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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 The aim of this study was to construct a recombinant lentivirus-mediated short hairpin RNA (shRNA) expression vector targeting the long non-coding RNA (lncRNA) BC002811 and establish a gastric cancer SGC-7901 cell line with stable BC002811 down-regulation. Methods Three small interfering RNAs (siRNA) were designed and synthesized. The best sequence for RNA interference was selected by real-time quantitative PCR (qPCR) and inserted into the lentiviral vector pLVX-shRNA2. After identification by DNA sequencing, the lentiviral vectors carrying BC002811 shRNA were packaged in HEK293T cells. The lentiviral particles were collected to infect human gastric cancer SGC-7901 cells. After screened by limiting dilution analysis, the SGC-7901 cell line with stable BC002811 down-regulation was established, the expression level of BC002811 detected by qPCR, and the effect of BC002811 on the proliferation of the cells analyzed by MTS. Results The results of qPCR showed that BC002811 siRNA-1 was the most effective siRNA sequence, with a knockdown efficiency of 87%. The recombinant lentiviral vector was packaged in the HEK293T cells with a viral titer of 3.7 × 108 TU/mL in the shRNA-1 group as compared with 4.5 × 108 TU/mL in the control. The expression of BC002811 in the shRNA-1 group was only 10% of that in the control group (P < 0.01), which indicated the successful establishment of the gastric cancer SGC-7901 cell line with stable BC002811 down-regulation. BC002811 knockdown significantly inhibited the proliferation of the SGC-7901 cells in the shRNA-1 group as compared with the control. Conclusion A recombinant lentiviral vector expressing BC002811 shRNA was successfully constructed and the gastric cancer cell line SGC-7901 with stable BC002811 silencing was established.
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Objective To construct and authenticate the lentiviral-mediated overexpression of mouse mitochondrial-targeted-8-oxoguanine DNA-glycosylase 1 (mito-OGG1) gene and the lentiviral-mediated short hairpin RNA (shRNA) down-regulation of OGG1 gene expression model in 661W cells.Methods Constructed the target plasmids,including pLenti-EF1a-EGFP-P2A-Puro-CMV-Mito-OGG1-3Flag (pLenti-OGG1-GFP) and pLKD-CMV-G&PR-U6-shRNA (pLKD-shRNA).293T cells were used to obtain green fluorescent protein (GFP)-tagged lentiviral vector of interest by using a second generation lentivirus packaging system.293T cells were also used for the virus titer estimation.The multiplicity of infection (MOI) of 661W cells was detected by fluorescence microscopy.A stable transfected cell line was screened by puromycin.Immunofluorescence was used to detect transfection efficiency and cytochrome C oxidase Ⅳ (COXⅣ)-OGG1 co-localization.OGG1 mRNA and protein expression levels were detected by real-time qantitative PCR (QPCR) and Western blot.Results Sequencing results showed that the inserted sequence in the over-expression plasmid was consistent with the mouse OGG1 (NM_010957.4) gene sequence in the gene library.The original lentiviral titer after packaging and purification was between 2.0× 107to 6.0× 107 TU/ml.The optimal MOI of 661W cells was 40,and puromycin with a concentration of 4.0 μg/ml successfully screened stable transformation.The transfection efficiency was up to 100％ after screening.Immunofluorescence demonstrated successful co-localization of OGG1 and COXⅣ.The relative expression levels of OGG1 mRNA in the blank control group,OGG1 group,overexpression control group,shRNA group and low expression control group were 1.000±0.000,41.581±12.206,0.888±0.056,0.239±0.121 and 1.081±0.083,and the relative expression levels of OGG1 protein were 1.029±0.153,1.657 ± 0.237,0.752 ± 0.143,0.471 ± 0.149 and 1.036 ± 0.185,respectively,with significant differences between them (F=44.654,30.948;both at P＜0.05),the relative expression levels of OGG1 mRNA and protein in the OGG1 group were significantly higher than those in the overexpression control group,the relative expression levels of OGG1 mRNA and protein in the shRNA group were significantly lower than those in the lower expression control group,with significant differences between them (all at P＜0.05).Conclusions The mitoOGG1 overexpression and OGG1 knockdown models of 661W cells are successfully constructed,which provides the preliminary experimental basis for follow-up study.
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Objective To construct and identify rat myosin heavy chain 14 (MYH14) gene recombined lentiviral vector by RNA interference technique. Methods Based on the MYH14 mRNA sequence, a single-stranded primer was designed to form a double-stranded oligonucleotide sequence, which was ligated into the GV298 lentiviral vector linearized by Ageand BamHdouble enzymes restriction, and then the bacterial liquid was verified by PCR and sequencing, respectively. The plasmid was extracted in the bacterial liquid with correct sequence and transfected into rat Schwann cells RSC96. The transfection efficiency was observed by immunofluorescence, the shRNA plasmid could effectively knock down MYH14 was screened by Western blotting, and the cell viability of RSC96 cells after transfection was detected by CCK-8. Results Three pairs of MYH14-shRNA sequences were synthesized and cloned into GV298 vector to construct recombinant plasmids MYH14-shRNA1, 2, and 3, and the vector MYH14-shRNA1 and MYH14-shRNA2 were screened by PCR and sequencing. Immunofluorescence showed that the cell fluorescence was the strongest at 72 h after transfection. Western blotting analysis showed that compared with the negative control (scramble sequence) group, the expression level of MYH14 protein in RSC96 cells was significantly decreased after MYH14-shRNA2 transfection (0.57±0.15 vs 1.11±0.06, P0.01), while there was no significant difference after MYH14-shRNA1 transfection (P0.05). There was no significant difference in cell viability of RSC96 cells between the negative control and MYH14-shRNA2 groups 24 h after transfection (1.09±0.16 vs 1.00±0.15, P0.05). Conclusion The rat MYH14 gene recombinant lentiviral vector has been successfully constructed, which can effectively down-regulate the expression of MYH14 in RSC96 cells.
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Objective@#To construct a recombinant short-hairpin RNA (shRNA) lentiviral vector targeting the β-catenin gene in cochlear precursor cells (CPCs) in mice, and to investigate its inhibitory effect.@*Methods@#PCR was used for the multiplication of the β-catenin gene, and shRNA oligo was designed based on the β-catenin gene to construct an interference vector. Gateway Technology was used to construct shRNA lentiviral vector which carried the β-catenin gene, and then 293FT cells were transfected with the constructed lentiviral vector and helper plasmids pLV/helper-SL3, pLV/helper-SL4, and pLV/helper-SL5. The virus supernatant was collected to obtain viral particles, and then mouse CPCs were transiently infected with the recombinant lentivirus with four different concentrations (0, 5, 10, and 20 μl) . The shRNA control group was established. Quantitative real-time PCR and Western blot were used to investigate the inhibitory effect of shRNA β-catenin lentiviral vector on β-catenin.@*Results@#The recombinant shRNA β-catenin lentiviral vector was successfully constructed, and the virus titers of shβ-catenin and shβ-catenin-control were 5.05×107 and 4.34×107, respectively. The results of in vitro experiments showed that in CPCs transfected with four different concentrations of recombinant lentivirus, the content of β-catenin protein gradually decreased with the increase in concentration, and there was a significant difference between groups (P<0.05) ; the CPCs transfected with shβ-catenin had significantly lower mRNA expression of β-catenin than those in the shβ-catenin-control group (P<0.05) .@*Conclusion@#The constructed lentiviral vector targeting the β-catenin gene has a high infection efficiency, and the successful construction of lentiviral vectors provides a technical support for analyzing the role of β-catenin in the differentiation of CPCs into auditory hair cells.
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Objective·To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods?·?Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay. The Raw 264.7 cells were randomly divided into four groups including adenovirus (-) LPS (-) group, adenovirus (-) LPS (+) group, adenovirus (+) LPS (-) group and adenovirus (+) LPS (+) group. The expression of intracellular IRF3 mRNA was detected by real-time PCR, and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting. Results?·?The titer of adenovirus was 2.2×1011PFU/mL and the best MOI was 300. The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state. The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA. Conclusion?·?Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3, but not affect the nuclear expression of Irak1bp1 protein.
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Objective To construct a recombinant lentiviral vector carrying the casitas B-lineage lymphoma (Cbl)-b short hairpin RNA (shRNA),and to evaluate its effect on the biological behavior of A375 melanoma cells in vitro.Methods Three specific shRNAs targeting Cbl-b gene and a negative control shRNA were designed and synthesized,and recombinant lentiviral vectors were constructed.A375 cells were divided into 5 groups to be transfected with 3 kinds of lentiviral vector expressing Cbl-b genespecific shRNAs (CBLB-shRNA-1 group,CBLB-shRNA-2 group and CBLB-shRNA-3 group),a lentiviral vector containing negative control shRNA (negative control group),and an empty vector (blank control group).Real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine the silencing efficiency at 72 hours after transfection.Cell counting kit-8 (CCK-8)assay was conducted to evaluate cellular proliferative activity at 24,48,72 and 96 hours after transfection,flow cytometry to detect cell apoptosis and cell cycle at 72 hours after transfection,and Transwell invasion assay to assess cellular invasive activity at 72 hours after transfection.Results Three recombinant lentiviral vectors containing Cbl-b shRNA were constructed successfully.As Western blot analysis revealed,the CBLB-shRNA-3 showed the highest silencing efficiency.CCK-8 assay indicated that the proliferative activity of A375 cells was significantly lower in the CBLB-shRNA-3 group than in the negative control group and blank control group at 72 and 96 hours after transfection(all P ＜ 0.01).Flow cytometry showed that the apoptosis rate of A375 cells was significantly higher in the CBLB-shRNA-3 group (22.73％ ± 6.58％) than in the negative control group (6.08％ ± 1.35％,P ＜ 0.01) and blank control group (6.34％ ± 1.07％,P ＜ 0.01).The CBLB-shRNA-3 group showed a significantly higher proportion of A375 cells at G1 phase,but a significantly lower proportion of A375 cells at S phase compared with the negative control group and blank control group(all P ＜ 0.01).Transwell assay showed that there were significant differences in the number of A375 cells crossing the artificial basement membrane (matrigel) at 72 hours after transfection among the negative control group,blank control group and CBLB-shRNA-3 group (76.60 ± 1.82,73.20 ± 3.83,19.60 ± 1.14,respectively;F =794.50,P ＜ 0.01).Conclusions A recombinant CBLB-shRNA-3-expressing lentiviral vector which can efficiently silence Cbl-b gene has been successfully constructed.It can inhibit the proliferation,cell cycle progression and invasive activity of A375 cells,but promote the apoptosis of A375 cells.
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Objective • To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods • Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay. The Raw 264.7 cells were randomly divided into four groups including adenovirus (-) LPS (-) group, adenovirus (-) LPS (+) group, adenovirus (+) LPS (-) group and adenovirus (+) LPS (+) group. The expression of intracellular IRF3 mRNA was detected by real-time PCR, and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting. Results • The titer of adenovirus was 2.2×1011 PFU/mL and the best MOI was 300. The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group. The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state. The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA. Conclusion • Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3, but not affect the nuclear expression of Irak1bp1 protein.
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Cancer is a manifestation of dysregulated gene function arising from a complex interplay of oncogenes and tumor suppressor genes present in our body. Cancer has been constantly chased using various therapies but all in vain as most of them are highly effective only in the early stages of cancer. Recently, RNA interference (RNAi) therapy, a comparatively new entrant is evolving as a promising player in the battle against cancer due to its post‑transcriptional gene silencing ability. The most alluring feature of this non‑invasive technology lies in its utility in the cancer detection and the cancer treatment at any stage. Once this technology is fully exploited it can bring a whole new era of therapeutics capable of curing cancer at any stage mainly due to its ability to target the vital processes required for cell proliferation such as response to growth factors, nutrient uptake/synthesis, and energy generation. This therapy can also be used to treat stage IV cancer, the most difficult to treat till date, by virtue of its metastasis inhibiting capability. Recent research has also proved that cancer can even be prevented by proper modulation of physiological RNAi pathways and researchers have found that many nutrients, which are a part of routine diet, can effectively modulate these pathways and prevent cancer. Even after having all these advantages the potential of RNAi therapy could not be fully tapped earlier, due to many limitations associated with the administration of RNAi based therapeutics. However, recent advancements in this direction, such as the development of small interfering RNA (siRNA) tolerant to nucleases and the development of non‑viral vectors such as cationic liposomes and nanoparticles, can overcome this obstacle and facilitate the clinical use of RNAi based therapeutics in the treatment of cancer. The present review focuses on the current status of RNAi therapeutics and explores their potential as future diagnostics and therapeutics against cancer.
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Objective To study the expressions of myeloid cell leukemia-1 (Mcl-1 ) gene in mouse macrophages Raw264.7 and human macrophages THP-1,to screen out the cell lines with high levels of expression as the experimental cells,and based on the screening results to construct the short hairpin RNA(shRNA)eukaryotic expression plasmid targeting mice Mcl-1 gene for transfection and further screen out the shRNA expression plasmid with the most obvious effect in silencing Mcl-1 gene.Methods Semi-quantitative PCR method was used to detect the expression of Mcl-1 mRNA in the two kinds of macrophages.Western blotting was adopted to detect the expressions of Mcl-1 proteins in the two kinds of macrophages.Three different gene loci for Mcl-1 shRNA fragments were designed with small molecules interfering RNA (siRNA)software.Eukaryotic expression plasmid Mcl-1 shRNA 1-3 carrying the shRNA fragments was constructed by a company.And the eukaryotic expression plasmid vector was transfected into scavenger cells Raw264.7 mice via through the liposome.The transfection results 24 h and 48 h after transfection were observed under the inverted fluorescence microscope;Mcl-1 mRNA and protein expression were detected by real time quantitative PCR and Western blot,respectively.Results The relative expression levels of Mcl-1 mRNA and protein in mouse macrophages Raw264.7 were significantly higher than those of human macrophages (P <0.05).The shRNA expression vector constructed within 24 h and 48 h could decrease Mcl-1 mRNA and protein levels in Raw264.7 cells,especially with the most obvious silencing effect at 48 h.The 48-h transfection group differed significantly from normal group,liposome group and negative control group (P <0.05).Compared with Mcl-1shRNA1and Mcl-1shRNA2,Mcl-1shRNA3 had the strongest effect in silencing Mcl-1mRNA and protein.Conclusion We have successfully screened out experimental Raw264.7 cells and Mcl-1 shRNA eukaryotic expression plasmid which has an obvious silencing effect targeting on Mcl-1 in mice macrophages Raw264.7.
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Objective To construct the recombinant adeno-associated virus vector carrying cancerous inhibitor of PP2A (CIP2A)short hairpin RNA (shRNA)for preparation of high-titer viruses.Methods The small hairpin RNA of CIP2A (CIP2A shRNA)was designed,synthesized and cloned into pDC31 6-EGFP-U6 plasmid which was double digested by Bam HⅠ and Hin dⅢ.The resultant plasmid pDC31 6-EGFP-shRNA was confirmed and served as template to appraise primers.EGFP-CIP2A shRNA sequence was amplified by PCR,double digested with Eco RⅠand Sal Ⅰ and ligated to pSNAV2.0 plasmid digested with the same enzyme pair.pSNAV2.0-EGFP-CIP2A shRNA plasmid DNA was prepared,purified,identified and transfected into BHK-21 cells.BHK-21 cells expressing CIP2A shRNA (BHK-21/CIP2A-shRNA ) were obtained and subsequently infected with VGTC’s proprietary AAV packaging system to package the rAAV2-CIP2A shRNA.After purification,the functional and infectious virus was obtained and the titer of virus was detected.Real-time PCR and Western blot methods were used to detect the expression of CIP2A after infection with HepG2 cells,and the empty viral vector rAAV2-EGFP was used as control. Results A recombinant adeno-associated virus-2 vector carrying CIP2A shRNA was constructed successfully.The presence of the target sequence in the vector was confirmed by double enzyme digestion and sequencing.By transfecting the pSNAV2.0-EGFP-CIP2A shRNA plasmid into BHK-21 cells,BHK-21/CIP2A shRNA cells were infected with helper virus HSV1-rc/ΔUL2 to package the rAAV2-CIP2A shRNA to obtain a functional and infectious virus.The titer of the recombinant virus was 0.25×10 1 2 v.g./mL.The expression of CIP2A mRNA and screening value of 1×10 5 MOI effected HepG2 cells.Conclusion A high-titer recombinant adeno-associated virus-2 vector carrying CIP2A shRNA has been constructed successfully.
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Objective To construct eukaryotic vectors expressing short hairpin RNAs ( shRNAs ) targeting at the CIP2 A gene and to explore its effects on gastric cell line BGC-823 .Methods Four oligonucleotides targeting the CIP2A gene were synthesized and cloned into the eukaryotic expression plasmid pGPU 6.The recombinant plas-mids, pGPU6/GFP/Neo-CIP2A-shRNA-1, 2, 3 and 4, were introduced into BGC-823 cells by lipofectamine-me-diated transfection and the infection rate was observed by fluorescence microscope .The gene silencing efficiency was measured by real-time PCR and Western blot .The effects on proliferation of BGC-823 cells were detected by CCK-8 .Results DNA sequencing and enzyme digestion analysis confirmed the identity of the four recombinant shRNA expression vectors .Immunofluorescsence demonstrated that transfection efficiency was above 70%.Trans-fection of shRNA-1, 2, 3 and 4, significantly knocked down the expression of CIP 2A mRNA and protein at 24, 48 and 72 h after transfection .Compared with the 2 , 3 and 4 , shRNA-1 had the more strong inhibitory effect on the expression of CIP2A mRNA and protein.The CCK-8 assay showed that the anti-proliferation effect on BGC-823 cells was significant ( P<0.05 ) .Conclusions The recombinant vector may effectively inhibit the expression of CIP2A in BGC-823 cells and depress the proliferation of BGC-823 cells.
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[Abstract ] Objective The purpose of this study was to construct a short hairpin RNA (shRNA) interference lentiviral vector targeting the humanβ-COP gene and to evaluate its inhibitory effect on β-COP in THP-1 cells. Methods We designed and synthesized 4 humanβ-COP-specific oligonucleotide sequences and inserted them into the pGMLV-SC1 vector to construct a recombinant vector fol-lowed by transfection of HEK 293T cells with the recombinant vector and Lenti-HG Mix to produce lentiviruses and detect the viral con-tent.After infecting the THP-1 cells with the packaged lentiviruses , we analyzed the inhibitory effect of β-COP-shRNA on the β-COP gene by quantitative PCR and Western blot . Results Sequencing confirmed that the β-COP-specific oligonucleotide sequences were in-serted into the lentiviral vector and the lentiviruses were packaged in the transfected HEK 293T cells, with the final viral content of 1 × 109 TU/mL.Quantitative PCR showed that the 4 β-COP-shRNA vectors significantly decreased the mRNA expression of β-COP (P<0.01), with interference rates of 16.9 %,32.5%, 74.0%, and 50.3%, respectively.Western blot also indicated their inhibitory effect on the protein expression of β-COP, with an inhibition rate of 76.4% onβ-COP-shRNA3. Conclusion Lentiviral shRNA interference vectors targeting human β-COP were constructed successfully , which could suppress the expression of the human β-COP gene.
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Objective To investigate the effect of short hairpin RNA(shRNA) eukaryotic expression vector‐mediated silen‐cing of the survivin‐gene on proliferation and apoptosis of human umbilical vein endothelial cells(HUVEC) .Methods The shRNA vector targeting the survivin gene and negative control vector were transfected into human umbilical vein endothelial cells(HUVEC) incubated with 50 ng/mL of recombinant VEGF in vitro by lipofectamine 2000 .Transfection after 48 h ,the expression of survivin mRNA and protein was detected by quantitative real‐time PCR and Western blot ,respectively .HUVEC proliferation was assayed by four methylthiazolyl tetrazolium(MTT) and cell apoptosis was detected by TUNEL .Results (1)Transfection with survivin‐shR‐NA vector significantly down‐regulated the expression of survivin mRNA and protein as compared with the control group ,after transfection of 48 h(P<0 .05) .(2)After survivin‐shRNA vector transfected ,the proliferation of HUVEC decreased significantly . After transfection 24 ,48 ,72 h ,the growth inhibition rate were (13 .53 ± 3 .91)% ,(38 .97 ± 1 .82)% ,(65 .75 ± 1 .83)% respective‐ly ,at 72 hours after transfection was the most significant .(3)The apoptosis rate of experimental group was (28 .07 ± 1 .71)% , which was higher than the negative control group (11 .45 ± 1 .52)% and blank control group (10 .04 ± 1 .46)% (P<0 .05) .Conclu‐sion The shRNA‐mediated mediated silencing of the survivin‐gene could significantly inhibit proliferation and promote the apopto‐sis of rheumatoid arthritis synovial fibroblasts by regulating survivin expression .
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Background and purpose:Gastric cancer is the most common malignant tumor of digestive tract, and the possibility of postoperative recurrence and metastasis is higher. Our previous studies showed that protein tyrosine phosphatase1B (PTP1B) gene is closely correlated with tumor size, lymph node metastasis and TNM stage of gastric cancer. The purpose of the present study was to explore the effect ofPTP1B gene on cell proliferation and migration of gastric cancer cell lines.Methods:Short hairpin RNA (shRNA) sequence targetingPTP1B gene and PTP1B cDNA were transfected into MKN28 and MKN45 cells, respectively. The expression of PTP1B mRNA and its protein in MKN28 and MKN45 cells were detected by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The effect of PTP1B on cell proliferation and migration was respectively assessed by cell counting kit-8 (CCK-8) assay, Transwell migration assay and wound healing assay.Results:Compared with blank and negative controls, the expressions of PTP1B mRNA and protein in MKN28 cells were successfully suppressed after the cells were transfected with shRNA (P<0.05). As CCK-8 test showed, the proliferation of MKN28 cells was successfully restrained at 48, 72 and 96 h after transfected with shRNA compared with blank control and negative control (P<0.05). Transwell and wound healing test were performed after PTP1B expression was interfered by shRNA. The result demonstrated that migration of MKN28 cells was signiifcantly inhibited (P<0.05). On the contrary, the expressions of PTP1B mRNA and protein in MKN45 cells were obviously enhanced after the cells were transfected with PTP1B cDNA. And the proliferation and migration of cells were significantly increased.Conclusion:PTP1B gene is an important enchancer for the proliferation and migration of gastric cancer.
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Objective To investigate the anti-fibrogenesis property and mechanism of short hairpin RNA (shRNA ) targeting heat shock protein 47 (HSP47) in schistosomiasis liver fibrosis mice model . Methods Sixty female BALB/c mice (SPF level) were infected percutaneously with (16 ± 1) Schistosoma japonicum cercariae . Twelve mice which were not infected with Schistosoma japonicum were set as uninfected control .The 60 mice were randomly divided into five groups including 1 mg/kg HSP47-shRNA intervention group ,2 mg/kg HSP47-shRNA intervention group ,4 mg/kg HSP47-shRNA intervention group ,unrelated shRNA intervention group and infected control group ,with 12 mice each group .From 6 to 14 weeks post infection ,mice in HSP47-shRNA intervention groups were intravenously injected with HSP47-shRNA weekly and those in unrelated shRNA intervention group were intravenously injected with 2 mg/kg of unrelated shRNA vector weekly .Survival rate ,liver and spleen in general and liver histology of mice in each group were observed at week 14 which was considered as end of intervention . The expressions of HSP47 mRNA and protein were determined by real-time polymerase chain reaction (PCR) and Western blot .Collagen type Ⅰ ,collagen type Ⅲ ,tissue inhibitor of metalloproteinase-1 (TIMP-1) , matrix metalloproteinase-9 (MMP-9) ,plasminogen activator inhibitor-1 (PAI-1) ,transforming growth factor-β1 (TGF-β1 ) , connective tissue growth factor (CTGF ) , interleukin (IL )-13 and IL-17 at transcriptional level were measured by real-time PCR .Means among groups were compared using student-t test .Results In vivo intervention of HSP47-shRNA mainly localized in hepatic stellate cells of mice . The survival rate of 1 mg/kg ,2 mg/kg and 4 mg/kg HSP47-shRNA intervention groups were 25 .00% , 25 .00% and 33 .33% , respectively .Mice received 4 mg/kg dose of HSP47-shRNA had significantly higher survival rate than the infected controls (χ2 = 4 .168 ,P= 0 .043) .In both 4 mg/kg HSP47-shRNA intervention group and the infected control group , hematoxylin and eosin staining showed chronic granuloma change ,of which ova were wrapped by the spindle-shaped collagen and inflammatory cell infiltrated .The percentage of positive staining for Masson trichrome and sirius red showed the quantity of the collagen deposition was significantly reduced by the HSP 47-shRNA intervention ,compared to the infected control group (t = 3 .191 ,P = 0 .039) .HSP47-shRNA significantly reduced HSP47 expression , and reduced the transcriptional levels of collagen type Ⅰ ,collagen type Ⅲ ,TIMP-1 ,PAI-1 ,TGF-β1 , CTGF ,IL-13 and IL-17 (all P < 0 .01) ,and increased the expression of MMP-9 mRNA ( P < 0 .01) . Conclusion Delivery of shRNA targeting HSP47 to the hepatic schistosomiasis mice model can silence the expression of HSP47 and improve the liver fibrosis .Collagen degradation and related cytokines release through upregulation of MMP-9 and downregulation of PAI-1 may be one of the anti-fibrotic mechanisms in HSP47-shRNA intervention .
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Background Age-related macular degeneration (AMD) is a group of vision-threatening eye disorders.Previous researches showed that the activation of Akt/mammalian target of rapamycin (Akt/mTOR) pathway closely related to the mechanism of AMD.A new specific method to inhibit Akt/mTOR pathway will become a breakthrough for the treatment of AMD.Objective This study was to establish a mouse fibroblast cell line (NIH/3T3) which can stably inhibit the expression of mTOR gene and provide a cell model for the study on the function of Akt/mTOR pathway in AMD and observe the influence of mTOR gene knockdown on related proteins.Methods Three short hairpin RNAs (shRNAs) targeting mTOR gene were designed and synthesized based on murine mTOR mRNA sequence.Double-strand shRNA hairpins were separately cloned into PIGZ-green fluorescent protein (GFP) +Puro vectors to produce recombination plasmids.The packaged lentiviral plasmids and RNAi plasmids were co-transfected into NIH/3T3 cells,a mouse fibroblast line.After puromycin selection and culture expansion,0.5 mg/L puromycin was added the culture medium to establish stable cell clones.The expressions of mTOR mRNA and protein in NIH/3T3 cells were detected by real-time PCR and Western blot respectively,and the inhibitory efficiency of interference was analyzed.Results Transfected GFP-labeled NIH/3T3 cells by lentiviral presented the green fluoresccence with the efficiency of infection of 90％ in the third day.Real-time PCR showed a distinct band of mTOR mRNA in 184 bp.The knockdown rate for sh1,sh2 and sh3 were respectively 31.3％,31.8％ and 45.3％ in the lower multipolicity of infection (MOI) group ;while in the higher MOI group,the knockdown rate for sh1,sh2 and sh3 were 47.1％,56.5％ and 71.6％ respectively.Western blot assay exhibited weakened expression band of mTOR protein in NIH/3T3 cell line for sh1,sh2 and sh3 in both lower and higher MOI groups with the weakest expression for sh3.Conclusions A stable mouse fibroblast cell line is established by the inhibition of mTOR with RNA interference technique,which can provide a cell model for studying the function of Akt/mTOR pathway in AMD.
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Objective To construct RNAi eukaryotic expressing vectors of human transcription factor glioma-associated oncogene homolog 1 (GLI1) with pGCsi-U6-GFP plasmid and to identify its activity in interfering GLI1.Methods Three GLI1siRNA targeting GLI1 were designed and synthesized according to the GLI1cDNA sequence in GeneBank,and then were cloned into pGCsi-U6-GFP to construct the recombinant plasmids,and transformed into E.coli DH5a,then it was amplified and plasmids were extracted,which were further confirmed by PCR reaction and DNA sequencing,pGCsi-U6-siRNA-C was negative as control wector.Then recombinant plasmids pGCs-U6-GLI1siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1siRNA-3 pGCsi-U6-siRNA-C and a eukaryotic over-expression vector pEGFP-N1-GLI1 were co-transfected into HEK293 cells by Lipofectamine 2000 respectively.The ceils were collected at 48 h after transfection.Semi-quantitative RTPCR and Western Blot were performed to detect the expression of GLI1 mRNA and protein to screen the optimal vector which had the best interfering effect.Results A 369 bp fragment was amplified from all three recombinant plasmids,(pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLIlsiRNA-3),showing that synthesized shRNA oligonucleotide fragments were correctly inserted into three recombinant plasmids,which were further confirmed by sequencing.Expression levels of GLIlmRNA and protein in cells in pGCs-U6-GLI1 siRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 were 0.290 ± 0.011,0.421 ± 0.018,0.373 ±0.018,and 0.318 ± 0.026,0.443 ± 0.021,0.381 ± 0.018,which were significantly lower than those in negative control group (0.834 ± 0.022,0.818 ± 0.024,P =0.000),the inhibitory rates were 65.8 ％,50.7％,55.7％,and 63.9％,48.3％,53.9％.The interfering efficacy of pGCs-U6-GLIlsiRNA-1 was the strongest among the three recombinant plasmids.Conclusions RNAi eukaryotic vectors pGCs-U6-GLIlsiRNA-1,pGCs-U6-GLI1 siRNA-2,pGCs-U6-GLI1 siRNA-3 are successfully constructed and the optimal vector is identified,and this can provide a solid experimental foundation for further functional study of GLI1 gene.
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Objective To investigate the effects of integrin β1 gene expression inhibited by short hairpin RNA (shRNA) on invasion of pancreatic carcinoma PANC1 cells in vitro,and investigate the mechanism.Methods The eukaryotic expression plasmid of shRNA targeting integrin β1 gene ( integrin β1 shRNA) and control eukaryotic expression plasmid shRNA (c-shRNA) was constructed and was transfected into PANC1 cells.The cells without plasmid transfection were used as control.The expressions of integrinβ1,MMP 2,MMP 9 mRNA and protein were detected by real-time PCR and Western blotting.The invasive ability of PANC1 cells was observed with Transwell cell culture chamber.Results Integrinβ1 mRNA expressions in integrinβ1 shRNA group,c-shRNA group and control group were 0.0029 ± 0.0004,0.0131 ± 0.0009,0.0138 ± 0.0005 ; the expressions of integrinβ1 protein were 0.0159 ± 0.0062,0.3215 ± 0.0126,0.3107 ±0.0094; the inhibitory rate of integrinβ1 mRNA and protein expression in integrinβ1 shRNA group was (78.6 ±7.2 ) ％ and (92.9 ± 3.2) ％ ( P ＜ 0.01 ).But there was no difference between the c-shRNA group and control group (P =0.2999).Number of penetrating cells in integrinβ1 shRNA group decreased from 52 ±5 to 21 ±4( P ＜ 0.01 ) ; the expression of MMP 2 and MMP 9 mRNA decreased from 0.592 ± 0.073,0.847 ± 0.069 to 0.102 ± 0.034,0.273 ± 0.071 ; the expression of M MP2 and MMP 9 protein decreased from 0.225 ± 0.046,0.416 ±0.081 to 0.059 ±0.013,0.106 ±0.022(P ＜0.05).Conclusions Recombinant integrinβ1 shRNA expression plasmid can effectively inhibit the expression of integrinβ1 gene and suppress the invasion of PANC1 cells in vitro by down-regulating MMP 2 and MMP 9 gene expression.
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Objective To compare the reversal effects of short hairpin RNA (shRNA) interference and tetrandrine on multidrug resistance (MDR) of human colorectal cancer cell line LoVo/5-fluorouracil(5-FU ).Methods An eukaryotic expression plasmid of shRNA targeting MDR1 was constructed and transfected into human colorectal cancer cell line LoVo/5-FU (transfection group).LoVo/5-FU was also pretreated with tetrandrine (tetrandrine group).Drug sensitivity was detected by methyl thiazolyltetrazolium colorimetric method.Cell cycle,apoptosis of cells and positive expression rate of P-glycoprotein (P-gp) were determined by flow cytometry assay.The expressions of MDR1 mRNA and P-gp were detected by real-time polymerase chain reaction and Western blot,respectively.All data were analyzed by analysis of variance and SNK-q test.Results (1)Drug sensitivity:the 50％ concentration of inhibition(IC50)of the control group,tetrandrine group and transfection group were (7.3 ± 0.3),(4.4 ±0.7) and (2.4 ±0.4) mmol/L,respectively,a significant difference between the 3 groups was found(F =65.27,P ＜ 0.05 ).There was a significant difference in the IC50 between the tetrandrine group and the transfection group (q =6.67,P ＜ 0.05 ).(2) Changes of cell cycle:the proportion of cells in the G1 phase and S phase of the control group,tetrandrine group and transfection group were 38.13％ ± 3.75％,51.36％ ± 2.76％,59.24％±4.31％ and 20.46％±2.23％,14.32％± 1.91％,9.40％± 1.65％,respectively,a significant difference between the 3 groups was found(F =25.23,24.37,P ＜ 0.05 ).There were significant differences in the proportion of cells in the G1 phase and S phase between the tetrandrine group and the transfection group(q =3.67,4.35,P ＜ 0.05 ).(3) Cell apoptosis:the cell apoptotic rates of the control group,tetrandrine group and transfection group were 1.32％ ± 0.47％,3.24％ ± 0.26％,5.88％ ±- 0.44％,respectively,a significant difference between the 3 groups was found(F =99.26,P ＜ 0.05 ).There was a significant difference in the cell apoptotic rate between the tetrandrine group and transfection group(q =11.48,P ＜ 0.05 ).(4)The expression of P-gp:the positive expression rates of P-gp of the control group,tetrandrine group and transfection group were 96.9％ ± 2.3％,61.6％ ± 4.9％,76.6％ ± 3.6％,respectively,a significant difference between the 3 groups was found(F =67.83,P ＜ 0.05 ).There was a significant difference in the positive expression rate of P-gp between the tetrandrine group and transfection group (q =6.97,P ＜ 0.05 ).(5)The mRNA expression of MDR1:the mRNA expressions of MDR1 of the control group,tetrandrine group and transfection group were 1462 ±161,570 ±85,233 ± 81,respectively,a significant difference between the 3 groups was found(F =90.59,P ＜ 0.05 ).There was a significant difference in the mRNA expression of MDR1 between the tetrandrine group and transfection group (q =5.12,P ＜ 0.05 ).Conclusions MDR1 shRNA and tetrandrine could reverse M DR1 gene-mediated m.ultidrug resistance in human colon cancer cell line LoVo/5-FU,but the effect of MDR1 shRNA is better than that of tetrandrine.MDR1 shRNA and tetrandrine might take effect by inhibiting P-gp expression and down-regulating mRNA expression of MDR1.