In silico Analysis of Two Novel Variants in the Pyruvate Carboxylase (PC) Gene Associated with the Severe Form of PC Deficiency.

Background: Inborne errors of metabolism are a common cause of neonatal death. This study evaluated the acute early-onset metabolic derangement and death in two unrelated neonates. Methods: Whole-exome sequencing (WES), Sanger sequencing, homology modeling, and in silico bioinformatics analysis were employed to assess the effects of variants on protein structure and function. Results: WES revealed a novel homozygous variant, p.G303Afs*40 and p.R156P, in the pyruvate carboxylase (PC) gene of each neonate, which both were confirmed by Sanger sequencing. Based on the American College of Medical Genetics and Genomics guidelines, the p.G303Afs*40 was likely pathogenic, and the p.R156P was a variant of uncertain significance (VUS). Nevertheless, a known variant at position 156, the p.R156Q, was also a VUS. Protein secondary structure prediction showed changes in p.R156P and p.R156Q variants compared to the wild-type protein. However, p.G303Afs*40 depicted significant changes at C-terminal. Furthermore, comparing the interaction of wild-type and variant proteins with the ATP ligand during simulations, revealed a decreased affinity to the ATP in all the variants. Moreover, analysis of Single nucleotide polymorphism impacts on PC protein using Polyphen-2, SNAP2, FATHMM, and SNPs&GO servers predicted both R156P and R156Q as damaging variants. Likewise, free energy calculations demonstrated the destabilizing effect of both variants on PC. Conclusion: This study confirmed the pathogenicity of both variants and suggested them as a cause of type B Pyruvate carboxylase deficiency. The results of this study would provide the family with prenatal diagnosis and expand the variant spectrum in the PC gene,which is beneficial for geneticists and endocrinologists.

Efficient CRISPR/Cas9-Mediated BAX Gene Ablation in CHO Cells To Impair Apoptosis and Enhance Recombinant Protein Production.

Background: Despite recent advances in recombinant biotherapeutics production using CHO cells, their productivity remains lower than industrial needs, mainly due to apoptosis. Objectives: Present study aimed to exploit CRISPR/Cas9 technology to specifically disrupt the BAX gene to attenuate apoptosis in recombinant Chinese hamster's ovary cells producing erythropoietin. Materials and Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 technique. The single guide RNAs (sgRNAs) targeting identified gene (BAX) were designed, and CHO cells were then transfected with vectors. Afterward, changes in the expression of the Bax gene and consequent production rates of erythropoietin were investigated in manipulated cells, even in the presence of an apoptosis inducer agent, oleuropein. Results: BAX disruption significantly prolonged cell viability and increased proliferation rate in manipulated clones (152%, P-value = 0.0002). This strategy reduced the levels of Bax protein expression in manipulated cells by more than 4.3-fold (P-value <0.0001). The Bax-8 manipulated cells displayed higher threshold tolerance to the stress and consequence apoptosis compared to the control group. Also, they exhibited a higher IC50 compared to the control in the presence of oleuropein (5095 µM.ml-1 Vs. 2505 µM.ml-1). We found a significant increase in recombinant protein production levels in manipulated cells, even in the presence of 1,000 µM oleuropein compared to the control cell line (p-value=0.0002). Conclusions: CRISPR/Cas9 assisted BAX gene ablation is promising to improve erythropoietin production in CHO cells via engineering anti-apoptotic genes. Therefore, exploiting genome editing tools such as CRISPR/Cas9 has been proposed to develop host cells that result in a safe, feasible, and robust manufacturing operation with a yield that meets the industrial requirements.

Targeting long non-coding RNA MALAT1 reverses cancerous phenotypes of breast cancer cells through microRNA-561-3p/TOP2A axis.

Non-coding RNAs, including Inc-RNA and miRNA, have been reported to regulate gene expression and are associated with cancer progression. MicroRNA-561-3p (miR-561-3p), as a tumor suppressor, has been reported to play a role in preventing cancer cell progression, and MALAT1 (Lnc-RNA) have also been demonstrated to promote malignancy in various cancers, such as breast cancer (BC). In this study, we aimed to determine the correlation between miR-561-3p and MALAT1 and their roles in breast cancer progression. The expression of MALAT1, mir-561-3p, and topoisomerase alpha 2 (TOP2A) as a target of miR-561-3p was determined in BC clinical samples and cell lines via qRT-PCR. The binding site between MALAT1, miR-561-3p, and TOP2A was investigated by performing the dual luciferase reporter assay. MALAT1 was knocked down by siRNA, and cell proliferation, apoptotic assays, and cell cycle arrest were evaluated. MALAT1 and TOP2A were significantly upregulated, while mir-561-3p expression was downregulated in BC samples and cell lines. MALAT1 knockdown significantly increased miR-561-3p expression, which was meaningfully inverted by co-transfection with the miR 561-3p inhibitor. Furthermore, the knockdown of MALAT1 by siRNA inhibited proliferation, induced apoptosis, and arrested the cell cycle at the G1 phase in BC cells. Notably, the mechanistic investigation revealed that MALAT1 predominantly acted as a competing endogenous RNA in BC by regulating the miR-561-3p/TOP2A axis. Based on our results, MALAT1 upregulation in BC may function as a tumor promoter in BC via directly sponging miRNA 561-3p, and MALAT1 knockdown serves a vital antitumor role in BC cell progression through the miR-561-3p/TOP2A axis.

K-Ras4A Plays a More Significant Role than K-Ras4B in Ductal Carcinoma of Breast.

Background: K-Ras mutations rarely occur in breast cancer. However, studies have supported that K-Ras upregulation is involved in breast cancer pathogenesis. Two main K-Ras transcript variants; K-Ras4A and K-Ras4B, originate from the alternative splicing of exon 4. In this study, we aimed to evaluate variations in the expression of K-Ras4A and K-Ras4B and their role in breast ductal carcinoma. Methods: Total RNA was extracted from breast tumors, and the NATs obtained via mastectomy. Patients were selected from new cases of breast cancer with no prior history of chemotherapy. Relative mRNA expression was calculated based on a pairwise comparison between the tumors and the NATs following normalization to the internal control gene. Predictive values of the transcript variants were examined by ROC curve analysis. Results: A statistically significant increase was found in the K-Ras4A and K-Ras4B expression with the mean fold changes of 7.58 (p = 0.01) and 2.47 (p = 0.001), respectively. The K-Ras4A/K-Ras4B ratio was lower in the tumors than that of the normal tissues. ROC curve analysis revealed the potential of K-Ras4A (AUC: 0.769) and K-Ras4B (AUC: 0.688) in breast cancer prediction. There was also a significant association between K-Ras4B expression and HER2 statues (p = 0.04). Furthermore, a significant link was detected between K-Ras4A expression and pathological prognostic stages (p = 0.04). Conclusion: Our findings revealed that expression levels of K-Ras4A and K-Ras4B is higher in the tumor compared to the normal breast tissues. Increase in K-Ras4A expression was more significant than that of K-Ras4B.

The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review.

Prenatal diagnosis of hereditary diseases has substantially altered the way medical geneticists are helping families affected by genetic disorders. However, the risk of miscarriage and fear of invasive diagnostic procedures may discourage many couples from seeking prenatal diagnosis. With the discovery of maternal plasma cell-free fetal DNA, prenatal diagnosis has entered a new era of progress. Cell-free DNA is released during normal physiological functions as well as through the cell death programs of apoptosis and necrosis. It can be found in the plasma and other body fluids. Although this method has the advantage of being noninvasive, it is still rather expensive and requires advanced hardware and comprehensive data analysis. Promising implications of noninvasive prenatal diagnosis methods for the diagnosis of common trisomy disorders have paved the way for the development of more complicated assays of single-gene disorders. Relative mutation dosage and relative haplotype dosage are the most widely implemented assays for noninvasive prenatal diagnosis of single-gene disorders. However, each assay has its own advantages and disadvantages. Relative mutation dosage is based on the droplet digital polymerase chain reaction (PCR) technique which includes quantification features of real-time PCR assays. Relative haplotype dosage is based on next-generation sequencing that includes analysis of the maternal and paternal genome followed by sequencing of maternal plasma cell-free DNA. Co-amplification at a lower denaturation temperature PCR is another approach that is based on forming heteroduplexes between alleles to selectively amplify paternal mutations. In this review, we have described the most common noninvasive prenatal diagnosis approaches and compared their applications in genetic disorder diagnosis with different inheritance patterns.

Targeting Caspase-3 Gene in rCHO Cell Line by CRISPR/Cas9 Editing Tool and Its Effect on Protein Production in Manipulated Cell Line.

Background: Chinese hamster ovary (CHO) cells are the widely used mammalian cell host for biopharmaceutical manufacturing. During cell cultures, CHO cells lose viability mainly from apoptosis. Inhibiting cell death is useful because prolonging cell lifespans can direct to more productive cell culture systems for biotechnology requests. Objectives: This study exploited a CRISPR/Cas9 technology to generate site-specific gene disruptions in the caspase-3 gene in the apoptosis pathway, which acts as an apoptotic regulator to extend cell viability in the CHO cell line. Methods: The STRING database was used to identify the key pro-apoptotic genes to be modified by CRISPR/Cas9 system. The guide RNAs targeting the caspase-3 gene were designed, and vectors containing sgRNA and Cas9 were transfected into CHO cells that expressed erythropoietin as a heterologous protein. Indel formation was investigated by DNA sequencing. Caspase-3 expression was quantified by real-time PCR and western blot. The effect of editing the caspase-3 gene on the inhibition of apoptosis was also investigated by induction of apoptosis in manipulated cell lines by oleuropein. Finally, the erythropoietin production in the edited cells was compared to the control cells. Results: The caspase-3 manipulation significantly prolongation of the cell viability and decreased the caspase-3 expression level of protein in manipulated CHO cells (more than 6-fold, P-value < 0.0001). Manipulated cells displayed higher threshold tolerance to apoptosis compared to the control cells when they were induced by oleuropein. They show a higher IC50 than the control ones (7271 µM/mL Vs. 5741 µM/mL). They also show a higher proliferation rate than the control cells in the presence of an apoptosis inducer (P-value < 0.0001). Furthermore, manipulated cell lines significantly produce more recombinant protein in the presence of 2,000 µM oleuropein compared to the control ones (P-value = 0.0021). Conclusions: We understood that CRISPR/Cas9 could be effectively applied to suppress the expression of the caspase-3 gene and rescue CHO cells from apoptosis induced by cell stress and metabolites. The CRISPR/Cas9 system-assisted caspase-3 gene ablation can potentially increase erythropoietin yield in CHO cells.

The role of miRNA-377 as a tumor suppressor in lung cancer by negative regulation of genes belonging to ErbB signaling pathway.

BACKGROUND: The ErbB signaling pathway plays important role in the pathogenesis of lung cancer. We explored the role of miRNA-377 as a tumor suppressor in NSCLC through silencing of some genes in the ErbB pathway. METHODS AND RESULTS: The targeting effect of miRNA-377 on EGFR, MAPK1, ABL2, and PAK2 was evaluated. The expression levels of these genes and miRNA-377 were surveyed in NSCLC and normal human tissues, Calu-6, and A549 cells. Real-time PCR was used to figure out whether miRNA-377 could decrease the target genes mRNAs in transfected lung cancer cell lines. The effects of miRNA-377 on apoptosis cell and proliferation were analyzed. We showed that miRNA-377 targets EGFR, MAPK1, and PAK2 mRNAs in in-silico and luciferase reporter assay. The expression of miRNA-377 was significantly downregulated in human NSCLC tissues, Calu-6 and A549 cells compared to their controls. We observed a negative correlation between EGFR, MAPK1, PAK2, and miRNA-377 expression in human NSCLC tissues. A significant reduction in EGFR, MAPK1, and PAK2 mRNA levels was detected, following miRNA-377 transfection in Calu-6 and A549 cells. The higher levels of miRNA-377 in Calu-6, and A549 cells induced apoptosis and reduced proliferation, significantly. CONCLUSIONS: All these data reveal that miRNA-377 functions as a tumor suppressor in NSCLC and may serve as a potential therapeutic target for the treatment of NSCLC.

Cell-free mtDNA level and its biomarker potency for ART outcome are different in follicular fluid of PCOS and non-PCOS women.

INTRODUCTION: Lack of reliable biomarkers for estimating the outcome is one of the current gaps in ART. In this study, we assessed whether cell-free mitochondrial DNA within the follicular fluid (FF cf-mtDNA) of PCOS patients has biomarker applicability or not. Furthermore, probable involved mechanisms in the FF cf-mtDNA pathway were evaluated. METHODS: The level of FF cf-mtDNA was compared between 50 PCOS patients and 50 women without any certain reproductive disorder, and analyzed for correlations with ART outcome. The associations between levels of FF cf-mtDNA and TFAM, POLG, and RNase H1 genes expression in mural granulosa cells (MGCs), as well as IL-6, and TNFα in follicular fluid (FF) were assessed. RESULTS: We identified that FF cf-mtDNA level was significantly lower in PCOS women and was accompanied by a reduction in the expression of mtDNA biogenesis genes in MGCs of the patients. Although a significant association between FF cf-mtDNA level and ART outcome was observed in the control group, no correlation could be proved in the PCOS group. Moreover, the expression level of TFAM was negatively associated, while amounts of IL-6 and TNFα were positively correlated with FF cf-mtDNA level in both groups. CONCLUSION: PCOS patients present a lower FF cf-mtDNA level in comparison with non-PCOS women. FF cf-mtDNA has biomarker applicability for ART outcome in women without any certain reproductive disorder, but not for those with PCOS. It seems that mtDNA packaging dysfunction results in elevated FF cf-mtDNA, and subsequent effects are triggered by increasing the inflammatory cytokines.

Cell-free DNA discoveries in human reproductive medicine: providing a new tool for biomarker and genetic assays in ART.

Cell-free DNAs (cfDNAs) are fragmented forms of DNA that are released into extracellular environments. Analyzing them, regarding either concentration or genetic/epigenetic status can provide helpful information about disorders, response to treatments, estimation of success rates, etc. Moreover, since they are presented in body fluids, evaluation of the aforementioned items would be achieved by less/non-invasive methods. In human reproduction field, it is required to have biomarkers for prediction of assisted reproduction techniques (ART) outcome, as well as some non-invasive procedures for genetic/epigenetic assessments. cfDNA is an appropriate candidate for providing the both approaches in ART. Recently, scientists attempted to investigate its application in distinct fields of reproductive medicine that resulted in discovering its applicability for biomarker and genetic/epigenetic analyses. However, due to some limitations, it has not reached to clinical administration yet. In this article, we have reviewed the current reported data with respect to advantages and limitations of cfDNA utilization in three fields of ART, reproduction of male and female, as well as in vitro developed embryos.

Pleiotropic effects of statins: A focus on cancer.

The statin drugs ('statins') potently inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase by competitively blocking the active site of the enzyme. Statins decrease de novo cholesterol biosynthesis and thereby reduce plasma cholesterol levels. Statins exhibit "pleiotropic" properties that are independent of their lipid-lowering effects. For example, preclinical evidence suggests that statins inhibit tumor growth and induce apoptosis in specific cancer cell types. Furthermore, statins show chemo-sensitizing effects by impairing Ras family GTPase signaling. However, whether statins have clinically meaningful anti-cancer effects remains an area of active investigation. Both preclinical and clinical studies on the potential mechanisms of action of statins in several cancers have been reviewed in the literature. Considering the contradictory data on their efficacy, we present an up-to-date summary of the pleiotropic effects of statins in cancer therapy and review their impact on different malignancies. We also discuss the synergistic anti-cancer effects of statins when combined with other more conventional anti-cancer drugs to highlight areas of potential therapeutic development.

Detection of Paternal IVS-II-1 (G>A) (HBB: c.315+1G>A) Mutation in Cell-Free Fetal DNA Using COLD-PCR assay.

Standardization of noninvasive prenatal diagnosis (PND) method that can identify common mutations in the population is of great value. The purpose of this study was to find the paternal HBB gene IVS-II-1 (G>A) (HBB: c.315+1G>A) mutation in maternal plasma cell-free DNA using the co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) method. We designed simulated circulating free DNA (cfDNA) in maternal plasma to optimize the COLD-PCR assay. Peripheral blood samples were collected from normal and IVS-II-1 heterozygous individuals as well as five heterozygous pregnant women whose husbands were carriers of IVS-II-1. The cfDNA was extracted from the plasma and subjected to optimized COLD-PCR followed by Sanger sequencing. The optimized protocol was tested on simulated cfDNA samples with proportions of 8.0, 6.0, 4.0 and 2.0%, and the results showed that the COLD-PCR is informative on samples containing 8.0% mutant alleles and above. The patients were undergoing invasive PND procedures via chorionic villi sampling (CVS) as scheduled at the 12th week of gestation. Paternal IVS-II-1 was detected in cfDNA samples of three patients who were in complete concordance with the outcome of CVS. Despite the limitations of the COLD-PCR method in noninvasive PND, it can be considered as a cost-effective screening option. The use of this approach for screening at-risk patients can prevent unnecessary invasive procedures identifying common mutations in high-prevalence diseases.

Genetic mutations and immunological features of severe combined immunodeficiency patients in Iran.

BACKGROUND: Severe combined immunodeficiency (SCID) is the most severe form of primary immunodeficiency disorders that is characterized by impaired early T lymphocyte differentiation and is variably associated with abnormal development of other lymphocyte lineages. SCID can be caused by mutations in more than 20 different genes. Molecular diagnosis in SCID patients contributes to genetic counseling, prenatal diagnosis, treatment modalities, and overall prognosis. In this cohort, the clinical, laboratory and genetic data related to Iranian SCID patients were comprehensively evaluated and efficiency of stepwise sequencing methods approach based on immunophenotype grouping was investigated METHODS: Clinical and laboratory data from 242 patients with SCID phenotype were evaluated. Molecular genetic analysis methods including Sanger sequencing, targeted gene panel and whole exome sequencing were performed on 62 patients. RESULTS: Mortality rate was 78.9% in the cohort with a median follow-up of four months. The majority of the patients had a phenotype of T-NK-B+ (34.3%) and the most severe clinical manifestation and highest mortality rate were observed in T-NK-B- SCID cases. Genetic mutations were confirmed in 50 patients (80.6%), of which defects in recombination-activating genes (RAG1 and RAG2) were found in 16 patients (32.0%). The lowest level of CD4+ and CD8+ cells were observed in patients with ADA deficiency (p = 0.026) and IL2RG deficiency (p = 0.019), respectively. CONCLUSION: Current findings suggest that candidate gene approach based on patient's immunophenotype might accelerate molecular diagnosis of SCID patients. Candidate gene selection should be done according to the frequency of disease-causing genes in different populations. Targeted gene panel, WES and WGS methods can be used for the cases which are not diagnosed using this method.

Graft versus host disease and microchimerism in a JAK3 deficient patient.

BACKGROUND: The lymphohematopoietic cells originating from feto-maternal trafficking during pregnancy may cause microchimerism and lead to materno-fetal graft versus host disease (GVHD) in severe combined immunodeficiency (SCID) patients. However, definitive diagnosis between GVHD and Omenn's syndrome is often difficult based on clinical and immunological phenotypes particularly in the patients with hypomorphic mutations. CASE PRESENTATION: A 3-year-old girl with a history of erythroderma and immunodeficiency was studied. The whole exome sequencing method was used to find the disease-causing variants, and T-A cloning and Quantitative Florescence Polymerase Chain Reaction (QF-PCR) methods were utilized to detect the presence of mosaicism or microchimerism. We identified a homozygous missense Janus Kinase 3 mutation (JAK3, c.2324G>A, p.R775H) as a new disease-causing variant in the patient, and the presence of microchimerism with maternal origin was proven as an underlying cause of her clinical presentation. CONCLUSION: The findings highlighted the importance of appropriate diagnostic approach in GVHD cases with hypomorphic JAK3 mutations. When analyzing the results of the next generation sequencing, the possibility of microchimerism should be considered based on the context of the disease.

Using chemical chaperones to increase recombinant human erythropoietin secretion in CHO cell line.

In recombinant protein production, over-expressed genes induce unfolded protein response (UPR), overloaded protein aggregation in endoplasmic reticulum and its expansion. In this study, we have used 16 chemicals to improve erythropoietin production in engineered CHO cells and tried to study the mechanism of reducing protein aggregation in each treatment. Endoplasmic reticulum expansion was studied through endoplasmic reticulum specific labeling with utilizing fluorescent glibenclamide and its molecular chaperones expression were studied by real-time polymerase chain reaction. The increase in the mRNA level of EPO and endoplasmic reticulum chaperones GRP78/BiP, XBP1, ATF6, and ATF4 in different chemical treatments were not related to ER expansion. On the other hand, ER expansion in beta alanine, beta cyclodextrin and taurine treatments resulted in increased EPO secretion. Dramatically increase in EPO expression in conjugated linoleic acid, spermidine, trehalose, and maltose (19, 20, 16, and 19-fold, respectively) did not increase erythropoietin productivity, but betaine which did not caused ER expansion, with minor increase in EPO gene expression increase EPO productivity. The results indicated that betaine increase EPO secretion in engineered CHO cell line without relation to ER expansion and molecular chaperones expression.

Effectiveness of Plasmocure™ in Elimination of Mycoplasma Species from Contaminated Cell Cultures: A Comparative Study versus other Antibiotics.

OBJECTIVE: Mycoplasmas spp. is among major contaminants of eukaryotic cell cultures. They cause a wide range of problems associated with cell culture in biology research centers or biotechnological companies. Mycoplasma are also resistant to several antibiotics. Plasmocin™ has been used to treat cell lines but Plasmocin™-resistant strains have been reported. InvivoGen has developed a new anti-Mycoplasma agent called Plasmocure™ in order to eliminate resistant Mycoplasma contamination. The aim of this study was the selection of the best antibiotics for treatment of Mycoplasma in cell cultures. MATERIALS AND METHODS: In this experimental study, a total of 100 different mammalian cell lines contaminated with different Mycoplasma species were evaluated by microbiological culture (as the gold standard method), indirect DNA fluorochrome staining, enzymatic (MycoAlert™), and universal or species-specific polymerase chain reaction (PCR) detection methods. In this study, animal and human cell lines available in National Cell Bank of Iran, were treated with Plasmocure™. The treatment efficacy and cytotoxicity of Plasmocure™ were compared with those of commonly used antibiotics such as BMcyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. RESULTS: Plasmocure™ is comprised of two antibiotics that act through various mechanisms of action than those in Plasmocin™. Two-week treatment with Plasmocure™ was enough to completely eliminate Mycoplasma spp. A moderate toxicity was observed during Mycoplasma treatment with plasmocure™; But, after elimination of Mycoplasma, cells were fully recovered. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. However, the outcome of the treatment process (i.e. the frequency of complete cure, regrowth or cell death) varied among different antibiotics. CONCLUSION: The highest number of cured cell lines was achieved by using Plasmocure™ which also had the lowest regrowth rate after a period of four months. As a conclusion; Plasmocure™ might be considered an effective antibiotic to treat Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells.

Factor VII Gene Defects: Review of Functional Studies and Their Clinical Implications

Coagulation factors belong to a family of plasma glycosylated proteins that should be activated for appropriate blood coagulation. Congenital deficiencies of these factors cause inheritable hemorrhagic diseases. Factor VII (FVII) deficiency is a rare bleeding disorder with variable clinical symptoms. Various mutations have been identified throughout the F7 gene and can affect all the protein domains. The results of previous experiments have partly revealed the correlation between genotype and phenotype in patients with FVII deficiency. Nevertheless, each particular variant may affect the coagulative function of FVII, mainly via altering its expression level, extra-cellular secretion, tissue factor binding affinity, or proteolytic activity. The pathogenicity of the variants and molecular mechanisms responsible for clinical symptoms in patients with FVII deficiency should be characterized via in silico and in vitro, as well as in vivo functional studies. This review has highlighted the most important functional studies reported on F7 gene variants, including relevant reports regarding Iranian FVII deficiency patients.

Analysis of Differential Expression of microRNAs and Their Target Genes in Prostate Cancer: A Bioinformatics Study on Microarray Gene Expression Data.

Early diagnosis of prostate cancer (PCa) as the second most common cancer in men is not associated with precise and specific results. Thus, alternate methods with high specificity and sensitivity are needed for accurate and timely detection of PCa. MicroRNAs regulate the molecular pathways involved in cancer by targeting multiple genes. The aberrant expression of the microRNAs has been reported in different cancer types including PCa. In this bioinformatics study, we studied differential expression profiles of microRNAs and their target genes in four PCa gene expression omnibus (GEO) databases. PCa diagnostic biomarker candidates were investigated using bioinformatics tools for analysis of gene expression data, microRNA target prediction, pathway and GO annotation, as well as ROC curves. The results of this study revealed significant changes in the expression of 14 microRNAs and 40 relevant target genes, which ultimately composed four combination panels (miR- 375+96+663/ miR- 133b+143- 3p + 205/ C2ORF72 + ENTPD5 + GLYAT11/LAMB3 + NTNG2+TSLP) as candidate biomarkers capable to distinguish between PCa tumor samples and normal prostate tissue samples. These biomarkers may be suggested for a more accurate early diagnosis of PCa patients along with current diagnostic tests.

miR-1266-5p and miR-185-5p Promote Cell Apoptosis in Human Prostate Cancer Cell Lines

Objective: Small non-coding RNA molecules are dysregulated in prostate cancer (PCa). In our previous study, downregulation of miR-1266 and miR-185 was demonstrated in PCa tissues and cell lines. The aim of the present study was to investigate whether miR-1266 and miR-185 are involved in the regulation of B-cell lymphoma (BCL) 2 and BCL2L1, respectively, and whether transfection of PCa cell lines with miR-1266 and miR-185 mimics can alter tumorigenic phenotypes. Methods: In order to investigate the regulation of BCL2 and BCL2L1 mRNA levels by miR-1266 and miR-185, respectively, a luciferase reporter assay was used. Real-time PCR was also used to analyze changes in the levels of BCL2 and BCL2L1 mRNAs in PCa cell lines following transfection with synthetic miR-1266 and miR-185. Cell apoptosis was determined by Annexin V protein expression analysis via flow cytometry. In addition to the MTT assay, a cell proliferation assay was performed. Result: A luciferase assay confirmed that the BCL2 and BCL2L1 genes may be targeted by miR-1266 and miR-185, respectively, through binding to their 3'UTR regions. Transfection of PC3 and DU145 cells with miR-1266 and miR-185 induced apoptosis and reduced proliferation, which also revealed an inverse correlation with BCL2 and BCL2L1 gene expression in the treated cells. Conclusion: Our data suggests that miR-1266 and miR-185 may be novel candidates for further research in PCa treatment through the anti-apoptotic pathway.

Downregulation of miR-1266-5P, miR-185-5P and miR-30c-2 in prostatic cancer tissue and cell lines.

Over the latest decade, the role of microRNAs (miRNAs/miRs) has received more attention. miRNAs are small non-coding RNAs that may serve a role as oncogenes or tumor suppressor genes. Certain miRNAs regulate the apoptosis pathway by influencing pro- or anti-apoptotic genes. We hypothesized that increases in the expression of B cell lymphoma 2 (BCL2) and BCL2-like 1 (BCL2L1) genes, which have been reported in various types of cancer tissues, may be due to the downregulation of certain miRNAs. The present study aimed to identify miRNAs that target BCL2 and BCL2L1 anti-apoptotic genes in prostate cancer (PCa) clinical tissue samples. Certain candidate miRNAs were selected bioinformatically and their expression in PCa samples was analyzed and compared with that in benign prostatic hyperplasia (BPH) tissue samples. The candidate miRNAs that targeted BCL2 and BCL2L1 genes were searched in online databases (miRWalk, microRNA.org, miRDB and TargetScan). A total of 12 miRNAs that target the 3'-untranslated region of the aforementioned genes and/or for which downregulation of their expression has previously been reported in cancer tissues. A total of 30 tumor tissue samples from patients with PCa and 30 samples tissues from patients with BPH were obtained and were subjected to reverse transcription-quantitative polymerase chain reaction for expression analysis of 12 candidate miRNAs, and the BCL2 and BCL2L1 genes. Additionally, expression of 3 finally selected miRNAs and genes was evaluated in prostate cancer PC3 and DU145 cell lines and human umbilical vein endothelial cells. Among 12 miRNA candidates, the expression of miR-1266, miR-185 and miR-30c-2 was markedly downregulated in PCa tumor tissues and cell lines. Furthermore, downregulation of these miRNAs was associated with upregulation of the BCL2 and BCL2L1 genes. An inverse association between three miRNAs (miR-1266, miR-185 and miR-30c-2) and two anti-apoptotic genes (BCL2 and BCL2L1) may be considered for interventional miRNA therapy of PCa.

Aberrant expression of miR-141 and nuclear receptor small heterodimer partner in clinical samples of prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the second most common cancer in men worldwide. Currently, prostate-specific antigen (PSA) test and digital rectal exam are the main screening tests used for PCa diagnosis. However, due to the low specificity of these tests, new alternative biomarkers such as deregulated RNAs and microRNAs have been implemented. OBJECTIVES: Aberrant expressions of small heterodimer partner gene (SHP, NR0B2) and mir-141 are reported in various cancers. The aim of this study was to investigate the SHP and miR-141 expression level in tissue samples of prostate cancer. METHODS: The expression level of SHP gene and miR-141 was assessed by real time PCR and their relative amounts were calculated by the Δ⁢ΔCT method. Also, IHC technique was used to determine the expression level of SHP protein. RESULTS: The miR-141 was significantly up-regulated in the samples of metastatic tumors compared to localized tumor samples (P< 0.001, 31.17-fold change). Tumor samples showed lower SHP mRNA expression levels than BPH samples (p= 0.014, 4.7-fold change). The results of paired t-test analysis showed there was no significant difference between the SHP gene expression in PCa samples and their matched tumor-adjacent normal tissue (p= 0.5). CONCLUSIONS: The data obtained in our study confirm the involvement of miR-141 in PCa progression and metastasis. These effects could be mediated by AR via down-regulation of its co-repressor protein, i.e., SHP.