An In Vitro Study for the Role of Schizophrenia-Related Potential miRNAs in the Regulation of COMT Gene.

This study aimed to analyze the possible association of miR-30a-5p, miR-30e-5p, and miR-34a-5p identified as potential candidate miRNAs in schizophrenia, with the COMT gene. Candidate miRNAs were obtained from the TargetScan database. The SH-SY5Y human neuroblastoma cell line was used as a cellular model for schizophrenia. miR-30a-5p, miR-30e-5p, and miR-34a-5p mimics were transfected into the SH-SY5Y cell line. Total RNA was isolated from transfected cells and RNA-IP samples and reverse transcripted for miRNA and mRNA analysis. RT-qPCR and western blot were performed to observe changes in expression levels of COMT. RNA-immunoprecipitation was performed to determine RNA-protein interactions after mimic transfection. In the study, it was observed that COMT gene expression levels decreased significantly after miR-30a-5p and miR-34a-5p expressions, whereas increased significantly as a result of miR-30e-5p transfection. RNA-IP data have shown that the amount of COMT pulled down by Ago2 was increased after miR-30a-5p and miR-34a-5p transfections. RNA-IP results revealed that miR-30a-5p and miR-34a-5p are direct targets for the COMT gene.

Exosome inhibition improves response to first-line therapy in small cell lung cancer.

Exosomes are recognized as important mediators of cell-to-cell communication, facilitating carcinogenesis. Although there have been significant advancements in exosome research in recent decades, no drugs that target the inhibition of sEV secretion have been approved for human use. For this study, we employed GW4869 and Nexinhib20 as inhibitors of exosome synthesis and trafficking combined. First, we found that Nexinhib20 and GW4869 effectively inhibited RAB27A and neutral sphingomyelinase 2 (nSMase2) nsMase2. Interestingly, the inhibition of nsMase2 and RAB27A decreased expression of CD9, CD63 and Tsg101, both at RNA and protein levels. We used a combination treatment strategy of cisplatin/etoposide plus GW4869 or Nexinhib20 on small cell lung cancer (SCLC) cell lines. The combination treatment of GW4869 or Nexinhib20 effectively enhanced the inhibitory effects of first-line chemotherapy on the SCLC cells. Furthermore, we demonstrated that reducing exosome release through GW4869 and Nexinhib20 treatment effectively reduced cellular proliferation and significantly induced apoptosis in SCLC cells. Also, we showed that combining exosome inhibition with chemotherapy has a significant synergistic effect on cellular proliferation. We also found increased p53 and p21 expressions with western blot and significantly changing Bax, BCL2, caspase-3 and caspase-9 expressions. Inhibiting the exosome pathway offers opportunities for developing novel, effective treatment strategies for SCLC.

Treatment of STING-associated vasculopathy with onset in infancy in patients carrying a novel mutation in the TMEM173 gene with the JAK3-inhibitor tofacitinib.

Objectives: This study aimed to reveal the genetic background of patients in the two-generation family suffering from rheumatoid arthritis, psoriatic arthropathy pain, scratches, and bruises. Patients and methods: A clinical exome sequencing analysis was performed in 10 individuals in the same family using the Sophia Genetics clinical exome solution kit. Results: A novel V194L mutation in the TMEM173 gene was identified in three members of the family. Two of the family members were treated with the JAK3 inhibitor tofacitinib and recovered completely one month after the treatment. Conclusion: The V194L mutation was reported for the first time in this study, and a positive response was achieved with tofacitinib.

Evaluation of Long Non-coding RNA Expression Profiles in Peripheral Blood Mononuclear Cells of Patients with Parkinson's Disease.

As in many biological processes, the long non-coding RNAs (lncRNA) are currently known to have important roles in Parkinson's disease (PD). The aim of the study is to evaluate differentiated expressions of lncRNAs and their target mRNAs in the peripheral blood cells of individuals with Parkinson's disease. The peripheral blood samples were taken from 10 Parkinson's diagnosed people aging 50 years and more and from 10 healthy people as for the control group. Total RNA was isolated from peripheral blood mononuclear cells (PBMC), and a total of 5 samples were selected and evaluated by microarray analysis. lncRNAs with high fold change (fc < 1.5/fc > 1.5) were determined as a result of the analysis. Following this, the expression changes of some lncRNAs and their target mRNAs were examined by quantitative simultaneous polymerase chain reaction (qRT-PCR) in all individuals in the patient and control groups. Also, in order to determine the molecular level basic activities of lncRNAs determined by microarray analysis and which biological process and biochemical pathway they were in, Gene Ontology (GO) analysis ( http://geneontology.org/ ) database was used. Thirteen upregulated and 31 downregulated lncRNAs whose expression changes were determined by microarray analysis and confirmed by qRT-PCR method were found in Parkinson's patients. As they were evaluated by GO analysis, lncRNAs were expressed differently in patient and control groups and they are found to be related with the processes such as macromolecule metabolic processes, immune system, gene expression, cell activation, ATPase activity, DNA packaging complex, signal receptor activity, immune receptor activity, and protein binding were found to be significant.

The effects of MYC on exosomes derived from cancer cells in the context of breast cancer.

MYC amplification and overexpression in breast cancer occur 16% and 22%, respectively, and MYC has a linchpin role in breast carcinogenesis. Emerging evidence has started to shed light on central role of MYC in breast cancer progression. On the contrary, tumor-derived exosomes and their cargo molecules are required for the modulation of the tumor environment and to promote carcinogenesis. Still, how MYC regulates tumor-derived exosomes is still a matter of investigation in the context of breast cancer. Here, we investigated for the first time how MYC affects the biological functions of normal breast cells cocultured with exosomes derived from MYC-expression manipulated breast cancer cells. Accordingly, exosomes were isolated from MCF-7 and MDA-MB-231 cells that MYC expression was manipulated through siRNAs or lentiviral vectors by using exosome isolation reagent. Then, normal breast epithelial MCF-10A cells were treated with breast cancer cell-derived exosomes. The cellular activity of MCF-10A was investigated by cell growth assay, wound healing assay, and transwell assay. Our results suggested that MCF-10A cells treated with exosomes derived from MYC-overexpressing breast cancer cells demonstrated higher proliferation and migration capability compared with nontreated cells. Likewise, MCF-10A cells treated with exosomes derived from MYC-silenced cancer cells did not show high proliferation and invasive capacity. Overall, MYC can drive the functions of exosomes secreted from breast cancer cells. This may allow exploring a new mechanism how tumor cells regulate cancer progression and modulate tumor environment. The present study clears the way for further researches as in vivo studies and multi-omics that clarify exosomal content in an MYC-dependent manner.

Whole Genome Sequencing and Phylogenetic Analysis of SARS­CoV­2 strains in Turkey.

INTRODUCTION: Coronaviruses which are single-stranded RNAs, are members of a large family of viruses that may be important pathogens for humans. SARS-CoV-2 was found to cause the severe respiratory syndrome, and on January 22, 2020 first human-to-human transmission was reported. We aimed to reveal the complete genomes of 19 SARS-CoV-2 isolates from Denizli province and identify Turkish patients' genetic similarities. METHODOLOGY: 15 samples with the highest viral loads resulting from RT-PCR were selected for NGS analysis. Fifteen SARS-CoV-2 complete genome sequences were then subjected to phylogenetic analysis and uploaded to the GISAID database. Phylogenetic trees were constructed by the Neighbor-Joining method using MEGAX software. RESULTS: Whole-genome sequencing of the viral RNA samples revealed 32 missense, 21 synonymous, and 4 non-coding alleles. In all samples c.1-25C>T (5'UTR), c.14144C>T (ORF1ab), c.2772C>T (ORF1ab) and c.1841A>G(S) mutations were detected. Phylogenetic analysis revealed that most of the present study's genomes are in 20B clade while the two are in 20A. The phylogenetic tree constructed with all complete SARS-CoV-2 genomes of Turkey showed that the viruses were spread nearly homogenous on eastern (around Kars) and western (around Istanbul) sides. CONCLUSIONS: Here, we reported the viral genomes in Denizli comprehensively for the first time. We identified 11 rare missense mutations in the virus compared to the reference genome. Phylogenetic analysis revealed that while most of our isolates were similar to European sequences, some had different sublineages depending on their genomic variants.

Bryonia multiflora Extract Induces Autophagy via Regulating Long Non-coding RNAs in Breast Cancer Cells.

Bryonia multiflora, one of the species of Bryonia L. (Cucurbitaceae) genus, is a perennial, dioecious, herbaceous plant with rhizome-shaped roots. Bryonia species have anti-inflammatory, antimicrobial, cytotoxic, antioxidant, etc., activities and their components consume antitumoral effects. Purpose of the study to investigate the effect of Bryonia Multiflora extract (BMST) on breast cancer cells. Our results revealed that MCF-7 and MDA-MB-231 cells underwent significant morphological changes leading to cell rounding. No significant changes were observed in the cell viability by MTT. Acridine orange staining of our cells gave rise to think that BMST might lead our cells to autophagy. Therefore, possible molecular mechanisms underlying morphological changes such as autophagy (LC-3B, Beclin, AMBRA1) and apoptosis (Bcl-2) were evaluated on mRNA and protein levels. BMST treated MCF-7 and MDA-MB-231 cells had increased levels of autophagy markers whereas decreased levels of Bcl-2. p21 levels were also found to be increased in both cells. Analysis of lncRNA expressions has shown that BMST treatment led to changes in the expression levels of several lncRNAs playing roles in autophagy. The current study has shown that BMST induces autophagy in MCF-7 and MDA-MB-231 cells via regulating the lncRNAs revealing that BMST could be a promising therapeutic agent.

Altered Expression of Long Non-coding RNAs in Peripheral Blood Mononuclear Cells of Patients with Alzheimer's Disease.

Long non-coding RNAs (lncRNAs) are involved in many neurological conditions, and neurodegenerative disorders including Alzheimer's disease (AD) regulate gene expression at transcriptional, post-transcriptional, and epigenetic levels. However, the roles of lncRNAs in the pathogenesis of AD remain unclear. In this study, we aimed to determine the expression of lncRNAs and also mRNAs in AD which may alter expression and contribute to the pathogenesis of the disease. Peripheral blood samples were obtained from patients admitted to the Neurology Department of Pamukkale University Medical Faculty (23 patients with AD, 33 control groups). Total RNA obtained from peripheral blood mononuclear cells (PBMC) of subjects with probable AD (n = 4) and healthy control groups (n = 4) was examined to determine the altered expression of lncRNAs and mRNAs in AD were evaluated by microarray analysis. Five lncRNAs with the highest end-to-end fold change (fc ≥ 2.0, p < 0.05) were identified and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). In our study, the profiles of lncRNAs and mRNAs that may be associated with Alzheimer's disease were determined. A total of 14 lncRNAs and 35 mRNAs were determined as upregulated, and 20 lncRNAs and 73 mRNAs determined as downregulated as a result of microarray analysis in patients with AD compared with control groups (fold change ≥ 2.0, p < 0.05). From lncRNAs, expression of lncRNA TTC39C-AS1, lnc-AL445989.1-2, LINC01420, lnc-CSTB-1, and LOC401557 was confirmed by qRT-PCR. When assessed by KEGG analysis of AD PBMC lncRNA and mRNA profiles, TNF signaling pathway, PI3K/AKT, Ras, and MAPK pathways; glutamatergic, dopaminergic, and cholinergic synapses; GABA, and neurotrophin signaling pathways are found to be significant. This is the first known study to investigate lncRNA profiles in AD PBMCs. We think that these results may open a door to the understanding of AD pathogenesis targeted by lncRNAs.

MYC-driven regulation of long non-coding RNA profiles in breast cancer cells.

AIM: MYC deregulation contributes to breast cancer development and progression. Deregulated expression levels of long non-coding RNAs (lncRNA) have been demonstrated to be critical players in development and/or maintenance of breast cancer. In this study we aimed to evaluate lncRNA expressions depending on MYC overexpression and knockdown in breast cancer cells. MATERIALS AND METHODS: Cells were infected with lentiviral vectors by either knockdown or overexpression of c-MYC. LncRNA cDNA was transcribed from total RNA samples and lncRNAs were evaluated by qRT-PCR. RESULTS: Our results indicated that some of the lncRNAs having tumor suppressor (GAS5, MEG3, lincRNA-p21) and oncogenic roles (HOTAIR) are regulated by c-MYC. CONCLUSION: We observed that c-MYC regulates lncRNAs that have important roles on proliferation, cell cycle and etc. Further studies will give us a light to identify molecular mechanisms related to MYC-lncRNA regulatory pathways in breast cancer.

Design of a Lentiviral Vector for the Inducible Expression of MYC: A New Strategy for Construction Approach.

Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.