Akhil Varshney

Non-coding RNAs: biological functions and applications

Analyses of the international human genome sequencing results in 2004 converged to a consensual number of ~20 000 protein‐coding genes, spanning over <2% of the total genomic sequence. Therefore, the developmental and physiological complexity of human beings remains unaccounted if viewed only in terms of the number of protein‐coding genes; the epigenetic influences involving chromatin remodelling and RNA interference and alternative precursor messenger RNA splicing of functional protein‐coding transcripts as well as post‐translational modifications of proteins increase the diversity and the functionality of the proteome and likely explain the increased complexity. In addition, there has been an explosion of research addressing possible functional roles for the other 98% of the human genome that does not encode proteins. In fact, >90% of the human genome is likely to be transcribed yielding a complex network of overlapping transcripts that include tens of thousands of long RNAs with little or no protein forming capacity; they are collectively called non‐coding RNA. This review highlights the fundamental concepts of biological roles of non‐coding RNA and their importance in regulation of cellular physiology under disease conditions like cancer. Copyright

Expression of Measles Virus Nucleoprotein Induces Apoptosis and Modulates Diverse Functional Proteins in Cultured Mammalian Cells

Background Measles virus nucleoprotein (N) encapsidates the viral RNA, protects it from endonucleases and forms a virus specific template for transcription and replication. It is the most abundant protein during viral infection. Its C-terminal domain is intrinsically disordered imparting it the flexibility to interact with several cellular and viral partners. Principal Findings In this study, we demonstrate that expression of N within mammalian cells resulted in morphological transitions, nuclear condensation, DNA fragmentation and activation of Caspase 3 eventuating into apoptosis. The rapid generation of intracellular reactive oxygen species (ROS) was involved in the mechanism of cell death. Addition of ascorbic acid (AA) or inhibitor of caspase-3 in the extracellular medium partially reversed N induced apoptosis. We also studied the protein profile of cells expressing N protein. MS analysis revealed the differential expression of 25 proteins out of which 11 proteins were up regulated while 14 show signs of down regulation upon N expression. 2DE results were validated by real time and semi quantitative RT-PCR analysis. Conclusion These results show the pro-apoptotic effects of N indicating its possible development as an apoptogenic tool. Our 2DE results present prima facie evidence that the MV nucleoprotein interacts with or causes differential expression of a wide range of cellular factors. At this stage it is not clear as to what the adaptive response of the host cell is and what reflects a strategic modulation exerted by the virus.

In vitro selected RNA aptamer recognizing glutathione induces ROS-mediated apoptosis in the human breast cancer cell line MCF 7

Glutathione (GSH) is an abundant natural tripeptide with antioxidant properties. Under different conditions, it can play protective as well as pathogenic roles. The redox state of the cell has an important role in the induction of apoptosis. Elevated level of glutathione in cancer cells provides resistance to a number of chemotherapeutic drugs. Inhibition of glutathione synthesis sensitizes the cells for apoptosis and enhances the activity of chemotherapeutic drugs. We have selected GSH-binding RNA aptamers by employing in vitro selection protocol SELEX. The Kd value of these aptamers with respect to GSH were determined by surface plasmon resonance (SPR) analysis and isocratic affinity chromatography. Two aptamers GSHapt 8.17 (class-III) and GSHapt 5.39 (class-IV) had Kd values of 4.18 and 4.89 x 10-8M, respectively and GSHapt class-I had a Kd value of 1.2 x 10-6M. CD spectra suggested conformational change in aptamers upon GSH binding. Cultured breast cancer cells (MCF7) responded to expression of GSH aptamers by accumulating ROS and undergoing morphological transition, nuclear condensation, and DNA fragmentation, with concurrent depletion of cellular GSH and activation of caspase 3 eventually leading to apoptosis. DTT and caspase-3 inhibitor partially rescued aptamer induced apoptosis. These aptamers exhibit high specificity to GSH over non specific competitor. The same aptamers did not induce apoptosis in 293T cells. The kinetic properties and pro-apoptotic effects suggest that glutathione-binding RNA aptamer could be developed into an effective anti-cancer chemotherapeutic agent.

Identification of an RNA aptamer binding hTERT-derived peptide and inhibiting telomerase activity in MCF7 cells

Human telomerase reverse transcriptase is an essential rate-limiting component of telomerase complex. hTERT protein in association with other proteins and the human telomerase RNA (hTR) shows telomerase activity, essential for maintaining genomic integrity in proliferating cells. hTERT binds hTR through a decapeptide located in the RID2 (RNA interactive domain 2) domain of N-terminal region. Since hTERT is essential for telomerase activity, inhibitors of hTERT are of great interest as potential anti-cancer agent. We have selected RNA aptamers against a synthetic peptide from the RID2 domain of hTERT by employing in vitro selection protocol (SELEX). The selected RNAs could bind the free peptide, as CD spectra suggested conformational change in aptamer upon RID2 binding. Extracts of cultured breast cancer cells (MCF7) expressing this aptamer showed lower telomerase activity as estimated by TRAP assay. hTERT-binding RNA aptamers hold promise as probable anti-cancer therapeutic agent.

Global expression profile of telomerase-associated genes in HeLa cells.

Telomerase is a specialized nucleoprotein enzyme complex that maintains the telomere length. The telomerase reverse transcriptase (TERT) is the catalytically active component of the telomerase complex. In humans, the protein component (hTERT) and RNA component (hTR) are found to differentially express in cancer cells. In contrast to differentiated cells, most of the cancer cells overexpress hTERT, which is needed to maintain the proliferative potential of cells. The overexpression of telomerase is not proportionate to telomere length in cancer cells, suggesting that the immortalizing phenotype can be mediated through other factors in addition to telomere length. To investigate the role of hTERT in immortalizing process, loss of gene function studies were carried out. Short interfering RNA (siRNA) and short hairpin RNA (shRNA) against hTERT showed the reduction of hTERT transcript, reduction of telomerase activity and alteration of gene expression in HeLa cells. The molecular basis of proliferative capacity of hTERT was investigated by gene expression microarray. Analysis of microarray data for HeLa cells following siRNA and shRNA mediated knockdown of hTERT showed that 80 genes were upregulated and 73 genes downregulated. Out of these, 37 genes are known to be involved in cancer. Further analyses of previously known genes involved in cancer like KLF4, FGF2, IRF-9 and PLAU by Real Time PCR showed their upregulation. We are documenting for the first time the effect of knocking down hTERT on expression of KLF4 and FGF2. Interestingly, it has been earlier reported that KLF4 and FGF2 up-regulate the expression of hTERT in cancer cells. This suggests that hTERT may be subject to its own auto-regulatory effects.

Expression of targeted ribozyme against telomerase RNA causes altered expression of several other genes in tumor cells

Telomeres are tandem repeat sequences present at chromosome end that are synthesized by RNA-protein enzyme called telomerase. The RNA component (TR) serves as template for telomerase reverse transcriptase (TERT) for generating telomere repeats. TERT is overexpressed in actively dividing cells including cancerous cells, absent in differentiated somatic cells whereas human telomerase RNA (hTR) is present in normal as well as in cancer cells. Telomerase overexpression in cancer cells ensures telomere length maintenance that actually provides proliferative advantage to cells. Stable expression of ribozyme against hTR in HeLa cells results in reduction of hTR levels, telomerase activity, and telomere length which is accompanied by altered cell morphology and expression of several specific cellular genes. The altered genes deduced from differentially display PCR and 2D gel electrophoresis upon hTR knockdown have function in ribosome biogenesis, chromatin modulation, cell cycle control, and p63-dependant pathways. Our observations shows hTR participates in diverse cellular functions other than telomere maintenance, validates as a possible drug targets in p53- and pRB-negative status, and indicated possible cross-talks between telomerase and other cellular pathways.

Diversity and functional evolution of the plasminogen activator system

The urokinase plasminogen activator system is a family of serine proteases which consists of uPA (urokinase plasminogen activator), uPAR (urokinase type plasminogen activator receptor) and PAI-1 (plasminogen activator inhibitor 1). In addition to their significant roles in activation, these proteases act as key regulators of the tumor microenvironment and are involved in the metastatic process in many cancers. High levels of uPA system proteases in many human cancer predicts poor patient prognosis and strongly indicated a key role of uPA system in cancer metastasis. Individual components of uPA system are found to be differentially expressed in cancer cells compared to normal cells and therefore are potential therapeutic targets. In this review, we present the molecular and cellular mechanisms underlying the role of uPA system in cancer progression. Epithelial to mesenchymal transitions (EMT) is the main cause of the cancer cell metastasis. We have also attempted to relate the role of uPA signaling in EMT of cancer cells.