Differential loading of the Argonaute complex in Epstein-Barr Virus (EBV)-infected cell lines derived from diffuse large B-cell lymphoma (DLBCL)

MicroRNAs sind essentielle post-transkriptionelle Regulatoren der Genexpression die an unterschiedlichen Prozessen wie Entwicklung, Signaltransduktion oder Wachstumskontrolle durch Bindung und Inhibition der Translation ihrer mRNA Ziel mRNAs beteiligt sind. Obwohl angenommen wird, dass ihre relative Expression in der Zelle das Ausmaß ihrer biologischen Funktion bestimmt, zeigt die Beladung der „Argonaute“ (Ago)-mRNA Komplexe mit miRNAs deren Relevanz besser an. In der vorliegenden Arbeit wurde die miRNA Beladung der Ago-Komplexe mit den miRNA Profilen der Gesamt-RNA zweier diffus-großzelliger B-Zell Lymphome (DLBCL) Linien nach Epstein-Barr Virus (EBV) -Infektion, verglichen. DLBCL ist ein hochmaligner Tumor, welcher in Aktivierte B-Zell (ABC-) und Germinal Center B-Zell (GCB-) DLBCL unterschieden wird. Etwa 10% aller DLBCLs sind mit dem onkogenen EBV, welches selbst 44 reife miRNAs exprimiert, infiziert. Die miRNA Profile aus Gesamtzellextrakten und des Ago2-Profils zweier DLBCL Linen wurden durch Hochdurchsatzsequenzierung (RNA-Seq) analysiert. In den Gesamt-Profilen konnten zwischen 713-851 humane miRNAs identifiziert werden, während die Ago2-Profile 1102-1372 humane miRNAs ergaben. Bei Anwendung eines 0.1% „cut-offs“ verringerte sich die Zahl der funktionellen miRNAs auf 56-62., welche jedoch immer noch 92-95% aller miRNAs-reads ausmachten. In Abhängigkeit von Latenztyp repräsentierten die viralen miRNAs 1.6% - 28% aller „reads“. In beiden Zelllinien hatte die EBV-Infektion den Verlust von miRNAs im Ago2-Komplex zur Folge. Die erhobenen Daten konnten für ausgewählte miRNAs durch RT-qPCR und im Northern Blot bestätigt werden. Auch die Analyse der Ago2-Komplexe der einzelnen Zellen ergab eine auffällige Umverteilung miRNAs zwischen Gesamt- und Ago2-assoziierten miRNAs. Zum Beispiel war die miRNA miR-423 in allen Zellen 6-8-fach im Ago2-Komplex angereichert, während die miR-142 im Ago2-Komplex depletiert vorliegt , was auf einen Funktionsverlust hinweist. Die hier erhobenen Daten werden zum weiteren Verständnis des Beitrags von miRNAs bei der Entstehung und Aufrechterhaltung von DLBCLs leisten.MicroRNAs are important post-transcriptional regulators of gene expression in all eukaryotic cells and play essential roles, i.e. in development, signal transduction or growth control by binding to and inhibiting the translation of their mRNA targets. While it is widely assumed that their overall level in a cell reflects their functional relevance, the loading and transfer of miRNAs to the “Argonaute” (Ago)-mRNA complex appears to give a more accurate indication of their activity in a cell. In this thesis, the miRNA loading of the Ago complex was compared with the total cellular miRNA profile of two cell lines derived from diffuse large B-cell lymphoma (DLBCL) in comparison with their Epstein-Barr Virus infected counterparts. DLBCL is a highly malignant tumor subdivided into Activated B-cell (ABC-) and Germinal Center B-cell (GCB-) subtypes. About 10% of DLBCL are infected with the oncogenic Epstein-Barr virus which itself encodes 44 mature miRNAs. The cellular and Ago2-bound miRNAs in two DLBCL lines were subjected to ultra-deep sequencing (RNA-seq). In the total profiles, 713-851 human miRNAs were detected while the Ago2-immunoprecipitation (Ago2-IP) resulted in 1102-1372 different miRNAs. When a 0.1% cut-off was applied which is considered to yield the functionally relevant miRNAs, only between 56-62 miRNAs were left. These, however, represented 92-95% of all reads. In the two EBV-infected counterparts, EBV-miRNAs represented between 1.6% - 28% of all reads depending on the viral latency type. In the case where EBV miRNAs accounted for 28 % of total reads cellular miRNAs were replaced from the Ago2-complex. The results could be confirmed for selected miRNAs by RT-qPCR and Northern blotting. Also within each cell line, various miRNAs were enriched or depleted from the Ago2 complex. For instance, mir-423-5p and mir-423-3p were highly enriched, by over 6-8-fold in Ago2-IP, compared to total cellular profile in all cell lines while miR142 was strongly depleted from the Ago2-complex indicating a functional loss in DLBCL. The data obtained will help to further understand the contribution of cell and viral miRNAs in induction and maintenance of DLBCL

Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma

Extracellular vesicles (EVs) are secreted by healthy and tumor cells and are involved in cell–cell communication. Tumor-released EVs could represent a new class of biomarkers from liquid biopsies. The aim of this study was to identify tumor-specific EV markers in clear cell renal carcinoma (ccRCC) using cell lines and patient-derived tissue samples. EVs from ccRCC cell lines (786-O, RCC53, Caki1, and Caki2) and patient tissues were isolated via ultracentrifugation. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting using exosome and putative tumor markers (epithelial cell adhesion molecule (EpCAM), carbonic anhydrase 9 (CA9), CD70, CD147). The tumor markers were verified using immunohistochemistry. CA9 was expressed in Caki2 cells and EVs, and CD147 was found in the cells and EVs of all tested ccRCC cell lines. In tumor tissues, we found an increased expression of CA9, CD70, and CD147 were increased in cell lysates and EV fractions compared to normal tissues. In contrast, EpCAM was heterogeneously expressed in tumor samples and positive in normal tissue. To conclude, we developed an effective technique to isolate EVs directly from human tissue samples with high purity and high concentration. In contrast to EpCAM, CA9, CD70, and CD147 could represent promising markers to identify tumor-specific EVs in ccRCC

Evaluation of IL-17A and IL-17F genes polymorphism in Iranian dyspeptic patients

Helicobacter pylori (H.pylori) colonize the gastric mucosa of approximately 50 of the world's population that involved in chronic gastritis. The relationship between Hp colonization and gastric inflammation is widely accepted. Polymorphisms in inflammation related genes such as cytokines were thought to partly determine the outcome of Hp infection and progression of gastritis. Interleukin IL -17A and IL-17F are inflammatory cytokines expressed by a novel subset of CD4+ Th cells, play important function in inflammation. Aimed: we evaluate association of IL-17A G197A and IL-17F A7488G polymorphisms with gastritis, Polymorphonuclear (PMN) and Monoculear (MN) infiltration in related to Hp. Methods: According to rapid urease test, PCR 16srRNA, urea and histological examination of biopsies, patients were classified Hp-infected and Hp-uninfected. The histological severity of gastritis was graded from normal to severe based on the degree of MN cell and PMN leukocyte infiltration, chronic gastritis and chronic active gastritis. Polymorphism in IL-17A G197A and IL-17F A7488G were evaluated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: AG, GG, AG/AA carriers of IL-17A G197A and AA, GA, GG, GA/GG carriers of IL-17F A7488G polymorphisms were not associated with MN infiltration, PMN infiltration, chronic gastritis and Chronic active gastritis in Hp-infected and Hp-uninfected groups (p > 0.05). AA genotype of IL-17A G197A was related to chronic gastritis and PMN infiltration in Hp-uninfected group. Conclusion: IL-17A G197A substitution may be a risk factor for development gastritis in Hp-uninfected patients, also affect the pathway MN cell production pathways

miRNA Expression Characterizes Histological Subtypes and Metastasis in Penile Squamous Cell Carcinoma

Although microRNAs are described as promising biomarkers in many tumor types, little is known about their role in PSCC. Thus, we attempted to identify miRNAs involved in tumor development and metastasis in distinct histological subtypes considering the impact of HPV infection. In a first step, microarray analyses were performed on RNA from formalin-fixed, paraffin-embedded tumor (22), and normal (8) tissue samples. Microarray data were validated for selected miRNAs by qRT-PCR on an enlarged cohort, including 27 tumor and 18 normal tissues. We found 876 significantly differentially expressed miRNAs (p ≤ 0.01) between HPV-positive and HPV-negative tumor samples by microarray analysis. Although no significant differences were detected between normal and tumor tissue in the whole cohort, specific expression patterns occurred in distinct histological subtypes, such as HPV-negative usual PSCC (95 differentially expressed miRNAs, p ≤ 0.05) and HPV-positive basaloid/warty subtypes (247 differentially expressed miRNAs, p ≤ 0.05). Selected miRNAs were confirmed by qRT-PCR. Furthermore, microarray data revealed 118 miRNAs (p ≤ 0.01) that were significantly differentially expressed in metastatic versus non-metastatic usual PSCC. The lower expression levels for miR-137 and miR-328-3p in metastatic usual PSCC were validated by qRT-PCR. The results of this study confirmed that specific miRNAs could serve as potential diagnostic and prognostic markers in single PSCC subtypes and are associated with HPV-dependent pathways

Characterization of CD147, CA9, and CD70 as Tumor-Specific Markers on Extracellular Vesicles in Clear Cell Renal Cell Carcinoma

Extracellular vesicles (EVs) are secreted by healthy and tumor cells and are involved in cell–cell communication. Tumor-released EVs could represent a new class of biomarkers from liquid biopsies. The aim of this study was to identify tumor-specific EV markers in clear cell renal carcinoma (ccRCC) using cell lines and patient-derived tissue samples. EVs from ccRCC cell lines (786-O, RCC53, Caki1, and Caki2) and patient tissues were isolated via ultracentrifugation. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting using exosome and putative tumor markers (epithelial cell adhesion molecule (EpCAM), carbonic anhydrase 9 (CA9), CD70, CD147). The tumor markers were verified using immunohistochemistry. CA9 was expressed in Caki2 cells and EVs, and CD147 was found in the cells and EVs of all tested ccRCC cell lines. In tumor tissues, we found an increased expression of CA9, CD70, and CD147 were increased in cell lysates and EV fractions compared to normal tissues. In contrast, EpCAM was heterogeneously expressed in tumor samples and positive in normal tissue. To conclude, we developed an effective technique to isolate EVs directly from human tissue samples with high purity and high concentration. In contrast to EpCAM, CA9, CD70, and CD147 could represent promising markers to identify tumor-specific EVs in ccRCC

Epstein-Barr Virus Infection of Cell Lines Derived from Diffuse Large B-Cell Lymphomas Alters MicroRNA Loading of the Ago2 Complex

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoid tumor which is occasionally Epstein-Barr virus (EBV)-positive and is further subtyped as activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL, which has implications for prognosis and treatment. We performed Ago2-RNA immunoprecipitation followed by high throughput RNA sequencing (Ago2-RIP-Seq) to capture functionally active miRNAs in EBV-negative ABC-DLBCL and GC-DLBCL cell lines and their EBV-infected counterparts. In parallel, total miRNomes of these cells were sequenced to capture the cellular miRNA profile for comparison with the functionally active profile. Selected miRNAs with differential abundance were validated using RT-qPCR and Northern Blot. We found 6 miRNAs with differential abundance (2 upregulated and 4 downregulated miRNAs) between EBV-neg. and pos. ABC-DLBCL, and 12 miRNAs with differential abundance (3 upregulated and 9 downregulated miRNAs) between EBV-neg and -pos GC-DLBCL. Eight and twelve miRNAs were confirmed using RT-qPCR in ABC-DLBCL and GC-DLBCL, respectively. Selected miRNs were analyzed in additional type I/II vs. type III EBV latency DLBCL cell lines. Furthermore, up regulation of miR-221-3p and down regulation of let-7c-5p in ABC-DLBCL and up regulation of miR-363-3p and down regulation of 423-5p in GC-DLBCL was verified using RIP-Northern blot. Our comprehensive sequence analysis of the DLBCL miRNomes identified sets of deregulated miRNAs in the Ago2-RIP-seq. Our Ago2-IP-seq miRNomes profile could be considered as an important data set for detection of deregulated functionally active miRNAs in DLBCL and could possibly lead to identification of miRNAs as biomarkers for classification of DLBCL or even as targets for personalized targeted treatment

New insight to IL-23/IL-17 axis in Iranian infected adult patients with gastritis : effects of genes polymorphisms on expression of cytokines

Background and Objective : Chronic inflammation is the hallmark of the pathogenesis of H. pylori-induced gastric cancer. IL-17A and IL-17F are inflammatory cytokines expressed by a novel subset of CD4(+)Th cells and play critical function in inflammation. We evaluated the relationship between IL-17A G197A, IL-17F A7488G and IL23R+2199 A/C polymorphisms with IL-6, IL-17, IL-21, IL-23 and TGF-beta 1 mRNAs expression in regard to H. pylori infection with chronic gastritis. Materials and Methods : Total RNA and genomic DNA were extracted from gastric biopsies of 58 H. pylori-infected patient with gastritis. Afterward, mucosal IL-6, IL-17, IL-21, IL-23 and TGF-beta 1 mRNAs expression and polymorphisms in IL-17A G197A, IL-17F A7488G and IL-23R +2199A/Cin gastric biopsies were determined by real-time PCR and PCR-RFLP. Results : Our results show that IL-17A G197A, IL-17F A7488G andIL23R +2199A/C polymorphisms have no effect on mucosal expression of IL-6, IL-17, IL-21 and TGF-beta 1 mRNAs expression in H. pylori-infected patients with chronic gastritis. Conclusion : These results suggest that IL-17A G197A, IL-17F A7488G and IL23R +2199A/C polymorphisms no alter mucosal cytokine pattern in Iranian patients with H. pylori-associated gastritis diseases

miRNA Expression Characterizes Histological Subtypes and Metastasis in Penile Squamous Cell Carcinoma

Although microRNAs are described as promising biomarkers in many tumor types, little is known about their role in PSCC. Thus, we attempted to identify miRNAs involved in tumor development and metastasis in distinct histological subtypes considering the impact of HPV infection. In a first step, microarray analyses were performed on RNA from formalin-fixed, paraffin-embedded tumor (22), and normal (8) tissue samples. Microarray data were validated for selected miRNAs by qRT-PCR on an enlarged cohort, including 27 tumor and 18 normal tissues. We found 876 significantly differentially expressed miRNAs (p ≤ 0.01) between HPV-positive and HPV-negative tumor samples by microarray analysis. Although no significant differences were detected between normal and tumor tissue in the whole cohort, specific expression patterns occurred in distinct histological subtypes, such as HPV-negative usual PSCC (95 differentially expressed miRNAs, p ≤ 0.05) and HPV-positive basaloid/warty subtypes (247 differentially expressed miRNAs, p ≤ 0.05). Selected miRNAs were confirmed by qRT-PCR. Furthermore, microarray data revealed 118 miRNAs (p ≤ 0.01) that were significantly differentially expressed in metastatic versus non-metastatic usual PSCC. The lower expression levels for miR-137 and miR-328-3p in metastatic usual PSCC were validated by qRT-PCR. The results of this study confirmed that specific miRNAs could serve as potential diagnostic and prognostic markers in single PSCC subtypes and are associated with HPV-dependent pathways