Construction and identification of a vector expressing TRAM siRNA in mammalian cells

Objective：To construct and identify a vector expressing TRAM siRNA in mammalian cells.Methods :It was constructed that the vector named R-pSUPER-EGFP used to transcribe functional TRAM siRNA. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. The recombinants were transformed into E. coli JM109, and finally their veracity was confirmed by double cutting with the enzymes and sequencing. R-pSUPER-EGFP was transfected into RAW264. 7 cell by using LipofectamineTM2000, and the expression of TRAM was detected by Western blotting. Results .. Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed, transfected into RAW264.7 cell line successively, which can specifically restrain expression of TRAM protein. Conclusion :The optimizing method in constructing the recombinant vector serves other plasmid-based RNA interference research. Therefore, the recombinant vectors establish the basis for research on the function of TRAM gene.     

Targeted silencing of heparanase gene by small interfering RNA inhibits invasiveness and metastasis of osteosarcoma cells

The effects of targeted silencing of heparanase gene by small interfering RNA(siRNA) on invasiveness and metastasis of osteosarcoma cells(MG63 cells) were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA(pGenesil-shRNA) was constructed successfully.MG63 cells were randomly allocated into 3 groups:blank group,empty vector(pGenesil) transfected group and expression vector(pGenesil-shRNA) transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%(P<0.01) and 75.3%(P<0.01) respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.     

Optimization of the construction of recombinant plasmids PPARγ- pSUPER-EGFP for RNA interference

Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.     

Protective effect of O6-methylguanine-DNA-methyltransferase on mammalian cells

Background O^6-methylguanine-DNA-methyltransferase （MGMT） is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells. Methods Mammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells （PBMCs） via liposome, then assayed for gene expression at RNA and protein levels. MIF assay was used to check the drug resistance of cells transfected with MGMT gene. Results MGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively. Results of Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC50 values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC. Conclusion The alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.     

Influence of silencing soluble epoxide hydrolase with RNA interference on cardiomyocytes apoptosis induced by doxorubicin

In order to investigate the influence of silencing soluble epoxide hydrolase(sEH) with double-stranded small interfering RNA(siRNA) on cardiomyocytes apoptosis induced by doxorubicin(DOX),two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents.The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively,and the plasmids that silenced sEH most significantly were selected,and renamed EH-R.The plasmids carrying a nonspecific siRNA coding sequence(PCN) served as the negative control.Cardiomyocytes were divided into four groups:control group,DOX group,PCN+DOX group,and EH-R+DOX group.Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L.Apoptosis rate of cardiomyocytes was determined by flow cytometery.The protein expression levels of Bcl-2 and Bax were detected by Western blotting.The results showed that the expression of sEH was down-regulated by EH-R plasmid.The expression levels of sEH mRNA and protein in the EH-R+DOX group were significantly decreased as compared with other groups(P<0.01).As compared with the control group,the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased,the expression levels of Bax increased,and those of Bcl-2 decreased(P<0.01).However,the expression levels of Bax were decreased,those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obvi-ously decreased in EH-R+DOX group when compared with those in the DOX group and the PCN+DOX group(P<0.01 for each).It was concluded that the recombinant plasmids could be successfully constructed,and transfected into the primary cultured cardiomyocytes.They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.     

Cloning and Identification of frc Gene from Oxalobacter Frmigenes

The cloning and identification of frc gene from Oxalobacter formigenes in the intestines of Chinese people were conducted. The genomic DNA of Oxalobacter formigenes was extracted. frc gene fragment was amplified by polymerase chain reaction (PCR) and linked with pEGFP-C1. The recombinant plasmid was designated pEGFP-frc and was identified by restriction-enzyme digestion and sequencing. Human embryo kidney 293 cells were transfected with pEGFP-frc, then RT-PCR and Western blotting were performed to detect the expression of frc gene. The length of frc gene was found to be 1287 bp, and the homology of nucleotides and amino-acid residue with the sequence in GenBank was 95.88% and 99.07%. Bright green fluorescent light could be observed in 293 cells transfected with the pEGFP-frc. frc mRNA and fusion protein FCoAT-EGFP were detected in the cells. It is concluded that frc gene cloned from the Oxalobacter formigenes in the intestines of Chi- nese people can be expressed in eucaryotic 293 cells and keep its enzyme activity.     

Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

Objective： To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide（LPS） in vitro. Methods： pShuttleHl-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH 1-siCas3 and pEGFPC 1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry, pShuttleHl-siCas3 was linearized and transformed into E. coli B J5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results： It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleHl-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×10^10pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion： The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.     

PI3K-like kinases restrain Pim gene expression in endothelial cells

Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.     

Influence of β-catenin small interfering RNA on human osteosarcoma cells

This study examined the effect of small interfering RNA-mediated β-catenin knockdown on the survival,invasion and chemosensitivity of human osteosarcoma cells(U2-OS cells).The siRNA against β-catenin was constructed and transfected into U2-OS cells.The expression of β-catenin was detected by qRT-PCR and Western blotting.Cell growth and apoptosis was detected in the presence or absence of doxorubicin by MTT and flow cytometry,respectively.Cell invasion ability was measured by transwell assay.The results showed that the transfection of β-catenin siRNA resulted in decreased expression of β-catenin,suppression of invasion and motility of U2-OS cells,reduced chemosensitivity to doxorubicin in vitro,and little change in cell growth and apoptosis.Additionally,down-regulated MT1-MMP expression was found after transfection.It was concluded that knockdown of β-catenin gene may decrease the invasive ability of human osteosarcoma cells through down-regulated MT1-MMP expression,and the chemosensitivity of osteosarcoma cells against doxorubicin.     

In vitro and in vivo evidence of metallopanstimulin-1 in gastric cancer progression and tumorigenicity

Purpose:The metallopanstimulin-1(MPS-1)gene is a growth factor-inducible gene,which is highly expressed in many human cancers and may be involved in the progression towards tumor malignancy.However,it is unclear whether MPS-1 plays any role in gastric cancer development or progression.Our studies were designed to clarify the MPS-1 expression pattern and to explore its potential role in gastric cancer.Experimental Design:The expression pattern of MPS-1 was determined in primary gastric cancer specimens and gastric cancer cell lines via immunohistochemistry and Western blotting.To investigate the functional significance of MPS-1 expression,three small interfering RNA(siRNA)expression plasmids were constructed and transfected into gastric cancer cell line SGC7901.The stable cell lines transfected with the siRNA targeting MPS-1 mRNA plasmids were selected and the biological features of these cells were examined.Results:MPS-1 was overexpressed in 86% of the gastric cancer tissues and all gastric cancer cells.In addition,MPS-1 expression was significantly increased and corresponded with the tumor-node-metastasis clinical stage,and was significantly higher in the late stage(P<0.01).The MPS-1 expression level was significantly decreased in the transfected cells with MPS-1-specific siRNA expression plasmid pRNAT-133.Furthermore,the stable transfected cancer cells exhibited an increase in the incidence of spontaneous apoptosis and a decrease in growth ability and tumorigenicity in nude mice.Conclusions:These results provide strong evidence that MPS-1 plays an important role in gastric cancer cell proliferation and development,and suggests that MPS-1 is a promising target for gastric cancer treatment.     

Effect of TRPC6 knockdown on puromycin aminonucleoside-induced podocyte injury

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.     

Small RNA interference-mediated gene silencing of TREK-1 potassium channel in cultured astrocytes

This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.     

Construction of Smac gene-containing and human prostate specific antigen promoter-regulated vector and its expression

To construct an eukaryotic expression vector containing Smac gene and study the expression efficiency and specificity of prostate specific antigen（PSA） enhancer/promoter in a possible targeted gene therapy scheme for prostate cancer. Methods： PSA enhancer （PSAE） and promoter （PSAP） sequences were amplified using PCR method. CMV and T7 promoters were deleted from pcDNA3.1-Smac and replaced by the two specific fragments to generate pPSAE-PSAP-Smac. After transfection into different cell lines, the status of cells was observed. And then, we determined the relative concentration of Smac mRNA in RT-PCR. Results： The recombinant plasmid of pPSAE-PSAP-Smac was successfully constructed. And only the prostate cancer cell line PC-3 was suppressed after transfection with pPSAE-PSAP-Smac. However, other nonprostate lines were not. Moreover, the concentration of Smac mRNA regulated by PSA promoter and enhancer was higher in comparison to the CMV promoter-driven control vectors. Conclusion： An expression vector containing the Smac gene （based on elements of the PSA gene regulatory sequences） has been developed and shown to function in prostate cancer cell lines which provides a solid platform for launching clinical studies.     

Down-regulation of Tissue Factor by siRNA Increased Doxorubi-cin-induced Apoptosis in Human Neuroblastoma

The effects of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblas-toma were investigated. The expression of TF was examined by Western blotting. TFsiRNA-pSUPER plasmid was constructed by inserting specific 19-nt silencing sequence targeting TF gene into pSU-PER vector. Transfection of TFsiRNA-pSUPER was performed using lipofectamine2000. The cytotox-icity of doxorubicin was determined by WST assay. The activation of Caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest33342 and counted under fluorescence inverted microscope. It was found that human neuroblastoma cell line SK-N-MC expressed high level of TF. Knockdown of the TF expression was achieved by trans-fection of TFsiRNA-pSUPER on SK-N-MC cells in a dose-dependent manner. Inhibition of TF sig-nificantly decreased the viability of transfected SK-N-MC cells treated with different concentrations of doxorubicin. Cleavage of Caspase-3 and PARP was enhanced in transfected SK-N-MC cells with down-regulation of TF. TFsiRNA treatment significantly increased the number of apoptotic cells in transfected SK-N-MC cells as compared with those control cells (P<0.05) when these cells were ex-posed to 1 μg/mL doxorubicin for 8 h. These results suggested that knockdown of the TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubi-cin-induced apoptosis in human neuroblastoma cells. Over-expression of TF might contribute to chemotherapy resistance in human neuroblastoma and its progression, at lest in part, by regulating doxorubicin-induced apoptosis.     

Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene

The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were ana- lyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04±0.10, 0.94±0.04 respectively and the expression of p-Akt protein ( 0.94±0.07) was lower than those in control groups (1.68±0.14, 1.66±0.10) (P< 0.05). The IC50 of DDP to C13K cells transfected with PTEN (7.2±0.3 μmol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7±0.4 μmol/l, 13.0±0.3 μmol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65±0.87)%, (18.61±0.70)% and (15.28 ±0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the ex- pression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt.     

Effect of siRNA Targeting Survivin Gene on the Biological Behavior of Hepatocellular Carcinoma

To study the influence of siRNA targeting survivin gene on the biological behavior of hepatocellular carcinoma (HCC), one pair of 21bp reverse repeated motifs of survivin target sequence with 9 spacers were synthesized and inserted into plasmid psilencer2.1 to generate siRNA eukaryotic expression vector. After stable transfection into HepG2 cells, the biological behaviors of the survivin siRNA transfected HCC cells were observed. After the recombinant plasmid Psilence( )-survivin was successfully constructed, survivin mRNA and protein expression inhibition ratio reached 73% and 75% respectively compared to control groups. Transfected cells with survivin siRNA demonstrated significantly inhibited cell growth and increased apoptosis. Subsequent study in nude mouse model demonstrated lower succeeding rate in cells transfected with survivin siRNA and slow growth rate. The results elucidated the siRNA targeting survivin gene could specially suppress its expression in HepG2 cells and inhibit tumor cells growth both in vivo and in vitro. This provides a theoretical basis to turn the drug resistance in tumor cells.