Browsing by Subject "CHRNA5"
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Item Open Access Analysis of CHRNA5 expression in breast cancer cell lines in response to serum starvation and estrogen treatment(Bilkent University, 2013) Açıkgöz, Azer AylinBreast cancer is a complex disease that can be classified into distinct molecular subtypes including Basal, Luminal A, Luminal B and HER2 positive. These molecular subtypes mainly differ in their hormone receptor expression and response to treatment. This makes the discovery of new molecular markers for further classification important. Cholinergic nicotinic acetylcholine receptors are ion channels involved in smoking behavior, neurodegenerative diseases and cancer. Cholinergic nicotinic receptor alpha 5 (CHRNA5) has been associated with nicotine addiction and recently with lung cancer yet its importance in breast cancer remains relatively unexplored. In the present study, a panel of 10 breast cancer cell lines were used for quantification of isoform-specific CHRNA5 expression using qPCR. Changes in CHRNA5 expression in response to serum starvation and estrogen treatment were assessed. qPCR showed that CHRNA5 was alternatively spliced, with at least five different isoforms in breast cancer cell lines. qPCR analysis for CHRNA5 expression in serum treated and serum starved cells were analyzed after outlier detection and exclusion; and statistical tests included ANCOVA using geometric mean of TPT1 and SDHA, as reference genes. Our results demonstrated that, CHRNA5 expression differed between different subtypes of breast cancer cell lines. CHRNA5 expression significantly responded to serum starvation in ZR75-1 and MDA-MB-157 cell lines, isoform specifically. Isoform expression of CHRNA5 exhibited significant alterations upon estrogen treatment in a dose and time-dependent manner. Expression of 1000bp variant, isoform2 and isoform3 of CHRNA5 significantly increased upon E2 treatment and total CHRNA5 and isoform2 CHRNA5 increased in expression at 24 hours when compared with 12 hours of treatment. Our findings show that CHRNA5 has multiple isoforms in breast cancer, with potential to be modulated by serum starvation and estrogen treatment in a cell-specific manner.Item Open Access CHRNA5 belongs to the secondary estrogen signaling network exhibiting prognostic significance in breast cancer(Springer, 2021-04) Shehwana, Huma; Keskus, Ayse Gokce; Ozdemir, E. Sila; Acikgöz, Azer Aylin; Biyik-Sit, Rumeysa; Cagnan, I.; Gunes, Damla; Jahja, Ermira; Cingir-Koker, Sahika; Olmezer, Gizem; Sucularli, Ceren; Konu, OzlenCholinergic signals can be important modulators of cellular signaling in cancer. We recently have shown that knockdown of nicotinic acetylcholine receptor subunit alpha 5, CHRNA5, diminishes the proliferative potential of breast cancer cells. However, modulation of CHRNA5 expression in the context of estrogen signaling and its prognostic implications in breast cancer remained unexplored.Item Open Access Development of a WEB application(Bilkent University, 2011) Kaya, Koray DoğanmicroRNAs, small non-coding RNA molecules with important roles in cellular machinery, target mRNAs for silencing by binding generally to their 3’ UTR sequences via partial base complementation. Thus, microRNAs with similar sequences also might exhibit expression and/or functional similarities. In this study, a modular tool, mESAdb (http://konulab.fen.bilkent.edu.tr/mirna/), was developed allowing for multivariate analysis of sequences and expression of microRNAs from multiple taxa. Its framework comprises PHP, JavaScript, packages in the R language, and a database storing mature microRNA sequences along with microRNA targets and selected expression data sets for human, mouse and zebrafish. mESAdb allows for: (i) mining of microRNA expression data sets for subsets of microRNAs selected manually or by a sequence motif; (ii) pair-wise multivariate analysis of expression data sets within and between taxa; and (iii) association of microRNA subsets with annotation databases, HuGE Navigator, KEGG and GO. mESAdb also permits user specified dataset upload for these analyses. Herein, utility of mESAdb was illustrated using different datasets and case studies. First, it was shown that microRNAs carrying the embryonic stem cell specific seed sequence, ‘AAGTGC’, were able to discriminate between normal and tumor tissues from hepatocellular carcinoma patients using dataset GSE10694. Second, mRNA targets of a set of liver specific microRNAs were annotated with human diseases based on HuGE Navigator. Third, the similarity between mouse and human tissue specificity of a given set of microRNAs was demonstrated. Forth, CHRNA5 targeting microRNAs were associated with estrogen receptor status in breast cancer using dataset GSE15885. Finally, a related tool under development for mRNA arrays planned for integration with mESAdb was presented.Item Open Access Development of novel tools for cancer diagnosis, prognosis and treatment using intra- or inter-species transcriptome metaanalysis(Bilkent University, 2017-08) Shehwana, HumaIn the past decades, a considerable number of studies have performed meta-analysis on large data collections to prioritize sets of genes, pathways or types/categories of disease focusing on either differential expression, survival analysis, or co-expression networks. However, not many web applications or databases have been developed from these studies thus findings largely remained restricted to the addressed questions and it was not possible for other researchers to use the collected data for the evaluation of novel hypotheses. In this thesis, transcriptomic meta-analysis strategies have been applied to untangle complexities in multiple aspects of cancer research including treatment, diagnosis, and prognosis. Furthermore, three different web-tools have been developed which are not limited to a single type of meta-analysis. In this context, in addition to interesting cancer related findings, novel methodologies have been proposed and tested in the field of meta-analysis and cancer research. First chapter of the thesis presented a general introduction on the concepts of the thesis. Second chapter focused on a pathway comparison strategy based on meta-analysis that was used to reveal concordant/discordant aspects of rapamycin-mediation on transcriptomes of zebrafish and mouse. Analysis has shown that ribosomal terms were significantly upregulated while proteasome was downregulated in both species. Zebrafish has undergone a whole-genome duplication event; I also found out that rapamycin treatment resulted in largely concordant behavior of duplicated gene pairs. In addition, an online database, CompariZome, was developed to evaluate the duplicate zebrafish gene pairs in multiple GEO datasets in zebrafish in comparison to respective human expression datasets. In the third chapter of this thesis, I focused on identification of correlation between a trio of genes, CDH1, HNF4A, and GRHL3, using Cancer Cell Line Encyclopedia (CCLE) dataset to reveal the significance of association between these genes in different cancers, including breast and other epithelial cancers. The findings indicated correlation within the module and has demonstrated the power of meta-analysis using CCLE dataset. In the fourth chapter of this thesis, I focused on understanding the association of CHRNA5, a subunit of cholinergic receptors, with epithelial-to-mesenchymal transition (EMT) as well as epithelial differentiation, TP53 induction, and estrogen (E2) signaling with respect to breast cancer. Meta-analysis of invitro and in-vivo microarray expression datasets showed that CHRNA5, itself, and its positively co-expressed neighbors, were likely secondary targets of E2-signaling; overexpressed in ER- breast cancer patients; and indicators of worse prognosis. Functional annotation revealed that CHRNA5 and its co-expression network was indeed associated with proliferation related pathways. Based on meta-analysis of different cohorts processed in the study, an online database E2S (Estrogen (E2) to Survival) was developed that can facilitate user to query any gene for evaluation of E2-mediated effects, regulation by estrogen receptor (ER), prognostic importance and co-expression network along with functional annotations. In the fifth chapter of this thesis I focused on deciphering the correlation and deregulation between a human parolog pair of genes, i.e., mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) in breast cancer. Meta-analysis of a separate normal/tumor cohort revealed that both genes were downregulated in breast cancer and their expressions were highly positively correlated. However, deregulation analysis predicted that expression of MR and GR was more tightly regulated in normal breast hence its regulation might be lost with the onset of tumorigenesis. Another Shiny database, DualExpBC, was developed to evaluate differential expression of a gene in breast cancer as well as correlation and deregulation of expression between any two input genes in the breast normal/cancer expression cohort. With this thesis, I have developed novel tools and approaches for intra- and inter-species comparative transcriptomics and meta-analysis providing potential diagnostic, prognostic and therapeutic biomarkers.Item Open Access Effects of Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi on apoptosis, DNA damage response, drug sensitivity, and HSA-MIR-495-3P overexpression in breast cancer(Bilkent University, 2018-12) Köker, Şahika CıngırCholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is associated with nicotine addiction and it has an important role in the prognosis of lung cancer. Despite its important cellular functions, its role in breast cancer remains to be elucidated. In this thesis, I aimed to identify the alterations in the important cancer signaling pathways occurring upon CHRNA5 depletion. Drug resistance is one of the major obstacles in breast cancer therapy. Heterogeneous nature of breast cancer necessitates identification of more biomarkers which aid in precise diagnosis and hence development of proper treatment options. In this study, by using more than one cell line which is representative of different subtypes of breast cancer, I showed the alterations occurred in cancer signaling pathways such as cell cycle and apoptosis upon CHRNA5 depletion, which could serve as a novel biomarker in breast cancer subtyping. Depending on mutation status of TP53, which is the gatekeeper protein during G1/S checkpoint, CHRNA5 depletion mostly exerted its effects over decreasing the levels of total CHEK1 and pCHEK1 (S345) which significantly altered the response of MCF7 cells to topoisomerase inhibitors in terms of enhanced drug sensitivity. Increases in apoptotic markers, such as BAX/BCL2 ratio along with increased FAS levels, further confirmed that this sensitization of MCF7 cells upon CHRNA5 depletion might have ended with apoptosis. So far in the literature, there is no study examining the regulation of CHRNA5 by small endogenous molecules such as miRNAs. Due to the predictive binding sites in 3’UTR of CHRNA5 and the importance of participating in tamoxifen resistance in breast cancer; I also examined the interplay between miR-15a family and CHRNA5 in MCF7 cells. I showed significant decrease in CHRNA5 levels upon using miR-15a mimic while demonstrating similar activity of miR-15a family mimics with CHRNA5 depletion using RT-qPCR. Another important implication of CHRNA5 depletion in MCF7 cells was the global change in miRNA expression prolife which was verified with independent microRNA arrays. Based on these in silico results, hsa-miR-495-3p appeared as the most downregulated miRNA which is known as a tumor suppressor miRNA. As stated in the literature, the role of miR-495 differs depending on the tumor type. Therefore, I tried to restore its expression by mimicking along with CHRNA5 depletion. The transcriptomic changes observed with CHRNA5 depletion was boosted with the restoration of miR-495 levels.Item Open Access Effects of depletion of CHRNA5 and/or TP53, and transient and stable overexpression of wildtype or mutant TP53 on expression of DLK1-MEG3 locus in MCF7 cells(Bilkent University, 2022-09) Arıcı, Burçin İremThe expression of CHRNA5 has a prominent role in lung cancer and nicotine addiction. Besides, depletion of CHRNA5 has been identified as being tumour suppressive in breast cancer. Moreover, CHRNA5 depletion causes increases in CDKN1A expression, downregulates the 14q32.31 miRNAs and decreases DLK1 expression. This thesis examined the effects of CHRNA5 and/or TP53 downregulation, as well as TP53 overexpression, on the expression of the DLK1-DIO3 region. My findings demonstrated that CHRNA5 depletion decreased the expression of the protein-coding DLK1 and the non-protein coding MEG3 genes in this locus in the presence or absence of TP53. Since these two genes have a vital role in tumour suppression and tumorigenesis, this provided more evidence for the importance of CHRNA5 in the modulation of expression in this locus in breast cancer. Since there is a relation between CHRNA5 and TP53 induction, the expression of one of the main TP53 regulators, MDM2, was also studied. Accordingly, it was found that combining CHRNA5 depletion with TP53 depletion caused a significant decrease in expression in both the MDM2 and MDM2 sequestering elements, PDLIM7 and CDH18. Additionally, the effects of both wild-type and mutant TP53 expression levels on DLK1-MEG3 locus and CHRNA5 were investigated in stable MCF7 breast cancer cells that we have generated. I have found that even if CHRNA5 depletion induced p53 expression, TP53 overexpression did not have CHRNA5-inducing effects regardless of the functionality of TP53. However, wild-type TP53 overexpressing MCF7 cells behaved differently than mutant TP53 overexpressing cells in their DLK1 expression levels. Future research should clarify the effects of CHRNA5 depletion on DLK1-MEG3 region for a given TP53 mutation status.Item Open Access Effects of miR-376 family miRNAs on CHRNA5 depleted MCF7 cell line model and co-culture competition studies(Bilkent University, 2019-07) Tiryaki, Rafed SaidCholinergic receptor nicotinic alpha 5 (CHRNA5) is a ligand-gated ion channel and one of the subunits of nicotinic acetylcholine receptors. Role of CHRNA5 in tumorigenesis has been initially shown in the lung tissue in which higher CHRNA5 expression has been significantly correlated with worse prognosis in lung cancer. In addition, our laboratory members recently shown that CHRNA5 depletion in breast cancer cell line MCF7 is antiproliferative (TUBITAK 111T316). In present study, effects of CHRNA5 depletion on miRNA expression profile were investigated and a significant decrease in the expressions of two members of the miR-376 family miRNAs, miR-376a-3p and miR-376c-3p, were identified. To test the effects of these two miRNAs, mimics were used in combination with CHRNA5 depletion on MCF7 cell line model. To investigate the synergism and/or antagonism of miR-376a mimic with CHRNA5 siRNA treatment a microarray study was performed and the signaling pathways involved were identified. Expressions of genes of interest were tested with RT-qPCR for both miRNAs. In addition, the effects of rescue on the cell phenotype and viability were also studied by using phalloidin staining and MTT experiments, respectively. Next a co-culture-based competition assay was developed using MCF7 cell lines expressing different fluorescent molecules to assess competition by both flow cytometer and fluorescent imaging. In summary, the results revealed that combinational treatments of si-CHRNA5 together with the miRNA mimics of two members from miR-376 family revealed enhancement of the antitumor effects. This study has been supported by TUBITAK (grant no. 114S367).Item Open Access hsa-miR-497 as a modulator of the expression in the presence or absence of chrna5 in breast cancer(Bilkent University, 2017-06) Özgürsoy, BaşakCHRNA5 is an important ligand-gated receptor with roles in addiction and in cancer. In lung cancer, CHRNA5 dysregulation is well known. There is also expression of CHRNA5 in breast cancer cell lines. microRNAs regulate mRNA expression; and different regulatory microRNAs are involved in different cancer types. microRNAs are thus potential biomarkers to diagnose the diseases (e.g. cancer). However, there is no study testing interactions between microRNAs and CHRNA5 in breast cancer. In the present study, mir-497 was found to be one of the most downregulated microRNAs with testable expression levels upon analysis of expression in the breast cancer cell line MCF7 when exposed to CHRNA5 siRNA. RT-qPCR was performed to test the expression level of mir-497. Validated target genes of mir-497 were found to be significantly related to a list of different KEGG pathways significantly (p value < 0.001) among which there were P53 and PI3K-Akt signalling pathways. Mimic-mir-497 treatment, alone or together with CHRNA5 siRNA, was applied on MCF7 cells to understand the interaction between the miRNA and siRNA under investigation. Selected target genes of mir-497 were tested; the most significantly modulated genes were involved in P53 pathway. The results indicated interactions between mir-497 and CHRNA5 however selected targets were not affected by mimicmir- 497. GSE41079 and GSE41074 public datasets containing mRNA and microRNA expression profiles of liver cancer cells treated with mimic-mir-497. Treatment were used to identify novel targets of mir-497 for future use. Immune system was detected in the second place upon REACTOME analysis of GSE41079 and GSE41074. Using multiple online microRNA-mRNA network tools mir-497 mRNA-miRNA networks were extracted for all or only immune genes. The results from network based analyses helped identify additional targets for later use in our mimic-siRNA system.Item 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(Bilkent University, 2021-09) Keşküş, Ayşe GökçeCholinergic 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.Item Open Access Investigation of novel RNAi and nanoparticle approaches for their anti-proliferative and drug-sensitizing effects in breast cancer(Bilkent University, 2017-08) Jahja, ErmiraDrug resistivity remains a major challenge in treating different cancer types. Among several strategies adapted to increase drug sensitivity in breast cancer cells, in the present thesis I studied an RNAi molecule targeting cholinergic receptor nicotinic alpha 5 subunit (CHRNA5) and a red-emitting oligomer nanoparticle, the two agents which I experimentally identified as negative regulators of cell proliferation. Cholinergic signaling is implicated in several different pathologies including cancer. Nicotinic acetylcholine receptors (nAChRs) are shown to be involved in regulation of cell proliferation, however they are mainly studied as mediators of nicotinic activity. CHRNA5 subunit has been shown to have roles in acetylcholine (ACh) production/stability, drug addiction and susceptibility to lung cancer. Few studies of lung and gastric cancers as well as high throughput RNAi screens show CHRNA5 as a modulator of cell proliferation. In the present study multiple CHRNA5 isoforms were cloned from MCF7 breast cancer cells (ER positive, TP53 positive) as in the case of lung cancer; moreover, a significant antimitotic effect of CHRNA5 RNAi application was demonstrated in MCF7 breast cancer cells. Similar effect of CHRNA5 silencing was only partially observed in BT-20 and MDA-MB-231 cells (ER negative, P53 mutant), yet in a seeding density-dependent manner. For the first time in literature the transcriptomic changes associated with CHRNA5 RNAi in the MCF7 cells were studied by microarrays from which differentially expressed gene lists were used to obtain the affected pathways. Additional assays confirmed the reduction in cell viability, DNA synthesis, G1 growth arrest, and changes in cytoskeleton complementing the microarray studies. Use of camptothecin (CPT) and doxorubicin (DOXO) in the absence or presence of CHRNA5 siRNA in MCF7, led to identification of CHRNA5’s role in drug sensitivity. Comparisons between CHRNA5 siRNA and public microarray datasets revealed common genes/networks between topoisomerase (TOPO)/cyclin-dependent kinase (CDK) inhibitors and CHRNA5 depletion profile in MCF7 cells. mRNA-miRNA network analysis of differentially expressed common gene sets between TOPO inhibitors and CHRNA5 RNAi treatment identified potential common regulatory miRNAs. In an independent study the anti-cancer as well as drug sensitivity associated effects of a novel CB7-capped, red-emitting conjugated oligomer nanoparticle (Red-CON) were characterized in MCF7 and MDA-MB-231 cells. Red-CON in its encapsulated form exhibited low toxicity and good efficacy as a drug delivery system. This nanoparticle formulation might serve well for future clinical and less toxic chemotherapeutic regimens.Item Open Access qPCR validation of in vivo diagnostic importance and regulation by estrogen for CHRNA5 isoform expression in breast cancer(Bilkent University, 2014) Özdemir, Emine SılaBreast cancer has multiple molecular subtypes; normal-like, basal-like, luminal A, luminal B and HER2 positive depending on receptor status of tumor cells. Cancer therapy is tailored according to the type of cancer; hence finding new diagnostic markers is important to decide on the best treatment approach. Cholinergic nicotinic receptor alpha 5 (CHRNA5) is one of the subunits of nicotinic acetylcholine receptors with significant roles in addiction and cancer. In the present study, CHRNA5 has been validated as an estrogen and/or Estrogen receptor (ER) modulated nicotinic acetylcholine receptor by qPCR in in vitro and in vivo in breast cancer samples. CHRNA5 isoform expression was measured using in vitro cell culture studies in which ER- and ER+ cell lines treated with different doses of estradiol (E2); MCF7 cell line was exposed to long-term E2 depletion, in another experiment it was treated with tamoxifen (4-OHT), an ER antagonist, and with or without E2. We found that all CHRNA5 isoforms exhibited increased expression in response to E2 dose-dependently in the ER+ MCF7 cell line while in the ER- MDAMB-231 cell line CHRNA5 isoform expression response was variable in direction and magnitude. CHRNA5 isoform expression in general steadily decreased in ER+ cell line MCF7 after 4-OHT treatment. After six months of E2 depletion, ER+ MCF7 cell line had increased CHRNA5_v3 isoform and ESR1 (ER gene) mRNA expression. In vivo, a human breast cancer cDNA panel was scanned with specially designed primers with qPCR using a custom-written GUI in MATLAB. It was found that CHRNA5, showing a statistically significant difference between normal and tumor cDNA, was a good candidate gene in diagnosis of breast cancer. CHRNA_v3 was able to distinguish between ER+ vs ER- breast tumor samples. We also addressed whether CHRNA5 isoforms exhibited differences in distinguishing tumor stage, and HER2 status. Our findings showed that expression of CHRNA5 isoforms were correlated with each other and regulated by E2 in breast cancer depending on ER receptor status.