siRNA的定量方法及其在药代动力学检测中的应用

siRNA作为新型药物已逐渐成为药物开发的新热点。了解siRNA在体内的吸收、分布、代谢等过程的动态变化有助于对siRNA药物进行临床前的药理、毒理及药效评价。siRNA较短,只有20～25个碱基,对其进行检测和准确定量比较困难。本文通过比较目前已报道的多种针对小核苷酸片段的定量方法,试图选择其中较为快捷、简单和准确的方法,并分析其在siRNA药代动力学定量中应用的可行性。

Clinical significance of human kallikrein 12 gene expression in gastric cancer

AIM:To investigate whether the expression of kallikrein 12(KLK12) is related to the development of gastric cancer(GC) and to determine the role of KLK12 in gastric cancer cells growth,invasion and migration.METHODS:Between September 2007 and March 2008,133 patients with histologically confirmed GC were recruited for the study.Expression of KLK12 was detected in samples from GC patients by quantitative real-time reverse transcription polymerase chain reaction and immunohistochemistry.The relationship between KLK12 protein expression and clinicopathological features of GC was analyzed.The difference in 5-year survival rates between the high KLK12 protein expression group and the low KLK12 expression group was compared.Additionally,the expression of KLK12 was examined in various human GC cell lines,including MKN-28,SGC-7901 and MKN-45.Small interfering RNA(siRNA) was used to inhibit KLK12 expression in MKN-45 cells.Cell clones stably transfected with KLK12 siRNA were tested for KLK12 expression by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting.Furthermore,a series of functional assays were performed in this study to assess the biological features of transfected cells.Cell proliferation was assessed using the methylthiazolyltetrazoliumassay.Finally,cell migration and invasion were assessed using transwell chamber assays.RESULTS:Of the 133 GC patients included in the study,126(94.7%) showed a higher expression level of KLK12 mRNA when compared to noncancerous tissue specimens.Expression of KLK12 mRNA was significantly higher in GC tissues than in normal tissue(P < 0.001).KLK12 protein expression was detected in 96 of 133(72.2%) GC samples with moderate or strong staining primarily in the cytoplasm.In contrast,negative immunostaining for KLK12 protein was observed in the corresponding normal gastric mucosal tissue.Overexpression of KLK12 protein was significantly associated with lymph node metastasis(P = 0.001),histological type(P < 0.001) and tumor-node-metastasis stage(P = 0.005),while no significant correlation was observed between expression of KLK12 protein and sex,age,depth of invasion,tumor size or lymphatic invasion.Furthermore,patients with high KLK12 expression had a significantly poorer 5-year survival rate than those with low KLK12 expression(P = 0.002).Expression of KLK12 mRNA was significantly higher in MKN-45 GC cells compared to normal mucosal cells or two other GC cell lines(P < 0.01).Expression of KLK12 in MKN-45 cells was downregulated after transfection with siRNA.Knockdown of KLK12 markedly decreased the proliferation of MKN-45 cells when compared with parent or mock-transfected cells(P = 0.001),especially from the 3rd to the 5th day of the assay.In migration assays,fewer KLK12 siRNA cells migrated through the chambers(22.00 ± 1.81) when compared to the parent(46.47 ± 2.42) or mock-transfected cells(45.40 ± 1.99);these differences were statistically significant(P < 0.001).However,in the invasion assay,the number of KLK12 siRNA cells that invaded the chambers was 18.40 ± 1.12,closely similar to both the parent(18.67 ± 0.98) and mock-transfected cells(18.53 ± 0.92).There was no significantly difference between the three groups in the invasion assay(P = 0.054).CONCLUSION:The KLK12 gene is markedly overexpressed in GC tissue,and its expression status may be a powerful prognostic indicator for patients with GC.KLK12 might serve as a novel diagnosis and prognosis biomarker in GC.

Function of chloride intracellular channel 1 in gastric cancer cells

AIM:To investigate the effect of chloride intracellular channel 1(CLIC1) on the cell proliferation,apoptosis,migration and invasion of gastric cancer cells.METHODS:CLIC1 expression was evaluated in human gastric cancer cell lines SGC-7901 and MGC-803 by real time polymerase chain reaction(RT-PCR).Four segments of small interference RNA(siRNA) targeting CLIC1 mRNA and a no-sense control segment were designed by bioinformatics technology.CLIC1 siRNA was selected using Lipofectamine 2000 and transfected transiently into human gastric cancer SGC-7901 and MGC-803 cells.The transfected efficiency was observed under fluorescence microscope.After transfection,mRNA expression of CLIC1 was detected with RT-PCR and Western blotting was used to detect the protein expression.Proliferation was examined by methyl thiazolyl tetrazolium and apoptosis was detected with flow cytometry.Polycarbonate membrane transwell chamber and Matrigel were used for the detection of the changes of invasion and migration of the two cell lines.RESULTS:In gastric cancer cell lines SGC-7901 and MGC-803,CLIC1 was obviously expressed and CLIC1 siRNA could effectively suppress the expression of CLIC1 protein and mRNA.Proliferation of cells transfected with CLIC1 siRNA3 was enhanced notably,and the highest proliferation rate was 23.3%(P = 0.002) in SGC-7901 and 35.55%(P = 0.001) in MGC-803 cells at 48 h.The G2/M phase proportion increased,while G0/G1 and S phase proportions decreased.The apoptotic rate of the CLIC1 siRNA3 group obviously decreased in both SGC-7901 cells(62.24%,P = 0.000) and MGC-803 cells(52.67%,P = 0.004).Down-regulation of CLIC1 led to the inhibition of invasion and migration by 54.31%(P = 0.000) and 33.62%(P = 0.001) in SGC-7901 and 40.74%(P = 0.000) and 29.26%(P = 0.002) in MGC-803.However,there was no significant difference between the mock group cells and the negative control group cells.

5-Ethynyl-2′-deoxyuridine as a molecular probe of cell proliferation for high-content siRNA screening assay by “click” chemistry

Labelling and identification of proliferating cells is important for the study of physiological or pathological processes in high-content screening (HCS) assays. Here we describe ethynyl deoxyuridine (EdU) as a biomarker for the assessment of cell proliferation and clearly demonstrate the feasibility of the EdU-labelling method for use in HCS assays. EdU detection is highly robust, reproducible, technically simple, and well suited for automated segmentation, which provides an excellent al- ternative for setting up multiplexed HCS assays of siRNA, miRNA and small-molecule libraries.