The long non-coding RNA TAZ-AS202 promotes lung cancer progression via regulation of the E2F1 transcription factor and activation of Ephrin signaling.

Long non-coding RNAs (lncRNAs) are transcripts without coding potential that are pervasively expressed from the genome and have been increasingly reported to play crucial roles in all aspects of cell biology. They have been also heavily implicated in cancer development and progression, with both oncogenic and tumor suppressor functions. In this work, we identified and characterized a novel lncRNA, TAZ-AS202, expressed from the TAZ genomic locus and exerting pro-oncogenic functions in non-small cell lung cancer. TAZ-AS202 expression is under the control of YAP/TAZ-containing transcriptional complexes. We demonstrated that TAZ-AS202 is overexpressed in lung cancer tissue, compared with surrounding lung epithelium. In lung cancer cell lines TAZ-AS202 promotes cell migration and cell invasion. TAZ-AS202 regulates the expression of a set of genes belonging to cancer-associated pathways, including WNT and EPH-Ephrin signaling. The molecular mechanism underlying TAZ-AS202 function does not involve change of TAZ expression or activity, but increases the protein level of the transcription factor E2F1, which in turn regulates the expression of a large set of target genes, including the EPHB2 receptor. Notably, the silencing of both E2F1 and EPHB2 recapitulates TAZ-AS202 silencing cellular phenotype, indicating that they are essential mediators of its activity. Overall, this work unveiled a new regulatory mechanism that, by increasing E2F1 protein, modifies the non-small cell lung cancer cells transcriptional program, leading to enhanced aggressiveness features. The TAZ-AS202/E2F1/EPHB2 axis may be the target for new therapeutic strategies.

Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors.

This study investigates the role of integrin ß4 (ITGB4) and stemness-associated factor SOX2 in platinum resistance in lung squamous cell carcinoma (LUSC). The expression of SOX2 and ITGB4 is found to be high in all LUSC subtypes, but the impact of ITGB4 expression on overall patient survival varies by subtype. Cancer stem cells (CSCs) isolated from LUSC patients were found to be resistant to cisplatin, but knocking down ITGB4 or SOX2 sensitized them to cisplatin. Carfilzomib (CFZ) synergized with cisplatin and suppressed CSC growth by inhibiting ITGB4 and SOX2 expression. Additionally, CFZ was found to inhibit SOX2 expression epigenetically by inhibiting histone acetylation at the SOX2 promoter site. CFZ also suppressed the growth of SOX2-dependent small cell lung cancer cells in vitro and in vivo. The study highlights the unique function of CFZ as a transcriptional suppressor of SOX2, independent of its proteasome inhibitory function.

Protocol for Bulk-ATAC sequencing in head and neck squamous cell carcinoma.

The transposase-accessible chromatin using sequencing (ATAC-seq) offers a simplified approach to detect chromatin changes in cancer cells after genetic intervention and drug treatment. Here, we present an optimized ATAC-seq protocol to elucidate chromatin accessibility changes at the epigenetic level in head and neck squamous cell carcinoma cells. We describe steps for cell lysate preparation, transposition, and tagmentation, followed by library amplification and purification. We then detail next-generation sequencing and data analysis. For complete details on the use and execution of this protocol, please refer to Buenrostro et al.,1 Chen et al.,2.

SNPs in Sites for DNA Methylation, Transcription Factor Binding, and miRNA Targets Leading to Allele-Specific Gene Expression and Contributing to Complex Disease Risk: A Systematic Review.

INTRODUCTION: The complex genetic diversity among human populations results from an assortment of factors acting at various sequential levels, including mutations, population migrations, genetic drift, and selection. Although there are a plethora of DNA sequence variations identified through genome-wide association studies (GWAS), the challenge remains to explain the mechanisms underlying interindividual phenotypic disparity accounting for disease susceptibility. Single nucleotide polymorphisms (SNPs) present in the sites for DNA methylation, transcription factor (TF) binding, or miRNA targets can alter the gene expression. The systematic review aimed to evaluate the complex crosstalk among SNPs, miRNAs, DNA methylation, and TFs for complex multifactorial disease risk. METHODS: PubMed and Scopus databases were used from inception until May 15, 2019. Initially, screening of articles involved studies assessing the interaction of SNPs with TFs, DNA methylation, or miRNAs resulting in allele-specific gene expression in complex multifactorial diseases. We also included the studies which provided experimental validation of the interaction of SNPs with each of these factors. The results from various studies on multifactorial diseases were assessed. RESULTS: A total of 11 articles for SNPs interacting with DNA methylation, 30 articles for SNPs interacting with TFs, and 11 articles for SNPs in miRNA binding sites were selected. The interactions of SNPs with epigenetic factors were found to be implicated in different types of cancers, autoimmune diseases, cardiovascular diseases, diabetes, and asthma. CONCLUSION: The systematic review provides evidence for the interplay between genetic and epigenetic risk factors through allele-specific gene expression in various complex multifactorial diseases.

Coding SNPs in hsa-miR-1343-3p and hsa-miR-6783-3p target sites of CYP2C19 modulates clopidogrel response in individuals with cardiovascular diseases.

AIMS: To investigate the impact of microRNA target SNPs (mirSNPs) and their interaction with miRNAs on important drug-metabolizing enzymes, transporters and target genes for prediction of clopidogrel drug response in cardiovascular disease individuals. MAIN METHODS: A prospective cross-sectional study was conducted on 292 individuals undergoing clopidogrel drug therapy. All the enrolled participants were administered 300 mg loading dose followed by 75 mg dose of maintenance therapy. Platelet aggregations were measured before administration of the loading dose and 2 h post fifth day dose of clopidogrel maintenance therapy. Clopidogrel carboxylic acid metabolite from plasma and urine were analyzed post maintenance therapy using the RP-HPLC method. Genotyping of mirSNP's shortlisted through in silico analysis was performed by tetra ARMS PCR and validated by Sanger DNA sequencing. The levels of selected miRNAs were estimated by the TaqMan-PCR assay. Functional validation of mirSNPs was performed in HepG2 cells after transfecting with the selected gene and miRNA mimics. Protein expressions were analyzed by western blot. KEY FINDINGS: 23% of enrolled individuals showed resistance to clopidogrel therapy. Out of 13 mirSNP's analyzed, CYP2C19 rs4244285 was associated with clopidogrel drug resistance and clopidogrel carboxylic acid metabolite in urine and plasma. hsa-miR-1343-3p and hsa-miR-6783-3p levels were significantly high in individuals with CYP2C19 rs4244285 mutant genotype and these miRNAs down-regulated the protein expression of CYP2C19. SIGNIFICANCE: We demonstrated the role of coding mirSNP (rs4244285) in the regulation of the CYP2C19 gene through miRNAs and its implications to clopidogrel drug response prediction in the Indian population.

Acute lymphoblastic leukemia and genetic variations in BHMT gene: Case-control study and computational characterization.

BACKGROUND: Remethylation of homocysteine is catalyzed by B12 dependent methionine synthase (MTR) in all types of cells and by B12 non-dependent betaine homocysteine methyltransferase (BHMT) in liver and kidney cells. Of many etiologies of cancer, an unexplored area is the variations of genes implicated in methylation reaction. OBJECTIVE: The study evaluated the association of BHMT (rs3733890) with acute lymphoblastic leukemia (ALL), followed by in-silico characterization of variations in BHMT gene. METHODS: BHMT [rs3733890; c.742G > A, which substitutes an arginine by a glutamine at codon 239 (R239Q)] was screened by Tetra-primer Amplification Refractory Mutation System PCR (T-ARMS-PCR) and confirmed using DNA sequencing. In-silico analysis was conducted using bioinformatics tools. RESULTS: BHMT (rs3733890) showed an insignificant association with both childhood and adult ALL. Bioinformatics analysis showed that 18 nsSNPs are deleterious, 3 SNPs in 3'-UTR (rs59109725, rs116634518 and rs138578732) alter the miRNA-binding site, and 11 CNVs are present in the BHMT gene. As consequence of BHMT (rs3733890) polymorphism the free energy changes from -101210.1 kJ/mol to -200021.8 kJ/mol. CONCLUSIONS: BHMT (rs3733890) polymorphism showed no association with ALL. Hence this investigation needs further evaluation in larger sample size and effect of other SNPs, CNVs and miRNA's is required to elucidate the role of BHMT gene in ALL development.