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Objective:To determine the molecular role of DNA methyltransferase (DNMT) in kidney tumorigenesis. Methods:Tissue samples consisted of 15 cancer tissues and 15 matched adjacent tissues from clear cell renal cell carcinoma (ccRCC) patients who had undergone nephrectomy in 2012 at the First Affiliated Hospital of Baotou Medical College, Science and Technology University of Inner Mongolia were collected. Real-time PCR, Western blot, combined bisulfite restriction analysis (COBRA) and methylation specific PCR were used in this study. Real-time PCR was used to examine the mRNA expression levels of DNMT. The global methylation level, DNA methylation level, and the expression of the antioncogene RASSF1A in ccRCC tissues were concurrently detected. Results:Both mRNA and protein levels of DNMT3B4, a splice variant of DNMT3B, were elevated in renal cell carcinoma tissue compared with those in con-troll tissue. Additionally, Alu was hypomethylated in ccRCC tissue (0.106±0.04) compared with control tissue (0.115±0.03) (P<0.05). Fur-thermore, the methylation of the promoter for RASSF1A, a tumor-suppressor gene, moderately increased in renal cell carcinoma tis-sue. By contrast, RASF1A expression decreased. Conclusion:DNMT3B4 overexpression may play an important role in human kidney tu-morigenesis via chromosomal instability and the decreased expression of RASSF1A.
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Objective To construct two vectors of small interfering RNA (siRNA) expressing α-Fodrin and investigate its therapeutic effects on mice model with primary Sj(o)gren's syndrome (non-obese diabetic mice,NOD mice).Methods Sixteen 8-week-old NOD mice were randomly divided into four groups:the control group,the vector group,the α-Fodrin-siRNA1 group and α-Fodrin-siRNA2 group,4 mice in each group.Four template DNA of α-Fodrin siRNA were chemically synthesized and annealed to two double stranded (dsDNA),then digested by BamH Ⅰ and Hind Ⅲ.The digested double strands oligos were inserted into the downstream of U6 promoter of linearized pGFP-V-RS vector.Recombinant were confirmed by restrictive enzyme digestion and sequencing.Then the vectors were injected throughtail veil once a week,two times in total,while mice in the control group were injected with the same dose of phosphate buffer saline (PBS)and the vector group were injected with the same dose of vector vehicle.pGFP-V-RS was labeled by green fluorescent protein(GFP) and lacriminal glands underwent pathological examination.In addition,the expression of α-Fodrin mRNA in lung were detected by reverse transcription-polymerase chain reaction (RTPCR),and α-Fodrin protein in lacriminal glands and lung were detected by immuno-histochemistry.Serum interferon (IFN-γ),interleukin-17 (IL-17) concentrations in each group were detected by enzyme linked immunosorbent assay (ELISA) in order to observe changes in cytokine levels.At the same time,the pathological changes of the lacriminal glands and organs with hematoxylin-eosin (HE) staining were observed.The repeat ANOVA was used for statistical analysis.Results ① We constructed two siRNA eukaryotic expression vector successfully; ② α-Fodrin-siRNA could target to the lacriminal glands.③ Compared with the control group and vector vehicle group,the expression of α-Fodrin mRNA and protein were significantly decreased in the treatment groups.④ Compared with the control group [(11.73±2.73) pg/ml] and vector vehicle group [(15.40±1.99) pg/ml],serum IL-17 levels in the treatment groups were [α-Fodrin-siRNA 1 group (4.38±1.02) pg/ml; α-Fodrin-siRNA 2 group (4.55±0.06) pg/ml] significantly decreased (P＜0.05),but IFN-γ levels in the αt-Fodrin-siRNA group were not decreased significantly (P＞0.05).⑤ Compared with the control group and vector vehicle group,lymphocyte infiltration of lacriminal gland and inflammatory cell infiltration of alveolar and interstitial were significantly reduced in α-Fodrin-siRNA groups.Conclusion Specific α-Fodrin siRNA can inhibit the inflammation,and suppress the inflammatory infiltration of lacriminal glands and lung in mice with primary Sj(o)gren's syndrome.So the constructed vectors may slow the progression of pSS.
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Objective To investigate the expressions of CD147,MMP-9 and TIMP-1 in human gliomas and analyze the correlations.Methods Expressions of CD147,MMP-9 and TIMP-1 were assessed in paraffin-embedded specimens collected from 78 gliomas and 12 benign brain lesion tissues by immunohistochemistry.Real time PCR was performed to detect CD147 mRNA expression.Results The positive rates of CD147,MMP-9 and TIMP-1 expression were 62%(48/78),71%(55/78),59%(46/78) respectively.We found a significant positive correlation between CD147,MMP-9,TIMP-1 expressions and poor gliomas differentiation by Spearman analysis(rs=0.2671-0.5631,Ps＜0.01).There was also a significant positive correlation between CD147 and MMP-9 expression(rs =0.3576,P＜0.01).In addition,the expressions of CD147(47% vs.80%,x2=9.510),MMP-9(56% vs.89%,x2=10.702),and TIMP-1(49% vs.71%,x2=4.138) were significantly higher in advanced gilomas than early gliomas(Ps＜0.05).The relative expression levels of CD147 mRNA in gliomas of Ⅰ to Ⅳ pathological grades were 0.15,0.27,0.46,0.78 respectively.Conclusion The expressions of CD147,MMP-9 and TIMP-1 were important characteristic of gliomas,which may serve as biomarkers in the glioma prognostic prediction.