten küçük olan veriler istatistiksel olarak anlamlı olarak değerlendirildi. Grafik çizimlerinde GraphPad Prism 5 ile Statistical Package for the Social Sciences 21 programlarından yararlanıldı.

RESULTS

Verification of Transfection Procedures

It was confirmed that the transfection occurred significantly for both miRNAs in miR-34a-5p and miR-145-5p transfected cells and control cells (Figure 1).

Effect of miR-34a-5p and miR-145-5p on Expression Level of Possible Target Genes in MDA-MB-231 Cells

As a result of the literature review and in silico studies, the expression of E2F1, MEK1 and CDK4 genes, which are among the genes that are predicted to be strongly associated with miR-34a-5p, were significantly suppressed in miR-34a-5p transfected cells compared to the control group (respectively p=0.009; p=0.001; p=0.005) and KLF4 gene, which is among the genes predicted to be strongly associated with miR-145-5p, was significantly suppressed in miR-145-5p transfected cells compared to the control group (p=0.007) (Figure 2, 3, 4, 5).

Effect of miR-34a-5p and miR-145-5p on Proliferation and Migration of MDA-MB-231 Cells

It was determined that proliferation in MDA-MB-231 cells transfected with both miR-34a-5p and miR-145-5p decreased in correlation with the literature (Figures 6, 7).

As a result of expression evaluation of epithelial-mesenchymal cell markers with qRT-PCR to understand whether selected miRNAs have any regulatory role in cell migration, it was determined that E-Cad expression level was increased significantly (p=0.01; p=0.0005 respectively), Vimentin expression level was decreased significantly (p=0.008;p=0.006, respectively), and N-Cad expression level was not changed significantly (p>0.05) (Figure 8, 9) in cells transfected with miR-34a-5p and miR-145-5p compared to control cells.

As a result, it was determined that miR-34a-5p and miR-145-5p contributed to the suppression of cell migration in MDA-MB-231 breast cancer cells by regulating through EMT markers.

DISCUSSION

MiRNAs, which can act as oncogene (onco-mir) or tumor suppressor (TSmir) according to the genes they target, are among the most interesting research topics in recent years¹⁹. Abnormal expression changes in miRNAs may be involved in the cancer process by changing the expression of many genes targeted by the relevant miRNAs. The fact that miR-34 was the first miRNA to be tested in phase 1 in relation to the use of miRNAs in therapy indicates that miR-34 may be one of the most promising miRNAs in the future²⁰.

Studies with miR-34a-5p localized at 1p36.22 have shown that ectopic expression of miR-34a-5p directly or indirectly causes a decrease in the expression of many proto-oncogenes, resulting in decreased cancer cell proliferation and elimination of metastasis⁵. The fact that p53, which is shown as the guardian of the genome, and its homologous p63 and p73 genes are controlled by miR-145-5p, and many important genes are targeted by this miRNA, indicates that miR-145-5p can have critical importance in early diagnosis and possible miRNA-based therapies⁴. Determining which genes control various cellular processes by regulating miRNAs is extremely important for understanding diseases at the molecular level²¹. miRNAs are involved in regulating the expression of most protein-coding genes at the post-transcriptional stage. Therefore, many bioinformatics tools have been created to predict the interaction between miRNA and target mRNA. Supporting in vitro and in vivo predictions based on base pairing between miRNA and mRNA in silico may be important for the development and progression of many diseases, especially cancer, and the development of biomarkers for these diseases². Figure 2 and Figure 4 show that miR-34a-5p and miR-145-5p can target hundreds of genes in silico. In the literature, it has been shown that the expression levels of miR-34a-5p and miR-145-5p are considerably reduced in breast cancer patient tissue samples and MDA-MB-231 cells, as in many other cancer types^22,23. Therefore, according to the results of our transfection study to investigate the effects of miR-34a-5p and miR-145-5p on MDA-MB-231 cells, miR-34a-5p and miR-145-5p have been found to have a significant effect on MDA-MB-231 cell proliferation. The effects of miR-34a-5p and miR-145-5p on cell proliferation may be related to decreased expression of various genes targeted by these miRNAs. The KLF4 gene is a gene that has been found to be highly expressed in more than 70% of breast cancers and has been reported to function as an oncogene²⁴. The E2F1 gene is also a gene with increased expression in breast cancer and associated with breast cancer metastasis²⁵. MEK1 and CDK4 genes, whose expression levels have been found to be increased in many cancers, including breast cancer, are important oncogenes that play critical roles in apoptosis, migration, invasion and cell proliferation processes^26,27. miR-34a-5p of the E2F1, MEK1 and CDK4 genes; despite the fact that KLF4 has been shown to be effective in various cancer processes by being targeted by miR-145-5p, there are no studies in the literature on this subject in breast cancer^28-32. In our study to fill this gap in the literature, it was determined that E2F1, MEK1, CDK4 and KLF4 gene expression levels were significantly decreased in MDA-MB-231 cells transfected with mimic miR-34a-5p and mimic miR-145-5p.

Study Limitations

Considering our study results and the information in the literature together, it can be predicted that miR-34a-5p may contribute to the cancer process by targeting E2F1, MEK1 and CDK4 genes, and miR-145-5p targeting KLF4 in breast cancer. Among the limitations of the study; the fact that the research was carried out in cell line, it was not confirmed in the tissue, and the genes whose expression level was examined were examined only at the mRNA level. It is obvious that more meaningful results can be obtained if the findings obtained in our study at the level of RNA expression are supported by methods such as Western-Blot and luciferase activity determination in new studies on the subject.

It was seen that CD44³³, NOTCH2³⁴, MDM4³⁵, IGF1R³⁶ genes, which have been shown as targets of miR-34a-5p both in silico and in vivo, and similarly the expression levels of EIF4E³⁷, EGFR³⁸, SOX2²¹, CDK4³⁷, CDK6³⁹, JAG1⁴⁰ genes which have been shown as targets of miR-145-5p did not have a statistically significant change in MDA-MB-231 cells in which transfection of miRNA mimics was carried out in our study.

TARTIŞMA

Hedefledikleri genlere göre onkogen (onko-mir) ya da tümör baskılayıcı (TSmir) olarak rol oynayabilen miRNA’lar son yılların en fazla ilgi duyulan araştırma konuları arasında yer almaktadır¹⁹. miRNA’lardaki anormal ifade değişimleri, ilgili miRNA’ların hedeflediği çok sayıdaki genin ekspresyonunu değiştirerek kanser sürecine dahil olabilmektedir. Tedavide miRNA’ların kullanımı ile ilişkili olarak Faz 1 denemesi yapılan ilk miRNA’nın miR-34 olması, miR-34’ün gelecekte umut vadeden miRNA’ların başında gelebileceğini göstermektedir²⁰. 1p36.22’de lokalize olan miR-34a-5p ile yapılan çalışmalarda miR-34a-5p ektopik ekspresyonunun direkt veya dolaylı yoldan birçok proto onkogenin ifadesinde düşüşe neden olduğu ve sonuçta kanser hücre proliferasyonunun azalmasını ve metastazın ortadan kalkmasını sağladığı gösterilmiştir⁵. Genomun gardiyanı diye gösterilen p53 ile bu genin homoloğu p63 ve p73 genlerinin miR-145-5p tarafından kontrol ediliyor olması, yine çok sayıda önemli genin bu miRNA tarafından hedefleniyor olması miR-145-5p’nin erken teşhis ve olası miRNA temelli terapilerde kritik öneme sahip olabileceğini göstermektedir⁴.

miRNA’ların hangi genleri regüle ederek çeşitli hücresel süreçleri kontrol ettiğinin belirlenmesi, hastalıkların moleküler düzeyde anlaşılması açısından son derece önemlidir²¹. miRNA’lar protein kodlayan genlerin büyük bir kısmının ifadelerinin post-transkripsiyonel aşamada düzenlenmesinde görev almaktadır. Bu nedenle miRNA ile hedef mRNA arasındaki etkileşimi tahmine yönelik çok sayıda biyoinformatik araç oluşturulmuştur. İn silico olarak miRNA-mRNA arasındaki baz eşleşmesine dayalı oluşturulan tahminlerin in vitro ve in vivo olarak da desteklenmesi kanser başta olmak üzere birçok hastalığın gelişimi, ilerlemesi ve bu hastalıklara yönelik biyobelirteçlerin geliştirilmesi bakımından önemli olabilir². İn silico olarak miR-34a-5p’nin ve miR-145-5p’nin yüzlerce geni hedefleyebileceği Şekil 2 ve Şekil 4’te gösterilmiştir. Literatürde diğer birçok kanser türünde olduğu gibi meme kanseri hasta doku örneklerinde ve MDA-MB-231 hücrelerinde miR-34a-5p ve miR-145-5p’nin ifade seviyelerinin oldukça azalmış durumda olduğu gösterilmiştir^22,23. Bu nedenle miR-34a-5p ve miR-145-5p’nin MDA-MB-231 hücreleri üzerindeki etkilerini araştırmak üzere yaptığımız transfeksiyon çalışmamızın sonuçlarına göre miR-34a-5p’nin ve miR-145-5p’nin MDA-MB-231 hücre proliferasyonu üzerinde belirgin şekilde etkili olduğu ortaya çıkmıştır. miR-34a-5p ve miR-145-5p’nin hücre proliferasyonu üzerindeki etkileri, bu miRNA’ların hedefledikleri çeşitli genlerin ekspresyonlarının azalmasıyla ilişkili olabilir. KLF4 geni, meme kanserlerinin %70’inden fazlasında yüksek oranda ifade edildiği tespit edilen ve onkogen olarak işlev gördüğü bildirilen bir gendir²⁴. E2F1 geni de yine meme kanserinde ekspresyon seviyesi artmış olarak tespit edilen ve meme kanseri metastazı ile ilişkilendirilmiş bir gendir²⁵. Meme kanseri de dahil çok sayıda kanserde ifade seviyesi artmış olduğu tespit edilen MEK1 ve CDK4 genleri apoptoz, migrasyon, invazyon ve hücre proliferasyon süreçlerinde kritik görevler üstlenen önemli onkogenlerdendir^26,27. E2F1, MEK1 ve CDK4 genlerinin miR-34a-5p; KLF4’ün ise miR-145-5p tarafından hedeflenerek çeşitli kanser sürecinde etkili olduğu yapılan farklı çalışmalarla gösterilmiş olmasına rağmen meme kanserinde bu konu ile ilgili literatürde herhangi bir çalışma bulunmamaktadır^28-32. Literatürdeki bu boşluğun doldurulması amacı ile yaptığımız çalışmada E2F1, MEK1, CDK4 ve KLF4 gen ifade düzeylerinin mimik miR-34a-5p ve mimik miR-145-5p transfeksiyonu yapılan MDA-MB-231 hücrelerinde anlamlı şekilde azalmış olduğu tespit edilmiştir.

Çalışmanın Kısıtlılıkları

Çalışma sonuçlarımız ve literatürdeki bilgiler birlikte düşünüldüğünde meme kanserinde de miR-34a-5p’nin E2F1, MEK1 ve CDK4 genlerini, miR-145-5p’nin ise KLF4’ü hedefleyerek kanser sürecine katkıda bulunabileceği öngörülebilir. Çalışmanın kısıtlılıkları arasında; araştırmanın hücre hattında yapılmış olması, dokuda doğrulanmamış olması ve ifade seviyesi incelenen genlerin sadece mRNA düzeyinde incelenmiş olması sayılabilir. Çalışmamızda RNA ekspresyon düzeyinde elde edilen bulguların konu ile ilgili yapılacak yeni çalışmalarda Western-Blot ve lusiferaz aktivite tayini gibi yöntemler ile desteklenmesi halinde daha anlamlı sonuçların elde edilebileceği ortadadır. Gerek in silico gerekse in vivo ortamda miR-34a-5p’nin hedefi olarak gösterilen CD44³³, NOTCH2³⁴, MDM4³⁵, IGF1R³⁶ genleri ile yine aynı şekilde miR-145-5p’nin hedefi olarak tespit edilmiş EIF4E³⁷, EGFR³⁸, SOX²¹, CDK4³⁷, CDK6³⁹, JAG1⁴⁰ genlerinin ekspresyon düzeylerinin, çalışmamızda miRNA mimiklerinin transfeksiyonunun yapıldığı MDA-MB-231 hücrelerinde istatistiki olarak anlamlı şekilde değişmediği görülmüştür.

CONCLUSION

The data obtained from our study may be evidence that the miRNA-mRNA pairing created by theoretical prediction or the miRNA-mRNA relationship shown in vivo media in different cancer types may not be seen likewise in all cancer types due to the complex nature of cancer.

Acknowledgement

We would like to thank our esteemed professors, Prof. Dr. Şükrü Palanduz, Prof. Dr. Şükrü Öztürk, Prof. Dr. Kıvanç Çefle and Prof. Dr. Uğur Gezer for the encouragement and support they have shown us in our work.

Ethics

Ethics Committee Approval: Ethics committee approval form was not received because it was studied with cell lines.

Informed Consent: Patient samples were not studied.

Peer-review: Externally and internally peer-reviewed.

Authorship Contributions

Concept-Design: İ.S., M.K., E.Ö., Data Collection or Processing: İ.S., M.K., E.Ö., Analysis or Interpretation: İ.S., M.K., E.Ö., Literature Search: İ.S., M.K., E.Ö., Writing: İ.S., M.K., E.Ö.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

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