Small Interfering RNA Molecules Research Articles

RNA Interference of Human Papillomavirus Type 18 E6 and E7 Induces Senescence in HeLa Cells

The human papillomavirus oncoproteins E6 and E7 promote cell proliferation and contribute to carcinogenesis by interfering with the activities of cellular tumor suppressors. We used a small interfering RNA molecule targeting the E7 region of the bicistronic E6 and E7 mRNA to induce RNA interference, thereby reducing expression of E6 and E7 in HeLa cells. RNA interference of E6 and E7 also inhibited cellular DNA synthesis and induced morphological and biochemical changes characteristic of cellular senescence. These results demonstrate that reducing E6 and E7 expression is sufficient to cause HeLa cells to become senescent.

Antisense technologies. Improvement through novel chemical modifications.

Antisense agents are valuable tools to inhibit the expression of a target gene in a sequence-specific manner, and may be used for functional genomics, target validation and therapeutic purposes. Three types of anti-mRNA strategies can be distinguished. Firstly, the use of single stranded antisense-oligonucleotides; secondly, the triggering of RNA cleavage through catalytically active oligonucleotides referred to as ribozymes; and thirdly, RNA interference induced by small interfering RNA molecules. Despite the seemingly simple idea to reduce translation by oligonucleotides complementary to an mRNA, several problems have to be overcome for successful application. Accessible sites of the target RNA for oligonucleotide binding have to be identified, antisense agents have to be protected against nucleolytic attack, and their cellular uptake and correct intracellular localization have to be achieved. Major disadvantages of commonly used phosphorothioate DNA oligonucleotides are their low affinity towards target RNA molecules and their toxic side-effects. Some of these problems have been solved in 'second generation' nucleotides with alkyl modifications at the 2' position of the ribose. In recent years valuable progress has been achieved through the development of novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. In addition, RNA-cleaving ribozymes and deoxyribozymes, and the use of 21-mer double-stranded RNA molecules for RNA interference applications in mammalian cells offer highly efficient strategies to suppress the expression of a specific gene.

Biological models and genes of tumor reversion: cellular reprogramming through tpt1/TCTP and SIAH-1.

Tumor reversion is the process by which some cancer cells lose their malignant phenotype. This study was aimed at defining some of the molecular and phenotypic properties of this process. Biological models of tumor reversion were isolated from human leukemia and breast cancer cell lines by using the H-1 parvovirus as a selective agent. Differential gene expression analysis was performed between the parental malignant cells and their revertants or alternatively between these parental cells and their SIAH-1 transfectant counterparts. These SIAH-1 transfectants have a suppressed malignant phenotype and were used as a control for a viral-free system. Two hundred sixty-three genes were found to be either activated or inhibited during the reversion process, as confirmed by Northern blot analysis or quantitative PCR. Of these, 32% were differentially expressed in all systems, irrespective of whether parvovirus-selected, SIAH-1 overexpressing, or p53 mutant or wild-type cell lines were used, suggesting the existence of a universal mechanism underlying tumor reversion. Translationally Controlled Tumor Protein (tpt1/TCTP) has the strongest differential expression, down-regulated in the reversion of U937- and SIAH-1-overexpressing cells. Inhibition of TCTP expression by anti-sense cDNA or small interfering RNA molecules results in suppression of the malignant phenotype and in cellular reorganization, similar to the effect of SIAH-1. Hence, tumor reversion can be defined at the molecular level, not just as the reversal of malignant transformation, but as a biological process in its own right involving a cellular reprogramming mechanism, overriding genetic changes in cancer, by triggering an alternative pathway leading to suppression of tumorigenicity.