慢病毒介导的甲胎蛋白敲减表达抑制肝癌细胞HepG2的增殖

甲胎蛋白（AFP）是原发性肝癌的标志物.本实验室前期研究表明,AFP的表达与肝癌细胞的增殖及细胞周期相关.为了建立高效稳定的AFPsiRNA细胞导入方法,本研究构建了AFP慢病毒siRNA干涉载体pRNAT-U6.2/AFPsiRNA.并将其转入HEK293细胞后,Western 印迹表明,pRNAT-U6.2/AFPsiRNA的干扰效率为88.7%（P<0.05）. pRNAT-U6.2/AFPsiRNA与4个包装质粒共同转染293T细胞包装成慢病毒后感染HepG2细胞,感染3 d时GFP的表达量达95%,感染35 d的子代细胞中GFP的表达量仍稳定在85%.GFP表达量的观察显示,慢病毒载体可以高效并稳定表达外源基因. Western 印迹结果也显示,HepG2细胞被病毒感染3 d和25 d后,AFP表达水平均有降低,抑制率分别为74.8%和63.4%(P<0.05).克隆形成实验表明,AFP沉默后,细胞克隆形成个数降低63%(P<0.01),表明HepG2细胞增殖能力受到抑制.     

Cigarette smoke extract induces HO‐1 expression in mouse cerebral vascular endothelial cells: Involvement of c‐Src/NADPH oxidase/PDGFR/JAK2/STAT3 pathway

Several chemicals present in cigarette smoke (CS) have been reported to induce heme oxygenase‐1 (HO‐1) expression, which represents a prime defense mechanism in protecting the cells from stress‐dependent adverse effects on peripheral vascular system. However, the effects of cigarette smoke extract (CSE) on HO‐1 induction and the mechanisms underlying CSE‐induced HO‐1 expression in brain vessels are not completely understood. Here, we used a mouse brain endothelial cell culture (bEnd.3) to investigate the effect of CSE on HO‐1 induction and the mechanisms underlying CSE‐induced HO‐1 expression in cerebral vessels. We demonstrated that sublethal concentrations of CSE (30 µg/ml) induced submaximal HO‐1 expression in bEnd.3 cells. NADPH oxidase‐dependent ROS generation played a key role in CSE‐induced HO‐1 expression. CSE‐induced HO‐1 expression was mediated through PDGFR/JAK2/STAT3 cascade, which was observed by pretreatment with the respective pharmacological inhibitors or transfection with PDGFR shRNA. CSE activated NADPH oxidase through c‐Src in bEnd.3 cells. Taken together, these results suggested that, in bEnd.3 cells, CSE‐induced HO‐1 expression was mediated through PDGFR/JAK2/STAT3 cascade, which was regulated by c‐Src or c‐Src activated‐NADPH oxidase/ROS. J. Cell. Physiol. 225: 741–750, 2010. © 2010 Wiley‐Liss, Inc.     

In trans interaction of hepatitis C virus helicase domains mediates protease activity critical for internal NS3 cleavage and cell transformation

Hepatitis C virus (HCV) internal non-structural protein 3 (NS3) cleavage can occur in trans in the presence of NS4A. In this study, we have further demonstrated a critical role of the helicase domain in the internal NS3 cleavage, different from HCV polyprotein processing which requires only the serine protease domain. The NTPase domain of NS3 helicase interacts with the RNA binding domain to facilitate internal NS3 cleavage. In addition, NS3 protease activity contributes to the transforming ability of the major internal cleavage product NS3(1-402). These findings imply important roles of the internal cleavage and protease activity of the NS3 protein in the pathogenesis of HCV.

Structured summary

MINT-7306465: NS3 (uniprotkb:P29846) physically interacts (MI:0915) with NS3 (uniprotkb:P29846) by anti tag coimmunoprecipitation (MI:0007).     

Selection and validation of suitable reference genes for miRNA expression normalization by quantitative RT-PCR in citrus somatic embryogenic and adult tissues

miRNAs have recently been reported to modulate somatic embryogenesis (SE), a key pathway of plant regeneration in vitro. For expression level detection and subsequent function dissection of miRNAs in certain biological processes, qRT-PCR is one of the most effective and sensitive techniques, for which suitable reference gene selection is a prerequisite. In this study, three miRNAs and eight non-coding RNAs (ncRNA) were selected as reference candidates, and their expression stability was inspected in developing citrus SE tissues cultured at 20, 25, and 30?°C. Stability of the eight non-miRNA ncRNAs was further validated in five adult tissues without temperature treatment. The best single reference gene for SE tissues was snoR14 or snoRD25, while for the adult tissues the best one was U4; although they were not as stable as the optimal multiple references snoR14?+?U6 for SE tissues and snoR14?+?U5 for adult tissues. For expression normalization of less abundant miRNAs in SE tissues, miR3954 was assessed as a viable reference. Single reference gene snoR14 outperformed multiple references for the overall SE and adult tissues. As one of the pioneer systematic studies on reference gene identification for plant miRNA normalization, this study benefits future exploration on miRNA function in citrus and provides valuable information for similar studies in other higher plants. Key message Three miRNAs and eight non-coding RNAs were tested as reference candidates on developing citrus SE tissues. Best single references snoR14 or snoRD25 and optimal multiple references snoR14?+?U6, snoR14?+?U5 were identified.     

Proteomic investigation of 5-fluorouracil resistance in a human hepatocellular carcinoma cell line

Multi-drug resistance (MDR) is a major obstacle towards a successful treatment of hepatocellular carcinoma (HCC). The mechanisms of MDR are intricate and have not been fully understood. Therefore, we employed a cell-line model consisting of the 5-fluorouracil (5-FU) resistant BEL7402/5-FU cell line and its parental BEL7402 cell line. Using relative and absolute quantification (iTRAQ)-coupled 2D LC-MS/MS, a successfully exploited high-throughput proteomic technology, in total, 660 unique proteins were identified and 52 proteins showed to be differentially expressed in BEL7402/5-FU compared with BEL7402. Several differentially expressed proteins were further validated by Western blot and real-time quantitative RT-PCR analysis. Furthermore, the association of MDR with ANXA3, one of the highly expressed proteins in BEL7402/5-FU, was verified. Our study represents the first successful application of iTRAQ technology for MDR mechanisms analysis in HCC. Many of the differentially expressed proteins identified had not been linked to MDR in HCC before, which provide valuable information for further understanding of MDR.     

The rice RAD51C gene is required for the meiosis of both female and male gametocytes and the DNA repair of somatic cells

The RecA/RAD51 family of rice (Oryza sativa) consists of at least 13 members. However, the functions of most of these members are unknown. Here the functional characterization of one member of this family, RAD51C, is reported. Knockout (KO) of RAD51C resulted in both female and male sterility in rice. Transferring RAD51C to the RAD51C-KO line restored fertility. Cytological analyses showed that the sterility of RAD51C-KO plants was associated with abnormal early meiotic processes in both megasporocytes and pollen mother cells (PMCs). PMCs had an absence of normal pachytene chromosomes and had abnormal chromosome fragments. The RAD51C-KO line showed no obvious difference from wild-type plants in mitosis in the anther wall cells, which was consistent with the observation that the RAD51C-KO line did not have obviously abnormal morphology during vegetative development. However, the RAD51C-KO line was sensitive to different DNA-damaging agents. These results suggest that RAD51C is essential for reproductive development by regulating meiosis as well as for DNA damage repair in somatic cells.     

The time course of photoinactivation of photosystem II in leaves revisited

Since photosystem II (PS II) performs the demanding function of water oxidation using light energy, it is susceptible to photoinactivation during photosynthesis. The time course of photoinactivation of PS II yields useful information about the process. Depending on how PS II function is assayed, however, the time course seems to differ. Here, we revisit this problem by using two additional assays: (1) the quantum yield of oxygen evolution in limiting, continuous light and (2) the flash-induced cumulative delivery of PS II electrons to the oxidized primary donor (P700(+)) in PS I measured as a 'P700 kinetics area'. The P700 kinetics area is based on the fact that the two photosystems function in series: when P700 is completely photo-oxidized by a flash added to continuous far-red light, electrons delivered from PS II to PS I by the flash tend to re-reduce P700(+) transiently to an extent depending on the PS II functionality, while the far-red light photo-oxidizes P700 back to the steady-state concentration. The quantum yield of oxygen evolution in limiting, continuous light indeed decreased in a way that deviated from a single-negative exponential. However, measurement of the quantum yield of oxygen in limiting light may be complicated by changes in mitochondrial respiration between darkness and limiting light. Similarly, an assay based on chlorophyll fluorescence may be complicated by the varying depth in leaf tissue from which the signal is detected after progressive photoinactivation of PS II. On the other hand, the P700 kinetics area appears to be a reasonable assay, which is a measure of functional PS II in the whole leaf tissue and independent of changes in mitochondrial respiration. The P700 kinetics area decreased in a single-negative exponential fashion during progressive photoinactivation of PS II in a number of plant species, at least at functional PS II contents ≥6?% of the initial value, in agreement with the conclusion of Sarvikas et al. (Photosynth Res 103:7-17, 2010). That is, the single-negative-exponential time course does not provide evidence for photoprotection of functional PS II complexes by photoinactivated, connected neighbours.     

Toward an understanding of the molecular basis of quantitative disease resistance in rice

Rice crops are severely damaged by diseases caused by bacterial, fungal, and viral pathogens. Application of host resistance to these pathogens is the most economical and environmentally friendly approach to solve this problem. Quantitative resistance conferred by quantitative trait loci (QTL) is a valuable resource for the improvement of rice disease resistance. Although numerous resistance QTL against rice diseases have been identified, these resources have not been used effectively in rice improvement because the genetic control of quantitative resistance is complex and the genes underlying most of the resistance QTL remain unknown. This review focuses on the latest molecular progress in quantitative disease resistance in rice. This knowledge will be helpful for characterizing more resistance QTL and turning the quantitative resistance into actual resources for rice protection.