Inhibitory Effect Of Antisense Oligodeoxynucleotides Against HTERT On Telomerase And Proliferation Of Human Tongue Squamous Cell Carcinoma

IntroductionTongue squmaous cell cancer (TSCC) is serious disease threatening human health. It is one of the most common worldwide malignant tumors. However, there is no effective radical treatment at present. It is very necessary to explore new therapy for TSCC. The developments in molecular biochemistry have provided the possibility for this purpose.Telomerase, a ribonucleoprotein enzyme, synthesizes DNA-protein complexes which is called telomere at the end of eukaryotic chromosomes that protects chromosome ends from fusion and degradation, makes sure for the complete replication of telomeric DNA. Telomerase is thought to be necessary for cellular immortality and carcinogenesis, and consists of human telomerase RNA (hTR),human telomerase protein(hTP), human telomerase reverse transcriptase subunit ( hTERT, also termed human telomerase catalytic component). Telomerase is a reverse transcriptase enzyme, Most normal somatic human cells lack telomerase and their telomeres shrink with each replication cycle by 30-100bp. Because short telomeres induce cellular senescence in tissueculture, it has been proposed that telomere shortening may limit the replicative potential of normal cells providing a powerful tumor-suppressive mechanism. Telomerase activity contributes, as part of a multistep process, to human carcinogenesis. According to study, >90% of examined tumors, including tongue cancers, show telomerase activity in connection with the expression of the activity-limiting component hTERT. hTERT was found in nearly all cancer types but not in most normal, somatic cells.The presence of hTERT mRNA was related to the presence of telomerase activity. Many studies showed that the expression of hTERT was restricted only to cells that exhibited telomerase activity, indicating that hTERT was the rate-limiting component of the telomerase. In tumors, hTERT is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. In contrast to the telomerase RNA subunit and other components, which commonly implicated in telomere maintenance, which are present in both normal tissues (without telomerase activity) and cancers, hTERT expression was found solely in tumor cells. Therefore, targeting the catalytic subunit hTERT represents a promising approach for diminishing telomerase function that will probably not cause substantial side effects on telomerase negative, somatic cells. Furthermore, ectopic expression of hTERT in telomerase-negative fibroblasts or endothelial cells is sufficient to restore telomerase activity and to stabilize telomere length, whereas overexpression of dominant-negative mutants of hTERT in tumor cells can inhibit telomerase activity and induce growth arrest.It was shown that transfection with antisense oligodeoxynucleotide (ASODN) specifically reduces the expression of target genes. This method was based on the complementarity of the constructs to the appropriate target mRNA. There were several mechanisms contributing to their antisense effects, such as inhibition of transcription, modulation of RNA processing, inhibition of translation, and selective cleavage of the target RNA by the cellular endonuclease RNase H. One of the limiting factors, discriminating between efficient and ineffective ASODNs, was the accessibility of the target RNA sequence. Experimental and theoretical procedures shouldincrease the probability to find effective ASODN target. Several studies about antisense-based telomerase inhibitors targeting hTR were reported recently, but few was used the activity-regulating subunit hTERT as a target for selected ASODN constructs to treat tongue cancer cells. In order to investigate whether hTERT inhibition by antisense oligodeoxynucleotides could act as an efficient strategy to specifically restrain the growth of tongue cancer cells, we designed this study. The study consisted of three parts. First part was quantitative detection of the expression of hTERT mRNA in oral cancer tissues, precancerous tissues and oral nomal tissues. The second was antisense oligodeoxynucleotides targ...