Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington's Disease.

Altered expression levels of protein-coding genes and microRNAs have been implicated in the pathogenesis of Huntington's disease (HD). The involvement of other ncRNAs, especially long ncRNAs (lncRNA), is being realized recently and the related knowledge is still rudimentary. Using small RNA sequencing and PCR arrays we observed perturbations in the levels of 12 ncRNAs in HD mouse brain, eight of which had human homologs. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. Transient knock-down of Meg3 and Neat1 in cell models of HD led to a significant decrease of aggregates formed by mutant huntingtin and downregulation of the endogenous Tp53 expression. Understanding Meg3 and Neat1 functions in the context of HD pathogenesis is likely to open up new strategies to control the disease.

Regulation of mitochondrial morphology and cell cycle by microRNA-214 targeting Mitofusin2.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the increase in CAG repeats beyond 36 at the exon1 of the gene Huntingtin (HTT). Among the various dysfunctions of biological processes in HD, transcription deregulation due to abnormalities in actions of transcription factors has been considered to be one of the important pathological conditions. In addition, deregulation of microRNA (miRNA) expression has been described in HD. Earlier, expression of microRNA-214 (miR-214) has been shown to increase in HD cell models and target HTT gene; the expression of the later being inversely correlated to that of miR-214. In the present communication, we observed that the expressions of several HTT co-expressed genes are modulated by exogenous expression of miR-214 or by its mutant. Among several HTT co-expressed genes, MFN2 was shown to be the direct target of miR-214. Exogenous expression of miR-214, repressed the expression of MFN2, increased the distribution of fragmented mitochondria and altered the distribution of cells in different phases of cell cycle. In summary, we have shown that increased expression of miR-214 observed in HD cell model could target MFN2, altered mitochondrial morphology and deregulated cell cycle. Inhibition of miR-214 could be a possible target of intervention in HD pathogenesis.

E2F1 activates MFN2 expression by binding to the promoter and decreases mitochondrial fission and mitophagy in HeLa cells.

Mitofusin-2 (MFN2) is primarily involved in mitochondrial fusion and participates in diverse biological processes. Several reports show that MFN2 is a target of different miRNAs; however, the transcriptional regulation of MFN2 has not been extensively studied. To gain insight into the transcriptional regulation of MFN2, we expressed E2F transcription factor 1 (E2F1) exogenously and observed that it increased the endogenous expression of MFN2 by binding to its putative promoter region. Although the levels of E2F1 were shown to vary during the cell cycle, the expression of MFN2 and its regulator SP1 did not change throughout the different phases, suggesting that E2F1 regulates MFN2 in a cell-cycle-independent manner. In the cell-cycle phases, where the expression of E2F1 was reduced, SP1 might act in its place to regulate the expression of MFN2. We showed that E2F1 and SP1 are present as a complex on the promoter of MFN2 during the S-phase as well as in E2F1 overexpressing cells, suggesting that they may regulate the expression of MFN2 synergistically. Furthermore, we found that E2F1 modulated mitochondrial fusion and mitophagy, likely through regulation of MFN2. Bioinformatic analysis revealed that several potential targets of E2F1 are localized in mitochondria and associated with autophagy. Collectively, these data identify the E2F1-MFN2 axis as a regulator of mitochondrial morphology and mitophagy, suggesting a potential therapeutic target for the treatment of mitochondrial disorders.

Cervical cancer subtypes harbouring integrated and/or episomal HPV16 portray distinct molecular phenotypes based on transcriptome profiling of mRNAs and miRNAs.

Heterogeneity in cervical cancers (CaCx) in terms of HPV16 physical status prompted us to investigate the mRNA and miRNA signatures among the different categories of CaCx samples. We performed microarray-based mRNA expression profiling and quantitative real-time PCR-based expression analysis of some prioritised miRNAs implicated in cancer-related pathways among various categories of cervical samples. Such samples included HPV16-positive CaCx cases that harboured either purely integrated HPV16 genomes (integrated) and those that harboured episomal viral genomes, either pure or concomitant with integrated viral genomes (episomal), which were compared with normal cervical samples that were either HPV negative or positive for HPV16. The mRNA expression profile differed characteristically between integrated and episomal CaCx cases for enriched biological pathways. miRNA expression profiles also differed among CaCx cases compared with controls (upregulation-miR-21, miR-16, miR-205, miR-323; downregulation-miR-143, miR-196b, miR-203, miR-34a; progressive upregulation-miR-21 and progressive downregulation-miR-143, miR-34a, miR-196b and miR-203) in the order of HPV-negative controls, HPV16-positive non-malignant samples and HPV16-positive CaCx cases. miR-200a was upregulated in HPV16-positive cervical tissues irrespective of histopathological status. Expression of majority of the predicted target genes was negatively correlated with their corresponding miRNAs, irrespective of the CaCx subtypes. E7 mRNA expression correlated positively with miR-323 expression among episomal cases and miR-203, among integrated cases. miR-181c expression was downregulated only among the episomal CaCx cases and negatively correlated with protein coding transcript of the proliferative target gene, CKS1B of the significantly enriched "G2/M DNA Damage Checkpoint Regulation" pathway among CaCx cases. Thus, the two CaCx subtypes are distinct entities at the molecular level, which could be differentially targeted for therapy. In fact, availability of a small molecule inhibitor of CKS1B, suggests that drugging CKS1B could be a potential avenue of treating the large majority of CaCx cases harbouring episomal HPV16.

Regulation of Cell Cycle Associated Genes by microRNA and Transcription Factor.

Cell cycle is a complex process and regulated at transcriptional, post-transcriptional and posttranslational levels. Large numbers of genes are implicated in the process. Abnormality at any stage of cell cycle may lead to diseases including cancer. To gain global view of genes associated with cell cycle, their regulation by transcription factors and microRNAs, we collected genes related to cell cycle from different databases. Experimentally validated targets of microRNAs are collected from miRTarbase. Transcription factors that bind to upstream sequences of cell cycle associated genes and microRNA genes were collected from published papers. We collected 3028 genes associated with cell cycle. These proteins belong to different protein classes like nucleic acid binding (594 proteins), transcription factors (305 proteins), cytoskeletal (232 proteins), kinases (174 proteins), phosphatase (111 proteins) and chaperones (84 proteins). Among 3028 cell cycle associated genes, 2125 genes are validated targets of 424 microRNAs; CDKN1A is a target of 46 miRNAs and miR-335 targets 301 genes. About 100 transcription factors had binding sites at potential promoter regions of 2722 genes and 329 microRNAs that target cell cycle associated genes. We presented the largest numbers of cell cycle associated genes. Many transcription factors regulate both cell cycle associated genes and the miRNAs that target cell cycle associated genes. These resources will be utilized to identify the co-regulation of cell cycle associated genes by transcription factors and miRNAs and to test specific hypothesis for cell cycle regulation and its alteration in different diseases.

Chaperone-like protein HYPK and its interacting partners augment autophagy.

To decipher the function(s) of HYPK, a huntingtin (HTT)-interacting protein with chaperone-like activity, we had previously identified 36 novel interacting partners of HYPK. Another 13 proteins were known earlier to be associated with HYPK. On the basis of analysis of the interacting partners of HYPK, it has been shown that HYPK may participate in diverse cellular functions relevant to Huntington's disease. In the present study, we identified additional 5 proteins by co-immunoprecipitation and co-localization. As of now we have 54 primary interactors of HYPK. From the database we collected 1026 unique proteins (secondary interactors) interacting with these 54 primary HYPK interacting partners. We observed that 10 primary and 91 secondary interacting proteins of HYPK are associated with two types of autophagy processes. We next tested the hypothesis that the hub, HYPK, might itself be involved in autophagy. Using mouse striatal STHdh(Q7)/Hdh(Q7) cell lines, we observed that over expression of HYPK significantly increased background cellular autophagy, while knock down of endogenous HYPK decreased the autophagy level as detected by altered LC3I conversion, BECN1 expression, cleavage of GFP from LC3-GFP, ATG5-ATG12 conjugate formation and expression of transcription factors like Tfeb, Srebp2 and Zkscan3. This result shows that HYPK, possibly with its interacting partners, induces autophagy. We further observed that N-terminal mutant HTT reduced the cellular levels of LC3II and BECN1, which could be recovered significantly upon over expression of HYPK in these cells. This result further confirms that HYPK could also be involved in clearing mutant HTT aggregates by augmenting autophagy pathway.