Investigation of the target genes of BLV miRNAs and the expression levels of miR-B4-3p and miR-B2-5p in cattle infected with Bovine Leukemia Virus.

Bovine Leukemia Virus (BLV) is an oncogenic retrovirus of the genus Deltaretrovirus. The genome of BLV encodes a cluster of 10 mature microRNAs (miRNAs). Considering the importance of miRNAs in regulating gene expression, it seems that each of the miRNAs of BLV plays a vital role in the process of pathogenesis and tumorigenesis of the virus. First, sequences of each of the miRNAs of BLV were selected and downloaded from the miRBase database. The sequences were then investigated using TargetScan and miRWalk to identify target genes of each of the mature miRNAs of the virus. Second, the expression levels of the two miRNAs with the highest number of target genes in B lymphocytes and lymphoid tissues were evaluated using qPCR and were compared between cattle with different forms of BLV infection: PL form was compared to aleukemic (AL) form (Group 1) and BLV+ with normal lymph nodes were compared to lymphosarcoma form (Group 2). We identified a total of 1595 target genes of the micro RNAs. The miRNAs with the highest target genes included miR-B4-3p with 760 and B2-5p with 102 target genes. In the second phase, miRNA expression in BLV-infected animals was investigated. The Fold Change (FC) values for miR-B4-3p and miR-B2-5p in group 1 were 22 and 67, respectively. In the second group, the FCs for miR-B4-3p and miR-B2-5p were 47 and 133, respectively. The expression was significantly higher in persistent lymphocytosis (PL) cattle in group one and lymphosarcoma cattle in group two.

Evaluation of the Function of a Rare Variant in the 3'-Untranslated Region of the ß-Globin Gene.

ß-Thalassemia (ß-thal) is an inherited genetic disease that occurs because of the absence or reduction of ß-globin chain synthesis. Genetic changes occur in different regions of the ß-globin gene, but these mutations are less reported in the 3' untranslated region (3'-UTR). The objective of the present investigation was to evaluate the functional effect of a rare variant in the 3'-UTR of the ß-globin gene. A variant at the first nucleotide of the 3'-UTR of the ß-globin gene (HBB: c.*1G > A) was identified by DNA sequencing in an individual with low hematological indices and a normal hemoglobin (Hb) electrophoresis pattern. To evaluate the functional effect of this variant, the normal and mutated 3'-UTR of the ß-globin gene was synthesized separately and sub cloned in the psiCHEK2 vector. Next, using the calcium phosphate method, the psiCHEK2 vectors containing normal and mutated 3'-UTR were transfected separately into the HEK293T cell line. Finally, the transfected cell line was analyzed by dual luciferase assay. The ratio of Renilla to firefly for the mutant sample was 1.26 ± 0.06, while for normal samples it was 1.12 ± 0.04. The results of the luciferase assay showed that there was no significant difference in the functional effect between the mutant and wild type construct. Therefore, it was concluded that this variant might not reduce the expression of the ß-globin gene. Future studies by globin chain synthesis or to evaluate the expression of the gene in erythroid cells, might be necessary to understand the regulatory function of this mutation.

Correction to: CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy.

[This corrects the article DOI: 10.1186/s12935-020-01546-8.].

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy.

Cancer immunotherapy has been emerged as a promising strategy for treatment of a broad spectrum of malignancies ranging from hematological to solid tumors. One of the principal approaches of cancer immunotherapy is transfer of natural or engineered tumor-specific T-cells into patients, a so called "adoptive cell transfer", or ACT, process. Construction of allogeneic T-cells is dependent on the employment of a gene-editing tool to modify donor-extracted T-cells and prepare them to specifically act against tumor cells with enhanced function and durability and least side-effects. In this context, CRISPR technology can be used to produce universal T-cells, equipped with recombinant T cell receptor (TCR) or chimeric antigen receptor (CAR), through multiplex genome engineering using Cas nucleases. The robust potential of CRISPR-Cas in preparing the building blocks of ACT immunotherapy has broaden the application of such therapies and some of them have gotten FDA approvals. Here, we have collected the last investigations in the field of immuno-oncology conducted in partnership with CRISPR technology. In addition, studies that have addressed the challenges in the path of CRISPR-mediated cancer immunotherapy, as well as pre-treatment applications of CRISPR-Cas have been mentioned in detail.

Designing new generation of potent inhibitors against membrane-type matrix metalloproteinase-2: a computational effort against multiple myeloma.

Matrix metalloproteinases (MMPs) play important roles in cancer progression and, despite their inhibitors have failed in the clinical trials, they have always been considered as suitable targets for the treatment of tumor. We have recently shown that membrane type (MT) 2-MMPs, is selectively expressed in multiple myeloma (MM) cells and mediates the metastatic characteristics of these cells. In this study, we designed efficient inhibitors against MT2-MMP using state-of-art molecular modeling methods. First, the 3D structure of MT2-MMP was predicted. Then, the proposed potent inhibitors against two regions of the catalytic domain of MT2-MMP (active site and MT-LOOP) were identified through molecular docking, QM-MM and molecular dynamics simulations from a set of compounds in Analyticon library, IBS library, Maybridge screening fragment library and drugbank library. Moreover, ADME estimation showed that pharmacokinetic properties of inhibitors are in the acceptable range for humans. Finally, our data suggested that compounds 'structures.722' (dobutamine) and 'M2' are suitable candidates to inhibit MT2-MMP for further examination in the laboratory.Communicated by Ramaswamy H. Sarma.

Expression of miR-9 and miR-200c, ZEB1, ZEB2 and E-cadherin in Non-Small Cell Lung Cancers in Iran.

MicroRNAs (miRNAs) exert a critical influence on physiological and pathological processes through posttranscriptional modification of their mRNA targets. They play important roles in tumorigenesis and are considered to be potential diagnostic and prognostic biomarkers with various cancers. MiR-200c and miR-9 are regulatory elements that can have dual impacts as oncogenes and/or tumor suppressor genes. MiR-200c regulates two transcription factors, ZEB1 and ZEB2, while miR-9 is a regulatory factor for the E-cadherin protein which has a critical function in cell-cell junctions and is inhibited by two transcription factors ZEB1 and ZEB2. In this study, expression levels of miR-200c and miR-9, ZEB-1, ZEB-2 and E-cadherin were assessed in 30 non-small cell lung cancers (NSCLCs) by real-time qPCR. MiR-9 was down-regulated significantly in tumor tissues compared to normal adjacent tissues, while there was no significant change in expression level of miR-200c. On the other hand, ZEB1 demonstrated significant increase and ZEB2a decrease at the mRNA level. These results indicate roles for miR-9 and ZEB1 in genesis of lung cancer, although clinico-pathological associations were not evident. Further studies are necessary to assess implications for treatment of lung cancer.

Clinically Significant Dysregulation of hsa-miR-30d-5p and hsa-let-7b Expression in Patients with Surgically Resected Non-Small Cell Lung Cancer.

BACKGROUND: The cyclin E2 (CYCE2) is an important regulator in the progression and development of NSCLC, and its ectopic expression promoted the proliferation, invasion, and migration in several tumors, including Non-Small Cell Lung Cancer (NSCLC). However, the upregulation of CYCE2 in NSCLC cells suggested that it has a key role in tumorigenicity. In addition, the RAS family proteins as oncoproteins were activated in many major tumor types and its suitability as the therapeutic target in NSCLC was proposed. Considering the crucial role of microRNAs, it was hypothesized that altered expression of hsa-miR-30d-5p and hsa-let-7b might provide a reliable diagnostic tumor marker for diagnosis of NSCLC. METHOD: Real-time RT-PCR approach could evaluate the expression alteration of hsa-miR-30d-5p and hsa-let-7b and it was related to the surgically resected tissue of 24 lung cancer patients and 10 non-cancerous patients. The miRNAs expression was associated with clinicopathological features of the patients. RESULTS: Hsa-miR-30d showed a significant downregulation (p=0.0382) in resected tissue of NSCLC patients compared with control group. Its expression level could differentiate different stages of malignancies from each other. The ROC curve analysis gave it an AUC=0.73 (p=0.037) which was a good score as a reliable biomarker. In contrast, hsa-let-7b was significantly overexpressed in tumor samples (p=0.03). Interestingly, our findings revealed a significant association of hsa-let-7b in adenocarcinoma tumors, compared to Squamous Cell Carcinomas (SCC) (p<0.05). Also, analysis of ROC curve of hsa-let-7b (AUC=0.74, p-value=0.042) suggests that it could be as a suitable biomarker for NSCLC. CONCLUSION: Together, these results suggest a possible tumor suppressor role for hsa-miR-30d in lung tumor progression and initiation. Moreover, upregulation of hsa-let-7b was associated with the tumor type.

Ral signaling pathway in health and cancer.

The Ral (Ras-Like) signaling pathway plays an important role in the biology of cells. A plethora of effects is regulated by this signaling pathway and its prooncogenic effectors. Our team has demonstrated the overactivation of the RalA signaling pathway in a number of human malignancies including cancers of the liver, ovary, lung, brain, and malignant peripheral nerve sheath tumors. Additionally, we have shown that the activation of RalA in cancer stem cells is higher in comparison with differentiated cancer cells. In this article, we review the role of Ral signaling in health and disease with a focus on the role of this multifunctional protein in the generation of therapies for cancer. An improved understanding of this pathway can lead to development of a novel class of anticancer therapies that functions on the basis of intervention with RalA or its downstream effectors.

Comparative Network Analysis of Patients with Non-Small Cell Lung Cancer and Smokers for Representing Potential Therapeutic Targets.

Cigarette smoking is the leading cause of lung cancer worldwide. In this study, we evaluated the serum autoantibody (AAb) repertoires of non-small cell lung cancer (NSCLC) patients and smokers (SM), leading to the identification of overactivated pathways and hubs involved in the pathogenesis of NSCLC. Surface- and solution-phase biopanning were performed on immunoglobulin G purified from the sera of NSCLC and SM groups. In total, 20 NSCLC- and 12 SM-specific peptides were detected, which were used to generate NSCLC and SM protein datasets. NSCLC- and SM-related proteins were visualized using STRING and Gephi, and their modules were analyzed using Enrichr. By integrating the overrepresented pathways such as pathways in cancer, epithelial growth factor receptor, c-Met, interleukin-4 (IL-4) and IL-6 signaling pathways, along with a set of proteins (e.g. phospholipase D (PLD), IL-4 receptor, IL-17 receptor, laminins, collagens, and mucins) into the PLD pathway and inflammatory cytokines network as the most critical events in both groups, two super networks were made to elucidate new aspects of NSCLC pathogenesis and to determine the influence of cigarette smoking on tumour formation. Taken together, assessment of the AAb repertoires using a systems biology approach can delineate the hidden events involved in various disorders.

Potential circulating miRNA signature for early detection of NSCLC.

Circulating microRNAs (c-miRNAs) are promising biomarkers for screening, early detection and prognosis of cancer. The purpose of this investigation was to identify a panel of c-miRNAs in plasma that could contribute to early detection of non-small cell lung cancer (NSCLC). We profiled the expression of 44 unique plasma miRNAs in training set of 34 NSCLC patients and 20 matched healthy individuals by miRCURY LNA™ Universal RT microRNA PCR Panel and calculated dysregulation fold changes using the 2-ΔΔCt equation. Selected plasma miRNAs were then validated by SYBR green q-RT PCR using an independent validation set of plasma samples from NSCLC patients (n: 72) and NC (n: 50). In the validation set, the receiver operating characteristic (ROC) curves were generated for four miRNAs. In the training set, 17 miRNAs were significantly up-regulated and nine were down-regulated in the plasma from NSCLC patients versus matched normal controls. Four miRNAs (miR-21, miR-328, miR-375 and miR-141) were selected for validating their diagnostic value in the testing set. ROC plot analysis showed that a high specificity (98%) and sensitivity (82.7%) in miR-141 in comparing early NSCLC patient and controls. So among these four plasma miRNAs only miR-141 could be promising biomarkers for early detection of NSCLC. In addition to, we found a significant positive correlation between stage and miR-21 expression level (95% CI: 0.687-0.863; p-value < 0.0001). Considering the accessibility and stability of circulating miRNAs, plasma miR-141 is a useful biomarker early detection of NSCLC as a supplement in future screening studies.

Abberent expression of oncogenic and tumor-suppressive microRNAs and their target genes in human adenocarcinoma alveolar basal epithelial cells.

CONTEXT: Lung cancer is one of the most serious types of cancer that often diagnosed at advanced stage. MicroRNAs (miRNAs) are small non-coding molecules which silence gene expression of target gene (s) at posttranscriptional level. They are key regulators of cell cycle, apoptosis, anti-cancer drug responsiveness and metastasis. AIMS: Identification of the differential expression level of miR-15a/16, miR-21, miR-34a, miR-126, miR-128 and miR-210 in A549 cell line versus normal tissues and their correlation with selected corresponding target genes. MATERIALS AND METHODS: A549 cell line was cultured in F-12K medium and miRNA was extracted from normal tissues (2-3 cm adjacent to tumor tissue) and A549 cell line. cDNA was synthesized with specific stem-loop primers for each miRNA, while OligodT primer was used for target genes cDNA synthesis. Real-time quantitative polymerase chain reaction. (RT-qPCR) was used to analyze the expression pattern of miRNAs and target genes in A549 and normal non-small cell lung carcinoma. (NSCLC) tissues. RESULTS: miR-15a/16, miR-34a, miR-126 and miR-128 were down-regulated significantly. (>2-fold change), while miR-21 and miR.210 were up-regulated in A549. Bcl-2 as miR-34a target gene was down-regulated while Hif-1α and Akt-3 were up-regulated that might be miR-210 and miR-34a target genes, respectively. CONCLUSION: The significant differential expression level of these miRNAs made them as candidate biomarkers in NSCLC tumor tissues of patients. Perhaps Bcl-2 down-regulation and Akt-3 up-regulation can be linked with survival signals in A549 cell line. We can conclude that Bcl-2 and Akt-3 might be therapeutic targets to inhibit cell proliferation in NSCLC.

Expression of miRNAs in non-small-cell lung carcinomas and their association with clinicopathological features.

Lung cancer is recognized as a leading cause of cancer-related deaths worldwide. Over the past several years, evidence emerged that microRNAs (miRNAs), a class of small non-coding RNA molecules regulating gene expression at posttranscriptional level, play an important role in cell functioning, as well as in human diseases. Here, we analyzed expression of miR-15a/16, miR-21, miR-34a, miR-126, miR-128, and miR-210 at transcriptional level in 30 non-small-cell lung carcinoma (NSCLC) tumor tissues compared to the matched adjacent normal tissues and their correlation with clinicopathological features of the patients. Samples were collected from the NSCLC patients undergoing surgery before radiotherapeutic or chemotherapeutic treatment. Expression levels of miRNAs were assessed by TaqMan RT-PCR assay. The data obtained in this study were processed using REST 2009 and SPSS statistical software. The graphs were designed by GraphPad prism 5.0. In tumor samples, we found downregulation of miR-15a/16 (50/83.3%), miR-34a (83.3%), miR-126 (70%), and miR-128 (63.3%). At the same time, miR-21 and miR-210 were upregulated by 53.3 and 66.6% in cancer tissue versus matched adjacent normal tissues, respectively. No significant correlation was found between the expression levels of miR-15a/16, miR-21, miR-34a, miR-126, miR-128, and miR-210 and lymph node, tumor size, sex, and smoking. However, the study demonstrated a correlation between a change in expression of miR-15, miR-16, miR-34a, miR-126, and miR-210 compared to normal tissues and TNM staging (P < 0.05). Furthermore, miR-126 expression level was different in adenocarcinomas and squamous cell carcinoma (SCC) subtype (P < 0.1). Detailed analysis revealed significant change in expression of miR-15a/16, miR-34a, miR-126, and miR-210 in NSCLC tumor samples indicating involvement of these miRNAs in lung cancer pathogenesis. miR-210 demonstrated the most consistent increase in tumor tissues between different patients, suggesting its potential significance for NSCLC.

Isolation and molecular identification of mycoplasma genitalium from the secretion of genital tract in infertile male and female.

BACKGROUND: Mycoplasmas can cause acute and chronic diseases at multiple sites with wide-range complications and have been implicated as cofactors in diseases. The infections influenced form genital mycoplasmas specifically Mycoplasma hominis and Mycoplasma genitalium potentially affect reproductive disorders, and infertility. OBJECTIVE: Isolation and molecular identification of Mycoplasma genitalium from the genital tract of infertile male and vaginal discharge of infertile female referred to Infertility Center of Kerman in 2013. MATERIALS AND METHODS: This study was a randomized, prospective study. We included 100 infertile male and 100 infertile female that were referred to the Infertility Center of Kerman. Then for isolation and molecular identification of Mycoplasma genitalium from urethral and vaginal discharge polymerase chain reaction was performed on Mycoplasma genus and genitalium. RESULTS: From a total of 100 semen samples 45 patients (45%) were mycoplasma-positive and 13 (28.8%) were genitalium species positive. Also, from a total of 100 women samples 43 women (43%) were mycoplasma-positive and 10 (23.2%) were genitalium species positive. Positive samples were sequenced and phylogenetic tree was drawn. CONCLUSION: According to the results of this study, a high percentage of infertile male and female were infected with the Mycoplasma genitalium. For prevention of harmful and significant consequences of this infection, we suggest a screening program in symptomatic infertile couples.

Inhibitory effects of ß-cyclodextrin-helenalin complexes on H-TERT gene expression in the T47D breast cancer cell line - results of real time quantitative PCR.

BACKGROUND: Nowadays, the encapsulation of cytotoxic chemotherapeutic agents is attracting interest as a method for drug delivery. We hypothesized that the efficiency of helenalin might be maximized by encapsulation in ß-cyclodextrin nanoparticles. Helenalin, with a hydrophobic structure obtained from flowers of Arnica chamissonis and Arnica Montana, has anti-cancer and anti-inflammatory activity but low water solubility and bioavailability. ß-Cyclodextrin (ß-CD) is a cyclic oligosaccharide comprising seven D-glucopyranoside units, linked through 1,4-glycosidic bonds. MATERIALS AND METHODS: To test our hypothesis, we prepared ß-cyclodextrin- helenalin complexes to determine their inhibitory effects on telomerase gene expression by real-time polymerase chain reaction (q-PCR) and cytotoxic effects by colorimetric cell viability (MTT) assay. RESULTS: MTT assay showed that not only ß-cyclodextrin has no cytotoxic effect on its own but also it demonstrated that ß-cyclodextrin- helenalin complexes inhibited the growth of the T47D breast cancer cell line in a time and dose-dependent manner. Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentration of ß-cyclodextrin-helenalin complexes increased. CONCLUSIONS: ß-Cyclodextrin-helenalin complexes exerted cytotoxic effects on T47D cells through down-regulation of telomerase expression and by enhancing Helenalin uptake by cells. Therefore, ß-cyclodextrin could be superior carrier for this kind of hydrophobic agent.

Molecular identification of Palearctic members of Anopheles maculipennis in northern Iran.

BACKGROUND: Members of Anopheles maculipennis complex are effective malaria vectors in Europe and the Caspian Sea region in northern Iran, where malaria has been re-introduced since 1994. The current study has been designed in order to provide further evidence on the status of species composition and to identify more accurately the members of the maculipennis complex in northern Iran. METHODS: The second internal transcribed spacer of ribosomal DNA (rDNA-ITS2) was sequenced in 28 out of 235 specimens that were collected in the five provinces of East Azerbayjan, Ardebil, Guilan, Mazandaran and Khorassan in Iran. RESULTS: The length of the ITS2 ranged from 283 to 302 bp with a GC content of 49.33-54.76%. No intra-specific variations were observed. Construction of phylogenetic tree based on the ITS2 sequence revealed that the six Iranian members of the maculipennis complex could be easily clustered into three groups: the An. atroparvus-Anopheles labranchiae group; the paraphyletic group of An. maculipennis, An. messeae, An. persiensis; and An. sacharovi as the third group. CONCLUSION: Detection of three species of the An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae, as shown as new records in northern Iran, is somehow alarming. A better understanding of the epidemiology of malaria on both sides of the Caspian Sea may be provided by applying the molecular techniques to the correct identification of species complexes, to the detection of Plasmodium composition in Anopheles vectors and to the status of insecticide resistance by looking to related genes.