Aberrant Expression of Breast Development-Related MicroRNAs, miR-22, miR-132, and miR-212, in Breast Tumor Tissues / 한국유방암학회지

PURPOSE: MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that posttranscriptionally regulate the expression of most genes in the human genome. miRNAs are often located in chromosomal fragile sites, which are suscept-ible to amplification or deletion. Chromosomal deletions are frequent events in breast cancer cells. Deletion and loss of heterozygosity at 17p13.3 have been reported in 49% of breast cancers. The aim of the current study was to evaluate potential expression alterations of miR-22, miR-132, and miR-212, which are located on the 17p13.3 locus and are required for mammary gland development. METHODS: A matched case-control study was conducted, which included 36 pairs of tumor and matched nontumor surgical specimens from patients diagnosed with breast invasive ductal carcinoma. Formalin-fixed paraffin-embedded samples from archival collections at the pathology department of Shariati Hospital were prepared for RNA extraction using the xylene-ethanol method before total RNA was isolated with TRIzol Reagent. Specific primers were designed for cDNA synthesis and miRNA amplification. The expression of miRNAs was then evaluated by real-time polymerase chain reaction (RT-PCR). RESULTS: According to our RT-PCR data, the miR-212/miR-132 family was downregulated in breast cancer (0.328-fold, p<0.001), and this reduced expression was the most prominent in high-grade tumors. In contrast, miR-22 exhibited a significant upregulation in breast tumor samples (2.183-fold, p=0.040). CONCLUSION: Consistent with the frequent deletion of the 17p13.3 locus in breast tumor cells, our gene expression data demonstrated a significant downregulation of miR-212 and miR-132 in breast cancer tissues. In contrast, we observed a significant upregulation of miR-22 in breast tumor samples. The latter conflicting result may have been due to the upregulation of miR-22 in stromal/cancer-associated fibroblasts, rather than in the tumor cells.

Identification of Reliable reference genes for quantification of MicroRNAs in serum samples of sulfur mustard-exposed veterans

Objective: In spite of accumulating information about pathological aspects of sulfur mustard [SM], the precise mechanism responsible for its effects is not well understood. Circulating microRNAs [miRNAs] are promising biomarkers for disease diagnosis and prognosis. Accurate normalization using appropriate reference genes, is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference gene for microRNA quantification in serum samples of SM victims

Materials and Methods: In this case and control experimental study, using quantitative real-time polymerase chain reaction [qRT-PCR], we evaluated the suitability of a panel of small RNAs including SNORD38B, SNORD49A, U6, 5S rRNA, miR-423-3p, miR-191, miR-16 and miR-103 in sera of 28 SM-exposed veterans of Iran-Iraq war [1980-1988] and 15 matched control volunteers. Different statistical algorithms including geNorm, Normfinder, best-keeper and comparative delta-quantification cycle [Cq] method were employed to find the least variable reference gene

Results: miR-423-3p was identified as the most stably expressed reference gene, and miR- 103 and miR-16 ranked after that

Conclusion: We demonstrate that non-miRNA reference genes have the least stability in serum samples and that some house-keeping miRNAs may be used as more reliable reference genes for miRNAs in serum. In addition, using the geometric mean of two reference genes could increase the reliability of the normalizers

[Optimization of extraction and amplification of microRNAs [mir-21, mir-141 and mir-205] in urine samples of patients with bladder cancer]

The current methods for bladder cancer diagnosis suffer from low sensitivity and specificity. Therefore, finding a novel tumor markers with high specificity and sensitivity is of great interest. MicroRNAs [miRNA, miR] are small endogenously-produced, non-coding RNAs with an important role in regulating gene expression. Recent studies show that miRNAs expression profiles represent significant tumor-specific changes that are unique for most cancers. The aim of this study was to optimize miRNA containing total RNA extraction from urine and use it as a reliable and repeatable technique for miRNA detection in urine of patients with bladder cancer. Total RNA was extracted from the urine of patients with bladder cancer and normal individuals using RNX and Trizol solutions with and without modifications of original protocols. Real-time quantitative RT-PCR was then used to detect miRNAs with a potential link to bladder tumorigenesis. RNX and the modified Trizol are practical methods for RNA extraction from urine samples. The mir-21 amplification of the extracted RNAs using modified Trizol method was more efficient than that of RNX method. It is noteworthy that, the levels of miRNAs expression were much higher in the frozen urines compared to the fresh ones. We have succeeded to set-up a protocol to easily amplify miRNAs in urine samples. Based on the data, microRNAs seem to be good biomarkers for early detection and screening of bladder cancer