Targeting miRNAs for drug discovery: a new paradigm.

The discovery of miRNAs and the establishment of it's clinical links with multiple diseases has led to a paradigm shift in the drug development pipeline of major pharmaceutical companies and has given birth to several biotechnology enterprises revolving around these magic molecules. The miRNA profiling studies over the last few years have indicated implicit involvement of miRNAs in the pathobiology of cancer, diabetes, infectious diseases as well as cardiovascular, neurological and immune system disorders. This information is currently being translated into tools for diagnosis, prognosis and predicting response to treatment. In addition, active and vigorous investigations are ongoing in several laboratories across academia and industry to decipher the precise molecular functions and mechanism of action for key miRNAs with therapeutic potential. Knowledge gained from these efforts will surely pave the way for designing effective R&D strategies and will revolutionize the way we diagnose and treat various diseases. The present review attempts to track the evolutionary progression of microRNA research from it's early infancy years to its maturity into a dynamic field of drug discovery.

Targeting human 8-oxoguanine DNA glycosylase to mitochondria protects cells from 2-methoxyestradiol-induced-mitochondria-dependent apoptosis.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite of 17beta-estradiol, is known to induce mitochondria-mediated apoptosis through several mechanisms. We sought to study the effect of mitochondrialy targeted hOGG1 (MTS-hOGG1) on HeLa cells exposed to 2-ME. MTS-hOGG1-expressing cells exposed to 2-ME showed increased cellular survival and had significantly less G(2)/M cell cycle arrest compared to vector-only-transfected cells. In addition, 2-ME exposure resulted in an increase in mitochondrial membrane potential, increased apoptosis, accompanied by higher activation of caspase-3, -9, cleavage of Bid to tBid and protein poly(ADP-ribose) polymerase (PARP) cleavage in HeLa cells lacking MTS-hOGG1. Fas inhibitors cerulenin or C75 inhibited 2-ME-induced caspase activation, PARP cleavage, apoptosis and reversed mitochondrial membrane hyperpolarization, thereby recapitulating the increased expression of MTS-hOGG1. Hence, MTS-hOGG1 plays an important protective role against 2-ME-mediated mitochondrial damage by blocking apoptosis induced through the Fas pathway.

Aberrant promoter methylation and tumor suppressive activity of the DFNA5 gene in colorectal carcinoma.

To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine (5-aza-dC). Out of 1776 genes that were initially 'absent (that is, silenced)' by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription-PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65%, 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3%, 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.

The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth.

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

Identification of differentially expressed genes in tobacco chewing-mediated oral cancer by differential display-polymerase chain reaction.

BACKGROUND: Identification of changes in gene expression that occur in oral squamous cell carcinoma (OSCC), after sufficient characterization, may yield novel molecular markers that may be useful in the diagnosis and disease management of oral cancer. MATERIALS AND METHODS: We used differential display-polymerase chain reaction (DD-PCR) to study critically the global gene expression profile of the oral tumour versus normal epithelium. The differential expression of fished out cDNA were confirmed by Northern blot and reverse transcription-PCR. The differentially expressed cDNA were cloned, sequenced and matched for homology in the GenBank database. RESULTS: We identified 13 cDNA that showed differential expression. Out of these we selected four cDNA showing consistent reproducibility. One of the cDNA expressed exclusively in tumour had a homology to DEK, a putative oncogene, and is linked to leukaemia, various cancers, HIV infection and several autoimmune disorders. Another cDNA expressed only in tumour had homology to sorcin protein. Sorcin is a 22-kDa calcium-binding protein and is associated with drug resistance in various cell lines. Apparently, sorcin expression might be responsible for drug resistance of OSCC and poor prognosis. Another cDNA showing 10 times overexpression in cheek tumour as compared to normal had homology to CDK6 gene. Hence, it seems from our results that CDK6 is dysregulated during oral carcinogenesis. The fourth cDNA was overexpressed in normal as compared to cheek tumour, but did not show any match in BLAST search. CONCLUSIONS: We conclude that there is an enormous significance of these differentially expressed cDNA in oral cancer progression as they can serve as cancer markers to be used for diagnosis and therapeutic intervention.

P53 codon 72 polymorphism and susceptibility to development of human papilloma virus-associated cervical cancer in Indian women.

BACKGROUND: Infection with human papilloma virus (HPV) is an important etiological factor in the development of cervical cancer and it has been proposed that individuals homozygous for Arg/Arg at codon-72 of p53 are seven times more susceptible to HPV-mediated cancer. In this study, we have analyzed the genetic predisposition of the India population to HPV infection and cervical carcinogenesis. METHODS: We investigated 71 cases of squamous cell carcinoma of the cervix, 14 cases of low-grade squamous intraepithelial lesions, 25 cases of high-grade squamous intraepithelial lesions and 29 noncancer controls for presence of HPV16/18 infections by L-1-specific PCR assay and Southern hybridization, and its association with polymorphism at p53 codon 72. RESULTS: We observed that 69.1% (76/110) of the cervical cancer patients were HPV positive, among which the presence of HPV16, 18 and 16/18 coinfection was 40.9%, 8.2% and 13.6%, respectively. The allele frequencies of the three p53 genotypes Arg/Arg, Arg/Pro and Pro/Pro in the HPV-positive tumour samples were 0.34, 0.57 and 0.09 in comparison with frequencies of 0.18, 0.44 and 0.38 for HPV-negative tumours. Hence, there is a significant difference in the allelic frequency of p53 Arg/Arg in high-risk HPV-infected cervical carcinoma cases (0.34) and HPV-negative carcinomas (0.18). CONCLUSION: Our results indicate a striking over-representation of homozygous arginine at codon 72 of p53 in HPV-associated cervical carcinogenesis. We conclude that women with Arg/Arg homozygous allele are more prone to infection by HPV16/18, which leads to cervical carcinomas having poor prognosis.