利用CRISPR/Cas9系统构建稳定敲除TrxR1基因的HCT-116细胞株北大核心CSCD

为更好地研究靶向硫氧还蛋白还原酶1的小分子化合物的细胞内靶点选择性,利用CRISPR/Cas9系统构建稳定敲除TrxR1基因(编码硫氧还蛋白还原酶1)的HCT-116细胞株。首先根据TrxR1基因序列和CRISPR/Cas9靶点设计原则,设计并选择合适的敲除位点,再根据敲除位点序列设计敲除TrxR1基因的sgRNA干扰序列,以pCasCMV-Puro-U6空质粒载体为骨架构建能表达该sgRNA干扰序列的重组质粒。质粒共转染至HCT-116细胞后,利用嘌呤霉素筛选TrxR1敲除的HCT-116细胞,通过DNA测序、免疫蛋白印迹、TRFS-green荧光探针和细胞内TrxR1酶活力检测等方法鉴定和验证HCT-116细胞的TrxR1基因敲除效果。进一步通过CCK-8实验初步研究靶向TrxR1小分子化合物对细胞内TrxR1酶活力和细胞增殖力抑制的相关性。结果显示,表达sgRNA干扰序列的重组质粒可以敲除HCT-116细胞中TrxR1基因,筛选获得的稳定敲除细胞HCT116-TrxR1-KO中无TrxR1蛋白表达,而靶向TrxR1小分子抑制剂对该细胞无TrxR1酶活力和细胞增殖力抑制效果。本研究利用CRISPR/Cas9系统成功构建了HCT-116的TrxR1基因敲除的稳定细胞株,为进一步研究TrxR1在相关疾病的发生机制和治疗中的作用奠定了基础。     

小干扰RNA对庚型肝炎病毒基因在人Huh-7细胞中表达的抑制作用

RNA干扰(RNAi)是由小干扰RNA(siRNA)引发的生物细胞内同源基因的转录后基因沉默现象, 是近年来兴起的一项研究生物基因调控与功能的崭新技术. 庚型肝炎病毒(HGV)是一单正链RNA病毒, 复制时不与宿主细胞基因组整合, 尤其适合用于RNAi的研究. 构建了含HGV完整结构基因并携带筛选标志潮霉素基因的真核表达载体pVAX.EH, 转染Huh-7细胞后, 筛选获得稳定表达HGV 结构蛋白的Huh-7细胞株(Huh-7-EH). RT-PCR和Western blot检测证实, HGV结构基因能在Huh-7-EH细胞中转录、表达, 并能进行剪切和翻译后修饰. 以体外转录法制备了2对靶向HGV E2基因的siRNA(1-E2 siRNA和2-E2 siRNA), 将其导入Huh-7-EH细胞中, 采用Western blot和克隆形成实验证实, HGV 1-E2 siRNA和2-E2 siRNA均能特异性抑制HGV结 构蛋白的表达, 抑制作用可维持1周以上. 其中2-E2 siRNA的抑制作用更强, 转染后对Huh-7-EH细胞潮霉素抗性克隆形成的抑制率达到了99%. Huh-7-EH细胞转染siRNA后对潮霉素敏感, 说 明HGV E2 siRNA不仅使HGV E2区的mRNA降解, 还可使融合在HGV E2区下游的潮霉素mRNA降解. 综上所述, 本实验建立的稳定表达HGV结构蛋白的Huh-7-EH细胞株, 能作为用于研究HGV复制和RNAi的细胞模型; HGV结构基因区的siRNA可同时抑制HGV结构蛋白及其下游的潮霉素基因的表达, 证明RNAi在真核细胞Huh-7-EH内可能存在放大作用.     

丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF-κB活性

将丙型肝炎病毒(HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3.1(-)CMV启动子下游,构建成重组质粒pCNS2.用脂质体LipoVecTM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κ B-Luc表达出的荧光素酶的活性比对照细胞降低了约2～4倍,并呈明显的剂量相关性.表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用.这可能与HCV慢性持续性感染的致病性有一定的相关性.     

丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF—κB活性

将丙型肝炎病毒（HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3．1(-)CMV启动子下游,构建成重组质粒pCNS2。用脂质体Lipo Vec^TM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κB-Luc表达出的荧光素酶的活性比对照细胞降低了约2—4倍,并呈明显的剂量相关性。表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用。这可能与HCV慢性持续性感染的致病性有一定的相关性。     

NPAS2基因敲除的HepG2细胞系构建及其对肝癌细胞凋亡的影响

目的:利用CRISPR/Cas9基因编辑技术构建生物节律基因NPAS2敲除的HepG2肝癌细胞系,并初步探讨NPAS2基因敲除对肝癌细胞凋亡的影响。方法:利用sgRNA在线设计工具,针对NPAS2设计两条sgRNA;利用PX459质粒构建分别含有两条sgRNA的敲除载体PX459-sgRNA1;PX459-sgRNA2;利用T7核酸内切酶I检测两条sgRNA活性;将活性较高的打靶载体瞬时转染HepG2细胞,经过药物筛选,克隆化培养及基因测序后得到NPAS2敲除的HepG2肝癌细胞系;利用Western blot检测NPAS2蛋白的表达和凋亡相关蛋白Caspase3的活化;利用流式细胞仪检测敲除细胞系的凋亡水平。结果:成功构建了针对NPAS2的打靶载体;并筛选得到了活性较高的打靶载体;经过药物筛选和克隆化培养得到的NPAS2敲除肝癌细胞系未检测到NPAS2蛋白的表达;进一步发现NPAS2敲除的肝癌细胞Caspase3明显活化,凋亡水平显著升高。结论:利用CRISPR/Cas9基因编辑技术成功构建了NPAS2基因敲除的HepG2肝癌细胞系,并发现NPAS2敲除可以促进肝癌细胞凋亡,为进一步研究生物节律基因NPAS2及其它相关基因在肝癌发生发展中的作用机制提供了有力的工具。     

Etk敲除及过表达细胞株构建并探讨Etk对细胞增殖的影响

目的:利用CRISPR-Cas9技术在人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVECs)中构建Etk(Epithelial and endothelial tyrosine kinase)敲除稳定细胞株以及利用慢病毒构建Etk过表达稳定细胞株,并初步探讨Etk基因对HUVECs细胞增殖的影响。方法:利用CRISPR-Cas9技术,使用在线工具设计针对Etk的sgRNA (https://chopchop.rc.fas.harvard.edu/)。将sgRNA利用连接酶整合到病毒载体中,包被病毒并感染HUVECs敲除HUVECs中的内源性Etk。利用嘌呤霉素筛选得到Etk敲除的稳定细胞株。PCR扩增Etk基因序列,将其整合到pLEX-MCS慢病毒过表达载体中构建Etk过表达重组质粒。包被病毒并感染HUVECs,在HUVECs中过表达Etk,利用嘌呤霉素筛选得到Etk过表达的稳定细胞株。利用qRT-PCR和Western-Blotting检测Etk的敲除和过表达情况。利用CCK-8盒子检测两种稳定细胞株细胞增殖情况。结果:利用CRISPR-Cas9技术有效的敲除HUVECs中内源性Etk,同对照相比,Etk的mRNA和蛋白水平显著地降低(P0.01)。同时利用慢病毒在HUVECs中过表达Etk,同对照相比,在过表达Etk稳定细胞株中Etk的mRNA和蛋白表达显著上调(P0.01)。CCK-8检测发现,Etk敲除降低细胞增殖能力;而Etk过表达增加细胞增殖能力。结论:通过CRISPR-Cas9技术成功在HUVECs中敲除Etk,利用慢病毒过表达系统成功的在HUVECs中过表达Etk,并且初步验证了Etk促进HUVECs细胞增殖。     

利用CRISPR/Cas9技术构建大鼠L2细胞α-ENaC基因敲除稳定细胞株

利用CRISPR/Cas9基因编辑技术构建大鼠L2细胞α-ENaC基因敲除的细胞株,研究α-ENaC基因对细胞增殖的影响。构建敲除α-ENaC基因的CRISPR/Cas9表达载体和筛选报告载体,通过转染和嘌呤霉素筛选获得单克隆细胞株,Western Blot、测序确定突变的细胞株,CCK-8检测突变细胞株的增殖活力。成功构建靶向α-ENaC基因第一外显子的CRISPR/Cas9表达载体和筛选报告载体,嘌呤霉素筛选后,挑选8个单细胞克隆中有两个单细胞克隆α-ENaC蛋白表达下降,一个单细胞克隆α-ENaC蛋白不再表达,测序结果显示3个单细胞克隆分别为2个单等位基因突变和1个双等位基因突变,且未发现脱靶现象。突变细胞株的增殖活力降低,其中双等位基因突变细胞株增殖活力降低更为显著。因此,利用CRISPR/Cas9结合SSA-RPG报告载体成功获得了α-ENaC基因敲除的L2细胞株,α-ENaC与细胞增殖有关。     

基于CRISPR/Cas9系统构建裸鼹鼠HIF-1α基因敲除质粒及其功能验证

目的 利用簇状规则间隔回文重复序列(CRISPR)/Cas9基因编辑系统构建质粒并敲除裸鼹鼠皮肤成纤维(NMR skin fibroblasts, NSF)细胞HIF-1α基因,为研究裸鼹鼠的耐低氧机制及缺氧相关疾病的发生发展机制提供体外细胞模型。方法 针对裸鼹鼠HIF-1α基因的1～4号外显子区域设计4对sgRNA序列,并成功构建表达质粒。筛选得到最优sgRNA后,转染HEK-293T细胞收集上清液测定病毒滴度。前期用pLenti-Cas9(blast)病毒感染NSF细胞,后用制备的HIF-1α-sgRNA病毒感染表达Cas9蛋白的NSF细胞。经药筛后观察荧光表达,同时提取细胞gDNA和蛋白。通过T7E1酶和Western Blot检测NSF细胞中HIF-1α基因变化及蛋白表达情况。结果Sanger测序显示,所设计的sgRNA成功插入到pX459和pKLV2-U6-sgRNA2载体上,序列验证正确,重组质粒构建成功;T7E1酶切实验成功切除3条带,sgRNA的打靶效率为54%,Western Blot结果表明,经药筛后的裸鼹鼠NSF细胞HIF-1α基因敲除成功,蛋白水平显著降低(P=...     

比较两种转录激活系统对生殖相关基因的表达调控

运用由CRISPR/Cas9技术延伸的两种转录激活系统Cas9-p300和dCas9-VPR分别激活生殖特化相关基因,比较它们针对生殖细胞特化基因的激活效率。实时荧光定量PCR结果表明,这两种激活系统均可激活大部分目的基因的表达。其中, Cas9-p300系统激活BLIMP1基因的效率更高, dCas9-VPR系统激活NANOS2、DAZL、SOX17基因的效率更高,两种系统在激活DDX4、TFAP2C基因时效率无显著性差异。由此,本研究通过比较Cas9-p300和dCas9-VPR转录激活系统调控生殖特化相关基因的表达,揭示了不同的转录激活系统在激活生殖特化相关基因时的不同效率,这将为利用转录激活系统研究生殖细胞分化提供理论基础,也将为研究其他发育系统中细胞命运的转变提供借鉴。     

CRISPR/Cas9慢病毒系统敲除胰岛β细胞PKA C-α的研究

利用CRISPR/cas9系统建立稳定敲除PKA C-α基因的INS-1细胞株,研究PKA C-α在胰岛β细胞中的功能。设计2个长25 bp且分别靶向PKA C-α基因的exon 5和exon 7的sgRNA,将其克隆至LentiCRISPRv2-sgRNA质粒并转染至293T细胞中制备sgRNA-Cas9慢病毒,慢病毒感染INS-1细胞,嘌呤霉素筛选出阳性细胞并采用有限稀释法筛选单克隆细胞,Western blotting印记法检测单克隆细胞中PKA C-α蛋白的表达水平,测序确认单克隆细胞中PKA C-α基因的突变位点。WB实验证实靶向Exon 5的sgRNA可成功敲除PKA C-α基因,得到稳定敲除PKA C-α基因的细胞株,测序结果表明该细胞株的PKA C-α基因发生1 bp碱基插入突变,并且敲除PKA C-α基因的INS-1细胞胰岛素分泌能力下降。本实验利用CRISPR/Cas9系统成功敲除INS-1细胞中的PKA C-α基因,为研究PKA C-α在胰岛β细胞中的功能奠定了基础。     

利用dCas9-FokI编辑大鼠Dnmt1基因

CRISPR/Cas9的发现为多种生物的基因编辑提供了强有力的工具。然而,该系统在提供靶向性基因修饰的同时,会产生一些不需要的突变,即脱靶现象。为提高CRISPR/Cas9的特异性,我们将野生型FokI核酸内切酶的功能结构域与催化功能区失活的Cas9蛋白(dCas9)进行融合,形成融合蛋白用于降低脱靶效应。FokⅠ是一种依赖于二聚化才能行使内切酶活性的核酸酶,在本研究中,通过将FokⅠ功能结构融合到dCas9的N端,构建表达质粒pST1374-dCas9-FokⅠ。我们前期研究中,发现一个sgRNA在介导Cas9编辑Dnmt1基因建立条件敲除大鼠时,存在显著的脱靶现象。以此为基础,我们利用dCas9-FokⅠ/sgRNA系统编辑大鼠Dnmt1基因,研究该系统是否能够进行基因编辑以及是否能够提高基因编辑特异性。将转录好的dCas9-FokⅠ mRNA和sgRNA显微注射到SD大鼠的受精卵中,用于产生基因编辑大鼠。通过显微注射以及胚胎移植,最终获得43只F0代大鼠,其中两只在靶点位置包含突变,突变效率达4.5%。对脱靶情况进行分析,结果显示,无脱靶现象存在。综上,表明dCas9-FokⅠ/sgRNA可以应用于编辑大鼠基因,并能显著提高特异性。尽管dCas9-FokⅠ/sgRNA系统相比于Cas9/sgRNA系统,基因编辑效率有所下降,但是该技术的发展为基因治疗提供了可供选择的潜在工具。     

Illuminating single genomic loci in live cells by reducing nuclear background fluorescence

The tagging of genomic loci in living cells provides visual evidence for the study of genomic spatial organization and gene interaction. CRISPR/dCas9(clustered regularly interspaced short palindromic repeats/deactivated Cas9) labeling system labels genes through binding of the dCas9/sgRNA/fluorescent protein complex to repeat sequences in the target genomic loci.However, the existence of numerous fluorescent proteins in the nucleus usually causes a high background fluorescent readout.This study aims to limit the number of fluorescent modules entering the nucleus by redesigning the current CRISPR/dCas9-SunTag labeling system consisting of dCas9-SunTag-NLS(target module) and scFv-sfGFP-NLS(signal module). We removed the nuclear location sequence(NLS) of the signal module and inserted two copies of EGFP into the signal module. The ratio of the fluorescent intensity of the nucleus to that of the cytoplasm(N/C ratio) was decreased by 71%, and the ratio of the signal to the background(S/B ratio) was increased by 1.6 times. The system can stably label randomly selected genomic loci with as few as 9 repeat sequences.     

Hepatoma‐derived growth factor promotes growth and metastasis of hepatocellular carcinoma cells

We aimed to elucidate the effects of hepatoma‐derived growth factor (HDGF) on growth and metastasis of hepatocellular carcinoma (HCC) cells. Tissue microarrays with 236 HCC specimens and 18 extrahepatic metastases were utilized to detect the HDGF expression by immunohistochemistry. Meanwhile, HDGF expressions in HCC cell lines with different metastatic potentials were examined using immunofluorescence staining, real‐time PCR and western blotting. After HDGF silencing, the growth and metastatic potentials of HCC cells were evaluated by soft agar assay, invasion assay, together with tumorigenicity assay in nude mice. The gelatin zymography was performed by detecting MMP‐2 and MMP‐9 levels. Additionally, western blotting was conducted to determine the levels of total and phosphorylated ERK1/2, JNK, p38 and Akt. The results showed that HDGF was overexpressed in HCC metastasis tumour, and the expression increased with the differentiation degree of tumours (Grade I 44.0%, Grade II 48.4% and Grade III 65.6%). Consistently, HDGF levels were positively associated with the metastatic capability of HCC cells (MHCC97L < MHCC97H < HCCLM3). The growth and metastasis were suppressed by HDGF‐siRNA. Gelatinolytic activities were enhanced in the three metastatic HCC cell lines, but had no significant difference among them. The tumourigenicity and metastatic capability of HCCLM3 cells in nude mice were inhibited after silencing HDGF. Meanwhile, HDGF‐siRNA specifically suppressed the total and phosphorylated protein levels of ERK1/2, while not JNK, p38 and Akt. In conclusion, HDGF was overexpressed in HCC patients and cells, and HDGF might be closely correlated with HCC metastasis via regulating ERK signalling pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

Increasing evidence suggests that the renin-angiotensin system (RAS) plays an important role in tumorigenesis. The interaction between Angiotensin II (AngII) and angiotensin type 1 receptor (AT1R) may have a pivotal role in hepatocellular carcinoma (HCC) and therefore, AT1R blocker and angiotensin I-converting enzyme (ACE) inhibitors may have therapeutic potential in the treatment of hepatic cancer. Although the involvement of AT1R has been well explored, the role of the angiotensin II Type 2 receptor (AT2R) in HCC progression remains poorly understood. Thus, the aim of this study was to explore the effects of AT2R overexpression on HCC cells in vitro and in mouse models of human HCC. An AT2R recombinant adenoviral vector (Ad-G-AT2R-EGFP) was transduced into HCC cell lines and orthotopic tumor grafts. The results indicate that the high dose of Ad-G-AT2R-EGFP–induced overexpression of AT2R in transduced HCC cell lines produced apoptosis. AT2R overexpression in SMMC7721 cells inhibited cell proliferation with a significant reduction of S-phase cells and an enrichment of G1-phase cells through changing expression of CDK4 and cyclinD1. The data also indicate that overexpression of AT2R led to apoptosis via cell death signaling pathway that is dependent on activation of p38 MAPK, pJNK, caspase-8 and caspase-3 and inactivation of pp42/44 MAPK (Erk1/2). Finally, we demonstrated that moderately increasing AT2R expression could increase the growth of HCC tumors and the proliferation of HCC cells in vivo. Our findings suggest that AT2R overexpression regulates proliferation of hepatocellular carcinoma cells in vitro and in vivo, and the precise mechanisms of this phenomenon are yet to be fully determined.     

microRNA-148a-3p enhances the effects of sevoflurane on hepatocellular carcinoma cell progression via ROCK1 repression

BackgroundSevoflurane (SEVO) inactivates the aggressiveness of hepatocellular carcinoma (HCC) cells by mediating microRNAs (miRNAs). Hence, we delved into the functional role of miR-148a-3p mediated by SEVO in HCC.MethodsLiver cells (L02) and HCC cells (HCCLM3 and Huh7) were exposed to SEVO to detect cell viability in HCC. HCCLM3 and Huh7 cells were treated with restored miR-148a-3p or depleted Rho-associated protein kinase 1 (ROCK1) to elucidate their roles in HCC cells' biological characteristics. HCCLM3 and Huh7 cells were treated with SEVO, and/or vectors that changed miR-148a-3p or ROCK1 expression to identify their combined functions in HCC cell progression. Tumor xenograft in nude mice was performed to determine growth ability of tumor. The target relationship between miR-148a-3p and ROCK1 was verified.ResultsSEVO inhibited proliferation, invasion and migration and enhanced apoptosis of HCCLM3 and Huh7 cells. MiR-148a-3p up-regulation or ROCK1 down-regulation inhibited HCCLM3 and Huh7 cell progression. ROCK1 was determined to be target gene of miR-148a-3p. Down-regulating miR-148a-3p or overexpressing ROCK1 mitigated cell aggressiveness inhibition caused by SEVO.ConclusionOur study elucidates that microRNA-148a-3p enhances the effects of sevoflurane on inhibiting proliferation, invasion and migration and enhancing apoptosis of HCC cells through suppression of ROCK1.     

Guide-target mismatch effects on dCas9–sgRNA binding activity in living bacterial cells

As an effective programmable DNA targeting tool, CRISPR–Cas9 system has been adopted in varieties of biotechnological applications. However, the off-target effects, derived from the tolerance towards guide-target mismatches, are regarded as the major problems in engineering CRISPR systems. To understand this, we constructed two sgRNA libraries carrying saturated single- and double-nucleotide mismatches in living bacteria cells, and profiled the comprehensive landscape of in vivo binding affinity of dCas9 toward DNA target guided by each individual sgRNA with particular mismatches. We observed a synergistic effect in seed, where combinatorial double mutations caused more severe activity loss compared with the two corresponding single mutations. Moreover, we found that a particular mismatch type, dDrG (D = A, T, G), only showed moderate impairment on binding. To quantitatively understand the causal relationship between mismatch and binding behaviour of dCas9, we further established a biophysical model, and found that the thermodynamic properties of base-pairing coupled with strand invasion process, to a large extent, can account for the observed mismatch-activity landscape. Finally, we repurposed this model, together with a convolutional neural network constructed based on the same mechanism, as a predictive tool to guide the rational design of sgRNA in bacterial CRISPR interference.