GENE SILENCING

Objectives: Cancer, the most complex group of genetic disorders results due to over expression or mutation of oncogenes/molecules involved in cell signaling pathways. KRAS is an oncogene that encodes a small GTPase protein with two isoforms KRasA & KRasB and is involved in the regulation of cell division. KRas is frequently found mutated in lung, pancreas, colorectal and many other cancers. Various studies have found that KRasB promotes cell proliferation and inhibits apoptosis whereas KRasA has negligible role in cell proliferation or rather is involved in apoptosis at times. Several experiments have shown tumor growth inhibition by silencing KRas in various tumor models having a differential allelic expression.The goal of our study was to determine the possible differential role of KRas A and B on MAPK Pathway. To examine the disparity in role of various isoforms of KRas on apoptosis, we evaluated the expression of these isoforms through different modalities in HeLa cells before and after silencing KRas through RNA interference. Study Design: In vitro study for isolation of protein molecules (Proteomics) and to study various genes (Genomics) through Polymerase chain reaction. Study Duration: December, 2011-September, 2014. Setting: Center for Research in Molecular Medicine, University of Lahore. Material & Methods: In present study, we studied the expression level and behavior of many sets of molecules such as KRasA, KrasB, Bad, Bcl2, BclxL and Mcl-1 through gene quantitation by Real Time PCR. We also analyzed the protein expression through Western blot immune-precipitation. All the tests were done before and after 48-hours of silencing of HeLa cells with shRNA designed for KRas. Results: We successfully downregulated KRasB (80%) but found upregulation of KRasA with continued cell proliferation. We also found overexpression of antiapoptotic genes, BclxL and Mcl1 and downregulation of proapoptotic molecule-Bad. Differences were considered significant at p< 0.01. Values were expressed as mean ± SEM from six separate experiments. Conclusion: We were able to show that in the absence of one proliferative gene, another sister gene upregulates and takes over the role of uncontrolled cell proliferation. This usually leads to failure of most cancer controltherapies.