MicroRNA-145 inhibits epithelial-mesenchymal transition in human renal cancer A-498 cells by ADAM28

AIM: To investigate the inhibitory effect of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) in renal cancer A-498 cells. METHODS: The A-498 cells were transfected with miR-145 mimics (M145) and mimic negative control(MNC), which served as M145 group and MNC group, respectively. Mock control (MC) group was set up using untreated A-498 cells. The expression level of miR-145 in each group was detected by RT-qPCR. Transwell assay was used to detect the invasion ability of the cells. The protein expression of vimentin, E-cadherin and ADAM28 was determined by Western blot. Bioinformatic method was used to predict the target genes of miR-145. Antagonistic effect of ADAM28 over-expression on the inhibition of EMT by miR-145 was detected by Western blot. The relationship between miR-145 and ADAM28 was analyzed by dual-luciferase reporter assay. RESULTS: The expression level of miR-145 in M145 group was significantly up-regulated than that in MC group (P<0.05). The number of invasive cells in M145 group was 12.78±3.37, which was significantly lower than that in MC group (P<0.05). ADAM28 may be the target gene of miR-145. Compared with MC group, the protein expression of vimentin and ADAM28 in M145 group was significantly decreased (P<0.05), while the protein expression of E-cadherin was significantly increased (P<0.05).After ADAM28 over-expression, the protein expression of vimentin in the A-498 cells of M145 group was significantly increased (P<0.05), and the protein expression of E-cadherin was significantly decreased (P<0.05). The results of dual-lucife-irasei reporter assay showed that ADAM28 was a downstream target gene of miR-145. CONCLUSION: miR-145 may inhibit the expression of EMT-related proteins through the downstream target gene ADAM28 and inhibit the EMT process of renal cancer A-498 cells.     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS: MMP⁺ remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP⁺ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 (P<0.05), and obvious increases in apoptosis (P<0.05) and ROS level (P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION: ZER protects SH-SY5Y cells against MPP⁺-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.     

小麦TaCYP78A5基因的克隆及生物信息学分析

细胞色素P450(Cytochromes P450)蛋白家族含有硫羟基和血红素结构域,参与多种生物合成,CYP78A家族成员对籽粒大小形成有重要的功能。利用同源克隆的方法,分别从小麦大粒品系‘P271’和小粒材料‘中国春’中扩增到位于2AS、2BS、2DS上的TaCYP78A5-2A、TaCYP78A5-2B和TaCYP78A5-2D基因,编码534、544、549个氨基酸。通过分析发现在‘P271’和‘中国春’克隆到的TaCYP78A5序列一致,说明TaCYP78A5基因在这2个粒型的小麦中是保守的。CYP78A家族在水稻和拟南芥中具有非自主性细胞表达模式,结合对CYP78A家族的生物进化关系研究发现,CYP78A家族的各个基因在单子叶植物中是保守的。‘P271’和‘中国春’籽粒大小的差异,可能是TaCYP78A5基因在‘P271’中的基因表达量高于‘中国春’,引起种子发育过程中内表皮细胞的增殖,从而促进种子表皮增大,使得‘P271’种子大小和质量增加。     

F-box domain is involved in regulating the proliferation of MCF-7 cells by FBXO39 protein

AIM: To investigate the effect of F-box domain on the regulation of MCF-7 cell proliferation by FBXO39 protein. METHODS: The effect of F-box domain on the localization of FBXO39 protein in the MCF-7 cells was investigated. MCF-7 cell cDNA library was used as the template resource. The full-length cDNA sequence of FBXO39 was amplified by PCR method and subcloned into eukaryotic expression vector pEGFP-C2. The pEGFP-FBXO39ΔF (F-box domain deletion mutation) plasmid was successfully constructed with the template resource of pEGFP-FBXO39 plasmid. The recombinant plasmids were transfected into the MCF-7 cells, and then the expression of FBXO39 and FBXO39ΔF were determined by Western blot. The cellular localization of FBXO39 and FBXO39ΔF were observed by confocal microscopy. The localization of endogenous FBXO39 in the MCF-7 cells was detected by immunofluorescence staining. In addition, MTT and EdU assays were used to measure the cell proliferation, flow cytometry was used to measure the cell cycle distribution, and immunohistochemical staining was used to observe the expression of FBXO39 in the breast cancer and para-carcinoma tissues. RESULTS: The eukaryotic expression vector pEGFP-FBXO39 and pEGFP-FBXO39ΔF were constructed successfully. F-box domain had no effect on the cell localization of FBXO39. FBXO39 promoted MCF-7 cell proliferation but FBXO39ΔF did not. FBXO39 was highly expressed in the breast cancer tissues. CONCLUSION: F-box domain had no effect on the cellular localization of FBXO39 protein. However, it plays an important role in the biological function of FBXO39. FBXO39 may be related to breast cancer tumorigenesis.     

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination...     

山葡萄VaCIPK18原核表达与多克隆抗体制备

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs) 作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK 信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E. coli Rosetta)中,经0.8 mmol·L^-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。     

三种酶制备牡蛎蛋白水解物的抗氧化活性和功能特性分析

目的：比较分析木瓜蛋白酶、胃蛋白酶和胰蛋白酶三种不同酶水解牡蛎获得的蛋白产物抗氧化活性和功能特性。方法：采用木瓜蛋白酶、胃蛋白酶和胰蛋白酶三种不同酶水解牡蛎蛋白获得水解物,并分别测定它们的在不同pH下的溶解度、乳化性以及不同浓度水解物的还原能力、DPPH自由基清除率和Fe^{2 +}螯合能力。结果：表明三种酶水解物都有较高的溶解度(p＜0.01),胰蛋白酶产生的水解物溶解高度达94.8％(p＜0.05)；水解物溶解度和乳化活性指数在pH4显著降低(p＜0.05)。牡蛎蛋白质水解产物的还原能力、DPPH自由基清除能力和金属螯合活性随浓度的增加而增强,其中胰蛋白酶和胃蛋白酶抗氧化能力较强(p＜0.01)。结论：三种蛋白酶水解牡蛎蛋白可获得抗氧化活性强、溶解度高的水解物,并受浓度和pH的影响,它们的水解物可作为一种潜在的抗氧化功能多肽利用。