The Biological Significance Of Telomerase Reverse Transcriptase And Telomerase Inhibitor PinX1in Esophagus Carcinoma Treating

Background:Esophageal cancer is a common malignant tumor, which has caused the highest risk of morbidity and mortality in China. The development and progression of esophageal cancer involves multi-factors, multi-genes and multi-stage. It is always the middle and advanced stage when diagnosed, so the prognosis is really inferior, due to which the survival rate in5years is only5%to20%. Therefore, exploring the pathogenesis and a safe and effective therapy for esophageal cancer has become the key point for the treatment of esophageal cancer. Telomeres and telomerase play an important role in the process of immortalization and evolution of tumor cells. The activation of telomerase can lead to unlimited proliferation of cells and the occuring of tumor. Telomere, as a molecular clock controlling the replication and caducity of human cells, is closely related to the lifespan of cells. During the division of normal cells, the length of the telomere DNA can be shortened by55-200bp. As a result of dividing and shortening to its lowest limit, the cell will result in apoptosis or death in the end. Telomerase is a reverse transcriptase based on its own RNA as a template. As its core component, human telomerase reverse transcriptase, is the most important approach to the activation of telomerase, of which the expression plays a key role in regulation of telomerase and is the determinant factor to present the activity of telomerase. A large number of studies have found that in the development of malignant tumor there is the length shortening of the telomere DNA and the activation of telomerase maintains the telomere length of most tissues, which counteracts the consumption of telomere DNA in the process of cell division and suggests that telomerase plays an important role in the procession of the malignant tumor. Existing studies have shown that except the normal cells of human body, such as spermatogonial, embryonic cells and stem cells, most of the telomerase of cells are almost inactive. In contrast, in the vast majority of malignant tumor, the activity of telomerase is of high expression, of which the rate is up to90%. So the activation of telomerase plays a vital role in the development of a variety of genetic changes in malignant tumor and can be used as a universal or a specific tumor molecular marker, which has an important position in the diagnosis of tumor. However, the inhibition the activity of telomerase may prevent the malignant proliferation of tumor cell, and telomerase is becoming a hot spot of current research of diagnosis and treatment of tumor. Telomerase is composed of three sigmasubunits:human telomerase RNA (hTR), human telomerase reverse transcriptase (hTERT) and the telomerase associated protein I (TEPI). We had made the antisense oligonucleotide (ASODN), complementary to the template region of hTR act on esophageal cancer cells (ECA109) and the results showed that it could inhibit the activity of telomerase and the growth of cells. But as a result of the fact that hTR is widely expressed in normal tissue cells, the antisense therapy aiming at hTR may bring with certain adverse reactions to normal tissue cells. Recent studies show that hTERT is the catalytic subunit of telomerase and the amount of expression quantity in the tissue cells is paralleled with telomerase activity. It is the most important part to effect the activity of telomerase and also the limiting factor of telomerase activity, so that antisense therapy aiming to hTERT maybe more effective than hTR, and apparently targeting hTERT gene therapy is more ideal. Among them the important approaches are blocking antisense technology, cutting hammerhead ribozymes and inhibiting the transcription of hTERT, etc. We synthesize a antisense oligonucleotides with the length of19bp, modify it with PS-ASDON, cover it with liposome and then transfer it into the esophageal cells so as to research the influence of it on telomerase activity and the growth of cancer cells.PinX1genes, as endogenous gene of telomerase, is regarded as a potential tumor suppressor gene in recent years and its expression may be related to the occurrence and development of a wide variety of tumor. There experiments confirmed that in patients with gastric cancer, colorectal cancer and leukemia, the expression level of PinXl decreased obviously and telomerase activity increased. But so far, biological significance and mechanism of PinXl genes in esophageal cancer are reported rarely at home and abroad, and there is no suitable esophageal cancer cell model for the research of PinXl gene. Because the gene transfection has become the effective way to treat tumor, then by exogenous overexpression, can PinX1genes suppress esophageal cancer cell proliferation and inhibit the telomerase activity? This experiment research uses RT-PCR, TRAP-argentation and MTT and flow cytometry to research the proliferation and apoptosis of cancer cell and the changes of telomerase with PinX1genes before and after the transfection. So as to understand the genetic biological influence on esophageal cancer cell preliminarily and further to explore the relationship between PinX1gene and telomerase. To prove that PinXl gene is a potential inhibitor to telomerase activity and may influence the development of tumor by targeting at inhibiting telomerase activity, hence, to open up new areas for targeted gene therapy of esophageal cancer.Objectives:We used PS-ASODN to transfer to Eca-109esophageal cancer cells with the aim of researching the influence of it on telomerase activity of esophageal cancer and the growth of cancer cell. Then we discussed its inhibitory effect and mechanism to cells of esophageal cancer so as to find new gene therapy for esophageal cancer.This experiment successfully built a PinX1gene expression vector--pEGFP-C3-PinXl and transfered it to Eca-109esophageal cancer cells high metastasis potential, to observe the influence of gene transfection PinXl on the proliferation and apoptosis of cells and the activity of telomerase of esophageal Eca109cell. At the same time, we used TRAP-argentation to test the changes of telomerase activity of Eca109cell before and after transfection to research the relationship between PinXl gene and telomerase, so as to preliminarily understand the biological influence on esophageal cancer cell. It was aimed to provide theoretical basis for the research of inner regulating mechanism of PinX1and gene therapy of esophageal cancer.Methods:1. The inhibiting effect of telomerase catalystic subunit hTERT antisense oligonucleotide on the cells of esophageal cancer; using MTT colorimetry to test the inhibition rate of PS-ASODN to the development of Eca-109cancer cell; adopting inverted microscope to observe the morphological changes of PS-ASODN of5μmol/L acting on10d of Eca-109cancer cell; adopting semi-quantitative TRAP-argentation to teat the telomerase activity.2. The influence of gene transfection PinX1on the proliferation and apoptosis of esophageal cancer Eca-109and the activity of telomerase; using moolecular biology techniques to construct gene expression vector pCDNA3.1-PinX1aiming at PinX1gene;using liposomes-RNA compounds after packaging liposomes LipofectamineTM2000with RNA, to transfer it into esophageal cancer cells Eca109so as to establish a stable Eca109cell model to express Eca-109. Laser confocal method is applied to determinate the transfection efficiency, MTT method to test cell proliferation before and after transfection, FCM method to test cell cycle and cell apoptosis.RT-PCR method to test the expression of PinXl gene and protein. TRAP-argentation method to test the telomerase activity of each group of cells before and after transfection, to clarify the relationship between PinX1gene and telomerase activation.ResultsThe inhibiting effect of telomerase catalystic subunit hTERT antisense oligonucleotide on the cells of esophageal cancer. PS-ASODN has a influence of dose, time dependence and sequence specificity on the inhibition of the development of Eca-109cell. After the role of PS-ASDON, the speed of Eca-109cell growth is slow, the connection between cells is loose, and the cells will be round and levitating and then become smaller. With the extension of action time, the inhibition of PS-ASODN to telomerase activity of Eca-109cell gradually strengthened, it becomes time-dependent; With the increase of drug concentration, the inhibition of PS-ASODN to telomerase activity of Eca-109cell gradually increased, it becomes concentration-dependent.Using liposome transfection method to test the influence of gene transfection PinX1on the proliferation and apoptosis and telomerase activity of esophageal Eca109cell, successfully transferring pCDNA3.1-PinXl recombinant plasmid into Eca109cells, and applying G418to screen successfully the stable transfection cells, according to the results determined by MTT method to test the condition of proliferation, after transfection of PinXl gene, Eca109cell grows more slowly significantly (P<0.05); however, comparing Eca-109cells with Eca-109only transferred into the empty carrier,there is no obvious difference between them.(P>0.05). It manifests that pCDNA3.1-Pinxl has significant inhibitory effect on the proliferation of Eca109cell, according to the growth curve of cells at different times (Oh24h!48h!72h) with OD490value, and growth inhibition rate calculated, it shows that after gene transfection into PinX1, there occurs the early apoptosis changes in the cell. According to the results of detection of RT-PCR, TRAP argentation and FCM, PinXl mRNA and protein expression level of Eca109/PinX1cells increased significantly compared with group of pCDNA3.1and the blank cell group(P<0.05). According to the results of detection of telomerase activity, after transfection of PinXl genes, telomerase activity of cells significantly decreased (P<0.05). Before and after transfection, the expression of telomerase activity and cell proliferation index (PI) of Eca109was positively correlated (rs=0.451, P=0.451).ConclusionAccording to the fact that hTERT antisense oligonucleotides can lead to apoptosis of esophageal cancer cells, inhibit telomerase activity and the synthesis of telomerase to telomerase so as to restrain the growth of the esophageal cancer cells, it suggests that telomerase antisense oligomeric DNA nucleotides can effectively inhibit the growth of tumor cells and provides an experimental basis of gene therapy to resist telomerase in tumor. After the transfection of PinXl gene to Eca-109cells, exogenous PinXl gene can significantly cut the telomerase activity and their hTERTmRNA expression in esophageal cancer cells, inhibit the proliferation and migration of tumor cell, and induce cell apoptosis. It shows that PinX1gene is a potential inhibitors of telomerase activity, considering that PinXl genes in esophageal cancer cells, by reducing the activity of telomerase, decrease the cell proliferation, it is expected to become the molecular targets of esophageal cancer treatment.