miR-15a 和miR-16-1 模拟物对人骨肉瘤细胞系SOSP-9607 凋亡 和增殖的影响

目的:探讨miR-15a和miR-16-1模拟物对于人骨肉瘤细胞系SOSP-9607凋亡和增殖的影响。方法:将SOSP-9607细胞分为实验组和对照组。实验组分为miR-15a组、miR-16-1组、miR-15a+miR-16-1组。以miR-15a组为例,采用miR-15a模拟物(hsa-miR-15a mimics)上调SOSP-9607细胞内的miR-15a表达量。对照组分为阴性对照组和空白对照组。采用流式细胞仪测定细胞凋亡率,四甲基偶氮唑蓝(MTT)法测定细胞增殖,并计算细胞增殖效率。结果:通过统计学分析,实验组凋亡率与阴性对照组凋亡率相比明显增高(P<0.05);实验组的细胞增殖率明显低于对照组(P<0.05)。结论:上调SOSP-9607细胞内miR-15a和miR-16-1的表达量可促进SOSP-9607细胞的凋亡并抑制其增殖。     

PIK3C3/VPS34, the class III PtdIns 3-kinase,plays indispensable roles in the podocyte

The mammalian homolog of yeast Vps34 (PIK3C3/VPS34) is implicated in the regulation of autophagy, and recent studies have suggested that autophagy is a key mechanism in maintaining the integrity of renal glomerular podocytes. To date, however, the role of PIK3C3 in podocytes has remained unknown. We generated a line of podocyte-specific Pik3c3-knockout (Pik3c3^pdKO/mVps34^pdKO) mice and demonstrated an indispensable role for PIK3C3 in the regulation of intracellular vesicle trafficking and processing to protect the normal cellular metabolism, structure and function of podocytes.     

Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Identification and characterization of a new 34 kDa MORN motif-containing sporozoite surface-exposed protein,Cp-P34, unique to Cryptosporidium

Despite the public health impact of childhood diarrhea caused by Cryptosporidium, effective drugs and vaccines against this parasite are unavailable. Efforts to identify vaccine targets have focused on critical externally exposed virulence factors expressed in the parasite s invasive stages. However, no single surface antigen has yet been found that can elicit a significant protective immune response and it is likely that pooling multiple immune targets will be necessary. Discovery of surface proteins on Cryptosporidium sporozoites is therefore vital to this effort to develop a multi-antigenic vaccine. In this study we applied a novel single-domain camelid antibody (VHH) selection method to identify immunogenic proteins expressed on the surface of Cryptosporidium parvum sporozoites. By this approach, VHHs were identified that recognize two sporozoite surface-exposed antigens, the previously identified gp900 and an unrecognized immunogenic protein, Cp-P34. This Cp-P34 antigen, which contains multiple Membrane Occupation and Recognition Nexus (MORN) repeats, is found in excysted sporozoites as well as in the parasite s intracellular stages. Cp-P34 appears to accumulate inside the parasite and transiently appears on the surface of sporozoites to be shed in trails. Identical or nearly identical orthologs of Cp-P34 are found in the Cryptosporidium hominis and Cryptosporidium tyzzeri genomes. Except for the conserved MORN motifs, the Cp-P34 gene shares no significant homology with genes of other protozoans and thus appears to be unique to Cryptosporidium spp. Cp-P34 elicits immune responses in naturally exposed alpacas and warrants further investigation as a potential vaccine candidate.     

AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR-96-5p-AIMP3-p53 axis

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) is a tumour suppressor, however, the roles of AIMP3 in non-small cell lung cancer (NSCLC) are not explored yet. Here, we reported that AIMP3 significantly inhibited the cell growth and metastasis of NSCLC (lung adenocarcinoma) in vitro and in vivo. We have firstly identified that AIMP3 was down-regulated in human NSCLC tissues compared with adjacent normal lung tissues using immunohistochemistry and western blot assays. Overexpression of AIMP3 markedly suppressed the proliferation and migration of cancer cells in a p53-dependent manner. Furthermore, we observed that AIMP3 significantly suppressed tumour growth and metastasis of A549 cells in xenograft nude mice. Mechanically, we identified that AIMP3 was a direct target of miR-96-5p, and we also observed that there was a negative correlation between AIMP3 and miR-96-5p expression in paired NSCLC clinic samples. Ectopic miR-96-5p expression promoted the proliferation and migration of cancer cells in vitro and tumour growth and metastasis in vivo which partially depended on AIMP3. Taken together, our results demonstrated that the axis of miR-96-5p-AIMP3-p53 played an important role in lung adenocarcinoma, which may provide a new strategy for the diagnosis and treatment of NSCLC.     

Long non-coding RNA ZFAS1 alleviates sepsis-induced myocardial injury via target miR-34b-5p/SIRT1

Long non-coding RNA ZFAS1 is down-regulated in sepsis. However, whether ZFAS1 participates in sepsis-induced cardiomyopathy (SIC) remains largely unknown. LPS injection to rats was used to establish an in vivo sepsis model, while LPS stimulation with H9C2 cell was used to mimic an in vitro sepsis-induced myocardial injury model. Western blots and quantitative RT-PCR were performed to evaluate protein and mRNA levels, respectively. ELISA was conducted to determine cytokine levels in supernatant. Flow cytometry was used to test apoptosis. Dual-luciferase assay was performed to validate binding between ZFAS1 and miR-34b-5p, miR-34b-5p and SIRT1. Our data revealed that ZFAS1 and SIRT1 were down-regulated, while miR-34b-5p was up-regulated in LPS-induced H9C2 cells. Inhibition of miR-34b-5p or overexpression of ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells. A mechanism study revealed that ZFAS1 sponged miR-34b-5p and thus elevated expression of SIRT1, which was prohibited by miR-34b-5p. ZFAS1 alleviated inflammatory response and cell apoptosis in LPS-stimulated H9C2 cells via the miR-34b-5p/SIRT1 axis, providing novel potential therapeutic targets for SIC.     

Long noncoding RNA DLGAP1-AS1 promotes cell proliferation in hepatocellular carcinoma via sequestering miR-486-5p

Hepatocellular carcinoma (HCC) is most prevalent tumor in liver and one of the most fatal cancers in the world. Long noncoding RNAs (lncRNAs) have been accepted as important regulators in carcinomas. But there are still many lncRNAs including DLGAP1-AS1 unannotated in HCC. First of all, GEPIA suggested that DLGAP1-AS1 presented high expression in HCC tissue samples relative to the normal tissues. Besides, overexpression of DLGAP1-AS1 was also proved in HCC cell lines. Moreover, DLGAP1-AS1 knockdown efficiently suppressed cell proliferation in HCC. Interestingly, miR-486-5p was predicted and validated to interact with DLGAP1-AS1, while the level of miR-486-5p was significantly increased In HCC after DLGAP1-AS1 knockdown. Moreover, we uncovered that ectopic expression of miR-486-5p induced suppression on HCC cell proliferation and that miR-486-5p inhibition offset the effect of DLGAP1-AS1 silence on HCC cell proliferation and apoptosis. Furthermore, H3F3B was identified as target of miR-486-5p and was therefore positively regulated by DLGAP1-AS1 in HCC. Of note, H3F3B upregulation partly revived the declined cell proliferative capacity in response to DLGAP1-AS1 knockdown. To conclude, DLGAP1-AS1 exerted its oncogenic role in HCC via miR-486-5p/H3F3B axis. Our new findings provided novel theoretical basis for discovery of therapeutic targets of HCC.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34^cdc2 protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34^cdc2, as well as several of the proteins that interact with and regulate p34^cdc2 function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34^cdc2 protein is entirely absent and is replaced by its human functional homologue p34^CDC2, We have used this strain to analyse aspects of the function of the human p34^CDC2 protein genetically. We show that the function of the human p34^CDC2 protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80^cdc25 that it responds to altered levels of both the mitotic inhibitor p107²³³¹ and the p34^cdc2-binding protein p13^suc1, and is lethal in combination with the mutant B-type cyclin p56^cdc13-117. In addition, we demonstrate that the human p34^CDC2 protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34^CDC2 proteins in fission yeast.