Tehran University Medical Journal

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Background: Colorectal Cancer (CRC) is the third common cancer in the world. One of the pathways in colorectal tumor genesis is Microsatellite Instability (MSI+). MSI is detected in about 15% of all colorectal cancers. Colorectal tumors with MSI have dis-tinctive features compared with Microsatellite Stable (MSS) tumors. Due to the high percentage of MSI+ in patients with CRC in Iran, screening of this type of CRC is im-perative. In current study, two markers (BAT-26 and BAT-25) were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose MSI status in patients with CRC. Methods: Allelic variation in two markers (BAT-26 and BAT-25) was analyzed in tis-sues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic colorectal cancer by Real Time PCR (Hy-bridization probe) and High-Performance Liquid Chromatography (HPLC) techniques. The sensitivity and specificity of Real Time PCR and HPLC compared with sequencing as gold standard. The data were statistically analyzed using Student’s t-test and 2 or fisher exact test, where applicable with (P<0.05). Receiver-operating-characteristic (ROC) curves were used to evaluate the sensitivity and specificity. Results: The sensitivity and specificity of BAT-26 with Real Time PCR method (Hy-bridization probe) were 100% in comparison with gold standard method. Whereas the sensitivity and specificity of BAT-26 and BAT-25 with HPLC were 83%, 100% and 50%, 97%, respectively. Neither HPLC nor Real time PCR could detect circulating DNA with MSI property in sera. Conclusion: The sensitivity and specificity of real time PCR in MSI detection is the same as sequencing method and more than HPLC. BAT-26 marker is more sensitive than BAT-25 and MSI detection with Real time PCR could be considered as an accu-rate method to diagnose MSI in CRC tissues not sera.

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Background: Colorectal cancer is the third most common cancer in the world. Non-coding RNA especially miRNAs have important regulatory roles in cancer. miRNAs are small non coding RNA 21-23 nucleotides long which have different levels of expression between tumors and normal tissues. This study was designed to compare expression level of miRNA-21 between Iranian population colorectal cancer tissues and normal tissue. Methods: This case-control study has performed in medical genetics department of Tehran University of Medical Sciences from January to November 2013. We used 35 samples. The samples were isolated from tumor and adjacent normal tissues of colon. Thirty-five samples were divided into different groups according to cliniopathologic features including tumor size (>4 and <4 cm), metastasis (+ and -) and stage. After small RNA extraction from tissues by small RNA purification kit the quality and quan-tity of extracted RNA was determined using spectrophotometry. cDNAs were synthe-sized and real-time polymerase chain reaction carried out. Finally expression levels were statistically analyzed by LinRegPCR and REST software. Results: miRNA-21 expression ratio in stages I, II and III were 1/804 and 4/574, re-spectively, the increase from stage III was statistically significant (P= 0.037). The ex-pression were also studied according to different clinicopathologic status of colon can-cer, tumor size (>4 and <4 cm) and metastatic (+ and -), miRNA-21 over expressed in both groups, however the increase was not statistically significant. Conclusion: In this study, we found miR-21 over-expression in advanced stage in tu-moral tissue comparing with normal adjacent tissue. This means perhaps in the future it would be possible to use miRNA-21 as an informative prognostic biomarker to guide for better treatment strategies for colorectal cancer patients. Our findings also indicate that miRNA-21 is a promising new molecular target for designing novel therapeutic strategies to control colorectal cancer.

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Background: Breast cancer is the second most common cancer in the world after lung cancer also is the fifth cause of cancer mortality. About 90 percent of cancer mortality is because of metastasis and devastating between cell attachments, especially tight cell junctions. Epithelial mesenchymal transition is a phenomena involved in metastasis and starts with cell detachment. Occludin is the integral membrane protein which is located in tight junctions. Obviously distressing tight junction, which facilitates the stages of metastasis in cancer cells are very critical step. The aim of this study was to demonstrate the importance of occludin expression and its relationship with invasiveness in human breast cancer. Methods: In a cross sectional study we evaluated 30 patients who were referred to Caner Institute of Imam Khomeini Hospital Complex, Tehran, Iran, from March 2013 to April 2013. Samples were derived from fresh frozen tumor of patients suffering from breast cancer after inform consent assignment in the Tumor Bank of Iran (TBI). RNA was extracted from tumor tissue followed by reverse transcription, polymerase chain reaction (PCR), conventional Real-time PCR and data analysis for the occludin gene expression. Data were analyzed based on clinical staging of breast cancer patients which were cited in data bank of TBI. Results: Results of this study have demonstrated that the occludin gene expression was increased with the advanced stage. In 22 of patients the expression of gene was elevated compared with normal samples. On the other hand, the expression was significantly increased in stage II in comparison with stage I. Conclusion: The expression of occludin has increased by elevation of stage compared with normal tissue. It is suggested that alteration in the expression of this gene might be a possible factor which could affect on patient’s prognosis the same as other factors which are belonging to the same family. Increasing in expression of this gene might be considered as one of the possible markers which predict the possibility of invasion and metastasis.

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Background: Breast cancer is the most common non- skin cancer among women and it’s the second leading cause of cancer related death in women. Ubiquitin and ubiquitin like proteins are member of signal transduction pathways which have several cellular functions. It has shown that Ubiquitin like protein D (UBD) has accelerated the cancer progress. The aims of this study is evaluation of UBD gene expression in women suffering from breast cancer and its correlation with disease progression. Methods: In this study 30 FFPE (Formalin-fixed, paraffin-embedded) samples 20 cases from breast cancer and 10 cases from mammoplasty were collected from Parsian and Kasra Hospitals in Tehran after confirmation by pathologist. For each sample collection characters included ER-positive, lymph node negative, tumor size less than 5 cm in diameter were considered. Samples belonged to May 2010 up to April 2012. At first paraffin was removed by adding xylene then xylene removed with replacing ethanol 98%. After removing ethanol, RNA was extracted from samples by using RNX plus solution and cDNA synthesis were performed by using Moloney murine leukemia virus (M-MuLV) enzyme. UBD gene expression were examined in all samples cDNA by relative Real Time PCR. In this study GAPDH gene expression was also used as internal control. Results: UBD gene expression was obtained by calculating ΔΔCT and RQ. The average incensement of UBD gene expression in comparison of normal samples was 11 times. The results have shown that the level of UBD expression was related to the development and extend of the disease. In patients with stage 1 of disease, UBD gene expression had 2.73 times increase (P=0.001) compared to the control samples. However in stage 4 of disease, this number has increased up to 19.4 times (P=0.0005) more than normal. Conclusion: Considering the results of this study, it could be said that UBD gene expression as useful biomarker has an important role in detection of breast cancer. In addition as UBD gene expression levels increased stages of disease increased too. So that evaluation of UBD gene expression can be useful in early detection of disease.

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Background: Bloodstream infections (BSI) have a high incidence and high mortality in the worldwide. The mortality rate is variable between 20-70%. Therefore, early and timely detection of BSI agent in clinical laboratories is necessary. The aim of this study was to determine an efficient diagnostic tool to septicemia in accompany of blood culture method by Real-time PCR (using panbacterial 23S rRNA gene).

Methods: This cross-sectional study was conducted in two analytical and clinical stages in Hamadan University of Medical Sciences, Iran, from October 2014 to June 2015. In analytical stage, sensitivity (by serial dilution from 104 to 1 CFU/ml) and specificity of the primer were evaluated with the Staphylococcus aureus (as Gram positive indicator bacteria) and Escherichia coli (as Gram-negative indicator bacteria), human genome (from Hella cell culture), Candida albicans yeast and Aspergillus fumigatus fungus. In clinical stage, 121 blood samples were collected from patients suspected to sepsis in intensive care unit (ICU) from Hamadan University Hospitals. Finally, the results of Real-time PCR and blood culture methods were compared.

Results: The Real-time PCR showed a sensitivity ranging from 2 to 10 target copies per reaction to the whole blood for Escherichia coli and Staphylococcus aureus respectively. The specificity of this method was evaluated and no false positive amplification was identified. 57.85% (70 cases) of the samples were positive by Real-time PCR and 13.22% (16 cases) of the samples were positive by blood culture. However, none of the cases that were positive by blood culture were negative in Real-time PCR. As well as, 44.62% (54 cases) of cases were positive by Real-time PCR but blood culture showed no bacteria in the samples, and 42.15% (51 cases) were negative by both methods. Correlation or agreement of Kappa was 0.20, that indicating poor agreement between the two methods.

Conclusion: Real-time PCR is more sensitive than blood culture and also, because of high sensitivity of this primer by Real-time PCR, we can use it for screening blood samples from suspected patients of sepsis.

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Background: Centaurea cyanus is an endemic and well-known herbal medicine in Iran, is an annual flowering plant in the family of Asteraceae. The flowers are the part used in modern herbal medicine and are considered to have tonic, stimulant and emmenagogue properties, with action similar to that of blessed thistle. The aim this study was to investigate the phytochemical constituents of C. cyanus extract, its antioxidant, anti-tumor and anti-bacterial activities.

Methods: This experimental study was conducted from June to January of 2015 in Islamic Azad University of Varamin, Iran. At first, the phytochemical components of C. cyanus extract was analyzed using gas chromatography–mass spectrometry (GC-MS) method. Subsequently, the antibacterial potential of the extract was evaluated against 4 pathogenic bacteria including Staphylococcus aureus, Streptococcus pyogenes, Psedomonas aeroginosa and Klebsiella pnemoniae via minimum inhibitory concentration (MIC) mathod. Moreover, the anti-oxidant and anti-tumor activities of extract on colon cancer cell line (HT29) were investigate using DPPH and MTT colorimetric methods, respectively. Finally, the Bax and Bcl2 apoptosis gene expression level was analyzed by quantitative Real-time PCR technique.

Results: GC-MS analysis of C. cyanus extract was shown 19 major components and the most frequent component was belonged to n-Hexadecanoic acid (36.4%) and Linoleic acid (19.3%). The maximum antibacterial activity of extract was observed on S. aureus and P. aeroginosa isolates. The antioxidant activity of the extract was 0.109±0.07 mg/ml. Moreover, the MTT results show that extract had IC50= 26.04±0.45 on HT29 cell line. The Real-time PCR results showed the expression level of Bax and Bcl2 was significantly increased and decreased respectively in colon cancer cell line (2.63±0.54 (P< 0.05), 0.38±0.72 (P< 0.05)).

Conclusion: The results of this study show that the extract had significant anti-bacterial and anti-cancer effects and it appear that the extract has potential uses for pharmaceutical industries.

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Results: The phytochemical analyses of T. cherleri extract showed the 20 major components and the most frequent component was belonged to hexadecanoic acid, ethyl ester (20.7%) and 2-Pentadecanone, 6,10,14-trimethyl (19.9%). The extract had maximum antibacterial effects against Staphylococcus aureus and Streptococcus pyogenes. There was a dose dependent increase in the cytotoxicity effect of extract against A549 cancer cell. Moreover, the Real-Time PCR results indicated that the caspase 3 and caspase 9 gene expression was significantly up-regulated 2.57±0.27 (P<0.05), and 3.3±0.46 (P<0.05), respectively.

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