Identification of novel potential drugs and miRNAs biomarkers in lung cancer based on gene co-expression network analysis.

Non-small cell lung cancer (NSCLC) is an important cause of cancer-associated deaths worldwide. Therefore, the exact molecular mechanisms of NSCLC are unidentified. The present investigation aims to identify the miRNAs with predictive value in NSCLC. The two datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs (DEmiRNA) and mRNAs (DEmRNA) were selected from the normalized data. Next, miRNA-mRNA interactions were determined. Then, co-expression network analysis was completed using the WGCNA package in R software. The co-expression network between DEmiRNAs and DEmRNAs was calculated to prioritize the miRNAs. Next, the enrichment analysis was performed for DEmiRNA and DEmRNA. Finally, the drug-gene interaction network was constructed by importing the gene list to dgidb database. A total of 3,033 differentially expressed genes and 58 DE miRNA were recognized from two datasets. The co-expression network analysis was utilized to build a gene co-expression network. Next, four modules were selected based on the Zsummary score. In the next step, a bipartite miRNA-gene network was constructed and hub miRNAs (let-7a-2-3p, let-7d-5p, let-7b-5p, let-7a-5p, and let-7b-3p) were selected. Finally, a drug-gene network was constructed while SUNITINIB, MEDROXYPROGESTERONE ACETATE, DOFETILIDE, HALOPERIDOL, and CALCITRIOL drugs were recognized as a beneficial drug in NSCLC. The hub miRNAs and repurposed drugs may act a vital role in NSCLC progression and treatment, respectively; however, these results must validate in further clinical and experimental assessments.

Altered expression of apoptosis-related genes in rheumatoid arthritis peripheral blood mononuclear cell and related miRNA regulation.

AIM: Impaired apoptosis and proliferation resulted in autoreactive lymphocyte development and inflammation in Rheumatoid arthritis (RA). TP53, BAX, FOXO1, and RB1 are related genes in cell survival, proliferation, and inflammation which could be important in RA development and disease severity. Here we investigated their expression in peripheral blood mononuclear cells (PBMCs) from RA patients in comparison to healthy controls. METHODS: Fifty healthy controls and 50 RA patients were selected. The quantitative real-time polymerase chain reaction was used to assess the gene expression level in PBMCs. RESULTS: The mRNA expression of TP53 (FC = 0.65, p = .000), BAX (FC = 0.76, p = .008), FOXO1 (FC = 0.59, p = .000) and RB1 (FC = 0.50, p = .000) were significantly reduced in RA PBMCs. TP53 expression was negatively correlated with miR-16-5p (p = .032) and FOXO1 expression was negatively correlated with miR-335-5p (p = .005) and miR-34a-5p (p = .014). A positive correlation was seen between TP53 expression and its downstream gene, BAX (p = .001). FOXO1 expression was also negatively correlated with disease activity, DAS28 (p = .021). CONCLUSION: All selected genes have downregulated expression in RA PBMCs which could be correlated with RA pathogenesis by regulating apoptosis, cell survival, inflammatory mediator production, and proliferation. Due to the correlation of miR-16-5p, miR-34a-5p, and miR-335-5p with TP53 and FOXO1 expression in RA PBMCs, they could be used as future therapeutic targets.

Combination of natural scaffolds and conditional medium to induce the differentiation of adipose-derived mesenchymal stem cells into keratocyte-like cells and its safety evaluation in the animal cornea.

Keratocytes are the main cellular components of the corneal stroma. This cell is quiescent and cannot be cultured easily. The aim of this study was to investigate differentiate human adipose mesenchymal stem cells (hADSCs) into corneal keratocyte cells by combining natural scaffolds and conditioned medium (CM) and evaluating their safety in the rabbit's cornea. Keratocytes were cultured in an optimal culture medium and this medium was collected and kept as a CM. hADSCs were cultured on the decellularized human small incision lenticule extraction (SMILE) lenticule (SL), amniotic membrane (AM), and collagen-coated plates, and were exposed to keratocyte-CM (KCM) for 7, 14, and 21 days. Differentiation was evaluated using Real-time PCR and immunocytochemistry (ICC). hADSCs were cultured on the SL scaffolds and implanted in the corneal stroma of 8 New Zealand male rabbits. Rabbits were followed for 3 months and the safety was evaluated by clinical and histological variables. Real-time PCR results showed a significant increase in the expression of keratocyte-specific markers on the 21 day of differentiation compared to the control group. ICC also confirmed the induction of differentiation. Implantation of SLs containing differentiated cells in the cornea of animals showed no serious complications including neovascularization, corneal opacity, inflammation, or signs of tissue rejection. Furthermore, the evaluation of the presence of keratocyte-like cells after three months in the rabbit stroma was confirmed by Real-time PCR and immunohistochemistry (IHC) analysis. Our results showed that combination of combination of corneal extracellular matrix and KCM can induced keratocytes differentiation of hADSC and can be introduced as a alternative method to supply the required keratocytes in corneal tissue engineering.

Evaluation of the current MicroRNAs expression levels as potential biomarkers in Oral Squamous Cell Carcinoma.

As the most common malignancy, oral squamous cell carcinoma (OSCC) is typically fatal. The survival of patients with oral cancer has not improved, and tumor recurrence remains high. During tumorigenesis, microRNAs (miRNAs) regulate gene expression. Patients' life expectancy can be determined by prognostic survival biomarkers, which can focus therapy on specific targets. This study evaluated five miRNAs associated with OSCC for their prognostic impact. It was determined through microarray analysis and quantitative reverse transcription polymerase chain reaction that there was a significant difference in the expression of miRNAs between OSCC patients and control patients in plasma. We used the unpaired t-tests and the Mann-Whitney test to conduct the statistical analysis. Based on the study's results, five miRNAs have been found to have significantly different expression levels in the plasma of patients with OSCC; in particular, miR-31 was found to have a significantly higher expression level in OSCC patients' plasma as compared with healthy controls. Aside from that, there was a significant reduction in the expression of miR-100, miR-199a, miR-203, and mir345 in the plasma of OSCC patients (P < 0.05). To better understand the importance of miRNAs in OSCC, various OSCC cases were analyzed. Detecting miRNAs in plasma may be a useful diagnostic tool for oral squamous cell carcinoma.

Investigation of the expression levels of miR-21, miR-132, miR-29a, miR-204, and miR-138 in patient's plasma with primary OSCCs.

This is the eighth most malignant tumor in the world, causing the highest incidence and malignancy rate of all cancers in the mouth and maxillofacial region. In cells, miRNAs regulate development, differentiation, proliferation, and differentiation, and miRNA expression is a better indicator of physiological status than DNA expression. miR-21, miR-132, miR-29a, miR-204, and miR-138 levels were measured in plasma from patients with primary OSCC and healthy controls. A Real Time-PCR technique was used to measure miR-21, miR-132, miR-29a, miR-29a, and miR-204 expression levels in plasma from 38 healthy and 38 people with primary OSCC. A standard distribution test and a CT unit were used to confirm the quantitative data on miRNA expression. Gene expression levels were compared between two groups of patients and healthy groups using a Mann-Whitney test and an unpaired t-test. MiR-21's median CT value was 29.68 in the OSCC group and 32.89 in the healthy group, and miR-29a's median CT value was 37.54 and 36.46 in the OSCC group and healthy group, respectively. Additionally, miR-132's CT values were 37.71 and 36.40, miR-138's CT value was 35.37 and 31.21, and miR-204's CT value was 36.44 and 36.17. The results showed that miR-21 expression levels increased significantly, while miR-29a, miR-132, and miR-138 (P < 0.05), and miR-204 expression levels did not differ significantly (P > 0.05). As a result of this study, the expression levels of microRNAs may be considered to diagnose OSCC at an early stage. It is essential to diagnose this disease early to improve treatment and patient health outcomes.

Alteration of TGFB1, GDF9, and BMPR2 gene expression in preantral follicles of an estradiol valerate-induced polycystic ovary mouse model can lead to anovulation, polycystic morphology, obesity, and absence of hyperandrogenism.

OBJECTIVE: In humans, polycystic ovary syndrome (PCOS) is an androgen-dependent ovarian disorder. Aberrant gene expression in folliculogenesis can arrest the transition of preantral to antral follicles, leading to PCOS. We explored the possible role of altered gene expression in preantral follicles of estradiol valerate (EV) induced polycystic ovaries (PCO) in a mouse model. METHODS: Twenty female balb/c mice (8 weeks, 20.0±1.5 g) were grouped into control and PCO groups. PCO was induced by intramuscular EV injection. After 8 weeks, the animals were killed by cervical dislocation. Blood serum (for hormonal assessments using the enzyme-linked immunosorbent assay technique) was aspirated, and ovaries (the right ovary for histological examinations and the left for quantitative real-time polymerase) were dissected. RESULTS: Compared to the control group, the PCO group showed significantly lower values for the mean body weight, number of preantral and antral follicles, serum levels of estradiol, luteinizing hormone, testosterone, and follicle-stimulating hormone, and gene expression of TGFB1, GDF9 and BMPR2 (p<0.05). Serum progesterone levels were significantly higher in the PCO animals than in the control group (p<0.05). No significant between-group differences (p>0.05) were found in BMP6 or BMP15 expression. CONCLUSION: In animals with EV-induced PCO, the preantral follicles did not develop into antral follicles. In this mouse model, the gene expression of TGFB1, GDF9, and BMPR2 was lower in preantral follicles, which is probably related to the pathologic conditions of PCO. Hypoandrogenism was also detected in this EV-induced murine PCO model.

Comparison of oncolytic virotherapy and nanotherapy as two new miRNA delivery approaches in lung cancer.

Lung cancer is known as the second leading cause of cancer death. Finding ways to detect early-stage lung cancer can remarkably increase the survival rate. Biomarkers such as microRNAs can be helpful in cancer diagnosis, predicting its prognosis, and patients' chances of survival. Numerous studies have confirmed the correlation between microRNA expression and the likelihood of patients surviving after treatment. Consequently, it is necessary to study the expression profile of microRNAs during and after treatment. Oncolytic virotherapy and nanotherapy are two neoteric methods that use various vectors to deliver microRNAs into cancer cells. Although these treatments have not yet entered into the clinical trials, much progress has been made in this area. Analyzing the expression profile of microRNAs after applying nanotherapy and oncolytic virotherapy can evaluate the effectiveness of these methods. This review refers to the studies conducted about these two approaches. The advantages and disadvantages of these methods in delivery and affecting microRNA expression patterns are discussed below.

LncRNA HSPC324 plays role in lung development and tumorigenesis.

Lung cancer is a leading cause of cancer-related death in the world. Therefore, identifying the genes and molecular pathways involved in lung development and tumorigenesis can help us improve the therapeutic strategies of lung cancer. Accumulating evidence confirms that long noncoding RNAs, as a novel layer of regulatory RNA molecules, play an important role in various aspects of the cells. Here, using available high throughput gene expression data, we identified an lncRNA (HSPC324) with high expression level in lung tissue that is distinctly expressed in lung tumor tissues relative to normal. Using GO enrichment and KEGG pathway analyses, we further analyzed the functions and pathways involving the HSPC324-correlated genes. Ectopic expression of lncRNA HSPC324 significantly inhibited proliferation, cell cycle and migration; on the other hand, increased apoptosis and ROS production in lung adenocarcinoma cells. Overall, this study introduces HSPC324 as a new player in the development of lung cancer.

Imatinib independent aberrant methylation of NOV/CCN3 in chronic myelogenous leukemia patients: a mechanism upstream of BCR-ABL1 function?

BACKGROUND: The NOV gene product, CCN3, has been reported in a diverse range of tumors to serve as a negative growth regulator, while acting as a tumor suppressor in Chronic Myelogenous Leukemia (CML). However, the precise mechanism of its silencing in CML is poorly understood. In the current study, we aimed to query if the gene regulation of CCN3 is mediated by the promoter methylation in the patients with CML. In addition, to clarify whether the epigenetic silencing is affected by BCR-ABL1 inhibition, we assessed the methylation status in the patients at different time intervals following the tyrosine kinase inhibition using imatinib therapy, as the first-line treatment for this type of leukemia. METHODS: To address this issue, we applied bisulfite-sequencing technique as a high-resolution method to study the regulatory segment of the CCN3 gene. The results were analyzed in newly diagnosed CML patients as well as following imatinib therapy. We also evaluated the correlation of CCN3 promoter methylation with BCR-ABL1 levels. RESULTS: Our findings revealed that the methylation occurs frequently in the promoter region of CML patients showing a significant increase of the methylated percentage at the CpG sites compared to normal individuals. Interestingly, this hypermethylation was indicated to be independent of BCR-ABL1 titers in both groups, which might suggest a mechanism beyond the BCR-ABL1 function. CONCLUSION: Despite suggesting that the CCN3 hypermethylation acts as a molecular mechanism independent of BCR-ABL1 function in CML patients, this scenario requires further validation by complementary experiments. In the case of acting upstream of BCR-ABL1 signaling, the methylation marker can provide early detection and a novel platform for targeted epigenetic modifiers for efficient treatment in imatinib resistant patients.

miR-9: From function to therapeutic potential in cancer.

Malignant neoplasms are regarded as the main cause of death around the world; hence, many research studies were conducted to further perceive molecular mechanisms, treatment, and cancer prognosis. Cancer is known as a major factor for health-related problems in the world. The main challenges associated with these diseases are prompt diagnosis, disease remission classification and treatment status forecast. Therefore, progressing in such areas by developing new and optimized methods with the help of minimally invasive biological markers such as circular microRNAs (miRNAs) can be considered important. miRNA interactions with target genes have specified their role in development, apoptosis, differentiation, and proliferation and also, confirm direct miRNA function in cancer. Different miRNAs expression levels in various types of malignant neoplasms have been observed to be associated with prognosis of various carcinomas. miR-9 seems to implement opposite practices in different tissues or under various cancer incidences by influencing different genes. Aberrant miR-9 levels have been observed in many cancer types. Therefore, we intended to investigate the precise role of miR-9 in patients with malignant neoplasms. To this end, in this study, we attempted to examine different studies to clarify the overall role of miR-9 as a prognostic marker in several human tumors. The presented data in this study can help us to find the novel therapeutic avenues for treatment of human cancers.

Evaluation of ten SNP Markers for Human Identification and Paternity Analysis in Persian Population.

BACKGROUND: DNA markers are inevitable tools of human identification in forensic science. Single Nucleotide Polymorphisms (SNPs) are one category of these markers which is concerned to use especially in the case of degraded DNA because of their short amplicons. OBJECTIVES: Detection of highly informative SNPs by the criteria is the essential step to develop a useful panel of SNP markers. The purpose of this work is to get high informative SNPs for human identification in Persian ethnic of the Iranian population. MATERIAL AND METHODS: Genotype and allele frequencies of 10 SNPs from the SNPforID browser were determined by a PCR-RFLP method on 100 samples that was taken from 100 unrelated Persian people. RESULTS: These ten SNPs were in Hardy-Weinberg equilibrium (P value > 0.1) except rs1355366 (P value = 0.02) and Heterozygosity of seven SNPs is greater than 0.45 but minor allele frequency of only four SNPs is more than 0.45. According to criteria only three SNPs rs1454361, rs2111980 and rs2107612 can pass all standards and are highly informative in population for forensic uses. CONCLUSIONS: Our data showed that the CPI (Combined probability of Identity) and CPE (Combined Power of Exclusion) for ten SNPs are 1.13 E-04 and 0.809 respectively. It was also showed based on the criteria only three SNPs (rs2107612, rs1454361 and rs2111980) are highly informative in Persian population. If we can find 39 SNPs with PE and PI close to PE and PI of these three SNPs (rs2107612, rs1454361 and rs2111980), we will be able to use of these 39 SNPs in human identification with sufficient power of discrimination.

A panel of noncoding RNAs in non-small-cell lung cancer.

Non-small-lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC could pave the way for effective therapies. Analysis of molecular genetic biomarkers in biological fluids has been proposed as a useful tool for cancer diagnosis. Here, we aimed to develop a panel of noncoding RNAs (ncRNAs) in sputum for NSCLC early detection. Expression of 11 ncRNAs were analyzed by real-time polymerase chain reaction in sputum samples of 30 NSCLC patients and 30 sex- and age-matched cancer-free controls. Stability of endogenous microRNAs (miRNAs) in sputum was evaluated after 3 and 6 days at 4°C, 6 months, and 1 year at -80°C. Nine ncRNAs showed significant differences of their expression in sputum between NSCLC patients and controls. A logistic regression model with the best prediction was built based on miR-145, miR-126, and miR-7. The composite of the three miRNAs produced 90% sensitivity and specificity in distinguishing NSCLC patients from the controls. Results indicate that miRNAs could be useful biomarkers based on their stability under various storage conditions and maintain differential changes between cancer and control groups. Moreover, measurement of miRNAs in sputum could be a noninvasive approach for detection of lung cancer.

Altered miR-223 Expression in Sputum for Diagnosis of Non-Small Cell Lung Cancer.

BACKGROUND: Diagnosis of Non-small Cell Lung Cancer (NSCLC) at an early stage is a daunting challenge due to the deficiency of specific noninvasive markers. MicroRNAs (miRNAs) play important roles in the initiation and progression of NSCLC. Measuring miRNA expression levels could provide a potential approach for the diagnosis of NSCLC. Our goals were to examine miR-223, miR-212, miR-192, miR-3074, SNORD33 and SNORD37 expression levels in tissue and sputum of NSCLC patients and cancer free subjects for molecular diagnosis of NSCLC. METHODS: Relative expressions of miR-223, miR-212, miR-192, miR-3074, SNORD33 and SNORD37 were examined with quantitative real-time RT-PCR assay in tissue and sputum obtained from 17 NSCLC patients and 17 controls. RESULTS: miR-3074 was upregulated in tissue samples of NSCLC patients compared with control group. miR-223 was upregulated, miR-212 and SNORD37 were downergulated in sputum samples of patients compared with controls. miR-223 quantification produced 82% sensitivity and 95% specificity with areas under the ROC curve at 0.90 in detection of NSCLC. CONCLUSION: miR-223 clearly discriminated cancer patients from cancer-free subjects and our results suggest that miR-223 could be a diagnostic useful biomarker. The measurement of altered miRNA expression in sputum samples manifested the potential noninvasive approach for detection of lung cancer.

Evaluation of miR-9 and miR-143 expression in urine specimens of sulfur mustard exposed patients.

Sulfur mustard (SM) or mustard gas is a chemical alkylating agent that causes blisters in the skin (blister gas), burns the eyes and causes lung injury. Some major cellular pathways are involved in the damage caused by mustard gas such as NF-κb signaling, TGF-ß signaling, WNT pathway, inflammation, DNA repair and apoptosis. MicroRNAs are non-coding small RNAs (19-25 nucleotides) that are involved in the regulation of gene expression and are found in two forms, extracellular and intracellular. Changes in the levels of extracellular microRNAs are directly associated with many diseases, it is thus common to study the level of extracellular microRNAs as a biomarker to determine the pathophysiologic status. In this study, 32 mustard gas injured patients and 32healthy subjects participated. Comparative evaluation of miR-9 and miR-143 expression in urine samples was performed by Real Time PCR and Graph Pad software. The Mann Whitney t-test analysis of data showed that the expression level of miR-143 and miR-9 had a significant decrease in sulfur mustard individuals with the respective p-value of 0.0480 and 0.0272 compared to normal samples, with an imbalance of several above mentioned pathways. It seems that reducing the expression level of these genes has a very important role in the pathogenicity of mustard gas injured patients.

Site-directed mutagenesis enhances the activity of NADH-FMN oxidoreductase (DszD) activity of Rhodococcus erythropolis.

Microbial desulfurization is potentially an alternative process to chemical desulfurization of fossil fuels and their refined products. The dibenzothiophene desulfurizing system of Rhodococcus erythropolis includes DszD which is an NADH-dependent FMN oxidoreductase with 192 residues that is responsible for supplying reducing equivalents in the form of FMNH(2) to monooxygenases, DszA and DszC. We performed amino acid sequence comparisons and structural predictions based on the crystal structure of available pdb files for three flavin reductases PheA2, HpaC(Tt) and HpaC(St) with the closest structural homology to IGTS8 DszD. The Thr62 residue in DszD was substituted with Asn and Ala by site-directed single amino acid mutagenesis. Variants T62N and T62A showed 5 and 7 fold increase in activities based on the recombinant wild type DszD, respectively. This study revealed the critical role of position 62 in enzyme activity. These results represent the first experimental report on flavin reductase mutation in R. erythropolis and will pave the way for further optimization of the biodesulfurization process.

Identification of a molten globule like state in HC-N fragment of botulinum neurotoxin A: shedding light on the poorly-known features of a conserved sub-domain.

C-terminal fragment of the Botulinum neurotoxin A comprises two sub-domains including H(C)-N and H(C)-C. Here, the conformational change of H(C)-N was studied by spectroscopic techniques. The results indicated that the partially unfolded state forms during unfolding of H(C)-N. This finding may shed light on poorly--known features of the protein.

Interaction between the two subdomains of the C-terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies.

The botulinum neurotoxin A C-terminal fragment (Hc), which mediates the binding of the toxin to neuronal cell surface receptors, comprises two subdomains, Hc-N (amino acids 873-1095) and Hc-C (amino acids 1096-1296). In order to define the minimal fragment of Hc carrying protective antigenic properties, Hc, Hc-N and Hc-C have been produced as recombinant proteins in Escherichia coli, and have been tested for their antigenicity in mouse protection assays. Hc, Hc-N and Hc-C induced similar antibody levels as shown by ELISA. However, a single immunization with Hc (10 microg) fully protected mice challenged with 10(3) mouse lethal dose 50 of toxin, whereas Hc-N, Hc-C, or Hc-N plus Hc-C did not give any protection. Triple immunizations with Hc-N or Hc-C were necessary to induce a higher level of protection. Circular dichroism and fluorescence studies showed that the isolated subdomains were folded and stable. However, an intense near-UV dichroic signal was only observed in the Hc spectrum, revealing a highly structured interface between both subdomains. Taken together, the results show that the generation of protective antibodies requires the whole Hc domain and especially the native structure of the interfacial region between Hc-N and Hc-C.