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Objective:To investigate the effects of telomerase reverse transcriptase (TERT) on neutrophils apoptosis in rats with acute necrotizing pancreatitis (ANP).Methods:Twenty-four Sprague Dawley (SD) rats were randomly divided into three groups including control group, ANP (3h、6h) group and TERT inhibitor(BIBR1532)group using random number method with 6 in each group. ANP rats were induced by retrograde injection of 5% sodium taurocholate into the pancreaticcobiliary duct. After 3 and 6 hours, the fresh neutrophils were collected and isolated from peripheral blood of ANP rats. Rats were intraperitoneally injected with 2 mg/kg BIBR1532. After ANP modeling for 3 h, rats were killed and peripheral neutrophils were collected. Subsequently, the expression of TERT mRNA in neutrophils was tested by real-time quantitative PCR; the protein levels of TERT, BCL-xL and Bax were determined by Western blotting; neutrophil apoptosis was detected by flow cytometry; TNF-α and IL-6 were assayed by Elisa; the rat pancreatic tissue was pathologically examined.Results:In neutrophils from control group, ANP 3 h group, ANP 6 h group and BIBR1532 group, TERT mRNA was 1.03±0.26, 3.31±1.07, 5.21±0.78 and 1.95±0.49; TERT protein expression was 0.09±0.03, 0.43±0.12, 0.58±0.11 and 0.22±0.07; Bcl-xL protein expression was 0.19±0.05, 0.50±0.07, 0.85±0.04 and 0.40±0.11; Bax protein expression was 0.29±0.08, 0.23±0.03, 0.17±0.02 and 0.43±0.12; apoptosis rate was 10.03±0.74%, 7.99±0.27%, 6.65±0.36% and 22.98±2.86%. TERT mRNA and protein expression and Bcl-xL protein expression in ANP 3 h and 6 h group were higher than those in control group, but Bax preotein expression and apoptosis rate were lower than those in control group; TERT mRNA and protein expression and Bcl-xL protein expression in BIBR1532 group were lower than those in ANP 3 h group, but Bax protein expression and apoptosis rate in BIBR1532 group were higher than those in ANP 3 h group; and all the differences were statistically significant (all P value <0.05). The level of TNF-α was [(96.67±27.12)ng/L, (382.30±46.33)ng/L and (206.88±36.42)ng/L], IL-6 was [(43.34±14.50)ng/L, (134.21±16.13)ng/L and (88.06±13.05)ng/L in control, ANP 3 h and BIBR1532 group; those in ANP 3 h group were all higher than those in control group, but those in BIBR1532 group were lower than those in ANP 3 h group; all the differences were statistically significant (all P value <0.05). Compared with ANP group, the morphology of pancreatic tissue was obviously alleviated in BIBER1532 group. Conclusions:TERT expression is obviously increased in peripheral neutrophils in ANP rats, and apoptosis rate of neutrophils from ANP rats is greatly increased after TERT inhibitor treatment, demonstrating that TERT could inhibit the apoptosis of peripheral neutrophil in ANP rats.
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Objective: To explore the relationship between TERT promoter mutations and BRAF V600E as well as their coexistence with the clinicopathological features of papillary thyroid cancer (PTC). Methods: A total of 728 patients enrolled in Shanxi Cancer Hospital from December 2014 to December 2016 with PTC were retrospectively analyzed. We reviewed and analyzed the clinical results, pathology records, ultrasound results, and BRAF V600E and TERT status. Results: BRAF V600E mutations were found in 42.3% (308 of 728) of patients, and TERT C228T and C250T promoter mutations were found in 10.7% (78 of 728) and 0.5% (4 of 728) of patients, respectively. The TERT promoter mutation was significantly associated with old age (P=0.034), extrathyroidal invasion (P=0.026), large tumor size (P=0.028), cervical lymph node metastasis (P=0.012), distant metastasis (P=0.001), advanced disease stages (P<0.001), histological type (P=0.003) and recurrence (P=0.002). The BRAF V600E mutation was significantly associated with extrathyroidal invasion (P= 0.001), large tumor size (P<0.001), Hashimoto thyroiditis (P<0.001), distant metastasis (P=0.010), advanced disease stages (P=0.009) and recurrence (P=0.001). The coexistence of BRAF V600E and TERT promoter mutations was particularly associated with high-risk clinicopathological features, such as old age (P=0.024), extrathyroidal invasion (P=0.022), Hashimoto thyroiditis (P=0.005), the cervical lymph node (P=0.018), and advanced disease stages (P=0.002). Conclusions: Our study demonstrates that BRAF V600E and TERT promoter mutations play a significant role in the aggressiveness of PTC, particularly when the two mutations coexist. The results reveal the significant role of these mutations in the treatment and prognosis prediction of PTC.
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BACKGROUND: Umbilical cord blood-derived mesenchymal stem cells can penetrate the ependyma into the brain tissue via the systemic circulation, migrate into the injury area and differentiate into neurons. They are as substitute cells applied in the treatment of central nervous system diseases OBJECTIVE: To explore the effects of umbilical cord blood-derived mesenchymal stem cell transplantation on the functional recovery of nerve and expression of telomerase reverse transcriptase in the brain tissue of cerebral infarction rats. METHODS: One hundred and thirty rats were enrolled and were randomly divided into control group (n=30), model group (n=30), umbilical cord blood-derived mesenchymal stem cell 1 group (n=35) and umbilical cord blood-derived mesenchymal stem cell 2 group (n=35). The control group was not given any treatment. The rat models of cerebral infarction were made by internal carotid artery suture method in the other groups. At 1 and 4 days after successful modeling, 2×106 umbilical cord blood-derived mesenchymal stem cells were transplanted into tail vein in the umbilical cord blood-derived mesenchymal stem cell 1 and 2 groups. The levels of reactive oxygen species and superoxide dismutase were detected at 24 hours after transplantation. The nerve function score was detected by Y-maze test at 7 and 14 days after transplantation. The neuronal apoptosis in the center of lesions was detected by TUNEL assay. The expression levels of Caspase-3, Bax, Bcl-2 and telomerase reverse transcriptase proteins around the infarct lesion were detected by western blot assay. The mRNA expression of telomerase reverse transcriptase around infarct lesions was detected by RT-PCR. RESULTS AND CONCLUSION: Compared with the control group, in the model group, the error number in the Y-maze test, nerve function scores, apoptotic index, expression levels of Caspase-3 and Bax, and level of reactive oxygen species were significantly increased (P < 0.05), the expression of Bcl-2 and telomerase reverse transcriptase protein and mRNA, and level of superoxide dismutase were significantly decreased (P < 0.05). Compared with the model group, in the umbilical cord blood-derived mesenchymal stem cell 1 and 2 groups, the error number in the Y-maze test, nerve function score, apoptotic index, expression of Caspase-3 and Bax, and level of reactive oxygen species were significantly decreased (P < 0.05), while the expression of Bcl-2 and telomerase reverse transcriptase protein and mRNA and level of superoxide dismutase were significantly increased (P < 0.05). Compared with the umbilical cord blood-derived mesenchymal stem cell 2 group, in the umbilical cord blood-derived mesenchymal stem cell 1 group, the error number in the Y-maze test, nerve function score, apoptotic index, expression of Caspase-3 and Bax, and level of reactive oxygen species were significantly decreased (P < 0.05), while the expression of Bcl-2 and telomerase reverse transcriptase protein and mRNA and level of superoxide dismutase were significantly increased (P < 0.05). These findings indicate that umbilical cord blood-derived mesenchymal stem cell transplantation can effectively promote the neurological recovery in rats with cerebral infarction, and up-regulate the expression of telomerase reverse transcriptase in rat brain tissue. Moreover, earlier cell transplantation indicates better effects.
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Background & objectives: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase enzyme that maintains telomere ends by the addition of telomeric repeats to the ends of chromosomal DNA, and that may generate immortal cancer cells. Hence, the activity of telomerase is raised in cancer cells including cervical cancer. The present study aimed to validate the unique siRNA loaded chitosan coated poly-lactic-co-glycolic acid (PLGA) nanoparticle targeting hTERT mRNA to knock down the expression of hTERT in HeLa cells. Methods: The siRNA loaded chitosan coated polylactic-co-glycolic acid (PLGA) nanoparticles were synthesized by double emulsion solvent diffusion method. The characterization of nano-formulation was done to determine efficient siRNA delivery. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate silencing efficiency of nano-formulation. Results: Size, zeta potential and encapsulation efficiency of nanoparticles were 249.2 nm, 12.4 mV and 80.5 per cent, respectively. Sustained release of siRNA from prepared nanoparticle was studied for 72 h by ultraviolet method. Staining assays were performed to confirm senescence and apoptosis. Silencing of hTERT mRNA and protein expression were analyzed in HeLa cells by RT-PCR and Western blot. Interpretation & conclusions: The findings showed that biodegradable chitosan coated PLGA nanoparticles possessed an ability for efficient and successful siRNA delivery. The siRNA-loaded PLGA nanoparticles induced apoptosis in HeLa cells. Further studies need to be done with animal model.
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Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is closely related to immortalization of cells, tumorigenesis and senescence of cells. hTERT has a great relationship with the occurrence and development of skin malignant tumors (melanoma, squamous cell carcinoma, basal cell carcinoma, cutaneous lymphoma, etc.). Abnormal expression of hTERT has important clinical application value in early diagnosis, individualized comprehensive treatment and prognosis evaluation of cutaneous malignant tumors.
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Objective To evaluation the value of single and combined detection of peripheral blood human telomerase reverse transcriptase (hTERT) mRNA and tumor markers carcinoembryonic antigen (CEA), carbohydrate antigen 72-4 (CA72-4), CA19-9 and CA125 in the diagnosis of gastric cancer. Methods A total of 48 patients diagnosed with gastric cancer from June 2017 to October 2018 in Baotou Tumor Hospital were enrolled in the study group. Fifty healthy subjects were selected as healthy control group. The peripheral venous blood samples were collected from all subjects. After the synthesis of hTERT cDNA by reverse transcription, real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used for the amplification of hTERT gene fragment. The tumor markers CEA, CA72-4, CA19-9 and CA125 were quantitatively detected by electrochemiluminescence instrument. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of individual markers or joint detection were calculated. The sensitivity and specificity of joint testing items were compared. Results There were statistically significant differences in the positive rates of hTERT mRNA (P< 0.01), CEA (P=0.002), CA72-4 (P=0.003), and CA19-9 (P=0.017) between the gastric cancer group and the healthy control group, while the positive rates of CA125 were not significantly different between the two groups (P> 0.05). The expression of hTERT mRNA was correlated with TMN stage, lymph node metastasis and distant metastasis (all P< 0.05), but was not correlated with age, sex, tumor location, invasion degree and differentiation degree (all P>0.05). The results of diagnostic indices showed that the hTERT mRNA had high sensitivity (84.8％), specificity (82.7％), accuracy (83.7％), positive predictive value (81.2％) and positive likelihood ratio (4.90). CA72-4 also had high specificity (61.2％), accuracy (64.3％), and positive predictive value (45.8％), which were second only to the hTERT mRNA. The diagnostic performance of CA125 combined with CEA, CA72-4, CA19-9 and peripheral blood hTERT mRNA was not significantly different from the latter four combined detection (P>0.05). The sensitivity and specificity of combined detection of traditional tumor markers CEA, CA72-4 and CA19-9 were 62.5％and 80.0％. The sensitivity and specificity of combined detection of hTERT mRNA, CEA, CA72-4 and CA19-9 could be change to 90.0％ and 67.5％. The difference in sensitivity between the two combined detection groups was statistically significant (P= 0.019), and the difference in specificity was not statistically significant (P> 0.05). Conclusion The comprehensive evaluation index of peripheral blood hTERT mRNA is better than the traditional gastric tumor markers, and it is expected to become a new marker for the diagnosis of gastric cancer.
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In recent years, the incidence of thyroid cancer has been increasing. Researchers around the world have begun to pay more attention to the exploration of its pathogenesis, disease evolution and prognosis. Among them, research in the field of gene molecules has become a hotspot, which includes the mutations of v-raf murine sarcoma viral oncogene homologue B1 (BRAF) and the telomerase reverse transcriptase (TERT) promoter. However, this field is not mature, and there are many problems and challenges need to be solved. This paper explores the value of BRAF mutation in the treatment, recurrence, mortality and prognosis of papillary thyroid carcinoma. In addition, we also explore the relationship between BRAF mutation and TERT promoter mutations and their influences in thyroid cancer. We hope this paper could help later scholars understand the current situation in this field and find a research direction in the future.
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Animals , Humans , Mutation , Promoter Regions, Genetic , Proto-Oncogene Proteins B-raf , Genetics , Telomerase , Genetics , Thyroid Neoplasms , GeneticsABSTRACT
OBJECTIVE@#This study aims to establish an effective and stable periodontal ligament cell line stably expressing human telomerase reverse transcriptase (hTERT) gene by using the adenovirus method.@*METHODS@#Polymerase chain reaction (PCR) was used to amplify the full length of hTERT gene to construct recombinant adenovirus plasmid pAd-pshuttle-cmv-hTERT. Packaged adenovirus particles were used for infection of human periodontal ligament cells. The expression levels of hTERT and osteogenic genes, such as alkaline phosphatase, Runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen Ⅰ mRNA, were detected by quantitative real-time PCR (qRT-PCR). The ability of osteogenic differentiation was observed by alizarin red staining, and the cell proliferation was determined by CCK-8.@*RESULTS@#Adenovirus particles containing the hTERT gene were successfully constructed and infected with periodontal ligament cells. The infected cells were similar to normal periodontal ligament cells. The qRT-PCR results showed that hTERT and osteogenesis-associated genes were highly expressed in the periodontal ligament cell lines constructed by adenoviruses. Alizarin red staining showed that the periodontal ligament cell line had strong osteogenic differentiation capability. CCK-8 showed that the periodontal ligament cell line had strong proliferation capability.@*CONCLUSIONS@#The human periodontal ligament cell line with high efficiency and stable expression of hTERT was established by the adenovirus method, thereby providing an ideal cell line for studying the mechanism of periodontal regeneration.
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Humans , Adenoviridae , Alkaline Phosphatase , Cell Differentiation , Cell Line , Cell Proliferation , Osteogenesis , Periodontal Ligament , TelomeraseABSTRACT
Human telomerase reverse transcriptase (hTERT)is a catalytic subunit of telomerase and is closely related to immortalization of cells,tumorigenesis and senescence of cells. hTERT has a great relation-ship with the occurrence and development of skin malignant tumors (melanoma,squamous cell carcinoma, basal cell carcinoma,cutaneous lymphoma,etc. ). Abnormal expression of hTERT has important clinical appli-cation value in early diagnosis,individualized comprehensive treatment and prognosis evaluation of cutaneous malignant tumors.
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Objective To explore the effect of age factor on the degree of pulmonary fibrosis and on the change in telomerase reverse transcriptase(TERT) activity in mice.Methods A total of 80 healthy male C57BL/6 mice aged 20-weeks were randomized into 4 groups:a young pulmonary fibrosis model group(n=20),a young control group(n=20),a senile pulmonary fibrosis model group(n=20),and an elderly control group (n =20).Two model groups were induced by an intratracheally injected bleomycin in 5 mg/kg,and two control groups by an intratracheally injected normal saline.The elderly were defined as 26 weeks old mice.Five mouse pulmonary fibrosis models and five mouse controls in four groups were randomly selected and killed at 7,14,21,28 days.Lung tissue specimens were stained with hematoxylin eosin (HE)and Masson trichrome (Masson)method,and then pathological changes were observed.In addition,immunohisto-chemical staining was used to observe the expression levels of epithelial cell marker protein E-cadherin(E-cad),stromal cell marker protein Vimentin,and alpha smooth muscle actin(α-SMA).Finally,the expression level of telomerase reverse transcriptase(TERT)protein was detected by Western blotting.Results A bleomycin-induced pulmonary fibrosis mice model was successfully prepared.The degree of pulmonary fibrosis was more severe in old mice than in young mice.Compared with the young pulmonary fibrosis model group,the expression level of E-Cad was decreased in the senile pulmonary fibrosis model group(P ＜0.05).Compared with the young control group and the elderly control group,the expression levels of E-Cad in the young pulmonary fibrosis model group and the senile pulmonary fibrosis model group were decreased (P ＜ 0.05),and decreased along with the prolongation of the modeling time.Compared with the young pulmonary fibrosis model group,the expression levels of alpha-SMA and vimentin were increased in the senile pulmonary fibrosis model group(P ＜ 0.05).The expression levels of alpha-SMA and vimentin were increased in the young pulmonary fibrosis model group and the senile pulmonary fibrosis model group,as compared to the young control group and the elderly control group(P＜0.05),and increased along with the prolongation of the modeling time.The activity of TERT in lung tissue of mice was increased at first and then decreased.Compared with the young pulmonary fibrosis model group,the activity of TERT in the senile pulmonary fibrosis model group significantly fluctuated(P＜0.05).Conclusions Age factor can affect the severity of pulmonary fibrosis by affecting the activity of telomerase reverse transcriptase in mouse models of pulmonary fibrosis.
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Objective To explore the effects of estradiol on the telomerase activity and apoptosis of lens epithelial cells and its mechanisms.Methods Fifty adult female Sprague-Dawley rats were randomly divided into five groups (n =10):ovariectomized group,estradiol group 1,estradiol group 2,estradiol group 3 and sham group.All rats were prepared for ovariectomized models except that of the sham group.After 2 weeks of the operation,rats in the 5 groups were given naphthalene solution through a stomach tube,and meanwhile,estradiol group 1,estradiol group 2,estradiol group 3 received estradiol valerate with the dose of 0.21 mg · kg-1 · d-1,0.42 mg · kg-1 · d-1 and 0.84 mg · kg-1 · d-1,accordingly,while rats in the ovariectomized group and sham group were given 9 g · L-1 Nacl by stomach perfusion with a dose of 0.42 mg · kg-1 ·d-1.Then,the opacity of the lens in each group was examined by a slit lamp microscope every week.After 12 weeks of naphthalene solution administration,all rats were sacririced and the serum estradiol concentration was determined by radioimmunoassay.Next,the lenses were taken out,and TERT mRNA expression of lens epithelial cells (LEC) was measured by RT-qPCR,and finally,the apoptotic rate was detected by TUNEL method.Results The opacity of lens in the ovariectomized group was different from that in the estradiol group 1,estradiol group 2,estradiol group 3 and sham group,with statistical significances (all P ＜ 0.05),and the opacity of lens in the estradiol group 1,2,3 and sham group were mild and occurred later.The serum estradiol concentration in the ovariectomized group (8.19 ± 1.45)ng · L-1 was significantly lower than that of estradiol groupl,2,3 and sham group,and there were significant differences (all P ＜ 0.05).Thc relative expression of TERT mRNA in LEC in the ovariectomized group (0.371 2-±0.056 4) was significantly lower than that in estradiol group 1,2,3,but the apoptotic rate of LEC (0.602 1 ±0.010 8) was obviously higher than that of estradiol group 1,2,3 in a dose-dependent manner,and there were significant differences (all P ＜ 0.05).Pearson correlation analysis showed the relative expression of LEC TERT mRNA in rats was negatively correlated with the apoptosis rate(r =-0.859,P ＜ 0.05).Conclusion Estradiol can up-regulate TERT mRNA expression and enhance telomerase activity of LEC in naphthalene-induced ovariectomized female rats in a dose-dependent manner.Estradiol can inhibit the LEC apoptosis in naphthalene-induced ovariectomized female rats,and the mechanism may be related to the increase of telomerase activity in the LEC.
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<p><b>Background</b>Idiopathic pulmonary fibrosis (IPF) is an age-related and progressive interstitial lung disease. Up to 20% of cases of IPF cluster in families, genetic factors contribute significantly to the pathogenesis of the disease. This study aimed to explore the association between rare genetic variants and IPF in Chinese Han families.</p><p><b>Methods</b>A Han family, comprising three IPF patients and five unaffected their first-degree relatives, and 100 ethnically matched control individuals from North China were enrolled in this study. Peripheral blood was collected, and genomic DNA was extracted. To elucidate if rare genetic variants are associated with the familial IPF, we performed whole-exome sequencing of affected members from a Chinese Han IPF family. Candidate rare variants were then confirmed by Sanger sequencing.</p><p><b>Results</b>We identified a potentially damaging rare variant-a heterozygous mutation c.2146G>A in exon 6 of the gene encoding for telomerase reverse transcriptase (TERT), which results in an amino acid substitution (p.Ala716Thr). We confirmed the missense mutation by Sanger sequencing in all the affected family members but did not detect this mutation in 100 ethnically matched healthy controls. Patients carried this mutation were characterized by the frequently acute exacerbation of IPF phenotype, with poor prognosis. The mean time to death was 2.8 years after diagnosis.</p><p><b>Conclusion</b>Using next-generation sequencing technology in familial IPF patients, we identified the heterozygous rare variant in TERT gene, and strengthened the importance of genetic variants in telomere-related pathogenesis in Chinese IPF patients.</p>
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Female , Humans , Male , Middle Aged , China , Idiopathic Pulmonary Fibrosis , Genetics , Mutation , Mutation, Missense , Pulmonary Fibrosis , Telomerase , Genetics , TelomereABSTRACT
Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.
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Objective To explore the effect of telomerase reverse transcriptase(TERT) gene transfected bone marrow stem cell(BMSC)on the memory function and hippocampal CA1 region synaptic plasticity in vascular dementia rat.Methods A total of 60 rats were randomly divided into the negative control group(group A),model group(group B),conventional BMSC group(group C) and transfected BMSC group(group D).The related indicators in each group were detected by using the Morris maze test,RTPCR and Western blot respectively.Results The escape latency period in the group C and group D was significantly longer than that in the group B,which in the group D was significantly longer than that in the group C.Compared with the group A,the expressions of brain-derived neurotrophic factor(BDNF)mRNA,TERT mRNA,SYP mRNA and protein in the group B,group C and group D were significantly decreased.The synaptic cleft arrange in group A was clear with more SYN positive ceils.The synaptic cleft in the group D was clearer,and the number of SYN positive cells was close to that in group A.Conclusion TERT transfected BMSC has obvious therapeutic effect on vascular dementia rats and its mechanism may be related to the promotion of BDNF,TrkB expression and the improvement of synaptic plasticity.
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The valproic acid (VPA)-induced animal model is one of the most widely utilized environmental risk factor models of autism. Autism spectrum disorder (ASD) remains an insurmountable challenge among neurodevelopmental disorders due to its heterogeneity, unresolved pathological pathways and lack of treatment. We previously reported that VPA-exposed rats and cultured rat primary neurons have increased Pax6 expression during post-midterm embryonic development which led to the sequential upregulation of glutamatergic neuronal markers. In this study, we provide experimental evidence that telomerase reverse transcriptase (TERT), a protein component of ribonucleoproteins complex of telomerase, is involved in the abnormal components caused by VPA in addition to Pax6 and its downstream signals. In embryonic rat brains and cultured rat primary neural progenitor cells (NPCs), VPA induced the increased expression of TERT as revealed by Western blot, RT-PCR, and immunostainings. The HDAC inhibitor property of VPA is responsible for the TERT upregulation. Chromatin immunoprecipitation revealed that VPA increased the histone acetylation but blocked the HDAC1 binding to both Pax6 and Tert genes. Interestingly, the VPA-induced TERT overexpression resulted to sequential upregulations of glutamatergic markers such as Ngn2 and NeuroD1, and inter-synaptic markers such as PSD-95, α-CaMKII, vGluT1 and synaptophysin. Transfection of Tert siRNA reversed the effects of VPA in cultured NPCs confirming the direct involvement of TERT in the expression of those markers. This study suggests the involvement of TERT in the VPA-induced autistic phenotypes and has important implications for the role of TERT as a modulator of balanced neuronal development and transmission in the brain.
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Animals , Female , Pregnancy , Rats , Acetylation , Autism Spectrum Disorder , Autistic Disorder , Blotting, Western , Brain , Chromatin Immunoprecipitation , Embryonic Development , Histones , Models, Animal , Neurodevelopmental Disorders , Neurons , Phenotype , Population Characteristics , Ribonucleoproteins , Risk Factors , RNA, Small Interfering , Stem Cells , Synaptophysin , Telomerase , Transfection , Up-Regulation , Valproic AcidABSTRACT
Telomere and telomerase maintain the stability of chromosome in structure and function.Studies have found that they are closely related with the development of human tumors.Telomeres are deoxyribonucleic acid in the terminal of chromossote of eukaryotes to maintain the integrity of chromosone.Telomerase is a kind of special structure of nucleoprotein,it prolongs the telomere and maintains the telomere' s stability,and it is directly related to the unlimited proliferation and canceration of cells.Human telomerase reverse transcriptase (hTERT) is both the important component and speed limit subunit of telomerase,it directly determines the telomerase activity.Further studies on structure,function and interaction of telomere,telomerase and hTERT help understand the developing mechanisms of tumors,they have important significance to the tumor diagnosis and treatment,and can provide new targets for cancer treatment.
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<p><b>OBJECTIVE</b>To construct a cell line of oral mucosa epithelial cells that stably express human telomerase reverse transcriptase (hTERT) by lentiviral vectors, approaches for the establishment of stable and efficient immortalized oral mucosa epithelial cell lines were explored.</p><p><b>METHODS</b>Whole RNA was extracted from 293T cells. The hTERT gene was amplified by polymerase chain reaction (PCR) and cloned into the lentiviral vector as pLVX-puro-hTERT. The lentivirus particles were successfully packaged and used to infect primary oral epithelial cells. The positive cell clones were selected by puromycin. Finally, the expression of hTERT was examined by real-time fluorescent quantitative PCR (qRT-PCR) and Western blot analysis.</p><p><b>RESULTS</b>The sequencing results confirmed the construction of the recombinant lentivirus pLVX-puro-hTERT. The morphology of infected cells was similar to that of normal oral mucosal epithelial cells, with a cobble stone-like appearance. The qRT-PCR and Western blot results showed that hTERT was overexpressed in infected cells compared with the normal group (P<0.05).</p><p><b>CONCLUSIONS</b>The oral epithelial cell line with stable expression of hTERT was successfully established by the lentivirus, which provides an experimental basis for the establishment of a highly efficient and stable oral epithelial immortalized cell line.</p>
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Humans , HEK293 Cells , Lentivirus , Mouth , Mucous Membrane , Real-Time Polymerase Chain Reaction , TelomeraseABSTRACT
Objective To detect the expression of human telomerase reverse transcriptase (hTERT) mRNA in the melanoma, and to analyze the relationship between the expression and subtypes and clinicopathological features of melanoma. Methods Expression of hTERT mRNA was detected by real-time quantitative PCR in 64 cases of melanoma and 30 cases of nevus. SPSS 17.0 software was used to analyze the relationship between hTERT mRNA expression and clinical pathological features of melanoma. Results The relative expression of hTERT mRNA in melanoma tissues was higher than that in nevus tissues [(52.43±5.42) vs (21.38±3.73), t= 4.72, P= 0.000]. The expression of hTERT mRNA in melanoma had no significant correlation with age, gender, ethnicity (all P> 0.05), but had relationship with subtypes, lymph node metastasis, Clark classification (all P 0.05). Conclusions The expression of hTERT mRNA in melanoma is high, especially in mucosal melanoma. hTERT may play an important role in the occurrence and development of melanoma.
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Previous studies have established a bovine mammary gland epithelia cells in vitro model by the adenovirus-mediated telomerase (hTERT-bMGEs). The present study was conducted to confirm whether hTERT-bMGEs were effective target cells to improve the efficiency of transgenic expression and somatic cell nuclear transfer (SCNT). To accomplish this, a mammary-specific vector encoding human lysozyme and green fluorescent protein was used to verify the transgenic efficiency of hTERT-bMGEs, and untreated bovine mammary gland epithelial cells (bMGEs) were used as a control group. The results showed that the hTERT-bMGEs group had much higher transgenic efficiency and protein expression than the bMGEs group. Furthermore, the nontransgenic and transgenic hTERT-bMGEs were used as donor cells to evaluate the efficiency of SCNT. There were no significant differences in rates of cleavage or blastocysts or hatched blastocysts of cloned embryos from nontransgenic hTERT-bMGEs at passage 18 and 28 groups (82.8% vs. 81.9%, 28.6% vs. 24.8%, 58.6% vs. 55.3%, respectively) and the transgenic group (80.8%, 26.5% and 53.4%); however, they were significantly higher than the bMGEs group (71.2%, 12.8% and 14.8%), (p < 0.05). We confirmed that hTERT-bMGEs could serve as effective target cells for improving development of somatic cell cloned cattle embryos.
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Animals , Cattle , Humans , Blastocyst , Clone Cells , Embryonic Structures , Epithelial Cells , In Vitro Techniques , Mammary Glands, Human , Muramidase , Telomerase , Tissue DonorsABSTRACT
AIM: The aim of this study was to assess visfatin expression and its effect on human telomerase gene expression in AGS gastric cancer cell line. MATERIALS AND METHODS: In this study, human gastric cancer (AGS) cell line was established as an in vitro model. Reverse transcription polymerase chain reaction (RT‑PCR) and enzyme‑linked immunosorbent assay was performed to show that visfatin expression in messenger ribonucleic acid (mRNA) and protein level respectively. After stimulating with increasing concentrations of visfatin for times of 24 h and 48 h, cell proliferation was assessed by 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT) assay. In order to investigate telomerase gene expression affected by visfatin in AGS cell line, total RNA was extracted and complementary deoxyribonucleic acid was synthesized buy using commercially available kits. Expression of human telomerase reverse transcriptase (hTERT) mRNA was carried out by real‑time RT‑PCR. RESULTS: After visfatin treatment gastric cell line proliferation was enhanced and was increased the expression of hTERT. CONCLUSIONS: The obtained data showed that visfatin induces endogenously gastric cancer cell proliferation and increases telomerase (hTERT) gene expression, as a cancer gene. Based on this study, it is suggested that expression of this adipocytokine protein in real samples could be biomarker for gastric cancer.