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Effect of inhibiting tyrosine kinase Src expression on protein phosphatase 2A and tau phosphorylation

LIU Rong, ZENG Ji, ZHOU Xinwen, WANG Jianzhi, PEI Jinjing

《医学前沿（英文）》 2008年 第2卷 第3期   页码 235-238 doi: 10.1007/s11684-008-0044-8

摘要： The aim of this study is to investigate the effect of tyrosine kinase Src on Tyrosine 307(Y307) phosphorylation, protein phosphatase 2A (PP2A) activity, and on tau phosphorylation. Specific Src siRNA was transfected into cultured mouse neuroblastoma N2a cells to inhibit the expression of Src protein, and the phosphorylation levels of PP2A Y307 and tau at different sites, as well as PP2A activity were detected at different time points after siRNA transfection. Twelve hours after siRNA transfection, the protein level of Src was dramatically decreased, with decreased PP2A Y307 phosphorylation. However, the total PP2A protein level was also decreased, together with a decreased PP2A activity. Tau was hyperphosphorylated at the Ser198/199/202 sites. Multiple factors may be involved in the cellular regulation of PP2A activity. Inhibiting Src expression could induce inactivation of PP2A and tau hyperphosphorylation.

关键词： hyperphosphorylation     PP2A activity     cellular regulation     siRNA     siRNA transfection    

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Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency

JI Yewei, NIE Bin, LI Ping, ZHOU Yuanguo, XU Xiaoyu

《医学前沿（英文）》 2007年 第1卷 第3期   页码 253-257 doi: 10.1007/s11684-007-0048-9

摘要： The purpose of this work was to construct the plasmid that could direct the synthesis of siRNA-like transcripts and thus mediate strong and specific repression of human heat shock protein 90β (Hsp90β) gene expression and to compare the transfection efficiency of the plasmids in varying conditions of transfection. Three 64 nt oligos corresponding to different regions of the target gene were chemically synthesized and annealed and were then ligated with pSUPER EGFP1 plasmid and double-digested with HindIII and Π. Recombinant plasmids were transformed into , DH5a, and the colonies were picked and grown in the Amp-agarose. The presence of positive clones was checked by the means of endodigestion and sequencing. Three cell strains, HepG2, Human umbilicus vein endothelium cells (HUVEC) and HeK293, were cultured. Then the plasmids were transfected into the cells at different ratios of plasmid to Lipofectamine. The transfection efficiency was measured by detection of enhanced green fluorescence protein (EGFP). The presence of positive recombinant clones were verified by double-digestion and sequencing. The bases inserted into the plasmids were correct and the positive colonies were named pSuper-Hsp90β1, pSuper-Hsp90β2 and pSuper-Hsp90β3. After optimizing the ratio of plasmid to Lipofectamine, we achieved high transfection efficiency in HeK293 cells. Transfection efficiency was still low in the HepG2 cells. In conclusion, the si-RNA-synthesizing plasmids targeting Hsp90β were constructed and transfected into cells with different transfection efficiency.

关键词： HindIII     transfection efficiency     detection     enhanced     different    

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Effects of podocin transfection on CD2AP distribution in HEK293 cells

SHA Yugen, HUANG Songming, ZHANG Aihua, ZHAO Fei, CHEN Ronghua

《医学前沿（英文）》 2008年 第2卷 第1期   页码 35-38 doi: 10.1007/s11684-008-0007-0

摘要： The aim of this paper is to construct a podocin fluorescence expression vector and observe the effects of podocin transfection on CD2AP distribution in HEK293 cells. The pGEMT-easy vector containing the full-length cDNA encoding human podocin was cloned and digested with HI and I. The digested full-length podocin was subcloned into pEGFP-C2. The constructed plasmids were transfected into HEK293 cells and its effects on CD2AP distribution were observed by immunofluorescence. The pEGFP-NPHS2 expression vector was successfully constructed and podocin exclusively located on the HEK293 cell membrane. After podocin transfection, CD2AP redistributed from the perinucleus to the cytoplasm in HEK293 cells. It can be concluded that podocin can recruit CD2AP to redistribute from the perinucleus to the cytoplasm in HEK293 cells.

关键词： pEGFP-NPHS2 expression     transfection     pEGFP-C2     cytoplasm     digested    

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Influence of Survivin-targeted siRNA on the biological features of colorectal carcinoma cells

XIONG Ying, GUO Wen, LI Ting, LI Ke

《医学前沿（英文）》 2007年 第1卷 第3期   页码 304-307 doi: 10.1007/s11684-007-0058-7

摘要： The transient transfection of survivin-targeted siRNA to Lovo cells and its influence on the biological features were studied. Two pairs of 19 base pairs (bp) siRNA-specific targeted survivin gene were designed and synthesized by transcription (Survivin-1, Survivin-2). After transient transfection of the two survivin-targeted siRNAs to Lovo cells by Lipofectamine™ 2000, the expression of survivin mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Apoptosis was detected by flow cytometry and cell proliferation was evaluated by MTT assay. We found that the expression levels of survivin mRNA of the two RNAi groups (Survivin-1 group and Survivin-2 group) respectively decreased by 70% and 39.1% compared with the control Lovo’s. Seventy-two hours after transfection, apoptosis rates of the two RNAi groups were 21.51% and 26.28%, both of which were higher than control Lovo’s (9.03%). The results at 72 h after transfection were that the optical density (OD) at 490 nm of the two RNAi groups was 0.581±0.070 and 0.681±0.104, both of which were much lower than the control Lovo’s (2.060±0.272). Based on the results, we can draw a conclusion that the two survivin-targeted siRNAs successfully suppressed the expression of survivin mRNA, inhibited cell growth and induce cell apoptosis. It provides a powerful evidence for colorectal carcinoma gene therapy.

关键词： control     therapy     influence     Survivin-1     colorectal carcinoma    

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Molecular engineering of dendrimer nanovectors for siRNA delivery and gene silencing

Yu Cao, Xiaoxuan Liu, Ling Peng

《化学科学与工程前沿（英文）》 2017年 第11卷 第4期   页码 663-675 doi: 10.1007/s11705-017-1623-5

摘要： Small interfering RNA (siRNA) therapeutics hold great promise to treat a variety of diseases, as long as they can be delivered safely and effectively into cells. Dendrimers are appealing vectors for siRNA delivery by virtue of their well-defined molecular architecture and multivalent cooperativity. However, the clinical translation of RNA therapeutics mediated by dendrimer delivery is hampered by the lack of dendrimers that are of high quality to meet good manufacturing practice standard. In this context, we have developed small amphiphilic dendrimers that self-assemble into supramolecular structures, which mimic high-generation dendrimers synthesized with covalent construction, yet are easy to produce in large amount and superior quality. Indeed, the concept of supramolecular dendrimers has proved to be very promising, and has opened up a new avenue for dendrimer-mediated siRNA delivery. A series of self-assembling supramolecular dendrimers have consequently been established, some of them out-performing the currently available nonviral vectors in delivering siRNA to various cell types and , including human primary cells and stem cells. This short review presents a brief introduction to RNAi therapeutics, the obstacles to their delivery and the advantages of dendrimer delivery vectors as well as our bio-inspired structurally flexible dendrimers for siRNA delivery. We then highlight our efforts in creating self-assembling amphiphilic dendrimers to construct supramolecular dendrimer nanosystems for effective siRNA delivery as well as the related structural alterations to enhance delivery efficiency. The advent of self-assembling supramolecular dendrimer nanovectors holds great promise and heralds a new era of dendrimer-mediated delivery of RNA therapeutics in biomedical applications.

关键词： gene therapy     RNAi therapeutics     dendrimer     nanovectors     gene silencing    

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The role of CDK1 siRNA interference in cell cycle and cell apoptosis

Hui XIAO PhD, Ming TIAN MM, Junna GE MM, Xin Wei MD, Zhaoming LI MM, Xiaolan LI MS, Deding TAO PhD, Junbo HU MD, Jianping GONG MD,

《医学前沿（英文）》 2009年 第3卷 第4期   页码 384-389 doi: 10.1007/s11684-009-0070-1

摘要： In the present report, cyclin-dependent kinase1 (CDK1) siRNA was transfected into cells to silence the CDK1 gene expression and study its role in the cell cycle and cell apoptosis. The siRNA targeting CDK1 gene was chemically synthesized and transfected into Hela cells by lipofectamine 2000. The expression levels of CDK1 gene and protein were examined by real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. The cell cycle was analyzed by using DNA content analysis by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The morphological changes of transfected cells were examined under the microscopy by Wright-Giemsa stain. CDK1 gene was successfully silenced by its siRNA, and the CDK1 protein expression level was decreased significantly, especially from 48thh to 60thh after transfection. The DNA content analysis showed that transfection of CDK1 siRNA led to cells accumulating in G/M phase. There was no significant difference in the apoptotic rate between transfected cells and the control cells after transfection of CDK1 siRNA for 48 or 60h. More double nucleus or multinucleus cells could be seen under the microscopy among the transfected cells. The decreased CDK1 expression by siRNA silencing gave rise to cell cycle arrest in G/M phase but did not induce apoptosis.

关键词： cyclin-dependent kinase1     siRNA interference     cell cycle     apoptosis    

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Effects of hypoxia inducible factor-1alpha siRNA on the invasion of human Hela cells and expression of

Bin YANG MS , Xianglin YUAN , Yanmei ZOU , Qingsong XI , Guoxian LONG , Qiang FU , Guangyuan HU MM ,

《医学前沿（英文）》 2009年 第3卷 第3期   页码 303-308 doi: 10.1007/s11684-009-0060-3

摘要： The effects of hypoxia on the invasion and the related protein expression of Hela cells and the role of hypoxia inducible factor-1α (HIF-1α) were investigated. The Hela cells were divided into three groups, namely, H (non-transfected Hela cells), H (pGenesil-1 empty plasmid-transfected Hela cells), and H (HIF-1α-shRNA plasmid-transfected Hela cells), and were cultured under hypoxia (1% O) and normoxia for 48h. The expression of HIF-1α, E-cadherin, β-catenin, and actin was detected using Western blot. The scratch test and the invasion assay were applied to examine the invasion in each group. The expression of HIF-1α, E-cadherin, and β-catenin in tumor grafts was assayed immunohistochemically. Western blot results revealed that the bands of HIF-1α, E-cadherin, β-catenin, and actin proteins were detected in the H and H groups under hypoxia for 48h. The expression of E-cadherin, β-catenin, and actin was detected in the H group under hypoxia for 48h, and normoxia. In the H, H, and H groups under normoxia, and the H group under hypoxia for 48h, no expression of HIF-1α was detectable. The scratch test showed that the invasive ability in the H group was significantly alleviated. Immunohistochemically, it was found that there was a significant difference in the expression of HIF-1α, E-cadherin, and β-catenin between the H and H groups (<0.05), but the difference was not significant between the H and H groups. It was concluded that the effects of hypoxia on the invasion of human cervical cancer Hela cells and the expression of related proteins (E-cadherin, β-catenin, and actin) depend on HIF-1α.

关键词： hypoxia     actin     β     -catenin     E-cadherin     invasion    

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Changes of phenotype and function of human CD4 CD25 T cells induced by transfection of Foxp3

WU Kui, BI Yutian, WANG Yaoli, WANG Changzheng

《医学前沿（英文）》 2008年 第2卷 第4期   页码 366-369 doi: 10.1007/s11684-008-0070-6

摘要： The aim of this paper is to explore the effects of transfection of Foxp3 gene on the phenotype and function of naive CD4 T cells. The pMSCV-Foxp3 retroviral vector encoding Foxp3 gene was transduced into the PT67 packaging cell line. Virus-containing supernatant was applied to differentiate CD4CD25 T cells. The resulting cells were sorted with flow cytometry. The expressions of CD25, CD127, CTLA-4 and the proliferation of transfected T cells were examined. The effect of transfected CD4 T cells on the proliferation and cytokine production of CD4CD25 T cells was examined. Foxp3-gene transfected CD4 T cells could express Foxp3 and transfection of Foxp3 gene up-regulated the expressions of CD25 and CTLA-4, but down-regulated CD127 expression. After transfection, the proliferation of CD4 T cells was eliminated. Transfected T cells inhibited the proliferation of CD4CD25 T cells. CD4CD25 T cells acquired a regulatory phenotype and function after it was transduced with the Foxp3 gene. This suggested a key role of Foxp3 in the generation of CD4CD25 regulatory T cells.
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Impact of siRNA targeting pirh2 on proliferation and cell cycle control of the lung adenocarcinoma cell

SU Yuan, JIN Yang, ZHANG Xiaoju, ZHOU Qiong, BAI Ming, ZHU Liping

《医学前沿（英文）》 2007年 第1卷 第4期   页码 359-363 doi: 10.1007/s11684-007-0069-4

摘要： The aim of this study was to investigate the role of pirh2 (p53-induced RING-H2) protein in the proliferation, apoptosis and cell cycle control of the lung cancer cell line A549. Pirh2 expression was detected by immunofluorescence, Western blot analysis and real-time polymerase chain reaction (PCR). Cell proliferation was assessed by cell counting kit-8 (CCK-8). Cell cycle control and apoptosis were analyzed by flow cytometry. The results showed that pirh2 was expressed in the cytoplasm of A549 cells. The inhibition of pirh2 expression by siRNA (psiRNA-pirh2) resulted in reduced cell proliferation and increased apoptosis. In addition, the number of G/G phase cells was increased but G/M cells were not affected significantly. Taken together, the inhibition of pirh2 expression in the lung adenocarcinoma cell line A549 resulted in reduced tumor cell growth via the inhibition of cell proliferation, the activation of apoptosis and the interruption of cell cycle transition.

关键词： control     interruption     cytoplasm     number     growth    

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Adenovirus-mediated tissue inhibitor of metalloproteinase-3 gene transfection inhibits rabbit intervertebral

Xudong YU MM, Zengwu SHAO MD, Liming XIONG MD, Weiwei XU MM, Hezhong WANG MM, Huifa XU MM,

《医学前沿（英文）》 2009年 第3卷 第4期   页码 415-420 doi: 10.1007/s11684-009-0072-z

摘要： The aim of this study was to investigate the inhibitory effects of recombinant adenovirus vector carrying tissue inhibitor of metalloproteinase-3 (RAdTIMP-3) against degeneration of rabbit intervertebral disc. Thirty Japanese white rabbits of 4 months old were randomly divided into 5 groups. Mild or moderate rabbit lumbar disc degeneration model was constructed with the controllable axial loading device by imposing 98N pressure at the discs for 2 weeks. Various doses of virus were injected into the degenerated discs as follows: 20μL of normal saline in group 1; 20μL of RAd66 (an empty adenovirus vector, 1.0×10OPU/mL) in group 2; and 20, 10, and 5μL of RAdTIMP-3 (1.0×10OPU/mL) in groups 3, 4, and 5, respectively. Two weeks after the injection, the discs were collected for investigations, including assessment of degeneration degrees according to the Thompson’s grading system, reverse-transcription polymerase chain reaction (RT-PCR) assay for TIMP-3 gene, Safranin O-Fast green staining, and immunohistochemical staining for TIMP-3 and type II collagen. According to Thompson’s criteria, the degeneration of groups 3, 4, and 5, especially group 3, was alleviated as compared with groups 1 and 2. RT-PCR revealed that the expression of TIMP-3 in groups 3, 4, and 5, especially in group 3, was significantly enhanced as compared with group 1 (<0.01). Both Safranin O-Fast green staining and type II collagen staining demonstrated better reserved integrity of disc matrix in groups 3, 4, and 5 than in groups 1 and 2. TIMP-3 staining exhibited an obvious increase of positive-staining rate in groups 3, 4, and 5 as compared with group 1. The positive-staining rate in group 3 (79.42%±1.35%) was about 3times that of group 1 (25.47%±5.46%, <0.01). RAdTIMP-3 can effectively protect the matrix of rabbit intervertebral disc against overloading-induced degeneration in a dose-dependent manner, resulting in the alleviation of disc degeneration.

关键词： tissue inhibitor of metalloproteinase-3     intervertebral disc     rabbit     gene therapy    

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Gene delivery into isolated

Nan Zheng, Ziyuan Song, Yang Liu, Lichen Yin, Jianjun Cheng

《化学科学与工程前沿（英文）》 2017年 第11卷 第4期   页码 521-528 doi: 10.1007/s11705-017-1612-8

摘要： The application of gene delivery materials has been mainly focused on mammalian cells while rarely extended to plant engineering. Cationic polymers and lipids have been widely utilized to efficiently deliver DNA and siRNA into mammalian cells. However, their application in plant cells is limited due to the different membrane structures and the presence of plant cell walls. In this study, we developed the cationic, -helical polypeptide that can effectively deliver DNA into both isolated protoplasts and intact leaves. The PPABLG was able to condense DNA to form nanocomplexes, and they exhibited significantly improved transfection efficiencies compared with commercial transfection reagent Lipofectamine 2000 and classical cell penetrating peptides such as poly(L-lysine), HIV-TAT, arginine9, and poly(L-arginine). This study therefore widens the utilities of helical polypeptide as a unique category of gene delivery materials, and may find their promising applications toward plant gene delivery.

关键词： α-helical polypeptide     plant gene delivery     protoplast     intact leaves     transfection    

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Multifunctional peptide conjugated amphiphilic cationic copolymer for enhancing ECs targeting, penetrating and nuclear accumulation

Xinghong Duo, Lingchuang Bai, Jun Wang, Jintang Guo, Xiangkui Ren, Shihai Xia, Wencheng Zhang, Abraham Domb, Yakai Feng

《化学科学与工程前沿（英文）》 2020年 第14卷 第5期   页码 889-901 doi: 10.1007/s11705-020-1919-8

摘要： Gene therapy has drawn great attention in the treatments of many diseases, especially for cardiovascular diseases. However, the development of gene carriers with low cytotoxicity and multitargeting function is still a challenge. Herein, the multitargeting REDV-G-TAT-G-NLS peptide was conjugated to amphiphilic cationic copolymer poly( -caprolactone-co-3(S)-methyl-morpholine-2,5-dione)- -polyethyleneimine (PCLMD- -PEI) via a heterobifunctional orthopyridyl disulfide-poly(ethylene glycol)- -hydroxysuccinimide (OPSS-PEG-NHS) linker to prepare PCLMD- -PEI-PEG-REDV-G-TAT-G-NLS copolymers with the aim to develop the gene carriers with low cytotoxicity and high transfection efficiency. The multitargeting micelles were prepared from PCLMD- -PEI-PEG-REDV-G-TAT-G-NLS copolymers by self-assembly method and used to load pEGFP-ZNF580 plasmids (pDNA) to form gene complexes for enhancing the proliferation and migration of endothelial cells (ECs). The loading pDNA capacity was proved by agarose gel electrophoresis assay. These multitargeting gene complexes exhibited low cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The high internalization efficiency of these gene complexes was confirmed by flow cytometry. The results of transfection demonstrated that these multitargeting gene complexes possessed relatively high transfection efficiency. The rapid migration of ECs transfected by these gene complexes was verified by wound healing assay. Owing to ECs-targeting ability, cell-penetrating ability and nuclear targeting capacity of REDV-G-TAT-G-NLS peptide, the multitargeting polycationic gene carrier with low cytotoxicity and high transfection efficiency has great potential in gene therapy.

关键词： gene carriers     multitargeting function     ECs     transfection efficiency    

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标题 作者 时间 类型 操作

Effect of inhibiting tyrosine kinase Src expression on protein phosphatase 2A and tau phosphorylation

LIU Rong, ZENG Ji, ZHOU Xinwen, WANG Jianzhi, PEI Jinjing

期刊论文

Construction of Hsp90β gene specific silencing plasmid and its transfection efficiency

JI Yewei, NIE Bin, LI Ping, ZHOU Yuanguo, XU Xiaoyu

期刊论文

Effects of podocin transfection on CD2AP distribution in HEK293 cells

SHA Yugen, HUANG Songming, ZHANG Aihua, ZHAO Fei, CHEN Ronghua

期刊论文

Influence of Survivin-targeted siRNA on the biological features of colorectal carcinoma cells

XIONG Ying, GUO Wen, LI Ting, LI Ke

期刊论文

Molecular engineering of dendrimer nanovectors for siRNA delivery and gene silencing

Yu Cao, Xiaoxuan Liu, Ling Peng

期刊论文

The role of CDK1 siRNA interference in cell cycle and cell apoptosis

Hui XIAO PhD, Ming TIAN MM, Junna GE MM, Xin Wei MD, Zhaoming LI MM, Xiaolan LI MS, Deding TAO PhD, Junbo HU MD, Jianping GONG MD,

期刊论文

Effects of hypoxia inducible factor-1alpha siRNA on the invasion of human Hela cells and expression of

Bin YANG MS , Xianglin YUAN , Yanmei ZOU , Qingsong XI , Guoxian LONG , Qiang FU , Guangyuan HU MM ,

期刊论文

Changes of phenotype and function of human CD4 CD25 T cells induced by transfection of Foxp3

WU Kui, BI Yutian, WANG Yaoli, WANG Changzheng

期刊论文

Impact of siRNA targeting pirh2 on proliferation and cell cycle control of the lung adenocarcinoma cell

SU Yuan, JIN Yang, ZHANG Xiaoju, ZHOU Qiong, BAI Ming, ZHU Liping

期刊论文

Adenovirus-mediated tissue inhibitor of metalloproteinase-3 gene transfection inhibits rabbit intervertebral

Xudong YU MM, Zengwu SHAO MD, Liming XIONG MD, Weiwei XU MM, Hezhong WANG MM, Huifa XU MM,

期刊论文

Gene delivery into isolated

Nan Zheng, Ziyuan Song, Yang Liu, Lichen Yin, Jianjun Cheng

期刊论文

Multifunctional peptide conjugated amphiphilic cationic copolymer for enhancing ECs targeting, penetrating and nuclear accumulation

Xinghong Duo, Lingchuang Bai, Jun Wang, Jintang Guo, Xiangkui Ren, Shihai Xia, Wencheng Zhang, Abraham Domb, Yakai Feng

期刊论文