FAM171B stabilizes vimentin and enhances CCL2-mediated TAM infiltration to promote bladder cancer progression.

BACKGROUND: Invasion and metastasis are the main causes of unfavourable prognosis in patients diagnosed with bladder cancer. The efficacy of immunotherapy in bladder cancer remains suboptimal due to the presence of an immunosuppressive microenvironment. The novel protein family with sequence similarity 171B (FAM171B) has been identified, but its precise role and mechanism in bladder cancer remain unclear. METHODS: In this study, we conducted an analysis to investigate the associations between FAM171B expression and the prognosis and clinicopathological stage of bladder cancer. To this end, we utilized RNA sequencing data from the TCGA and GEO databases, as well as tumor tissue specimens obtained from our clinical centre. RNA sequencing analysis allowed us to examine the biological function of FAM171B at the transcriptional level in bladder cancer cells. Additionally, we used immunoprecipitation and mass spectrometry to identify the protein that interacts with FAM171B in bladder cancer cells. The effects of FAM171B on modulating tumor-associated macrophages (TAMs) and vimentin-mediated tumor progression, as well as the underlying mechanisms, were clarified by phalloidin staining, immunofluorescence staining, ELISA, RNA immunoprecipitation, flow cytometry and a bladder cancer graft model. RESULTS: FAM171B expression exhibits strong positive correlation with poor survival outcomes and advanced clinicopathological stages in patients with bladder cancer. FAM171B significantly promoted bladder cancer growth and metastasis, accompanied by TAM accumulation in the microenvironment, in vivo and in vitro. Through studies of the molecular mechanism, we found that FAM171B contributes to tumor progression by stabilizing vimentin in the cytoplasm. Additionally, our research revealed that FAM171B enhances the splicing of CCL2 mRNA by interacting with heterogeneous nuclear ribonucleoprotein U (HNRNPU), ultimately leading to increased recruitment and M2 polarization of TAMs. CONCLUSIONS: In this study, we identified FAM171B as a potent factor that promotes the progression of bladder cancer. These findings establish a solid theoretical foundation for considering FAM171B as a potential diagnostic and therapeutic biomarker for bladder cancer.

Cyclooxygenase-2 inhibitor NS398 enhances radiosensitivity of radioresistant esophageal cancer cells by inhibiting AKT activation and inducing apoptosis.

This study aimed to evaluate the radiosensitizing effect of a COX-2 inhibitor, NS398, and its mechanism in radioresistant esophageal cancer Eca109R50Gy cells. NS398 enhanced radiosensitivity of Eca109R50Gy cells, characterized by redistribution of cell cycle, inhibition of DNA-dependent protein kinase catalytic subunit expression and induction of apoptosis. NS398 also reduced phospho-AKT level, upregulated expression of Bax and both procaspase-3 and active caspase-3, and downregulated Bcl-2 expression. Finally, NS398-induced radiosensitization was partly reversed by insulin-like growth factor-1, but not by prostaglandin E2. Our results suggest that NS398 may enhance radiosensitivity of Eca109R50Gy cells through blocking AKT activation and inducing apoptosis.

[The protective roles of tiangui gengnian soft capsule on brain in aged female rats and its mechanism].

OBJECTIVE: To investigate the brain-protective and anti-aging effects and the mechanism of action of Tiangui Gengnian Soft Capsule (TGSC), a Chinese herbal preparation composed of sea buckthorn fatty acids. METHODS: Sixteen-month-old female rats were administered via gastric perfusion with low, medium and high doses (0.72 g/kg, 1.80 g/kg and 4.5 g/kg) of TGSC for 180 days. The 3-month and 22-month old rats were taken as controls. Expression of estrogen receptor beta(ERbeta), tyrosine hydroxylase (TH), granulocyte colony-stimulating factor (G-CSF) and vascular endothelial growth factor (VEGF) in the brain of rats were observed and analyzed using immunohistochemical technique, and changes in the number of neurons and glial cells were counted with Nissl's staining. RESULTS: Immunohistochemistry showed the number of ERbeta- and TH-positive neurons in the hypothalamus was significantly higher (P < 0.01 and P < 0.05), G-CSF-positive astrocyte stain was much weaker (P < 0.01) and the expression of VEGF-positive cerebral vessel was stronger (P < 0.05) in the TGSC treated groups than those in the control. Nissl's staining showed no significant difference in the number of neurons and glial cells among groups. CONCLUSION: TGSC could up-regulate the expressions of ERbeta, TH and VEGF in the brain of aged female rats, modulate the responsibility of astrocyte and its G-CSF expression, suggesting that TGSC may have certain neuro-protective and anti-aging effects.

[The reproductive system impairment of adult male rats induced by cocaine].

OBJECTIVE: To investigate the reproductive system impairment induced by cocaine in adult male rats and the possible underlying mechanism. METHODS: Thirty adult male rats were randomly divided into experimental and control groups, with 15 rats in each group. Rats of the experimental group were injected cocaine hydrochloride (15 mg/kg body weight) subcutaneously daily for four weeks. The weight of body and testis, as well as the level of serum hormone of the rats were examined. In addition, the apoptosis rate of testicular tissue by TUNEL and the expression of Fas gene in testicular tissue were examined by immunohistochemistry. RESULTS: Compared with the control, the weight of testis in the cocaine exposed group decreased significantly (P<0.05), and the serum testosterone level decreased significantly (P<0.05). Moreover, both the apoptosis rate and the expression of Fas gene increased in the testicular tissue of rats in the cocaine exposed group in comparison to the control group (P<0.05). The apoptosis rate was significantly correlated with the expression of Fas gene (r=0.9012, P<0.05). CONCLUSION: Cocaine may cause reproductive system injury in adult male rats, and Fas-mediated apoptosis may be one of the functional mechanisms involved in the reproductive system injuried by cocaine.

Prenatal restraint stress impairs learning and memory and hippocampal PKCbeta1 expression and translocation in offspring rats.

Prenatal stress results in various learning, behavioral and emotional alterations observed in later life. However, the mechanisms underlying these effects of prenatal stress are not fully understood. In the present study we examined the impact of prenatal stress (an unpredictable restraint stress) during gestational days 13 to 20 on the performance in Morris water maze and passive avoidance training in 1- and 3-month-old rat offspring. The expression and translocation/activation of protein kinase C (PKC) beta1 in the hippocampus of prenatally stressed offspring were also investigated. One-month-old female and male and 3-month-old female prenatally stressed offspring showed longer latency to find the platform and used the inefficient search strategy in the water maze task and showed lower memory score in the passive avoidance training compared with controls. The expression of PKCbeta1 protein and mRNA in the hippocampus of prenatally stressed offspring was dramatically weakened. In the control offspring hippocampus, passive avoidance training induced the PKCbeta1 translocation from the cytosol to the membrane, which, however, was not observed in prenatally stressed offspring. Our results suggest that deficient signal transduction of PKCbeta1 in the hippocampus resulting from prenatal restraint stress may play an important role in the impairment of learning and memory abilities of offspring.

[Gender-dependent difference of NF-kappaB expression in the hippocampus of prenatally stressed offspring rats].

In this study, immunohistochemistry and Western blot were used to determine whether the expression of NF-kappaB in the hippocampus of prenatally stressed offspring rats is gender-dependent. The results were as follows: In the female offspring rats, the expressions of p65 in the hippocampal dentate gyrus in mid-term stress (MS) and late-term stress (LS) groups were significantly less than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly more than that in the control group (P<0.01). A significant difference was also present between MS and LS groups (P<0.01). In the male offspring rats, the expressions of p65 in the hippocampal dentate gyrus in MS and LS groups were evidently more than that in the control group (P<0.01). There was a significant difference between MS and LS groups (P<0.01). The expressions of p50 in all regions of hippocampus in MS and LS groups were significantly less than that in the control group (P<0.05, P<0.01). There was also a significant difference in p65 expression between MS and LS groups (P<0.01). In addition, in the control group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly more than that of male ones (P<0.01). However, in LS group the expressions of p65 in the hippocampal dentate gyrus of female offspring rats were significantly less than that of male ones (P<0.01). Moreover, there was no significant difference in p65 expression between female and male offspring rats in MS group. In the control group the gender difference in the expression of p50 was only observed in hippocampal CA1 (P<0.01). The expressions of p50 in all regions of hippocampus of female offspring rats were significantly more than that of male ones in LS group (P<0.01). There was no significant difference in p50 expression between female and male offspring rats in MS group. The results of Western blot were similar to those of immunohistochemical study. These results indicate that prenatal stress in different gestational periods significantly affects the expressions of p65 and p50 in hippocampus, and this effect is gender-dependent. This may be one of the mechanisms underlying the gender difference in the ability of learning and memory of the prenatally stressed offspring rats.

Effect of vector-expressed shRNAs on hTERT expression.

AIM: To study the effect of short hairpin RNAs (shRNAs) expressed from DNA vector on hTERT expression. METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expression vector pUC18U6 to form pUC18U6ht1-4, which were then introduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR. RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P<0.05). CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.

[Detection of HPV 58 and cloning of its E7 gene in cervical cancer].

OBJECTIVE: To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer. METHODS: HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3) cells, and induced to express 58 E7 protein by IPTG. RESULTS: Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M(r) 16 x 10(3) was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins. CONCLUSION: HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.

Vasoactive intestinal peptide increases VEGF expression to promote proliferation of brain vascular endothelial cells via the cAMP/PKA pathway after ischemic insult in vitro.

Vasoactive intestinal peptide (VIP) enhances angiogenesis in rats with focal cerebral ischemia. In the present study, we investigated the molecular mechanism of the proangiogenic action of VIP using an in vitro ischemic model, in which rat brain microvascular endothelial cells (RBMECs) are subjected to oxygen and glucose deprivation (OGD). Western blotting and immunocytochemistry were carried out to examine the expression of VIP receptors and vascular endothelial growth factor (VEGF) in cultured RBMECs. The cell proliferation was assessed by the MTT assay. Cyclic adenosine monophosphate (cAMP) and VEGF levels were measured by using the enzyme-linked immunosorbent assay. The cultured RBMECs expressed VPAC1, VPAC2 and PAC1 receptors. Treatment with VIP significantly promoted the proliferation of RBMECs and increased OGD-induced expression of VEGF, and this effect was antagonized by the VPAC receptor antagonist VIP6-28 and VEGF antibody. VIP significantly increased contents of cAMP in RBMECs and VEGF in the culture medium. The VIP-induced VEGF production was blocked by H89, a protein kinase A (PKA) inhibitor. These data suggest that treatment with VIP promotes VEGF-mediated endothelial cell proliferation after ischemic insult in vitro, and this effect appears to be initiated by the VPAC receptors leading to activation of the cAMP/PKA pathway.

Vasoactive intestinal peptide protects against ischemic brain damage induced by focal cerebral ischemia in rats.

Vasoactive intestinal peptide (VIP) exerts neuroprotective effects under various neurotoxic conditions in vitro. In the present study, we investigated the effects of VIP on transient ischemic brain damage. Focal cerebral ischemia was induced using middle cerebral artery occlusion (MCAO) for 120 min in the adult rat brain. Either a single intracerebroventricular injection of VIP or saline was given at the beginning of reperfusion. Forty-eight hours after MCAO, the rats were sacrificed for evaluation of the infarct volume and histological analysis. ELISA was performed to assay levels of serum S100B before being sacrificed. We also evaluated the blood-brain barrier (BBB) permeability using Evans blue dye injection method. In contrast to the cases treated with vehicle, the infarct volume was significantly (P<0.05) reduced, and terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining and immunoreactivity for S100B were also significantly (P<0.05) decreased in the ischemic hemisphere with VIP treatment. In addition, the elevations of serum S100B were significantly (P<0.01) attenuated in VIP-treated rats compared with those of control rats. Treatment with VIP did not result in a significant reduction of Evans blue leakage, although it tended to be lower than that in the control rats. Our data suggest that treatment with VIP reduces brain damage in ischemic rats, and this effect may be associated with the attenuation of apoptosis and S100B expression.

Bisphenol A exposure induces apoptosis and upregulation of Fas/FasL and caspase-3 expression in the testes of mice.

There are controversies about adverse effects of bisphenol A (BPA), a ubiquitous xenoestrogen, on reproduction and development of male animals. To understand BPA action and assess its risk more completely, we examined the impact of BPA at high doses on the testes of pubertal male Kunming (China) mice. BPA at 0 (control), 160, 480, and 960 mg/kg/day was given by gavage to mice from postnatal days (PND) 31-44, followed by observation of morphology and detection of apoptosis and expressions of Fas/FasL and active caspase-3 on PND 45, 60, and 90 by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling, immunohistochemistry, and Western blotting. There was no effect of BPA at 160 mg/kg/day, however, at 480 and 960 mg/kg/day there was underdevelopment of testes and disruption of spermatogenesis. There were many apoptotic Leydig and germ cells in the testes with apoptotic indices being significantly increased compared with controls. The expression of Fas and active caspase-3 was localized in the same cell types as apoptosis occurred, and expression levels of Fas, FasL, and active caspase-3 were significantly increased compared with controls. The disturbed spermatogenesis, apoptosis and upregulation of Fas, FasL, and active caspase-3 expression persisted to PND 90. The results suggest that high-dose BPA induces apoptosis of Leydig and germ cells in the mouse testis through the Fas-signaling pathway. Therefore, there is concern about reproductive health for humans occupationally exposed to high levels of BPA.

Caspase-3 antisense oligodeoxynucleotides inhibit apoptosis in gamma-irradiated human leukemia HL-60 cells.

To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODNs) on apoptosis, we designed four ASODNs targeting different regions of caspase-3 mRNA and transfected them into human leukemia HL-60 cells. The transfected cells were given 10 Gy gamma-irradiation followed by incubation for 18 h and measurement of apoptosis and caspase-3 expression. Our results showed that ASODN-2 targeting the 5' non-coding region of sites -62 to -46, and ASODN-3 targeting the 5' coding region of sites -1 to 16, both reduced apoptosis measured by gel electrophoresis and flow cytometry. Hoechst 33258 staining and TUNEL assay revealed that apoptotic indexes in the ASODN-2 and ASODN-3 groups were significantly lower than those in the untransfected and mismatched oligodeoxynucleotide (MODN) groups. Immunocytochemistry, Western blotting and RT-PCR showed that expression levels of caspase-3 protein and mRNA in both ASODN-2 and ASODN-3 groups were decreased compared with those in the untransfected and MODN groups. In conclusion, caspase-3 mRNA ASODNs can inhibit gamma-radiation-induced apoptosis of HL-60 cells and reduce expression of caspase-3 protein and mRNA. The results suggest that antisense approach may be useful for therapeutic treatment of certain neurodegenerative diseases in which apoptosis is involved.

Overexpression of PTEN suppresses growth and induces apoptosis by inhibiting the expression of survivin in bladder cancer cells.

The tumor suppressor gene PTEN, which encodes a multifunctional phosphatase protein, is mutated in a variety of human cancers. Several reports have indicated that it has growth-suppressive and proapoptosis properties and displayed an altered expression pattern during human oncogenesis. Overexpression of PTEN leads to decreasing cell growth and tumorigenicity in vitro and in vivo. In the present study, we further demonstrated that overexpression of PTEN mediated by adenovirus suppressed bladder cancer cell growth and significantly induced apoptosis, through downregulating of survivin and activating of caspase cascades. Our results indicate that Ad-PTEN exerts its tumor suppressive effect on bladder cancer cells through inhibiting survivin and upregulating caspase-related proteins. Thus Ad-PTEN may be potentially therapeutic for the treatment of bladder cancers.

[Expression of TRAIL gene eukaryotic expression vector modulated by the human telomerase reverse transcriptase gene core promoter in ovarian cancer cell line SKOV3].

AIM: To construct the TRAIL gene eukaryotic expression vector modulated by the human telomerase reverse transcription gene core promoter and to evaluate the expression of the TRAIL gene in ovarian cancer cell line SKOV3 in vitro. METHODS: The amplified TRAIL gene fragment was subsequently cloned into hTERT promoter-pIRES2-EGFP vector and CMVpromoter-pIRES2-EGFP vector. The hTERT promoter-pIRES2-EGFP-TRAIL and CMVpromoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were obtained, respectively. The plasmids were transfected into human ovarian carcinoma SKOV3 cell line by lipofeclin mediation and the positive clones were screened by G418. The expression of TRAIL gene was examined by RT-PCR, Western blot, immunocytochemistry and flow cytometry. RESULTS: All the constructed vectors were verified by enzyme digestion. The TRAIL gene of transfected cells was increased significantly (P<0.01). CONCLUSION: An eukaryotic expression vector of the TRAIL gene modulated by the human telomerase reverse transcription gene core promoter has been constructed successfully and its steady expression in human ovarian cancer cell line SKOV3, which will be beneficial to further research into its role in regulating the biological behavior of ovarian cancer cells.

[Construction of tumor cell-specific expression vector modulated by human telomerase reserse transcription gene core promoter].

AIM: To construct the tumor cell-specific expression vector modulated by human telomerase reserse transcription gene (hTERT) core promoter. METHODS: The hTERT gene core promoter fragment was amplified by PCR using the total genomic DNA from SKOV3 cells as template. The amplified gene fragment was subsequently cloned into pIRES2-EGFP vector. Then the expression vector modulated by hTERT core gene promoter was transfected into tumor cell lines, HO-8910, Hela and normal ECV304 cells through lipofectamine, respectively. The activity of reporter gene GFP was detected 48 h after transfection. RESULTS: There was significant transcriptional activity in 3 kinds of transfected tumor cells, while no transcriptional activity was detected in transfected normal cells. CONCLUSION: The hTERT core gene promoter has the tumor specificity, suggesting that the specific expression vector modulated by hTERT core gene promoter may be a novel and promising approach to the tumor treatment.

[Caspase-3 antisense oligodeoxynucleotides inhibit caspase-3 expression and apoptosis of gamma-irradiated HL-60 cells].

It has been shown that gamma -irradiation induces apoptosis of the human promyeloid leukemia cell line HL-60, but the mechanism remains unclear. To explore the effect of caspase-3 in this apoptotic model, antisense oligodeoxynucleotides (ASODNs) targeting 5'-noncoding region (ASODN-1) and initial translation region (ASODN-2) of caspase-3 mRNA were designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by gamma-irradiation in the present study. The TUNEL assay was used for morphological analysis of HL-60 cell apoptosis. Immunocytochemical staining, Western blotting and RT-PCR were, respectively, performed for detecting expression of caspase-3 and its mRNA. HL-60 cells transfected with mismatched oligodeoxynucleotide (MODN) or untransfected were taken as the control groups. The TUNEL assay showed that the percentages of HL-60 cell apoptosis induced by gamma-Irradiation in both ASODN-1 and ASODN-2 groups were significantly reduced compared with those in the control group (P<0.01) when the final transfection concentration was > or = 3 micromol/L. Immunocytochemistry demonstrated that in the ASODN-1 and ASODN-2 groups, caspase-3 positive cell percentages were reduced and average gray values of the positive cells increased significantly compared with those in the control group (P<0.01). Western blotting found that procaspase-3 expression in HL-60 cells of the ASODNs groups was decreased,and it was lower in the ASODN-1 group than in the ASODN-2 group. RT-PCR revealed marked expression of caspase-3 mRNA in HL-60 cells of the control group. Expression of caspase-3 mRNA was decreased after ASODNs transfection. Furthermore, ASODN-1 was more effective in inhibiting HL-60 cell apoptosis (P<0.05) and caspase-3 expression (P<0.01) than ASODN-2. These results indicate that caspase-3 mRNA ASODNs prevent HL-60 cells from apoptosis induced by gamma-radiation,and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.

[Expression, purification, and serum antibody detection of HPV16 E6 in cervical cancer patients].

BACKGROUND & OBJECTIVE: Human papillomavirus type 16 (HPV16) is the predominant high-risk type of HPV in cervical cancer tissues. Serum antibody responded to HPV16-related proteins is associated with the development of cervical cancer. This study was to construct and purify recombinant HPV16 E6 protein, detect its corresponding serum antibody among different populations, and explore the correlation of HPV16 E6 serum antibody reaction to cervical cancer. METHODS: HPV16 E6 early gene was constructed into pRSET-A expression vector. The plasmids were transfected into BL21 (DE3) cells, which were induced to express HPV16 E6 protein by isopropylthio-beta-D-galactoside (IPTG). E6 inclusions were denatured, purified through Ni column, and renatured. After the activity of HPV16 E6 protein being identified, the antibodies against HPV16 E6 in serum samples from 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients were determined by ELISA using the fusion protein as antigen. HPV DNA genotype was estimated in cervical cancer tissues by fluorescence polarization. RESULTS: HPV16 E6 fusion protein of Mr 24x10(3) was expressed in pRSET-16E6 after induction of IPTG. The fusion protein was accounted for 22.3% of total bacterial proteins, and expressed as inclusive body. After purification with Ni-NTA agarose resin, the purity of the recombinant protein was over 95%, and its activity was identified by ELISA. The antibody-positive rate was significantly higher in cervical cancer patients than in healthy women and chronic cervicitis patients (31.2% vs. 5.0% and 6.5%, P<0.01). In the 32 cervical cancer patients, the positive rates of HPVs DNA and HPV16 DNA in cancer tissues were 90.61% and 46.88%. The antibody-positive rate of HPV16 E6 was higher in HPV16 DNA-positive cervical cancer patients than in HPV16 DNA-negative patients (46.7% vs. 17.6%), but the difference was not significant. CONCLUSIONS: HPV16 E6 fusion protein obtained from pRSET-A/BL21 can be used in serologic studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E6 is more common in cervical cancer patients than in healthy women and chronic cervicitis patients.

[Construction of TRAIL gene eukaryotic expression vector modulated by hTERT gene core promoter and its effect on apoptosis of ovarian cancer cells].

AIM: To construct the TNF-related apoptosis inducing ligand(TRAIL) gene eukaryotic expression vector modulated by human telomerase reserse transcriptase (hTERT) gene core promoter and to study its effect on apoptosis of ovarian cancer cells. METHODS: Genomic RNA was extracted from human placenta tissues and the fragment of TRAIL was obtained by RT-PCR. The amplified gene fragment was subsequently cloned into hTERTpromoter-pIRES2-EGFP vector and CMV promoter-pIRES2-EGFP vector after sequencing. The hTERTpromoter-pIRES2-EGFP-TRAIL and CMV promoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were constructed respectively. The recombinant plasmids were transfected into ovarian carcinoma cell line, SKOV3, and the levels of mRNA were determined by RT-PCR. The cell cycle and apoptosis rate of SKOV3 cells were determined by FCM. RESULTS: The constructed two recombinant vectors were verified by restriction enzyme digestion analysis and DNA sequencing. After being transfected with two recombinant vectors, the growth of SKOV3 cells was strongly inhibited and apoptotic features appeared. CONCLUSION: The recombinant eukaryotic expression vector has been constructed successfully. TRAIL gene driven by hTERTpromoter can be obviously expressed in ovarian carcinoma SKOV3 cells, suggesting that the specific expression vector modulated by hTERT core gene promoter may be a novel and promising approach to the tumor treatment.

[The effect of short hairpin RNA on hTERT expression].

AIM: To study the effect of short hairpin RNA (shRNA) on hTERT expression. METHODS: Oligonucleotides encoding shRNA against hTERT was cloned into a mammalian shRNA expression vector pUC18U6 to form pUC18U6ht which was transfected into HepG2 cells by using liposome. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir which expressed shRNA against green fluorescent protein were used as controls. hTERT mRNA in the transfected cells was quantified by using real-time fluorescent RT-PCR. RESULTS: Compared with the controls, the short hairpin RNA against hTERT decreased the hTERT mRNA level by 49% (P<0.05). CONCLUSION: hTERT expression was reduced by the shRNA.

[Quantitation of human telomerase reverse transcriptase mRNA with real-time fluorescent RT-PCR].

AIM: To establish a real-time fluorescent RT-PCR assay to quantify human telomerase reverse transcriptase (hTERT) mRNA. METHODS: Total cellular RNA was isolated from HepG2 cells using Trizol reagent, which was then reverse-transcribed into cDNA. By using a pair of gene-specific primers and a TaqMan MGB probe, cDNA of hTERT was quantified with real-time fluorescent PCR. Human beta-actin (hBA) mRNA was quantified at the same time as an endogenous control. Serial dilutions of cloned human beta-actin gene were used to construct a standard curve. RESULTS: The correlation coefficient of the standard curve was 1.00. The mean coefficient of variation of the assay was 7.1%. The ratio of hTERT mRNA to hBA mRNA of HepG2 cells was (1.3+/-0.3)x10(-4). CONCLUSION: A real-time fluorescent RT-PCR assay to quantify hTERT mRNA was established, which will facilitate the study on the biological function of telomerase.