Browsing by Subject "miRNA"

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Open Access
Development and validation of methods for the diagnosis of lung cancer via serological biomarkers
(2019-02) Akçay, Abbas Güven
Over 10% of all new cancer cases are lung cancer. Moreover, estimates till 2030 indicate that already increasing lung cancer incidences will keep increasing, especially in developing countries like Turkey. Lung cancer, the leading cause of cancer deaths, has two large divisions: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC). SCLC is the most aggressive subtype of lung cancer. And although, the treatment options and median survival time is more favorable in Limited Disease (LD), high tumor growth rate and metastatic tendency of SCLC even in the early stages, makes the diagnosis troublesome. Similarly, if NSCLC is diagnosed in early stages, surgery option is open and this increases the patient survival rate. However, current methods in screening and diagnosis, such as computed tomography (CT) and positron emission tomography (PET), are all limited by false positivity rates. Additionally, biopsy methods used in histological evaluations are both invasive and prone to false negativity. Therefore, new diagnostic tools which are cheap, accurate and non-invasive are in high demand. Autologous antibodies are abundantly elicited and stably exist in patient sera years before the clinical diagnosis of disease. Several such antibodies were reported by our group and other groups in lung cancer. Therefore, new diagnostic methods incorporating autologous antibodies can be a huge step forward in early diagnosis of lung cancer. Moreover, miRNAs, with their unique hormone like features such as circulation in serum and their regulatory effects in cell, are another good candidate for the early diagnosis of lung cancer. Therefore, in this study I aimed to develop a reliable, robust and automated evaluation method to re-evaluate custom Protein Array (cPA) screenings previously performed in our lab, and to determine the autologous antibodies with highest discriminatory power between SCLC patients & healthy controls. Moreover, I aimed to develop a Quartz Crystal Microbalance with Dissipation (QCM-D) based immunoassay to be incorporated later in the validation of cPA results. Lastly, in a parallel study I aimed to identify and validate novel miRNA biomarkers NSCLC. My results indicate that cPAs can have better sensitivity and specificity than ELISA and that QCM-D can be developed as an alternative to ELISA. miRNAs identified in silico, can also be validated ex vivo. Previously, Protein Arrays (PAs) and cPAs were screened using 49 SCLC patient’s and 50 healthy serums in our laboratory, incorporating visual and manual evaluations. Sensitivity and specificity values were calculated for individual autologous-antibodies and a number of autologous-antibody panels. Moreover, validations of cPA results were carried via ELISA. However, large discrepancies between cPA and ELISA results, as well as inconsistencies among ELISA results urged me to consider re-evaluation of cPA results with a more robust way, and to focus on developing a method superior to ELISA in autologous-antibody evaluations. Therefore, I incorporated AIDA to generate numeric values out of cPA screening images and filtered low quality data with optimized cut-off values. Several Receiver Operating Characteristic (ROC) curves were plotted using evaluated data. Improved results were evident by the increased Area Under Curve (AUC) values in both individual and combined ROC curves. Moreover, I developed a QCM based immunosensor for detection of anti-SOX2 antibody to be incorporated later in validation of cPA results. Binding interaction between anti-SOX2 antibody and SOX2 protein was modelled using 1:1 Langmuir Isothermal Binding and standard curves generated in QCM. In a parallel study, I also investigated miRNAs significantly upregulated in NSCLC when compared to high risk controls. For that purpose, miRNA expression datasets were gathered from GEO. Selected 2 datasets with the same sample type were analyzed for common significantly upregulated miRNAs among these two datasets. Significantly upregulated miRNAs were subjected to logistic regression analysis with LASSO regularization (error metrics: AUC and MSE) to select best panel of miRNAs that can distinguish NSCLC patients from healthy controls in given datasets. Moreover, selected miRNAs were analyzed with qRT-PCR to validate the panel. I was able to re-evaluate cPA results by eliminating low quality data from numeric values generated via AIDA software from cPA images. I identified a panel of 4 autologous antibodies (FKBP8 – P53 – SOX2 – POLB) which resulted in 60% sensitivity at 100% specificity in discrimination of SCLC from controls. ROC of this autologous antibody panel had an AUC of 95.04%. Given panel surpassed diagnostic power of the only commercially available diagnostic kit of the same kind; EarlyCDT-Lung. Moreover, proof of concept for measurements of anti-protein antibodies were carried successfully in QCM, using anti-SOX2 antibody-SOX2 protein pair in PBS buffer as an example for it. Early results of anti-SOX2 mAb QCM indicate a linear assay range comparable to ELISA. Langmuir Isothermal Binding model revealed a strong interaction between antibody and protein in our QCM anti-SOX2 measurement experiments. Lastly, I was able to select 5 miRNAs using logistic regression and LASSO regularization that can best discriminate between NSCLC patients and high risk controls. However, validation experiments using qRT-PCR needs to be repeated as low Ct values and prominent hemolysis in serum samples prevented drawing meaningful conclusions.
Open Access
Discovering regulatory non-coding RNA interactions
(2019-09) Olgun, Gülden
The vast majority of eukaryotic transcriptomes comprise noncoding RNAs (ncRNAs) which are not translated into proteins. Despite the accumulating evidence on the functional roles of ncRNAs, we are still far from understanding the whole spectrum of molecular functions ncRNAs can undertake and how they accomplish them. In this thesis we develop computational methods for discovering interactions among ncRNAs and tools to analyze them functionally. In the first part of the thesis, we present an integrative approach to discover long non-coding RNA (lncRNA) mediated sponge interactions where lncRNAs can indirectly regulate mRNAs expression levels by sequestering microRNAs (miRNAs), and act as sponges. We conduct partial correlation analysis and kernel independence tests on patient gene expression profiles and further refine the candidate interactions with miRNA target information. We use this approach to find sponge interactions specific to breast-cancer subtypes. We find that although there are sponges common to multiple subtypes, there are also distinct subtype-specific interactions with high prognostic potential. Secondly, we develop a method to identify synergistically acting miRNA pairs. These pairs have weak or no repression on the target mRNA when they act individually, but when together they induce strong repression of their target gene expression. We test the combinations of RNA triplets using non-parametric kernel-based interaction tests. In forming the triplets to test, we consider target predictions between the miRNAs and mRNA. We apply our approach on kidney tumor samples. The discovered triplets have several lines of biological evidence on a functional association among them or their relevance to kidney tumors. In the third part of the thesis, we focus on functional enrichment analysis of noncoding RNAs while some non-coding RNAs (ncRNAs) have been found to play critical regulatory roles in biological processes, most remain functionally uncharacterized. This presents a challenge whenever an interesting set of ncRNAs set needs to be analyzed in a functional context. We develop a method that performs cis enrichment analysis for a given set of ncRNAs. Enrichment is carried out by using the functional annotations of the coding genes located proximally to the input ncRNAs. To demonstrate how this method could be used to gain insight into the functional importance of a list of interesting ncRNAs, we tackle different biological questions on datasets of cancer and psychiatric disorders. Particularly, we also analyze 28 different types of cancers in terms of molecular process perturbed and linked to altered lncRNA expression. We hope that the methods developed herein will help elucidate functional roles of ncRNAs and aid the development of therapies based on ncRNAs.
Open Access
Early detection and staging of colorectal cancer using a panel of micro RNAs
(OMICS, 2018) Shapira, R.; Ilyayev, N.; Attali, R.; Westrich, G.; Halle, D.; Speter, C.; Stavropoulos, A. V.; Roistacher, M.; Pavlov, V.; Grinbaum, R.; Protic, P.; Güre, Ali O.; Bilchik, A. J.; Stojadinovic, A.; Mitrani-Rosenbaum, S.; Nissan, A.
Purpose: To improve lymph node (LN) staging in patients with colon cancer (CC). The present study describes the selection of CC-specific miRNAs and assesses their utility as a micro metastases detection assay. Methods: 30 miRNAs have been selected from a microarray assay and 16 miRNAs from database mining for their specific upregulation in colon cancer tissues as compared to normal adjacent tissues. Differential expression was validated by RT-qPCR in a larger cohort of samples (n=20) and compared to normal lymphatic tissues (n=6) and normal peripheral blood lymphocytes (PBLs, n=14). The selected miRNA panel was then used for the screening of 84 lymph nodes (LN) obtained from colon cancer patients (n=20) Results: After validation, a panel of 8 miRNAs was found to be significantly upregulated in CC compared to normal adjacent tissues and to normal lymphatic tissues: miR-96, miR-183, miR-194, miR-200a, miR-200b, miR200c, miR-203 and miR-429. A total of 84 LNs were analysed: 12 LN metastases were detected by H&E, 18 by CK staining whereas 32 were detected by the CC-specific miRNA analysis. This represents an increase of 40% in the detection rate. Conclusion: This study demonstrated the ability of a CC-specific 8 miRNA panel in detecting micro metastases in CC patients.
Open Access
Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients
(Springer, 2021-11-18) Cagnan, I.; Keles, M.; Keskus, Ayse Gokce; Tombaz, Melike; Sahan, O. B.; Aerts-Kaya, F.; Uckan-Cetinkaya, D.; Konu, Ozlen; Gunel-Ozcan, A.
Fanconi anemia (FA) is a rare genetic disorder characterized by genomic instability, developmental defects, and bone marrow (BM) failure. Hematopoietic stem cells (HSCs) in BM interact with the mesenchymal stem/stromal cells (MSCs); and this partly sustains the tissue homeostasis. MicroRNAs (miRNAs) can play a critical role during these interactions possibly via paracrine mechanisms. This is the first study addressing the miRNA profile of FA BM–MSCs obtained before and after BM transplantation (preBMT and postBMT, respectively). Non-coding RNA expression profiling and quality control analyses were performed in Donors (n = 13), FA preBMT (n = 11), and FA postBMT (n = 6) BM–MSCs using GeneChip miRNA 2.0 Array. Six Donor-FA preBMT pairs were used to identify a differentially expressed miRNA expression signature containing 50 miRNAs, which exhibited a strong correlation with the signature obtained from unpaired samples. Five miRNAs (hsa-miR-146a-5p, hsa-miR-148b-3p, hsa-miR-187-3p, hsa-miR-196b-5p, and hsa-miR-25-3p) significantly downregulated in both the paired and unpaired analyses were used to generate the BM–MSCs’ miRNA—BM mononuclear mRNA networks upon integration of a public dataset (GSE16334; studying Donor versus FA samples). Functionally enriched KEGG pathways included cellular senescence, miRNAs, and pathways in cancer. Here, we showed that hsa-miR-146a-5p and hsa-miR-874-3p were rescued upon BMT (n = 3 triplets). The decrease in miR-146a-5p was also validated using RT-qPCR and emerged as a strong candidate as a modulator of BM mRNAs in FA patients.
Open Access
Identification of modulatory functions of TP53, estrogen signaling, and 14q32.31 miRNA cluster on CHRNA5 knock-down expression profile and development of syneRgy APP
(2021-09) Keşküş, Ayşe Gökçe
Cholinergic receptor subunit alpha 5 (CHRNA5) is a ligand-gated ion channel expressed in not only the nervous system but also other tissues. Differential expression and the polymorphisms of CHRNA5 have been associated with addiction, particularly nicotine and various cancer types. The tumor-suppressive properties of CHRNA5 depletion, i.e., decrease in cell proliferation, induction of DNA damage response, and drug sensitivity, have been identified in breast cancer cell lines. This thesis focuses on identifying critical factors modulating or modulated by the knock-down of CHRNA5 in breast cancer cell lines using both wet-lab and bioinformatics approaches. Here I have first found the significant correlation between CHRNA5 and DNA damage response in breast cancer tumor datasets. Moreover, I discovered that the introduction of siTP53 antagonized the actions of siCHRNA5 and reverted the siCHRNA5-mediated cell cycle inhibition and drug sensitivity in the MCF7 breast cancer cell line. Furthermore, siCHRNA5 was found to inhibit the secondary signaling of estrogen/ESR1 in time and dosage-dependent manners. CHRNA5 depletion also downregulated the conserved 14q32.31 miRNA cluster expression. Among those miRNAs, miR495-3p appeared to be the most prominent candidate, exhibiting a similar expression profile with selective estrogen degraders and partially with siCHRNA5. However, the inhibitory effect of the combinatorial treatment with siCHRNA5 and miR495-3p on the secondary targets of estrogen signaling indicated that siCHRNA5 and miR495-3p might target converging pathways, evidenced by the antagonism (rather than addictiveness) between them. In addition, the Shiny-based syneRgy app was developed to analyze the transcriptome-based synergy between treatments and/or genetic modifications. As a case study, syneRgy analysis using novel MDM2 inhibitor and/or temozolomide treated neuroblastoma cell lines revealed that although the tumor-suppressor effect of combination therapy was more than each individual treatment, it was less than additive. syneRgy was applied to understand the combinatorial treatment of siCHRNA5 with siTP53 as well as siCHRNA5 with miR495-3p mimic and enhanced our understanding of the TFs that might have a role in the crosstalk. To our knowledge, syneRgy is the first-ever online tool to perform statistical synergy analysis using RNAseq count or logFC transcriptomic data and synreg, i.e. our novel methodology allowing for statistical tests of TF target enrichment using regression models.
Open Access
Mirna based identification of prostate cancer by investigation of urinary exosomes
(2020-08) Bozbeyoğlu, Naz
Prostate cancer is one of the most incident cancer subtypes with high mortality rate. Currently, diagnosis of prostate cancer is based on rectal examination, Prostate Specific Antigen (PSA) testing and biopsy. Normal range of PSA is defined as 0-4 ng/ml and individuals with higher PSA levels are considered as potential prostate cancer patients. However, PSA fluctuates as a result of many factors and it is shown to increase with age. Thus, PSA testing causes significantly high false positive results and many healthy men have biopsy unnecessarily due to high PSA levels or they even get overtreated. This situation has huge psychological as well as financial effects on these people. Herein, we investigated the diagnostic potential of urinary exosomal microRNAs (miRNA) in prostate cancer. Rather than investigation of cellular miRNAs, we focused on exosomal miRNAs because of high integrity of exosomes and their abundance in many biofluids including urine. Development of a sensitive diagnostic method from urine would be advantageous because accurate diagnosis of prostate cancer would be possible by a non-invasive procedure. miRNAs are the small non-coding RNAs and they suppress expression of target genes via degradation of mRNA or post-translational regulation. miRNAs have high potential as cancer biomarkers because they can act as tumor suppressor and repress oncogenic gene expression or function as oncogenic miRNA and suppress tumor suppressor gene expression. For this reason, we identified several candidate exosomal tumor suppressor and oncogenic miRNAs and continued our study with the most potent ones. At the beginning of the study, we validated that we efficiently isolated exosomes via several techniques such as flow cytometry, Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM). Then, we studied differential expression of candidate miRNAs in prostate cancer PC-3 cell line. Similar to our literature search findings, we found that expression levels of miR-107, miR-139, miR-145 and miR-204 were significantly lower in PC-3 exosomes in comparison to healthy urinary exosomes. On the other hand, oncomiRs; miR-21-5p, miR-375-5p and miR-574 3p were upregulated in PC-3 cell line exosomes. Of note, expression of another candidate miRNA; miR-30a was almost the same in PC-3 exosomes with healthy controls. We used these results as preliminary data and collected urine specimens from 17 prostate cancer patients. We firstly analyzed their PSA level-age and PSA Level- Gleason Score correlations. Our analyses revealed that PSA level was increasing with age and PSA was not correlated with Gleason Score. We concluded that PSA was being affected by several reasons in addition to tumor formation and it was not correlated with disease progression. Again, this was a finding which supported that a more sensitive diagnosis method than PSA testing was necessary for prostate cancer. When we detected expression levels of candidate miRNAs in urinary exosomes of prostate cancer patients and healthy controls, we observed that miR-107, miR-139, miR-145 and miR-204 were downregulated whereas miR-375-5p was upregulated in patients’ urinary exosomes. For miR-21, there was a very slight upregulation and miR-30a-5p and miR-574-3p levels were almost the same with healthy controls. Further, we wondered the diagnostic potential of these miRNAs in our patient cohort and we performed Receiver Operator Characteristic (ROC) Curve analysis for them. When they were used as combination, our miRNAs had 77% (AUC=0.7731) accuracy in distinguishing patients from healthy controls. After that, we examined miRNA dysregulations in patients with different PSA levels with the hypothesis that they may have different miRNA expression profiles. We saw that expression of miRNAs in exosomes patients with PSA<10 ng/ml and PSA=10-15 ng/ml were very similar with our expectations; downregulation of tumor suppressor and upregulation in oncogenic miRNAs whereas patients with PSA>15 ng/ml had a unique profile and all miRNAs were downregulated. Due to sample size limitations, we only tested diagnostic potential of candidate miRNAs in exosomes of patients with PSA<10 ng/ml and observed that AUC was 0.8500 so we could discriminate patients and healthy controls with 85% accuracy by using our candidate miRNA panel in testing. Taken together, our findings indicated that candidate urinary exosomal tumor suppressor and oncogenic miRNAs which we suggested in thesis are very potent in diagnosis of prostate cancer with differential expressions in prostate cancer patients with different PSA levels and this study opens the way for development of a noninvasive prostate cancer diagnosis method.
Open Access
RNA based biomarkers for prediction of the endometrial window of implantation
(2020-12) Dedeoğlu, Ege
Early reproductive failure is the most common issue related to successful pregnancies, as around 30% of all conceptions reach live birth. The path to a successful pregnancy is reliant on the successful implantation of the embryo to the endometrium. This event requires three major components; a viable embryo ready for implantation, a receptive endometrium in which the implantation will occur, and healthy crosstalk between the embryo and receptive endometrium. It is estimated that two out of all three implantation failures are related to endometrial origin. This has led many researchers to attempt to elucidate the mechanism behind endometrial receptivity and generate a prediction of successful implantation of endometrial origin. Although there have been plenty of articles on this subject, there is still no consensus regarding standard endometrial receptivity biomarkers. Additionally, most of these articles’ findings cannot find their way into clinics. This is highlighted by the fact that the success rate of embryo implantation in ART applied in clinics is only around 10%. This study aimed to identify novel methods and biomarkers to predict the endometrium's receptivity, which could be applied in clinics easier and faster than the current kits in the market. We took several different approaches to achieve this aim. The first was to identify and validate particular miRNAs that showed a change in expression levels of the different days of the endometrial cycle in a healthy women’s serum. Our bioinformatical analysis has yielded ten miRNAs that show statistical differences in the human endometrium and being expressed in the serum. Downstream RNA-Seq and qPCR experiments have validated specific miRNAs previously predicted and identified novel miRNAs used for this purpose. The second was using in silico methods, identifying novel genes present in the endometrium that can predict the optimal point of receptivity. If considered and validated in vitro, this novel gene-list will be a cheaper but still as powerful alternative to the current endometrial test kit used in clinics today. Further validation RNA-Seq experiments on healthy and infertile females will elucidate our novel biomarkers' strength, designed to be used in ART clinics worldwide. Furthermore, upon building on these findings, it is possible to uncover previously overlooked mechanisms leading to women's implantation success.
Open Access
Role of non-coding RNAs as novel biomarkers for detection of colorectal cancer progression through interaction with the cell signaling pathways
(Elsevier, 2020) Esmaeili, M.; Keshani, M.; Vakilian, M.; Esmaeili, M.; Peymani, M.; Forootan, F. S.; Chau, Tieu Lan; Göktuna, Serkan İsmail; Zaker, S. R.; Esfahani, M. H. N.; Ghaedi, K.
Colorectal cancer (CRC) is one of the most common types of cancer which affects the colon and the rectum. Approximately one third of annual CRC mortality occurs due to the late detection of this type of cancer. Therefore, there is an urgent need for more powerful diagnostic and prognostic tools for identification and treatment of colorectal tumorigenesis. Non-coding RNAs (ncRNAs) have been implicated in the pathology of CRC and also linked to metastasis, proliferation, differentiation, migration, angiogenesis and apoptosis in numerous cancers. Recently, attention has turned towards ncRNAs as specific targets for diagnosis, prognosis and treatment of various types of cancers, including CRC. In this review, we have tried to outline the roles of ncRNAs, and their involvement in signaling pathways responsible for the progression of CRC.