Long noncoding RNA CASC9 promotes LIN7A expression via miR-758-3p to facilitate the malignancy of ovarian cancer

The long noncoding RNA cancer susceptibility 9 (CASC9) has been reported to be a pivot modulator in growth and metastasis of breast cancer, liver cancer, esophageal squamous cell carcinoma, lung adenocarcinoma, gastric cancer, and nasopharyngeal cancer. However, its potential roles in ovarian cancer remain unclear. In this study, we aimed at its functions and molecular mechanism in ovarian cancer progression. We showed that CASC9 was highly expressed in ovarian cancer tissues and cell lines. An elevated level of CASC9 predicts an unfavorable prognosis in patients with ovarian cancer. Loss-of-function and gain-of-function assays illustrated that CASC9 promotes ovarian cancer cell proliferation, migration, and invasion in vitro, and accelerates tumor growth in vivo. We showed that CASC9 works as a competing endogenous RNA (ceRNA) for miR-758-3p which targets LIN7A. CASC9 inhibits the level of miR-758-3p, and in turn stimulates LIN7A expression in ovarian cancer. Overexpression of LIN7A reverses the suppressive roles of CASC9 depletion on ovarian cancer. In sum, our findings reveal a novel undefined regulatory signaling pathway, namely CASC9/miR-758-3p/LIN7A axis, involved in ovarian cancer progression.     

Structure and functionality of a multimeric human COQ7:COQ9 complex

1. Download : Download high-res image (298KB)
2. Download : Download full-size image

     

Preparation of polyclonal antibody specific for BRD7 and detection of its expression pattern in the human fetus.

BRD7 is a novel bromodomain gene. It plays critical role in cell growth, cell cycle progression, and signal-dependent gene expression. Overexpression of the BRD7 gene in nasopharyngeal carcinoma cells is effective to inhibit cell growth and cell cycle progression from G1 to S phase. However, little is known about its bio-functions because of the unavailability of a specific BRD7 antibody. In this study, for the first time, we generated a highly specific BRD7 antibody. It is able to specifically recognize recombinant GST-BRD7N protein with a molecular mass of 65 kDa and recognize BRD7-Myc and endogenously expressed BRD7 protein with an approximate molecular mass of 75 kDa, which corresponds well with the calculated molecular mass of the BRD7 protein. More importantly, with these antisera, we analyzed BRD7 distribution in the human fetus by Western blot and immunohistochemistry assays. Obvious nuclear expression of BRD7 protein presents in human cerebellum, pancreas, intestines, liver, and kidney. Cardiomyocyte shows high cytoplasm expression of the BRD7 protein. Weak nuclear expression of the BRD7 protein is found in human cerebrum, lung, and stomach. These data may help to further study the cellular role of the BRD7 gene. In particular, the prepared BRD7 antibody will be helpful for studying the bio-functions of endogenously expressed BRD7 protein.     

人溶酶体酸性b-葡萄糖脑苷脂酶基因的克隆及其在COS7细胞中的表达

从人胎盘组织提取总RNA, 采用RT-PCR扩增人溶酶体酸性b-葡萄糖脑苷脂酶(Lysosomal acid b-glucosidase, GlcCerase)基因编码区的全部序列, 并克隆到pMD-19T载体上, 构建克隆载体pMD-GlcCerase。经测序验证后, 将GlcCerase亚克隆至表达载体pEGFP-C1上, 构建了人GlcCerase绿色荧光蛋白真核表达载体pEGFP-GlcCerase。采用脂质体法将其瞬时转染至COS7细胞系后, 在细胞中检测到了GlcCerase基因, 并在细胞裂解产物中检测到了GlcCerase生物活性的表达。GlcCerase基因的克隆及其表达, 为进一步了解GlcCerase基因的功能以及利用转基因动物乳腺生物反应器高效生产GlcCerase奠定了基础。     

The effects of dehydroepiandrosterone (DHEA) and its metabolites on the polycystic ovarian condition (PCO): cystogenic changes of rat granulosa cells in vitro

During mammalian folliculogenesis, granulosa cells (GCs) are initially steroidogenically quiescent, later proliferate, and subsequently commence to hormonally differentiate, first producing estrogen and later, in the preovulatory stage, secreting both estrogen and progesterone. In this study and elsewhere, we have used follicle-stimulating hormone with a combination of growth factors in vitro to simulate the above in vivo conditions. In a previous study, we used dehydroepiandrosterone (DHEA) to accomplish the polycystic ovary condition (PCO) in rats. In the latter model, there were high circulating levels of DHEA and its metabolite, androstenedione. In the present study, we investigated the effects of high levels of DHEA (10⁻⁵M) and its metabolites, androstenedione, androstenediol and dehydroepiandrosterone sulfate on the quiescent, proliferative, and steroidogenically differentiating stages of GCs cultured in a serum-free medium for up to 10 days. In addition to possessing the regularly occurring organelles, when cultured with the aforementioned androgens, the GCs acquired endoplasmic reticulum of the smooth variety which is associated with steroidogenesis. The radioimmunoassay data showed that GCs cultured in the quiescent and proliferative stages in the presence of the androgens, no longer remain in these stages but proceed to differentiate in a preovulatory direction by producing both estrogen and progesterone. This study supports our hypothesis that high circulating levels of DHEA and/or its metabolites have most effect during the quiescent and proliferative stages of granulosa cells, with regard to their structure and their steroidogenic activities.     

Effects of different sources of copper on Ctr1, ATP7A,ATP7B,MT and DMT1 protein and gene expression in Caco-2 cells

Copper sulfate (CuSO₄), micron copper oxide (micron CuO) and nano copper oxide (nano CuO) at different concentrations were, respectively, added to culture media containing Caco-2 cells and their effects on Ctr1, ATP7A/7B, MT and DMT1 gene expression and protein expression were investigated and compared. The results showed that nano CuO promoted mRNA expression of Ctr1 in Caco-2 cells, and the difference was significant compared with micron CuO and CuSO₄. Nano CuO was more effective in promoting the expression of Ctr1 protein than CuSO₄ and micron CuO at the same concentration. Nano CuO at a concentration of 62.5 μM increased the mRNA expression levels of ATP7A and ATP7B, and the difference was significant compared with CuSO₄. The addition of CuSO₄ and nano CuO to the culture media promoted the expression of ATP7B proteins. CuSO₄ at a concentration of 125 μM increased the mRNA expression level of MT in Caco-2 cells, and the difference was significant compared with nano CuO and micron CuO. Nano CuO at a concentration of 62.5 μM inhibited the mRNA expression of DMT1, and the difference was significant compared with CuSO₄ and micron CuO. Thus, the effects of CuSO₄, micron CuO and nano CuO on the expression of copper transport proteins and the genes encoding these proteins differed considerably. Nano CuO has a different uptake and transport mechanism in Caco-2 cells to those of CuSO₄ and micron CuO.     

Slow freezing versus vitrification technique for human ovarian tissue cryopreservation: An evaluation of histological changes,WNT signaling pathway and apoptotic genes expression

This study compared slow freezing and vitrification of ovarian tissue by evaluation of histological changes, WNT signaling pathway and apoptotic genes expression. Ovarian tissue was obtained from women aging 27–38 years old. Ovarian cortex from each patient was divided into three pieces and randomly grouped as slow freezing, vitrification and control groups for investigation of WNT signaling gene expression and β-CATENIN presence as well as histological studies. The stromal structure of all ovaries were preserved. The number of secondary follicles decreased in vitrified group (P < 0.05). WNT-3, β-CATENIN, FZD-2 and GSK-3β expressions were significantly higher in slow frozen and vitrified groups, compared to control group (P < 0.05). On the contrary, AXIN1 expression in slow frozen samples were significantly lower than that of the vitrified and control group. The expression of apoptotic genes, excluding CASP3, was significantly decreased in slow-frozen samples (P < 0.05). Conversely, BAX:BCL-2 percentage significantly increased in vitrification versus slow freezing and control(P < 0.05). Follicles in slow frozen samples displayed nuclear and cytoplasmic β-CATENIN staining, while control and vitrification groups only showed β-CATENIN protein in the cytoplasm. The presented data show that slow freezing results in a better preservation regardless of the type of follicle. Therefore, it is concluded that slow freezing is still an ideal method for ovary cryopreservation.     

人H7N9禽流感病毒与H1N1流感病毒感染小鼠病理学损伤的比较

目的比较分析H7N9病毒与H1N1病毒感染小鼠病理学损伤特点,初步探讨两种病毒感染致小鼠急性肺损伤的致病机制。方法 H7N9病毒与H1N1病毒分别感染小鼠,观察不同病毒感染后小鼠生存率,并于不同时间点取心、肝、脾、肺、肾、脑、肠等组织,伊红-苏木素染色并进行组织病理学分析,免疫组化检测病毒抗原分布及中性粒细胞浸润。综合分析肺组织病理损伤与病毒复制、宿主免疫反应之间的关系。结果 H7N9病毒感染小鼠肺及脾脏损伤较轻,存活率较高。H1N1病毒感染的小鼠肺及脾脏损伤较重,感染后9 d全部死亡;两种病毒抗原主要分布于支气管上皮细胞、少量间质细胞和肺泡上皮细胞,病毒复制水平无明显差异。但H1N1病毒感染后肺及脾脏中均有大量中性粒细胞浸润,小鼠机体炎症反应明显强于H7N9病毒感染后小鼠炎症反应。结论 H7N9病毒与H1N1病毒感染后小鼠病理学损伤特点及程度均不同,病毒复制是小鼠肺损伤的诱发因素但并非决定因素,宿主针对病毒感染产生的免疫反应程度与急性肺损伤密切相关。     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.     

Alterations in mRNA expression of ribosomal protein S9 in hydrogen peroxide-treated neurotumor cells and in rat hippocampus after transient ischemia

This study was designed to isolate new genes related to apoptosis in rat pheochromocytoma (PC12) cells treated with hydrogen peroxide (H₂O₂), and to characterize the roles of the genes using both in vitro and in vivo models of oxidative injury. cDNA libraries were prepared from H₂O₂-treated and -untreated PC12 cells, and a ribosomal protein S9 (RPS9) clone was isolated by a differential screening method. Increase of RPS9 expression in both H₂O₂-treated PC12 and neuroblastoma (Neuro-2A) cells was shown by Northern blot analysis. Viability of the antisense-transfected Neuro-2A (RPS9-AS) cells following H₂O₂ treatment was significantly reduced in a dose-dependent manner. In an in vivo model of transient forebrain ischemia, an increase in RPS9 expression was prominent by 1 day postischemia in the granule cell layer neurons of the dentate gyrus. Both activation of caspase-3 and significant recovery of viability following pretreatment with cycloheximide were shown in RPS9-AS cells treated with H₂O₂. These data suggest that RPS9 plays a protective role in oxidative injury of neuronal cells.     

高糖对原代培养人肾小球系膜细胞表达MMP-9、TIMP-1的影响

目的了解高糖对原代培养的人肾小球系膜细胞表达MMP-9、TIMP-1和MMP-9/TIMP-1的影响,进一步探讨糖尿病肾病的发病机制。方法取自愿水囊引产的胎儿肾,解剖取肾皮质剪碎,应用肾皮质组织块法合优生选择法对人肾小球系膜细胞进行原代培养。ELISA方法检测MMP-9、TIMP-1的表达变化。结果与正常组相比较,高糖组MMP-9在24h、48h、72h均显著降低（P〈0.01）,TIMP-1在24h、72h可显著升高（P〈0.05）,48h表达降低;MMP-9/TIMP-1的比值在24h、48h、72h均显著降低（P〈0.01）。结论高糖能够降低MMP-9/TIMP-1,与肾小球系膜细胞细胞外基质降解能力降低密切相关。     

Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody: possible role of the protein in cytoskeleton rearrangements during differentiation

Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.     

Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4‐7

Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race‐specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B. napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co‐segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re‐sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5‐9 to reflect the dual specificity of this locus. Avrlm5‐9 transgenic isolates were avirulent when inoculated on the B. napus cultivar Goéland. The expression of AvrLm5‐9 during infection was monitored by RNA sequencing. The recognition of AvrLm5‐9 by Rlm9 is masked in the presence of AvrLm4‐7, another Lm effector. AvrLm5‐9 and AvrLm4‐7 do not interact, and AvrLm5‐9 is expressed in the presence of AvrLm4‐7. AvrLm5‐9 is the second Lm effector for which host recognition is masked by AvrLm4‐7. An understanding of this complex interaction will provide new opportunities for the engineering of broad‐spectrum recognition.     

卡尼鄂拉蜂锌转运蛋白-7相似蛋白（ZnT-7-like）的基因克隆、 抗体制备及差异表达

【目的】本研究旨在克隆卡尼鄂拉蜂Apis mellifera carnica锌转运蛋白 7相似蛋白（zinc transporter 7-like, ZnT-7-like）基因, 制备ZnT-7-like多克隆抗体, 了解该基因在卡尼鄂拉蜂王浆腺中不同时期的差异表达情况。【方法】运用RT PCR法从卡尼鄂拉蜂王浆腺总RNA中扩增ZnT-7-like基因, 进行生物信息学分析, 为避开跨膜结构的干扰, 选择克隆其部分序列（273 bp）作为多肽免疫序列。将其亚克隆入原核表达载体pGEX-4T-1, 转入大肠杆菌Escherichia coli BL21 （DE3）中诱导表达获得融合蛋白, 然后将融合蛋白纯化后免疫新西兰大白兔制备多克隆抗体, 分别用间接ELISA和Western blot检测抗体的效价和特异性, 最后采用实时荧光定量RT-PCR以及Western blot技术检测该基因在不同日龄成虫王浆腺中的相对表达量。【结果】克隆得到卡尼鄂拉蜂ZnT-7-like基因, 大小为1 065 bp。SDS-PAGE电泳结果显示融合蛋白成功表达; 制备的多克隆抗体效价高达1∶64 000, 且具有很高的特异性。ZnT-7-like在卡尼鄂拉蜂5个日龄成虫中的转录情况存在较大差异, 表现为3日龄成虫中的表达量极显著高于其他日龄（P<0.01）, 12日龄表达量最低, 其他日龄间表达量两两差异显著(P<0.05)或极显著（P<0.01）; ZnT-7-like蛋白表达与转录水平基本一致。【结论】成功克隆了卡尼鄂拉蜂ZnT-7-like基因, 制备了兔抗蜂ZnT-7-like多克隆抗体, 并在转录和翻译两个水平上测定了ZnT-7-like在卡尼鄂拉蜂王浆腺中不同时期的相对表达量。这些结果为深入研究卡尼鄂拉蜂ZnT-7-like基因的功能奠定了基础。