Effect Of Mutant Telomerase RNA On HeLa Cell Growth

Telomere is a DNA-protein complex in the chromosome ends, protecting the chromosome ends from degradation. Telomere matainnence and telomerase activation is necessary for most tumor formation. Mutation of the telomere associated proteins and telomerase RNA subunits can cause losing of the telomere end capping function and the genome instability. In such conditions, genome ends fusions and breakage/fusion/bridge (B/F/B) cycles arise with the cell division. It is proposed that telomerase may be a critical factor. The concept of telomerase have been proposed in the 20th century, 80 years.Telomerase is a large ribonucleoprotein (RNP) reverse transcriptase, adding telomeric DNA repeats onto the chromosome ends to maintain the telomere length. The essential telomerase components include the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC), and the TERC-binding protein dyskerin which binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. In recent years, it is found that telomerase activity and telomere stability have related with the tumor formation. Telomerase activity is undetectable in most human cells, while up-regulated in 85% of human cancer cells becomes a hallmark of cancers.This study targets the telomerase RNA subunits and inducing the mutated telomerase RNA into the telomerase positive cancer cells. This is a new method to suppress the growth of the cancer cell. This study aims to construct recombinant pcDNA3.1 (-)-hTER eukaryotic expressing vector, then observed the influence on the the growth of the cancer cell. The recombinants expressing wild type hTER (WT-hTER) or MT-hTER were constructed by molecular cloning and PCR-based mutagenesis technique. HeLa cells were transfected with recombinants expressing WT-hTER or MT-hTER. Stable cell clones expressing extrinsic genes were obtained by G418 screening. The hTER expression level was detected by RT-PCR and the cell growth velocity was measured by MTT. The transfected cells are stained with Hoechst 33342, and then the apoptotic morphology is observed.The results revealed the recombinants could express WT-hTER or MT-hTER successfully in HeLa cells. The two mutant-transfection groups grew slower than the control group. The results indicate that the expression of the MT-hTER could inhibit the growth of the HeLa cell. In one hand, the matutation of the template region can cause the mutated telomeric repeats to be added to the chromosome ends. It may reduce the affinity of the telomere associated protein of the telomere DNA and losing of the telomere end capping function, followed by the chromosome fusion and the genome instability. The cell cycle checkpoint thus is activated and cells are led to senescence and death. On the other hand, telomerase RNA subunit mutation may affect its combination with hTERT, or affect the telomerase stability in the cell, or the way how telomerase extension.Recombinant adenovirus vector of the mutations in human telomerase RNA subunit, which controled by the hTERT promoter, was successfully constructed with the molecular cloning method. In the latter part of the experiment, we intended to use the constructed vector to package the adenovirus.So that the targeted gene only expressed in the telomerase positive cancer cells, thereby increasing the specificity of cancer gene therapy.The research is useful to not only understand the role of telomerase mechanism to and tumor diagnosis, but also genen therapy.