Assessment of telomerase as drug target in breast cancer

Telomerase is a specialized enzyme which maintains telomere length at the extreme end of the chromosome.Over 90% of all cases of cancer show high expression of telomerase while in normal cells, its expression isextremely low or undetectable. Detection of telomerase activity in a wide range of breast cancer makestelomerase an interesting target for diagnosis and therapy. In this review, we have aimed to describe telomeraseas a therapeutic and accurate diagnostic target in breast cancer. Telomerase performs many extracurricularactivities apart from maintaining telomere length; here, we have also tried to address its role in epithelialmesenchymal transition (EMT) of breast cancer progression.

Serum hTERT Level as Sensitive Biomarker With Prognostic Implications in Breast, Lung, Gastric and Liver Cancers

@#Human telomerase reverse transcriptase (hTERT) plays an important role in telomere restitution and gene regulation. Evidences suggest that hTERT is linked with the risk and progression of several types of malignancies. Detection of hTERT mRNA levels, as one of tumor markers, may reflect the tumor burden and the clinical status of the patient. Present paper emphasizes the potency of hTERT mRNA detection in serum as a sensitive tumor biomarker in different types of cancer. Detection of serum hTERT mRNA levels has been found highly sensitive and specific for varied cancers. A number of reports reflect its superiority to other conventional tumor markers including alfa-fetoprotein, EGFR, lens culinaris agglutinin-reactive AFP and Des-gamma carboxy prothrombin. Serum hTERT has been found linked with the risk and progression of different cancer types. hTERT levels in combination with other tumor markers may be used to improve cancer detection, tumor size and level of cancer progression.

Study on Inhibitory Effects of Recombinant Adenovirus Ad-GFP-C197 on 3 Kinds of Tumor Cells / 中国药房

OBJECTIVE:To study the inhibitory effects of recombinant adenovirus Ad-GFP-C197, which prepared by adenovirus vector system-loading human telomerase reverse transcriptase(hTERT)C fragment(C197),on the proliferation of 3 kinds of tumor cells in vitro. METHODS:Ad-GFP-C197 was amplified and purified with HEK293 cells. Human gastric cancer cells SGC7901,human breast cancer cells MCF7 and human colorectal cancer cells CaCO2 were infected by Ad-GFP-C197 respectively. Using blank adenovirus carrier (Ad-GFP) as reference,the protein expression of C197 in 3 kinds of tumor cells infected by Ad-GFP-C197 was detected by Western blot assay. The inhibitory effects of Ad-GFP-C197 on 3 kinds of tumor cells were detected by MTT assay. The cell proliferation curve was drawn and the proliferation inhibition rate was calculated. RESULTS:The protein expression of C197 was not detected in 3 kinds of tumor cells infected by Ad-GFP,while significant protein expression of C197 was found in above cells infected by Ad-GFP-C197. The proliferation curves of the 3 kinds of tumor cells infected by Ad-GFP-C197 were significantly inhibited with the time extended,and the proliferation inhibitory rate reached 37.31%-41.42%. CONCLUSIONS:Ad-GFP-C197 shows significant inhibitory effects on the proliferation of SGC7901,MCF7 and CaCO2 cells, which is rapid to make up for the slow effect of other telomerase inhibitors.

TGF-beta receptor mediated telomerase inhibition, telomere shortening and breast cancer cell senescence

Human telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene.

L-carnitine Effectively Induces hTERT Gene Expression of Human Adipose Tissue-derived Mesenchymal Stem Cells Obtained from the Aged Subjects

BACKGROUND AND OBJECTIVES: Human mesenchymal stem cells (hMSCs) are attractive candidates for cell therapy and regenerative medicine due to their multipotency and ready availability, but their application can be complicated by the factors such as age of the donors and senescence-associated growth arrest during culture conditions. The latter most likely reflects the fact that aging of hMSCs is associated with a rise in intracellular reactive oxygen species, loss of telomerase activity, decrease in human telomerase reverse transcriptase (hTERT) expression and finally eroded telomere ends. Over-expression of telomerase in hMSCs leads to telomere elongation and may help to maintain replicative life-span of these cells. The aim of this study was to evaluate of the effect of L-carnitine (LC) as an antioxidant on the telomerase gene expression and telomere length in aged adipose tissue-derived hMSCs. METHODS: For this purpose, cells were isolated from healthy aged volunteers and their viabilities were assessed by MTT assay. Quantitative gene expression of hTERT and absolute telomere length measurement were also performed by real- time PCR in the absence and presence of different doses of LC (0.1, 0.2 and 0.4 mM). RESULTS: The results indicated that LC could significantly increase the hTERT gene expression and telomere length, especially in dose of 0.2 mM of LC and in 48 h treatment for the aged adipose tissue-derived hMSCs samples. CONCLUSION: It seems that LC would be a good candidate to improve the lifespan of the aged adipose tissue-derived hMSCs due to over-expression of telomerase and lengthening of the telomeres.

Progress of microRNA and telomere in tumors / 肿瘤研究与临床

In recent years, tumor has become one of the major diseases that endanger human health. It is of great significance to explore the pathogenesis of tumors in the prevention, diagnosis, treatment and prognosis of tumors. microRNA (miRNA) is a class of small non-coding RNA, which is involved in post transcriptional processing, epigenetic inheritance, cell growth and individual development and other important life activities. miRNA, charactered by oncogenes and anti-oncogenes function, participates in the development, invasion and metastasis of tumors. Telomeres, located at the end of chromosomes, are closely related to the malignant transformation of cells and the aging of human. To understand the pathogenesis of tumors and to provide a novel direction for the diagnosis, treatment and prognosis of tumors, the role and the relationship of miRNA and telomere in tumors are reviewed in this paper.

Chinese herbal extracts inhibit the proliferation and induce the apoptosis of human lung cancer A549 cells / 医学研究生学报

Objective Cancer, a disease induced by abnormally regulated cell growth and apoptosis, is imposing a global threat to human health.This study was to explore the effects of Chinese herbal extracts ( CHE) in inducing the apoptosis and inhibiting the proliferation of human lung cancer cells. Methods Human lung cancer A549 cells were divided into a negative control, a high-dose CHE (680 ng/mL), a medium-dose CHE (340 ng/mL), and a low-dose CHE (170 ng/mL) group.The inhibitory effect of CHE on the proliferation of the lung cancer cells was detected by CCK8 and LDH assays, the apoptosis of the cells was assessed by fluorescence microscopy and flow cytometry, and the expressions of hTERT mRNA, cleaved caspase-3 and cleaved PARP were deter-mined by RT-PCR and Western blot. Results CHE inhibited the proliferation of the A549 cells with an IC50 value of 510 ng/mL. Treatment with high-dose CHE for 48 hours significantly suppressed the proliferation of the cells, induced the release of LDH, and promo-ted the apoptosis of the cells by 72.3%.RT-PCR and Western blot showed that 24-hour treatment with medium-dose CHE reduced the expression of hTERT mRNA by 4 times that of the negative control and up-regulated the expressions of cleaved caspase-3 and cleaved PARP. Conclusion Chinese herbal extracts can induce cell apoptosis by decreasing the expression of hTERT mRNA and increasing those of the cleaved caspase 3 and cleaved PARP proteins.

RNAi-silenced Survivin and hTERT gene inhibits proliferation and induces apoptosis of human colorectal carcinoma cell line SW 480 / 基础医学与临床

Objective To investigate the Influence of Survivin and hTERT gene on cell proliferation and apoptosis in human colorectal carcinoma cell line SW 480 and to find experiment evidence for gene therapy of colorectal carci-noma .Methods Plasmids carrying shRNAs targeting survivin and hTERT were designed , constructed and trans-fected into SW480 cells.SW480 cells were then divided into blank group , blank Plasmid control group , survivin RNAi group , hTERT RNAi group and Survivin-hTERT RNAi group .The telomerase activity was examined by TRAP-PCR-ELISA analysis 48h after hTERT-shRNA transfection.Survivin and hTERT mRNA and protein expres-sion was analyzed by RT-PCR and Western blot .Cell apoptosis , proliferation were measured by flow cytometry , CCK-8 assay.Results Telomerase activity of SW480 cells in Survivin-hTERT RNAi groups were significantly decreased compared with the blank group ( P<0.01 ) .The expression of survivin and hTERT mRNA, proteins in the Survivin-hTERT RNAi group was reduced by 82.8%and 73.6%( P<0.01 ) ,79.2%and 66.7%( P<0.01 ) respectively .The inhibitory rate of cell proliferation of Survivin-hTERT RNAi group was 43.6% ±0.1%( P <0.01 ) .The apoptosis rate was 39.2%±2.3%( P<0.01 ) in the Survivin-hTERT RNAi group .Conclusions The Survivin-hTERT RNAi group could significantly reduces the protein expression of survivin and hTERT mRNA, in-hibit cell proliferation and induces cell apoptosis in human colorectal carcinoma cell line SW 480 .

The effect of RNA interference induced by inhibition of hTERT and Bi-1 gene expression / 重庆医学

Objective In this study ,we constructed a series of recombinant plasmids carriers expressing shRNA targeting hTERT and Bi‐1 gene .These recombinant plasmids carriers were transfected into CNE‐2Z cell lines using Lip and continuously in‐duced the expression of shRNAs .Furthermore ,the shRNAs caused the degradation of mRNAs homologous in sequence with the target genes ,which lead to a sequence‐specific gene silencing .Methods The CNE‐2Z cells was divided into untreated group ,pEG‐FP‐N1 group and pEGFP‐N1/Lip group .Flow cytometry(FCM ) was applied to determine the transfection efficiency .The changes of hTERT and Bi‐1 gene expression were detected by Real‐time RT‐PCR and Western blotting .Results The best transfection effi‐ciency between plasmid and Lip was 2 .5 μg plasmid and 6 .25μL Lip .Conclusion We constructed several shRNA recombinant eu‐karyotic expression plasmids successfully .The recombinant plasmid can inhibit the expression of hTERT and Bi‐1 gene specifically and effectively .

Inhibitory Effect of siRNA Expression Vector Inhibiting IGF2 Gene on the Proliferation of Hepatoma Cell Line Huh-7 / 中国药房

OBJECTIVE:To investigate the inhibitory effect of siRNA expression vector inhibiting human insulin-like growth factor 2(IGF2)gene on the proliferation of hepatoma cell line Huh-7. METHODS:siRNA expression vector pGL3-hAFP-hTERT-siRNA3(“siRNA3”)which inhibited IGF2 gene by dual promoter regulation of recombinant human alpha-foetoprotein(hAFP)and human telomerase reverse transcriptase(hTERT)was transfected into the Huh-7 cell and normal hepatocyte L-02,and then a nega-tive control group(vector pGL3-hAFP-hTERT)and a blank control group were set up. IGF2 mRNA expression was detected by re-al-time fluorescent quantitative polymerase chain reaction 48 h after transfection into the cells in all groups;the activity of the cells by the microplate reader 0,24,48 and 72 h thereafter;and the cell cycle and apoptosis by the flow cytometer 48 h thereafter,and the changes in the protein levels of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in the cell were detected by Western blot. RESULTS:Compared with the negative control group and blank control group,IGF2 mRNA expression in the Huh-7 cell transfected with siRNA3 was obviously weaker;at 48 and 72 h after transfection,the activity of Huh-7 cell signigicantly reduced, Huh-7 cells at G1 phase obviously increased and those at S phase markedly decreased;the occurrence of early,late and total apopto-sis in Huh-7 cells apparently increased,and the protein expression of IGF2,PCNA,Cyclin E2,Cyclin D2,Cdc2 and Bcl-2 in cells significantly weakened,with statistically significance(P0.05). CONCLUSIONS:siRNA which inhibited IGF2 gene by dual promoter regulation of recombinant hAFP and hTERT can specially inhibit IGF2 gene expression and the prolifer-ation of Huh-7 cells,which may be involved with down-regulated protein expression of cell proliferation-associated gene PCNA, cell cycle control-associated genes Cyclin E2,Cyclin D2 and Cdc2 and apoptosis regulation-associated gene Bcl-2 as a result of down-regulated IGF2 mRNA expression and protein expres-sion.

Effect of human telomerase reverse transcriptase-tumstatin combined with human telomerase reverse transcriptase-thymidine kinase/ganciclovir system for liver cancer cell line HepG2 / 肿瘤研究与临床

Objective To observe the effect of human telomerase reverse transcriptase-thymidine kinase/ganciclovir (phTERT-TK/GCV) system combined human telomerase reverse transcriptase-tumstatin (phTERT-tumstatin) system on apoptosis of human HepG2 and mRNA expression and protein content of AFP,RhoC related with cancer.Methods Fluorescence microscopy was used to observe expression of EGFP and MCHERRY in transfected HepG2 and L-02.Real-time PCR and Western blot were used to detect AFP and RhoC mRNA and protein content.Flow cytometry was used to detect the apoptosis of transfected HepG2.Results phTERT-tumstatin and phTERT-TK/GCV genes expressed in transfected HepG2.Real-time PCR showed that AFP and RhoC mRNA expression in different group were 0.76±0.09 and 0.80±0.04 (TK/GCV group),0.62±0.09 and 0.40±0.02 (TM group),0.49±0.07 and 0.54±0.03 (MK group).The differences were significant (P ＜ 0.01) except TK/GCV group compared with empty plasmid group.Western blot test results showed that protein content of AFP and RhoC were higher in TK/GCV group (0.97±0.02/1.17± 0.01),TM group (0.83±0.02/0.99±0.02),MK group (0.69±0.01/0.77±0.02) than in empty plasmid group (1.19±0.03/1.32±0.05) and non-transfected group (1.15±0.05/1.29±0.30) (P ＜ 0.01).Additionally,protein content of AFP and RhoC in MK group were significant difference with TK/GCV group and TM group (P ＜ 0.01).Flow cytometry showed that phTERT-tumstatin,phTERT-TK/GCV co-transfected HepG2 cells apoptosis rate was significantly higher than both individually transected group.Cells apoptosis rate of alone and co-transfected groups was significant difference compared with empty vector group and untransfected group.Conclusions The effect of phTERT-TK/GCV and phTERT-tumstatin on pro-apoptotic of HepG2 cells is significant.phTERT-TK/GCV combined with phTERT-tumstatin has strong therapeutic function.

Mechanism of ouabain inducing the apoptosis of T lymphoblastic leukemia Jurkat cells / 医学研究生学报

Objective Ouabain is a cardiotonic steroid that can induce the apoptosis of many tumorous cells .This study was to investigate the anti-tumor mechanisms of ouabain by observing its effects on the apoptosis of T lymphoblastic leukemia Jurkat cells and the expressions of hTERT and c-myc mRNA and protein . Methods Jurkat cells were treated with ouabain at the concentrations of 50 and 100 nmol/L for 24 and 48 hours, and those treated with 1 ×PBS served as the control .Then the apoptosis rate of the cells was detected by flow cytometry after Annexin V/PI staining, the expressions of hTERT and c-myc mRNA determined by RT-PCR, and those of hTERT and c-myc protein by Western blot . Results The apoptosis rates of the Jurkat cells in the 50 and 100 nmol/L oua-bain groups were (5.67 ±3.71)%and (9.63 ±4.83)%respectively at 24 hours, and (19.67 ±4.55)%and (37.60 ±11.89)%at 48 hours, significantly higher than (4.23 ±1.01)%in the PBS control group at 48 hours (P<0.05).Compared with the control, the expressions of hTERT and c-myc mRNA were decreased by 200%and those of hTERT and c-myc protein by 224%and 400%, re-spectively, at 48 hours (P<0.05).There was a positive correlation between the reduction of the mRNA levels and that of the protein levels of hTERT and c-myc (P<0.05). Conclusion Ouabain can down-regulate the mRNA and protein expressions of hTERT and c-myc, which may be one of the mechanisms of its induction of the apoptosis of Jurkat T lymphocyte leukemia cells .

Conditionally replicative adenovirus under the control of glial fibrillary acidic protein and human telomerase reverse transcriptase dual-promoters direct sodium iodide symporter expression for malignant glioma radioiodine therapy / 中华放射医学与防护杂志

Objective To explore the possibility of using 131I as a targeted therapy method for malignant glioma by infecting U87 and U251 cells with conditionally replicative adenovirus Ad-Tp-E1a-Gp-NIS.Methods Human telomerase reverse transcriptase (hTERT) promoter and glial fibrillary acidic protein (GFAP) promoter were cloned and their transcriptional activities were detected by luciferase assay.The conditionally replicative adenovirus Ad-Tp-E1 a-Gp-NIS was constructed,purified,and transfected into U87 and U251 glioma cells.For these transfected cells,the selective replication ability was evaluated by plaque forming assay,and protein expression was detected by Western blot assay.125I-iodide uptake and exflux,the clonoy formation of 131I-iodide treated cells were also measured.Results Transcriptions activity of the GFAP and hTERT promoters was 59.75％-62.10％ (F =11.89,P ＜ 0.01) in U87 cells and 37.31％-49.00％ (F =5.87,P ＜ 0.05) in U251 cells.The Ad-Tp-E1a-Gp-NIS could be selectively replicated and the hNIS gene was successfully expressed in the hTERT-positive and GFAP-positive glioma cells which showed two protein bands with relative molecular mass of 120 × 103 and 49 × 103 in Western blot assay.After infection with Ad-Tp-E1a-Gp-NIS,the cell ability of 125I uptake was increased by 78.80 (F =2 914.58,P ＜0.01) and 92.48 (F =2 275.91,P ＜0.01) times in U87 and U251 cells,respectively.The GFAP-negative MRC-5 cells could not take in 125I.The in vitro clonogenic assay indicated that,after 131I treatment,more than 90％ of the transfected cells were killed,while only about 65％ (t =11.73-78.33,P ＜ 0.01) of control cells were killed.Conclusions The Ad-Tp-E1a-Gp-NIS has a good ability in selective replication and the enhancement of antitumor therapy effect by increasing tumor-specific iodide uptake in malignant glioma cells.

Immortalization of human fallopian tube epithelial cells / 中国癌症杂志

10.3969/j.issn.1007-3969.2013.04.00X

Mitochondrial Dysfunction of Immortalized Human Adipose Tissue-Derived Mesenchymal Stromal Cells from Patients with Parkinson's Disease

Mitochondrial dysfunction in dopaminergic neurons of patients with idiopathic and familial Parkinson's disease (PD) is well known although the underlying mechanism is not clear. We established a homogeneous population of human adipose tissue-derived mesenchymal stromal cells (hAD-MSCs) from human adult patients with early-onset hereditary familial Parkin-defect PD as well as late-onset idiopathic PD by immortalizing cells with the hTERT gene to better understand the underlying mechanism of PD. The hAD-MSCs from patients with idiopathic PD were designated as "PD", from patients with Parkin-defect PD as "Parkin" and from patients with pituitary adenomas as "non-PD" in short. The pGRN145 plasmid containing hTERT was introduced to establish telomerase immortalized cells. The established hTERT-immortalized cell lines showed chromosomal aneuploidy sustained stably over two-years. The morphological study of mitochondria in the primary and immortalized hAD-MSCs showed that the mitochondria of the non-PD were normal; however, those of the PD and Parkin were gradually damaged. A striking decrease in mitochondrial complex I, II, and IV activities was observed in the hTERT-immortalized cells from the patients with idiopathic and Parkin-defect PD. Comparative Western blot analyses were performed to investigate the expressions of PD specific marker proteins in the hTERT-immortalized cell lines. This study suggests that the hTERT-immortalized hAD-MSC cell lines established from patients with idiopathic and familial Parkin-defect PD could be good cellular models to evaluate mitochondrial dysfunction to better understand the pathogenesis of PD and to develop early diagnostic markers and effective therapy targets for the treatment of PD.

Human telomerase reverse transcriptase expression in ovarian tumors.

Background : Ovarian cancer is the 6 th most common cancer among women. In ovarian tumors, the borderline category is not well defined due to the difficulty in assessing stromal invasion. The World Health Organization (WHO) defined it as tumor that lacks obvious invasion of the stroma with mitotic activity and nuclear abnormalities intermediate between clearly benign and unquestionably malignant. Telomerase is expressed in many human cancers and is hence a potential biomarker for cancer. Immunohistochemical study of anti-human telomerase enzyme reverse transcriptase (hTERT) antibody allows direct visualization of its expression. The aim of this study was to determine the expression of hTERT and serum CA-125 level in ovarian epithelial tumors, and their ability to distinguish borderline tumor from malignancy. Materials and Methods : This was a retrospective study on 68 ovarian epithelial tumors, comprising of 41 cystadenocarcinoma, 22 borderline tumor and five cystadenoma. By immunohistochemistry, hTERT expression was graded as negative (0-10%), focal (11-25%), regional (26-75%) and diffuse (>75%) positivity. Results : hTERT protein expression in ovarian cystadenocarcinoma, borderline tumor and cystadenoma were 71.4%, 59.1% and 0%, respectively. hTERT and CA-125 had a linear relationship with tumor grade and stage. hTERT protein is detected as large granules/speckled in the cytoplasm and nuclei of ovarian tumors. Conclusions : hTERT protein was highly expression in ovarian epithelial carcinoma. However, the difference between carcinoma and borderline tumor was not statistically significant (P-value = 0.51). It is not an independent biomarker to differentiate borderline tumor from malignant tumor. We suggest using the combination of hTERT immunohistochemistry and serum CA-125 to evaluate difficult situations where histological evaluation fails to distinguish malignant from borderline ovarian tumor.

Empirical study of hepatocellular carcinoma's gene therapy using hTERTp-TK/GCV in HepG2 which was mediated by chitosan nanoparticles vehicles / 肿瘤研究与临床

Objective To compare the transfection efficiency of galactosylated chitosan nanoparticle vehicle with chitosan nanoparticles vehicle,and observe the therapeutic effect of pGL3-hTERTp-TK on HCC cell line HepG2. Methods Preparing the chitosan and galaetosylated chitosan. Constructing the pGL3-hTERTp-TK plasmid and the Ch/DNA and GC/DNA complexes.Transfecting the HepG2 and the normal hepatic cell L-02 with chitosan/DNA and galactosylated chitosan/DNA complexs.Detecting the fluorescence and the expression of luciferase gene using the fluorescent microscope and the scintillation counter.Detecting the cell growth and apoptosis through the Caspase-3 and the flow cytometry. Results Two clear straps appeared in the agarose gel.The locations were 300 bp and 1100 bp.The relative lueiferase activity of pGL3-hTERTp-Luc+mediated by galactosylated chitosan was powerful than which of pGL3-hTERTp-Luc+mediated by chitosan in HepG2 by the scintillation counter.However,the relative luciferase activity was very weak in L-02.The same results were observed by fluorescent microscope.When the concertration of the GCV was 10 μg/ml(t=51.40,P=0.000),the HepG2 cell inhibition which was transfected by GC-pGL3-hTERTp-TK was obviously different from the L-02 cell inhibition which was transfected by GC-pGL3-hTERTp-TK in the statistics.The significant apoptotic rate was 65.28%in HepG2 which was transfected with GC/DNA,whereas it was only 10.80% in L-02. The significant apoptotic rate in HepG2 which was transfected with GC/DNA was very higher than the other groups (LSD, P ＜0.05). The significant apoptotic rate (10.80%) in L-02 which was transfected with GC/DNA was very higher than the group which was Ch-pGL3-control (LSD, P =0.000). The average fluorescence intensity of the HepG2 which is transfected by the Ch-pGL3-hTERTp-TK was 168.02± 3.68. The average fluorescence intensity of the HepG2 which is transfected by the GC-pGL3-hTERTp-TK was 204.45 ±3.45. The two groups had a significant difference in the statistics (t=-12.504, P＜0.05). Conclusion Galactosylated chitosan has higher transfection efficiency than chitosan. GC-pGL3-hTERTp-TK could specially attack HCC cell line and almost has no influence on normal hepatic cells.

The expression and significance of hTERT and P53 in thyroid carcinoma / 西安交通大学学报·英文版

Objective: To determine the expression of human telomerase reverse transcriptase (hTERT) and P53 in thyroid carcinoma and its relationship with development and prognosis of the carcinoma. Methods: Totally 90 cases of thyroid specimens (60 thyroid carcinomas, 10 thyroid adenomas, 10 goitres and 10 normal thyroid tissues) were studied by SP immunohistochemical method. Results: Positive immunoreactivity of hTERT and P53 was higher in thyroid carcinoma (P<0.05). The positive rates of hTERT and P53 were higher in undifferentiated carcinomas, carcinomas with lymph nodes metastasis or at stage III + IV than in well-differentiated carcinomas, carcinomas without lymph nodes metastasis or at stage I + II (P<0.05). The expression of hTERT was significantly related with that of P53 (P<0.05). Conclusion: Over-expressed hTERT and P53 may be related to the carcinogenesis and progression of thyroid carcinoma and hTERT expression is related to P53 protein. Examination of expression of hTERT and P53 proteins may be helpful to judge the thyroid cancer's behavior and prognosis.

Effects of Combined siRNA-TR and-TERT on Telomerase Activity and Growth of Bladder Transitional Cell Cancer BIU-87 Cells / 华中科技大学学报（医学）（英德文版）

The effects of combined RNA interference(RNAi)of human telomerase RNA(hTR)and human telomerase reverse transcriptase(hTERT)genes on telomerase activity in a bladder cancer cell line(BIU-87 cells)were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer(BTCC).Three TR-specific double-stranded small interfering RNAs(siRNAs)and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA.The phTR-siRNA,pbTERT-siRNA,and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells.The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction,and a telomeric repeat amplification protocol was applied to detect telomerase activity.Growth inhibition of BIU-87 cells was measured by MTT assay.Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro.The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,and pRNAT-hTR-Ⅲ+hTERT-Ⅲ in BIU-87 cells.The inhibition efficiency of pRNAT-hTERT-Ⅲ,pRNAT-hTR-Ⅲ,pRNAT-hTERT-Ⅲ+pRNAT-hTR-Ⅲ was 67% for TERT mRNA,41% for TR mRNA,57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively.The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased,especially in the cells treated with combined RNAi-hTR and-hTERT.Gene chip analysis revealed that 21 genes were down-regulated(ATM,BAX,BCL2,BCL2L1,B1RC5,CD44,CTNNB1,E2F1,JUN,MCAM,MTA1,MYC,NFKB1,NFKBIA.NME4,PNN,PNN,SERPINE1,THBS1,TNFRSF1A,and UCC1).The results indicated that hTR-siRNA and hTERT-siRNA,especially their combination,siRNA hTR+hTERT,specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity.Molecular biological mechanism by which combined siRNA-TR and-TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation ofthe 21 genes.

The expression and significance of hTERT and P53 in thyroid carcinoma / 药物分析学报

Objective To determine the expression of human telomerase reverse transcriptase (hTERT) and P53 in thyroid carcinoma and its relationship with development and prognosis of the carcinoma. Methods Totally 90 cases of thyroid specimens (60 thyroid carcinomas, 10 thyroid adenomas, 10 goitres and 10 normal thyroid tissues) were studied by SP immunohistochemical method. Results Positive immunoreactivity of hTERT and P53 was higher in thyroid carcinoma (P<0.05). The positive rates of hTERT and P53 were higher in undifferentiated carcinomas, carcinomas with lymph nodes metastasis or at stage Ⅲ+Ⅳ than in well-differentiated carcinomas, carcinomas without lymph nodes metastasis or at stage Ⅰ+Ⅱ (P<0.05). The expression of hTERT was significantly related with that of P53 (P<0.05). Conclusion Over-expressed hTERT and P53 may be related to the carcinogenesis and progression of thyroid carcinoma and hTERT expression is related to P53 protein. Examination of expression of hTERT and P53 proteins may be helpful to judge the thyroid cancer's behavior and prognosis.