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Background: MicroRNAs (miRNAs) are a class of naturally occurring small noncoding RNAs that regulate gene expression, cell growth, differentiation and apoptosis by targeting mRNAs for translational repression or cleavage. Present study is conducted to study miRNAs in breast cancer (BC) and their relation to metastasis, tumor invasion and apoptosis in addition to their association with the ER and PR statuses.
Methods: In the present study, we performed Real Time RT-PCR EvaGreen to identify the miRNA expression level of eight miRNAs and eight of their targeted genes in 40 breast cancer samples and their adjacent non-neoplastic tissues. The expression levels of each miRNA relative to U6 RNA were determined using the 2-Î”CT method. Also, we evaluate miRNA expression profiles of the BC and the corresponding ANT.
Results: We found suppression in the miRNAs tumor suppressor (miR-17-5p, miR-335 and miR-126) with the suppression of TIMP-1, TIMP-3, TMP1, PDCD4 and cyclin D1 genes. There was over expression of certain oncogenic miRNAs (miR-21, miR-155, miR-10b and miR-373) with over-expression of MMP-2, MMP-9, and VEGF genes.
Conclusion: Tumor size, histological grade, and the presence of lymph node or distant metastases alone, or in combination with each other, enable the identification of individuals who are at the increased risk of dying owing to BC but they may benefit also from aggressive treatment.
Breast cancer (BC) is the leading cause of cancer related death in women . Over the past years, the worldwide incidence of BC has nearly doubled. More than one million new breast cancer cases occur worldwide annually, with nearly half million cases in developed countries . As part of this situation, BC has become a serious threat to the health of women, with noticeably increased mortality. In Egypt, it is the most common malignancy among Egyptian females accounting about 37.6% of all malignancies [2,3]. Recently, the importance of low-molecular-weight (LMW) RNAs, such as small interfering RNAs (siRNAs) and small nuclear RNAs (snRNAs), has been outlined in different studies. In particular, the importance of a new class of small RNA, miRNAs, has been highlighted. Each miRNA is thought to regulate multiple genes, and as hundreds of miRNA genes are predicted to be present in higher eukaryotes, the potential regulatory circuitry afforded by them is enormous.
MiRNAs are 20~25 nucleotide, the smallest, functional non-coding RNA, that plays important roles in post-transcriptional regulation . There may be thousand of miRNA genes in the human genome, transcribed by RNA polymerase as longer primary-miRNA molecules, then processed in the nucleus forming pre-miRNAs [4,5]. These pre-miRNAs transported from the nucleus to the cytoplasm for further processing [6,7].
During oncogenesis, dysregulated or dysfunctional miRNA can result in increased translation of oncoprotein and/or decreased translation of tumor suppressor protein . By binding to the 3â€² UTR region of targeted genes, miRNA can rapidly inhibit the translation of the mRNA transcript and subsequently, through formation of RNA-induced silencing complex, cause degradation of the transcript . MiRNA can promote the degradation of the targeted mRNA . The quantitative and qualitative (mutational) changes in miRNA and their target binding sites can promote the development and progression of tumors [8, 11-13]. MiRNA profiling studies have revealed their differential expression in various carcinomas compared to that in normal tissue counterparts [12,14] and have further been linked to the repression of tumor suppressor genes or the upregulation of oncogenes at the protein product level [15-18].
Several miRNAs are associated with breast cancer, miR-155 is up-regulated in breast cancer and may act as an oncogene [19, 20]. Up-regulation of miR-373 and miR-520c promotes metastasis. The increased expression of gene encoding miR-10b can promote tumor invasion . MiR-21 up-regulated in breast cancer causing down-regulation in programmed cell death 4 and tropomyosin 1 genes [21, 22]. We investigated the role of microRNA in the initiation and progression of Egyptian breast cancer.
Materials and methods:
This study was conducted on 40 breast cancer cases and their matched adjacent non-neoplastic tissues collected from National Cancer Institute, Cairo University during May 2007 to August 2008. The clinico-pathological features of the participants are shown in table (1). All involved patients gave a written informed consent. All practical work was done in collage of pharmacy, pharmacology department, King Saud University, Saudi Arabia.
Nucleic acid extraction and reverse transcription step:
The total RNA and genomic DNA were isolated from 100mg of frozen tissue with TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The quality and quantity of the isolated RNA and DNA were analyzed using UV spectrophotometer. Total RNA (700ug) was loaded to the microRNA isolation column (Qiagene, Germany) for isolation of low molecular weight (LMW) RNA following the manufacturer's protocol. One microgram aliquot of DNase-treated total RNA and LMW RNA were reverse transcribed to cDNA using antisense of gene-specific primers, U6 and thermoscript, thermostable reverse transcriptase (Invitrogen). One microgram RNA was incubated with 1.5µl of a cocktail containing 10mM each of the antisense gene specific and U6 primers. The reaction was denatured at 800C for 5 min, incubated for 5 min at 600C to anneal the primers, followed by cooling to room temperature and the remaining reagents [5x buffer, dNTPs, DTT, RNase inhibitor, Thermoscript] were added as specified in the Thermoscript protocol and the reaction proceeded for 60 min at 450C. Finally, reverse transcriptase was inactivated by incubating the reaction at 85°C for 5 min.
MiRNA expression by EvaGreen Real-Time PCR:
The expression of the miRNA precursors was determined by using real-time quantitative PCR . Master mix contained 0.5 ul of 10x PCR buffer, 0.7ul of 25mM MgCl2, 0.1µl of 12.5mM, 0.5 Eva Green (Jena Bioscience, Germany) dNTPs, 0.01ul UNG, 0.5µl of DNA Taq polymerase, 0.5µl of dilute cDNA (1:50) and completed with water to 3 ul. Three microliters of the master mix containing all of the reaction components except the primers was dispensed into a 96-well real-time PCR plate (Applied Biosystems, 7500 ). A 2µM of each pair of primers listed in table 2 was stored in 12-well PCR strip tubes. Each primer (2µl) was dispensed into duplicate wells of the 96-well plate. All the reactions were run in triplicate and included no template and no reverse transcription controls for each miRNA. The reactions were amplified for 15s at 950C and 1 min at 600C for 40 cycles. Melting curve and agarose gel electrophoresis analysis following amplification, melting curve analysis was performed to verify the correct product according to its specific melting temperature (Tm). Amplification plots and Tm values were routinely analyzed to confirm the specificities of the amplicons for EvaGreen PCR amplification. Real-time PCR is a sensitive and reproducible gene expression quantitation technique which is now being used to profile miRNA expression in cells and tissues. To correct for systematic variables such as amount of starting template, RNA quality and enzymatic efficiencies, the data is commonly normalized to a universal endogenous control gene, which ideally, is stably-expressed across the test sample set. The expression of each miRNA relative to U6 RNA was determined using the 2-Î”CT where Î”CT = (CTmiRNA-CTU6RNA) relative gene expression was multiplied by 105 in order to simplify the presentation of the data.
Eva-Green real time PCR for target genes expression:
Real-time PCR was performed on an Applied Biosystems 7500 to detect the expression of TIMP1, TIMP3, MMp2, MMp9, TPM1, PDCD4, VEGF, and cyclin-D1 genes. All reactions were run in triplicate and included no template and no reverse transcription controls for each gene. The reactions were amplified for 15 s at 950C and 1 min at 600C for 40 cycles. The thermal denaturation protocol (dissociation curve) was run at the end of the PCR. The expression of each gene relative to GAPDH was determined using the 2 -Î”CT.
Melting Curve and Agarose Gel Electrophoresis Analysis
Following amplification, melting curve analysis was performed to verify the specific product according to its specific melting temperature (Tm). The results were analyzed by the melting curve analysis software of Applied biosystem. Amplification plots and Tm values were analyzed to confirm the specificities of the amplicons for EvaGreen -based PCR amplification.
Validation of miRNA precursor primers by EvaGreen PCR:
Each pair of primers included in this study was validated on extracted genomic DNA, mouse genomic DNA and no template control reaction. All of the primers worked successfully on genomic DNA by the presence of single peak on the thermal melting curve but not on mouse genomic DNA.
Over-expression of miR-155, mir-10, mir-21 and mir-373 were increased by 25, 26, 75, 15 folds respectively in cancer compared to the matched adjacent non-neoplastic tissues. On the other hand, miR-17p, miR-126, miR-335 and miR-30b were down regulated in cancer compared to ANT tissues.
Tumor characteristics and their associations with the expression levels of the miRNAs and genes, are summarized in table 2. According to the tumor size, MiR-10b and MiR-21, mir-155 and mir373 were significantly associated with increasing in tumor size (P < 0.05), whereas, mir30b, mir-126 and mir-335 were equally expressed in tumor tissues in both large and small tumor size. In relation to pathological grade, mir-10b and mir373 were significantly associated with increasing in the pathological grade (P < 0.05). MiR-155 was significantly over-expressed in cancer tissues positive for lymph node metastasis compared to the negative one.
MiRNAs (mir-10, mir-21, mir-155 and mir-373) were insignificantly over-expressed in breast cancer cases having both ERâˆ’/PRâˆ’ whereas there were no difference observed in the down-regulation of mir30b, mir-126 and mir335 in patients with positive or negative ER/PR. The down-regulation of miR-30b was significantly high in cancer 87.5% (7/8) with ER/PR negative patients (P < 0.05).
In cancer tissues having up-regulation for the miR-21, mir-155 and miR-373, TIMP1 , TMP1and TIMP3 were down-regulated in 90%, 90% and 88% respectively. On the other hand, Both MMP2 and MMP9 were up-regulated, programmed cell death 4 (PDCD4) was suppressed.
Over expression of VEGF was observed in 90% (18/20) of cancer tissues having over expression for miR-10b. Cyclin-D1 was down regulated with miR-17-5p.
MicroRNAs (miRNA) are a recently discovered family of short non-protein-coding RNAs that negatively regulate gene expression. Recent studies of miRNAs highlight a requirement for cell viability. Post transcriptional silencing of target genes by miRNAs occurs either by targeting specific cleavage of homologous mRNAs, or by targeting specific inhibition of protein synthesis. Involvement of miRNA in tumor initiation and progression has come under intense study in recent years. In normal cells, miRNAs control normal rates of cellular growth, proliferation, differentiation and apoptosis. Down-regulation of some miRNAs may play a role in the development or progression of cancer since miRNAs inhibit cell cycle progression and drive terminal differentiation . By targeting and controlling the expression of mRNA, miRNAs can control highly complex signal transduction pathways and other biological pathways. The biologic roles of miRNAs in cancer may correlate with diagnosis, prognosis and therapeutic outcome. In this study, 40 breast cancers and their adjacent non-neoplastic tissues were studied for detection the expression of miRNAs and some genes.
MiRNAs regulate a variety of cellular pathways through regulation of expression of multiple target genes . In the present study, BC miRNA expression was quantified by using real-time PCR in which miR-21, miR-373, miR-10b and miR-155 were up-regulated, whereas, miR-17p, miR-335 and miR-126 were down-regulated in tumor tissue compared to ANT. Their expressions in tumor tissues were accompanied by up-regulation in MMP-2, MMP-9 and VEGF and down-regulation in TIMP-1, TIMP-3, TMP-1, PDCD-4 and cyclin-D1.
Tumor-suppressor programmed cell death 4 (PDCD4) targets translation by inhibiting transformation and invasion in cancers. Tropomyosin 1 (TPM1) is a member of the tropomyosin family of proteins, which are associated with actin and serve to stabilize microfilaments .
The miR-21 gene is located on chromosome 17q23.2, which is located within the common fragile site FRA17B. In the present study, we identify increased expression of miR-21 in 75% of cancer as compared to ANT tissues. MiR-21 was significantly up-regulated in BC, similar previous study on the expression of miRNA . In addition, miR-21 over-expression is widespread in many types of cancer, including malignant cholangiocytes , glioblastomas , and malignancies of the colon, lung, pancreas, prostate, and stomach. Our results show that the large tumors size increases the miR-21 expression. These data are consistent with reports indicating that miR-21 expression increased with advanced clinical stage [29, 30] .
MiR-21 functions as an oncogene because it is over-expressed in tumor compared with the normal tissues [31-34] and its suppression inhibits cell growth through activation of apoptosis pathways [31, 32]. In this study, for the up-regulated miR-21, the genes that belonged to the class of tumor suppressor genes (TIMP1, TIMP3 and PDCD4) were affected. In the cancer tissues, the up-regulation of miR-21 has been found to be associated with down-regulation of TIMP1 and TIMP3 genes in 90% and 88% respectively. Similar to our data, others found suppression in the tumor suppressor genes, TIPM1 and TIPM3 that played a role in the malignant phenotype [35, 36]. The down-regulation of TIPM1 and TIPM3 with the up-regulation of miR-21 may confirm that the suppression of miR-21 and can inhibit tumor growth  supporting the notion that miR-21 functions as an oncogene.
MiR-155 is up-regulated in breast cancer, suggesting that it may act as an oncogene . In this study, miR-155 was over-expressed in 80% of cancer compared to 10% in ANT tissues respectively. In similar studies on breast cancer, the miR-155 was up regulated by using microarray technique [19, 39, 40]. Classes of tumor suppressor genes were the targeted genes for the miR-155 over-expression. In our study, MMP2, MMP9 and VEGF genes were found up-regulated.
MiR-30 family members include miR-30a, -30b, -30c, -30d and -30e. They all have the same "seed sequence" in their 5â€² terminuses. Our present work shows that miR-30b is down-regulated in tumor compared to ANT tissues. Similarly, others found that miR-30 family are able to suppress apoptosis . To our knowledge, there has been no publication delineating the relationship between miR-30 and metastasis.
The over expression of miR-10b can promote tumor invasion and is associated with the increase of VEGF that play role in the angiogenesis. In vivo ectopic expression of miR-10b conferred invasive properties on otherwise non-invasive breast cancer cells; miR-10b over-expressing tumors exhibited an invasive behavior and were highly vascularized. In this study, miR-10b was over-expressed by 26 fold in 50% (20/40) of cancer comparing to the ANT tissues. Similarly, Ma and coworkers  found that miR-10b initiates breast cancer invasion and metastasis . In another study, miR-10b was found to be down-regulated in about 50% of the metastatic breast cancers in comparison with normal breast tissue [19, 20]. MiR-10b can promote metastasis in otherwise non-metastatic breast cancer cells . Other results by using miRNA microarray analysis differ from our BC study in which Iorio et al. reported that miR-10b was down-regulated and both miR-21 and miR-155 were up-regulated. This difference might be due to the difference in the different technical methods used.
MiR-373 is metastasis-promoting micro-RNAs . Upregulation of miR-373 promotes metastasis by inhibiting Cyclin-D1 expression. In the cancer tissues, the up-regulation of miR-373 has been found to be associated with down-regulation of TIMP1 and TIMP3 genes in 90% and 88% respectively. Upregulation of miR-373 promotes metastasis by inhibiting the expression of TIMP1 and TIMP3 and increase the expression of MMP2 and MMP9. Similarly, others reported that miR-373 stimulates cancer cell migration and invasion [43, 44]. In vitro studies have sown that over-expressing miR-373 developed metastatic nodules, which were absent in the control cells. MiR-373 was upregulated in cancer, in particular in tumors exhibiting lymph node metastasis Negrini et al. .
In this study, we found down-regulation in the miR-335 and miR-126 in the breast cancer tissues compared to ANT. Similar to our study, Tavazoie et al  found that miR-335 and miR-126 are metastasis-suppressor miRNAs. They found that miR-335 and miR-126 were consistently down-regulated in metastatic foci and restoring the expression of them significantly decreased the number of metastatic foci and significantly associated with poor metastasis-free survival. Thus, these two miRNAs were markers for the likelihood of developing metastasis suggesting the potential use of them in prognostic stratification of breast cancer patients.
Conclusion: Specific miRNAs have been associated with tumor metastasis, and other clinical characteristics for breast cancer, unlike most other biomarkers that are currently available. The PDCD4 is negatively regulated by miR-21. Our study confirms that up-regulation of miRNA-21 expression in metastatic cell associated with the suppression of both TIMP1 and TIMP3 expression. It is the first report to demonstrate that miRNA are related to invasion and metastasis. Therapeutic implications of this work may be explored