A Comprehensive Overview of NF1 Mutations in Iranian Patients.

Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutations in the NF1 gene. This disorder shows nearly complete penetrance and high phenotypic variability. We used the whole-exome sequencing technique to identify mutations in 32 NF1 cases from 22 Iranian families. A total of 31 variants, including 30 point mutations and one large deletion, were detected. In eight cases, variants were inherited, while they were sporadic in the remaining. Seven novel variants, including c.5576 T > G, c.6658_6659insC, c.2322dupT, c.92_93insAA, c.4360C > T, c.3814C > T, and c.4565_4566delinsC, were identified. The current study is the largest in terms of the sample size of Iranian NF1 cases with identified mutations. The results can broaden the spectrum of NF1 mutations and facilitate the process of genetic counseling in the affected families.

Promoter Methylation of Two HOXA9 and NISCH Genes in Opium Users.

Background: Opiate abuse has been critically increased in the world, especially in Iran. Owing to the association of opiate use with multiple human cancers and neurological disorders, seeking for genetic and epigenetic effects of opium can pave the way for early diagnosis of major health defects in addicted users. Accordingly, the present study aimed to determine the methylation status of the promoter of two genes, which are actively involved in neurodevelopment and cancer evolution. Methods: DNA was isolated from peripheral blood of 28 opium abusers and 19 healthy controls and then subjected to sonication. Sonicated DNAs undergone methylated DNA immunoprecipitation-real time polymerase chain reaction (MeDIP-Real Time PCR) using specific primer pairs designed for HOXA9 and NISCH genes. Obtained data were analyzed using SPSS software. Findings: HOXA9 and NISCH genes were found to be significantly methylated in addicted users compared to controls (P<0.001) which was significantly associated with the mean of the age regarding HOXA9 gene (P=0.002). Neither opium amount nor duration or route of using was associated with the methylation status of HOXA9 or NISCH genes. Conclusion: Hypermethylation of HOXA9 and NISCH genes as tumor suppressor in opium-addicted individuals can be considered as confirmatory evidence for carcinogenesis of opium. Further studies are required to figure out the role of epigenetic alterations in cancer evolution among opium users.

Identification of Potential Key Genes Linked to Gender Differences in Bladder Cancer Based on Gene Expression Omnibus (GEO) Database.

Background: Growing evidence strongly indicates pivotal roles of gender differences in the occurrence and survival rate of patients with bladder cancer, with a higher incidence in males and poorer prognosis in females. Nevertheless, the molecular basis underlying gender-specific differences in bladder cancer remains unknown. The current study has tried to detect key genes contributing to gender differences in bladder cancer patients. Materials and Methods: The gene expression profile of GSE13507 was firstly obtained from the Gene Expression Omnibus (GEO) database. Further, differentially expressed genes (DEGs) were screened between males and females using R software. Protein-protein interactive (PPI) network analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Kaplan-Meier survival analyses were also performed. Results: We detected six hub genes contributing to gender differences in bladder cancer patients, containing IGF2, CCL5, ASPM, CDC20, BUB1B, and CCNB1. Our analyses demonstrated that CCNB1 and BUB1B were upregulated in tumor tissues of female subjects with bladder cancer. Other genes, such as IGF2 and CCL5, were associated with a poor outcome in male patients with bladder cancer. Additionally, three signaling pathways (focal adhesion, rheumatoid arthritis, and human T-cell leukemia virus infection) were identified to be differentially downregulated in bladder cancer versus normal samples in both genders. Conclusion: Our findings suggested that gender differences may modulate the expression of key genes that contributed to bladder cancer occurrence and prognosis.

Using chitosan-stabilized, hyaluronic acid-modified selenium nanoparticles to deliver CD44-targeted PLK1 siRNAs for treating bladder cancer.

Aims: Achieving an effective biocompatible system for siRNAs delivery to the tumor site remains a significant challenge. Materials & methods: Selenium nanoparticles (SeNPs) modified by chitosan (CS) and hyaluronic acid (HA) were fabricated for PLK1 siRNAs (siPLK1) delivery to the bladder cancer cells. The HA-CS-SeNP@siPLK1 efficacy was evaluated using in vitro and in vivo models. Results: HA-CS-SeNP@siPLK1 was selectively internalized into T24 cells through clathrin-mediated endocytosis. Treatment with HA-CS-SeNP@siPLK1 successfully silenced the PLK1 gene, inhibited cell proliferation and induced cell cycle arrest in vitro. HA-CS-SeNP@siPLK1 could also inhibit tumor growth in vivo without causing systemic toxicity. Conclusion: Our results suggest that HA-CS-SeNPs may provide a good vehicle for delivering siPLK1 to the bladder tumor site.

siRNAs are small biomolecules shown as novel insights in cancer gene therapy because of their capability to silence target genes. However, achieving an effective biocompatible system for siRNA delivery to the tumor site remains a significant challenge. This work aimed to develop a nanoparticle-based delivery system consisting of selenium nanoparticles modified by chitosan and hyaluronic acid to sustain the release of siRNAs to bladder cancer cells. The results of this study demonstrated that this nanosystem successfully silenced the PLK1 gene and reduced the proliferation in vitro and in vivo. These findings suggest that hyaluronic acid-chitosan-selenium nanoparticles may open a new insight for targeted gene therapy for bladder cancer.

Roles of non-coding RNAs in cell death pathways involved in the treatment of resistance and recurrence of cancer.

Considering the burden of cancer, a number of methods have been applied to control or stop it. However, because of drug resistance or cancer recurrence, these treatments usually face failure. Combination of modulation of expression of non-coding RNAs (ncRNAs) with other treatments can increase treatment-sensitivity of tumors but these approaches still face some challenges. Gathering information in this field is a prerequisite to find more efficient cures for cancer. Cancer cells use ncRNAs to enhance uncontrolled proliferation originated from inactivation of cell death routs. In this review article, the main routes of cell death and involved ncRNAs in these routes are discussed. Moreover, extant information in the role of different ncRNAs on cell death pathways involved in the treatment resistance and cancer recurrence is summarized.

Evaluation of IGF2, KRT14, and KRT20 as Urinary Biomarkers in Patients with Bladder Cancer.

Background: Many researchers have tried to identify bladder cancer biomarkers to reduce the need for cystoscopy. The aim of this study was to identify and measure appropriate transcripts in patient urine to develop a non-invasive screening test. Methods: From February 2020 to May 2022, 49 samples were obtained from Velayat Hospital, Qazvin University of Medical Sciences, Qazvin, Iran. Twenty-two samples were obtained from bladder cancer patients and 27 from bladder cancer-free subjects. RNA was extracted from participant samples, quantitative RT-PCR was performed, and TNP plots were used to assess IGF2 (NCBI Gene ID: 3481), KRT14 (NCBI Gene ID: 3861) and KRT20 (NCBI Gene ID: 54474) expression. For UCSC Xena analysis, Dataset ID: TCGA-BLCA was used to compare transitional cell carcinoma (TCC) and normal samples for survival rates. Results: IGF and KRT14 were more greatly expressed in patient urine samples than in those of the normal group. However, KRT20 expression did not significantly differ between the two groups. IGF2 had 45.45 and 88.89% sensitivity and specificity, respectively, for detecting TCC in urine samples while KRT14 had 59 and 88.89% sensitivity and specificity, respectively. Also, these results infer that overexpression of IGF would be prognosticators of poor TCC outcomes. Conclusion: Our study showed that IGF2 and KRT14 are overexpressed in bladder cancer patient urine, and IGF2 could be a potential biomarker for poor prognoses in TCC.

Identification of TSGA10 and GGNBP2 splicing variants in 5' untranslated region with distinct expression profiles in brain tumor samples.

Introduction: Brain tumors (BTs) are perceived as one of the most common malignancies among children. The specific regulation of each gene can play a critical role in cancer progression. The present study aimed to determine the transcripts of the TSGA10 and GGNBP2 genes, considering the alternative 5'UTR region, and investigating the expression of these different transcripts in BTs. Material and methods: Public data on brain tumor microarray datasets in GEO were analyzed with R software to evaluate the expression levels of TSGA10 and GGNBP2 genes (the Pheatmap package in R was also used to plot DEGs in a heat map). In addition, to validate our in-silico data analysis, RT-PCR was performed to determine the splicing variants of TSGA10 and GGNBP2 genes in testis and brain tumor samples. The expression levels of splice variants of these genes were analyzed in 30 brain tumor samples and two testicular tissue samples as a positive control. Results: In silico results show that the differential expression levels of TSGA10 and GGNBP2 were significant in the GEO datasets of BTs compared to normal samples (with adjusted p-value<0.05 and log fold change > 1). This study's experimental results showed that the TSGA10 gene produces four different transcripts with two distinct promoter regions and splicing exon 4. The relative mRNA expression of transcripts without exon 4 was higher than transcripts with exon 4 in BT samples (p-value<001). In GGNBP2, exon 2 in the 5'UTR region and exon 6 in the coding sequence were spliced. The expression analysis results showed that the relative mRNA expression of transcript variants without exon 2 was higher than other transcript variants with exon 2 in BT samples (p-value<001). Conclusion: The decreased expression levels of transcripts with longer 5'UTR in BT samples than in testicular or low-grade brain tumor samples may decrease their translation efficiency. Therefore, decreased amounts of TSGA10 and GGNBP2 as potential tumor suppressor proteins, especially in high-grade brain tumors, may cause cancer development by angiogenesis and metastasis.

Extreme ßHCG levels in first trimester screening are risk factors for adverse maternal and fetal outcomes.

Multiples of the normal median (MoM) of free ßHCG is a valuable parameter in evaluation of risk of adverse pregnancy outcomes. In the current retrospective study, we assessed the maternal and fetal outcomes in pregnant women having free ßHCG MoM levels < 0.2 or > 5 in their first trimester screening (FTS). Relative risk of trisomy 21 was significantly higher in patients having free ßHCG MoM > 5. On the other hand, relative risk of trisomies 13 and 18 and Turner syndrome were higher in those having free ßHCG MoM < 0.2. Other chromosomal abnormalities were nearly equally detected between those having free ßHCG MoM < 0.2 or > 5. Relative risk of hydrocephaly and hydrops fetalis was higher when free ßHCG MoM was below 0.2. On the other hand, relative risk of low birth weight was higher when free ßHCG MoM was above 5. Moreover, frequency of gestational diabetes mellitus, preeclampsia, preterm delivery and vaginal bleeding increased with levels of free ßHCG MoM. However, polyhydramnios had the opposite trend. Frequencies of premature rupture of membranes and pregnancy induced hypertension were highest among pregnant women having levels of free ßHCG MoM < 0.2. The current study indicates importance of free ßHCG MoM in identification of at-risk pregnancies in terms of both fetal and maternal outcomes. In fact, ßHCG MoM < 0.2 or > 5 can be regarded as risk factors for adverse maternal or fetal outcomes irrespective of the presence of other abnormalities in the FTS results.

CRISPR-Based Fluorescent Reporter (CBFR) Assay for Sensitive, Specific, Inexpensive, and Visual Detection of a Specific EGFR Exon 19 Deletion in NSCLC.

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Mutations in the EGFR gene, including deletions in exon 19 and the mutation L858R, induce responsiveness of non-small cell lung cancer (NSCLC) to a group of drugs known as tyrosine kinase inhibitors. Here, we report the development of the CRISPR-based fluorescent reporter (CBFR) assay including a two-step strategy combining PCR amplification and Cas12a-driven cleavage to detect the delE746_A750 subtype of EGFR exon 19 deletions. Sensitivity and specificity of the CBFR assay were analyzed with different concentrations of fluorescence reporter and different amounts of PCR product. The results demonstrated that increasing the fluorescent reporter to 4 µM and the PCR product to 5 µl enhanced sensitivity. The CBFR assay could detect EGFR exon 19 deletion even with a frequency of 1% in samples. In clinical NSCLC samples, optimized CBFR assay enabled visual detection of the delE746_A750 subtype in less than 1 h. The CBFR assay provides a sensitive, specific, and simple strategy designed based on a straightforward and inexpensive process. We suggest that the CBFR assay could serve as a diagnostic approach to detect mutations, deletions, and pathogens in underequipped laboratories and promote personalized therapeutic approaches.

Antiproliferative effects of AAV-delivered CRISPR/Cas9-based degradation of the HPV18-E6 gene in HeLa cells.

Human papillomavirus infections are associated with most cervical cancers, which are the fourth most common cancer in women. HPV-E6 protein binds to protein p53 and inhibits its function, leading to the switching of normal cells toward cancer cells. Here, we disrupted the HPV-E6 gene and investigated its effects on the proliferation and apoptosis of HeLa cells. The HPV18-E6 gene was targeted with two designed sgRNAs cloned into an AAV-CRISPR-based plasmid. The AAV-E6-CRISPR/Cas9 virions were prepared and titrated in HEK293t cells. The cleavage created in the HPV-E6 gene was detected using the T7E1 assay. Cell cycle profiling, MTT assay, and annexin V/PI staining were performed. Also, the p53 protein level was measured by Western blotting. Our data showed that disruption of the HPV-E6 gene led to increased cell apoptosis and decreased cell proliferation. A significant accumulation of infected cells in sub-G1 phase was observed in the cell profiling assay. Also, HPV-E6 gene disruption resulted in a significant increase in the level of P53 protein. Our findings indicated that AAV-mediated delivery of CRISPR/Cas9 can effectively target the HPV-E6 gene in HeLa cells, and its antiproliferative effects may provide therapeutic benefits of local administration of this gene-editing system for HPV-related cervical cancers.

Antimicrobial and anti-biofilm potencies of dermcidin-derived peptide DCD-1L against Acinetobacter baumannii: an in vivo wound healing model.

BACKGROUND: The global emergence of Acinetobacter baumannii resistance to most conventional antibiotics presents a major therapeutic challenge and necessitates the discovery of new antibacterial agents. The purpose of this study was to investigate in vitro and in vivo anti-biofilm potency of dermcidin-1L (DCD-1L) against extensively drug-resistant (XDR)-, pandrug-resistant (PDR)-, and ATCC19606-A. baumannii. METHODS: After determination of minimum inhibitory concentration (MIC) of DCD-1L, in vitro anti-adhesive and anti-biofilm activities of DCD-1L were evaluated. Cytotoxicity, hemolytic activity, and the effect of DCD-1L treatment on the expression of various biofilm-associated genes were determined. The inhibitory effect of DCD-1L on biofilm formation in the model of catheter-associated infection, as well as, histopathological examination of the burn wound sites of mice treated with DCD-1L were assessed. RESULTS: The bacterial adhesion and biofilm formation in all A. baumannii isolates were inhibited at 2 × , 4 × , and 8 × MIC of DCD-1L, while only 8 × MIC of DCD-1L was able to destroy the pre-formed biofilm in vitro. Also, reduce the expression of genes involved in biofilm formation was observed following DCD-1L treatment. DCD-1L without cytotoxic and hemolytic activities significantly reduced the biofilm formation in the model of catheter-associated infection. In vivo results showed that the count of A. baumannii in infected wounds was significantly decreased and the promotion in wound healing by the acceleration of skin re-epithelialization in mice was observed following treatment with 8 × MIC of DCD-1L. CONCLUSIONS: Results of this study demonstrated that DCD-1L can inhibit bacterial attachment and biofilm formation and prevent the onset of infection. Taking these properties together, DCD-1L appears as a promising candidate for antimicrobial and anti-biofilm drug development.

A Computational Framework to Infer Prostate Cancer-Associated Long Noncoding RNAs and Analyses for Identifying a Competing Endogenous RNA Network.

Background: Prostate cancer (PC) is the second leading cause of cancer death after lung cancer in men. Current biomarkers are ineffective for the treatment and management of the disease. Long noncoding RNAs (lncRNAs) are a heterogeneous group of transcripts that are involved in complex gene expression regulatory networks. Although lncRNAs have been suggested to be promising as future biomarkers, the connection between the majority of lncRNAs and human disease remains to be elucidated. One approach to elucidate the roles of lncRNAs in disease is through the development of computational models. For example, a novel computational model termed HyperGeometric distribution for LncRNA-Disease Association (HGLDA) has been developed. Such models need to be developed on a tumor-specific basis to better suit the particular problem. Methods: In this study, we constructed a potential pipeline through two models, HGLDA and pathway-based using data from several databases. To validate the obtained data, the expression levels of selected lncRNAs were investigated quantitatively in the DU-145, LNCaP, and PC3 PC cell lines using quantitative real-time PCR. Results: We obtained a number of lncRNAs from both models, many of which were filtered through several databases that ultimately resulted in identification of six high-value lncRNA targets. Their expression was correlated with one important component of the PI3K pathway, known to be related to PC. Conclusion: Through the assembly of a lncRNA-miRNAs-mRNA competing endogenous RNA network, we successfully predicted lncRNAs interfering with miRNAs and coding genes related to PC.

JPX and LINC00641 ncRNAs expression in prostate tissue: a case-control study.

BACKGROUND AND PURPOSE: Prostate cancer (PC) is the second most prevalent cancer in men. Prostate-specific antigen (PSA) is the main biomarker for screening PC. An increase in PSA could lead to false-positive results. Thus, more appropriate markers should be investigated. In the present study, JPX and LINC00641 expression levels were measured in tumoral prostate tissue compared with the non-tumor tissue. EXPERIMENTAL APPROACH: 43 pairs of prostate tumoral and non-tumor tissue were prepared. The expression levels of JPX and LINC00641 were investigated by RT-qPCR. FINDINGS/RESULTS: Significant upregulation of LINC00641 (2.47 ± 0.5 vs 1.41 ± 0.2) and downregulation of JPX (1.42 ± 0.6 vs 2.83 ± 1.0) were observed in PC tissues compared with the normal tissues (their adjacent non-tumoral tissues). CONCLUSION AND IMPLICATIONS: Dysregulation of JPX and LINC00641 in PC patients could be used in the future as a prognostic biomarker in PC.

New evidence on tumor suppressor activity of PTEN and KLLN in papillary thyroid carcinoma.

This study aimed to address the hypothesis that the expression of PTEN and KLLN tumor suppressor genes could diminish in papillary thyroid cancer (PTC) compared to paired normal tissue (PNT) and multinodular goiter (MNG). PTEN and KLLN expressions were assessed at both mRNA and protein levels in 82 tissue samples, including 30 PTC, 30 PNT, and 26 MNG using SYBR-Green Real-Time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Bioinformatics studies were performed to evaluate the genomic location and the genes promoter region. The mRNA expression of PTEN and KLLN in PTC was significantly lower than PNT (PTEN, P = 0.0033; KLLN, P = 0.0005). A significant decrease in the mRNA level of KLLN was also observed in PTC than MNG (P = 0.0304). Decreased level of PTEN mRNA (odds ratio=0.391; P = 0.013) or KLLN mRNA (odds ratio=0.023; P = 0.025) was associated with an increased risk of PTC tumorigenesis. Areas under the ROC curve for PTEN and KLLN were 0.69 and 0.78, respectively. PTEN and KLLN protein expressions in PTC compared to PNT or MNG were not significantly different. The bioinformatics studies revealed the sequence near the promoter region is lowly conserved across species. Four GC boxes were found upstream of the PTEN transcription start site (TSS), and one TATA box and one GC box were found upstream of KLLN TSS. The results suggest PTEN and KLLN are the two tumor suppressor genes that decreasing or loss of both of them occurs in sporadic PTC tumorigenesis. It appears they could have a promising application in both diagnostic and therapeutic areas.

Efficacy of co-immunization with the DNA and peptide vaccines containing SYCP1 and ACRBP epitopes in a murine triple-negative breast cancer model.

Multiepitope cancer vaccines have gained lots of attention for prophylactic and therapeutic purposes in cancer patients. In our previous study, multiepitope DNA and peptide cancer vaccines consisted of the most immunodominant epitopes of ACRBP and SYCP1 antigens were designed by bioinformatic tools. In this study, the effect of prophylactic co-immunization with these DNA and peptide cancer vaccines in the 4T1 breast cancer animal model was assessed. Serum levels of the peptide-specific IgG total, IgG2a and IgG1 were measured by enzyme-linked immunosorbent assay (ELISA). Also, the efficacy of the immunized mice splenocytes' for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) was evaluated. The co-immunization caused a significant (P < .05) increase in the serum levels of IgG1 and IgG2a. The co-immunized mice splenocytes exhibited significantly enhanced IL-4 (6.6-fold) and IFN-γ (19-fold) production. Also, their lymphocytes exhibited higher proliferation rate (3-fold) and granzyme B production (6.5-fold) in comparison with the control. The prophylactic co-immunization significantly decreased the breast tumors' volume (78%) and increased the tumor-bearing mice survival time (37.5%) in comparison with the control. Taking together, prophylactic co-immunization with these multiepitope DNA and peptide cancer vaccines can activate the immune system against breast cancer. However, further experiments are needed to evaluate their efficacy from different angles.

Expression profiling revealed up-regulation of three lncRNAs in breast cancer samples.

Long non-coding RNAs (lncRNAs) have been vastly investigated for their critical roles in the pathogenesis of breast cancer. Yet, the expression pattern and clinical significance of three lncRNAs namely CTBP1AS2, LINC-ROR and SPRY4-IT1 in breast cancer are not completely clarified. In the present investigation, we assessed expression of these lncRNAs in breast cancer tissues and paired non-cancerous specimens from the same patients using quantitative real time PCR. Notably, expression of CTBP1AS2, LINC-ROR and SPRY4-IT1 were upregulated in breast cancer tissues compared with non-cancerous tissues (ER = 17.62, P value<0.000; ER = 4.62, P value = 0.001 and ER = 3.47, P value = 0.005, respectively). Relative expression of LINC-ROR in tumoral tissues compared with non-tumoral tissues was associated with a history of hormone replacement therapy (P = 0.04). Expression levels of CTBP1AS2, LINC-ROR and SPRY4-IT1 were significantly correlated with each other in both tumoral and non-tumoral tissues. The strongest correlations were detected between CTBP1AS2/ LINC-ROR and CTBP1AS2/ SPRY4-IT1 pairs in non-tumoral tissues. CTBP1AS2 and SPRY4-IT1 had the best sensitivity (80%) and specificity (64%) values, respectively. Based on AUC values, the best diagnostic power belonged to CTBP1AS2. The current study potentiates CTBP1AS2, LINC-ROR and SPRY4-IT1 as putative contributors in the pathogenesis of breast cancer and suggests these lncRNAs as candidates for functional analysis in this kind of cancer.

Evaluation of the Epigenetic Demethylation of NRF2, a Master Transcription Factor for Antioxidant Enzymes, in Colorectal Cancer.

BACKGROUND: Epigenetic changes in CpG islands of the promoter regions of homeostasis-related genes, including nuclear factor erythroid 2-related factor 2 (NRF2), have been shown to hold a significant role in the development of colorectal cancer. Therefore, we aimed to examine the DNA demethylation pattern of the NRF2 promoter region in cancerous lesions from patients with colorectal cancer and the association of methylation status with clinicopathological features in the Iranian population. METHODS: In this cross-sectional study, 114 colorectal tissue samples were collected. These samples included: 34 tumour tissue samples, 60 precancerous polyps, and 20 normal tissue samples. The promoter methylation status of the NRF2 gene was examined using methylation-specific PCR. Additionally, the relationship between the methylation status and the clinicopathological features was investigated. RESULTS: The frequency of NRF2 demethylation in the tumour samples was significantly higher compared to the polyp tissues (p= 0.003) and normal tissue (p= 0.009), indicating that cancerous colorectal tissues exhibit increased demethylation of the NRF2 promoter. After examining the demethylation status of tissue samples, the clinicopathological features were compared to the demethylation results. No significant association was found between NRF2 promoter demethylation and the clinicopathological features of patient samples. CONCLUSION: Our findings suggest that the epigenetic modifications leading to NRF2 demethylation found in colorectal tumour samples may contribute to cancer progression from precancerous polyps to cancerous lesions.

Anti-angiogenic effects of testis-specific gene antigen 10 on primary endothelial cells.

Growing evidence indicates the antitumor and antiangiogenesis activities of testis-specific gene antigen 10 (TSGA10). However, the underlying mechanisms and precise role of TSGA10 in angiogenesis are still elusive. In this study, we isolated human umbilical cord vein endothelial cells (HUVECs) and stably transfected with pcDNA3.1 carrying TSGA10 coding sequence. We demonstrated that TSGA10 over-expression significantly decreases HUVEC tubulogenesis and interconnected capillary network formation. HUVECs over-expressing TSGA10 exhibited a significant decrease in migration and proliferation rates. TSGA10 over-expression markedly decreased expression of angiogenesis-related genes, including VEGF-A, VEGFR-2, Ang-1, Ang-2, and Tie-2. Our ELISA results showed the decrease in VEGF-A mRNA expression level is associated with a significant decrease in its protein secretion. Additionally, over-expressing TSGA10 decreased expression levels of marker genes of cell migration (MMP-2, MMP-9, and SDF-1a) and proliferation (PCNA and Ki-67. Furthermore, ERK-1 and AKT phosphorylation significantly reduced in HUVECs over-expressing TSGA10. Our findings suggest a potent anti-angiogenesis activity of TSGA10 in HUVECs through down-regulation of ERK and AKT signalling pathways, and may provide therapeutic benefits for the management of different pathological angiogenesis.

Rapamycin Reduces Cervical Cancer Cells Viability in Hypoxic Condition: Investigation of the Role of Autophagy and Apoptosis.

BACKGROUND: Rapamycin has been known as an anti-cancer agent that affects different malignancies such as glioblastoma and prostate cancer. However, there are few studies concerning rapamycin effects on the cervical cancer cells. In this study, it was aimed to investigate the possible effect of rapamycin on a cervical cancer cell line and explored the possible mechanism(s) and pathway(s) for this agent. MATERIALS AND METHODS: To do so, HeLa cells as cervical cancer cell line were used and treated with different concentrations of rapamycin under both normoxic and hypoxic conditions. Then, cell viability assays, Western blot, quantitative real-time polymerase chain reaction (QR-PCR), acridine orange and acridine orange/propidium iodide staining were performed to evaluate rapamycin effect on the mentioned cell line. RESULTS: The results showed that autophagy and apoptosis-related genes increased significantly in rapamycin-treated HeLa cells compared to controls. Moreover, cervical cancer cell death by rapamycin-induced autophagy in hypoxia was greater than normoxia compared with controls. In this study, it was showed that autophagy induction by rapamycin can mediate programmed cell death of cervical cancer cells, especially in hypoxic condition. CONCLUSION: These findings provide a new evidence that rapamycin may inhibit hypoxic HeLa cell proliferation through the trigger of programmed cell death, facilitating the development of novel anti-cancer therapy.

Investigation of promoter methylation of MCPH1 gene in circulating cell-free DNA of brain tumor patients.

INTRODUCTION: Despite advanced diagnostic and therapeutic techniques, many brain tumors are still diagnosed at high grades and, therefore finding novel molecular markers may assist in early detection and reducing brain tumors-related mortality rate. Owing to the previous reports on the importance of MCPH1 gene in tumorigenesis, the present study was aimed to study the promoter methylation of MCPH1 gene in paired circulating cell-free DNA (cfDNA) and tumor tissues of brain tumor patients. MATERIALS AND METHODS: Fourteen fresh paired serum and tumor tissue samples in addition to 18 isolated serum samples were collected from patients affected by different grades of brain tumor. Genomic DNA and cfDNA was isolated from tissue and serum samples using QIAamp DNA Mini Kit Norgen Bioteck Kit, respectively. Methylation DNA immunoprecipitation Real-time polymerization chain reaction (MeDIP-Real-time PCR) was performed on isolated DNA samples using EpiQuik MeDIP Ultra Kit and specific primer pairs. cfDNA quantity was determined through Real-time PCR analysis using specific primer pairs designed for GAPDH gene. RESULTS: MCPH1 was methylated in 54% of cfDNA samples which was significantly associated with tumor grade, as well (P-value = 0.02). The methylation rate of MCPH1 was found as 78% in the tissue samples which was meaningfully associated with tumor grade (P-value = 0.03). Moreover, methylation of the MCPH1 gene was consistent in 57% of the same cfDNA and tissue samples. Methylation of MCPH1 gene in neither tumor tissues nor cfDNA was not correlated with age and sex of the patients. DISCUSSION AND CONCLUSION: Due to the conformity of methylation of MCPH1 gene in cfDNA and tissue samples in more than half of the enrolled patients, especially in higher grades of tumors, it seems that MCPH1 promoter methylation could be a potential epimarker in not only detection of brain tumors but also in response to chemo- and radiotherapy which warranted further assessment.