笃斯越橘响应低温和短光周期的蛋白质组分析

以3年生笃斯越橘苗为材料,将苗木分别置于对照(23℃,日照长度14 h),低温短日照(4℃,日照长度10 h)和低温长日照(4℃,日照长度14 h)的人工气候室中。处理21 d后,采用同位素标记相对和绝对定量(iTRAQ)蛋白质组学技术测定枝条蛋白质表达变化情况。结果显示:通过质谱鉴定出5 972个蛋白质,600个差异表达蛋白,其中在低温短日照与对照比对组中,丰度显著上调蛋白有140个,丰度显著下调蛋白有114个;在低温长日照与对照比对组中,丰度显著上调蛋白有255个,丰度显著下调蛋白有122个;在低温短日照与低温长日照比对组中,丰度显著上调蛋白有39个,丰度显著下调蛋白有187个。这些蛋白主要参与(1)RNA代谢,(2)蛋白质翻译后修饰,(3)碳水化合物转运和代谢,(4)能量代谢和转换,(5)脂质代谢,(6)次生代谢产物合成,(7)抗氧化与胁迫防御,(8)光合作用,(9)无机离子转运和代谢。结果表明与抗冻性有关的蛋白差异表达主要受低温诱导,少数蛋白表达受低温和光周期共同影响,低温短光周期更有利于抗冻性形成。低温锻炼可显著提高RNA代谢、蛋白质翻译后修饰、抗氧化和防御反应、次生代谢产物合成以及脂质代谢过程中许多蛋白质的丰度,提示这些代谢途径和相关蛋白可能在笃斯越橘抗冻机制中起重要作用。     

Progress in epigenetic study of clear cell renal cell carcinoma

The epigenetic changes of clear cell renal cell carcinoma (ccRCC) are considered to be the main molecular mechanisms of its pathogenesis, including DNA methylation, histone modification, microRNA change, and so on. DNA methyltransferases (DNMTs) catalyze the occurrence of DNA methylation. DNA methylation changes are manifested in the overall low methylation of the genome and the high methylation of specific sites, which were involved in the development of ccRCC by affecting the expression of tumor suppressor genes. Due to histone-modified enzyme involvement, histone modification is shown as possible genetic reversal. MicroRNA plays an important role in the abnormal expression of ccRCC genes. With the studies of epigenetic mechanism and molecular pathology, it is important to explore the mechanisms and to seek effective early diagnosis, treatment and prognosis intervention of ccRCC.     

基于银杏花芽3个分化时期转录组测序的相关基因筛选与表达分析

鉴定银杏花芽分化调控的关键基因,揭示银杏花芽分化调控的主要分子机制,为缩短银杏童期和选育银杏早花品种提供理论指导。本研究中采用高通量测序技术对银杏花芽分化3个时期（花芽未分化期、花芽分化始期、花芽分化盛期）的样品进行转录组测序,并分析数字表达谱,筛选开花调控相关基因并进行荧光定量PCR（RT-qPCR）表达验证。转录组测序共产生27.52 Gb原始数据,注释到8大功能数据库（GO、COG、KEGG、KOG、NR、Pfam、Swiss-Prot、eggNOG）上的 unigene 总数为35 179个。通过GO分类和KEGG Pathway 富集性分析,将unigene分别归于55个GO类别和126个代谢途径。差异表达基因分析显示,花芽未分化期较花芽分化始期有2 253个基因上调,2 032个基因下调;花芽分化始期较花芽分化盛期有1 770个基因上调,1 901个基因下调;花芽未分化期较花芽分化盛期有1 865 个基因上调,2 042个基因下调。发掘出大量的开花相关的基因涉及5个开花调控途径（光周期途径、春化途径、赤霉素途径、自主途径和年龄途径）。筛选出gene.Gb_17618（GI序列）、gene.Gb_19790（FT/TFL1序列）、gene.Gb_16301（AG序列）、gene.Gb_28337（花发育MADS-box序列）、gene.Gb_01884（SOC1序列）和gene.Gb_41704（CO序列）等6个银杏花芽分化差异表达关键基因序列,荧光定量PCR检测表达水平与转录组结果一致。     

High expression of BIRC5 enhances cell viability,inhibits apoptosis and is associated with poor prognosis in gastric cancer

AIM To investigate the expression of baculoviral inhibitor of apoptosis protein repeat-containing protein 5 （BIRC5） in gastric cancer tissue and its relationship with prognosis of gastric cancer patients, and to explore the effect of BIRC5 knock-down on the viability and apoptosis of gastric cancer cells. METHODS The expression of BIRC5 was detected by immunohistochemistry in 67 cases of gastric cancer tissues and paracancerous tissues for analyzing the relationships with clinicopathological characteristics. The mRNA and protein expression levels of BIRC5 in gastric carcinoma cell lines （AGS, MKN-1 and MGC-803） and normal gastric epithelial cell line GES-1 were detected by RT-qPCR and Western blot. The AGS cells were divided into blank group （no treatment）, Ctr-sh group （blank plasmid transfection） and BIRC5-sh group （BIRC5-shRNA plasmid transfection）. The interference efficiency of BIRC5-shRNA was evaluated by Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry, and the levels of apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. RESULTS BIRC5 was mainly expressed in cytoplasm, and the positive expression rate of BIRC5 in the gastric cancer tissues was higher than that in the adjacent tissues （P<0.01）. The positive rates of BIRC5 in the gastric cancer patients at TNM Ⅲ~Ⅳ stages and with lymph node metastasis were higher than those in the patients at TNM Ⅰ~Ⅱ stages and without lymph node metastasis, respectively （P<0.05）. The survival time of the patients with positive BIRC5 expression was shorter than that of the patients with negative BIRC5 expression （P=0.011 2）. The cell viability in BIRC5-sh group was lower than that in blank group and Ctr-sh group at time points of 48, 72 and 96 h. The apoptotic rate in BIRC5-sh group was increased compared with blank group and Ctr-sh group. The protein levels of cleaved caspase-3 and Bax in BIRC5-sh group were higher than those in blank group and Ctr-sh group, while the protein expression of Bcl-2 in BIRC5-sh group was lower than that in blank group and Ctr-sh group （P<0.05）. CONCLUSION High expression of BIRC5 in gastric cancer indicates poor prognosis. BIRC5 promotes the growth of gastric cancer cells and inhibits apoptosis.     

Functional analysis of differential microRNA expression profile in mouse fibrotic liver tissues induced by CCl4

AIM:To discover the expression profile of microRNAs (miRNAs) in mouse fibrotic liver tissues induced by carbon tetrachloride (CCl4), and to investigate the functions of these differential miRNAs based on the gene ontology (GO) analysis and KEGG Pathway analysis. METHODS:The mice were randomly divided into normal group and model group. Liver fibrosis was induced by subcutaneous injection of CCl4. miRNA expression profile of the liver tissues was assayed by a mouse miRNA microarray (Agilent 12.0). The differential expression of miRNAs between the normal and model mice was screened, and GO analysis and KEGG Pathway analysis were performed to determine the functions of these differential miRNAs. RESULTS:Thirty-nine miRNAs with differential expression were discovered in the model mice compared with the normal mice, among which 23 were up-regulated and 16 were down-regulated. GO analysis and KEGG Pathway analysis indicated that most pathological processes of liver fibrosis regulated by miRNAs included cell proliferation and activation, cell apoptosis, cell cycle, cell adhesion, inflammatory reaction, cell migration, transforming growth factor β (TGF-β) signaling pathway, Wnt signaling pathway and proteometabolism process. GO analysis revealed that the key up-regulated miRNAs were mmu-miR-322, mmu-miR-15b, mmu-miR-195, mmu-miR-200b and mmu-miR-214, and the key down-regulated miRNAs were mmu-miR-16, mmu-miR-130a, mmu-miR-101b, mmu-miR-30a and mmu-miR-30e. Analyzing the target genes screened out by GO analysis and Pathway analysis simultaneously, we found that the key up-regulated miRNAs included mmu-miR-200b, mmu-miR-322, mmu-miR-106b, mmu-miR-23a and mmu-miR-15b, and the key down-regulated miRNAs included mmu-miR-16, mmu-miR-30e, mmu-miR-30c, mmu-miR-30a and mmu-miR-130a. CONCLUSION: Differential expression of miRNAs is discovered in mouse fibrotic liver tissues induced by CCl4 compared with the normal liver tissues. Most of the pathological processes involved in liver fibrosis may be regulated by miRNA, such as cell proliferation and activation, cell adhesion and apoptosis, cell migration and differentiation, metabolism, TGF-β receptor signaling pathway and so on.     

葡萄着色期果皮蛋白质组分析

为了研究葡萄不同着色期果皮中蛋白质表达特征,以着色初期、中期和后期等3个着色时期的葡萄果皮为研究对象,运用2-DE、MALDI-TOF/TOF-MS质谱技术以及生物信息学方法对差异蛋白进行分析,结果表明:(1)双向凝胶电泳显示,果皮着色3个时期有1050个高度重复的蛋白点,差异显著的蛋白点有162个,其中108个蛋白得到鉴定,有87个蛋白映射到葡萄蛋白质组数据库中;3个时期均表达的差异蛋白有20个,且随着果皮颜色加深,差异蛋白数量呈上升趋势。(2)GO分析显示,ATP合成酶β亚基(ATPβ)极显著富集于磷酸核糖代谢过程,对着色初期果皮生理代谢的能量需求具有重要意义。(3)KEGG分析表明,碳代谢、生物固碳、磷酸戊糖、氨基酸生物合成等代谢通路在果皮着色的不同时期显著富集。(4)qRT-PCR分析显示,S–腺苷甲硫氨酸合成酶基因(VvMETK4)在果皮着色后期表达量最高。     

柑橘抗溃疡病转录因子CsAP2-09互作蛋白筛选与分析

以前期获得的柑橘抗溃疡病的正调控转录因子基因 CsAP2-09 超表达植株为材料，利用 GST融合蛋白沉降技术（GST pull-down）联合液相色谱串联质谱（LC–MS/MS）方法筛选并鉴定 CsAP2-09的互作蛋白。首先原核表达并纯化 GST-CsAP2-09 作为诱饵蛋白，然后与 CsAP2-09 超表达植株叶片总蛋白孵育、洗脱后进行 SDS-PAGE 验证，最后进行 LC–MS/MS 鉴定。得到 17 个互作蛋白，将蛋白进行注释、GO 分析、KEGG 分析，发现其中 5 个可能与植物抗病相关（如过氧化氢酶 Cs3g27280）。构建这 17个蛋白的互作网络，其中 14 个蛋白的互作关系得到已有数据的支持。对 CsAP2-09 与互作蛋白的共表达分析发现 CsAP2-09 超表达引起了 16 个蛋白表达上调和 1 个蛋白表达下调。     

TMT-tagged quantitative proteomics analysis of differentially expressed proteins and enrichment of signaling pathways in nasopharyngeal carcinoma cells with or without SATB1 over-expression

AIM: To analyze the effects of special AT-rich sequence binding protein 1 (SATB1) expression on the protein expression profiles in human nasopharyngeal carcinoma (NPC) cells, and to enrich the differential signaling pathways through bioinformatics analysis. METHODS: SATB1 over-expressing lentivirus and negative control lentivirus were used to infect the CNE1 cells, and then the cell lines were obtained by puromycin stressed method. The total proteins of the 2 cells were extracted, and the differentially expressed proteins were screened by TMT-labeled protein quantification technique and tandem mass spectrometry. The mRNA levels of the differential protein-coding genes were verified by RT-qPCR. GO analysis was used to annotate and enrich the differentially expressed proteins, and the KEGG database was used to enrich and analyze the signaling pathways of differential proteins. RESULTS: SATB1 over-expressing CNE1 cells were established through infected with associated lentivirus. Compared with the control group, 278 differentially expressed proteins were identified in SATB1 over-expressing CNE1 cells, in which 115 were up-regulated and 163 were down-regulated. 10 representative differential protein-coding genes were verified by RT-qPCR, which showed the consistence with the proteomic results. GO analysis indicated differentially expressed proteins were mainly involved in cellular processes, single-organism processes, biological regulation, metabolic processes, protein binding and catalysis. Cell components of differentially expressed proteins mainly existed in cell part, cells and organelles. KEGG analysis showed that differentially expressed proteins were involved in signaling pathways closely related to tumors, includeing MAPK, PI3K-Akt, AMPK, JAK-STAT, p53, PPAR, Hippo and HIF-1 signaling pathways. CONCLUSION: Over-expression of SATB1 significantly alters the protein expression profiles in the NPC cells and affects multiple signaling pathways closely related to tumors. Proteomics also provides a possible macro approach to the screening of molecular mechanisms, therapeutic and prognostic targets for NPC.     

A 3D cell culture system to study epithelial-mesenchymal transition and drug resistance of lung cancer

AIM To explore the molecular mechanism of transforming growth factor-β1 （TGF-β1）-induced epithelial-mesenchymal transition （EMT） and cisplatin resistance in three-dimensionally cultured lung cancer cells. METHODS Under three-dimensional culture condition, the morphological changes and protein expression changes of human non-small-cell lung cancer 95D cells were observed by inversed fluorescence microscopy, scanning electron microscopy, laser scanning confocal microscopy and Western blot before or after TGF-β1 stimulation. The cisplatin sensitivity was determined by MTT assay. RESULTS Under the three-dimensional culture condition, the structure of 95D cell spheroids after TGF-β1 stimulation collapsed, the cells were dispersed and migrating, and the spheroids merged with each other. The results of laser confocal microscopy showed that E-cadherin protein expression in the 95D cells did not changed after TGF-β1 stimulation, and the protein expression of N-cadherin and vimentin was significantly up-regulated. The results of Western blot showed that the expression of E-cadherin was down-regulated after TGF-β1 stimulation, and the protein levels of N-cadherin, vimentin, phosphorylated AKT and phosphorylated mTOR were up-regulated. LY294002 and rapamycin reversed TGF-β1-induced expression of the above proteins. The results of MTT assay showed that TGF-β1 reduced the sensitivity of three-dimensionally cultured 95D cells to cisplatin, while LY294002 and rapamycin reversed the cisplatin resistance of the 95D cells stimulated by TGF-β1. CONCLUSION TGF-β1 induces the EMT and cisplatin resistance of three-dimensionally cultured lung cancer cells through the activation of PI3K/AKT/mTOR signaling pathway.     

Effect of abnormal expression of PDK4 on glycolysis and proliferation in prostate cancer cells

AIM To investigate the expression of pyruvate dehydrogenase kinase 4 （PDK4） in prostate cancer tissue and its effect on glycolysis and growth of prostate cancer cells. METHODS Immunohistochemistry was used to compare the expression differences of PDK4 protein in benign prostatic hyperplasia （BPH） and prostate cancer tissues. The expression levels of PDK4 in normal prostatic epithelial cells （RWPE-1） and different prostate cancer cell lines （PC3, LNCaP, DU145 and C4-2） were detected by RT-qPCR and Western blot. Recombinant plasmid carrying PDK4-shRNA was constructed, and the expression of PDK4 in prostate cancer PC3 cells was down-regulated by transfection with PDK4-shRNA. The changes in glycolysis level of PC3 cells before and after transfection were determined by cell glycolysis kit, and the effects of PDK4 on the viability and cell cycle distribution of PC3 cells were detected by CCK-8 assay and flow cytometry. RESULTS In prostate cancer tissues, the expression level of PDK4 protein was significantly higher than that in BPH tissues （P<0.05）, and the analysis of immunohistochemical score showed that prostate cancer tissues with high Gleason score displayed significantly higher PDK4 expression than those with low Gleason score （P<0.05）. Compared with normal prostatic epithelial cells, RT-qPCR and Western blot results indicated that the expression level of PDK4 was also significantly increased in prostate cancer cell lines （P<0.05）. In addition, CCK-8 assay results showed that the viability of prostate cancer PC3 cells was significantly decreased after knockdown of PDK4 expression （P<0.05）. The results of flow cytometry demonstrated that knockdown of PDK4 expression in PC3 cells resulted in a notable increase in G₀/G₁ phase arrest （P<0.05）. CONCLUSION PDK4 is highly expressed in prostate cancer tissues and cell lines, and significantly increases in prostate cancer with high Gleason score. In addition, down-regulation of PDK4 expression significantly inhibits glycolysis and growth of prostate cancer cells, resulting in cell cycle arrest at G₀/G₁ phase.     

辣椒转录组SNP挖掘及多态性分析

 利用SNP分析软件从辣椒（Capsicum annuum L.）251 068条Unigenes中筛选出18 159个SNP,其中有1 781个SNP位点被匹配在1 291个注释基因上,基因功能分类和代谢途径分析表明,其中有853个基因参与初生代谢（28.7%）、细胞代谢（17.3%）、生物合成过程（15.7%）,另有125个（9.7%）基因序列参与新陈代谢途径,53条（4.1%）序列参与次生代谢产物合成途径,31条（2.4%）序列参与植物激素合成途径。 EST-SNP序列中4 172条（22.9%）满足设计CAPS引物条件,为了验证EST-SNP正确性,并选取了15对CAPS引物对5份辣椒材料进行扩增,结果发现有8对（53.3%）引物表现出多态性。表明筛选出这些EST-SNP标记可作为辣椒基因分型、图谱构建等的候选分子标记。     

广叶绣球菌子实体不同发育阶段的比较蛋白质组学分析

利用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)标记结合二维液相色谱串联质谱(two-dimensional liquid chromatography tandem mass spectrometry,2D LC-MS/MS)技术,研究广叶绣球菌(Sparassis latifolia)原基期、幼菇期和成熟子实体阶段的差异表达蛋白质组.采用Q-Exactive质谱鉴定并经ProteinPilot软件搜库,对所获得的差异蛋白进行GO (gene ontology)、KEGG(kyoto encyclopedia of genes and genomes)和转录因子注释分析.结果共鉴定到可信蛋白2305个,其中2219个蛋白具有相对定量信息.与原基期相比,幼菇期显著上调蛋白104个,下调蛋白142个,子实体阶段显著上调蛋白155个,下调蛋白460个.GO分子功能提示这些差异性蛋白主要参与催化活性、蛋白结合和水解酶活性.KEGG代谢通路分析结果显示,差异蛋白主要涉及碳代谢、氨基酸合成、核糖体、糖酵解/糖衍生等代谢过程.差异蛋白中与信号传导和转录因子相关的蛋白数量分别为27个和7个.     

miR-92b-5p attenuates renal injury and inflammatory response in diabetic nephropathy rats by targeting HMGB1

AIM To investigate the effect of microRNA-92b-5p （miR-92b-5p） on renal injury and inflammatory response in diabetic nephropathy （DN） rats and its mechanism. METHODS The rats were divided into control group， DN group， lentiviral negative control （LV-NC） group， LV-miR-92b group， LV-high mobility group protein B1 （LV-HMGB1） group and miR-92b+HMGB1 group， with 15 rats in each group. After fasting for 12 h， the model rats were intraperitoneally injected with streptozotocin at dose of 60 mg/kg， and the control rats were intraperitoneally injected with an equal volume of citrate buffer. Three days later， the rats in each treatment group were intravenously injected with 100 μL LV-NC， LV-miR-92b and LV-HMGB1 （1×10¹¹ U/L） twice a week for 8 consecutive weeks. Urinary protein， blood glucose， blood urea nitrogen and serum creatinine were detected by an automatic biochemical analyzer. The expression of miR-92b-5p and HMGB1 mRNA was detected by RT-qPCR. The targeting relationship between miR-92b-5p and HMGB1 was verified by dual-luciferase reporter assay. HMGB1 expression in kidney tissue was detected by Western blot. The kidney damage was observed by HE staining. The apoptosis was detected by flow cytometry. The levels of interleukin-6 （IL-6）， IL-1β and tumor necrosis factor-α （TNF-α） in renal tissues were detected by ELISA. RESULTS In DN model rats， miR-92b-5p was down-regulated， while HMGB1 was highly expressed. There was a binding site between miR-92b-5p and HMGB1 3'-untranslated region. High expression of miR-92b-5p inhibited the luciferase activity of the wild-type HMGB1 plasmid （P<0.01）， but had no effect on the luciferase activity of the mutant HMGB1 plasmid. Compared with DN group， urinary protein， blood glucose， serum creatinine and blood urea nitrogen in LV-miR-92b group were significantly reduced （P<0.01）. The degree of hyperplasia， swelling and inflammatory cell infiltration of glomerular mesangium and basement membrane tubules， the apoptosis rate of renal tissues， and the content of IL-6， IL-1β and TNF-α in renal tissues were significantly decreased （P<0.01）. Co-transfection of LV-HMGB1 significantly reversed the effect of miR-92b-5p on DN rats. CONCLUSION miR-92b-5p reduces renal injury and inflammatory response in DN rats by targeting HMGB1 and down-regulating its expression.     

枳感染柑橘衰退病病毒后的应答基因分析

 枳（Poncirus trifoliata L.）高抗柑橘衰退病病毒（Citrus tristeza virus,CTV）,但相关抗性机理仍不为人所知。以嫁接健康枝条的无毒枳作为对照,用Affymetrix柑橘基因芯片分析了感染CTV后枳叶片中基因的表达变化情况。结果共检测到表达量差异倍数≥ 2的基因295个,其中216个表达上调,79个表达下调。BLAST2GO分析发现,差异表达的基因中与抗逆反应相关的基因最多,乙烯、茉莉酸、赤霉素、脱落酸、生长素、水杨酸等植物激素代谢和调节相关基因为数不少,一些编码细胞壁形成或组分相关蛋白的基因差异表达明显。本研究可为揭示枳抗CTV的机理提供了转录组学线索。     

Effects of oxidative stress and inflammatory response on kidney injury in mice with hyperthyroidism

AIM To explore the effects of oxidative stress and inflammatory response on kidney injury induced by hyperthyroidism in mice. METHODS Forty male Kunming mice were randomly divided into control group （n=20） and L-thyroxine （T4） group （n=20）. The mice in T4 group were intraperitoneally injected with T4 diluent at a dose of 1 mg/kg to induce hyperthyroidism, and those in control group were injected with normal saline of the same volume. After 7 weeks, the mice were weighed and dissected, the kidneys were removed and weighed, and the length of tibia was also measured. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the kidney tissues were detected. The pathological changes of the kidney tissues were observed by HE staining. The levels of 4-hydroxynonenal （4-HNE）-modified proteins, interleukin-1 receptor-associated kinase 1 （IRAK1） and tumor necrosis factor receptor-related factor 6 （TRAF6） were determined by Western blot and immunohistochemistry. RESULTS Compared with control group, the body weight of the mice was decreased, while the kidney size and weight were increased significantly in T4 group. In addition, the ratios of kidney weight/body weight and kidney weight/tibia length were also increased （P<0.05）. In T4 group, the renal tubules were enlarged, and the epithelial cells of renal tubules were swollen and exfoliated, with vacuolar degeneration. Furthermore, reduced SOD activity, and increased MDA content and 4-HNE-modified proteins were found in T4 group, all of which were related to oxidative stress （P<0.05）. The levels of inflammation-related proteins IRAK1 and TRAF6 were significantly increased in T4 group （P<0.05）. CONCLUSION Excessive T4 may lead to kidney hypertrophy and injury in mice, and the mechanism may be related to oxidative stress and inflammatory response.     

Relationship between rat hepatic fibrosis and regulation of hepatic stellate cell autophagy by microRNA-181a

AIM To observe the changes of liver structure, the levels of transforming growth factor-β1 （TGF-β1）, microRNA-181a, LC3-II/-I, beclin-1 and collagen deposition in hepatic fibrosis （HF） rats induced by carbon tetrachloride （CCl₄）, and the effect of microRNA-181a on autophagy of rat hepatic stellate cells （HSCs） induced by TGF-β1, and to explore the possible mechanism of microRNA-181a in regulating HSC activation and HF. METHODS Wistar rats （n=40） were randomly divided into 5 groups （with 8 in each）： control group （subcutaneous injection of olive oil, 3 mL/kg, twice a week）, and CCl₄-induced HF groups of 2, 4, 6 and 8 weeks （subcutaneous injection of 40% CCl₄, 3 mL/kg, twice a week for 2, 4, 6 and 8 weeks, respectively）. Masson staining was used to evaluate the changes of HF in rats. The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA. The level of microRNA-181a in rat liver tissues was detected by RT-qPCR. The protein levels of LC3-II/-I, beclin-1, α-smooth muscle actin （α-SMA）, collagen type I （Col I） and collagen type Ⅲ （Col Ⅲ） in rat liver tissues were measured by Western blot. HSC-T6 cells were transfected with microRNA-181a inhibitor, or pretreated with the autophagy inhibitor 3-methyladenine （3-MA）, before treatment with TGF-β1 to stimulate autophagy. The expression of microRNA-181a, LC3-II/-I, beclin-1, α-SMA, Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot. RESULTS The levels of TGF-β1, microRNA-181a, LC3-II/-I ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF, and microRNA-181a expression showed a positive correlation with autophagy-associated proteins （P<0.01）. MicroRNA-181a level was significantly increased, which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181a inhibitor, along with suppression of autophagy and cell activation （P<0.01）, which were similar to the effects of 3-MA treatment. CONCLUSION CCl₄ promotes rat HF, the microRNA-181a expression of liver tissue, and autophagy in a time-dependent manner. Reducing the expression of microRNA-181a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1. The regulation of HSC autophagy by microRNA-181a may be involved in rat HF.     

PPARγ affects epithelial-mesenchymal transition in renal tubular epithelial cells under high glucose by regulating AKT/FAK signaling pathway through PTEN

AIM To investigate the role of peroxisome proliferator-activited receptor γ （PPARγ） in the regulation of PTEN/AKT/FAK signaling pathway and epithelial-mesenchymal transition （EMT） in renal tubular epithelial cells grown in high-glucose environment. METHODS Renal tubular epithelial cells （NRK52E cells） cultured in high glucose were used as an in vitro model system. PPARγ was over-expressed or knocked down in these cells, and its effect on PTEN expression was determined by RT-qPCR, immunofluorescence and Western blot. The changes of EMT-related proteins were also measured. The PPARγ inhibitor GW9662 and the PPARγ agonist rosiglitazone were used along with PTEN over-expression or knockdown to determine whether the effects of PPARγ were mediated through PTEN. RESULTS PPARγ over-expression resulted in the increased expression of PTEN at mRNA and protein levels, the up-regulation of E-cadherin, and the down-regulation of vimentin and α-SMA. Knockdown of PPARγ expression reduced the mRNA and protein levels of PTEN, down-regulated E-cadherin, and up-regulated vimentin and α-SMA （P<0.05）. Treatment of the NRK-52E cells with GW9662 decreased PTEN expression and increased the protein levels of p-AKT （Thr308）, FAK and p-FAK （Tyr397）. These effects were rescued by PTEN over-expression. Treatment of the NRK-52E cells with rosiglitazone increased PTEN expression and decreased the protein levels of p-AKT （Thr308）, FAK and p-FAK （Tyr397）. These effects were rescued by PTEN knockdown. These changes were all statistically significant （P<0.05）. CONCLUSION PPARγ regulates the mRNA and protein expression of PTEN in renal tubular epithelial NRK52E cells, and affects EMT in renal tubular epithelial cells. The regulation of AKT/FAK signaling pathway by PPARγ is primarily mediated by PTEN.     

Preliminary study on miRNA expression spectrum related to chemotherapy resistance in colon cancer

AIM: To screen the chemotherapy resistance-related microRNAs (miRNAs) of colon cancer using gene chip technique, and to explore the mechanism of miRNAs regulating chemotherapy resistance. METHODS: Gene chip technique was used to analyze the expression of miRNAs in colon cancer cell line HCT8 and vincristine-resistant cell line HCT8/v, and screen the miRNAs with significantly different expression. The results were verified by RT-qPCR. The target genes of these miRNAs were predicted, and the Gene Ontology (GO) analysis and the signaling pathway analysis of the predicted genes were carried out. RESULTS: Altogether 342 miRNAs with significantly differential expression were selected, in which 190 were up-regulated, and 152 were down-regulated. The verification results of RT-qPCR showed that the expression of miR-125-5p, miR-181c-5p and miR-153-3 was consistent with the results of chip detection. The expression of miR-130a-3p and miR-149-3p was not consistent with the results of chip detection. The results of GO analysis showed that the main pathway of chemotherapy resistance-related genes was RNA polymerase II regulatory region sequence-specific DNA binding. The chemotherapy resistance-related genes played roles mainly through positive regulation and are mainly located in intracellular membrane-bound organelles. The results of KEGG analysis showed that the pathways associated with the most enriched chemotherapy resistance-related genes were axon guidance pathway, insulin signaling pathway, and phospholipase D signaling pathway.CONCLUSION: miRNAs are closely related to chemotherapy resistance in colon cancer. Through the researches on miRNAs, we can have a deeper understanding of the mechanism of chemotherapy resistance and provide new ideas for reversing chemotherapy resistance in colon cancer.     

Expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and its clinical significance

AIM: To evaluate the expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and the significance. METHODS: The patients with breast cancer (n=150) in our hospital from January 2015 to January 2017 were selected as study object. The tumor tissue samples of these patients were obtained from paraffin section of breast cancer by surgical resection with complete clinicopathological data. The corresponding paracancerous tissue sam-ples were taken from the non-tumor tissue samples from the above breast cancer patients, which were 0.5~1 cm away from the tumor tissue. The methods of real-time PCR and Western blot were performed to examine the expression of Wnt-1 and β-catenin at mRNA and protein levels. Human breat cancer MCF-7 cells were divided into 3 groups:control group (MCF-7 cells without treatment), agonist group[MCF-7 cells+Wnt3a (1 mg/L)] and antagonit group[MCF-7 cells+DKK1 (16 μmol/L)]. The expression of Wnt-1 and β-catenin at mRNA and protein levels was detected by real-time PCR and Western blot. RESULTS: Compared with the paracancerous tissues, the expression levels of Wnt-1 and β-catenin were higher in tumor tissues at mRNA and proteins levels (P<0.05). Notably, the positive expression rates of Wnt-1 and β-catenin were significantly higher in tumor tissues than that in the paracancerous tissues. Furthermore, Wnt-1 expression was associated with tumor metastasis (χ²=5.352, P=0.021), tumor stage (χ²=9.412, P=0.002) and tumor size (χ²=9.412, P=0.002). In addition, β-catenin expression was also associated with tumor metastasis (χ²=9.851, P=0.002) and tumor stage (χ²=5.661, P=0.017). Compared with control group, the expression of Wnt-1 and β-catenin at mRNA and protein levels in agonist group was increased (P<0.05),while that in antagonist group was decreased (P<0.05). CONCLUSION: The expression levels of Wnt-1 and β-catenin related with Wnt/β-catenin signaling pathway are increased in the breast cancer, which are closely related to the malignant state of the tumor.