Effect Of HTER Site-mutants On Cell Proliferation

Telomeres were composed of specific DNA repeat sequence and associated specific binding proteins in eukaryotes chromosome ends. The length of telomere gradually shortened as the number of cell divisions increase, and telomerase which used its RNA template to synthesis telomere fragments can compensate for deletions caused by replication. Binding proteins were also needed to participate in the regulation of telomere function. Telomere-binding proteins were a class of proteins which were specific binding on the different sites of telomere involved in telomere functions. The function of telomeres interacting with telomere-binding protein was to protect the chromosome from being degradation by nuclease and prevent chromosome from end fusion, also to overcome ends of chromosomes shorten caused by DNA replication defects and ensure the integrity of chromosomes.Telomerase played an important role in the telomere function of synthesis telomere DNA and stabilize chromosome structure. Thus site-directed mutagenesis near the template sequence of telomerase may have a great impact on telomerase function, also have great significance and reference value for cancer treatment.By cloning human telomerase RNA gene and site-directed mutagenesis of A61A62/GG and G63G64/AA near the template sequence of telomerase RNA through overlap extension PCR method (OE-PCR), two mutant fragments named T1 and T2 were obtained. Recombinant plasmids of pMD18-hTER were constructed respectively by pMD18T-vector with T1 and T2. Two connection products were transformed into E.coli DH5αcompetent cells and recombinant plasmids were identified by PCR and enzyme digestion after plasmid DNA were extracted. Digestion products were connected with eukaryotic expression vector of pcDNA3.1(-) after they were purified. Connection products were transformed into E.coli DH5αcompetent cells again. The vector of pcDNA-hTER was obtained by extraction of recombinant plasmid and was successfully constructed confirmed by identification of enzyme digestion. In the process of constructing eukaryotic expression vector, recombinant plasmid which was constructed by pcDNA3.1(-) with telomerase RNA gene without mutations was set as positive control and pcDNA3.1(-) empty vector was set as a negative control. Recombinant plasmids of T1, T2, positive control and negative control were transfected into HeLa cells. After G418 screening, monoclones of cells were formed by transfecting with each recombinant plasmid. Extraction of total RNA from culture clones which were expanded, and then detecting the target gene was integrated into the genome of HeLa cells through reverse transcriptase PCR method (RT-PCR). It was indicated that recombinant plasmids are expressed stably in cells.The growth curve of HeLa cells transfected with different plasmids was drawn by the MTT method. The results showed that T1 promoted cell growth to some extent and T2 inhibited cell growth to some extent. Analysis of causing these results may be due to the T1 mutant made the combination between telomerase and telomere becomes more stable playing a catalytic role in the synthesis of telomeres, and T2 mutant weakened the specific binding between telomerase and telomere therefore playing an inhibitory effect cell division and growth.