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Differential expressions of cancer-associated genes and their regulatory miRNAs in colorectal carcinoma.

Colorectal cancer is one of the frequently seen malignancies in the world. To date, several oncogenes and tumor suppressor genes have been identified and linked to colorectal cancer pathogenesis. Although recent advances in the diagnosis and therapy of colorectal cancer are promising, identifying novel genetic contributors is still high priority. In the present study, expression profile of some cancer-related genes and their regulatory miRNA molecules were evaluated by using a high-throughput real-time PCR method. For the study, a total of 54 patients diagnosed with CRC and normal colon tissue samples of 42 healthy controls were included. For the expression analysis, total RNA was extracted from FFPE tissue samples and converted to cDNA. All expression analyses were assessed by using Fluidigm Microfluidic Dynamic Array chips for 96 samples and the reactions were held in Fluidigm BioMark™ HD System Real-Time PCR. As a result of the study, expression of the ADAMTS1, FHIT, RUNX1, RUNX3 and WWOX genes was shown to be significantly altered in CRC tissues in contrast to normal tissue samples. Moreover, miR-378a-3p, miR-155-5p, miR-193b-3p, miR-96-5p, miR-17-5p, miR-27a-3p, miR-133b, miR-203a, miR-205-5p, miR-34c-5p, miR-130a-3p, miR-301a-3p, miR-132-3p, miR-222-3p, miR-34a-5p, miR-21-5p, miR-29a-3p and miR-29b-3p were found to be significantly deregulated in CRC. Consequently, results of the current study strongly suggest the involvement of novel cancer-related genes and their regulatory miRNAs in CRC physiopathology.

The association of the expression of miR-122-5p and its target ADAM10 with human breast cancer

MicroRNAs can regulate many biological functions. miR-122-5p has a tumor suppressor function through different molecular pathways. Also, our second hit, ADAM10, targeted by miR-122-5p, is a major determinant of HER2 shedding causing that trastuzumab cannot bind to HER2 receptors. Therefore, our analysis upon ADAM10 expression and miR-122-5p was a good point to understand molecular mechanism of breast cancer. In our study, we investigated the expression profiles of miR-122-5p and its target ADAM10 in 71 breast cancer patients. Immunohistochemical analysis of ER, PR and HER2 gene products was used to categorize tumors in patients. Expression data and immunohistochemical findings were evaluated to comment on the relationship between miR-122-5p and ADAM10. ADAM10 expression was higher in tumor than that of normal tissue but miR-122-5p expression was lower in tumor than that of normal tissue. The expression pattern in HER2+ patients was reverse of the overall result. It can be explained like that miR-122-5p expression increases especially in HER2+ cancer cell to suppress ADAM10 shedding activity on HER2 receptor. However, increase in expression of tumor suppressor miR-122-5p is not enough to inhibit ADAM10. All in all, we can think miR-122-5p as potential regulator of ADAM10 and trastuzumab resistance. Since if we increase miR-122-5p activity together with trastuzumab administration, then HER2+ breast cancer cells may overcome trastuzumab resistance by inhibiting ADAM10 shedding activity on HER2 receptors and increase the efficiency of trastuzumab.

Expression patterns of miR-221 and its target Caspase-3 in different cancer cell lines

Caspases are important initiators and most well-known finishers of apoptosis. By changing the death propagation homeostatic equilibrium, their different expression patterns might trigger the progression of hazardous diseases like cancer. miR-221 is an oncogenic miRNA. It is known to have both anti-angiogenic and angiogenic effect. The aim of this work was to compare the expression levels of miR-221 and its target caspase-3 in different cancer cell lines and to find out a relationship between these two. We also tried to establish a prominent relationship between miR-221 and its role in apoptosis by studying their expression levels. Our results indicate that expression of caspase-3 is quite lower as compared to miR-221 expression in all of the selected cancer cell lines. As a result, we conclude that miR-221 may have a crucial role in repressing the expression of caspase-3 which may contribute to a lower apoptotic rate, thus supporting the selection of more aggressive cancer cells. To our knowledge, this is the first study related to the expression levels of caspase-3 and miR-221 in different cell lines at the same time. We expect that our study might pave the way for better understanding the role of miR-221 in apoptotic regulation of caspase-3.

MTUS1 and its targeting miRNAs in colorectal carcinoma: significant associations

Deregulated microRNA (miRNA) expression has been shown to be involved in the pathogenesis of several types of cancers including colorectal cancer (CRC). Thus, determining miRNA targets of genes that play critical role in the malignant transformation is very important. Here, expression levels of tumor suppressor microtubule-associated tumor suppressor 1 (MTUS1) and its regulatory miRNAs were reported. Predicted and validated targets of MTUS1 gene was determined by a computational approach. Expressions of MTUS1 and miRNAs were determined by using 96.96 Dynamic Array™ integrated fluidic circuit (Fluidigm). As a result, MTUS1 levels were found to be diminished in formalin-fixed, paraffin-embedded (FFPE) tissue samples of CRC patients compared to controls. Also, several of MTUS1 targeting miRNAs were found to be upregulated in CRC samples (miR-373-3p, 183-5p, 142-5p, 200c-3p, 19a-3p, -20a-5p, -181a-5p, -184, -181d-5p, -372-3p, 27b-3p, 98-5p, -let-7i-5p, -let-7d-5p, -let-7g-5p, -let-7b-5p, and -let-7c-5p). Of these miRNAs, miR-135b-5p, -373-3p, 183-5p, 142-5p, 200c-3p, 19a-3p showed marked expression levels. In contrast, expression levels of let-7a-5p, 7e-5p, 7f-5p, hsa-miR-125a-5p, and 125b-5p were found to be downregulated in CRC tissues. Accordingly, some of the overexpressed miRNAs especially the miR-135b-5p, -373-3p, 183-5p, 142-5p, 200c-3p, and 19a-3p may play key roles in CRC pathophysiology through MTUS1. In contrast, let-7a-5p, 7e-5p, 7f-5p, miR-125a-5p, and 125b-5p may play important roles in CRC carcinogenesis independent from the MTUS1. In conclusion, MTUS1 targeting miRNAs may play key roles in the development of CRC by downregulating tumor suppressor MTUS1.

Insecticide imidacloprid influences cognitive functions and alters learning performance and related gene expression in a rat model.

The potential toxic effects of several pesticides, including imidacloprid on non‐target organisms have not been clearly established. Also, the chronic effects of non‐toxic doses on cognitive function in mammals are unknown. In this study, the effects of different doses of imidacloprid on learning and memory of infant and adult rats were evaluated, and the expressions of genes synthesizing proteins known to be associated with learning in brain tissues were also documented. 0.5, 2 and 8 mg/kg doses of imidacloprid were administered to newborn infant and adult Wistar albino rats by gavage. Their learning activities were evaluated, and the expression levels of the inotropic glutamate receptor GRIN1, synoptophysin, growth‐associated protein 43 and the muscarinic receptor M1 in hippocampus were determined by real‐time PCR method. Learning activities were diminished significantly at 2 and 8 mg/kg doses in the infant model groups and at 8 mg/kg dose in adult rats. Also, expression levels of GRIN1, SYP and GAP‐43 were found to be insignificantly altered. Only the expression of M1 were significantly changed in high doses of adult group. Thus imidacloprid in high doses causes deterioration in cognitive functions particularly in infant rats, and this deterioration may be associated with changes in the expressions of related genes.

A novel variable exonic region and differential expression of LINC00663 non-coding RNA in various cancer cell lines and normal human tissue samples

Long non-coding RNAs (lncRNAs) are found to play crucial roles in several biological processes and have been associated with many complex human diseases including cancers. Several lines of evidences indicate that lncRNAs deregulated in many cancer tissues. In this particular study, differential expression of long intergenic non-coding RNA 663 (LINC00663) was demonstrated in various cancer cell lines and healthy human tissues by using RT-PCR and qPCR methods. While expression level of LINC00663 was most prominent in thyroid gland and uterus, it is least expressed in skeletal muscle tissues. Moreover, LINC00663 was found to be differentially expressed in various cancer cells. Particularly, its expression was highly diminished in DU-145, PC3, HGC-27, CRL-1469, A549, MCF7, and BCPAP cancer cells. Also, LINC00663 expression was most prominent in A172 glioblastoma cells. Additionally, a novel splice variant of LINC00663 RNA was also detected. The sequence and Basic Local Alignment Search Tool (BLAST) analysis results revealed the presence of a novel exonic region between exons 2 and 3. Subsequently, five potential splice variants showing high level of variation have been identified. Secondary structures of these variants with minimum free energy were also demonstrated. Furthermore, putative microRNA (miRNA) binding sites to these variants have been shown. In conclusion, LINC00663 was shown to be differentially expressed in various human tissues and cancer cell lines. Also, LINC00663 undergoes alternative splicing and the novel exonic region alters its secondary structure and its interactions with potential targeting miRNAs. The role of LINC00663 in cancer formation further needs to be investigated with a wide range of studies.

The role of the UTS2 gene polymorphisms and plasma Urotensin-II levels in breast cancer

Breast cancer is the most common malignancy predominantly affecting women. To date, numerous numbers of studies were reported novel genetic contributors with diagnostic, prognostic, and therapeutic potential for the breast carcinogenesis. However, the role of urotensin-II in breast carcinogenesis has not been elucidated yet. Urotensin-II is a somatostatin-like cyclic tiny peptide identified by its potent vasoconstrictor activity. Soon after its discovery, its involvement in many disease states as well as its expression in various tissues including the tumors have been demonstrated. Moreover, there is strong evidence that suggest urotensin-II as the significant contributor of angiogenesis as well as cell proliferation and tumor biology. In this study, enzyme-linked immunosorbent assay (ELISA) and restriction fragment length polymorphism analysis were used to evaluate plasma levels of urotensin-II and Thr21Met and Ser89Asn polymorphisms of UTS2 gene in breast cancer patients. In the present case-control study, we noticed a significant decrease in the levels of urotensin-II protein in the plasma of the breast cancer patients (p < 0.05). Also, Thr21Met polymorphism in the UTS2 gene was associated with the risk of developing breast cancer (p < 0.0001), whereas the genotype frequency of Ser89Asn was found to be similar in patients and controls (p > 0.05). In addition, we demonstrated the gradual decreasing of urotensin-II protein levels from TT and TM to MM genotypes. In conclusion, these results strongly suggest that urotensin-II could contribute to breast carcinogenesis and Thr21Met polymorphism can be an important risk factor in developing breast tumors.

MTUS1 tumor suppressor and its miRNA regulators in fibroadenoma and breast cancer.

Breast cancer is major public health problem predominantly effects female population. Current therapeutic approaches to deal with breast cancer are still lack of effectiveness. Thus, identifying/developing novel strategies to fight against breast cancer is very important. The frequent deletions at 8p21.3-22 chromosomal location nearby D8S254 marker enabled the discovery of a novel tumor suppressor gene, MTUS1. Subsequently, MTUS1 was demonstrated to be less expressed in a variety cancer types including breast cancer. Also, it is obvious that gene expression is widely regulated by miRNAs. Here, we aimed to report differential expression of MTUS1 and its regulatory miRNAs in breast cancer and fibroadenoma tissues. Dynamic analysis of MTUS1 expression levels and its miRNAs regulators were attained by Fluidigm 96×96 Dynamic Array Expression chips and reactions were performed in Fluidigm BioMark™ HD System qPCR. Consequently, MTUS1 mRNA levels were significantly diminished in breast cancer tissues and elevated in fibroadenoma tissues. Also, among MTUS1 targeting miRNAs, miR-183-5p was identified to be overexpressed in breast cancer and down-regulated in fibroadenoma tissues. Also, expression levels of MTUS1 and miR-183-5p were well correlated with clinical parameters. In particular, MTUS1 expression was found to be diminished and miR-183-5p expression was elevated with the advancing stage. In conclusion, as a potential therapeutic target, miR-183-5p can be a chief regulator of MTUS1 and MTUS1-miR-183-5p axis may have significant influence in the pathology of breast cancer.

Differential expression of the UGT1A family of genes in stomach cancer tissues

Uridine 5′-diphospho-glucuronosyltransferases (UGT) are the key players in the biotransformation of drugs, xenobiotics, and endogenous compounds. Particularly, UDP-glucuronosyltransferase 1A (UGT1A) participates in a wide range of biological and pharmacological processes and plays a critical role in the conjugation of endogenous and exogenous components. Thirteen alternative splicing products were produced from UGT1A gene locus designated as UGT1A1 and UGT1A3–10. A growing amount of evidence suggests that they have important roles in the carcinogenesis which is well documented by colon, liver, pancreas, and kidney cancer studies. Here, we report differential expressions of UGT1A genes in normal and tumor tissues of stomach cancer patients. Total numbers of 49 patients were enrolled for this study, and expression analysis of UGT1A genes was evaluated by the real-time PCR method. Accordingly, UGT1A1, UGT1A8, and UGT1A10 were found to be upregulated, and UGT1A3, UGT1A5, UGT1A7, and UGT1A9 were downregulated in stomach tumors. No expression changes were observed in UGT1A4. Also, UGT1A6 transcription variants were significantly upregulated in stomach cancer tissues compared to normal stomach tissue. Additionally, UGT1A7 gene showed highest expression in both normal and tumoral tissues, and interestingly, UGT1A7 gene expression was significantly reduced in stage II patients as compared to other patients. In conclusion, UGT1A genes are differentially expressed in normal and tumoral stomach tissues and expression changes of these genes may affect the development and progression of various types of cancer including the cancer of the stomach.

Differential expression of PIWIL2 in papillary thyroid cancers

Thyroid cancer is the most common type of endocrine malignancy and a leading cause of death among endocrine organ-related cancers. Similar to other types of cancers, early diagnosis of thyroid cancer is important to increase the survival and treatment of this disease. Several immunohistochemical markers are used in the differential diagnosis of thyroid papillary carcinoma. Also, increasing evidence indicates that P-element induced wimpy testis like 2 (PIWIL2) is an RNA-binding protein involved in the induction and progression of numerous types of human malignancies such as lung, breast, colon, prostate and cervix cancers. However, the role of PIWIL2 was poorly investigated in thyroid cancers. Accordingly, aim of the present study was to elucidate the relationship between PIWIL2 and thyroid cancers. The expression level of PIWIL2 was determined by analyzing both protein and mRNA levels in papillary and micropapillary carcinoma tissues by using immunohistochemistry and real-time PCR methods, respectively. Immunohistochemical analysis of HBME-1, galectin-3 and CK-19 was also performed. Similar to other immune markers of HBME-1, galectin-3 and CK-19, protein expression levels of PIWIL2 was significantly up-regulated in both papillary and micropapillary thyroid cancers (p < 0.01). Moreover, consistent with protein expression levels, mRNA expression levels of PIWIL2 was elevated in both papillary and micropapillary thyroid cancer tissues. Yet, mRNA expression changes were statistically insignificant. In conclusion, results of the current study suggest that PIWIL2 can be involved in thyroid cancer tumorigenesis and can be used as a novel predictive biomarker and/or therapeutic target.