To Select the Appropriate Reference Gene for Normalizing the Quantitative Data to Assess MicroRNAs in Plasma Samples of Patients with Gastric Cancer

Abstract Background: Circulating microRNAs are promising biomarkers in diagnosis and assessment of cancerous patients. Quantitative Real-time PCR assay is a sensitive test for evaluating the levels of miRNAs expression. Nevertheless, there is no concurrence on selecting appropriate reference genes for qPCR analysis of miRNAs in circulation. Therefore, the current study aimed to select a suitable reference gene for normalizing the RT-qPCR assay results in plasma samples of patients with gastric cancer. Materials and Methods: Based on previously published studies, three molecules SNORD47, U6 RNA, and miR-103 were selected as the candidate reference genes. After RNA extraction from plasma samples of 40 patients with gastric cancer and 40 healthy individuals, expression levels of these molecules were evaluated using Real-time PCR method. Results: The results showed that the developed assays are able to diagnose their specified targets by a suitable linear range. By comparing patients and control groups, although the expression levels of miR-103 molecule were not equal between the two groups (p= 0.017), SNORD47 and U6 RNAs had similar expression levels. However, the variations of SNORD47 expression were lower that U6 RNA. Conclusion: Based on the results of the current study, the SNORD47 molecule has a stable expression levels in plasma samples of patients with gastric cancer and normal individuals and can be used as an appropriate reference gene for normalizing the quantitative data of qPCR assay

Comparative Analyses of Villin and HER-2 Genes Expression in Breast Cancer

Background: It has been previously demonstrated that HER-2 (human epidermal growth Factor receptor 2) positive breast cancers are associated with an aggressive nature. Villin is an actin bundling protein that plays a key role in actin reorganization and cell remodeling during stress. In this study, we aimed to investigate the correlation of Villin gene expression with HER-2 in breast cancer patients. Methods: Samples of 42 patients with breast cancer, and 3 controls were collected. Expression of Villin and HER-2 genes were monitored with real-time PCR using pre-designed primers. Student T-test was used to compare the means between the groups. Results: The mean age of the patients was 50±4.11years. Expression of the Villin gene was decreased in 28 samples (18 and 10 samples with negative and strongly HER-2 positive, respectively). Villin gene expression was increased in 14 samples (7, 2 and 5 samples with negative, weakly positive, and strongly HER-2 positive, respectively). The expression of Villin was significantly correlated with HER-2 positive status (P = 0.00057) Conclusions: We found that Villin gene expression is associated with HER-2 positivity and may be a predicting factor in aggressive breast cancer

Fibroblast growth factor-10 and epithelial-mesenchymal transition in colorectal cancer

As an inducer of epithelial-mesenchymal transition (EMT), fibroblast growth factor-10 (FGF-10) has a role in cell proliferation and differentiation in the embryo in addition to invasion and metastasis during carcinogenesis. In this study, we aimed to investigate the FGF-10 gene expression in tumor tissues based on the pathological feature of tumor related to EMT and metastasis. 62 tumors were obtai ned from 62 colorectal cancer patients during surgery. The pathological characteristics of the patients were carefully collected and classified by Iran National Tumor Bank. To quantify FGF-10 gene expression, RNA extraction, reverse transcription-PCR and real-time PCR were respectively performed. In addition, three colorectal cancer cell lines including LS174T, SW-948 and SW-480 were collected and cultured for further molecular analysis. Consequently, FGF-10 gene expression showed increased expression level in LS174T and SW-948 while it displayed decreased level in SW-480. Considering the tumor samples, we found an upregulation of FGF-10 gene expression in 52.1 % of all tumors in stage III and only in 9.09 % of all tumors in stage I. Also, there were an upregulation of FGF-10 gene expression in 50 % of all positive lymph invasion patients. Besides, FGF-10 gene upregulation was observed in 50 % of all tumors with a size larger than 5 cm (P value < 0.05) and 69 % of all tumors located in the colon (P value < 0.05). To our knowledge, this is the first time that FGF-10 expression is reported based on pathological features of colorectal cancer

Determination of E-cadherin and vimentin genes expression on the tumor specimens of ovarian cancer patients

Background: Ovarian cancer is a leading metastatic disease. The epithelial ovarian cancer is one of the most common malignant cancers that usually remains asymptomatic up to metastasis stages, and most patient when diagnosed are in the advanced stage of the disease. Studies have shown that in the majority of epithelial cancers mesenchymal factor expression such as Vimentin increases, and the epithelial factor expression such as E-cadherin decreases, as a result, it causes an epithelial-mesenchymal transition (EMT). The aim of this study was to determine the expression level of these genes and association between EMT phenomenon and development of ovarian cancer based on clinical and morphological findings. Methods: In the present case series study, 70 samples were chosen from the tumor Bank of Cancer Institute taken from patients at Imam Khomeini Hospital, Tehran, Iran. The amount of expression of two genes, E-cadherin and vimentin, was investigated by real-time PCR method from February 2016 to September 2017. The RNA extraction was done manually, and then cDNA synthesis was performed; In each sample the expression level of vimentin and E-cadherin was measured with real-time PCR method. The patient&rsquo;s clinical information with other data were analyzed with nonparametric statistical methods in SPSS software, version 19 (SPSS Inc., Chicago, IL, USA). Results: There was a significant relationship between expression of vimentin gene and the stage (P=0.026) of the disease and metastasis (P=0.009), There was no significant relationship between vimentin gene expression and tumor grade (P=0.207), age (P=0.11), tumor size (P=0.71) and family history (P=0.6). There was a significant correlation between E-cadherin gene expression and metastasis (P=0.027), no significant correlation was found between E-cadherin gene expression with tumor grade (P=0.690), stage (P=0.753), age (P=0.09), tumor size (P=0.537) and family history (P=0.56). Conclusion: According to the changes in expression of vimentin and E-cadherin genes in ovarian tumor cells, and association between these two genes with clinical and morphological findings and the role of these genes in the migration and invasion, we can use the both genes, vimentin and E-cadherin, as genes involved in the EMT process to assess disease progression and incidence of cell invasion in ovarian cancer

Expression of miR-127, miR-154, and miR-183 in Medullary Thy-roid Carcinoma Tumors

Background: Medullary thyroid cancer (MTC) accounts for 5%–10% of all thyroid cancers, but causes 13% of all thyroid cancer related deaths. MicroRNAs (miRs) have key functions in the development and progression of MTC. Altered expression of some miRs has been reported in many human cancers, including Thyroid cancer. Therefore, we aimed to analyze the expression of miR-154, miR-183 and miR-127 in MTC tumor tissues. Methods: In this case-control study, 15 MTC Formalin-fixed, paraffin-embedded (FFPE) tissue samples and 15 adjacent normal thyroid FFPE tissues, as a control group, were collected from Taleghani, and Loghman Hakim Hospitals, Tehran, Iran since 2005 till 2015. After RNA extraction and cDNA synthesis, the expression of miR-127, miR-154 and miR-183 was measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Results: Our data showed a significant increase in the expression of miR-127 in MTC samples in comparison with the control group (P<0.05). Although miR-154 and miR-183 expression levels had increase expression in MTC tumors, this change was not statistically significant. Conclusion: The miR-127 could be considered as a prognostic, diagnostic and therapeutic marker for the management of MTC, and it is proposed for further investigation to fully establish the role of this miRNA in MTC