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.

Analysis of KLF7 and KLF5 transcription factors gene variants in coronary artery disease.

INTRODUCTION AND OBJECTIVE: Genetic susceptibility has a key role in the pathogenesis of coronary artery disease (CAD). KLF5 and KLF7 are transcriptional factors essential to cell development and differentiation. Their genetic variants have been associated with the risk of metabolic disorders. The present study aimed to evaluate the possible correlation of KLF5 (rs3812852) and KLF7 (rs2302870) single nucleotide polymorphisms (SNPs) with the risk of CAD for the first time in the world. METHODS: The clinical trial study comprised 150 patients with CAD and 150 control subjects without CAD from the Iranian population. After blood sampling, deoxyribonucleic acid was extracted and genotyped using the Tetra Primer ARMS-PCR method and confirmed by Sanger sequencing. RESULTS: The KLF7 A/C genotypes and C allele frequency were meaningfully higher in the control group compared to the CAD+ group (p<0.05). No obvious association has been observed between KLF5 variants and CAD risk. However, the distribution of the AG genotype of KLF5 was statistically lower in CAD+ patients with diabetes than in CAD+ patients without diabetes (p<0.05). CONCLUSION: This study identified KLF7 SNP as a causative gene contributing to CAD, which presents novel insight into the molecular pathogenesis of the disease. It is, however, unlikely that KLF5 SNP has an essential role in the risk of CAD in the studied population.

Association between The SIRT1 and SIRT3 Levels and Gene Polymorphisms with Infertility in War Zones of Kermanshah Province, Iran: A Case-Control Study.

OBJECTIVE: War toxin, mustard gas, alkylating agent results in male infertility via inducing reactive oxygen species (ROS) production and DNA mutagenesis. SIRT1 and SIRT3 are multifunctional enzymes that involve in the DNA repair, oxidative stress responses. This study aim is to assess the correlation between serum levels of SIRT1, SIRT3 and both rs3758391T>C and rs185277566C>G gene polymorphisms with infertility in the war zones of Kermanshah province, Iran. MATERIALS AND METHODS: In this case-control study based on the semen analysis, samples were divided into two groups infertile (n=100) and fertile (n=100). High-performance liquid chromatography (HPLC) method was used to determine the malondialdehyde level, and also a sperm chromatin dispersion (SCD) test was used to evaluate the DNA fragmentation rate. Using the colorimetric assays, superoxide dismutase (SOD) activity was measured. SIRT1 and SIRT3 protein levels were determined by using ELISA. The genetic variants of SIRT1 rs3758391T>C, and SIRT3 rs185277566C>G, were detected by polymerase chain reaction-restriction fragment length (PCR-RFLP) technique. RESULTS: Malondialdehyde (MDA) level and the percentage of DNA fragmentation were higher in infertile samples, but serum levels of SIRT1 and SIRT3, and SOD activity was lower in infertile compared to fertile samples (P<0.001). The TC+CC genotypes and the C allele from SIRT1 rs3758391T>C polymorphism, and CG+GG genotypes and the G allele from SIRT3 rs185277566C>G polymorphism could increase risk of infertility (P<0.05). CONCLUSION: The results of this study suggest that war toxins through the impact on genotypes, decreasing levels of SIRT1 and SIRT3 and increasing levels of oxidative stress, lead to defects in the concentration, motility and morphology of sperms and thus, infertility in men.

Intercalative binding of two new five-coordinated anticancer Pt(II) complexes to DNA: experimental and computational approaches.

Binding interaction of two organoplatinum complexes, [Pt(C^N)Cl(dppa)], 1, and [Pt(C^N)Cl(dppm)], 2, (C^N = N(1), C(2')-chelated, deprotonated 2-phenylpyridine, dppa = bis(diphenylphosphino)amine, dppm = bis(diphenylphosphino)methane), as anti-tumor agents, with calf thymus DNA (CT-DNA) under pseudo-physiological conditions has been investigated using various biophysical techniques viz., UV-Vis and fluorescence spectroscopies, viscosity measurements, and thermal denaturation experiments. A hypochromic shift in UV-Vis absorption titration, fluorescence enhancement of Pt(II) complexes in the presence of CT-DNA, fluorescence quenching in competitive ethidium bromide displacement assay, and an uptrend in the viscosity (η) and melting temperature (Tm) indicated the existence of a tight intercalative interaction of Pt(II) complexes with CT-DNA. The fluorescence quenching of CT-DNA was a combined quenching of static and dynamic with Stern-Volmer quenching constants of 7.520 × 103 M-1 for complex 1 and 5.183 × 103 M-1 for complex 2, at low concentrations of Pt(II) complexes. Besides the experimental studies, computational studies were done. Molecular modeling studies confirmed the intercalation of the studied complexes by the phenyl groups of dppa and dppm, leading to π-π interactions but with a certain steric hindrance because of the size and shape of the considered complexes. The combination of experimental and computational data showed that reported Pt(II) complexes are promising structures and could be developed for cancer therapeutic applications.Communicated by Ramaswamy H. Sarma.

CRISPER/CAS System, a Novel Tool of Targeted Therapy of Drug-Resistant Lung Cancer.

Lung cancer (LC) is the most common cause of cancer-related death worldwide. Patients with LC are usually diagnosed at advanced phases. Five-year survival rate in LC patients is approximately 16%. Despite decades of research on LC treatments, clinical outcomes are still very poor, necessitating to develop novel technologies to manage the disease. Considering the role of genetic and epigenetic changes in oncogenes and tumor-suppressor genes in cancer progression, gene therapy provides a hot spot in cancer treatment research. Gene therapy offers less side effects compared to conventional methods such as chemotherapy. Unlike the traditional approaches of gene therapy that have temporary effects, using genetic modification tools can offer persistent cure. Over the past a few years, many studies have effectively used the CRISPR-Cas9 approach to modify gene expression in cells. This system is applied to induce site-specific mutagenesis and epigenetic modifications and regulate gene expression. In this review, we discuss recent applications of the CRISPR-Cas9 technology in treating LC.

Anticancer Effects of Valproic Acid via Regulation of Epigenetic Mechanisms in Non-small-cell Lung Cancer A549 Cell Line.

Epigenetic mechanisms are the most important factors contributing to both the development and metastasis of cancer cells. We aimed to scrutinize the role of epigenetic alternations of genes involved in cancer metastasis, including CD44v6 (metastasis indicator) and Nm23-H1 (a novel tumor suppressor), in the A549 lung cancer cell line. The A549 cells were cultured in the DMEM medium. Valproic acid (VPA) was used as a histone deacetylase inhibitor. Caspase-3 activity was assessed by adding DEVD-pNA substrate to the cell lysate. Gene expression was determined by real-time PCR. Finally, protein expression was assessed by western blot. The results showed that VA significantly decreased the expression of the CD44v6 gene and its protein level. This was further accompanied by lower expressions of MMP-2 and MMP-9 genes. On the other hand, the expression of Nm23-H1 and its protein were significantly increased in the cells accompanied by higher activity of caspase-3 (P ˂ 0.05). Our results showed that epigenetic regulation of CD44v6, Nm23-H1, MMP-2, and MMP-9 might be involved in the pathogenesis and metastasis of lung cancer. Therefore, the use of histone deacetylase inhibitors can be effective in the suppression of metastases and the treatment of these tumors.

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.

Association of Rs61764370 polymorphism within let-7 microRNA-binding site with lung cancer in Iranian population.

INTRODUCTION: Polymorphisms within miRNAs binding sites are associated with miRNAs function. The aim of this study was to investigate the relationship between rs61764370 polymorphism within let-7 miRNA binding site in KRAS gene and the risk of lung cancer in Iranian population. METHODS: This case-control study was conducted with 100 lung cancer patients and 100 healthy persons. The rs61764370 polymorphism was analyzed using PCR-RFLP technique and direct sequencing. RESULTS: We found a significant relationship between rs61764370 (T / G) polymorphism and lung cancer risk, the GT genotype (OR: 6.25; 95% CI = 2.605-15.00; P= 0.000) and G allele (OR: 5.25; 95% CI = 2.259-12.208; P= 0.000) were significantly associated with an increased risk of lung cancer. CONCLUSION: According to our findings, there is a significant relationship between the KRAS rs61764370 polymorphism and lung cancer risk in Iranian population and this polymorphism may be used as a marker in detection of lung cancer in the future.

Considering smoking status, coexpression network analysis of non-small cell lung cancer at different cancer stages, exhibits important genes and pathways.

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer among smokers, nonsmokers, women, and young individuals. Tobacco smoking and different stages of the NSCLC have important roles in cancer evolution and require different treatments. Existence of poorly effective therapeutic options for the NSCLC brings special attention to targeted therapies by considering genetic alterations. In this study, we used RNA-Seq data to compare expression levels of RefSeq genes and to find some genes with similar expression levels. We utilized the "Weighted Gene Co-expression Network Analysis" method for three different datasets to create coexpressed genetic modules having relations with the smoking status and different stages of the NSCLC. Our results indicate seven important genetic modules having important associations with the smoking status and cancer stages. Based on investigated genetic modules and their biological explanation, we then identified 13 newly candidate genes and 7 novel transcription factors in association with the NSCLC, the smoking status, and cancer stages. We then examined those results using other datasets and explained our results biologically to illustrate some important genes in relation with the smoking status and metastatic stage of the NSCLC that can bring some crucial information about cancer evolution. Our genetic findings also can be used as some therapeutic targets for different clinical conditions of the NSCLC.

The anti-cancer effect of amygdalin on human cancer cell lines.

Derived from rosaceous plant seed, amygdalin belongs to aromatic cyanogenic glycoside group, and its anticancer effects have been supported by mounting evidence. In this study, we objected to investigate amygdalin effect on two antiapoptotic genes (Survivin, XIAP) and two lncRNAs (GAS5, MALAT1) in human cancer cells (A549, MCF7, AGS). Employing RT-qPCR analysis, we compared the mRNA levels of the genes related to apoptosis in A549, MCF7, and AGS cancer cells between amygdalin-treated (24, 48 and 72 h) and un-treated groups. RNA was extracted from both cell groups and then cDNAs were synthesized. The changes in the gene expression levels were specified using ΔΔCt method. RT-qPCR analysis has revealed that the expression of Survivin, XIAP, GAS5 and MALAT1 in amygdala-treated cancer cells were significantly different, compared to the un-treated cells. However, these expressions were different depending on the treatment time. According to the results, amygdalin significantly inhibited the expression level of Survivin, and XIAP genes in treated via untreated group. Our findings suggest that amygdalin might have an anticancer effect due to the various gene expressions in A549, MCF7, and AGS human cancer cells, showing it's potential as a natural therapeutic anticancer drug.

Genotyping of 49-plex autosomal SNP panel in Iranian Turkmens ethnic group.

A total of 94 unrelated individuals from Turkmens ethnic group in Iran were typed for forty-nine of the autosomal single nucleotide polymorphisms (SNPs) in the SNPforID 52plex using the SNaPshot assay. Allele frequencies are presents for the 49 SNPs. No deviation from Hardy-Weinberg equilibrium (HWE) was observed in all but one of the 49 SNP systems and no significant linkage disequilibrium was detected for any SNP pairs. FIS and FST were estimated. A statistically significant global FST value was obtained when Turkmens ethnic group were compared with other 20 populations in Turkey, Israel, Pakistan, India, China, Taiwan, Japan, Thailand, Siberia, Algeria, Somali, Uganda, Mozambique, Angola, Nigeria, Russia, Slovenia, Sweden, France and Spain. All but 11 pairwise FST values were statistically significant. Multidimensional scaling plot drawn based on the pairwise FST values showed that the Turkmens ethnic grouped with populations geographically close to Iran and other Middle-Eastern populations. The cumulative values for the match probability using the 49 SNPs was 5.65 × 10-19 consistent to a combined power of discrimination of >99.99999% and the mean exclusion probability was 99.95%.

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.

Nebulette Expression Is Associated with Lymph Node Metastasis in Patients with Colorectal Cancer.

BACKGROUND Colorectal cancer (CRC) is one of the most common cancers among men and women worldwide. Cancer metastasis is the main cause of death in patients with cancer. NEBL (nebulette, Gene ID: 10529) protein interacts with thin filaments in the cell and may functionally destabilize focal adhesion composition. There are some studies on NEBL gene expression alteration in cancer. In the presented study we aimed to analyze NEBL gene expression in patients with colorectal cancer to explore possible association of this gene with clinicopathological features in CRC. METHODS Sixty-seven fresh samples of colorectal tumors and adjacent normal tissues were collected from Iranian patients with CRC. Real time polymerase chain reaction was performed to measure the level of NEBL gene expression and its association with clinico-pathological features. RESULTS A significant overexpression with 3 fold increse was seen in NEBL mRNA level in tumoral tissues compared with the adjacent normal tissues. In addition there was a significant association between NEBL gene expression with lymph node metastasis in patients with CRC. CONCLUSION The overexpression of NEBL has the capacity to be considred as a prognostic biomarker in patients with CRC.

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.

PD-1 and cancer: molecular mechanisms and polymorphisms.

The programmed cell death protein 1 (PD-1) is expressed by activated T cells that act as an immunoregulatory molecule, and are responsible for the negative regulation of T cell activation and peripheral tolerance. The PD-1 gene also encodes an inhibitory cell surface receptor involved in the regulation of T cell functions during immune responses/tolerance. Beyond potent inhibitory effects on T cells, PD-1 also has a role in regulating B cell and monocyte responses. An overexpression of PD-1 has been reported to contribute to immune system avoidance in different cancers. In particular, PD-1 over-expression influences tumor-specific T cell immunity in a cancer microenvironment. Blocking the PD-1/PD-1 ligand (PD-L1) pathway could potentially augment endogenous antitumor responses. Along these lines, the use of PD-1/PD-L1 inhibitors has been applied in clinical trials against diverse forms of cancer. It was believed that antibodies targeting PD-1/PD-L1 might synergize with other treatments that enhance endogenous antitumor immunity by blocking inhibitory receptor-ligand interactions. However, in all cases, the host genetic status (as well as that of the tumor) is likely to have an impact on the expected outcomes. Various investigations have evaluated the association between PD-1 polymorphisms and the risk of various types of cancer. Frequently studied PD-1 polymorphisms, PD-1.1 (rs36084323), PD-1.3 (rs11568821), PD-1.5 (rs2227981), PD-1.9 (rs2227982), and PD-1 rs7421861, and their associations in the risk of susceptibility to different types of cancer are mentioned in this review, as are studies highlighting the significance of conducting genetic association studies in different ethnic populations.

Association of endothelial nitric oxide synthase gene variants (-786 T>C, intron 4 b/a VNTR and 894 G>T) with idiopathic recurrent pregnancy loss: A case-control study with haplotype and in silico analysis.

OBJECTIVE(S): Many lines of evidence suggest that reduced production of nitric oxide (NO) due to single nucleotide polymorphisms in endothelial nitric oxide synthase (eNOS) gene may affect the implantation and maintenance of pregnancy. Accordingly, our objective was to investigate whether the eNOS polymorphisms (-786 T>C, intron 4 b/a VNTR and 894 G>T) and haplotypes may be associated with increased susceptibility to recurrent pregnancy loss (RPL). STUDY DESIGN: A total of 130 women with a history of two or more unexplained consecutive first trimester miscarriages and 110 ethnically matched women with at least two normal pregnancies and no history of pregnancy loss were included in the study as cases and controls, respectively. To identify the genotypes, we used polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) methods In addition, an in silico analysis was conducted to predict the possible effects of the eNOS 894 G>T polymorphism on the structure and function of eNOS mRNA and protein using prediction servers. RESULTS: Our findings revealed that the prevalence of eNOS -786 T>C polymorphism, eNOS -786C allele and TC+CC genotype in cases were significantly higher than those in healthy controls (p<0.05). Also, the combination genotypes -786TT/4b4a and -786TT/894GG were significantly associated with reduced risk of RPL. We also found that the C-4a-G haplotype of the eNOS gene studied polymorphisms was significantly associated with a predisposition to RPL (odds ratio, 3.219; 95% confidence interval, 1.649-6.282; p=0.0003). The in silico analysis showed that the eNOS 894 G>T polymorphism couldn't affects eNOS mRNA and protein significantly. CONCLUSION: Our findings provide evidence to support the hypothesis that eNOS -786 T>C polymorphism and the -786C-4a-894G haplotype are associated with the high risk of RPL.

A systems biology approach for miRNA-mRNA expression patterns analysis in non-small cell lung cancer.

Non-small cell lung cancers (NSCLCs) is a prevalent and heterogeneous subtype of lung cancer accounting for 85 percent of patients. MicroRNAs (miRNAs), a class of small endogenous non-coding RNAs, incorporate into regulation of gene expression post-transcriptionally. Therefore, deregulation of miRNAs' expression has provided further layers of complexity to the molecular etiology and pathogenesis of different diseases and malignancies. Although, until now considerable number of studies has been carried out to illuminate this complexity in NSCLC, they have remained less effective in their goal due to lack of a holistic and integrative systems biology approach which considers all natural elaborations of miRNAs' function. It is able to reliably nominate most affected signaling pathways and therapeutic target genes by deregulated miRNAs during a particular pathological condition. Herein, we utilized a holistic systems biology approach, based on appropriate re-analyses of microarray datasets followed by reliable data filtering, to analyze integrative and combinatorial deregulated miRNA-mRNA interaction network in NSCLC, aiming to ascertain miRNA-dysregulated signaling pathway and potential therapeutic miRNAs and mRNAs which represent a lion' share during various aspects of NSCLC's pathogenesis. Our systems biology approach introduced and nominated 1) important deregulated miRNAs in NSCLCs compared with normal tissue 2) significant and confident deregulated mRNAs which were anti-correlatively targeted by deregulated miRNA in NSCLCs and 3) dysregulated signaling pathways in association with deregulated miRNA-mRNAs interactions in NSCLCs. These results introduce possible mechanism of function of deregulated miRNAs and mRNAs in NSCLC that could be used as potential therapeutic targets.