Effects of Gax gene transfection on proto-oncogene expression and proliferation of PASMCs in rats under hypoxia conditions

AIM: To explore the effects of Gax gene transfection on expressions of c-fos and c-jun mRNA and proliferation of pulmonary arterial smooth muscle cells (PASMCs) in rat under hypoxia.METHODS: PASMCs were transfected with Gax gene by Ad-Gax.Under normal oxygentention (21% O₂) or hypoxia (2.5% O₂) for 12 h condition,expressions of Gax mRNA and protein in PASMCs were detected by RT-PCR and immunocytochemistry.The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR.［³H］-TdR incorporation was used to measure the PASMCs proliferation.RESULTS: The Gax overexpression in transfection group was confirmed by RT-PCR and immunocytochemistry.Under normal oxygentention or hypoxia,the c-fos and c-jun mRNA levels in transfection group were lower than those in the non-transfection group,respectively (P<0.05).［³H］-TdR incorporation in the transfection group was lower than that in non-transfection group (P<0.05,P<0.01).CONCLUSION: Gax overexpression might inhibit the PASMCs proliferation induced by hypoxia through downregulating the expressions of c-fos and c-jun.     

Effect of ginsenoside Rb1 on proliferation and serotonin transporter and 5-HT1B R expression in hypoxia-induced rat pulmonary artery smooth muscle cells via Rho/Rho-kinase pathway

AIM: To observe the effect of ginsenoside Rb1 on the proliferation and the expression of serotonin transporter (SERT), 5-hydroxytryptamine 1B receptor (5HT_1BR) in rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia condition and the relationship with Rho/Rho-kinase signal pathway.METHODS: PASMCs were isolated from the adult male SD rats and primarily cultured. The subcultured cells from the 4th generation to the 6th generation were harvested and divided into normal group, and hypoxia group, different concentrations of Rb1 incubation groups treated with 50, 100 and 200 mg/L ginsenoside Rb1 under hypoxia (HR50, HR100 and HR200 groups, respectively). The viability of the PASMCs was measured by CCK-8 assay. BrdU positive cells were determined using flow cytometry. The expression of serotonin transporter and 5HT_1BR at mRNA and protein levels was detected by RT-PCR and Western blot, respectively. The PASMCs were randomly divided into normal group, hypoxia group, HR200 group and hypoxia+Y-27632 incubation group (HY group). The mRNA expression of Rho-kinase and phosphorylated myosin phosphatase target subunit 1 (p-MYPT1) protein level were investigated by RT-PCR and Western blot, respectively.RESULTS: Compared with normal group, the proliferation of PASMCs in hypoxia group was significantly increased (P<0.01). The cell viability and the expression of SERT and 5HT_1BR at mRNA and protein levels in all different concentrations of Rb1 groups were obviously decreased compared with hypoxia group (P<0.05). The mRNA expression of Rho-kinase and protein level of p-MYPT1 were markedly decreased in HR200 group, and no significant difference compared with HY group was observed (P<0.01).CONCLUSION: Treatment with ginsenoside Rb1 might prevent hypoxia-induced proliferation of PASMCs and over-expression of SERT and 5HT_1BR through inhibiting the Rho/Rho-kinase pathway.     

Effects of SOCS3 gene on the expression of c-myc mRNA and proliferation of rat pulmonary arterial smooth muscle cells under hypoxia conditions

AIM: To explore the effect of SOCS3 gene on the expression of c-myc mRNA and proliferation of rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxia conditions. METHODS: PASMCs was cotransfected with pEFSOCS3 and pSV2neo by lipofectamine, and positive cell clones were obtained after being screened with G418. Expressions of SOCS3 protein in PASMCs before and after transfection were detected by immunocytochemistry, respectively. Before and after transfection, PASMCs were exposed to normoxic and hypoxia conditions at various time points, respectively, and the expressions of c-myc mRNA were assessed by semi-quantitive RT-PCR. ［3H］-TdR incorporation method was used to detect the cell proliferation. RESULTS: The expression of SOCS3 protein was confirmed by immunocytochemistry in PASMCs transfected with SOCS3 gene. c-myc mRNA level in the SOCS3 gene-transfected cells exposed to hypoxia were remarkablely lower than that in the control cells, respectively (P<0.01). Compared with the control groups at the same time points, ［3H］-TdR incorporation in SOCS3 gene-transfected cells was significantly low. CONCLUSION: SOCS3 protein may inhibit the proliferation of PASMCs by downregulating the c-myc gene expression under hypoxia conditions.     

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.     

Role of translationally controlled tumor protein in PRL-3-promoted metastasis of colon cancer cells

AIM: To study the role of translationally controlled tumor protein (TCTP) in the proliferation, migration and invasion of phosphatase of regenerating liver-3(PRL-3)-promoted colon cancer cells.METHODS: The vectors pAcGFP-C3 and pAcGFP-C3-PRL-3 were constructed and transfected into the colon cancer cell line LoVo.LoVo-PRL-3 cells stably expressing PRL-3 and LoVo-control cells were established. The expression levels of PRL-3 and TCTP in both cells were detected by Western blotting and real-time PCR. The specific siRNA sequence for TCTP mRNA and control-siRNA were synthesized and transfected into the LoVo-PRL-3 cells. TCTP expression at mRNA and protein levels in LoVo-PRL-3 was detected by Western blotting and real-time PCR 24 h, 48 h and 72 h after transfection. The proliferation, migration and invasion abilities of LoVo-control cells, LoVo-PRL-3 cells, TCTP-siRNA and control-siRNA cells were detected by CCK-8 assay and the method of Transwell cell culture chambers.RESULTS: The expression of TCTP at mRNA and protein levels in LoVo cells was significantly increased after PRL-3 transfection (P<0.05). TCTP mRNA was significantly inhibited 24 h, 48 h and 72 h after transfection of TCTP-siRNA (P<0.01). TCTP protein was also significantly inhibited 48 h and 72 h after transfection (P<0.01). Compared with LoVo-control cells, the proliferation, migration and invasion abilities of LoVo-PRL-3 cells were significantly enhanced (P<0.05). However, lowering the up-regulated expression of TCTP in LoVo-PRL-3 cells inhibited the proliferation, migration and invasion abilities (P<0.05). CONCLUSION: PRL-3 promotes proliferation, migration and invasion of colon cancer cells by up-regulating the TCTP expression. siRNA targeting TCTP may be an effective method for prevention and treatment of colon cancer cell metastasis.     

miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

Urotensin Ⅱinduces pulmonary arterial smooth muscle cell proliferation and up-regulates Egr-1 expression through ERK1/2 pathway in rats

AIM: To investigate the effect of urotensinⅡ (UⅡ) on the proliferation of cultured rat pulmonary arterial smooth muscle cells (PASMCs), and to explore whether mitogen-activated protein kinase (MAPK) signaling pathways and early growth response factor-1 (Egr-1) involved in the regulation of the PASMCs proliferation stimulated by UⅡ. METHODS: The rat PASMCs were isolated and cultured in vitrowith explant culture technique. The proliferation of cultured PASMCs stimulated by different doses of UⅡwas detected by BrdU incorporation. The mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase (SAPK), p38 MAPK and Egr-1 in cultured PASMCs treated with UⅡ, UⅡ-specific antagonist urantide, and ERK1/2 inhibitor PD98059 was detected by real-time PCR. The protein levels of phosphorylated ERK1/2 (p-ERK1/2), p-SAPK, p-p38 and Egr-1 in cultured PASMCs were determined by Western blotting. RESULTS: UⅡ at concentrations of 1 μmol/L, 0.1 μmol/L and 0.01 μmol/L increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.01 or P<0.05), with the maximal effect at a concentration of 1 μmol/L. However, urantide inhibited the promotion effect of UⅡ on PASMC proliferation (P<0.05). UⅡ up-regulated the mRNA expression of ERK1/2, SAPK and Egr-1 (P<0.01 or P<0.05), but not the p38 MAPK. However, the up-regulatory effect of UⅡ on ERK1/2 and Egr-1 expression was inhibited by PD98059 and/or urantide (P<0.01 or P<0.05). UⅡ also increased the protein levels of p-ERK1/2, p-SAPK and Egr-1 (P<0.01 or P<0.05), but the promotion effect was also inhibited by PD98059 and/or urantide (P<0.01 or P<0.05).CONCLUSION: UⅡ increases the proliferation of PASMCs, and U Ⅱand Egr-1 participates in UⅡ-mediated proliferation of cultured PASMCs through activation of ERK1/2 signal pathway.     

Role of reactive oxygen species and ERK1/2 signaling pathway in proliferation and apoptosis of hypoxic rat pulmonary arterial smooth muscle cells

AIM:To investigate the change of reactive oxygen species (ROS) production in hypoxic pulmonary arterial smooth muscle cells (PASMCs) of rats, the effect of ROS on the expression of extracellular signal-regulated kinase (ERK)1/2 protein, and the role of ROS and ERK1/2 in the imbalance between proliferation and apoptosis of PASMCs.METHODS: Primary cultures of PASMCs were established and cells between passages 2 to 3 were used for experiments. PASMCs were treated with tiron, a membrane permeable ROS scavenger, and PD98059, an ERK1/2 inhibitor, under normoxia or hypoxia condition. The ROS production was measured by DCFH-DA and NBT reduction. The expression of phosphorylated-ERK1/2 (p-ERK1/2) protein was detected by immunofluorescence. Cell proliferation was examined by MTT colorimetric assay and the expression of PCNA. Cell apoptosis was detected by TUNEL.RESULTS: (1)Compared with control group, the ROS levels in hypoxia group were significantly increased (P<0.01). (2) In hypoxia group, the proliferative capacity was higher and the apoptosis index was lower than those in control group (P<0.01). Tiron significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). (3) The expression of p-ERK1/2 in hypoxia group were higher than that in control group (P<0.01), which were significantly suppressed by tiron (P<0.01)．(4) PD98059 significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). The proliferative capacity and apoptosis index was similar in hypoxia+tiron+PD98059 group to those in hypoxia+tiron group (P>0.05).CONCLUSION:The hypoxia-mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2. ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.     

Effects of Kv1.5 on proliferation and apoptosis of rat PASMCs under hypoxia+hypercapnia condition and relationship with MAPK pathway

AIM：To investigate the effects of voltage-dependent K＋ channel 1.5 (Kv1.5) on the proliferation and apoptosis of rat pulmonary artery smooth muscle cells (PASMCs) under hypoxia+hypercapnia condition and the relationship with mitogen-activated protein kinase(MAPK) signal pathway. METHODS：The PASMCs isolated from the male SD rat were cultured under hypoxia+hypercapnia condition, and randomly divided into normal group (N group), hypoxia+hypercapnia group (HH group), hypoxia+hypercapnia+DMSO incubation group (HD group), hypoxia+hypercapnia+U0126 (an extracellular signal-regulated kinase 1/2 inhibitor) incubation group (HU group), hypoxia+hypercapnia+SB203580 (a p38 mitogen-activated protein kinase inhibitor) incubation group (HS group), and hypoxia+hypercapnia+anisomycin (an agonist of MAPK) incubation group (HA group). Cell Counting Kit-8 was used to detect the cell viability. The protein expression of Kv1.5, PCNA and Bax was detected by Western blotting. RESULTS：Compared with N group, the cell viability and PCNA protein expression in HH group and HD group were significantly raised (P<001), but Kv1.5 and Bax proteins were significantly decreased (P<0.01). No difference between HH group and HD group was observed (P>005). Compared with HD group, the cell viability and PCNA protein expression in HU group, HS group and HA group were decreased (P<0.05 or P<0.01), but Kv1.5 protein and Bax protein were raised (P<0.01), with the most significant changes in HA group. ^{CONCLUSION：}The regulation of Kv1.5 to the proliferation and apoptosis of PASMCs under hypoxia+hypercapnia condition might have a relationship with the activation of MAPK signal pathway.     

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.     

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.     

Effect of hPTTG1 siRNA on proliferation and apoptosis of ovarian cancer cells

AIM: To observe the proliferation and apoptosis of ovarian cancer cells by silencing the expression of human pituitary tumor-transforming gene 1 ( hPTTG1 ) using RNA interference technique.METHODS: The chemically synthesized siRNA targeting hPTTG1 was transfected into ovarian cancer cell line A2780 in vitro. The expression levels of hPTTG1 and c-myc were examined by RT-PCR and Western blotting. Cell proliferation was measured by MTT colorimetric assay and -TdR incorporation test. Cell apoptosis was detected by flow cytometry with annexin V/PI and TUNEL labeling.RESULTS: The expression of hPTTG1 at mRNA and protein levels was inhibited after transfection of hPTTG1 siRNA. The inhibitory efficiency was 70.5%±3.9% and 63.8%±4.5%, respectively. The absorbance began to decrease 24 h after transfection of hPTTG1 siRNA,and the highest inhibitory rate was 42.9%±5.2% at 48 h post-transfection. Radioactive incorporation of -TdR in hPTTG1 siRNA group was lower than that in normal and negative groups. The survival rate declined while the apoptotic rate and necrotic rate increased in hPTTG1 siRNA group. Apoptotic index in hPTTG1 siRNA group was higher than that in normal and negative groups. The expression of c-myc at mRNA and protein levels was down-regulated.CONCLUSION: Cell proliferation is inhibited and cell apoptosis is induced by hPTTG1 siRNA through down-regulating the expression of c-myc. hPTTG1 can be regarded as a candidate gene for ovarian cancer gene therapy.     

Effects of NOB1 gene silencing on drug sensitivity and invasion and migration abilities of human colon cancer SW480 cells

AIM:To investigate the effect of NOB1 gene expression knock-down by transfection of small interfering RNA (siRNA) on the viability, drug sensitivity, apoptosis, cell cycle distribution, and invasion and migration abilities of human colon cancer SW480 cells. METHODS:NOB1 siRNA was transfected into SW480 cells using Lipofectamine 3000. The mRNA and protein levels of NOB1 in the SW480 cells were determined by real-time PCR and Western blot. The cell viability and sensitivity to different chemotherapeutic drugs (cisplatin, 5-fluorouracil, oxaliplatin and capecitabine) were detected by MTT assay after knock-down of NOB1 gene expression in the SW480 cells. The apoptosis and cell cycle distribution of SW480 cells were analyzed by flow cytometry. The invasion and migration abilities of SW480 cells were detected by Transwell assay. RESULTS:After transfection with NOB1 siRNA, the mRNA and protein levels of NOB1 in the SW480 cells were significantly decreased (P<0.05). Compared with control group and control siRNA group, the viability of SW480 cells in NOB1 siRNA group was significantly decreased at 24~72 h. The half maximal inhibitory concentrations of the chemotherapy drugs cisplatin, 5-fluorouracil, oxaliplatin and capecitabine were significantly decreased. The apoptotic rate was significantly increased and the cell cycle were blocked. The cell invasion and migration abilities were significantly reduced (P<0.05). CONCLUSION:Knock-down of NOB1 gene expression inhibits the viability and invasion and migration abilities of colon cancer SW480 cells, and promotes drug sensitivity and apoptosis. NOB1 may be a new target for diagnosis and treatment of colon cancer.     

Effect of cigarette smoking condensate on HDAC2 and inflammatory mediators in mouse myoblast C2C12 cells

AIM: To investigate the effect of cigarette smoking condensate on histone deacetylase 2 (HDAC2) and inflammatory mediators in mouse myoblast C2C12 cells. METHODS: C2C12 cells were treated with cigarette smoke extract (CSE). HDAC2 siRNA was transfected into the cells using Lipofectamine^TM 2000. The levels of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in the culture supernatants were measured by ELISA, and the expression of HDAC2 at mRNA and protein levels was determined by real-time PCR and Western blotting. RESULTS: The expression of HDAC2 at mRNA and protein levels in CSE group was lower than that in control group (P<0.05). The supernatant levels of IL-8 and TNF-α in CSE group were significantly higher than those in control group (P<0.05). When the cells were transfected with HDAC2 siRNA followed by CSE stimulation, the expression of HDAC2 at mRNA and protein levels was decreased, and the supernatant levels of IL-8 and TNF-α were significantly increased as compared with CSE group and control group (P<0.05).CONCLUSION: Under the oxidative stress condition, C2C12 cells generate high levels of IL-8 and TNF-α by down-regulating the expression of HDAC2.     

Relationship between EGFR/HER2 gene knocked down and the downstream signal pathway in SPC-A-1

AIM: RNAi technique was applied to explore the relationship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 RNA interference. METHODS: Sequence-specific siRNA for EGFR and HER2 were designed as literatures described. “Tuschl rules” and BLAST on the full length of EGFR/HER2 cDNA were used to ensure the sequence-specific siRNA for EGFR/HER2 joint interference. Based on the above sequence-specific siRNA, recombinant plasmids with GFP and neomycin resistance marker were constructed. Six groups including mock, negative control, shRNA-EGFR, shRNA-HER2, shRNA-EGFR/HER2 and shRNA-EGFR+shRNA-HER2 were established by transient transfection. Real time quantitative RT-PCR was used to detect the silencing of the EGFR/HER2 gene level. Western blotting was used to measure the levels of EGFR/HER2 protein and protein phosphorylation expression. Transfected cells were stimulated with EGF 15 min before protein extraction. MTT assay and flow cytometry were used to evaluate the cell proliferation, apoptosis and cell cycle distribution after RNAi. The protein expression levels of downstream signaling pathway proteins including Akt, p-Akt, p-Erk1/2, p-p38 were measured by real time quantitative RT-PCR and Western blotting. Randomized block analysis of variance and SNK methods were used to compare the differences between groups. RESULTS: Cell proliferation was inhibited in the groups of shRNA-EGFR, shRNA-HER2, shRNA-EGFR+shRNA-HER2 and shRNA-EGFR/HER by MTT assay. Cell cycle analysis by flow cytometry showed that apoptosis ratio in shRNA-HER2 (P<0.01), shRNA-EGFR/HER2 (P<0.01) and shRNA-EGFR+shRNA-HER2 (P<0.05) groups were significantly higher than those in negative control group, while there was no statistical difference between shRNA-EGFR and negative control (P>0.05), and that the distributions in phase G1 and phase S in shRNA-EGFR (P<0.01), shRNA-HER2 (P<0.01), shRNA-EGFR/HER2 (P<0.01) and shRNA-EGFR+shRNA-HER2 (P<0.01) were significantly different compared with the negative control. The level of EGFR/HER2 protein and protein phosphorylation expression were down regulated. The cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference showed no significant relationship with downstream signal pathway molecular in EGFR family. CONCLUSION: EGFR gene knockdown may not cause significant apoptosis in SPC-A-1 cell line. The variations of cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference were not found to have significant relationship with downstream signal pathway molecules in EGFR family.     

Expression of Gab2 in human osteosarcoma U2-OS cells

AIM: To investigate the expression of Grb2-associated binding protein 2 (Gab2) in human osteosarcoma cells and its relationship with the invasion and metastases of human osteosarcoma cells. METHODS: The technique of small RNA interference was used to transfect human osteosarcoma U2-OS cell lines. Western blotting and RT-PCR were used to detect the protein and mRNA expression of Gab2 in transfected U2-OS cells. After transfection, through chemotaxis and invasion assays in vitro, the cell migration and invasion abilities were detected. RESULTS: After transfection, the expression of Gab2 at mRNA and protein levels in Gab2 siRNA transfected cells (SiGab2/U2-OS) was lower than that in scrambled siRNA transfected cells (Scr/U2-OS) and U2-OS cells. After stimulation with epidermal growth factor (EGF) at concentration of 10 μg/L, the migration SiGab2/U2-OS cells was significantly less than Scr/U2-OS cells and U2-OS cells (P<0.01). The number of invasion cells of SiGab2/U2-OS group was significantly lower than the other 2 control groups (P<0.01). CONCLUSION: Inhibition of Gab2 expression obviously attenuates the migration and invasion abilities of human osteosarcoma U2-OS cell line.     

Effects of EGLN1 siRNA on growth of rat pulmonary artery smooth muscle cells under hypoxic condition

AIM: To observe whether EGLN1 gene is involved in the growth of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EGLN1 gene expression was interference by siRNA. METHODS: The rat primary pulmonary arterial smooth muscle cells were cultured, and the specific lipidosome of EGLN1 siRNA was constructed and transfected into the PASMCs. The transfected PASMCs were cultured under hypoxia or normoxia conditions, respectively. The viability of the PASMCs was detected by CCK-8 assay. The protein expression of EGLN1 and vascular endothelial growth factor (VEGF) was determined by Western blot. RESULTS: The viability of the PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition in a time-dependent manner. In hypoxia or normoxia condition, the viability and VEGF protein expression of the PASMCs were suppressed by EGLN1 siRNA. CONCLUSION: EGLN1 gene may involve in the growth of rat PASMCs by regulating VEGF protein level under hypoxic condition.     

Role of caveolin-1 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of caveolin-1 in bone marrow mesenchymal stem cells (MSCs) differentiation into neurons. METHODS: The MSCs used in the experiment were divided into non-transfected group, transfected group (transfected with Rn-caveolin-1 siRNA), positive control group (transfected with Rn-MAPK-1 control siRNA) and negative control group (transfected with negative control siRNA). The MSCs were induced by β-ME to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The mRNA expression of caveolin-1 and MAPK-1 was detected by RT-PCR. NSE, NF-M and GFAP, the neural cell specific markers, were detected by immunocytochemistry staining. The survival ratio of MSCs was detected by MTT method. RESULTS: The fluorescence of MSCs was mostly displayed at 72th h after transfection and the efficiency of transfection was up to 81.5%±2.8%. Meanwhile, the mRNA expression of caveolin-1 in transfected MSCs was decreased (P<0.05). No significant difference of the survival MSCs ratio between transfected group and other groups was observed by MTT method. β-ME induced MSCs into neural cells. The differentiation efficiency of MSCs transfected with Rn-caveolin-1 siRNA was the highest and the expression of NSE and NF-M was increased significantly compared to the other groups (P<0.01). The expression of caveolin-1 was increased persistently with time and the highest expression was observed 6 d after induction. Furthermore, there was significant difference between before induction and 6 d after induction. CONCLUSION: Lipid raft labeled with careolin as marker protein has important role in regulating MSCs differentiation into neurons.