Knockdown of survivin expression using siRNA inhibits the growth of PC-3M cells

AIM: To study the inhibitory effects of survivin siRNAs on the growth of PC-3M cells. METHODS: Two pairs of DNA template coding siRNA against survivin were synthesized to construct two recombinant plasmids, pSi-sur1 and pSi-sur2. The two recombinants and the two controls, lipofectin and vacant plasmid were transfected into PC-3M cells. The expressions of survivin mRNA and protein were detected respectively by RT-PCR and Western blotting. Proliferation abilities were measured by MTT, and the cell cycle and apoptosis were assayed by FCM. RESULTS: After 72 h of transfection, the level of cell survivin mRNA in the two siRNA groups was 48%±6% (n=3) and 30%±5% (n=3) of that in lipofectin group, and expression of survivin protein were 38%±4% (n=3) and 36%±4% (n=3) respectively of that in lipofectin control. The proliferation rate of cells in pSi-sur1 and pSi-sur2 groups was also inhibited according to MTT, about 44.20%±2.08% (n=3) and 39.20%±1.93% (n=3) of that in lipofectin group. Cell numbers of G1 phase in two siRNA groups were significantly higher than that in two controls, while cells of G2 phase and S phase were much lower. Cell apoptosis was found in both siRNA groups. CONCLUSION: The two survivin siRNA significantly inhibit the expression of survivin in mRNA and protein levels, arrest the cell cycle in G1 phase, and suppress the growth of PC-3M cells and induce apoptosis in vitro.     

Inhibitory effect of survivin-siRNA on prostatic subcutaneous xenografts in nude mice

AIM: To investigate the inhibitory effect of survivin-siRNA recombinant plasmid on prostate cancer xenografts. METHODS: Prostatic cancer DU145 cells were cultured and subcutaneously injected into nude mice. When the tumor grew to 8 mm in diameter, it was aseptically removed and divided into about 2 mm blocks through surgery and subcutaneously implanted into another nude mice. After the prostatic cancer xenograft model was reconstructed, the mice were treated with survivin-siRNA plasmid and control scrambled siRNA plasmid using electric transfection method. The tumor growth curve was plotted and the inhibitory rate was calculated. HE staining, immunohistochemical staining and TUNEL assay were applied to observe the effect of survivin-siRNA on the xenografts. RESULTS: The prostatic cancer xenograft model was successfully constructed in vivo. Compared with mock and scrambled siRNA groups, transfection of survivin-siRNA recombinant plasmid obviously inhibited the tumor growth with the inhibitory rates of 61.81% and 62.87%, respectively. Compared with both controls, survivin-siRNA depressed the protein expression of survivin and promoted the cell apoptosis. CONCLUSION: Survivin-siRNA recombinant plasmid significantly inhibits the growth of prostatic tumor xenografts by inhibiting the protein expression of endogenous survivin and promoting cell apoptosis.     

Construction of siRNA-survivin and GRIM-19 coexpression plasmid and its growth inhibitory effect on prostatic cancer DU145 cells

AIM: To construct the recombinant plasmid that expresses siRNA-survivin and GRIM-19 simultaneously, and to identify the validity of the recombinant plasmid and observe its effect on expression of survivin and GRIM-19 and proliferation ability of prostate cancer DU145 cells. METHODS: The recombinant plasmid coexpressing siRNA-survivin and GRIM-19 was constructed using gene cloning technique. The prostatic cancer DU145 cells were transfected with the coexpression plasmid and control plasmids. Survivin and GRIM-19 mRNA expression was detected by semi-quantitative RT-PCR. The proliferation ability affected by coexpression plasmid was measured by MTT assay. RESULTS: The coexpression plasmid pGRIM-19-si-survivin was successfully constructed according to DNA recombinant technique and identified through restriction enzyme digestion and plasmid sequencing. Compared with the mock, survivin mRNA expression levels were 0.55?0.05,0.62?0.08 and 0.35?0.05 in psi-survivin, pGRIM-19 and pGRIM-19-si-survivin groups, respectively. Compared with psi-survivin and pGRIM-19, pGRIM-19-si-survivin inhibited survivin mRNA expression markedly (P<0.05), while the expression levels of GRIM-19 mRNA were 1.93?0.14, 2.57?0.20 and 4.12?0.21 in psi-survivin, pGRIM-19 and pGRIM-19-si-survivin groups, respectively (P<0.01). Compared with pGRIM-19 group, pGRIM-19-si-survivin enhanced GRIM-19 mRNA expression more obviously (P<0.05). After transfection for 48 h, the proliferation rates were 58.0%?7.2%, 62.1%?6.1% and 50.2%?4.8% in the 3 experiment groups compared with the mock (P<0.05). After transfection for 72 h, the proliferation rate were 43.4%?4.3%, 51.3%?6.7% and 26.8%?7.1% in experiment groups compared with the mock (P<0.05). Compared with psi-survivin and pGRIM-19, pGRIM-19-si-survivin significantly inhibited the cell growth (P<0.05). CONCLUSION: Transfection of coexpression plasmid pGRIM-19-si-survivin dramatically changes the mRNA expression of survivin and GRIM-19 and inhibits the cell growth.     

Effect of NEU3 on biological behaviors of human prostate cancer DU145 cells

AIM:To explore the effects of neuraminidase 3 (NEU3) on the viability, invasion and apoptosis of human prostate cancer DU145 cells and the molecular mechanism. METHODS:The human prostate cancer DU145 cells were divided into blank control group and treatment group. The cells in treatment group were treated with either neuraminidase inhibitor DANA, or NEU3 small interfering RNA (siRNA) to knock down the expression of NEU3. The cell viability was measured by CCK-8 assay. The cell invasion ability was detected by Transwell assay. The effects of the treatments on the mRNA level of Bcl-2 were detected by qPCR. The effects of the treatments on the protein levels of matrix metalloproteinase 2 (MMP2) and apoptotic inhibitory protein Bcl-2 were determined by Western blot. Apoptosis of the cells was analyzed by flow cytometry. RESULTS:The protein level of NEU3 and the apoptotic rate in DANA group were not significantly different from those in blank control group. The viability of DANA-treated DU145 cells was increased, and the invasion ability, MMP2 protein level, and Bcl-2 mRNA and protein levels were all decreased in these cells, compared with blank control group. On the other hand, the levels of NEU3 protein and Bcl-2 mRNA and protein in NEU3 siRNA group were significantly decreased compared with blank control group, while the viability and apoptotic rate of the cells with NEU3 siRNA transfection were increased (P<0.05). However, the protein expression of MMP2 and the invasion ability of the cells were not significantly changed after NEU3 siRNA treatment. CONCLUSION:The inhibition of NEU3 in enzyme activity and expression decreases the viability, and enhances the apoptosis of human prostate cancer DU145 cells. However, it has no obvious effect on the invasion ability of DU145 cells.     

Molecular mechanism of miR-29a inhibiting malignant phenotypes in prostate cancer cells

AIM:To investigate the biological functions of microRNA-29a (miR-29a) in prostate cancer and the molecular mechanism of miR-29a over-expression inhibiting malignant phenotypes of prostate cancer cells. METHODS:The levels of miR-29a expression in the prostate cancer tissues and cells were detected and analyzed using gene microarray and bioinformatics. The expression levels of miR-29a and lysine (K)-specific demethylase 4B (KDM4B) mRNA in prostate cancer tissues, paracarcinomatous tissues, 4 prostate cancer cell lines (PC3, DU145, LNCaP and ArCaP) and normal prostate epithelial cell line (RWPE-1) were measured by real-time PCR. PC3, DU145, LNCaP and ArCaP cells were transfected with pGenesil-1-miR-29a plasmid using transient transfection. The cell viability, colony formation rate and apoptotic rate were analyzed by MTT assay, colony formation assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The protein expression of KDM4B was determined by Western blot. RESULTS:The results of gene microarray and bioinformatic analysis indicated that differential expression of miR-29a was found in the prostate cancer tissues and the paracarcinomatous tissues. The levels of miR-29a in the prostate cancer tissues and prostate cancer cells were significantly decreased, while the mRNA levels of KDM4B were notably increased compared with the paracarcinomatous tissues and RWPE-1 cells, respectively (P<0.05). Compared with negative control (pGenesil-1) group, the cell viability and colony formation rate were significantly decreased, the apoptotic rate was significantly increased, and the protein expression of KDM4B was notably inhibited in the prostate cancer cells with miR-29a over-expression (P<0.05). The cell viability was significantly enhanced, and the apoptosis was significantly inhibited in the prostate cancer cells with KDM4B over-expression (P<0.05). CONCLUSION:Low expression of miR-29a was found in the prostate cancer tissues and cells. miR-29a over-expression inhibits the growth of prostate cancer cells and induces apoptosis. The mechanism may be associated with inhibiting the protein expression of KDM4B.     

Effects of baicalin on CA46 cell proliferation inhibition and apoptosis induction

AIM: To investigate the effects of baicalin on proliferation inhibition and apoptosis induction in human Burkitt lymphoma cell line CA46 and to explore its underlying mechanisms. METHODS: CA46 cells were exposed to baicalin at different dosages and its proliferation inhibition was detected by MTT assay. The ability of baicalin to induce CA46 cell apoptosis was examined by Annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation. The mRNA expressions of c-myc and bcl-2 were detected by RT-PCR, and the protein expressions of c-Myc, Bcl-2, caspase-3 precursor (procaspase-3) and poly ADP-ribose polymerase (PARP) were detected by Western blotting. RESULTS: Baicalin remarkably inhibited the CA46 cell proliferation, with an IC50 value of 10 μmol/L. Apoptosis was remarkably induced by baicalin in a dose-dependent manner, and its earlier and later stages were detected by annexin V-FITC/PI double staining analysis, TUNEL labeling method and DNA fragmentation, respectively. Furthermore, RT-PCR showed that the mRNA expressions of c-myc and bcl-2 in treated CA46 cells decreased in a time-dependent manner. Western blotting showed that the protein expressions of c-Myc, Bcl-2, procaspase-3 and PARP (116 kD) in baicalin treated CA46 cells were down-regulated in a time-dependent manner, while the expression of PARP(85 kD) was up-regulated. CONCLUSION: Baicalin efficiently induces proliferation inhibition and apoptosis in CA46 cells, which may be related with the down-regulation of c-Myc and Bcl-2 expressions, as well as the up-regulation of caspase-3 activity.     

Value of endonuclease domain containing 1 in progression of prostate cancer

AIM: To analyze the difference of endonuclease domain containing 1 (ENDOD1) expression between benign prostatic hyperplasia (BPH) tissues and prostate cancer (PCa) tissues and to investigate the effect of ENDOD1 on the biological function of human prostate cancer cells. METHODS: The BPH samples (n=20) and PCa samples (n=21) were processed and analyzed according to the instruction of immunohistochemical (IHC) staining. The mRNA and protein levels of ENDOD1 in the normal prostate epithelial cells and prostate cancer cells were evaluated by RT-qPCR and Western blot, respectively. The recombinant plasmids pCMV-N-Flag-ENDOD1 was constructed and was transfected into the human prostate cancer cells. The proliferation, apoptosis, migration and invasion abilities of the prostate cancer cells were evaluated by MTT assay, flow cytometry, Transwell migration and Matrigel invasion assays, respectively. RESULTS: The analysis of variance of the immunoreactivity score showed that PCa tissues with high Gleason score displayed significantly lower ENDOD1expression than that with low Gleason score and BPH (P<0.05). The expression of ENDOD1 at mRNA and protein levels in PC3 cells and DU145 cells was significantly lower than that in the LNCap cells (P<0.05). The proliferation of DU145 transfected with ENDOD1 was inhibited. The flow cytometry indicated that ENDOD1 over-expression in the DU145 cells resulted in a notable increase in G₀/G₁ phase arrest (P<0.05), but the apoptotic rates showed no statistical difference. The results of Transwell assay showed that migration and invasion abilities of the cells were also inhibited after transfection with over-expressing ENDOD1 plasmid (P<0.05). CONCLUSION: The expression of ENDOD1 significantly decreased in prostate cancer with high Gleaon score. Meanwhile, the ENDOD1 is specifically down-regulated in androgen independent prostate cancer (AIPC) cell lines. Over-expression of ENDOD1 remarkably inhibits the proliferation, migration and invasion abilities of AIPC.     

Influence of siRNA-mediated MIF knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids

AIM：To investigate the influence of siRNA-mediated macrophage migration inhibitory factor (MIF) knockdown on inhibition of inflammatory lipid mediator release by glucocorticoids.
METHODS：Mouse macrophage cell line RAW2647 was transiently transfected with MIF siRNA and control siRNA by liposome method. The transfection efficiency was assessed by immunofluorescence technique. The expression of MIF mRNA and protein was examined by RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production in cell culture supernatants was measured by ELISA, and the protein expression of Annexin 1, cytosolic phospholipase A2α (cPLA2α) and phospho-cPLA2α were evaluated by Western blotting.
RESULTS：MIF siRNA significantly inhibited MIF expression both at mRNA and protein levels in RAW2647 cells and subsequently enhanced the inhibitory effect of dexamethasone (Dex) on PGE2 and LTB4 production. MIF siRNA also increased Annexin 1 expression becreased by Dex, and strengthened the inhibitory effect of Dex on the phosphorylation of cPLA2α.
CONCLUSION：MIF siRNA enhances the inhibitory effect of Dex on PGE2 and LTB4 production from RAW2647 cells partly via increasing Annexin 1 expression and inhibiting cPLA2α phosphorylation. Intracellular MIF knockdown mediated by siRNA may enhance the sensitivity of RAW2647 cells to the anti-inflammatory effect of glucocorticoids.     

Effect of RelB on proteasome inhibitor-induced maspin expression

AIM: To investigate the effects of RelB on proteasome inhibitor-induced maspin expression in prostate cancer cells. METHODS: Western blotting analysis was performed to examine endogenous and proteasome inhibitor(MG-132)-induced expression of RelA, RelB and maspin in prostate cancer cells. The expression profiles of RelB and maspin in human prostate cancer tissues were obtained by immunohistochemistry assay. RNA interference targeting RelB was performed in DU145 cells. The effects of RelB-silencing on maspin expression induced by MG-132 were detected by Western blotting. The cell viability was determined by PI staining and FACS analysis. RESULTS: RelB expression was increased in androgen-independent prostate cancer cell line DU145, while maspin expression was minimally detected. Among 10 tissue samples tested, a strong nuclear RelB staining and an absence of maspin expression were found in high-grade specimens (Gleason scores 4-5). RelB expression was reduced upon treatment with MG-132 for 24 h, which was coincided with the induction of maspin expression. RelB-silencing in DU145 cells by siRNA didn't influence the proteasome inhibitor-induced maspin expression. CONCLUSION: The expression of RelB is inversely correlated to maspin expression in androgen-independent prostate cancer cells and prostate cancer tissues. RelB expression is critical to the proteasome inhibitor-induced maspin expression.     

Effects of AZD8055 on autophagy and apoptosis in cholangiocarcinoma cells

AIM: To explore the effects of mammalian target of rapamycin (mTOR) double inhibitor AZD8055 on autophagy and apoptosis of human cholangiocarcinoma cell line HuCCT1. METHODS: The effect of AZD8055 on the viability of HuCCT1 cells was detected by MTT assay. Autophagosome was detected by acridine orange (AO) staining. After treated with AZD8055, the expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3 and auto-phagy marker proteins beclin 1, LC3 and p62 were determined by Western blot. Apoptotic rate was analyzed by flow cyto-metry with Annexin V-FITC/PI double staining. RESULTS: AZD8055 significantly inhibited the viability of HuCCT1 cells (P<0.05). AO staining showed that AZD8055 significantly increased orange granules in the cytoplasm. After treated with AZD8055, compared with the control group, the protein level of beclin 1 and the ratio of LC3-Ⅱ/LC3-I were enhanced, while p62 was attenuated (P<0.05). The protein expression level of pro-apoptotic regulator Bax was down-regulated and anti-apoptotic regulator Bcl-2 was increased. The protein level of cleaved caspase-3 was reduced (P<0.05). The results of flow cytometry showed that AZD8055 inhibited cell apoptosis. CONCLUSION: AZD8055 inhibits the viability of cholangiocarcinoma cells, and the mechanism is closely related with autophagy induced by AZD8055.     

Inhibition of survivin expression in BGC-823 cells by RNA interference

AIM: To investigate the feasibility and specificity of gastric carcinoma gene therapy by utilizing RNA interference (RNAi) to inhibit survivin expression in vitro and in vivo. METHODS: Small interference RNA (siRNA) homologous to survivin was designed. pTZU6+1-siRNA-survivin vector was constructed and transfected into BGC-823 cells. The transplanted BGC-823 tumor in nude mice was established to induce RNAi. The changes of survivin gene expression, tumor cell cycle and cell apoptosis were detected by flow cytometry, RT-PCR, Western blotting, immunochemistry and TUNEL. RESULTS: The expression of survivin was obviously inhibited by RNAi in vitro. The phase of cell cycle indicated the reduction of S phase, while G1/G0 phase increased. Cell apoptosis was obvious. Both the mRNA level and the protein expression of survivin decreased obviously. The tumor size reduced after treated with pTZU6+1-siRNA-survivin vector in vivo. The expression of survivin decreased in siRNA treatment group. In contrast, little change in control group in vitro and in vivo was observed. CONCLUSION: RNA interference down-regulates survivin gene expression, inhibits BGC-823 cell proliferation and induces cell apoptosis with good specificity, which may be a possible new approach for neoplasm gene therapy.     

Effect of siRNA for survivin gene on growth and apoptosis in A549 cells

AIM:To observe the influence of lentiviral vectors expressing siRNA for survivin gene knockdown in A549 cells, sequentially as tools to explore the molecule pathogenesis and new gene therapy of lung adenocarcinoma. METHODS:The lentiviral vectors, which express survivin siRNA, were constructed and transfected into A549 cell strain. The titers of the lentiviruses were determined by 293T cells. The expressions of survivin and caspase-3 were detected by Western blotting and RT-PCR. The cell cycle and cell growth of A549 cells were examined by MTT and FCM．RESULTS:The expression of survivin was suppressed effectively by siRNA targeting survivin. The expression of survivin mRNA decreased by 97%. The expression of survivin protein decreased by 94%. The rate of cell growth was decreased. The G1 phase cells were increased, whereas S phase cells were decreased. CONCLUSION:The lentivirus vectors expressing siRNA for survivin can significantly inhibit gene expression and the cell growth, and markedly induce the apoptosis. It is hopeful to be a new gene therapy of lung adenocarcinoma.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

Effects of G6PD silencing by siRNA on expression of HIF-1α in human colon cancer cells

AIM: To study the effects of glucose-6-phosphate dehydrogenase (G6PD) silencing by small interference RNA(siRNA) on the levels of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and hypoxia-inducible factor 1α(HIF-1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells. The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD. LoVo cells were cultured in vitro under hypoxic condition. NADPH levels were determined.The mRNA and protein levels of HIF-1α were analyzed by RT-PCR and Western blotting,respectively. RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells. Compared with untransfected cells,the mRNA expression of G6PD in transfected cells was decreased by 43% and G6PD activity was decreased by 63.5%. Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased (41% vs 100%, P<0.05).HIF-1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells. CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF-1α stability in cancer cells under hypoxic condition. By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.     

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 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.     

Mechanism responsible for inhibitory effect of recombinant rat augmenter of liver regeneration on renal turbular epithelial cell apoptosis by gentamycin

AIM: To investigate the effects of recombinant rat augmenter of liver regeneration (rrALR) on apoptosis of renal tubular cells (NRK-52E cells) induced by gentamycin sulfate (GM). METHODS: The cultured NRK-52E cells were divided into four groups: normal control cells, cells with GM (GM 1.6 g/L) or GM and rrALR (15 mg/L or 25 mg/L) treatments. The apoptosis of cultured cells were assessed at 24 h, 48 h by AO/EB staining, DNA agarose gel electrophoresis analysis and flow cytometry using Annexin V-FITC and propidium iodide (PI) staining. The protein and mRNA expressions of Bcl-2 and Bax were detected by Western blotting and RT-PCR, respectively. RESULTS: (1) rrALR inhibited NRK-52E cells apoptosis induced by GM (P＜0.05). (2) rrALR promoted the expression of Bcl-2 protein and mRNA, but inhibited the Bax protein and mRNA expression (P＜0.05) in cultured NRK-52E cells in a dose-dependent manner. The value of Bcl-2/Bax increased. CONCLUSION: rrALR inhibits renal tubular epithelial cell apoptosis and ameliorates cell injury induced by nephrotoxic drug GM presumably via the regulation of Bcl-2 and Bax protein and mRNA expressions.     

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 ruthenium-polypyridine complex on apoptosis of gastric cancer SGC-7901 cells

AIM:To study the effect of ruthenium-pyridine complex Ru1 on apoptosis of gastric cancer SGC-7901 cells. METHODS:MTT assay and crystal violet staining method were used to detect the viability and cell number of SGC-7901 cells treatment with Ru1. Annexin V-FITC and PI staining was performed to test the apoptosis rate of SGC-7901 cells. The protein expression of Bax and Bcl-2 was detected by Western blot. RESULTS:The results of MTT assay and crystal violet staining showed that the ruthenium-pyridine complexes significantly reduced the viability and cell number of SGC-7901 cells. Treatment with Ru1 for 24 h significantly increased the apoptotic rate of SGC-7901 cells (P<0.05). Ru1 up-regulated the expression of Bax protein and down-regulated the expression of Bcl-2 protein in the SGC-7901 cells (P<0.05). CONCLUSION:Ru1 induces apoptosis of SGC-7901 cells by affecting the expression of Bax and Bcl-2 proteins.