Effect of small interfering RNA targeting survivin gene on biological behaviour of bladder cancer

Background Bladder cancer is the most common type of urinary system tumours. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin, a newly discovered member inhibitor of apoptosis protein （IAP） family in several human cancers, by inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. We studied the influence of small interfering RNA （siRNA） targeting survivin on the biological behaviour of bladder cancer cells. Methods A double strand survivin target sequence specific siRNA was designed and synthesized. After transfection of bladder cancer cell line T24 by siRNA/liposome complex with increasing concentrations （50-200 nmol/L）, the transfectant cells were intratumourally injected at different doses （5 μg or 50 μg）. The effects were measured in vitro and in vivo. Results The selected siRNA efficiently down-regulated survivin mRNA expression in a dose and time dependent manner. The maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%. The inhibition rate of cell growth was 55.29% （P〈0.01） and the markedly increased apoptotic rate was 45.70% （P〈0.01）. In vivo intratumoural injection of 50 μg siRNA-survivin could notably orevent the growth of bladder cancer （P〈0.01） in xenografted animals. Conclusion The application of siRNA-survivin could markedly inhibit survivin expression in bladder cancer cell line by inducing apoptosis and inhibiting the growth of the tumour. It may become a new gene therapy tool for bladder cancer.     

Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.     

Heparanase antisense oligodeoxynucleotide inhibits angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice

Objective： To evaluate the inhibitory effect of heparanase antisense oligodeoxynucleotide （AS-ODN） on the angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice. Methods： The AS-ODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxynucleotide （NS-ODN） were designed and synthesized. A subcutaneous growth model and an acute hematogenous metastasis model of human mammary carcinoma were established in nude mice and were treated with ODNs. The heparanase expression in tumor was evaluated by RT-PCR and Western blot. The microvessel density （MVD） was measured by immunohistochemistry for factor VS. The tumor volume was calculated and lung metastatic nodules were counted. Results ： The heparanase expression, MVD, tumor volume and lung metastatic nodules in AS-ODN treated group were significantly decreased compared with that in NS-ODN treated group and that in PBS group （P〈0.01）. Conclusion ： Heparanase AS-ODN has significant inhibitory effect on the angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice.     

Construction of Smac gene-carrying and human uroplakin Ib promoter-Regulated Genetic Vector and its Expression

Objective： To construct an eukaryotic expression vector that contains Smac gene, which is regulated by human Uroplakin Ib （UpIb） promoter. Methods： For the directionality of Smac expression in the transitional cell carcinoma of bladder, internal CMV and T7 promoter sequences in eukaryotic expression vector pcDNA3.1-Smac were replaced with UpIb promoter to construct a new plasmid. The plasmid DNA was identified by gel electrophoresis after being double digested at respective sites, and then the sequence was analyzed. The expression of Smac mRNA and protein in BIU87 cell line were detected after the transfection by using the newly constructed vector. Results： The Smac gene-carrying and UpIb promoter-regulated eukaryotic expression vector pcDNA3-UpIb-promoter-Smac was successfully constructed. The expression of Smac mRNA was approximately increased by 2.1 times and the expression of Smac protein was increased in about 71% BIU87 cells. Conclusion： The new vector can be effectively expressed in bladder cancer cells and be of great significance for bladder cancer-targeted gene therapy.     

Comparison of seven screening methods in the diagnosis of bladder cancer

Background We compared the validity （evaluated by sensitivity and specificity）, reliability （evaluated by reproducibility） and yield （evaluated by predictive value, examining complexity and cost） of individual and combined tests for bladder tumour antigen stat （BTAstat）, nuclear matrix protein 22 （NMP22）, hyaluronic acid （HA）, survivin, CD44v6, vascular endothelial growth factor （VEGF）, and voided urine cytology （VUC） in detecting bladder cancer. And at the same time we evaluated the clinical value of these seven detecting methods in the diagnosis of bladder cancer. Methods The six markers and VUC were detected in the urine of cancer group （151 patients with bladder cancer） and two control groups （50 patients with benign urological diseases and 50 healthy controls）. The sensitivity, specificity, predictive value, reproducibility, examining complexity and checking cost of each marker and combined markers were calculated. Results There was a significant difference between bladder cancer group and the two control groups. The sensitivity, specificity and positive predictive value were as follows： VUC （36.4%, 100.0%, 100%）, BTAstat （76.8%, 87.0%, 89.9%）, NMP22 （77.5%, 81.0%, 86.0%）, HA （82.8%, 83.0%, 88.0%）, survivin （70.2%, 85.0%, 87.6%）, CD44v6 （50.3%, 79.0%, 78.4%）, and VEGF （68.2%, 93.0%, 93.6%）. The highest sensitivities were 91.4% for NMP22＋BTAstat and HA＋NMP22, whereas the combined marker with the lowest sensitivity （62.3%） was VUC＋CD44v6. The highest specificity was 93.0% for the combined use of VUC＋VEGF and HA＋CD44v6 had the lowest specificity （73.0%）. The most convenient examining method was the detection for BTAstat, the lowest cost was the detection for HA, and the best reproducibility were the detection for BTAstat and VUC. Conclusions All the markers have obvious clinical value in diagnosis of bladder cancer. The use of BTAstat＋HA or NMP22＋BTAstat are better examining methods in terms of validity, reliability, and yield.     

Expression of X-linked Inhibitor of Apoptosis Protein and Its Effect on Chemotherapeutic Sensitivity of Bladder Carcinoma

The expression of X-linked inhibitor of apoptosis protein (XIAP) gene and its effect on chemotherapeutic sensitivity in bladder carcinoma was explored. By using immunohistochemistry, the expression of XIAP was detected in 47 bladder carcinomas and 5 normal bladder tissues. The XIAP gene was transfected into bladder cancer cell line T24 by liposome and the positive clone was screened by G418. Cellular XIAP mRNA level was detected by RT-PCR. Low-dose mitocycin C was administered to induce the apoptosis of T24 cells. The in vitro growth of bladder carcinoma cells was analyzed by MTT colorimetry, and the apoptosis rate was assayed by TUNEL methods. It was found XIAP was moderately expressed in bladder carcinomas with the the positive rate being 78.73% (37/47), but the positive rate was not correlated with carcinoma stages and grades (P<0.05). XIAP mRNA level in transfected T24 cells was significantly increased by 3.8 times as compared with that in the cells not transfected with XIAP. After treatment with low-dose mitomycin C (0.005 and 0.05 mg/mL), the growth rate in XIAP no-transfected control group was increased by (11.60±0.25)% and (16.51±0.87)% (P<0.05), and the apoptosis rate was decreased by (10.1±0.2)% and (11.9±0.2%) (P<0.05) respectively as compared with XIAP transfected group. It was concluded that XIAP was expressed in most of bladder carcimoma samples. Overexpression of XIAP in T24 could significantly reduce the MMC-induced apoptosis of bladder carcinoma, suggesting its effect on the chemothera- peutic sensitivity of T24 cells.     

Establishment of an Organic Model of SMC Proliferation with Cultured Aorta of Rats

Objective To study the mechanism of proliferous vascular disease as well as its prevention and treatment, an organic model was established with rat aorta. Methods The aorta segments of rats were cultured in vitro for 5, 8 and 13 days respectively. The proliferation of smooth muscle celI（SMC） was observed by HE staining and bromodeoxyuridine（Brdu） DNA labeling, and the expressions of hypertension-related gene- 1 （HRG- 1 ） and smooth muscle 22 alpha（SM22ct） mRNA was detected by RT-PCR. Results After being cultured 5 days in vitro, various degrees of proliferation of SMC on cultured artery segments was observed by HE staining and the conspicuous plaques were developed after being cultured 13 days. The nuclei of proliferous cells labeled by Brdu were seen under microscope. The expressions of HRG-1 and SM22amRNA decreased with prolonging of culture time and it completely disappeared after being cultured 13 days. Conclusion The results demonstrate that the culturing of rat aorta segments in vitro can induce the proliferation of SMC and the transformation of phenotype from contractile to synthesize type. This may be a good organic model that supplies a good experimental platform for us to research the mechanism of proliferous vascular disease as well as its prevention and treatment.     

Inhibition of PCNA Antisense Oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells

Pro1iferative vitreoretinopathy (PVR), as a blind- ness-causing condition, is a common complication of retina detachment and the main reason that leads to the failure of recovery operation of retina detachment. PVR is essentially an intraocular reactive course of wound healing and principally relevant to pathological prolifera- tion of retina pigment epithelia (RPE). Proliferating cell nuclear antigen (PCNA), expressed in many proliferative tissues, play an important role in the cell cycle…     

Association of polymorphisms in angiotensin II receptor genes with aldosterone-producing adenoma

This study examined the association of polymorphisms in angiotensinⅡreceptor genes(AT1R and AT2R) with the risk for aldosterone-producing adenoma(APA) in a Chinese Han population.Four polymorphisms including rs5182(573T/C) in exon 4,rs5186(1166A/C) in 3’-untranslated region(3’-UTR) in AT1R gene and rs5194(2274G/A) in 3’-UTR,rs1403543(1675G/A) in intron 1 in AT2R gene were detected in 148 APA patients and 192 normal subjects(serving as control) by using a MGB-Taqman probe.The distribution of genotypes of each locus was in accordance with Hardy-Weinberg Equilibrium(HWE) in the APA and control groups(P>0.05).The allele A frequency at rs5194 was significantly higher in the APA group(0.49) than in the control group(0.35)(χ2=12.08,P=0.001).Subjects with homozygotic genotype AA and heterozygotic genotype GA were at an increased risk for APA as compared to those with GG genotype(OR=2.66,95% CI=1.45-4.87;OR=1.67,95% CI=1.02-2.74).Furthermore,rs5194 single-nucleotide polymorphism(SNP) at AT2R gene was significantly associated with APA in additive(OR=1.64,95% CI=1.21-2.20,P=0.001),dominant(OR=1.94,95% CI=1.23-3.06,P=0.003),and recessive model(OR=2.01,95% CI=1.17-3.45,P=0.01).It was concluded that rs5194 polymorphism at AT2R gene was associated with the risk for APA,which may constitute a genetic marker of APA.     

Expression and significance of SHP-2 in human papillomavirus infected cervical cancer

This study investigated the expression and prognostic value of SHP-2 in cervical cancer caused by human papillomavirus (HPV) infection. Forty-five specimens from patients with cervical can-cer (stageⅠ-Ⅲ), 32 specimens from patients with cervical intraepithelial neoplasia (CIN) (Ⅰ, Ⅱ) and 20 normal cervical samples from patients with hysteromyoma were collected in Department of Pathol-ogy for comparison. The expression levels of SHP-2 and IFN-β proteins were detected by using immu-nohistochemistry. The mRNA expression level of SHP-2 was detected by using quantitative real-time polymerase chain reaction (PCR). HPVs were detected by HPV GenoArray Test. The Spearman corre-lation was used to compare the expression level of SHP-2 in HPV infected cervical cancer vs non-HPV infected normal cervix. The level of SHP-2 protein expression in the cancer tissues (88.8%) was sig-nificantly higher than in CIN tissues (62.5%) and normal cervixes (45%) (P<0.05 and P<0.05, respec-tively). The SHP-2 mRNA levels in the cancer tissues were upregulated as compared with those in the normal cervixes (P<0.05). Twenty-one (46.7%) cervical cancers, 25 (78.1%) CINs and 17 (85%) normal cervixes showed IFN-β positive staining in cytoplasm. There was statistically significant difference in the expression rate of IFN-β between cervical cancer and normal cervix (χ2=8.378, P<0.05) as well as between cervical cancer and CIN (χ2=7.695, P<0.05). HPV16/18 infections could be found in normal cervixs (15%), CINs (68.7%) and cervical cancers (84.4%). There was a correlation between HPV in-fection and SHP-2 expression in cervical cancer (rs=0.653, P<0.05). SHP-2 may be a useful prognostic and diagnostic indicator for HPV infected cervical cancer. In cervical cancers, SHP-2 mRNA and pro-tein overexpression was associated with IFN-β lower-expression.     

Clinial Implication of tThe Expression of Aurora B in Normal Endometrium and Endometrial Carcinoma

The expression of Aurora B in normal endometria and endometrial carcinomas and its relation with clinicopathologic parameters of endometrial carcinomas were investigated.Streptavidin-biotin peroxidase(SP) immunohistochemical technique was used to detect the expression of Aurora B in 10 cases of normal proliferative phase endometria,10 cases of normal secretory phase endometria and 72 cases of endometrial carcinomas respectively.According to the 1988 International Federation of Gynecology and Obstetrics(FIGO) grade,there were 37 patients in grade 1,23 in grade 2 and 12 in grade 3 respectively.According to the FIGO stage,there were 59 patients in stage Ⅰ-Ⅱ and 13 patients in stage Ⅲ-Ⅳ.Aurora B was expressed in both normal proliferative phase endometria,secretory phase endometria and endometrial carcinomas,but its positive labeling index(PLI) in proliferative phase endometria was significantly higher than that in secretory phase endo-metria(P<0.01) and endometrial carcinomas(P<0.01).The PLI of Aurora B was lower in tumors with well differentiation(G1),low surgical staging(Ⅰ-Ⅱ),and ≤1/2 myometrial invasion than that in tumors with moderate and low differentiation(G2-G3),higher surgical staging(Ⅲ-Ⅳ),and >1/2 myometrial invasion(all P<0.01).Aurora B exerts its functions in the replication of normal endometrial glandular cells;Expression of Aurora B is significantly correlated with biologic behavior of endo-metrial carcinoma,indicating that Aurora B may be a promising prognostic factor in endometrial carcinoma.     

HDAC1 expression and effect of curcumin on proliferation of Raji cells

Summary Histone deacetylase (HDAC₁) has a high expression in many cancer cells and curcumin can inhibit the growth of cancer cells. This paper was designed to investigate the expression of HDAC₁ of Raji cells and the effect of curcumin on their proliferation and apoptosis. Raji cells were treated with 3. 125–50 μmol/L curcumin for 8–48 h and the growth inhibition rates of Raji cells were measured by MTT. The expression of HDAC₁ on Raji cells were examined by mRNA, Western blot at 24 h various concertrations (1.6–50 μmol/L). Curcumin could selectively inhibit the proliferation of Raji cells in a dose and time dependent manner, with the inhibition rate being 52.47%–82.18% (P<0.01). The up-regulation of HDAC₁ expression was observed within 24 h after the treatment with curcumin as shown by RT-PCR and Western blot. With the increase of concentration, the expression was down-regulated in a dose dependent manner. It is concluded that the expression of HDAC₁ plays an important role in the proliferation and apoptosis of Raji cells and curcumin can inhibit the growth of Raji cells at various concentrations and promote the apoptosis of Raji cells. WU Qing, female, born in 1970, M. D., Ph. D. This project was supported by a grant from the National Natural Sciences Foundation of China (No. 30271672).     

Apoptosis of human trabecular meshwork cells induced by transforming growth factor-β<Subscript>2</Subscript><Emphasis Type="Italic">in vitro</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Summary Whether transforming growth factor-β₂ (TGF-β₂) induces apoptosis of human trabecular meshwork cells was investigatedin vitro. Cultured 3–5 passage human trabecular meshwork cells were treated with 0 (control). 0. 32. 1, 3. 2 ng/ml TGF-β₂ for 48 h and divided into control group and experimental group. The apoptosis of human trabecular meshwork cells was examined by transmisson electron microscopy. TUNEL technique and flow cytometry. The results showed character istic morphologic changes of apoptotic cells were observed under transmission electron microscopy. DNA fragmentation of human trabecular meshowork cells was found by TUNEL technique. Quantitative analysis of flow cytometry showed that percentages of apoptotic human trabecular meshwork cells were (2.79±0.44)%. (4.43±1.17)% and (9.60±2.05)% respectively with different concentrations [1 ng/ml (P<0.05), 3.2 ng/ml (P<0.01)] of TGF-β₂ with the difference being significant between experimental group and control group [(1.41±0.34)%]. It was concluded that TGF-β₂ can induce apoptosis of human trabecular meshwork, cellsin vitro and may be involved in the decrease of trabecular meshwork cells in the patients with primary open angle glaucoma and aging of normal people. CAO Yang, male, born in 1972, M. D., Ph. D., Associate Professor This project was supported by a grant from the National Natural Sciences Foundation of China (No. 38970758).     

Effect of modified Wuzi Yanzong Granule (加味五子衍宗颗粒) on patients with mild cognitive impairment from oxidative damage aspect

Objective:To observe the effects of modified Wuzi Yanzong Granule(加味五子衍宗颗粒,WYG)on memory function and the activity of serum superoxide dismutase(SOD), malondialdehyde(MDA)levels,leukocyte mitochondrial DNA(mtDNA)deletion rate andβ-amyloid protein_(1-28)(Aβ_(1-28))in patients with mild cognitive impairment(MCI).Methods:Thirty-six patients with MCI were selected based on the internationally recognized Petersen's criteria,and equally and randomly assigned to two groups.The treated group was treated with WYG and the control group was treated with placebo for 3 months.In addition,20 healthy subjects were included in the study as the normal control group.Changes of memory function,SOD activity,MDA content,leukocyte mtDNA deletion rate and Aβ_(1-28)content were observed before and after treatment.Results: Compared with the normal control group,the memory quotient and SOD activity in patients with MCI decreased significantly(P<0.01),while MDA,Aβ_(1-28)levels and the leukocyte mtDNA deletion rate increased significantly(P<0.01).After treatment,levels of memory quotient and serum SOD activity increased while the serum MDA level,leukocyte mtDNA deletion rate and Aβ_(1-28)level decreased in the treated group compared with those before treatment(P<0.01,P<0.05).Meanwhile,leukocyte mtDNA deletion rate and Aβ_(1-28)content in the treated group were all lower than those in the control group(P<0.05).Conclusion:WYG could improve memory function in patients with MCI and the therapeutic mechanism is possibly related to the increased activity of anti-oxidase,the improved free radical metabolism and the alleviation of leukocyte mtDNA oxidation damage.WYG shows clinical significance in delaying the progression of MCI.     

Inhibitory effect of PPARγ agonist on the proliferation of human pterygium fibroblasts

The effects of DK2,a peroxisome proliferator-activated receptor γ agonist,on cultured human pterygium fibroblasts (HPFs) in virto were studied.The HPFs were incubated with 0-200 μmol/L DK2 for 12-72 h.The MTT method was used to assay the bio-activity of DK2 at different doses and time.The cytotoxic effect of DK2 was measured by LDH release assay.The cell cycle distribution and apoptosis were flow cytometrically detected.The expression of proliferating cell nuclear antigen (PCNA) in each group was detected by real-time PCR (RT-PCR) and Western blotting.The results showed that administration of 1-75 μmol/L DK2 for 12-72 h could significantly inhibit HPF proliferation in a dose-and time-dependent manner.DK2-treated cells did not release significant amount of LDH as compared with rosiglitazone-treated cells.After treatment with DK2 at concentrations of 15,25 μmol/L for 24 h,the number of HPFs in G 0 /G 1 phase was significantly increased while that in S phase was significantly decreased (P<0.05),leading to arrest at G 0 /G 1 phase.The apoptosis rates of HPF cells in drug-treated groups were significantly higher than the rate of control group (P<0.05).At the dosage range between 15-25 μmol/L,DK2 could inhibit the expression of PCNA mRNA and protein in HPFs in a dose-dependent fashion (P<0.05).It was concluded that PPARγ agonist can significantly inhibit HPF proliferation,resulting in the arrest at G 0 /G 1 phase,induce the apoptosis of HPFs,and suppress the synthesis of PCNA,in dose-and time-dependent manners.     

剖宫产术中娩出胎儿前膀胱下推法在凶险性前置胎盘手术中的应用效果

目的评价剖宫产手术时娩出胎儿前膀胱下推法在凶险性前置胎盘手术中的应用效果。方法选择2016年1月至2017年12月安徽医科大学第一附属医院产科收治凶险性前置胎盘孕产妇89例,根据剖宫产时下推膀胱的时机不同,分为A组(47例)与B组(42例),A组为娩出胎儿前膀胱下推组,B组为娩出胎儿后下推膀胱组。比较两组孕产妇一般情况(年龄、孕次、产次、孕周、胎盘植入率)、平均手术出血量、平均输血量、手术时间、术后住院时间、产褥病率、子宫切除、膀胱损伤和新生儿窒息方面的差异。结果 A、B组孕产妇术中出血量分别为(1 927. 76±356. 31) mL、(2 859. 55±477. 80) mL,输血量分别为(861. 72±91. 58) mL、(1 285. 73±162. 69) mL,手术时间分别为(1. 83±0. 87) h、(2. 71±0. 94) h,术后住院时间分别为(5. 37±1. 72) d、(6. 83±1. 56) d,产褥病率分别为10. 64%、28. 57%,子宫切除率分别为14. 89%、35. 71%,新生儿窒息发生率分别为4. 26%、19. 05%,两组差异有统计学意义(P <0. 05)。结论凶险性前置胎盘娩出胎儿前膀胱下推法疗效满意,值得临床推广应用。