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Objective To evaluate the impact and clinical significance of NOD-like receptor pyrin domain-containing protein 3(NLRP3)in the activation of lipopolysaccharide(LPS)-induced BV2 microglia cells.Methods shRNA plasmids were devised for BV2 microglia transfection,and the most effective transfection segment was identified via RT-PCR and Western blot assays.NLRP3 expression in the cell line was detected by Western blotting,while light microscopy was used to observe morpho-logical alterations in BV2 cells transfected with NLRP3-shRNA.Furthermore,enzyme-linked immunosorbent assay(ELISA)was used to quantify levels of inflammatory cytokines IL-18,IL-1β,TNF-α and NO in cell supernatants.Immunofluorescence staining was used to observe the expression and localization of microglial activation markers iNOS,Arg-1,Iba1,and NLRP3.Results NLRP3 was highly expressed in LPS-induced BV2 cells.The Western blot result showed that the mRNA expression level was the lowest and transfection was the least effective in NLRP3-mus-727 group.Microscopic examination revealed a round or short spindle-shaped morphology in BV2 cells transfected with shNLRP3,which was akin to resting state cells in the blank control group.ELISA showed that pro-inflammatory mediators IL-18,IL-1β,TNF-α and NO levels were decreased in BV2 cells trans-fected with shNLRP3(all P＜0.05).Immunofluorescence staining indicated a relative decrease in iNOS and Iba1 expression,with an upregulation of Arg-1 in BV2 cells transfected with shNLRP3.Conclusion NLRP3 is highly expressed in LPS-induced BV2 cells.Inhibition of NLRP3 expression can suppress the inflammatory response of BV2 cells induced by LPS,promoting their polarization towards the M2 phenotype.

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Aim To determine the effect of FATP5 gene silencing on fatty hepatic cell inflammation and to explore its possible mechanism. Methods Five shR-NA sequences were designed and synthesized. The efficient FATP5-shRNA was screened by the siCHECK™ system. After preparing the FATP5-shRNA lentivirus, the FATP5 gene silence hepatic cell lines was obtained by HepG2 cell infection and puromycin screening. The FATP5 silencing efficiency was detected by Western blot. Then the oleic acid induced ROS and MDA generation, TNF-a and IL-6 protein expression and secretion, and NF-kB activation in FATP5 gene silence cells were analyzed by the detection kit, Western blot, nucleo-plasmic separation and reporter gene system. Results In the gene silence cells, FATP5 protein expression was reduced by 90% and the lipid accumulation was also significantly inhibited. Moreover, the FATP5 knockdown could reduce the oleic acid induced ROS and MDA generation, and suppress the NF-kB activation, thereby inhibiting the protein expression and secretion of TNF-a and IL-6. Conclusions FATP5 gene silence inhibits fatty hepatic cell inflammation, and its mechanism may be related to the inhibition of oxidative stress and lipid peroxidation.

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Preventing the replication of adenovirus could have practical uses, such as controlling infection with wild-type virus or in applications involving recombinant vectors. Mainly transient methods have been used to inhibit adenovirus replication, including siRNA or drugs. Here, we tested whether stable expression of shRNA designed to target hexon, Iva2, or pol can inhibit the replication of a recombinant adenoviral vector, Ad-LacZ (serotype 5, E1/E3 deleted), in 293T cells. Significant knockdown correlating with reduced Ad-LacZ replication was achieved only when hexon was targeted. Cell sorting and isolation of cellular clones further accentuated knockdown of the hexon transcript, reduced protein levels by more than 90%, and diminished adenovirus production. As visualized by transmission electron microscopy, the cellular clone expressing the hexon-specific shRNA yielded 89.2% fewer particles compared to the parental 293T cells. Full scale production followed by purification revealed a 90.2% reduction in Ad-LacZ biological titer. These results support the notion that stable expression of shRNA can be used as a means to control adenovirus replication.

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Objective:To evaluate the effect of down-regulation of Sp1 expression on the radiosensitivity of glioma cells.Methods:The oligonucleotide sequence encoding shRNA was designed and synthesized, and cloned into LV3 (H1/GFP & Puro) vector to construct the recombinant. U251 and U87 cells were infected with recombinant lentivirus, then the stably-transfected cell lines were obtained by puromycin screening. The expression levels of Sp1 mRNA and protein were detected by RT-PCR and Western blot. Cell survival was detected by clonal survival assay, cell cycle was determined by flow cytometry, and DNA damage was measured by immunofluorescence assay, respectively.Results:At 72 h after infection, high expression of Sp1 lentiviral vector was observed in two cell lines under fluorescence microscope. RT-PCR and Western blot confirmed that the expression levels of Sp1 mRNA and protein were significantly down-regulated in both transfected cells (both P<0.01) and the silencing rates of Sp1 were above 90%. The sensitization enhancement ratio (SER) of shRNA-U251 and shRNA-U87 cells at 10% cell survival level were 1.39 and 1.18, respectively. After irradiation, the G 2/M phase ratio and the number of γ-H2AX foci in two Sp1 knockout groups were significantly increased. Conclusion:shRNA silencing of Sp1 increases the G 2/M phase arrest induced by X-ray, aggravates the degree of DNA double-strand breaks, and improves the radiosensitivity of glioma cells.

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@#[摘 要] 目的：评价口服携带HPV16 E7 shRNA和IL-12基因的重组短双歧杆菌在小鼠体内抗宫颈癌移植瘤的效果。方法：将pMG36e-E7 shRNA、pMG36e-mIL-12D质粒分别转化短双歧杆菌，经筛选鉴定并扩增获得携带HPV16 E7 shRNA和IL-12 基因的重组短双歧杆菌。通过小鼠皮下宫颈癌细胞移植建立荷瘤小鼠模型。口服重组短双歧杆菌1、7 d后，检测小鼠主要器官（心、肝、脾、肺、肾）和肿瘤组织匀浆液或血清在PYG培养基中形成的菌落数量，评价短双歧杆菌的肿瘤靶向性，以小鼠体内肿瘤生长曲线评估重组短双歧杆菌的抗肿瘤效果，通过主要器官切片H-E染色和检测荷瘤小鼠血清相关细胞因子水平评价口服重组短双歧杆菌的安全性。结果：成功制备重组短双歧杆菌和宫颈癌TC-1细胞移植瘤小鼠。7 d后，移植瘤组织匀浆液和血清的菌落数量证实短双歧杆菌具有靶向体内瘤组织的定殖能力，口服重组短双歧杆菌明显抑制荷瘤小鼠的肿瘤生长（P<0.05或P<0.01），但联合使用携带HPV16 E7 shRNA和IL-12基因的重组双歧杆菌的肿瘤抑制率与单独使用的并无显著差异，治疗后未见对荷瘤小鼠主要器官的损伤和血清中IL-12及IFN-γ的水平明显变化。结论：短双歧杆菌可用作靶向肿瘤的治疗性基因分子递送载体，其对宫颈癌移植瘤的疗效明显且安全可控。

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Objective@#To investigate the effect of brain and muscle arant-like-1 (Bmal1) and miRNA-155-5p on the proliferation ability and aging of bone marrow mesenchymal stem cells (BMMSCs) to provide an experimental basis for elucidating the mechanism of bone senescence.@*Methods @# BMMSCs were extracted from the femur medullary cavity of 1-month-old mice, purified and cultured via the whole bone marrow mesenchymal adherent method and passed to P3. The characteristics of BMMSCs were detected by flow cytometry. BMMSCs were transfected with lentivirus to construct stable miR-155-5p and Bmal1 overexpression/interference BMMSCs. shRNA-transfected BMMSCs were identified by qRT-PCR. The proliferation activities of miR-155-5p and Bmal1 overexpression/interference BMMSCs were detected via CCK-8 assay. The apoptosis rates were measured by flow cytometry. The aging status of BMMSCs was identified with the senescence-associated β-galactosidase (SA-β-Gal) test. The expression of senescence-related genes P16 and P53 was detected by qRT-PCR.@*Results@#The shRNA-transfected BMMSCs were successfully generated. The proliferation ability decreased, and the apoptosis rates, the activity of SA-β-Gal and the relative expression levels of P53 and P16 increased when miRNA-155-5p was overexpressed. The proliferation ability increased, and the apoptosis rates, the activity of SA-β-Gal and the relative expression levels of P53 and P16 decreased when miRNA-155-5p was inhibited. The effect of Bmal1 is opposite to that of miRNA-155-5p.@*Conclusions @# The expression of Bmal1 promotes the proliferation and antiaging ability of BMMSCs, while miRNA-155-5p inhibits the proliferation and accelerates the aging of BMMSCs.

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Objective:To investigate the effect of ANXA on biological behavior of papillary thyroid carcinoma (PTC) cells by interfering with the expression of annexin A1 (ANXA1) in PTC cell lines by short hairpin RNA (shRNA) .Methods:The shRNA with specific and high efficiency was designed to specifically interfere with the expression of ANXA1 in TPC-1 and BCPAP cell lines, and transfect the TPC-1 and BCPAP cell lines respectively, including specific ANXA1 interference and negative control virus transfection, and they were divided into shANXA1 group and negative control virus group. Semi-quantitative reverse transcription PCR (Q-PCR) and Western Blot were employed to verify gene expression. The shANXA1 group was used as the experimental group, the untransfected virus group and the negative control virus group were set as the control groups. The expression levels of ANXA1 in the three groups were compared and the shRNA interference efficiency was verified. The effects of ANXA1 knockdown on the proliferation, migration and invasion of TPC-1 and BCPAP cell lines were investigated by scratch, CCK8 and Transwell invasion experiments. Independent sample t test was used to compare the means between the two groups, and one-way analysis of variance was employed to compare multiple groups, with P<0.05 as statistically significant. Results:shRNA could efficiently silence the expression of ANXA1 at the transcription and translation level in PTC cell lines. Compared with the negative control cells, the cells proliferated after successful lentiviral transfection of TPC-1 and BCPAP (BCPAP, 24h: F= 25.15, P<0.001; 48h: F=6.44, P<0.001; 48h: F=46.94, P<0.001; TPC-1, 24h: F=207.50, P<0.001; 48h: F=202.45, P<0.001; 48h: F=55.89, P<0.001) , its migration (BCPAP, F=12511.10, P<0.001; TPC-1, F=3966.10, P<0.001) and invasion ability (BC-PAP: F=94.65, P<0.001; TPC-1: F=681.74, P<0.001) significantly decreased. Conclusion:After shRNA knock-down of ANXA1 gene, the proliferation, migration and invasion ability of TPC-1 and BCPAP cell lines decreased significantly, indicating that silencing this gene can reduce tumor aggressiveness, and initially reveals that ANXA1 may be an important potential in PTC biotherapy Target.

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Objective To construct SLC6A4-shRNA lentiviral vector, establish PC12 cells stable transformation cell line，and detect the effect of SLC6A4 gene silencing on hypoxia induced PC12 cells apoptosis. Methods Three specific targets sequence of SLC6A4 were designed and short hairpin RNA was synthesized, and then were recombined into shRNA expression vector GV248 plasmid, with non-homology shRNA sequence as negative control. The connection products were switched to competent cells. After dentification and sequencing, the vectors were co-transfected with the auxiliary vectors into 293T cells in order to produce recombinant shRNA lentiviral particles. Then, PC12 cells were infected with the recombinant lentiviral and screened by puromycin. The PC12 cells were divided into two groups: lentiviral negative control group (NC-shRNA) and SLC6A4-silenced group (SLC6A4-shRNA). The expression of SLC6A4 mRNA was detected by real-time fluorescence quantitative and the 5-HTT protein level was assayed by Western blot, The effect of SLC6A4 gene silencing on hypoxia induced apoptosis was detected by flow cytometry. Results The SLC6A4-shRNA lentiviral expression vector was constructed and the recombinant lentiviral particles by packaging the 293T cells were obtained, the stably infected PC12 cells were established after filtering. Compared with negative control group, the expression level of SLC6A4 gene and 5-HTT protein in SLC6A4-shRNA group was suppressed notably (P<0.01). It was confirmed that lentiviral vector could effectively silence SLC6A4 gene in PC12 cells and SLC6A4 gene silencing could decrease apoptosis rate of PC12 cells under hypoxia condition. Conclusion The SLC6A4 gene of PC12 cells could be effectively silenced by shRNA lentivirus vector，which could reverse hypoxia induced apoptosis.

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【Objective】 To investigate the effects of silencing AEG-1 gene by shRNA on vasculogenic mimicry (VM) of a glioma xenograft model. 【Methods】 U87 glioma cells were infected with AEG-1 shRNA lentivirals. Real-time PCR and Western blotting were used to detect the mRNA and protein expressions of AEG-1 in U87 cells after infected by the AEG-1 shRNA lentivirals. A glioma xenograft model was generated and CD 34/PAS double-staining was performed to detect the VM channels in vivo. The immunohistochemical assay was performed to evaluate the expressions of MMP-2, MMP-9, VE-cadherin, and VEGF in glioma xenograft models. 【Results】 AEG-1 shRNA lentivirals could significantly inhibit the AEG-1 expression in glioma cells (P<0.01). Meanwhile, they also decreased the number of VM in the glioma xenograft model (P<0.01). Furthermore, the expressions of MMP-2, MMP-9, VE-cadherin, and VEGF in glioma significantly decreased in vivo (P<0.01). 【Conclusion】 These results suggest that silencing AEG-1 gene by shRNA can significantly inhibit VM of glioma in vivo, the mechanism of which may partly be through regulating MMP-2, MMP-9, VEGF, and VE-cadherin expressions.

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NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.

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Objective To investigate the effect of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) on the radiosensitivity of esophageal squamous carcinoma cells and explore its mechanism. Methods The shRNA sequence was designed with human UHRF1 as the target, and the UHRF1-shRNA was transfected into Eca-109 cells mediated by lentivirus. The expressions of UHRF1 mRNA and protein before and after transfection were determined by fluorescence quantitative PCR and Western blotting. The radiosensitivity of Eca-109 cells after down-regulation of UHRF1 gene was assessed by colony formation assay. The effect of silencing UHRF1 on the apoptosis of Eca-109 cells before and after X-ray irradiation was detected by flow cytometry. Western blotting was used to detect the effects of UHRF1 silencing and X-ray irradiation on the expressions of apoptosis-related proteins Bcl-2, caspase-3 and proteins related to AKT/mTOR signaling pathway in Eca-109 cells. Results We successfully constructed an Eca-109 shUHRF1 cell line with UHRF1 stablely down-regulated. Colony formation assays showed that the radiosensitivity of Eca-109 cells was increased after down-regulation of UHRF1 expression compared with the NC group. The proportion of apoptotic cells in Eca-109 cells was increased, while this change was more obvious after irradiation. Compared with NC group, down-regulation of UHRF1 expression increased the level of active caspase-3 protein and decreased the level of the apoptosis inhibitory protein Bcl-2; the expression levels of p-AKT and p-mTOR protein were decreased. The changes in these four proteins in Eca-109 cells with down-regulation of UHRF1 combined with 6 Gy X-ray irradiation were more obvious compared with the NC group. Conclusion UHRF1 knock-down increased the radiosensitivity of ESCC via inhibiting the Akt/mTOR signaling pathway activity and inducing the apoptosis of the cells.

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Abstract Introduction: Serum- and glucocorticoid-inducible kinase 3, a serine/threonine kinase that functions downstream of the PI3K signaling pathway, plays a critical role in neoplastic processes. It is expressed by various tumors and contributes to carcinogenesis. Objective: The objective was to investigate serum- and glucocorticoid-inducible kinase 3 expression in nasopharyngeal carcinoma, to study the anti-tumor effects of serum- and glucocorticoid-inducible kinase 3 shRNA by inhibiting its expression in nasopharyngeal carcinoma cells and to discuss the potential implications of our findings. Methods: Serum- and glucocorticoid-inducible kinase 3 protein expression in nasopharyngeal carcinoma cell lines (CNE-1, CNE-2, HNE-1, HONE-1, and SUNE-1) and the human immortalized nasopharyngeal epithelium cell line NP69 were assayed by western blotting. Serum- and glucocorticoid-inducible kinase 3 expression in 42 paraffin-embedded nasopharyngeal carcinoma tissues were performed by immunohistochemistry. MTT assay, flow cytometry, and scratch tests were performed after CNE-2 cells were transfected with the best serum- and glucocorticoid-inducible kinase 3 shRNA plasmid selected by western blotting using lipofectamine to study its effect on cell proliferation, apoptosis, and migration. Results: Serum- and glucocorticoid-inducible kinase 3 was overexpressed in human nasopharyngeal carcinoma tissues and cells. Serum- and glucocorticoid-inducible kinase 3 expression decreased markedly after CNE-2 cells were transfected with the serum- and glucocorticoid-inducible kinase 3 shRNA, leading to strong inhibition of cell proliferation and migration. In addition, the apoptosis rate increased in CNE-2 cells after serum- and glucocorticoid-inducible kinase 3 knockdown. Conclusion: Serum- and glucocorticoid-inducible kinase 3 expression was more frequently observed as the nasopharyngeal epithelium progresses from normal tissue to carcinoma. This suggests that serum- and glucocorticoid-inducible kinase 3 contributes to the multistep process of NPC carcinogenesis. Serum- and glucocorticoid-inducible kinase 3 represents a target for nasopharyngeal carcinoma therapy, and a basis exists for the further investigation of this adjuvant treatment modality for nasopharyngeal carcinoma.

Resumo Introdução: A quinase 3 sérica induzida por glicocorticoide, uma serina/treonina quinase que funciona downstream da via de sinalização PI3K, desempenha um papel crítico nos processos neoplásicos. É expressa por vários tumores e contribui para a carcinogênese. Objetivo: Investigar a expressão de quinase 3 sérica induzida por glicocorticoide no carcinoma nasofaríngeo, estudar os efeitos antitumorais do shRNA da quinase 3 sérica induzida por glicocorticoide, que inibem sua expressão em células de carcinoma nasofaríngeo, e discutir as implicações potenciais de nossos achados. Método: A expressão de proteína quinase 3 sérica induzida por glicocorticoide em linhagens de células de carcinoma nasofaríngeo (CNE-1, CNE-2, HNE-1, HONE-1 e SUNE-1) e a linhagem de células humanas imortalizadas do epitélio nasofaríngeo NP69 foram avaliadas por Western blot. A expressão da quinase 3 sérica induzida por glicocorticoide em 42 tecidos de CNF embebidos em parafina foi feita por imuno-histoquímica. Testes com MTT, citometria de fluxo e testes de raspagem foram feitos após as células CNE-2 terem sido transfectadas com o melhor plasmídeo shRNA da quinase 3 sérica induzida por glicocorticoide selecionado por Western blot, com o uso de lipofectamina para estudar seu efeito na proliferação, apoptose e migração celular. Resultados: Foi observada uma sobre-expressão da quinase 3 sérica induzida por glicocorticoide em tecidos e células de carcinoma nasofaríngeo humanas. A expressão de quinase 3 sérica induzida por glicocorticoide diminuiu acentuadamente após as células CNE-2 terem sido transfectadas com o shRNA da quinase 3 sérica induzida por glicocorticoide, conduzindo a forte inibição de proliferação e migração celular. Além disso, a taxa de apoptose aumentou nas células CNE-2 após o knockdown da quinase 3 sérica induzida por glicocorticoide. Conclusão: A expressão de quinase 3 sérica induzida por glicocorticoide foi observada com maior frequência à medida que o epitélio nasofaríngeo progride de tecido normal para carcinoma. Isso sugere que a quinase 3 sérica induzida por glicocorticoide contribui para o processo multietapas da carcinogênese do carcinoma nasofaríngeo. A quinase 3 sérica induzida por glicocorticoide representa um alvo para a terapia do carcinoma nasofaríngeo e há uma base para a investigação adicional dessa modalidade de tratamento adjuvante para o carcinoma nasofaríngeo.

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Objective To investigate the differential expression of HK2 in liver cancer tissues and its effects on cell proliferation ,cell cycle and apoptosis of liver cancer cells . Methods 48 cases of liver cancer tissues and corresponding para cancer tissues were collected,and the expression of HK2 was detected by immunohistochemistry. At the same time,Western blot was used to detect the expression of HK2 in human hepatoma cell HepG2 and in normal liver cell L-02. The relationship between HK2 expression and clinic pathological characteristics of hepatocellular carcinoma was analyzed statistically. Four HK2 shRNA vectors were constructed,Western blot was used to detect the interference efficiency,and the best HK2 shRNA was selected for transfecting cell. For blank group (normal culture,non-transfected plasmid),control shRNA group(transfected control shRNA),HK2 shRNA group (transfected shRNA_3) HepG2 cells,MTT was used to detect cell proliferation activity,flow cytometry was used for cell cycle,Annexin V-FITC /PI double labeling method was used to detect cell apoptosis. Results The expression of HK2 increased significantly in HCC tissues,and the expression was correlated with tumor diameter,TNM stage and histopathological grade. The decrease of HK2 expressionsignificantly reduced the proliferation activity of HepG2 cells,obviously changed the cell cycle and make cells less stagnant at S stage,significantly increased the apoptosis of HepG2 cells. Conclusion HK2 shows strong anti-tumor potential and has a certain clinical significance. It provides a new idea and theoretical basis for the clinical diagnosis of liver cancer,prognosis judgment and molecular targeting therapy based on HK2.

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Objective To investigate the effect of Mucin 3A (Muc3A) gene silencing by shRNA mediated with lentivirus vector on human extrahepatic cholangiocarcinoma.Methods Short hairpin RNA (shRNA) interference sequence targeting Muc3A gene was designed and synthesized.Recombinant lentiviral plasmids were packaged to produce virus venom and their titers were determined.After transfected with QBC939 cells of extrahepatic cholangiocarcinoma,stable positive cell lines were obtained by optimal drug screening concentration.QBC939 cells were divided into three groups:lentivirus mediated shRNA transfected cells (transfected group),empty virus transfected cells (negative control group),and untransfected cells (blank group).ShRNA silencing efficiency of Muc3A gene was detected with Western blot.Cell growth was assessed by MTS assay,cell colony formation was detected with plate clonogenesis assay,and cell cycle distribution were detected by flow cytometry.Results Lentivirus was successfully packaged and titer of virus suspension was 1 × 108 TU/ml.Western blot confirmed that shRNA worked well in QBC939 cells.3 μg/ml puromycin concentration was added for table cell lines selection.Western blot results showed that the expression of Muc3A in the transfection group was significantly decreased comparing with negative control group and the blank group (P ＜ 0.05).The MTS results showed that the value of OD490nm in the transfected group was significantly lower than that in the negative control group and the blank group,and the differences were statistically significant (P ＜ 0.05).The number of clone formation in the transfection group was significantly lower than that in the negative control group and the blank control group,and the differences were statistically significant (P ＜ 0.05).Cell cycle of the experimental group was in G2/M is more,but S phase is less,but there is no statistical difference compared with blank group (P ＞ 0.05).Conclusion Lentivirus mediated shRNA transfection can significantly inhibit the growth,proliferation and colony formation of QBC939 cells of extrahepatic cholangiocarcinoma after interfering with Muc3A gene expression,which sugests that Muc3A can promote the growth of cholangiocarcinoma cells.

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Objective The shRNA library was used to screen the tumor suppressor genes related to lung epithelial cells,and its function of inhibiting malignant transformation in lung cells was preliminarily verified,which provided a theoretical basis and a new therapeutic target for tumor prevention and treatment. Methods The shRNA retrovirus library was constructed by GP2 -293 virus packaging cell line to infect the immortalized lung bronchial epithelial BEAS-2B cells. The transfected epithelial cell clones were screened by soft agar colony formation,and a single transformed cell clone with a diameter greater than 1. 0 mm was selected. The in-serted shRNA fragment was amplified by PCR,and the target candidate gene corresponding to shRNA was determined by the conven-tional DNA sequencing and blast alignment. A candidate tumor suppressor gene INPP4B was verified by soft agar cloning and tumor formation in nude mice. MTT assay was used to detect the cell proliferation. Results Six lung epithelial malignant transformation in-hibitory factors were screened by soft agar colony formation. The candidate INPP4B gene was selected for functional experiments. Si-lencing INPP4B gene in BEAS-2B cells promoted the formation of clones in the soft agar plates,and the cell proliferation rate was accelerated. The silencing cell line showed the enhanced tumorigenicity in nude mice,indicating that INPP4B was involved in tumor formation. Conclusion shRNA library and soft agar colony formation assays are a powerful tool for screening tumor suppressor genes, and INPP4B is a malignant transformation inhibitor of lung epithelial cells.

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Objective To construct lentiviral vector of late endosomal／lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 ( lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection.Methods Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage.There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control.The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction ( RT-qPCR ) and Western blot.The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-αsecreted by the cells were detected by RT-qPCR.T test was used for comparison between groups.Results The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully.The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000 ±0.000, 0.596 ±0.125 and 0.120 ±0.080, respectively.The expression of lamtor2 in the sh2 group was significantly lower than that in the sh 1 group (t＝3.399, P＝0.015), and they were both significantly lower than the control group ( t ＝3.333 and 9.734, respectively, both P ＜0.05).After infection with Klebsiella pneumoniae, expression levels of IL-1β( t ＝15.20), IL-6 (t＝43.30) and TNF-α(t＝12.67) were significantly higher than those in the control group (all P＜0.01).Conclusion The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism , which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

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Objective@#To construct lentiviral vector of late endosomal/lysosomal adaptor, mitogen-activated protein kinase and mammalian target of rapamycin activator 2 (lamtor2) gene, and to explore its regulatory role on inflammatory response of macrophages after Klebsiella pneumoniae infection.@*Methods@#Two pairs of mouse lamtor2 short hairpin RNA (shRNA) were designed and sub-cloned into PLKO.1-puro to construct lentiviral vector, and were transfected into the murine RAW264.7 macrophage. There were two experimental groups including pLKO.1-puro shlamtor 2-1(sh1 group) and pLKO.1-puro shlamtor 2-2 (sh2 group), and the RAW264.7 cells transfected with non-treated pLKO.1-puro was set as control. The expession of lamtor2 were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. The levels of inflammatory factors including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α secreted by the cells were detected by RT-qPCR. T test was used for comparison between groups.@*Results@#The recombinant lentiviral vector PLKO.1-shlamtor 2 transfected RAW264.7 cells successfully. The relative expressions of lamtor2 mRNA in the control group, the sh1 group and the sh2 group were 1.000±0.000, 0.596±0.125 and 0.120±0.080, respectively. The expression of lamtor2 in the sh2 group was significantly lower than that in the sh1 group (t=3.399, P=0.015), and they were both significantly lower than the control group (t=3.333 and 9.734, respectively, both P< 0.05). After infection with Klebsiella pneumoniae, expression levels of IL-1β (t=15.20), IL-6 (t=43.30) and TNF-α (t=12.67) were significantly higher than those in the control group (all P<0.01).@*Conclusion@#The lentiviral vector of lamtor2 can stably down-regulate the expression of lamtor2 gene in macrophages through RNA interference mechanism, which has a significant effect on the secretion of inflammatory factors of macrophages that are infected with Klebsiella pneumoniae.

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CD36, the major scavenger receptor, is intimately involved in the uptake of oxLDL in macrophages. To further study the function of CD36 in macrophages, we constructed CD36 gene silence cell lines (J774A.1) by lentivirus-mediated RNA interference technique, and analyzed the effect of CD36 in caveolin-1 protein expression. At first, 5 shRNA fragments were designed and synthesized according to the coding sequence (CDS) region of CD36 gene. Next, the CD36-shRNA was inserted into lentiviral vector to yield pLKO.1-CD36-shRNA plasmid. After DNA sequencing, the pLKO.1-CD36-shRNA plasmid and psiCHECK-II-CD36 were co-transfected into the 293T cells to screen the efficient CD36-shRNA. The efficient CD36-shRNA plasmid and the helper plasmid were co-transfected into the 293T cells to package the lentivirus, and then infected the J774A.1 cells. After screening by puromycin, CD36 gene silence cell lines (J774A.1) was established. Western blotting and confocal fluorescence microscopy results showed that the CD36 silencing efficiency in the gene silence cell line was 90%. Accompanied by a decrease in CD36 protein on cell surface, oxLDL binding to CD36 was significantly inhibited, indicating that the CD36 gene silence cell line is successfully established. Finally, the oxLDL stimulation and inhibitor experiments results showed that the CD36 knockdown significantly suppresses the phosphorylation of JNK and ERK, thereby inhibiting the oxLDL-induced caveolin-1 protein expression, demonstrating that CD36 modulates the caveolin-1 protein expression through the JNK/ERK-mediated signaling transduction.

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Objective Little is known about the effect of RNAi on mitochondrial apoptotic pathways. This study aims to explore the effects of the Survivin shRNA-APC double-gene on colon cancer mitochondrial apoptosis pathway-related factors survivin,cytochrome C (Cytc),second mitochondria-derived activator of caspases (Smac),and cysteine aspartic acid specific protease 9 (Caspase-9) as well as on the apoptosis of colon cancer transplanted tumor (CCTT) cells. Methods Thirty nude mice were randomly divided into five groups of equal number,Survivin shRNA-APC double-gene,survivin shRNA,APC,empty vector and blank transfection. The CCTT model was established in the nude mice by subcutaneous injection of the colon cancer cell strains stably transfected with the Survivin shRNA-APC double-gene,survivin shRNA,APC,an empty vector and HT-29,respectively,into the mid-posterior part of the left armpit of the nude mice. The rate of tumor growth inhibition was calculated by measuring the volume and weight of the CCTTs in the nude mice. The mRNA and protein expressions of survivin,Cytc,Smac and Caspase-9 in the tumor tissue were detected by real time PCR and immunohistochemistry,respectively,and the apoptosis rate of the CCTT cells was detected by TUNEL. Results The model of CCTT was successfully established in the nude mice. Com-pared with the empty vector and blank transfection groups,the mice in the double-gene,survivin shRNA and APC groups showed sig-nificantly decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). In comparison with the survivin shRNA and APC groups,the double-gene group exhibited remarkably decreased average volume and weight of the tumor tissue (P<0.05) but increased inhibition rate of its volume and weight (P<0.05). The mRNA and pro-tein expressions of survivin were significantly lower while those of Cytc,Smac and Caspase-9 markedly higher in the double-gene,sur-vivin shRNA and APC groups than in the empty vector and blank transfection groups (P<0.05),the former even lower (P<0.05) and the latter even higher in the double-gene than in the survivin shRNA and APC groups (P<0.05). The apoptosis rate of the CCTT cells was significantly increased in the double-gene ([56.78±3.04]%),survivin shRNA ([33.61±2.02]%) and APC groups ([30.16± 1.72]%) as compared with the empty vector ([10.05±0.42]%) and blank transfection groups ([9.87±0.30])% (P<0.05),even higher in the double-gene group than in the survivin shRNA and APC groups (P<0.05). Conclusion The Survivin shRNA-APC double-gene may induce apoptosis of colon cancer transplanted tumor cells by down-regulating the expression of the apoptosis inhibitor survivin,upregulating the expressions of Cytc,Smac and Caspase-9,and suppressing the growth of the colon transplanted tumor,with more significant abilities than a single gene in regulating apoptosis-related factors,inducing cell apoptosis and inhibiting the growth of the transplanted tumor.

ABSTRACT

Objective:To establish a mouse macrophage line lacking NLRP3.Methods:A GFP and neomycin dual selection marker vector which contains an efficient shRNA-coding insert for mouse NLRP3,was constructed and transfected into macrophages (RAW264.7) to select the stable clone cells in G418-contained medium.Then,the expanded clone cells that retain strong GFP expression were further sorted using the popular flow cytometry.The obtained cell mix (herein termed RAWNKD) were passaged and maintained for further identification,including observation of GFP marker,especially quantitative PCR (RT-qPCR) to confirm knockdown of NLRP3 in the generated RAWNKD cells which were challenged with LPS and ATP or not.Results:Over 80% of RAWNKD cells expressed GFP,and little NLRP3 mRNA was detected in RAWNKD cells,notably no obvious increase in NLRP3 mRNA was observed when the RAWNKD cells were challenged by LPS and ATP.Conclusion:The macrophage line lacking NLRP3 was successfully established,and such macrophage deficient in NLRP3 inflammasome is a valuable cell model for investigating the activation of NLRP3 inflammasome,especially signaling in inflammation mediated by NLRP3 inflammasome.