FAK gene silencing enhances chemotherapeutic drug sensitivity in leukemic cells XU Lü-hong1,

AIM: To investigate the effect of focal adhesion kinase (FAK) gene silencing on chemotherapeutic drug sensitivity in leukemic cells. METHODS: Lentiviral-FAK-shRNA was transfected into BCR/ABL-BaF3 leukemic cells. The protein expression of FAK was detected by Western blotting. BCR/ABL-BaF3 leukemic cells were treated with different concentrations of imatinib in vitro, and the apoptosis was determined by labeling with Annexin V. A murine model of leukemia was established and the mice were treated with FAK shRNA and imatinib. Survival time and distribution of leukemic cells in bone marrow and spleen of the mice were monitored. RESULTS: FAK shRNA was successfully constructed and effectively inhibited FAK gene expression. With 5 μmol/L imatinib treatment, the percentages of apoptotic cells in vector control group and FAK shRNA group were (9.76±1.97)% and (21.90±3.20)%, respectively, and significant difference between these 2 groups (P<0.05) was observed. With 50 μmol/L imatinib treatment, the percentages of apoptotic cells in vector control group and FAK shRNA group were (56.10±6.00)% and (82.10±5.70)%, respectively,also with significant difference between these 2 groups (P<0.05). Compared with vector control group, the mice in FAK gene silencing group displayed significantly prolonged survival time. Moreover, 60 days after injection of leukemic cells, the percentages of leukemic cells in bone marrow and spleen of the mice were significantly decreased in FAK gene silencing group as compared with those in vector control group. CONCLUSION: FAK gene silencing promotes the efficacy of chemotherapeutic drug in leukemic cells, indicating that FAK gene silencing might be considered as a new therapeutic strategy for leukemia.     

FAK shRNA inhibits growth of leukemic cell line BCR/ABL-BaF3

AIM: To investigate the effect of focal adhesion kinase (FAK) shRNA on the growth of leukemic cells.METHODS: Lentiviral-FAK-shRNA was transfected into BCR/ABL-BaF3 cells, while empty vector was transfected into the same cells for control. The proteins of FAK and other molecules were detected by Western blotting. Cell growth was observed by culturing the leukemic cells in RPMI-1640 medium in vitro, and colony formation was observed by culturing the leukemic cells in methylcellulose medium. To establish a murine model of leukemia, BCR/ABL-BaF3 cells were injected into BALB/c mice through tail vein. Survival time of the leukemic mice was monitored, and the distribution of the leukemic cells in spleen of the mice was also detected. RESULTS: FAK shRNA inhibited the protein expression of FAK, reduced STAT5 phosphorylation and induced caspases-3 activation in BCR/ABL-BaF3 cells. FAK shRNA inhibited the cell growth in vitro. Colony formation experiment showed that the number of colony in vector control group and FAK shRNA group was 215.60±13.01 and 125.00±9.06, respectively, and the difference was statistically significant (P<0.01). The mice in vector control group died between day 21 and day 27, while the mice in FAK shRNA group died between day 52 and day 60, and the difference was statistically significant (P<0.05). Moreover, 25 days after injection of leukemic cells, the percentage of leukemic cells in spleen of the leukemic mice in vector control group and FAK shRNA group was (82.40±6.13)% and (14.50±3.70)%, respectively (P<0.01). CONCLUSION: FAK shRNA inhibits the growth of leukemic cells in vitro and in vivo, indicating that FAK gene silencing might be a new therapeutic strategy for leukemia.     

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells

AIM:To investigate the inhibitory effect of vector-based RNA interference ( RNAi) on the expression of melanoma associated antigen A3 (MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells. METHODS:A vector for transcribing specific small hairpin RNA ( shRNA) targeting MAGEA3 gene was constructed ,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000. The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively. The cell apoptosis was studied by DNA fragmentation, electron microscopy ，TUNEL assay, and annexin V/PI staining. RESULTS:The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells. After the shRNA expression vector was transfected into the MEL-ED1 cells, the expression of MAGEA3 gene was inhibited significantly ( by 90% ). DNA fragmentation，electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ±1.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group (both P<0.01). CONCLUSION:The specific small hairpin RNA targeting MAGEA3 mRNA can inhibit the expression of MAGEA3 and cause apoptosis of hepatocellular carcinoma cells , which suggests inhibitory effect of MAGEA3 on apoptosis in cancer and provides an experimental basis for treating human tumors with RNAi.     

Construction of FAK-shRNA retroviral vector and screening of stable HCC-LM3 cell line with persistent knockdown of FAK

AIM: To construct a recombinant retroviral vector of short interfering RNA targeting focal adhesion kinase (FAK) gene and to establish a cell line with stable knockdown of FAK.METHODS: The oligonucleotides that transcribed to short hairpin RNA (shRNA) targeting FAK gene were synthesized in vitro, cloned into retroviral vector pSuper.retro and transfected into Phoenix cell line. The stable clones were screened and high-titer virus was produced. The human hepatocellular carcinoma cell line HCC-LM3 was infected with the virus-rich supernatant. The stable LM3 cell line, which showed significantly to silence FAK and associated proteins, was selected by puromycin.RESULTS: The recombinant retroviral vector was successfully constructed. Persistent knockdown of FAK in the LM3 cell line infected with the supernatant containing the retrovirus was confirmed by Western blotting. Down-regulation of FAK resulted in the inhibition of p-Akt and p-MAPK1/2 expression and led to decreased migration and invasion of the cells. The cell cycle was blocked at G₀/G₁ phase, and apoptosis was increased. The proliferation rate also decreased significantly.CONCLUSION: FAK-shRNA virus generated by recombinant retroviral vector pSuper-FAK can inhibit the protein expression of FAK and phosphorylation of Akt and MAPK1/2 in HCC-LM3 cells. Down-regulation of FAK shows a significant impact on biological behaviors of tumor cells.     

Effect of shRNA-mediated survivin gene silencing on sensitivity of lymphoma cells to adriamycin

AIM: This study was designed to use RNA interference technique to down-regulate the expression of survivin gene in human Burkitts lymphoma cell line Daudi and to explore the effect on sensitivity of Daudi cells to adriamycin. METHODS: The survivin-shRNA expression vector was constructed and transfected into Daudi cells. Expression of survivin mRNA and protein were assessed by RT-PCR and Western blotting analysis, respectively. Apoptosis index of transfected Daudi cells was quantified by flow cytometry. The sensitivity of Daudi cells to adriamycin (ADR) before and after transfection was detected by MTT test. RESULTS: The mRNA and protein levels of survivin were down-regulated by 62.32% and 61.88%, respectively, compared to those in control-shRNA treated group and PBS treated group (P<0.05). Meanwhile, the apoptosis index was significantly increased (19.10%±2.15%), compared to that in control group (4.48%±1.54%) and PBS group (4.35%±1.37%, P<0.05). The 50% inhibition concentration (IC50) of ADM to Daudi cells was significantly decreased (0.25±0.43) μmol/L, compared to that in control group (0.87±0.21) μmol/L and PBS group (0.91±0.36) μmol/L, P<0.05. CONCLUSION: Down-regulation of survivin expression in Daudi cells by shRNA effectively induces apoptosis and increases the sensitivity of Daudi cells to ADR.     

Effect of CREB-shRNA on mitochondrial morphology and cell apoptosis in OGD/R-induced cortical neurons

AIM: To construct recombinant lentiviral vector with short hairpin RNA (shRNA) of CREB gene, and to investigate the effect of CREB gene silencing on mitochondrial morphology and cell apoptosis in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cortical neurons. METHODS: Three lentiviral vectors pLentiLox3.7 (PLL) inserted shRNA fragments targeting CREB gene were co-transfected with the packaging plasmids psPAX2 and pMD2.G to the 293T cells, and the virus particles, which was infected with the primary cortical neurons, was encapsulated. The protein expression of CREB was detected by Western blot. The mitochondrial morphology, cell apoptosis and the expression of Bcl-2 and Bax were evaluated by the methods of MitoTracker red, TUNEL and Western blot in OGD/R induced cortical neurons after CREB gene silencing. RESULTS: The pLL-CREB-shRNA1 was the most effective shRNA, which inhibited 80% CREB gene expression in the cortical neurons. The mitochondrial was appeared dot and fragment morphology in OGD/R induced cortical neurons with transfected pLL-CREB-shRNA1 plasmid. In addition, the expression of Bcl-2 was decreased, the expression of Bax, and the apoptosis of the neurons were increased by tranfected with pLL-CREB-shRNA1. CONCLUSION: CREB shRNA recombinant lentiviral vector specifically inhibits the expression of CREB gene. CREB gene silencing promotes the cell apoptosis and mitochondrial morphological changes in the cortical neurons induced by OGD/R.     

Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.     

Effects of TSG101 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM:To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human neuroblastoma cell line SH-SY5Y.METHODS:The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination,then transfected into SH-SY5Y cells.Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blotting analysis.The growth curve was made using MTT assay.Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferative indexes were calculated.The apoptosis after CDDP treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses.The expression of Bcl-2,Bax,P-gp and MRP were analyzed by Western blotting.RESULTS:mU6pro-TSG101 siRNA was successfully constructed and transfected into SH-SY5Y cells.As detected by MTT and flow cytometry,down-regulation of TSG101 significantly suppressed the proliferation of SH-SY5Y cells with a G1 cell cycle arrest,compared with that in control (P<0.05).As detected by DNA ladder and Annexin V/propidium iodide binding analyses,down-regulation of TSG101 significantly enhanced the sensitivity of SH-SY5Y cells to CDDP-induced apoptosis,compared with that in control (P<0.05).The expression of P-gp and Bcl-2 in transfected cells were decreased as compared with that in the control,while MRP and Bax were not.CONCLUSIONS:Down-regulation of TSG101 suppresses the proliferation of SH-SY5Y cells,and enhances the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents to a degree,suggesting TSG101 may be useful for gene therapy in the future.     

Caspase-8 small hairpin RNA attenuates apoptosis of human bone marrow mesenchymal stem cells under conditions of serum deprivation and hypoxia

AIM：To investigate the effect of caspase-8 small hairpin RNA (shRNA) on attenuating apoptosis of human mesenchymal stem cells (hMSCs). METHODS：Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed. Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR. The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid, which was linearized and transfected into HEK293 cells for packaging and amplification of the recombinant adenovirus rAd-Cap8 shRNA. The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting. Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hMSCs under the conditions of serum deprivation and hypoxia. The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR. RESULTS：The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR. The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant adenovirus (rAd)-Cap8 shRNA successfully. rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 expression in hMSCs. Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the apoptotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia, with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2. CONCLUSION：Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia.     

Effect of FAK-related non-kinase on apoptosis in hepatic stellate cells

AIM: To evaluate the inhibitory effect of FRNK on the phosphorylation of FAK and apoptosis in hepatic stellate cells (HSCs). METHODS: After stimulated with fibronectin, HSCs was transfected with FRNK plasmid by cationic liposome method. The apoptosis of FRNK-induced HSCs was examined by Annexin-V/propidium iodide double-labeled flow cytometry (FCM), gel electrophoresis and transmission electron microscope. The protein levels of FRNK, FAK and p-FAK (Tyr397) in HSCs were assayed by Western blotting, and RT-PCR was used to detect the expression of mRNA. RESULTS: The expression of FRNK was enhanced and the phosphorylation of FAK was inhibited after FRNK was transiently transfected into HSCs in vitro. The apoptotic rate in HSCs exposed to FRNK plasmid for 48 h was higher than that in the non-FRNK plasmid group ［(25.37±1.92) % vs (9.28±1.05) %, P<0.01］, and accompanied by a significant increase in caspase-3 activity both in the protein and in the mRNA level ［(264.17±12.60 vs 185.82±9.69), P<0.01; (4.19±0.48 vs 1.07±0.27), P<0.01］. CONCLUSION: In HSCs, the expression of FRNK is enhanced and the phosphorylation of FAK is inhibited after FRNK transfection. FRNK induces the HSCs apoptosis.     

Construction of Pax-8 gene recombinant plasmid and study of its function

AIM: To investigate the effects of over-expression of Pax-8 gene on the proliferation and apoptosis of H9c2 cells(a cardiomyocyte cell line). METHODS: The full length of rat Pax-8 gene was restrictively digested by Kpn I and Not I from the pCMV sport6-Pax-8 vector, and then inserted into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)-Pax-8 was confirmed by restriction endonuclease digestion and sequencing. The pcDNA3.1(+)-Pax-8 was transfected into H9c2 cells. The expression of Pax-8 at mRNA and protein levels was identified after transfection by RT-PCR and Western blotting. The cell proliferation was measured by CCK-8. Cell apoptosis was induced by serum deprivation in H9c2 cells transfected with Pax-8 gene. The apoptosis rate of the cells was determined by flow cytometry with annexin V-FITC and propidium iodide double staining. The protein expression of activated caspase-3 was measured by Western blotting. RESULTS: The full length of Pax-8 gene was successfully cloned into pcDNA3.1(+) expression vector and over-expression of Pax-8 at mRNA and protein levels was observed in H9c2 cells transfected with Pax-8 gene as compared to the wild-type cells and the cells transfected with an empty vector (both P<0.05). Transfection of Pax-8 gene promoted the proliferation of the cardiomyocytes (P<0.05) and inhibited the apoptosis rates induced by serum deprivation (P<0.01). The expression level of activated caspase-3 was increased by serum deprivation and attenuated by Pax-8 transfection (P<0.01). CONCLUSION: The pcDNA3.1(+)-Pax-8 expression vector was successfully constructed and over-expression of Pax-8 gene in cardiomyocytes is obtained. Pax-8 gene acts as an anti-apoptotic factor in cardiomyocytes by promoting cell proliferation and inhibiting apoptosis.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effects of fibulin-3 expression on malignant pleural mesothelioma cells

AIM: To observe the effects of over-expression and silencing of fibulin-3 on malignant pleural mesothelioma (MPM) cell line SMC-1, and to search for a new method for the treatment of MPM. METHODS: The vectors for fibulin-3 over-expression and short hairpin RNA (shRNA) were constructed, and control vector, fibulin-3 over-expression vector (Exp), over-expression control vector (Exp-NC), interference vector (shRNA1, shRNA2, shRNA3 and shRNA4), and interference control vector (shRNA-NC) were transfected into the SMC-1 cells. The cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression of fibulin-3 before and after interference was determined by real-time PCR and Western blot. RESULTS: Compared with control group, the number of the SMC-1 cells in G₂ phase in Exp group was increased (P<0.05), while that in shRNA2 group was decreased (P<0.05). The results of apoptosis analyzed by flow cytometry showed that compared with control group, the apoptotic rate in Exp group was decreased (P<0.05), while that in shRNA2 group was increased (P<0.05). The mRNA and protein expression levels of fibulin-3 and mesothelin in Exp group were up-regulated, while those in shRNA group were down-regulated (P<0.05).CONCLUSION: The vectors for over-expression and silencing of fibulin-3 are successfully constructed, proving that transfection of Exp and shRNA effectively changes fibulin-3 expression and the SMC-1 cell growth. Fibulin-3 may be a target molecule for MPM treatment.     

Construction and identification of eukaryotic expression vector of bcl-2 siRNA

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.     

Effect of AMPKα2 gene silencing by shRNA recombinant plasmid on chloride-mediated anoxia/reoxygenation injury in rat cardiomyocytes

AIM: To explore the role of AMP-activated protein kinase α2 subunit (AMPKα2) gene in chloride-mediated anoxia/reoxygenation (A/R) injury by transfection of short-hairpin RNA (shRNA) expression vector targeting to AMPKα2 gene into H9c2 cardiomyocytes. METHODS: Recombinant shRNA expression vector pSuper-AMPKα2 targeting to AMPKα2 gene was constructed and transfected into H9c2 cardiomyocytes. The protein expression of AMPKα2 was determined by Western blotting. The cells were divided into 5 groups: control group, A/R group, Cl^--free A/R group, pSuper+Cl^--free A/R group and pSuper-AMPKα2 shRNA+Cl^--free A/R group. After treatment, the cell viability was detected by MTT assay. LDH activity was analyzed with an automatic biochemical analyzer. The apoptotic rate and the level of intracellular ROS was measured by flow cytometry. The activity of SOD and GSH-Px was analyzed by a colorimetric method. RESULTS: The result of sequencing proved that the recombinant plasmid pSuper-AMPKα2 shRNA was correctly constructed. The protein level of AMPKα2 significantly decreased after the plasmid was transfected into the cardiomyocytes. Compared with A/R group, the cell viability and the activity of SOD and GSH-Px were significantly increased, while the activity of LDH, apoptotic rate and ROS production were significantly decreased in Cl^--free A/R group. The protective effect of Cl^--free solution on the A/R-induced injury of cardiomyocytes was abolished, and the ROS production was increased and the activity of SOD and GSH-Px was decreased after the cells were transfected with pSuper-AMPKα2 shRNA. CONCLUSION: Recombinant plasmid pSuper-AMPKα2 shRNA is successfully constructed, and silencing of AMPKα2 gene abolishes the protective effect of Cl^--free solution on A/R injury.     

Effect of rAAV2-PEDF on migration and apoptosis of human retinal capillary endothelial cells

AIM: To construct a rAAV2-pigment epithelium-derived factor (PEDF) vector and to explore the effect of rAAV2-PEDF on the migration and apoptosis of human retinal capillary endothelial cells (HRCECs). METHODS: PEDF gene was cloned into an adeno-associated virus vector pSNAV. The recombinant pSNAV-PEDF was transfected into BHK cells. The G418 resistant cells were selected and infected with recombinant HSV which packaged the recombinant pSNAV-PEDF. The high-titer rAAV2-PEDF was obtained by purification. After transfected the rAAV2-PEDF into HRCECs, the protein expression of PEDF were detected by Western blotting, the migration of HRCECs was assessed by a boyden chamber, and the apoptosis of HRCECs was analyzed by flow cytometry. RESULTS: The evaluation results of PCR, enzyme digestion, and DNA sequencing showed that the rAAV2-PEDF was constructed correctly. The GFP positive cells were observed under laser scanning confocal microscope 48 h after rAAV2-GFP transfection. The expression level of PEDF protein was higher in experimental group than that in control group. The number of migrated cells was 33.0±2.7 in normal control group, 35.0±3.6 in rAAV2-GFP treatment group, and 12.0±2.1 in rAAV2-PEDF treatment group (P<0.05). In normoxic condition, the apoptotic cells were 2.10%±0.53% in control group, 3.40%±0.62% in rAAV2-GFP group and 1.60%±0.47% in rAAV2-PEDF group (P>0.05). In hypoxic condition, the apoptotic cells were 4.00%±0.55% in CoCl₂ treatment group, 6.10%±0.71% in CoCl₂+rAAV2-GFP treated group and 40.00%±2.10% in CoCl₂+rAAV2-PEDF treatment group (P<0.05). CONCLUSION: rAAV2-PEDF is successfully constructed and PEDF gene is stably expressed in HRCECs after rAAV2-PEDF transfection. Over-expression of PEDF inhibits the migration of HRCECs and induces the cell apoptosis obviously in hypoxia.     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.