| Lung cancer is among the tumors of high malignancy and high incidence. Tumorigenesis involves a complicated multi-step process and is related to activation of various proto-oncogenes and / or inactivation of various antioncogenes at different stages. These gene changes will eventually lead to limitlessness of tumor cell growth. Therefore, gene therapy targeting a specific oncogene, antioncogene or cytokine cannot obtain satisfactory results considering the whole process of tumorigenesis. The ideal target for tumor treatment is a cell constituent that is indispensable for tumor cell growth but is absent from normal cells. Telomerase, an enzyme with special activities, resides in some immortalized cell lines and in most tumor cells, contributing to transformation of cell immortality and tumorigenesis as well. Telomerase is expressed in 84-95% of human malignant cells and immortalized cells and is inactivated in human normal cells but is expressed at low levels in germinal cells and a few hematopoietic cells. A high level of telomerase expression in malignant tumor makes it an ideal potential target for gene therapy of tumors.Telomere, a DNA cap with special functions that locates at the terminal end of a chromosome, is a ribonucleoprotein complex composed of a G-abundant DNA repetitive sequence of TTAGGG and telomere binding proteins. Although telomere involves no genes, it plays significant roles in maintaining the integrity and stability of chromosome. Along with mitoses, the telomere shortens by degrees. As the telomere becomes shorter than a critical value, the mechanism of stability of chromosome terminals breaks down, and the somatic cells exit cell cycle, becoming senescent and dying off, i.e., replicative senescence.Telomerase, a ribonucleoprotein with reverse transcriptase activity, is composed of RNAs and proteins. Telomerase activation maintains the length of telomere and, thus, the cell eludes death and becomes immortal, i.e., a tumor cell with limitless proliferative capability. Telomerase activity has significant implications in cell malignancy andtumorigenesis. If telomerase activity in tumor cells can be specifically inhibited, tumor cell cycle will be blocked, losing its limitless proliferative capability, ceasing tumor growth and even resulting in spontaneous remission. Telomerase is composed mainly of three parts: telomerase, RNA constituent and telomerase-associated protein and catalytic submit. Telomere is synthesized according to the template of telomerase RNA by reverse transcription of telomerase catalytic subunit. RNA constituent of human telomerase has been cloned. Since telomerase RNA sequence involves the template sequence complementary to telomere DNA sequence, telomerase RNA, protein and catalytic subunit can be investigated for telomerase inhibition: 1. to inhibit telomerase using nucleoside analogues; 2. to inhibit telomerase using antisense techniques; 3.to inhibit telomerase using ribozyme techniques; 4. to inhibit telomerase using differentiation inductors; 5. other means. Previous researches were mainly based on telomerase RNA template. The templates were exhausted as a result of binding with antisense ribonucleotide or antisense thioribonucleotide, consequently disabling telomere sequence proliferation. However, there have been few reports on antisense catalytic subunit study although the catalytic subunit acts as an essential constituent of telomerase.The constitutional unit of peptide nucleic acid (PNA) is (2-aminoethanoic acid) glycine that is completely different from phosphoribosome in chemical property, and the basic groups are linked to the backbone via methyene carbonyl. Peptide nucleic acid (PNA) is extremely similar to nucleotide in structure but possesses many advantages over nucleotide. Phosphodiester bond of ligonucleotide is easily degradable and is not sensitive to binding to DNA or RNA but is susceptible to external conditions. However, peptide nucleic acid (PNA) keeps stable in vivo, and cannot be degraded by nuclease or protease. Due to its advantages of high specificity and stability, peptide nucleic acid (PNA) may substitute oligonucleotide as an antisense drug in the study of telomerase.In the study, the promoter upstream of hTERT mRNA template was used as a target site and was inhibited using artificially synthesized and fluorescin-labeled antisense peptide nucleic acid (PNA) and was cotransfected into lung cancer A549 cell line using liposome. The results of transfection was observed and identified under an inverted fluorescence microscope. Cell growth status was observed, values of telomerase activity measured at different time points and concentrations, cell survival rate detected using MTT assay, cellcycle ratio assessed using flow cytometry. Changes of expression of mRNA and protein of P14 and PI6 (both are cell cycle genes of lung cance) were investigated using RT-PCR, Western blot and immunohistochemistry following antisense PNA action. The conclusions of the study are as follow:1. The expression of the telomerase activity is higher in lung cancer tissues than in its adjacent noncancerous tissues, and it's abcent in benign pulmonary tissues; It's related to stage and metastasis and of lung cancer. The telomerase activity may be an useful marker for diagnosing lung cancer.2. Antisense PNA was successfully transfected into lung cancer A549 cell line and manifested inhibitory effects on the cells.3. The activity of telomerase in A549 cell transfected with antisense PNA is decreased in a time- and concentration-dependent manner with the increase in time and concentration.4. Antisense PNA inhibits the growth of lung cancer cell. The inhibitory effect is increased in a time- and concentration-dependent manner with the increase in time and concentration.5. Antisense PNA increases the expression of mRNA and protein of cell cycle genes P14 and P16 in lung cancer cell, blocking the cell at Gl/S stage and leading to apoptosis of lung cancer cell.
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