A protocol to isolate and characterize pure monocytes and generate monocyte-derived dendritic cells through FBS-Coated flasks.

This study explores methods to isolate high-pure monocytes and optimize the best growth factor concentration to generate monocytes-derived dendritic cells (mo-DCs), subset DC1, which is crucial in immune responses. Three protocols for monocyte isolation from peripheral blood mononuclear cells (PBMCs) were evaluated: three-hour incubation on FBS-coated flasks; an overnight incubation on FBS-coated flasks; and Magnetic Activated Cell Sorting (MACS). Additionally, five different concentrations of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and human recombinant interleukin-4 (hrIL-4) were compared. We used Flow cytometry to assess the isolation, purification, and generation of pure monocytes characterized as CD14+, and expression of mo-DC classical markers (HLA-DR, CD80, CD83, and CD86). The obtained results show that monocytes isolated with the second method (overnight incubation) had the highest purity (P < 0.0001) but the lowest yield (P > 0.05), balancing purity and cost-effectiveness. A combination of hrGM-CSF and hrIL-4 at 400 U/mL produced the most favorable outcomes, leading to the highest rate of mo-DC generation (P < 0.05). Notably, this concentration resulted in increasing expression of HLA-DR, CD80, and CD86 surface markers in the generated DCs (P < 0.0001), with no changes in CD83 expression levels. In conclusion, this study offers valuable insights into selecting the optimal approach for monocyte isolation and mo-DC generation in various research contexts, providing a foundation for more effective immunological studies.

Epigenetic modulation of cytokine expression in Mycobacterium tuberculosis-infected monocyte derived-dendritic cells: Implications for tuberculosis diagnosis.

BACKGROUND: To delineate alterations in DNA methylation at high resolution within the genomic profile of monocyte-derived-dendritic cells (mo-DCs) in connection with Mycobacterium tuberculosis (MTB) infection, with particular emphasis on pro/ anti-inflammatory genes. METHODS: In the context of this investigation, mo-DCs were infected by various active strains of MTB (Rifampicin-resistant [RIFR], H37Rv, multidrug-resistant [MDR], and extensively drug-resistant [XDR]). Subsequently, the pro/anti-inflammatory hub gene expression levels within the IL-6, IL-12, IFN-γ, IL-1ß, TNF-α, and IL-10 pathways were evaluated employing real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, the effects of MTB infection on mo-DC protein expression were examined through western blot analysis. The methylation status (%) of TNF-α and IL-10 was considered through Methylation Sensitive-High Resolution Melting (MS-HRM). RESULTS: The results revealed an up-regulation of all pro-inflammatory genes among all groups, with TNF-α exhibiting the highest expression level. Conversely, the anti-inflammatory gene (IL-10) showed a down-regulated expression level. Furthermore, the DNA methylation status (%) of TNF-α decreased significantly among all the groups (P < 0.001), although there were no notable distinctions in the DNA methylation status (%) of IL-10 when compared to the control group (P > 0.05). CONCLUSION: MTB infection induces DNA methylation changes in mo-DCs. The hypo-methylation of TNF-α may induce the up-regulation of this gene. This correlation revealed that the more resistant the MTB strain (XDR) is, the lower the methylation status (%) in the TNF-α gene.

Interference of Bisphenol A on Cumulus Cells Development and Number of Retrieved Mature Oocytes in Unexpected Poor Ovarian Response Women: A Prospective Cohort Study.

OBJECTIVE: This study aimed to investigate the relationship between follicular fluid Bisphenol A (BPA) concentrations with alterations in the expressions of NOTCH1-3, CASPASE 3/7, HLA-G, and ICAM-1 genes and the number of retrieved mature oocytes (MII oocyte) in the cumulus cells of infertile poor ovarian response stimulates women. MATERIALS AND METHODS: In this prospective cohort study, 80 infertile unexpected poor ovarian response (POR) subjects were selected on the basis of subgroup 1a of the POSEIDON classification. They were divided into two groups: group 1 consisted of 40 women, each with a higher number of metaphase II (MII) oocytes (G1, 3-4 oocytes retrieved), while group 2 comprised of 40 women, each with a lower number of MII oocytes (G2, ≤2 oocytes retrieved). The expressions of the studied genes were evaluated by quantitative-real time polymerase chain reaction (PCR). The concentration of BPA in follicular fluid was measured with HPLC. RESULTS: The expression levels of NOTCH1-3, HLA-G, and ICAM-1 genes were significantly lower in G2 than G1 (P<0.05). Meanwhile, CASPASE 3/7 expression levels were higher in unexpected POR patients in G2 compared to G1 (P<0.05). There was a significant direct correlation between the levels of NOTCH1-3, HLA-G and ICAM-1 gene expressions and there was also a significant inverse correlation (P<0.05) between the levels of CASPASE 3/7, with the number of MII oocytes and embryo development between the two groups. The concentration of BPA in the follicular fluids of G2 was higher compared to G1 (P<0.05). CONCLUSION: A higher concentration of BPA was associated with a lower number of mature oocytes and oocyte quality in these patients. Also, alterations of NOTCH1-3, CASPASE 3/7, HLA-G, and ICAM-1 transcript levels in unexpected POR women were associated with BPA concentration.

Methylation Status of miR-34a and miR-126 in Non-Small Cell Lung Cancer (NSCLC) Tumor Tissues.

Background: MiR-34a and miR-126 mainly act as tumor suppressors and are often downregulated in various cancers, including non-small cell lung cancer (NSCLC). We aimed to determine the methylation status of miR-34a and miR-126 in NSCLC patients. Methods: The current study included 63 paraffin-embedded NSCLC and paired adjacent normal tissues. After DNA extraction and bisulfite treatment, the methylation status of miR-34a and miR-126 were evaluated using the MSP method. Results: There was no statistically significant difference between tumor and normal tissues regarding the methylation status of miR-34a and miR-126 (p > 0.05). Moreover, we found no significant correlation between the methylation status of miR-34a and miR-126 with patients' demographic parameters, including gender, age, and pathology subtype (p > 0.05). Conclusion: Considering the low expression of mir-126 and mir-34 in NSCLC, more sensitive methods are recommended to be exploited for detecting the level of methylation or underlying mechanisms other than promoter hypermethylation in silencing these genes in NSCLC.

CRISPR-Cas Technology as a Revolutionary Genome Editing tool: Mechanisms and Biomedical Applications.

Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.

Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions.

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify -α3.7, -α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

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.

Overexpression of miR-32 in Chinese hamster ovary cells increases production of Fc-fusion protein.

The demand for industrial genetically modified host cells were increased with the growth of the biopharmaceutical market. Numerous studies on improving host cell productivity have shown that altering host cell growth and viability through genetic engineering can increase recombinant protein production. During the last decades, it was demonstrated that overexpression or downregulation of some microRNAs in Chinese Hamster Ovary (CHO) cells as the host cell in biopharmaceutical manufacturing, can improve their productivity. The selection of microRNA targets has been based on their previously identified role in human cancers. MicroRNA-32 (miR-32), which is conserved between humans and hamsters (Crisetulus griseus), was shown to play a role in the regulation of cell proliferation and apoptosis in some human cancers. In this study, we investigated the effect of miR-32 overexpression on the productivity of CHO-VEGF-trap cells. Our results indicated that stable overexpression of miR-32 could dramatically increase the productivity of CHO cells by 1.8-fold. It also significantly increases cell viability, batch culture longevity, and cell growth. To achieve these results, following the construction of a single clone producing an Fc-fusion protein, we transfected cells with a pLexJRed-miR-32 plasmid to stably produce the microRNA and evaluate the impact of mir-32 overexpression on cell productivity, growth and viability in compare with scrambled control. Our findings highlight the application of miRNAs as engineering tools and indicated that miR-32 could be a target for engineering CHO cells to increase cell productivity.

A de novo TINF2, R282C Mutation in a Case of Dyskeratosis Congenital Founded by Next-Generation Sequencing.

Background: Dyskeratosis congenita (DC), an inherited and rare disease prevalent in males, is clinically manifested by reticulate hyperpigmentation, nail dystrophy, and leukoplakia. DC is associated with the increased risk of malignancy and other potentially lethal complications such as bone marrow failure, as well as lung and liver diseases. Mutations in 19 genes were found to be correlated with DC. Herein, we report a 12-year-old boy carrying a de novo mutation in TINF2 gene. Methods: Whole exome sequencing (WES) was performed on DNA sample of the proband, and the variant was investigated in the family by Sanger sequencing. Population and bioinformatics analysis were performed. Results: The NM_ 001099274.3(TINF2): c.844C>T (p.Arg282Cys) mutation was found by WES. Conclusion: There was no history of the disease in the family, and the variant was classified as a de novo mutation.

Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP.

BACKGROUND: Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli). METHODS: Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum. RESULTS: Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (˃1.6-fold), and binding activity (˃2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability. CONCLUSION: We were able to produce  21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies.

Mutations in COL6A Gene Family Responsible for Muscular Dystrophies in Three Unrelated Families.

Background: Muscular dystrophy is an inherited disease with clinical and genetic heterogeneity. Muscle weakness is the primary symptom of these disorders that often leads to disability and death. The overall prevalence for all types of muscular dystrophies worldwide is 19.8-25.1 per 100,000 population. Autosomal recessive types of muscular dystrophies are more common in Iran, likely due to the high rate of consanguineous marriage. We aimed at deciphering molecular defects in three unrelated families with muscular dystrophies not related to Duchene MD or limb girdle muscular dystrophies. We are reporting families having affected children with MD owing to the mutations in three genes related to the COL6A (collagen type VI, alpha subunit) gene family. Methods: Three unrelated families, who had at least one member affected with MD and for whom a definite molecular diagnosis was not provided by routine methods, were investigated by WES and confirmed by Sanger sequencing. Results: In the first family, a homozygous variant was found in the COL6A3 gene (NM_004369.4:c.4390C>T:p.Arg1464Ter), which explains the clinical symptoms observed in this family. In the second family, two homozygote missense variants with possible relevance to the patient's phenotype were identified in COL6A1 and COL6A2 genes (NM_001848.2:c.803A>G: p.Glu268Gly and NM_001849.3:c.2489G>A:p.Arg830Gln). Also, a heterozygous pathogenic variant in the COL6A2 gene (NM_001849.3: c.1053+1G>T) was detected in the third family. Conclusion: WES can serve as an effective method for detecting the causative mutations in families with unresolved cases of MD. The data provided herein broadens the spectrum of mutations causing MD in Iran.

RNA Expression Analysis of Mycobacterial Methyltransferases Genes in Different Resistant Strains of Mycobacterium tuberculosis.

Background: Tuberculosis infection still represents a global health issue affecting patients worldwide. Strategies for its control may be not as effective as it should be, specifically in case of resistant strains of Mycobacterium tuberculosis (M.tb.) In this regard, the role of mycobacterial methyltransferases (MTases) in TB infection can be fundamental, though it has not been broadly deciphered. Methods: Five resistant isolates of M.tb were obtained. M.tb H37Rv (ATCC 27249) was used as a reference strain. Seven putative mycobacterial MTase genes (Rv0645c, Rv2966c, Rv1988, Rv1694, Rv3919c, Rv2756c, and Rv3263) and Rv1392 as SAM synthase were selected for analysis. PCR-sequencing and qRT-PCR were performed to compare mutations and expression levels of MTases in different strains. The 2-ΔΔCt method was employed to calculate the relative expression levels of these genes. Results: Only two mutations were found in isoniazid resistance (INHR) strain for Rv3919c (T to G in codon 341) and Rv1392 (G to A in codon 97) genes. Overexpression of Rv0645c, Rv2756c, Rv3263, and Rv2966c was detected in all sensitive and resistant isolates. However, Rv1988 and Rv3919c decreased and Rv1694 increased in the sensitive strains. The Rv1392 expression level also decreased in INHR isolate. Conclusion: We found a correlation between mycobacterial MTases expression and resistance to antibiotics in M.tb strains. Some MTases undeniably are virulence factors that specifically hijack the host defense mechanism. Further evaluations are needed to explore the complete impact of mycobacterial MTases within specific strains of M.tb to introduce novel diagnosis and treatment strategies.

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.

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.

Mitochondrial DNA Copy Number Variations in Gastrointestinal Tract Cancers: Potential Players.

Alterations of mitochondria have been linked to several cancers. Also, the mitochondrial DNA copy number (mtDNA-CN) is altered in various cancers, including gastrointestinal tract (GIT) cancers, and several research groups have investigated its potential as a cancer biomarker. However, the exact causes of mtDNA-CN variations are not yet revealed. This review discussed the conceivable players in this scheme, including reactive oxygen species (ROS), mtDNA genetic variations, DNA methylation, telomere length, autophagy, immune system activation, aging, and infections, and discussed their possible impact in the initiation and progression of cancer. By further exploring such mechanisms, mtDNA-CN variations may be effectively utilized as cancer biomarkers and provide grounds for developing novel cancer therapeutic agents.

Comparing mRNA expression and protein abundance in MDR Mycobacterium tuberculosis: Novel protein candidates, Rv0443, Rv0379 and Rv0147 as TB potential diagnostic or therapeutic targets.

Tuberculosis (TB) is a sizable public health threat in the world. This study was conducted to determine the differential protein composition between susceptible and MDRTB strains. Tuberculosis proteins were extracted by Triton™ X-114 and ammonium sulfate. Two-dimensional gel electrophoresis protein spots were selected for identification by mass spectrometry and mRNA expression levels were measured by real- time PCR. 2DE-Western blot and T cell epitope prediction for identified proteins were made by the IEDB server. The result shows at least six protein spots (Rv0147, Rv3597c, Rv0379, Rv3699, Rv1392 and Rv0443) were differentially expressed in MDRTB isolates. However, difference in mRNA gene expression was not found in the six mRNA genes. 2DE-Western blot procedures indicated strong reaction against MDRTB proteins corresponds to 13, 16 and 55 kDa areas that might be used as new diagnostic tools. In conclusion, these MDRTB proteins identified in this study could be reliable TB diagnostic candidates or therapeutic targets.

Effect of bisphenol A on alterations of ICAM-1 and HLA-G genes expression and DNA methylation profiles in cumulus cells of infertile women with poor response to ovarian stimulation.

This study aimed to investigate the relationship between follicular fluid Bisphenol A (BPA) concentrations with alterations of ICAM-1 and HLA-G genes and proteins expression as well as methylation profiles in the cumulus cells of poor ovarian response (POR) women based on their healthy lifestyle habit. Eighty women under the age of 35 were divided into two groups: 1-POR without using plastic containers (n = 40) and 2-POR with using plastic containers (n = 40). The ICAM-1 and HLA-G genes and protein expressions were examined by the quantitative PCR and western blotting technique. The methylation pattern was investigated by the methylation-specific PCR. Total BPA in follicular fluid was measured with high-performance liquid chromatography technique and the detection limit was 1.14 ng/ml. ICAM-1 and HLA-G genes were differentially expressed between the two groups studied. ICAM-1, HLA-G genes, and protein expressions in group 1 were up-regulated compared to the second group (P < 0.05). While DNA methylation status in group 1 were decreased compared to the other group (P < 0.05). The concentration of BPA in the follicular fluid of group 1 was lower compared to the second group (P < 0.05). The oocyte quality and clinical pregnancy ratio showed significantly higher in group 1 than in the other ones (P < 0.05). The alteration of ICAM-1 and HLA-G gene expressions in POR women is probably related to BPA concentration. As a result Lifestyle habits may also affect the methylation pattern and protein levels in the cumulus cells of POR women. Additionally, lifestyle habits may be considered as a marker for ovulation, oocyte maturation, preimplantation, and clinical pregnancy process.

Investigation the Role of Autophagy in Non-Small Cell Lung Cancer.

OBJECTIVE: Recent studies have shown the role of autophagy in different types of cancer including lung cancer. MicroRNAs are considered as key factors in regulation of autophagy related genes. miR-30d, miR-204-5p and miR-20a are regulatory markers which can suppress the expression of beclin1, LC3, bcl2 and ULK1 as their target genes and they lead to decrement of autophagy in human cancer cells. Moreover, epigenetic modifications DNA methylation has been indicated in regulation of autophagy in different stages of cancer. METHODS: In this study, the expression levels of miR-30d, miR-204-5p and miR-20a as well as their target genes were analyzed in 30 non-small cell lung cancers (NSCLCs) patients sample and adjacent normal tissues by real-time qPCR. In addition, DNA methylation of beclin1, LC3, bcl2 and ULK1 genes were assessed by MS-HRM method. RESULTS: MiR-30d (p value= 0.01) and miR-204-5p (P=0.048) significantly down-regulated in tumor samples compared to normal adjacent tissues, while there was no significant change in expression level of miR-20a. On the other hand, target genes expression level was significantly increased in NSCLC tissues, however methylation pattern of the target gene promoters, did not show any significant alteration. CONCLUSION: These results indicate roles for miR-30d, miR-204-5p as tumor suppressor genes as well as target genes as oncogenes in NSCLC patients. Although these factors may have a significant role in NSCLC progression, further studies are necessary to investigate the implications of these findings for treatment of lung cancer. 
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Dynamic Changes of Mitochondrial DNA Copy Number in Gastrointestinal Tract Cancers: A Systematic Review and Meta-Analysis.

We have performed a systematic review and meta-analysis for evaluation of mitochondrial DNA copy number (mtDNA-CN) alterations in peripheral blood leukocytes (PBL), and tumor tissues of gastrointestinal tract (GIT) cancers. Analysis of the PBL demonstrated a significant decrease [OR: 0.6 (0.5, 0.8)] and increase [OR: 1.4 (1.1, 1.9)] prior to and following GIT cancer development, respectively. This trend was more evident in CRC, and GC subgroups. Analysis of tissue yielded high levels of heterogeneity. However, the mean difference for the CRC subgroup was statistically significant [1.5 (1.0, 2.2)]. Our analysis suggests mtDNA-CN deserves further investigations as a GIT-cancer screening tool.