白藜芦醇抑制肠癌细胞焦亡的作用*

目的: 研究白藜芦醇(Res)对肠癌细胞焦亡的影响。方法: ①葡聚糖硫酸钠(DSS)诱发小鼠结肠癌(CRC)实验:30只C57BL/6小鼠随机分为正常对照组(Contro组),氧化偶氮甲烷(AOM)组,AOM/DSS组,AOM/DSS+Res组和Res组,每组6只,造模周期共70 d。第1周第1日对AOM组、AOM/DSS组和AOM/DSS+Res组小鼠AOM(10 mg/kg)腹腔注射一次,无菌水饮水,1% DSS水供AOM/DSS组和AOM/DSS+Res组饮用,对AOM/DSS+Res组和Res组小鼠灌胃给予Res(50 mg/kg),造模结束后,取小鼠结肠组织固定、包埋、切片; IHC和Western blot检测小鼠结肠组织NLRP3、Caspase-1、IL-18蛋白表达情况。②离体实验:HCT 116细胞给予Res(2.4 μg/L)以及转染miR-31,加Res实验分为4组,分别标记0 h、12 h、24 h、48 h组;细胞转染分组为5组,即control组、miR-31 mimic组、miR-31 mimic+Res组、miR-31inhibitor组、miR-31inhibitor+Res组,48 h后收集细胞,每组设置三个复孔,并通过Western blot检测细胞NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达情况。结果: 动物实验:与control组相比较,AOM/DSS组NLRP3、Caspase-1、IL-18蛋白表达显著升高(P<0.01),AOM/DSS+Res组NLRP3、Caspase-1、IL-18蛋白表达水平相较于AOM/DSS组有显著下降(P<0.01);细胞实验:与control组相比,miR-31 mimic组NLRP3(P<0.01)、GSDMD-N(P<0.05)、IL-18(P<0.01)蛋白表达显著升高, miR-31 inhibitor组NLRP3、GSDMD-N、IL-18蛋白表达显著降低(P<0.05)。结论: Res可通过细胞焦亡抑制结肠癌。     

过表达miR-31对结肠炎模型小鼠TLR4/NF-κB信号通路及凋亡蛋白的调控

目的: 探讨miR-31对DSS诱发结肠炎小鼠TLR4/NF-κB信号通路和凋亡相关蛋白的影响。方法: ①小鼠结肠炎实验:用1%葡聚糖硫酸钠(DSS)诱发小鼠溃疡性结肠炎(UC)。14只FVB非转基因小鼠随机分为control组(n=6),DSS组(n=8),16只FVB miR-31转基因小鼠随机分为miR-31过表达组(n=8),miR-31过表达+DSS 组(n=8),DSS溶于水后通过饮水给予小鼠。DSS组和miR-31+DSS组第一周饮用1%DSS水,第二周饮用正常无菌水,第三周饮用1%DSS水,如此5周后造模完成,之后留取小鼠的结肠组织,通过Western blot和IHC检测小鼠结肠组织NF-κB p65、TLR4、Bax、Bcl-2蛋白的表达;TUNEL检测小鼠结肠组织细胞凋亡。②细胞培养实验:在人结肠上皮细胞系HCT 116细胞中通过脂质体转染的方法转染miR-31 mimic和inhibitor,使miR-31过表达或敲低,每组均进行三次重复,48 h后收取细胞,通过Western blot检测NF-κB p65、TLR4蛋白的表达。结果: ①动物实验中,与control组相比,小鼠结肠组织中DSS组和miR-31过表达组NF-κB p65、TLR4蛋白表达水平和凋亡细胞指数均显著升高(P＜0.05或P＜0.01),Bcl-2/Bax比值显著降低(P＜0.05或P＜0.01);且与DSS组相比,miR-31+DSS组NF-κB p65、TLR4蛋白表达水平和凋亡细胞指数也显著升高(P＜0.01),Bcl-2/Bax比值显著降低(P＜0.01)。②细胞实验中,与control组相比, HCT 116细胞过表达miR-31组的NF-κB p65、TLR4蛋白表达水平均显著升高(P＜0.05或P＜0.01),敲低miR-31组的NF-κB p65、TLR4蛋白表达水平下降(P＜0.05)。结论: miR-31通过促进TLR4/NF-κB信号通路和介导肠上皮细胞凋亡促进结肠炎的发展。     

乙酰哈巴苷通过Wnt信号通路诱导结肠癌HCT116细胞凋亡

本文探讨乙酰哈巴苷诱导结肠癌HCT116细胞凋亡机制。采用流式细胞术检测乙酰哈巴苷对HCT116细胞周期及凋亡的影响,发现细胞周期被阻滞在G1期,0.75 mmol/L乙酰哈巴苷处理48 h后,细胞凋亡明显增加。转录组测序显示乙酰哈巴苷引起HCT116细胞1 921个mRNA上调和2 208个mRNA下调,可显著影响Wnt信号通路。RT-qPCR和Western blot验证实验表明,乙酰哈巴苷处理后,HCT116细胞中β-catenin、CyclinD1、Survivin、Bcl-2和c-Myc蛋白显著降低(P<0.05),凋亡相关蛋白Bax和cleaved-caspase3表达增加(P<0.05)。上述结果表明,乙酰哈巴苷主要通过Wnt信号通路抑制结肠癌细胞增殖并诱导细胞凋亡。     

氯化两面针碱对小鼠溃疡性结肠炎的干预作用及其机制*

目的:探讨氯化两面针碱(NC)通过靶向miR-31对葡聚糖硫酸钠(DSS)诱发小鼠结肠炎的保护作用及其机制。方法:用1%DSS诱发小鼠溃疡性结肠炎(UC)。30只雄性C57BL/6小鼠随机分为正常对照组(Control)(n=7),DSS组(n=8),DSS+NC组(7.27 mg/kg)(n=8)和NC组(n=7),饮水给予DSS,灌胃给予氯化两面针碱。造模周期为3周,分别为Control组和NC组每天饮用无菌水,DSS组和DSS+NC组第一周饮用1% DSS水,第2周正常饮水,第3周1% DSS水。造模最后一周给予Control组和DSS组小鼠0.5% 羧甲基纤维素钠(CMC-Na)灌胃,DSS+NC组和NC组给予NC灌胃。造模完成后,观察小鼠结肠炎相关的疾病活动指数(DAI),HE染色进行结肠组织病理评分,qPCR检测小鼠结肠组织miR-31的表达水平,Western blot检测小鼠结肠组织炎症蛋白NF-κB和COX-2的表达情况。结果:①与DSS组相比,DSS+NC组的 DAI 显著降低(P＜0.01),结肠病理损伤明显改善;②与Control组相比,DSS组小鼠结肠组织miR-31表达显著升高(P＜0.01),DSS+NC组miR-31的表达水平显著低于DSS组(P＜0.05);③与DSS组相比,DSS+NC组中的炎症蛋白NF-κB和COX-2表达水平显著下降(P＜0.05)。结论:氯化两面针碱对DSS诱导的小鼠溃疡性结肠炎有明显的治疗作用,其抗炎机制与下调miR-31的表达有关。     

苦荞凝集素靶向抑制结肠癌细胞HCT116增殖的机制

苦荞凝集素（tartary buckwheat lectin,TBL）是一类兼具核糖体失活蛋白N-糖苷酶活性和芦丁水解酶活性的糖蛋白,具有显著的抑制结肠癌细胞增殖的功能。先前研究表明,TBL可以下调结肠癌细胞HCT116中包括miR-135a 和miR-135b在内的一些oncomiRs的表达,并且可以抑制结肠癌细胞HCT116的增殖。因此,我们推测TBL可能通过调控oncomiRs从而抑制HCT116细胞的增殖。本研究中,我们构建了包含miR-135a 和miR-35b结合位点的腺瘤性结肠息肉病基因（adenomatous polyposis coli,APC）的3′-UTR区。双荧光素酶报告系统表明,经过TBL处理后,实验组的荧光素酶活性较对照组高,而且用miR-135a&b的反义核酸anti-miR-135a&b处理后和实验组有相似的结果。Western 印迹分析表明,TBL处理HCT116细胞24 h后,细胞中APC和p-β-catenin的表达上调,总β-catenin的表达下调,且存在剂量依赖效应,而对GSK-3β的表达无明显影响。进一步探究了TBL进入细胞的方式,我们发现,加入TBL作用2 h后,其主要存在于HCT116细胞的表面,部分进入细胞内部,在细胞表面可以同半乳糖凝集素galectin-3竞争结合细胞膜表面受体,并且存在剂量和时间依赖性。这些结果表明：TBL以HCT116细胞表面的galectin-3受体为靶点,通过内吞作用进入细胞,进而调控HCT116细胞中miR-135a&b的表达,影响Wnt信号通路而抑制结肠癌细胞的增殖。     

FBXO31基因对宫颈癌细胞增殖的影响

为了探讨FBXO31在宫颈癌中的表达情况及其对宫颈癌细胞增殖的影响及其可能机制,本研究采用实时定量PCR法检测FBXO31在宫颈癌组织中的表达水平;MTT法检测Hela细胞增殖能力;流式细胞术检测Hela细胞周期分布;Western blotting检测Hela细胞FBXO31、β-catenin、CyclinD1和c-Myc蛋白的表达水平。研究结果表明,FBXO31在宫颈癌组织中表达明显下调(p0.05)。FBXO31过表达能够明显抑制宫颈癌Hela细胞增殖能力。与空载质粒组比较,过表达FBXO31组的G1期细胞数显著增加,S期细胞数明显降低(p0.05)。本研究还发现FBXO31过表达能明显下调β-catenin蛋白、cyclin D1和c-Myc蛋白水平(p0.05)。本研究结论表明,FBXO31基因在宫颈癌中低表达;过表达FBXO31基因可通过抑制Wnt/β-catenin通路从而抑制宫颈癌细胞增殖。     

棉铃虫转录因子c-Myc基因在滞育和非滞育蛹脑中的表达分析及多克隆抗体制备

【目的】c-Myc是近年来研究较多的转录因子,也是受Wnt/β-catenin信号通路调节的重要靶标。本研究旨在克隆棉铃虫 Helicoverpa armigera c-Myc基因,从核酸水平初步调查 c-myc 在滞育和非滞育蛹脑中的表达情况,同时制备其蛋白的多克隆抗体。【方法】通过RACE方法克隆棉铃虫 c-myc 基因的cDNA,运用RT-PCR方法比较滞育和非滞育蛹脑中Har-c-myc基因的表达情况。根据获取的序列构建原核表达载体,在大肠杆菌 Escherichia coli 中进行表达,纯化后免疫新西兰兔,制备了多克隆抗体。【结果】克隆了棉铃虫 c-myc 基因,核酸水平的研究表明滞育蛹脑中 c-myc 表达水平明显低于非滞育蛹脑。成功地在大肠杆菌中表达了c-Myc部分肽段并通过镍柱纯化获得了较纯的重组蛋白。制备的c-Myc抗体效价达到了1:125 000。【结论】滞育蛹脑中 Har-c-myc 的表达下调。获得了抗棉铃虫c-Myc的多克隆抗体。本研究的成果为后续进一步深入研究棉铃虫Wnt/β-catenin信号通路在棉铃虫发育中的作用奠定了基础。     

4-1BBL通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移

4-1BB和4-1BB配体(4-1BBL),又被称为CD137和CD137配体,分别属于肿瘤坏死因子(TNF)受体和配体家族的成员。4-1BBL 与4-1BB相互作用可以激活T细胞免疫应答。因此,4-1BBL一直在抗肿瘤免疫应答中发挥经典的免疫共刺激分子作用。近期研究发现,4-1BBL在肿瘤细胞中另有其他的生物学功能,但4-1BBL在胃癌进展过程中的功能尚不明确。本文探讨了4-1BBL在人胃癌细胞中的生物学功能和分子作用机制。首先,通过检索TCGA和Kaplan Meier plotter数据库发现,4-1BBL在胃癌组织中的表达显著高于癌旁组织（P<0.001）,且4-1BBL的高表达与胃癌的不良预后正相关（P<0.05）。细胞生物学的结果显示,敲除4-1BBL明显抑制胃癌细胞的增殖（P<0.05）、侵袭和迁移（P<0.05）,促进胃癌细胞的凋亡（P<0.05）;另外,蛋白质免疫印迹结果表明,敲除4-1BBL可使β-联蛋白、c-Myc和细胞周期蛋白D1(cyclin D1)的蛋白质表达水平下降,抑制Wnt/β-catenin信号通路。相反,过表达4-1BBL则显著促进胃癌细胞增殖（P<0.05）、侵袭和迁移（P<0.05）,减少胃癌细胞的凋亡（P<0.05）;且过表达4-1BBL促进β-联蛋白(β-catenin)、c-Myc和细胞周期蛋白D1的蛋白质表达,激活Wnt/β-catenin信号通路。综上所述,4-1BBL可通过激活Wnt/β-catenin信号通路促进人胃癌细胞的增殖和迁移。     

苦荞凝集素靶向抑制结肠癌细胞HCT116增殖的机制北大核心CSCD

苦荞凝集素(tartary buckwheat lectin,TBL)是一类兼具核糖体失活蛋白N-糖苷酶活性和芦丁水解酶活性的糖蛋白,具有显著的抑制结肠癌细胞增殖的功能。先前研究表明,TBL可以下调结肠癌细胞HCT116中包括miR-135a和miR-135b在内的一些oncomiRs的表达,并且可以抑制结肠癌细胞HCT116的增殖。因此,我们推测TBL可能通过调控oncomiRs从而抑制HCT116细胞的增殖。本研究中,我们构建了包含miR-135a和miR-135b结合位点的腺瘤性结肠息肉病基因(adenomatous polyposis coli,APC)的3'-UTR区。双荧光素酶报告系统表明,经过TBL处理后,实验组的荧光素酶活性较对照组高,而且用miR-135a&b的反义核酸anti-miR-135a&b处理后和实验组有相似的结果。Western印迹分析表明,TBL处理HCT116细胞24 h后,细胞中APC和p-β-catenin的表达上调,总β-catenin的表达下调,且存在剂量依赖效应,而对GSK-3β的表达无明显影响。进一步探究了TBL进入细胞的方式,我们发现,加入TBL作用2 h后,其主要存在于HCT116细胞的表面,部分进入细胞内部,在细胞表面可以同半乳糖凝集素galectin-3竞争结合细胞膜表面受体,并且存在剂量和时间依赖性。这些结果表明:TBL以HCT116细胞表面的galectin-3受体为靶点,通过内吞作用进入细胞,进而调控HCT116细胞中miR-135a&b的表达,影响Wnt信号通路而抑制结肠癌细胞的增殖。     

The Low-density Lipoprotein Receptor-related Protein 6 Pathway in the Treatment of Intestinal Barrier Dysfunction Induced by Hypoxia and Intestinal Microbiota through the Wnt/β-catenin Pathway

Our study is to explore the key molecular of Low-density lipoprotein receptor-related protein 6 (LRP6) and the related Wnt/β-catenin pathway regulated by LRP6 during the intestinal barrier dysfunction. Colorectal protein profile analysis showed that LRP6 expression was decreased in dextran sulfate sodium (DSS)-induced colitis mice, and mice received fecal bacteria transplantation from stroke patients. Mice with intestinal hypoxia and intestinal epithelial cells cultured in hypoxia showed decreased expression of LRP6. Overexpression of LPR6 or its N-terminus rescued the Wnt/β-catenin signaling pathway which was inhibited by hypoxia and endoplasmic reticulum stress. In mice overexpressing of LRP6, the expression of β-catenin and DKK1 increased, Bcl2 decreased, and Bax increased. Mice with LRP6 knockout showed an opposite trend, and the expression of Claudin2, Occludin and ZO-1 decreased. Two drugs, curcumin and auranofin could alleviate intestinal barrier damage in DSS-induced colitis mice by targeting LRP-6. Therefore, gut microbiota dysbiosis and hypoxia can inhibit the LRP6 and Wnt/β-catenin pathway, and drugs targeting LRP6 can protect the intestinal barrier.     

血浆miRNA-1、miRNA-21对冠心病合并糖尿病患者PCI术后支架内再狭窄的影响

目的：观察血浆miRNA-1、miRNA-21在冠心病（CHD）与无冠脉病变患者中含量的差异,探讨其在CHD合并糖尿病（DM）患者的经皮冠状动脉介入治疗（PCI）后发生支架内再狭窄（ISR）与非支架内再狭窄（NISR）患者的含量差异及其对ISR的预测价值。方法：入选CHD支架置入术后患者（CHD组,n=187）、无冠脉病变患者（对照组,n=195）。根据指南,将对照组分为正常对照组（n=150）、单纯DM组（n=45）;将CHD组分为单纯CHD组（n=119）、CHD合并DM组（n=68）,又将CHD组分为ISR组（n=48）及NISR组（n=139）;再将ISR组分为单纯ISR组（n=26）和ISR合并DM组（n=22）。收集各组患者血浆,提取总miRNA,检测miRNA-1、miRNA-21含量并分析各组的含量差异。结果：与对照组比较,CHD组miRNA-1、miRNA-21含量均升高（P<0.05）;与NISR组比较,ISR组miRNA-1、miRNA-21含量均升高（P<0.05）。与单纯CHD组比较,CHD合并DM组ISR发生率明显升高;而且与单纯ISR组比较,ISR合并DM组miRNA-21含量升高（P<0.05）,而单纯ISR组与ISR合并DM组miRNA-1含量无明显差异（P<0.05）。Logistics分析表明,与CHD患者相关危险因素DM、miRNA-1、miRNA-21的OR值分别为2.132,3.066,1.924（P<0.05,P<0.01）;ISR患者相关危险因素DM、miRNA-21的OR值分别为3.091,5.654（P<0.05,P<0.01）;CHD合并DM患者PCI术后发生ISR相关危险因素miRNA-21的OR值为9.148（P<0.01）。ROC曲线评价表明,miRNA-1,miRNA-21与CHD患者发生ISR的AUC分别为0.854,0.857;miRNA 21与CHD合并DM患者发生ISR的AUC为0.783。结论：血浆miRNA-1、miRNA-21对诊断CHD患者支架置入术后ISR发生有重要临床意义,miRNA-21对诊断CHD合并DM患者支架置入术后ISR发生尤其有重要临床意义。     

Resveratrol restores sensitivity of glioma cells to temozolamide through inhibiting the activation of Wnt signaling pathway

Malignant gliomas are aggressive primary neoplasms that originate in the glial cells of the brain or the spine with notable resistance to standard treatment options. We carried out the study with the aim to shed light on the sensitization of resveratrol to temozolomide (TMZ) against glioma through the Wnt signaling pathway. Initially, glioma cell lines with strong resistance to TMZ were selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then, the glioma cells were subjected to resveratrol, TMZ, Wnt signaling pathway inhibitors, and activators. Cell survival rate and inhibitory concentration at half maximum value were detected by MTT, apoptosis by flow cytometry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, in vitro proliferation by hanging drop method and β-catenin translocation into nuclei by TOP/FOP-FLASH assay. The expressions of the Wnt signaling pathway-related and apoptosis-related factors were determined by western blot analysis. Nude mice with glioma xenograft were established to detect tumorigenic ability. Glioma cell lines T98G and U138 which were highly resistant to TMZ were selected for subsequent experiments. Resveratrol increased the efficacy of TMZ by restraining cell proliferation, tumor growth, and promoting cell apoptosis in glioma cells. Resveratrol inhibited Wnt2 and β-catenin expressions yet elevated GSK-3β expression. Moreover, the Wnt signaling pathway participates in the sensitivity enhancing of resveratrol to TMZ via regulating O ⁶-methylguanine-DNA methyltransferase (MGMT) expression. Resveratrol sensitized TMZ-induced glioma cell apoptosis by repressing the activation of the Wnt signaling pathway and downregulating MGMT expression, which may confer new thoughts to the chemotherapy of glioma.     

Resveratrol Inhibits Breast Cancer Stem-Like Cells and Induces Autophagy via Suppressing Wnt/β-Catenin Signaling Pathway

Resveratrol, a natural polyphenolic compound, is abundantly found in plant foods and has been extensively studied for its anti-cancer properties. Given the important role of CSCs (Cancer Stem Cells) in breast tumorigenesis and progression, it is worth investigating the effects of resveratrol on CSCs. The article is an attempt to investigate the effects of resveratrol on breast CSCs. Resveratrol significantly inhibits the proliferation of BCSCs (breast cancer stem-like cells) isolated from MCF-7 and SUM159, and decreased the percentage of BCSCs population, consequently reduced the size and number of mammospheres in non-adherent spherical clusters. Accordingly, the injection of resveratrol (100 mg/kg/d) in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice effectively inhibited the growth of xenograft tumors and reduced BCSC population in tumor cells. After the reimplantation of primary tumor cells into the secondary mice for 30 d, all 6 control inoculations produced tumors, while tumor cells derived from resveratrol-treated mice only caused 1 tumor of 6 inoculations. Further studies by TEM (Transmission electron microscopy) analysis, GFP-LC3-II puncta formation assay and western blot for LC3-II, Beclin1 and Atg 7, showed that resveratrol induces autophagy in BCSCs. Moreover, resveratrol suppresses Wnt/β-catenin signaling pathway in BCSCs; over-expression of β-catenin by transfecting the plasmid markedly reduced resveratrol-induced cytotoxicity and autophagy in BCSCs. Our findings indicated that resveratrol inhibits BCSCs and induces autophagy via suppressing Wnt/β-catenin signaling pathway.