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Objective To investigate the associations between expression of Tensin1 protein and clinicpathological characteristics and prognoses of gastric cancer (GC) patients.Methods The retrospective casecontrol study was conducted.The clinicopathological data of 163 GC patients who were admitted to the Affiliated Hospital of Jiangsu University between July 31,2011 and December 31,2013 were collected.The GC tissues and adjacent normal tissues were taken to paraffin imbedding,and then were detected by immunohistochemistry.Observation indicator:(1) expressions of Tensinl protein in GC tissues and adjacent normal tissues;(2) association between expression of Tensinl protein in GC tissues and clinicopathological characteristics;(3) followup and survival situations;(4) prognostic factors analysis.Follow-up using telephone interview was performed to detect survival up to January 1,2017.Measurement data with skewed distribution were described as M (range).Count data were analyzed using the chi-square test or pairing chi-square test.The survival curve and rate were respectively drawn and calculated using the Kaplan-Meier method,and Log-rank test was used for survival analysis.The univariate analysis and multivariate analysis were done using the COX roportional hazard model.Results (1) Expressions of Tensinl protein in GC tissues and adjacent normal tissues:immunohistochemistry showed that Tensinl protein in GC tissues and adjacent normal tissues mainly expressed in cytoplasm.Of 163 patients,154 (66 with high expression and 88 with low expression) and 9 had respectively positive and negative expressions of Tensinl protein in GC tissues;79 (37 with high expression and 42 with low expression) and 84 had respectively positive and negative expressions of Tensinl protein in adjacent normal tissues,with statistically significant differences in positive expression ratio and expression levels (x2=64.65,12.93,P＜0.05).(2) Association between expression of Tensinl protein in GC tissues and clinicopathological characteristics:high expression rate of Tensinl protein in GC tissues were respectively 31.34％ (21/67) in GC patients with tumor metastases and 46.88％ (45/96) in GC patients without tumor metastasis,with a statistically significant difference (x2 =3.95,P＜0.05).(3) Follow-up and survival situations:all the 163 patients were followed up for 3.3-64.7 months,with a median time of 28.7 months.The 3-year cumulative disease-free survival rate and cumulative overall survival rate in GC tissues were 63.12％,74.22％ in 66 patients with high expression of Tensinl protein and 47.30％,55.74％ in 97 patients with low and negative expressions of Tensin1 protein,showing statistically significant differences in above indicators (x2 =4.58,4.11,P＜0.05).Survival analysis of subgroups showed that 3-year cumulative disease-free survival rate in GC tissues of patients with maximum tumor dimension ≥ 5 cm,nerve and / or vascular invasions and stage Ⅲ of TNM staging were 45.98％,62.79％,52.75％ in patients with high expression of Tensin1 protein and 18.11％,31.10％,32.80％ in patients with low and negative expressions of Tensin1 protein,with a statistically significant difference (x2 =5.85,7.89,4.96,P＜0.05).The 3-year cumulative overall survival rate was respectively 66.00％,75.75％,67.93％ in patients with high expression of Tensin1 protein and 30.74％,40.15％,44.67％ in patients with low and negative expressions of Tensinl protein,with statistically significant differences (x2 =7.59,9.62,4.32,P ＜ 0.05).(4) Prognostic factors analysis:results of univariate analysis showed that maximum tumor dimension,histological grade,nerve and / or vascular invasions,postoperative TNM staging,postoperative adjuvant chemotherapy and expression of Tensin1 protein were related factors affecting prognoses of GC patients [hazard ratio (HR) =3.66,2.45,2.17,3.36,0.41,0.54,95％ confidence interval (CI):2.09-6.41,1.43-4.19,1.17-4.04,1.52-7.41,0.23-0.72,0.31-0.96,P＜0.05].Results of multivariate analysis showed that maximum tumor dimension ≥ 5 cm and grade Ⅲ of histological grade were independent risk factors affecting prognoses of GC patients (HR=3.21,2.17,95％CI:1.63-6.32,1.18-3.99,P＜0.05),and postoperative adjuvant chemotherapy and high expression of Tensin1 protein were independent protective factors affecting prognoses of GC patients (HR=0.50,0.44,95％CI:0.28-0.90,0.24-0.82,P＜0.05).Conclusion High expression of Tensin1 protein may inhibit GC metastasis,and it is also an independent protective factor affecting prognoses of GC patients.
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Objective To evaluate the effect of try psin on the proliferation,migration and adhesion of a skin squamous cell carcinoma cell line A431.Methods Cultured A431 cells were divided into several experimental groups treated with trypsin at concentrations of 0.1,1,10 and 100 nmol/L for 24,48 and 72 hours respectively,and a control group treated with DMEM complete medium only.Cell counting kit-8 (CCK8) assay was conducted to evaluate cellular proliferative activity to select the optimal concentration of trypsin.Then,some A431 cells treated with trypsin at the selected concentration for 24,48 and 72 hours respectively (or 48 hours only) served as the experimental groups (or group),and other A431 cells treated with DMEM complete medium served as the control group.Flow cytometry was performed to assess cell cycle distribution and proliferation index,fibronectin-based adhesion assay to estimate cell adhesive capacity,and wound healing assay and Transwell assay were conducted to evaluate the migratory capacity of cells in two-and three-dimensional space.Statistical analysis was carried out by using analysis of variance,paired samples t test and chi-square test.Results The proliferative activity of A431 cells increased along with the increase of trypsin concentrations,with the strongest increasing effect observed at 100 nmol/L.After treatment with 100 nmol/L trypsin,the experimental group showed a decrease in the percentage of G1-phase cells,but an increase in the percentage of S-phase cells,proliferation index,migratory and adhesive capacity compared with the control group (all P ＜ 0.05).Conclusion Trypsin can promote the proliferation,migration and adhesion of A431 cells.
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Objective To observe the effect of short hairpin RNA (shRNA)-mediated vascular endothelial growth factor (VEGF) gene silencing on the growth of human skin squamous cell carcinoma(SCC) xenografts in nude mice.Methods Two eukaryotic expression plasmids targeting VEGF gene,including psilencer-VEGF1-shRNA (VEGF-s1) and psilencer-VEGF2-shRNA (VEGF-s2),as well as one negative control plasmid containing random target sequence (psilencer-Target-off-shRNA,T-off),were chemically synthesized,and transfected into a human skin SCC cell line A431 to develop stably transfected cell lines.Real time quantitative PCR (RT-qPCR) and double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) were carried out to measure the expression of VEGF mRNA and protein respectively in A431 cells.Twelve nude mice were divided into 4 goups to be subcutaneously inoculated in the axillary region with untransfected A431 cells as well as A431 cells transfected with VEGF-s1,VEGF-s2 and T-off,respectively.The tumor growth was observed in nude mice every 5 days.Twenty days after the inoculation,the mice were sacrificed,and transplanted tumors were obtained from the mice and subjected to an immunohistochemical study for the measurement of VEGF,proliferating cell nuclear antigen (PCNA) and CD34 expression.Data were statistically analyzed by using the Stata 7.0 software,and t test was conducted to compare the differences between groups.Results The mRNA and protein expression levels of VEGF were significantly lower in A431 cells transfected with VEGF-s1 and VEGF-s2 than in untransfected A431 cells (27.85 ± 3.95 and 24.69 ± 2.83 vs.54.06 ± 6.38,t =6.05,7.29,both P＜ 0.01; 32.67 ± 2.52 and 29.27 ± 1.10 vs.52.85 ± 2.23,t =8.04 and 11.53,both P＜0.01 ).Twenty davs after the inoculation,the volume and weight of xenografted tumors in mice inoculated with VEGF-sl- and VEGF-s2-transfected A431 cells were significantly lower than those in mice with untransfected A431 cells ( ( 192.50 ± 10.90) mm3 and (203.67 ± 3.21 )mm3 vs.(272.00 ± 21.07) mm3,t =5.80 and 5.55,both P＜ 0.01; (0.05 ± 0.03) g and (0.13 ± 0.04) g vs.(0.25 ± 0.02) g,t =9.60 and 4.64,both P＜ 0.01 ).Decreased expression rate of VEGF,PCNA and number of CD34-positive vessels were observed in the xenografted tumor tissue from mice inoculated with VEGF-sl- and VEGF-s2-transfected A431 cells compared with that from mice with untransfected A431 cells (52.00％ ± 2.00％ and 56.67％ ± 3.06％ vs.70.00％ ± 2.00％,both P ＜ 0.01;37.01％ ± 2.41％ and 33.94％ ± 3.25％ vs.72.11％ ± 3.02％,both P＜ 0.01; 2.05 ± 0.07 and 1.72 ± 0.10 vs.4.01± 1.27,both P ＜ 0.01).No significant differences were observed in the above parameters between cells transfected with VEGF-s1- and VEGF-s2-transfected A431 cells,between untransfected and T-off-transfected A431 cells,between tumor xenografts derived from VEGF-sl- and VEGF-s2-transfected A431 cells,or between tumor xenografts derived from untransfected and T-off-transfected A431 cells (all P ＞ 0.05).Conclusions The shRNA targeting VEGF gene can significantly inhibit the expression of VEGF in A431 cells and A431-derived tumor xenografts in nude mice,in turn suppress the growth and attenuate the malignant phenotype of tumor.
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Objective To evaluate the effect of low-molecular weight heparin (LMWH) on biological behavior of a human cutaneous squamous cell carcinoma cell line A431.Methods To optimize the concentration of LMWH,A431 cells were treated with different concentrations (12.5,25,50,100 and 200 IU/ml) of LMWH for 24,48 and 72 hours followed by CCK-8 assay for the detection of cell viability.Then,A431 cells were cultured with or without the presence of LMWH at 200 IU/ml for 24,48 and 72 hours.Subsequently,flow cytometry was performed to assess cell cycle,real time quantitative PCR (RT-qPCR) and Western blot to quantify the expression of vascular endothelial growth factor (VEGF) mRNA and protein respectively,double-antibody sandwich enzyme linked immunosorbent assay (ELISA) to determine the expression level of VEGF protein in the supernatant of A431 cells,wound-healing assay,Transwell assay,and adhesion assay to observe the migration and adhesivity of A431 cells.Analysis of variance and t test were carried out for statistical analysis.Results The optimal concentration of LMWH was determined as 200 IU/ml according to the CCK-8 assay,and used in the following experiment.The LMWH of 200 IU/ml resulted in a decrease in cell viability,cell cycle arrest,an increase in cell percentage in G1 phase,and a reduction in cell percentage in S phase.The proliferation index was 23.41 ± 5.51 and 11.76 ± 5.13 respectively in A431 cells at 48 and 72 hours after treatment with LMWH of 200 IU/ml,significantly lower than that in untreated A431 cells (48.62 ± 4.50,t =6.14,P ＜ 0.05; 46.86 ± 3.51,t =9.78,P ＜ 0.05).A significant decrease was observed in LMWH-treated A431 cells at 48 and 72 hours compared with the untreated A431 cells in the expression level of VEGF mRNA (10.16 ±0.07 vs.18.77 ± 0.11,4.11 ± 0.01 vs.17.39 ±0.05,t=114.38,451.10,both P＜ 0.05),VEGF protein (0.16 ± 0.01 vs.0.20 ± 0.01,0.12 ± 0.01 vs.0.21 ± 0.01,t =4.90,11.02,both P ＜ 0.05),and in the supernatant level of VEGF protein ((67.17 ± 3.34) ng/L vs. ( 122.63 ± 23.17) ng/L, (28.14 ± 3.14) ng/L vs.(86.76 ± 1.18) ng/L,t =4.10,30.27,both P＜ 0.05).The percentage of adherent cells was 29.7％ ± 1.92％ and 17.5％± 0.79％ in LMWH-treated A431 cells at 48 and 72 hours,respectively,significantly lower than that in untreated A431 cells (36.9％ ± 0.35％,34.6％ ± 0.96％,respectively,both P＜ 0.05).The migration of A431 cells was also obviously inhibited by the treatment with LMWH for 24,48 and 72 hours.Conclusion LMWH may suppress the proliferation,migration and adhesion of A431 cells via downregulating cellular viability and VEGF expression.