ABSTRACT
Prostaglandins are a class of poly-unsaturated fatty acids-derived bioactive lipids with important physiological function by binding to specific receptors. Prostaglandin receptors lack specific antibodies, which greatly impedes the research on our understanding of the signaling of prostaglandins. The aim of this study was to identify nine mouse lines with amino terminal (-NH2, -N) HA-tagged prostaglandin receptors by using the combination of artificial sperm and CRISPR-Cas9 technology. The guide RNA expression plasmid and labeled targeting vector plasmids were transferred into "artificial sperm cells". The "artificial sperm cells" containing labeled proteins were selected and injected into mouse oocytes, and implanted into pseudopregnant mice to obtain labeled mice. The genomic DNA of the prostaglandin receptor tagged mice was extracted, and the genotypes of mice were detected by PCR method. We also isolated mouse peritoneal macrophages to verify the protein expression of HA-labeled prostaglandin receptor by Western blot. Specific DNA bands were amplified in prostaglandin receptor labeled mice, and specific HA protein bands were detected in macrophage proteins, which was not detected in wild type mice. In summary, we successfully constructed 9 mouse lines with HA-tagged prostaglandin receptors, providing a powerful tool for further study of the pathophysiological functions of prostaglandin signaling both in vivo and in vitro.
Subject(s)
Animals , Mice , Oocytes , Plasmids , Receptors, ProstaglandinABSTRACT
Objective To investigate the influence of proline-rich tyrosine kinase 2 (Pyk2)gene RNA interference on proliferation,invasion and migration of Hep3B hepatocellular carcinoma cells.Methods The Pyk2 gene RNA interference vector was transfected in Hep3B hepatocellular carcinoma cells by lipofectamine. The Hep3B cells divided into three groups:siRNA group (the vector with Pyk2 RNAi gene was transfected), negative control group (the vector without Pyk2 RNAi gene was transfected),and blank control group (no vectors was transfected).Pyk2 mRNA and protein were detected using reverse transcription reverse transcription-poly-merase chain reaction (RT-PCR)and Western blotting.The biological behavior including cell proliferation,inva-sion and migration were detected by 3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl-2H-tetrazolium bromide (MTT), transwell and wound healing assay,respectively.Results The expression of Pyk2 mRNA of Hep3B cell line in siRNA group (0.1 6 ±0.03)was significantly decreased than those in negative group (0.74 ±0.1 3)and blank control group (0.77 ±0.1 6),with statistically significant differences (t=51 .46,P=0.000;t=53.21 ,P=0.000).The expression of Pyk2 protein of Hep3B cell line in siRNA group (0.24 ±0.06)was significantly decreased than those in negative group (0.83 ±0.05)and blank control group (0.91 ±0.06),with statisti-cally significant differences (t=57.29,P=0.000;t=68.53,P=0.000).The cell proliferation inhibition rate at 48 hours in siRNA group (26.1 7%±0.28%)was significantly raised than those in negative group (9.28%± 0.22%)and blank control group (6.47%±0.31%),with statistically significant differences (t=31 .45,P=0.004;t=34.64,P=0.002).The number of transmembrane cells in siRNA group (32.5 ±8.5)/1 0 HP was significantly declined than those in negative group (98.4 ±1 2.3 )/1 0 HP and blank control group (1 1 2.6 ± 1 1 .3)/1 0 HP,with statistically significant differences (t=95.64,P=0.000;t=1 05.1 7,P=0.000).The wound healing assay in siRNA group (28.1 7%±1 .46%)was significantly lower than those in negative group (77.38%±2.24%)and blank control group (79.41%±3.1 7%),with statistically significant (t=85.86,P=0.000;t=89.37,P=0.000).Conclusion Pyk2 gene involves the proliferation,invasion and migration of Hep3B cells,which has close correction with development and metastasis of hepatocellular carcinoma.Pyk2 gene is very helpful to become a molecular target for the diagnosis and treatment of hepatocellular carcinoma.
ABSTRACT
Objective To evaluate the efficacy of extended-field intensity-modulated radiotherapy (IMRT) in the treatment of patients with early-stage NK/T cell lymphoma (NKTCL),and to examine the clinical characteristics and the effect of treatment factors on the prognosis of these patients.Methods The clinical data of 165 patients with early-stage NKTCL who underwent extended-field IMRT with (n=158,95.8%) or without chemotherapy (n=7,4.2%) were reviewed.Of these 165 patients,140(84.8%) received a radiation dose of ≥50 Gy to the primary lesion,and 25 patients (15.2%) received a radiation dose of<50 Gy.Most patients (n=147,89.1%) were treated with L-asparaginase-based chemotherapy regimens,whereas only 11 patients (6.7%) were treated with doxorubicin-based CHOP/CHOP-like regimens.In addition,109 patients (66.1%) received ≥4 cycles of chemotherapy.Locoregional control (LRC),overall survival (OS),and progression-free survival (PFS) rates were calculated using the Kaplan-Meier method,and the log-rank test was used for survival comparison and univariate prognostic analysis.A multivariate prognostic analysis was performed using the Cox model.Results The 5-year sample size 55.The 5-year OS,PFS,and LRC rates of all patients were 74.2%,72.5%,84.4%,respectively.The patients who received a dose of ≥50 Gy had a significantly higher 5-year LRC rate than those with<50 Gy (91.8% vs.39.7%,P=0.000).The 5-year OS was significantly higher in the low-risk early-stage group than in the high-risk early-stage group (P=0.002).For the high-risk early-stage NKTCL group,patients who received ≥4 cycles of chemotherapy had significantly higher 5-year OS and PFS than those who received<4 cycles of chemotherapy (5-year OS:71.3% vs.59.5%,P=0.032;5-year PFS:70.4% vs.54.4%,P=0.009).In addition,multivariate analysis showed that ECOG≥2,primary tumor invasion (PTI),and Ann Arbor stage Ⅱ were associated with poor OS (P=0.006,0.002,0.014),and ECOG≥2 and PTI were associated with reduced LRC (P=0.004,0.016).Furthermore,ECOG≥2,PTI,Ann Arbor stage Ⅱ,and extranasal primary site were associated with lower PFS (P=0.045,0.003,0.030,0.032).Conclusions Extended-field IMRT at a dose of ≥50 Gy can lead to favorable LRC,OS,and PFS in patients with early-stage NKTCL.However,it is less effective against distant early-stage NKTCL in patients with poor prognosis.Nevertheless,≥4 cycles of chemotherapy can significantly improve the OS and PFS of patients with early-stage NKTCL.
ABSTRACT
Objective To investigate the expressions of phosphatidylinositol 3-kinase (PI3K), Akt and E-cadherin and their clinical significance in thyroid papillary carcinomas.Methods Expressions of PI3K, Akt, and E-cadherin were detected in 62 cases of thyroid papillary carcinomas,30 cases of thyroid goiter and 30 cases of normal thyroid by immunohistochemistry (EnVison),and simultaneously compared with age, sex, tumor size, clinical tumor node metastasis( TNM) stages, and lymph node metastasis in thy-roid papillary carcinomas.Results The expression rate of PI3K, Akt, and E-cadherin was 74.2%(46/62), 66.1%(41/62), 16.1%(10/62),respectively.Expressions of three proteins in thyroid papillary carcinomas were significantly different from those in thyroid goiter and normal thyroid tissues ( P <0.05). The lower positive rates of PI3K and Akt proteins were obtained in the group of stageⅠ~Ⅱthan that in the group of stageⅢ~Ⅳ(χ2 =4.976, P =0.026;χ2 =6.233, P =0.013).Higher positive rates of PI3K and Akt proteins were obtained in the group of lymph-node metastasis than that in group of non-lymph-node metastasis (χ2 =6.675, P =0.010;χ2 =7.511, P =0.006).Higher positive rate of E-cadherin protein was obtained in the group of stage Ⅰ~Ⅱ than that in the group of stage Ⅲ ~Ⅳ (χ2 =6.558, P =0.010 ) .Higher positive rate of E-cadherin proteins was obtained in the group of non-lymph-node metastasis than that in the group of lymph node metastasis(χ2 =5.678, P =0.017).There was significant positive correlation between expressions of PI3K and Akt through Spearman correlation analysis ( r =0.423, P <0.05).PI3K was negatively correlated with E-cadherin with Spearman correlation analysis ( r =-0.527, P <0.05).Akt was also negatively correlated with E-cadherin ( r =-0.417, P <0.05).Conclusions PI3K/Akt pathway might regulate thyroid papillary carcinoma cells proliferation, invasion and metastasis.
ABSTRACT
Objective To investigate the expressions of cancerous inhibitor of protein phosphatase 2A (CIP2A) and vascular endothelial growth factor (VEGF) in hepatocellular carcinomas and their clinical significance.Methods The expressions of CIP2A and VEGF proteins were tested with immunohistochemistry in 60 cases of hepatocellular carcinomas and adjacent paracancerous tissues.Results The positive rate of CIP2A in hepatocellular carcinomas was significantly higher than adjacent paracancerous tissues (83.3％ vs 9.5％,P ＜ 0.05).Significant differences were also observed in the expression rate of VEGF between the patients with hepatocellular carcinomas and paracancerous tissues (75.0％ vs 14.3％,P ＜0.05).The expression of CIP2A in hepatocellular carcinomas was significantly positively correlated with its pathological grading,differentiation,and tumor node metastasis (TNM) stage (P ＜ 0.05).The expression of VEGF in hepatocellular carcinomas was significantly positively correlated with its pathological grading,differentiation,and TNM stage (P ＜ 0.05).Significantly positive correlation was found between expressions of CIP2A and VEGF with Spearman correlation analysis (rs =0.465,P ＜ 0.01).Conclusions The abnormal expressions of CIP2A and VEGF gene may promote tumor angiogenesis and progression of a hepatocellular carcinoma.The study supports positive regulation between expressions of CIP2A and VEGF in a hepatocellular carcinoma.
ABSTRACT
Objective To study the expression of Survivin and COX-2 in ampullary carcinoma and their clinical significance.MethodsThe expression of Survivin and COX-2 proteins were tested by EnVision immunohistochemistry in 40 cases of ampullary carcinomas,and 8 cases of normal ampulla of vater as the controls.ResultsThe positive rate of Survivin in ampullary carcinonas was significantly higher than that of the controls(82.5％ vs 0,P ＜ 0.01 ). The expression of Survivin in ampullary carcinoma was correlated with duodenal invasion,pancreatic invasion and lymph node metastasis ( P ＜ 0.05 ).Significant difference was also observed in the expression rate of COX-2 between the patients with ampullary carcinoma and the normal controls (67.5％ vs 0,P ＜ 0.01 ).The expression of COX-2 in ampullary carcinoma was correlated with duodenal invasion,pancreatic invasion and lymph node metastasis (P ＜ 0.05). Significantly positive correlation was found between the expression of Survivin and COX-2 by using spearman correlation analysis ( r =0.383,P =0.015).ConclusionThe specific up-regulation of COX-2 gene and Survivin gene may play an important role in the genesis and development of ampullary carcinoma.COX-2 and Survivin may be used as early diagnosis markers and potential therapeutic targets in ampullary carcinoma.