Expression of eEF1A2 in hepatocellular carcinoma

AIM: To study the expression of eukaryotic elongation factor 1A2 (eEF1A2) in the hepatocellular carcinoma (HCC) tissues and the effects of eEF1A2 over-expression on the biological behaviors of the HCC cells. ME-THODS: The expression of eEF1A2 at mRNA and protein levels in the HCC tissues and matched liver tissues from 62 HCC patients, and 20 normal liver tissues were detected by the methods of real-time PCR and immunohistochemical staining, respectively. The mRNA and protein expression of eEF1A2 in the HCC cells was also determined by real-time PCR and Western blot, respectively. The lentivirus containing eEF1A2 gene was constructed, and was used to infect the HCC cells with low eEF1A2 expression. The expression of eEF1A2 at mRNA and protein levels in the infected cells was detected by real-time PCR and Western blot, respectively. The cell activity, cell cycle and mRNA expression of albumin were measured by MTT assay, DNA ploid analysis and real-time PCR, respectively.RESULTS: The mRNA expression levels and protein expression positive rates of eEF1A2 in the 62 cases of HCC tissues, were significantly higher than those of 62 matched liver tissues and 20 normal liver tissues (P<0.01). eEF1A2 mRNA and protein were highly expressed in SMMC-7721 cells and BEL-7402 cells, and expressed in SK-HEP-1 cells at low level. The expression of eEF1A2 at mRNA and protein levels in the SK-HEP-1 cells was significantly enhanced by infection of GV287-eEF1A2 expression lentivirus.Compared with negative control group (transfected with negative control lentivirus), the cell activity in eEF1A2 over-expression group (transfected with GV287-eEF1A2 expression lentivirus) was significantly enhanced, the mRNA expression of albumin was remarkably reduced, and the cells in G₀/G₁ phase were significantly decreased with increased percentage of the cells in S and G₂/M phases.CONCLUSION: eEF1A2 is selectively over-expressed in human HCC cancer tissues. eEF1A2 might be a putative oncoprotein in HCC. eEF1A2 over-expression has noticeable effects on the HCC cell proliferation enhancement, differentiation inhibition, and cell cycle acceleration through the G₀/G₁ phase to S phase and G₂/M phases.     

Mechanism of AKT1 negatively regulating breast cancer cell migration

AIM: To investigate the molecular mechanism and downstream signaling pathway by which AKT1 inhibition regulates breast cancer cell migration. METHODS: RNA interference was used to knockdown the expression of AKT1. Western blot assay was performed to examine the expression of AKT1 total protein, β-catenin total protein and β-catenin nuclear protein. Immunofluorescence was used to examine the cellular localization of β-catenin. Transwell assay was used to investigate whether β-catenin nuclear accumulation as an alternative pathway was responsible for breast cancer metastasis induced by AKT1 inhibition. RESULTS: The total protein expression of AKT1 was decreased in MCF-7 and MDA-MB-231 cells treated with AKT1 siRNA. A significant increase in the protein expression of β-catenin was observed in MCF-7 cells and MDA-MB-231 cells treated with AKT1 siRNA. Immunofluorescence staining showed that MCF-7 cells and MDA-MB-231 cells displayed strong β-catenin staining in the cytoplasm and nucleus after knockdown of AKT1 expression. The ability of tumor cell migration increased dramatically after treated with AKT1 specific siRNA in the breast cancer MCF-7 cells and MDA-MB-231 cells in Transwell assay. XAV-939 reversed breast cancer cell migration induced by knockdown of AKT1 expression. CONCLUSION: β-catenin nuclear accumulation contributes to AKT1 inhibition-mediated breast cancer cell migration.     

Effect of RNAi-mediated IGF1R gene silencing on growth,migration, and invasion of hepatocellular carcinoma cells

AIM: To investigate the effect of RNA interference (RNAi)-mediated insulin-like growth factor 1 receptor (IGF1R) gene silencing on the growth, migration, and invasion of hepatocellular carcinoma cells. METHODS: The most effective siRNA targeting IGF1R gene was designed and screened. After lentiviral expression vector pLVX-shRNA2-IGF1R carrying the most effective siRNA sequence was constructed, it was transfected into 293T cells and packed into pLVX-shRNA2-IGF1R lentivirus. Huh7 and Hep3B cells were infected with the pLVX-shRNA2-IGF1R lentivirus to screen the positive clone Huh7 cells and Hep3B cells with the lentivirus. These Huh7 cells and Hep3B cells were cultured to analyze the mRNA level of IGF1R, cell proliferation, cell cycle, cell apoptosis, cell migration/invasion, and the protein levels of IGF1R, Ki-67, p-AKT, p-ERK1, Gli1, β-catenin, cyclin D1, p21 and BCL-XL. RESULTS: The mRNA expression of IGF1R in Huh7 cells and Hep3B cells with pLVX-shRNA2-IGF1R lentivirus was significantly reduced. The proliferation of these cells was remarkably inhibited, and the number in G₁ phase was increased significantly. The percentages of apoptotic cells were increased markedly, and the number of cell migration/invasion was decreased markedly. The protein levels of IGF1R, Ki-67, p-AKT, p-ERK1, Gli1, β-catenin, cyclin D1, p21 and BCL-XL were decreased significantly compared with the blank control group and negative control group. CONCLUSION: The RNAi-mediated IGF1R gene silencing significantly suppresses the growth and the malignant biological characteristics of Huh7 cells and Hep3B cells, which may be involved in the reduced protein levels of the above genes induced by down-regulation of IGF1R expression.     

Effect of ABCE 1 gene silencing on proliferation and migration of human bladder cancer cell line T24

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24. METHODS: The siRNA against ABCE1 was constructed and transfected into the T24 cells with Lipofectamine^TM 2000. The expression of ABCE1 was detected by RT-PCR and Western blot. Flow cytometry was used to detect the cell cycle. The effects of ABCE1 gene silencing on proliferation, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion assay, respectively. RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA. The cell cycle was arrested at G₀/G₁ phase and the cell number in S phase was decreased in the T24 cells. Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly. CONCLUSION: Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

ATPase inhibitory factor 1-a host cell protein molecule interacting with human cytomegalovirus pUL23 protein

AIM: pUL23, the product of human cytomegalovirus (HCMV) gene UL23 was identified as one of tegument proteins. The aim of this research is to investigate the function of pUL23 during HCMV life cycle.METHODS: GAL4 yeast two-hybrid assay was performed to screen the human fetal kidney cDNA library to obtain host cell protein molecules which interact with pUL23 of HCMV. Then the GST-pulldown experiment was applied to confirm the protein interactions identified by yeast two-hybrid.RESULTS: ATPase inhibitory factor 1 (ATIF1) was selected from host cells using yeast two-hybrid assay. GST-pulldown experiments in vitro further confirmed the interaction between ATIF1 and pUL23.CONCLUSION: pUL23 of HCMV can interact with ATIF1 in host cell, which may provide the evidence for understanding the function of pUL23 in the life cycle of HCMV.     

Role of caveolin-1 on down-regulation of LPS-induced monocyte chemotactic protein 1 by 17β-estradiol in vascular smooth muscle cells

AIM: To elucidate the effect of caveolin-1 on the down-regulation of LPS-induced monocyte chemotactic protein 1 (MCP-1) by 17β-estradiol (E₂) in vascular smooth muscle cells (VSMCs).METHODS: The primary-cultured VSMCs were exposed to E₂ at concentrations of 10^-9-10^-6 mol/L. LPS-induced MCP-1 production was assayed by ELISA. The protein expression of caveolin-1 was determined by Western blotting and was silenced by β-methyl cyclodextrin(β-MCD) or caveolin-1 specific siRNA. RESULTS: LPS significantly enhanced MCP-1 production. E₂ at concentrations of 10^-9-10^-6 mol/L inhibited LPS-induced MCP-1 production. The use of caveolin-1 inhibitor β-MCD or silencing the protein expression of caveolin-1 by specific siRNA largely impaired LPS-enhanced MCP-1 production, while E₂ markedly inhibited caveolin-1 expression. CONCLUSION: Inhibition of LPS-induced MCP-1 production by E₂ is related to the suppression of caveolin-1.     

Klotho: a protein molecule interacting with FPC protein

AIM: To investigate the role of fibrocystin/polycystin (FPC) in autosomal recessive polycystic kidney disease (ARPKD) development by means of screening the protein interaction using yeast two-hybrid approach. METHODS: The constructed pGBKT7-FPC was used as the bait to screen the pre-transformed human fetal kidney cDNA expression library by yeast two-hybrid assay to obtain the host cell protein which interacted with C-terminal region of FPC. The sequence transformation screening experiment was applied to confirm the protein interactions in yeast. RESULTS: After yeast mating and co-transformation screening analysis, Klotho (KL) was selected from the host cells and the interaction between KL and FPC was further confirmed. CONCLUSION: C-terminal region of FPC can interact with KL, which probably provide the approach for further studying the role and biochemistry mechanism of FPC protein in ARPKD.     

Screening of proteins interacting with activation domain of macrophage migration-inhibitory factor

AIM: To screen the proteins that interact with the catalytic thiol-protein oxidoreductase (TPOR) domain of macrophage migration-inhibitory factor (MIF). METHODS: Yeast two-hybrid system was used to study the proteins that interacted with MIF. The bait vector pBTM116-MIF was constructed and transfected into L40 yeast strain. L40 competent cells expressing the TPOR domain were prepared and used to screen the proteins interacting with the TPOR domain of MIF in a human osteosarcoma cDNA library. The proteins interacting with TPOR domain were identified by HIS3 reporter gene and β-galactosidase assay. The techniques of co-immunoprecipitation and immunofluorescence were also used to verify the proteins interacting with TPOR domain. RESULTS: Wild-type pBTM116-MIF and pACT2-TXNL2 (thioredoxin-like 2 protein) were constructed and co-transfected into L40 yeast strain. The activation of HIS3 reporter gene and the positive result of β-galactosidase assay indicated that TXNL2 was the candidate protein that interacted with the catalytic TPOR domain of MIF. The vector of pcDNA3.1-Myc-TXNL2 was constructed and transfected into MCF-7 cell line. The result of co-immunoprecipitation assay showed that the catalytic TPOR domain of MIF co-immunoprecipitated with Myc-TXNL2 protein. The result of immunofluorescence assay demonstrated that the catalytic TPOR domain of MIF and TXNL2 co-localized in the cytoplasm of the cells. CONCLUSION: TXNL2 is the protein that interacts with the catalyzed TPOR domain of MIF.     

Knockdown of astrocyte elevated gene-1 by siRNA inhibits cell proliferation and induces apoptosis in human neuroblastoma cell line

AIM: To investigate the effect of siRNA-induced astrocyte elevated gene-1 ( AEG-1 ) down-regulation on the proliferation, apoptosis and cell cycle of neuroblastoma cells. METHODS: An siRNA targeting to AEG-1 mRNA (AEG-1 siRNA) was constructed and transfected into neuroblastoma cells with Lipofectamine 2000. A non-specific siRNA (control siRNA) and non-treatment were used as negative control and blank control,respectively . The cell proliferation was detected by MTT method and colony formation assay. The apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Compared with control groups, the expression of AEG-1 mRNA was evidently declined in the cells transfected with AEG-1 siRNAs (P<0.05). AEG-1 siRNA significantly decreased the cell proliferation. After treated with AEG-1 siRNA for 48 h, the percentage of apoptotic cells was significant increased and the cell cycle was arrested in G₀/G₁ phase compared with the control cells (P<0.05). CONCLUSION: The mRNA expression of AEG-1 is down-regulated by AEG-1 siRNA in neuroblastoma cells. Knockdown of AEG-1 expression in human neuroblastoma cells significantly inhibits cell proliferation and apoptosis, and induces cell arrest in G₀/G₁ phase of the cell cycle.     

Effects of Ku70 on protein expression of HTLV-1 in HTLV-1 positive T cells

AIM:To explore the effects of Ku70 on the protein expression of human T-lymphotrophic virus 1 (HTLV-1) in HTLV-1 positive T cells. METHODS:The expression level of Ku70 in HTLV-1 positive T cells was exa-mined by Western blot. The siRNA targeting Ku70 was constructed and the effect of the siRNA on knockdown of Ku70 expression was determined by Western blot. After knockdown of Ku70 expression in the HTLV-1 positive T cells by siRNA, the expression of HTLV-1-related proteins at mRNA and protein levels was examined by real-time PCR and Western blot, and the expression levels of interferons and proinflammatory cytokines were examined by real-time PCR. RESULTS:The HTLV-1 positive T cells, including MT2, MT4 and C8199 cells, displayed a higher expression level of Ku70. The protein expression of HTLV-1 was increased in Ku70-silencing MT2 cells and MT4 cells. After knockdown of Ku70 expression in the MT2 cells and MT4 cells, the production of interferon (IFN)-α, IFN-γ and tumor necrosis factor-α was reduced.CONCLUSION:The HTLV-1 positive T cells have a higher expression level of Ku70. In HTLV-1 positive T cells, Ku70 promotes the production of interferons and proinflammatory cytokines and inhibits HTLV-1-related protein expression.     

Effects of lentivirus-mediated siRNA targeting IGF-1R on migration and invasion abilities of endometrial carcinoma cells

AIM:To investigate the down-regulation of insulin-like growth factor tgpe 1 receptor(IGF-1R) on the migration and invasion abilities of human endometrial cancer cell HEC-1B. METHODS:The siRNAs targeting IGF-1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC-1B cells(HEC-1B-KD group). The control cells(without virus transfection, HEC-1B-CON group) and negative virus transfection control cells(HEC-1B-NC group) were also set up. The gene silencing effect of siRNA targeting IGF-1R was determined by real-time PCR and Western blotting at mRNA and protein levels,respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experiment. The mRNA levels of matrix metalloproteinase(MMP)-2 and MMP-9 were measured by real-time PCR. RESULTS:The mRNA and protein levels of IGF-1R in HEC-1B-KD cells were significantly reduced by 81% and 91.5%, respectively(P<0.05). In anchorage-dependent growth by colony formation assay, HEC-1B-KD cells showed much less colonies than HEC-1B-CON cells and HEC-1B-NC cells. Compared with the control cells, knockdown of IGF-1R in HEC-1B cells resulted in significant reduction of cell motility. Down-regulation of IGF-1R in HEC-1B cells also significantly reduced the invasion potential(P<0.05). Down-regulation of IGF-1R substantially reduced the expression of MMP-2 and MMP-9 compared with the control cells. CONCLUSION:Knockdown of IGF-1R reduces the migration and invasion abilities of human endometrial cancer cells in vitro accompanied with a decrease in MMP-2 and MMP-9 expression.     

Using folic acid-modified polyethylenimine-SPIO nanoparticles for PD-L1 siRNA delivery to target gastric cancer

AIM: To synthesis and characterize a multi-functional siRNA delivery agent with effective therapeutic effects and MR-tracing ability for programmed death ligand-1 (PD-L1) positive gastric cancer SGC-7901 cell line. METHODS: The characterization, binding ability, cytotoxicity, transfection efficiency and cellular internalization of the polyplex were determined. The PD-L1 knockdown effect was analyzed, and cytokines secreted by cocultured T cells were measured.RESULTS: We developed folic acid (FA)-PEG-SS-PEI-SPION as siRNA delivery agent for PD-L1 knockdown. At N/P ratio of 10, the FA-PEG-SS-PEI-SPION bound PD-L1 siRNA to form polyplex in a diameter of (116.7±2.5) nm with zeta potential of (9.14±0.80) mV. Transfection efficiency of the targeted polyplex was (95.06±0.44)%, compared with (93.87±1.05)% of the untargeted polyplex. Mean fluorescence intensity of the targeted polyplex was 1 892.67±81.51, significantly higher than 1 324.33±186.58 of the untargeted. The cellular magnetic resonance (MR) imaging showed the polyplex also acted as T₂ weighted contrast agent for cancer MR imaging. The relative mRNA level of PD-L1 in polymer/siRNA-2 treatment group was (9.07±0.79)%. Decreased protein expression of PD-L1 was showed by Western blot. The secretion levels of IFN-γ and TNF-α in cocultured T cells increased, while that of IL-10 decreased. CONCLUSION: Our findings highlighted the potential of the multifunctional theranostic nanoparticles for effective targeting PD-L1 knockdown therapy and MR imaging diagnosis in gastric cancers.     

Regulatory effects of Rock2 on cell cycle checkpoint Cdc25A

AIM: To investigate the regulatory effects of Rho-associated coiled-coil-containing protein kinase 2(Rock2) on the cell cycle checkpoint cell division cycle 25A(Cdc25A). METHODS: The protein expression levels of Rock2 and Cdc25A in 51 pairs of hepatocellular carcinoma and the adjacent tissues were detected by Western blotting. shRock2 plasmids were constructed, selected and stably transfected into hepatocellular carcinoma Huh-7 and HepG2 cells. The protein expression of Cdc25A in the cells was determined by Western blotting. Based on the Rock2 interfering sequences, we designed the primers and changed the 4 indicated bases via site-specific mutagenesis. The Rock2-mutant plasmid was verified by sequencing and was transfected into stable Rock2-knockdown cells. The protein expression of Cdc25A was detected by Western blotting, and the cell proliferation was measured by MTT assay. The protein levels of checkpoint kinase(Chk)1/Chk2 were also detected in stable Rock2-knockdown cells. The interaction between Rock2 and Cdc25A was measured by co-immunoprecipitation, and the co-localization of Rock2 and Cdc25A was detected by confocal laser scanning microscopy. RESULTS: Rock2 and Cdc25A were apparently up-regulated in hepatocellular carcinoma,with a significantly positive correlation. The protein expression of Cdc25A was significantly down-regulated in stable Rock2-knockdown cells. The expression of Chk1 and Chk2 was not changed following knockdown of Rock2. The co-immunoprecipitation results verified that Rock2 bound to Cdc25A. The results of confocal laser scanning microscopy showed that Rock2 and Cdc25A were co-localized in hepatocellular carcinoma cells. CONCLUSION: Rock2 positively regulates the cell cycle checkpoint Cdc25A, which is independent of Chk1/Chk2 and this may provide a new target gene for treatment of hepatocellular carcinoma.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.     

Down-regulation of lncRNA PCAT1 suppresses oral squamous cell carcinoma cell proliferation,growth, invasion,migration and epithelial-mesenchymal transition

AIM: To investigate the effects of the long non-coding RNA (lncRNA) PCAT1 on the oral squamous cell carcinoma (OSCC) cell proliferation, growth, invasion and migration, and to explore the underlying mechanisms. METHODS: The PCAT1 siRNA was transfected by Lipofectmine 2000, and RT-qPCR and Western blot were performed to determine the mRNA and protein expression of relevant genes, respectively. CCK-8 assay and colony formation assay were used to measure OSCC cell proliferation and growth, respectively. The cell invasion and migration assays were used to measure the invasive and migratory abilities of the OSCC cells, respectively. RESULTS: PCAT1 was significantly up-regulated in OSCC tissues and cells compared with normal adjacent tissues and normal human oral keratinocyte cells, respectively (P<0.05). PCAT1 siRNA transfection suppressed the expression of PCAT1 in Tca8113 and TSCCa cells (P<0.05). Knockdown of PCAT1 in Tca8133 cells and TSCCa cells significantly suppressed the cell proliferation, invasion and migration abilities (P<0.05). In addition, knockdown of PCAT1 in Tca8133 cells and TSCCa cells also suppressed the mRNA and protein levels of ZEB-1, N-cadherin and vimentin, and increased the mRNA and protein expression of E-cadherin (P<0.05). CONCLUSION: Knockdown of PCAT1 suppresses cell proliferation and migration abilities, and the effect of PCAT1 on OSCC cells may be associated with epithelial-mesenchymal transition.     

Knockdown of IK1 potassium channel inhibits expression and function of ClC-3 chloride channel

AIM: To investigate whether the ClC-3 chloride channel is an acting target of the IK1 potassium channel, and to study the action of IK1 potassium channel on the functional activities and expression of ClC-3 chloride channels. METHODS: IK1 gene was silenced by IK1 siRNA in poorly-differentiated nasopharyngeal carcinoma cells (CNE-2Z). Real-time PCR and Western blot were used to detect the expression of ClC-3 at mRNA and protein levels. The distribution of ClC-3 protein in the cells was observed under confocal immunofluorescence microscope. The chloride current was recorded by the patch-clamp technique. RESULTS: IK1 siRNA was successfully transfected into the CNE-2Z cells and knocked down the expression of IK1 potassium. The mRNA expression of ClC-3 was increased by the IK1 siRNA. IK1 siRNA inhibited the expression of ClC-3 protein. A chloride current was activated by hypotonic challenges, and the hypotonicity-induced current was reduced in the cells which successfully transfected with IK1 siRNA. CONCLUSION: The knockdown of IK1 potassium channels inhibits the expression and function of ClC-3 chloride channel.     

Effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells

AIM:To investigate the effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells. METHODS:Transient transfection of NANOG targeting siRNA into HepG2 cells was performed. The expression of NANOG and cyclin D1 at mRNA and protein levels was detected by real-time PCR and Western blotting. Cell proliferation was examined by CCK-8 assay and colony formation assay, and cell cycle was tested by flow cytometry. RESULTS:After transfection with NANOG-targeting siRNA, the inhibition of NANOG expression was observed. Compared with mock group, the mRNA and protein expression levels of NANOG and cyclin D1 were decreased (P<005). In addition, knockdown of NANOG expression inhibited the cell proliferation and increased the proportion of G 0/G 1-phase cells (P<0.05). CONCLUSION:Silencing of NANOG expression in HepG2 cells causes down-regulation of cyclin D1 expression and decreases the cell proliferation ability.     

Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133+ cell subset by promoting formation of Apaf-1/caspase-9 complex

AIM:To investigate the synergistic effect of resveratrol and 5-fluorouracil on osteosarcoma CD133⁺ cell subset.METHODS:Human osteosarcoma cell line MG-63 CD133⁺ cell subset and the corresponding CD133^- cell subset were treated with resveratrol and 5-fluorouracil.After treatment,the viability of MG-63 cells was measured by MTT assay.The apoptosis of MG-63 cells was analyzed by flow cytometry.Activation of caspase-9 and caspase-3,the expression of Apaf-1,and the release of cytochrome C were evaluated by Western blot.The interaction between Apaf-1 and pro-caspase-9 was detected by co-immunoprecipitation.RESULTS:The cell death and apoptosis of MG-63 CD133⁺ cell subset induced by 5-fluorouracil were significantly weaker than those in the corresponding MG-63 CD133^- cell subset.However,co-treatment with resveratrol significantly enhanced the effect of 5-fluorouracil on inhibiting the viability of MG-63 CD133⁺ cell subset.Mechanically,treatment with resveratrol upregulated the expression of Apaf-1.Transfection with Apaf-1 siRNA abolished the synergistic effect of resveratrol and 5-fluorouracil in MG-63 CD133⁺ cell subset.In addition,the results of co-immunoprecipitation indicated that the combination of resveratrol and 5-fluorouracil significantly induced the formation of Apaf-1/pro-caspase-9 complex,leading to the activation of caspase-9 in MG-63 CD133⁺ cell subset.CONCLUSION:Resveratrol enhances 5-fluorouracil-induced apoptosis of osteosarcoma CD133⁺ cell subset by promoting the formation of Apaf-1/caspase-9 complex.