Influence of Angiotensin II on α1-Adrenergic Receptors Function in Rat Aorta and Expression in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) is the main mediator of the Renin-Angiotensin-System acting on AT1 and other AT receptors. It is regarded as a pleiotropic agent that induces many actions, including functioning as a growth factor, and as a contractile hormone, among others. The aim of this work was to examine the impact of Ang II on the expression and function of α1-adrenergic receptors (α1-ARs) in cultured rat aorta, and aorta-derived smooth muscle cells. Isolated Wistar rat aorta was incubated for 24 h in DMEM at 37˚C, then subjected to isometric tension and to the action of added norepinephrine, in concentration-response curves. Ang II was added (1 × 10−5 M), and in some experiments, 5-Methylurapidil (α1A-AR antagonist), AH11110A (α1B-AR antagonist), or BMY-7378 (α1D-AR antagonist), were used to identify the α1-AR involved in the response. Desensitization of the contractile response to norepinephrine was observed due to incubation time, and by the Ang II action. α1D-AR was protected from desensitization by BMY-7378;while RS-100329 and prazosin partially mitigated desensitization. In another set of experiments, isolated aorta-derived smooth muscle cells were exposed to Ang II and α1-ARs proteins were evaluated. α1D-AR increased at 30 and 60 min post Ang II exposure, the α1A-AR diminished from 1 to 4 h, while α1B-AR remained unchanged over 24 h of Ang II exposure. Ang II induced an increase of α1D-AR at short times, and BMY-7378 protected α1D-AR from desensitization.

Comparative Analysis of Ki-67 Protein as a Proliferative Expression Index in Cutaneous Basal and Squamous Cell Carcinoma in Federal Medical Centre Umuahia, Nigeria

Background: Evaluating the tumor proliferative index helps predict clinical behavior and provides prognostic insights for cutaneous basal cell carcinoma (cBCC) and squamous cell carcinoma (cSCC). Objective: This study aimed to identify differences in the proliferative indices among variants of cBCC and cSCC diagnosed at a tertiary healthcare center. Method: Skin biopsies histologically diagnosed as cBCC and cSCC between 2012 and 2018 at the Federal Medical Centre (FMC) Umuahia, Abia State, Nigeria, were analyzed. Archival formalin-fixed, paraffin-embedded (FFPE) tissue blocks were retrieved along with clinical data, and were prepared on charged microscope slides and the immunohistochemical staining was carried out. The primary antibody used in this study was clone BioCare CRM325C (RM) and adenotonsillar tissue blocks/slides served as positive controls. Ki-67 immunohistochemistry was performed on fresh 4µm sections of the tumor specimens. Results: The application of Ki-67 immunoperoxidase on both BCC and SCC cohort, yielded an intense observable brownish nuclear stain in areas of dense proliferating tumour cells on both cutaneous tumours. The average Ki-67 index for all cSCC cases was 24.7%, with a range of 2.3% - 80%, while the mean for cBCC was 15.8%, ranging from 1.2% - 45.6%. Variants with high proliferative indices were observed in 11.9% of cBCC cases and 29.1% of cSCC cases. Among the low proliferative index category, cSCC accounted for 5.4%, while cBCC represented 14.3%. For mild proliferative indices, cSCC cases made up 7.3% and cBCC, 11.9%. The majority of cases showed moderate proliferative indices, with 61.9% for cBCC and 58.2% for cSCC. Overall, there was a significant difference in proliferative indices between cSCC, cBCC, and their variants. Conclusion: The study found a significantly higher rate of cell proliferation, measured by Ki-67 immunostaining, in cSCC and its variants compared to cBCC. However, certain variants of cBCC also exhibited high Ki-67 expression, indicating they can be as aggressive as some cSCC variants.

Molecular Cloning and Expression of RSSG58 Gene in Rice Sperm Cells

Myosins, a large family of structurally diverse mechanoenzymes, which, upon interaction with actin filaments, convert energy from ATP hydrolysis into mechanical force, play an important role in male reproductive processes. In this study we report the rice ( Oryza sativa L.) RSSG58 gene, which was cloned from the cDNA library of rice sperm cells by using sperm cell mainly expression subtractive clone as probe. This gene encodes a putative 66.7 W polypeptide, which shows similarity to the myosin heavy chain of Arabidopsis thaliana, and consists of 579 amino acids with an isoelectric point (pI) of 4.885. RSSG58, which is a member of a divergent gene family, generates transcripts of 2 278 bp and 2 437 bp that differ only in their polyadenylation sites. Southern hybridization showed that RSSG58 has only one copy in rice genome and RSSG58 transcripts are most abundant in sperm cells, with two distinct signals. The RT-PCR analysis indicated that the transcriptions of the RSSG58 gene were various in the different development stages and tissues. The greatest accumulation of RSSG58 mRNA was detected in sperm cells, while weaker expression was detected in leaves, microspore mother cells, unicellular microspore pollen stage, two-cell stage pollens, mature pollens and pollinated ovaries. These results suggest that RSSG58 is especially abundantly expressed in rice sperm cells.

Differential Expression of APOEGene in Porcine Fetal Fibroblast Cells

[Objective]The aim was to study the differential expression ofAPOEgene in different generations of porcine fetal fibroblasts cells.[Method]The first,tenth,fifteenth,twentieth,twenty-fifth,fiftieth generations of porcine fetal fibroblast cells,which were normally grown and passed,were collected before total RNA was extracted respectively.The expression ofAPOEgene in different generations of porcine fetal fibro-blast cells was detected by RT-PCR technique.[Result]The expression level of porcine APOE mRNA in the first generation of porcine fetal fi-broblast cells was the highest,and then it gradually decreased with the increase of cell generations and was the lowest in the fiftieth generation.[Conclusion]The expression ofAPOEgene had the selective trend in different generations of porcine fetal fibroblast cells.

EFFECT OF rhTGF-β1 AND rhGM-CSF ON RECEPTOR EXPRESSIONS IN J6-1 AND J6-2 LEUKEMIC CELLS

ABC immunoperoxidase was used to test the effects of rhTGF-β1 and rhGM-CSF on receptor expressions in J6-1 and J6-2 leukemic cell lines. Computer assisted image analysis system was introduced to evaluate positive index of time-and dose-dependent specimens. The expression of c-kit was elevated both in positive rate and positive index by TGF-01 in both time- and dose-dependent manners. Ing/ml rhTGF-β1 simultaneously enhanced the expression of c-fms and PDGF-R which is not detected in 50 ng / ml GM-CSF treatment. Endoglin was down-regulated after TGF-β treatment and up-regulated in J6-2 cells after GM-CSF treatment, c-kit Expression was elevated by TGF-β in J6-1 cells while decreased by both in J6-2 cells.

Astrocytic endothelin-1 overexpression impairs learning and memory ability in ischemic stroke via altered hippocampal neurogenesis and lipid metabolism

Vascular etiology is the second most prevalent cause of cognitive impairment globally.Endothelin-1,which is produced and secreted by endothelial cells and astrocytes,is implicated in the pathogenesis of stroke.However,the way in which changes in astrocytic endothelin-1 lead to poststroke cognitive deficits following transient middle cerebral artery occlusion is not well understood.Here,using mice in which astrocytic endothelin-1 was overexpressed,we found that the selective overexpression of endothelin-1 by astrocytic cells led to ischemic stroke-related dementia(1 hour of ischemia;7 days,28 days,or 3 months of reperfusion).We also revealed that astrocytic endothelin-1 overexpression contributed to the role of neural stem cell proliferation but impaired neurogenesis in the dentate gyrus of the hippocampus after middle cerebral artery occlusion.Comprehensive proteome profiles and western blot analysis confirmed that levels of glial fibrillary acidic protein and peroxiredoxin 6,which were differentially expressed in the brain,were significantly increased in mice with astrocytic endothelin-1 overexpression in comparison with wild-type mice 28 days after ischemic stroke.Moreover,the levels of the enriched differentially expressed proteins were closely related to lipid metabolism,as indicated by Kyoto Encyclopedia of Genes and Genomes pathway analysis.Liquid chromatography-mass spectrometry nontargeted metabolite profiling of brain tissues showed that astrocytic endothelin-1 overexpression altered lipid metabolism products such as glycerol phosphatidylcholine,sphingomyelin,and phosphatidic acid.Overall,this study demonstrates that astrocytic endothelin-1 overexpression can impair hippocampal neurogenesis and that it is correlated with lipid metabolism in poststroke cognitive dysfunction.

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period

Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immun-ofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary sperma-tocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion: GCNF may play important roles in spermatogenesis, capacitation and fertilization.

Prognostic value of programmed death.1, programmed death-ligand 1, programmed death-ligand 2 expression, and CD8(+) T cell density in primary tumors and metastatic lymph nodes from patients with stage T1.4N+M0 gastric adenocarcinoma

Background: Anti-programmed death-1/programmed death-ligand 1(PD-1/PD-L1) immunotherapy has been proved to be effective on gastric cancer in ongoing clinical trials. However, the value of PD-L1 in predicting responses of patients with gastric cancer to anti-PD-1/PD-L1 immunotherapy is controversial. Some studies suggested that intra-and inter-tumoral heterogeneity of PD-L1 expression might explain the controversy.This study aimed to analyze the expression of PD-L1, PD-L2, and PD-1 as well as CD8(+) T-cell density in primary tumors and lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma to explore the heterogeneity of PD-1 signaling pathway molecules.Methods: In primary tumors and metastatic as well as non-metastatic lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma, we detected PD-L1 and PD-L2 expression with immunohistochemistry. CD8(+)T-cell density in primary tumors and PD-1 expression on CD8(+)T cells were detected with immunofluorescence. Univariate analysis was used to determine the prognostic values of them. Cox proportional hazard regression model was used to identify independent risk factors that affect patients' overall survival and disease-free survival.Results: Among 119 eligible patients who had undergone surgical resection, the positive rate of PD-L1 was higher in metastatic lymph nodes than in primary tumors(45.4% vs. 38.7%, P = 0.005); the positive rate of PD-1 on CD8(+)T cells was significantly higher in primary tumors and metastatic lymph nodes than in tumor-free lymph nodes(both P < 0.001). The intensity of PD-1 expression on CD8(+) T cells in primary tumors and in metastatic lymph nodes were stronger than that in tumor-free lymph nodes from the same patient. Beside, the positive rate of PD-L2 did not show any differences between primary tumors and metastatic lymph nodes. In multivariate analysis, PD-L1 expression,PD-L2 expression, a low density of CD8(+) T cells in primary tumors, and PD-1 expression on CD8(+) T cells in primary tumors were associated with poor prognosis.Conclusion: The expression of PD-L1 is heterogeneous in primary tumors and in metastatic lymph nodes from patients with stageT1-4 N+M0 gastric adenocarcinoma, which might explain the inconsistent results in assessing the prognostic value of PD-L1 expression in previous studies.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

AIM To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3. CHO cells were transfected by pMSG-ns3 using calcium phosphate precipitation method and cultivated for 12 h-24 h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS After treated with 3×10-8mol/ L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.

Overexpression of p27^(KIP1)induced cell cycle arrest in G_1 phase and subsequent apoptosis in HCC-9204 cell line

AIM We have previously reported that inducible over-expresaion of Bak may prolong cell cycle in G1 phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27KIP1 plays an important role in this process. MEHODS In order to elucidate the exact function of p27KIP1 in this process, a zinc inducible p27KIP1 stable transfectant and transient p27KIP1- GFP fusion transfectant were constructed. The effects of inducible p27KIP1 on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells. RESULTS This p27KIP1-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27KIP1 induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72 h of p27KIP1 expression, p27KIP1 caused cell cycle arrest after 24 h of induction, with 40% increase in G1 population. Prolonged p27KIP1 expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27KIP1 expression showed a characteristic DNA ladder on agarose gel electrophoresis.

Expression of the Human Growth Hormone Genein Insect Cells

The insect cellvirus (AcNPV) system was used to express heterologous protein. The recombinant transfer vector pVL1393/hGH was reconstructed in which the human Growth Hormone (hGH) gene was inserted under the control of the polyhedron gene promoter. The Spodoptera frugiperda (Sf9) cells was cotransfected with the plasmid DNA containing hGH gene and wildtype AcNPV DNA. The hGH gene was transferred to the AcNPV genome DNA through homologous recombination, and the recombinant virus rAcVhGH was obtained by multiple plaque purification. The high level of production of hGH (40 μg/mL) in supernatant of the infected monolayer culture was determined by immunochemiluminescent assay.

Gene expression changes in dorsal root ganglia following peripheral nerve injury: roles in inflammation,cell death and nociception

Subsequent to a peripheral nerve injury, there are changes in gene expression within the dorsal root ganglia in response to the damage. This review selects factors which are well-known to be vital for inflammation, cell death and nociception, and highlights how alterations in their gene expression within the dorsal root ganglia can affect functional recovery. The majority of studies used polymerase chain reaction within animal models to analyse the dynamic changes following peripheral nerve injuries. This review aims to highlight the factors at the gene expression level that impede functional recovery and are hence are potential targets for therapeutic approaches. Where possible the experimental model, specific time-points and cellular location of expression levels are reported.

Senescence-like changes induced by expression of p21^(Waf1/Cipl)in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/Cip1 involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21Waf1/Cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1 is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/Cip1 expression. Induction of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21Waf1/Cip1-transduced NIH3T3 cells expressed β-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p21Waf1/Cip1 can also induce senescence-like changes in murine cells.

Differences in phenotype and gene expression of prostate stromal cells from patients of varying ages and their influence on tumour formation by prostate epithelial cells

Prostate cancer （PCa） is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone （PZ） of donors of varying ages and found that cultured stromal cells from old donors （PZ-old） were more enlarged and polygonal than those from young donors （PZ-young）. Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage（〉100） and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor （HGF）, fibroblast growth factor 5 （FGF5）, insulin-like growth factor 2 （IGF2）, insulin-like growth factor-binding protein 4 （IGFBP4）, IGFBP5 and matrix metallopeptidase 1 （MMP1）. Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction （PCR）, Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.

Spatial and temporal expression of germ cell nuclear factorin murine epididymis

Aim: To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period. Methods: The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope. Results: GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis. The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis. In adults, GCNF exhibited a region-specific expression pattern, i.e., it was expressed predominantly in the initial segment, caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis. GCNF could be found in the nuclei of the principal, apical, narrow, clear and halo cells. Conclusion: GCNF may play an important role in epididymal differentiation and development and in sperm maturation.

RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%~46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

Expression of midkine and its clinical significance in esophageal squamous cell carcinoma

AIM： To investigate the expression of midkine in eso- phageal squamous cell carcinoma （ESCC） and analyze its relationship with clinicopathological features. METHODS： RT-PCR and immunocytochemical staining were used to detect the expression of midkine mRNA and protein in EC109 cells, respectively. Then the expression of midkine in 66 cases of ESCC samples were detected by immunohistochemistry using monoclonal antibodies against human midkine. RESULTS： Midkine was expressed in EC109 cell by RT- PCR and imrnunocytochemistry. The immunoreactivity was detected in 56.1% （37/66） of the ESCC samples. The expression of midkine was found in cytoplasm of tumor cells. Notably, the intensity of midkine was stronger at the area abundant in vessels and the invading border of the tumors. Midkine was more intensely expressed in well differentiated tumors （76.9 %） than in moderately and poorly differentiated tumors （43.1% and 41.2%, respectively） （P〈0.05）. There was no statistically significant correlation between midkine expression and gender, age, clinical stage, lymph node metastasis or survival in ESCC. CONCLUSION： IVlidkine is overexpressed in ESCC. It may play a role in tumor angiogenesis and invasion. The expression of midkine is correlated with tumor cell differentiation in ESCC. The more poorly tumor cells differentiate, the weaker midkine expresses.

Transient Expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine （PEI） nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMl205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEl and DNA is in the range of 5：1-1：4. Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P〈5 and reached the highest at N/P=5, and began to decrease beyond N/P〉5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.

Soluble expression of recomb inant cMyc,Klf4,Oct4,and Sox2 proteins in bacteria and transduction into living cells

AIM：To develop a new method to produce recombinant reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,in soluble format with low cost for the generation of induced pluripotent stem cells（i PSCs）.METHODS：A short polypeptide sequence derived from the HIV trans-activator of transcription protein（TAT） and the nucleus localization signal（NLS） polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into p Cold-SUMO vector which can extremely improve the solubility of recombinant proteins.Then these vector plasmids were transformed into E.coli BL21（DE3） Chaperone competent cells for amplification.The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining.The recombinant proteins were purified by NiNTA resin and identified by Western blot.The transduction of these proteins into HEK 293 T cells were evaluated by immunofluorescence staining.RESULTS：These four reprogramming proteins could be produced in soluble format in p Cold-SUMO expression vector system with the assistance of chaperone proteins in bacteria.The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells.CONCLUSION：The results in the present study indicate the four important reprogramming proteins,c Myc,Klf4,Oct4,and Sox2,can be produced in soluble format in bacteria with low cost.Our new method thus might be expected to greatly contribute to the future study of i PSCs.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.