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[Abstract ] Objective The role of serum Pygo2 expression in children with non-alcoholic fatty liver disease(NAFLD) in the disease process and whether it can be used to evaluate the degree of liver fibrosis still remain unknown .The article aimed to detect Pygo 2 expression in the peripheral blood of children with NAFLD and ana-lyze its relationship with traditional serum hepatic fibrosis index in or-der to evaluate the clinical significance of Pygo 2 measurement in chil-dren with NAFLD . Methods We enrolled 120 cases of childhood obesity and 18 healthy controls in Anhui Provincial Hospital and the First Affiliated Hospital of Nanjing Medical University from September 2014 to February 2016.The cases of childhood obesity were di-vided into simple obesity group ( n=44, no diffuse fatty liver under liver ultrasound detection ) , NAFL group ( n=35, diffuse fatty liver with normal liver function under liver ultrasound detection ) and NASH group ( n=41, diffuse fatty liver with abnormal liver function under liver ultrasound detection ) .The peripheral serum was collected from all patients and healthy controls .ELISA was used to detect serum Pygo2 expression and radioimmunoassay was used to detect the serum hyaluronia acid (HA), procollagen type Ⅲ(PCⅢ), pro-collagen type Ⅳ(CⅣ) and laminin(LN) levels.Finally the serum ALT and γ-GT levels were measured with totally automatic enzymat-ic method. Results Pygo2 expression in NAFL group [52.1(12.3)μg/L] and NASH group[78.3(50.0)μg/L] increased signifi-cantly compared with simple obesity group [43.2(18.7)μg/L](P<0.05).Pygo2 expression in NASH group increased significantly compared with control group [41.7(16.8)μg/L] and NAFL group (P<0.001).The serum hepatic fribrosis and inflammation markers ( HA, PC, ALT, C III and IV gamma-GT) levels gradually increased in the obese children .There were statistically positive correla-tions between serum Pygo2 and HA, PCⅢ, CⅣ, ALT or γ-GT (P<0.001).In particular, more significant positive correlations were found between serum Pygo2 and HA, CⅣor γ-GT (r=0.708, P<0.001;r=0.589, P<0.001;r=0.674, P<0.05). Conclusion The degree of liver fibrosis worsens with the increase of Pygo 2 expression and Pygo 2 is in remarkable correlation with conventional he-patic fibrosis serum makers ( HA, C IV) orγ-GT, which shows Pygo 2 expression can be taken as the clinical evaluation index of liver fibrosis in children with NAFLD .
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Objective To study the expression and the clinical significance of Wnt signal pathway regulation factor Pygo2 in human renal cell carcinoma.Methods Using RFQ-PCR and immunohistochemical SP methods to detect Pygo2 mRNA and protein expression in 42 cases renal clear cell carcinoma and their own normal kidney tissue specimens.All specimens had a definite diagnosis by pathologic.All were renal clear cell carcinoma.The tumor diameter was 1.2-15.5 cm.The average was 7.2 cm.Among all patients,there were 7 cases with diameter ＜ 4.0 cm,9 cases 4.0-7.0 cm,26 cases ＞ 7.0 cm.Fuhrman classification:grade Ⅰ 6 cases,grade Ⅱ 17 cases,grade Ⅲ 17 cases,grade Ⅳ 2 cases.AJCC TNM stages:stage Ⅰ 16 cases,stage Ⅱ 7 cases,stage Ⅲ 7 cases,stage Ⅳ 12 cases.Statistics was done to analyze the expression difference of pygo2 between normal kidney and renal cell carcinoma,and among renal cell carcinoma within each group.Results There was higher expression of Pygo2 in renal cell carcinoma,and in the adjacent lower expression.The pygo2 mRNA expression were 2.88 ± 1.26 and 1.00 ± 0.00 in respective specimens (P ＜ 0.0001).The pygo2 protein expression were 45.53 + 24.54 and 11.02 + 1.39 in respective specimens (P ＜ 0.0001).Pygo2 expression in grading and staging were statistically significant (P ＜0.0001),but in gender,age was not statistically significant (P ＞ 0.05).Conclusions High expression of Pygo2 was found in Fuhrman high grade,high clinical staging,lympho-metastasized renal clear cell carcinoma.Pygo2 might play an important role in the occurrence and development process in renal clear cell carcinoma.
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Objective: To construct recombinant vectors for RNA interference(RNAi) targeting Pygo2, and to assess its influence on the proliferation, invasion of glioblastoma U251 cells and the related mechanism. Methods: A pair of oligonucleotides containing short hairpin structure targeting Pygo2 cDNA sequences were designed and synthesized, and their negative control sequences were also synthesized. After annealed, they were inserted into pSuper vector to generate the recombinant plasmids. Then the recombinant plasmids were digested with EcoR I and Hind III for identification, and the sequence was assayed by DNA sequencing. The recombinant plasmids were transfected into cultured glioblastoma U251 cells using Lipofectamine™ 2000. The effect of Pygo2 shRNA on Pygo2 mRNA and protein in U251 cells was detected by real-time PCR and Western blotting analysis, respectively. MTT assay was used to detect the cell proliferation; cell cycle was analyzed by flow cytometry; Bromodeoxyuridine (BrdU) incorporation analysis was used to examine DNA synthesis; and cell invasion assay was performed using Transwell chambers. The effect of Pygo2 shRNA on the protein level and subcellular location of cyclin D1 and β-catenin was detected by Western blotting analysis and immunofluorescent staining. Results: The recombinant plasmids were completely coincided with the design by the restriction map and the sequence analysis. Pygo2 mRNA and protein expression was significantly suppressed by Pygo2 shRNA. Furthermore, the proliferation of cells in Pygo2 shRNA group was notably inhibited, cell cycle was arrested at the G1 phase, and BrdU incorporation and migrating cells were significantly inhibited. In addition, Pygo2 knockdown significantly down-regulated cyclin D1 expression without altering the subcellular location, and the expression level and subcellular location of β-catenin had no noticeable changes. Conclusion: The recombinant vectors for specific suppression of Pygo2 expression have been constructed successfully. Inhibition of Pygo2 expression can suppress cell proliferation and invasion of glioma U251 cells, decrease DNA synthesis, arrest cell cycle at the G1 phase, and decrease expression of the Wnt target gene cyclin Dl.