miR-145 supports cancer cell survival and shows association with DDR genes, methylation pattern, and epithelial to mesenchymal transition.

BACKGROUND: Despite several reports describing the dual role of miR-145 as an oncogene and a tumor suppressor in cancer, not much has been resolved and understood. METHOD: In this study, the potential targets of miR-145 were identified bio-informatically using different target prediction tools. The identified target genes were validated in vitro by dual luciferase assay. Wound healing and soft agar colony assay assessed cell proliferation and migration. miR-145 expression level was measured quantitatively by RT-PCR at different stages of breast tumor. Western blot was used to verify the role of miR-145 in EMT transition using key marker proteins. RESULT: Wound healing and soft agar colony assays, using miR-145 over-expressing stably transfected MCF7 cells, unraveled its role as a pro-proliferation candidate in cancerous cells. The association between miR-145 over-expression and differential methylation patterns in representative target genes (DR5, BCL2, TP53, RNF8, TIP60, CHK2, and DCR2) supported the inference drawn. These in vitro observations were validated in a representative set of nodal positive tumors of stage 3 and 4 depicting higher miR-145 expression as compared to early stages. Further, the role of miR-145 in epithelial-mesenchymal (EMT) transition found support through the observation of two key markers, Vimentin and ALDL, where a positive correlation with Vimentin protein and a negative correlation with ALDL mRNA expression were observed. CONCLUSION: Our results demonstrate miR-145 as a pro-cancerous candidate, evident from the phenotypes of aggressive cellular proliferation, epithelial to mesenchymal transition, hypermethylation of CpG sites in DDR and apoptotic genes and upregulation of miR-145 in later stages of tumor tissues.

Mapping of PARK2 and PACRG overlapping regulatory region reveals LD structure and functional variants in association with leprosy in unrelated indian population groups.

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae, where the host genetic background plays an important role toward the disease pathogenesis. Various studies have identified a number of human genes in association with leprosy or its clinical forms. However, non-replication of results has hinted at the heterogeneity among associations between different population groups, which could be due to differently evolved LD structures and differential frequencies of SNPs within the studied regions of the genome. A need for systematic and saturated mapping of the associated regions with the disease is warranted to unravel the observed heterogeneity in different populations. Mapping of the PARK2 and PACRG gene regulatory region with 96 SNPs, with a resolution of 1 SNP per 1 Kb for PARK2 gene regulatory region in a North Indian population, showed an involvement of 11 SNPs in determining the susceptibility towards leprosy. The association was replicated in a geographically distinct and unrelated population from Orissa in eastern India. In vitro reporter assays revealed that the two significantly associated SNPs, located 63.8 kb upstream of PARK2 gene and represented in a single BIN of 8 SNPs, influenced the gene expression. A comparison of BINs between Indian and Vietnamese populations revealed differences in the BIN structures, explaining the heterogeneity and also the reason for non-replication of the associated genomic region in different populations.

Insulin enhances metabolic capacities of cancer cells by dual regulation of glycolytic enzyme pyruvate kinase M2.

BACKGROUND: Insulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating glycolytic enzyme pyruvate kinase M2 (PKM2) was examined. RESULTS: We observed that insulin up-regulated PKM2 expression, through PI3K/mTOR mediated HIF1α induction, but significantly reduced PKM2 activity independent of this pathway. Drop in PKM2 activity was attributed to subunit dissociation leading to formation of low activity PKM2 oligomers, as assessed by density gradient centrifugation. However, tyrosine 105 phosphorylation of PKM2, known for inhibiting PKM2 activity, remained unaffected on insulin treatment. Interestingly, insulin-induced ROS was found responsible for PKM2 activity reduction. The observed changes in PKM2 status led to augmented cancer metabolism. Insulin-induced PKM2 up-regulation resulted in enhanced aerobic glycolysis as confirmed by PKM2 knockdown studies. Further, PKM2 activity reduction led to characteristic pooling of glycolytic intermediates and increased accumulation of NADPH; suggesting diversion of glucose flux towards macromolecular synthesis, necessary for cancer cell growth. CONCLUSION: The study identifies new PKM2-mediated effects of insulin on cancer metabolism, thus, advancing the understanding of insulin's role in cancer.

tREPs-A New Class of Functional tRNA-Encoded Peptides.

We asked if transfer RNA (tRNA) ever got an opportunity of translating its own sequence during evolution, what would have been the function of such tRNA-encoded peptides (tREPs)? If not, could one artificially synthesize tREPs to study the corresponding functional outcomes? Here, we report a novel, first-in-the-class, chemically synthesized tREP-18 molecule originating from the Escherichia coli tRNA sequence showing potent antileishmanial property. As a first step, E. coli tRNAs were computationally translated into peptide sequence equivalents and a database of full-length hypothetical tREPs was created. The tREP sequences were sent into sequence, structure, and energy filters to narrow down potential peptides for experimental validation. Based on the functional predictions, tREPs were screened against antiparasitic targets, leading to the identification of tREP-18 as a potential antiparasitic peptide. The in vitro assay of chemically synthesized tREP-18 on the Ag83 strain of Leishmania donovani showed its potent antileishmanial property (IC50 value of 22.13 nM). The atomic force microscopy and scanning electron microscopy images indicated significant alteration in the cytoskeletal architecture of tREP-18-treated parasites. Also, tREP-18 seems to destabilize the mitochondrial membrane potential of parasites, disrupting their cellular integrity and leading to parasitic death. The cellular assays of the tREP-18 peptide on the BS12 strain, a clinical isolate of post-kala azar dermal leishmaniasis, demonstrated its significant efficacy at an IC50 value of 15 nM. The tREP-18 peptide showed a toxic effect on the amastigote stage of the parasite, showing macrophage pathogen clearance at a concentration of 22.5 nM. This study provides the proof of the concept of making a new class of functional peptides from tRNA sequences. It also opens a huge untapped tRNA-peptide space toward novel discoveries and applications. In the future, it would be interesting to perform tREP edits and redesign tREPs toward specific applications.

miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.

INTRODUCTION: New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored. METHODS: Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2. RESULTS: It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2. CONCLUSIONS: mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

Coumarin: An emerging antiviral agent.

Viral infections are responsible for many illnesses, and recent outbreaks have raised public health concerns. Despite the availability of many antiviral drugs, they are often unsuccessful due to the generation of viral mutants and less effective against their target virus. Identifying novel antiviral drugs is therefore of critical importance and natural products are an excellent source for such discoveries. Coumarin is one such natural compound that is a potential drug candidate owing to its properties of stability, solubility, and low toxicity. There are numerous evidences showing its inhibitory role against infection of various viruses such as HIV, Influenza, Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). The mechanisms involve either inhibition of proteins essential for viral entry, replication and infection or regulation of cellular pathways such as Akt-Mtor (mammalian target of rapamycin), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and anti-oxidative pathway including NrF-2 (The nuclear factor erythroid 2 (NFE2)-related factor 2). This review summarizes the present state of understanding with a focus on coumarin's antiviral effect and their possible molecular mechanisms against Influenza virus, HIV, Hepatitis virus, Dengue virus and Chikungunya virus.

MicroRNA (hsa-miR-19b-2-5p) targets key mitochondrial biogenesis genes-a bioinformatics analysis.

The present study on the basis of a detailed bioinformatics analysis proposed a potential role of a miRNA, hsa-miR-19b-2-5p, in regulating the mitochondrial biogenesis. The miRNA has shown to be involved in important biological processes of cellular metabolic, cellular macromolecule biosynthetic processes and gene expression pathways. The miRNA, hsa-miR-19b-2-5p, was predicted to regulate the molecular function of nucleic acid, organic/heterocyclic compound, nucleic acid binding transcription factor activity. The pathway enrichment analysis suggested that this miRNA participated in several metabolic pathways which could be a key to the regulation of the mitochondrial gene expression and biogenesis. In addition, this miRNA targets a total of 112 mitochondria-related genes, establishing further the crucial role of the candidate miRNA in mitochondrial biology.

Deciphering the role of microRNA - A step by step guide.

MicroRNAs (miRNAs), are small non-coding RNAs of approximately 22 nucleotides in length, playing an important role in regulating gene expression post-transcriptionally. Understanding the effect of miRNA regulation in a pathway-specific manner unravels the approaches adopted to apprehend biological mechanisms, the information, which is scanty for researchers, not primed already for miR related research. Here, we describe a quick perspective in 5 steps with probable approaches and assays at every level to unravel the specific role of a microRNA, miR-145a-5p, as an example. This perspective as a guide would help in identifying novel targets for a microRNA, as shown for miR-145a-5p, which down-regulated the mRNA expression of ADD3 and BRCA2, using bioinformatic tools and experimental assays.

ERK2-ZEB1-miR-101-1 axis contributes to epithelial-mesenchymal transition and cell migration in cancer.

Regulation of metastasis continues to remain enigmatic despite our improved understanding of cancer. Identification of microRNAs associated with metastasis in the recent past has provided a new hope. Here, we show how microRNA-101 (miR-101) regulates two independent processes of cellular metastasis by targeting pro-metastatic upstream regulatory transcription factors, ZEB1 and ZEB2, and downstream effector-actin modulators, RHOA and RAC1, providing a single target for therapeutic intervention. Further, we depict how down-regulation of miR-101 by extracellular signal-regulated kinase-2 (ERK2) is vital for MAP kinase pathway induced cellular migration and mesenchymal transition. Importantly, EKR2 induced expression of ZEB1 seems essential for down-regulation of miR-101-1 and induction of EMT. Given the role of EMT in metastasis, we also observe a significant correlation between miR-101 expression and lymph node metastasis; and identify the ERK2-ZEB1-miR-101-1 pathway active in breast cancer tissues, with an apparent clinicopathological implication.

miR-24-2 regulates genes in survival pathway and demonstrates potential in reducing cellular viability in combination with docetaxel.

MicroRNAs the small (18-22 in length) noncoding RNA molecules are negative regulators of gene expression, modulating biological processes of cell differentiation, survival and death. The latter two phenomena are critical in tumour biology. We provide here the results of human genome wide target prediction of one such microRNA, hsa-miR-24-2, shown to target genes essential for initiating cellular stability and cell survival. The protein-protein interaction study showed important nodes which could affect cell cycle progression and differential oncogenesis. An analysis of hsa-miR-24-2 in sporadic breast tumours showed a negative correlation with metastasis and increasing nodes. The conclusion drawn of hsa-miR-24-2 targeting the genes of cell survival correlated with the methylation profile and resultant transcription factor binding site gain or loss in support of absence of cell survival. In order to accentuate the potential of hsa-miR-24-2 to reduce cellular viability under experimental conditions, in vitro studies in the presence and absence of anti-cancer drugs, such as docetaxel resulted in a significant decrease in cellular viability even at a 200-fold reduced dose of the drug in combination with hsa-miR-24-2.

Combined effect of microRNA, nutraceuticals and drug on pancreatic cancer cell lines.

AIM: We proposed to investigate the combination effect of microRNA, nutraceuticals and drug (MND), in two pancreatic cancer cell lines to assess the therapeutic potential. MATERIALS AND METHODS: MIA PaCa-2 and PANC-1 cells transfected with miR-101 or miR-24-2 were treated with Betulinic acid or Thymoquinone and gemcitabine independently and in combination and assessed for the extent of synergism in both experimental and control conditions, considering significance at the p value of <0.05. RESULTS: miR-101 or miR-24-2 over-expressing cells when treated with lower than IC50 doses of the dietary compounds and drug showed a reduced (37-50%) viability in two cell lines with differential synergistic effect and the outcome for Pro-caspase3, Poly (ADP-ribose) polymerase (PARP) cleavage and PKM2 expression. CONCLUSION: Two independent microRNA backgrounds showed promise in therapeutic intervention of gemcitabine sensitive, MIA PaCa-2 and resistant, PANC-1 pancreatic cancer cells, in combination with dietary agents and drug.

MiR-101 induces senescence and prevents apoptosis in the background of DNA damage in MCF7 cells.

Moderately increased DNA damage due to the exogenous miR-101 (4 fold) over-expression in MCF7 cells was substantiated by an increase in the number of γ-H2AX foci, correlating with a simple-to-do Halo-assay. miR-101 induced mild/moderate DNA damage favoured senescence rather than apoptosis. An experimental support emanated from the induced mild/moderate DNA damage with 1 µM/5 µM etoposide in MCF7 cells, which resulted in an endogenous miR-101 over-expression (10/4 fold, respectively), followed by senescence. On the other hand, the severe DNA damage induced with 10 µM etoposide, resulted in a low (<1 fold) endogenous expression of miR-101 and an elevated percentage of apoptotic cells. Using bioinformatics tools along with in-vitro and in-vivo validations, miR-101 was found to target and downregulate the mRNA expression of UBE2N and SMARCA4, involved in DNA damage repair (DDR) pathways. Recovery of the expression of the two novel targets in anti-miR-101 transfection validated the results. We conclude that a threshold range of over-expressed miR-101, capable of inducing mild/moderate DNA damage, is sensed by cells to become senescent. The observation derives further support from in-silico protein-protein network analysis where the two novel targets showed their involvement in senescence pathway.

mtDNA germ line variation mediated ROS generates retrograde signaling and induces pro-cancerous metabolic features.

mtDNA non-synonymous germ line variation (G10398A; p.A114T) has remained equivocal with least mechanistic understanding in showing an association with cancer. This has necessitated showing in-vitro how an over-expression within mitochondria of either of the variants produces higher intracellular ROS, resulting in differential anchorage dependent and independent growth. Both these features were observed to be relatively higher in ND3:114T variant. An elevated amount of intracellular carbonylated proteins and a reduced activity of a key glycolytic enzyme, Pyruvate kinase M2, along with high glucose uptake and lactate production were other pro-cancerous features observed. The retrograde signaling through surplus ROS was generated by post-ND3 over-expression regulated nuclear gene expression epigenetically, involving selectively the apoptotic-DDR-pathways. The feature of ND3 over-expression, inducing ROS mediated pro-cancerous features in the cells in in vitro, was replicated in a pilot study in a limited number of sporadic breast tumors, suggesting the importance of mitochondrial germ-line variant(s) in enabling the cells to acquire pro-cancerous features.

Synergistic combination of gemcitabine and dietary molecule induces apoptosis in pancreatic cancer cells and down regulates PKM2 expression.

Gemcitabine, an effective agent in treatment of cancer of pancreas, has undergone failures in many instances after multiple cycles of therapy due to emergence of drug resistance. Combination of dietary compounds with clinically validated drugs has emerged as an effective therapeutic approach to treat pancreatic tumors, refractory to gemcitabine therapy. In order to optimize a possible synergistic combination of Gemcitabine (GCB) with dietary molecules, Betuilnic acid (BA) and Thymoquinone (TQ), stand-alone IC50 dose of GCB, BA and TQ was calculated for pancreatic cancer cell lines. Fixed IC50 dose ratio of the dietary molecules in combination with reduced IC50 dose of GCB was tested on GCB resistant PANC-1 and sensitive MIA PaCa-2 cells for synergism, additive response and antagonism, using calcusyn. Combination index (CI) revealed that pre-treatment of BA and TQ along with GCB synergistically inhibited the cancer cell proliferation in in-vitro experiments. Pyruvate kinase (PK) M2 isoform, a promising target involved in cancer cell metabolism, showed down-regulation in presence of TQ or BA in combination with GCB. GCB with BA acted preferentially on tumor mitochondria and triggered mitochondrial permeability transition. Pre-exposure of the cell lines, MIA PaCa-2 and PANC-1, to TQ in combination with GCB induced apoptosis. Thus, the effectiveness of BA or TQ in combination with GCB to inhibit cell proliferation, induce apoptosis and down-regulate the expression of PKM2, reflects promise in pancreatic cancer treatment.

Replication of type 2 diabetes candidate genes variations in three geographically unrelated Indian population groups.

Type 2 diabetes (T2D) is a syndrome of multiple metabolic disorders and is genetically heterogeneous. India comprises one of the largest global populations with highest number of reported type 2 diabetes cases. However, limited information about T2D associated loci is available for Indian populations. It is, therefore, pertinent to evaluate the previously associated candidates as well as identify novel genetic variations in Indian populations to understand the extent of genetic heterogeneity. We chose to do a cost effective high-throughput mass-array genotyping and studied the candidate gene variations associated with T2D in literature. In this case-control candidate genes association study, 91 SNPs from 55 candidate genes have been analyzed in three geographically independent population groups from India. We report the genetic variants in five candidate genes: TCF7L2, HHEX, ENPP1, IDE and FTO, are significantly associated (after Bonferroni correction, p<5.5E-04) with T2D susceptibility in combined population. Interestingly, SNP rs7903146 of the TCF7L2 gene passed the genome wide significance threshold (combined P value = 2.05E-08) in the studied populations. We also observed the association of rs7903146 with blood glucose (fasting and postprandial) levels, supporting the role of TCF7L2 gene in blood glucose homeostasis. Further, we noted that the moderate risk provided by the independently associated loci in combined population with Odds Ratio (OR)<1.38 increased to OR = 2.44, (95%CI = 1.67-3.59) when the risk providing genotypes of TCF7L2, HHEX, ENPP1 and FTO genes were combined, suggesting the importance of gene-gene interactions evaluation in complex disorders like T2D.