Effects of silencing of COX-2 gene expression by siRNA on cell proliferation,cell cycles,apoptosis and tumorigenicity of human pancreatic cancer Capan-2 cells

AIM: To investigate the effects of silencing of cyclooxygenase-2 (COX-2) gene expression by siRNA on the proliferation, apoptosis, cell cycle and tumorigenicity of human pancreatic cancer Capan-2 cells.METHODS: The gene transfection was performed using Lipofectamine 2000 (Lipo). The proliferation, apoptosis and cell cycle of Capan-2 cells were tested by the methods of cell counting, microscopy and FCM. The mRNA expression of COX-2 was determined by RT-PCR and real-time PCR. The protein level of COX-2 was detected by Western blotting. The tumorigenicity of Capan-2 cells transfected with siRNA-COX-2 was determined using the model of nude mice. RESULTS: Transfection efficiency of 96.47% was obtained under the conditions that the transfection volume was 2 mL, concentration of Lipo was 5 μL and that of siRNA-COX-2 was 50 nmol/L. The best sequence of siRNA-COX-2 for silencing of COX-2 gene expression was siRNA006 with the silencing rate of up to 73% 24 h after tansfection. siRNA-COX-2 slowed down the growth of Capan-2 cells 48 h after transfection (P<0.05). At time points of 48 h and 72 h after transfection, the protein expression of COX-2 was down-regulated to 67% and 61% of the normal level, the proliferation inhibition rate was 35.48% and 56.32%, and the apoptotic rate was 2.03% and 3.27%, respectively. At time points of 24 h, 48 h and 72 h after transfection, the proportion of the cells in G₀/G₁ phrase was 58.03%, 63.31% and 65.66%, and that of the cells in S phase was 30.27%, 24.87% and 22.2%, respectively. The mean volume and weight of tumor tissues were remarkably decreased due to the transplantation of Capan-2 cells transfected with siRNA-COX-2.CONCLUSION: siRNA-COX-2 effectively silences the expression of COX-2 gene, inhibits the growth and decreases the tumorigenicity of Capan-2 cells.     

Effect of transketolase-like protein 1 on proliferation of human nasopharyngeal carcinoma cells

AIM: To investigate the effect of transketolase-like protein 1 (TKTL1) on proliferation of human nasopharyngeal carcinoma cells in vitro. METHODS: The siRNA against TKTL1 mRNA was constructed and transfected into human nasopharyngeal carcinoma cells (CNE cell line). The activity of transketolase was detected before and after RNA interference.Real-time PCR was used to determine the mRNA expression of transketolase (TKT) gene family in the CNE cells.Flow cytometry and MTT test were used to detect the effect of anti-TKTL1 siRNA on cell proliferation and cell cycle in the CNE cells. RESULTS: The total transketolase activity was significantly decreased in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector or untransfected CNE cells. No significant difference in the expression level of TKT and TKTL2 gene between the CNE cells transfected with siRNA TKTL1 construct and the cells transfected with control vector or untransfected CNE cells was observed (P>0.05). However, the expression level of TKTL1 gene was significantly downregulated in the CNE cells transfected with siRNA TKTL1 construct compared with the cells transfected with control vector.Cancer cells were arrested in G0/G1 phase, and cancer cell proliferation was significantly inhibited in the CNE cells transfected with siRNA TKTL1 construct. CONCLUSION: TKTL1 plays an important role in the total transketolase activity and cell proliferation of human nasopharyngeal carcinoma. TKTL1 may be considered as a potential target for novel anti-cancer therapy.     

Effects of siRNA targeting PCNA gene on nasopharyngeal carcinoma CNE2 cell cycle

AIM: To explore the effects of small interfering RNA (siRNA) targeting PCNA gene on nasopharyngeal carcinoma CNE2 cells growth and cycle.METHODS: Three synthesized siRNA targeting PCNA gene was transfected into CNE2 cells by using LipofectamineTM reagent. The PCNA mRNA and PCNA protein were detected by real-time polymerase chain reaction (RT-PCR) and immunohistochemical method. Inverted phase contrast microscope was used to determine the CEN2 cells growth before and after PCNA-siRNA transfected. Flow cytometry was used to observe the cell cycle.RESULTS: In CNE2 cells after PCNA-siRNA transfection, the expressions of PCNA mRNA and protein were down-regulated at different degree. Inhibition ratio of PCNA mRNA was 98.5%. Meanwhile, the cell cycle was suffocated at G0/G1 stage.CONCLUSION: The synthesized PCNA-siRNA effectively interferes nasopharyngeal carcinoma cells by down-regulating the expressions of the PCNA mRNA and its protein, therefore inhibits the growth of CNE2 cells. Future application of PCNA-siRNA in the gene therapy of nasopharyngeal carcinoma might be expected．     

Effects of inhibition of Cripto gene siRNA on vascular endothelial growth factor of colon cancer cell line LS-174T

AIM:To study the effects of Cripto gene on vascular endothelial growth factor (VEGF) of colon carcinoma cells.METHODS:Cripto siRNA was designed and constructed. Colon cancer LS-174T cells were divided into 4 groups: control group and different dose (3.125, 6.25 and 12.5 nmol/L) of siRNA groups. After transfected for 24, 48 and 72 h, colon cancer cells were harvested to carry on the next tests. Expression of Cripto mRNA was determined with real-time PCR, and immunofluorescence isothiocyanate (FITC) labeling assay and Northern blotting were performed to examine the expression of protein and mRNA of VEGF, respectively. The cells in control group and cells transfected with 12.5 nmol/L siRNA were inoculated into nude mice respectively. 30 days after inoculated, the mice of two groups were executed, and immunohistochemical (ICH) assay was used to evaluate the VEGF protein of mice tumor. RESULTS:siRNA down-regulated the Cripto mRNA in a dose and time dependent manner. Protein and mRNA of VEGF in transfected cells reduced in a dose and time dependent manner. Compared to control, the expression of VEGF protein from ICH assay was lowered significantly (P<0.05).CONCLUSION:Cripto gene might contribute to the regulation of angiogenesis of colon carcinoma. The down-regulation of Cripto gene by siRNA can suppress angiogenesis of human colon cancer.     

Effect of silencing hHBrk1 on proliferation and migration of lung carcinoma cells

AIM: To observe the effect of hHBrk1 gene on proliferation and migration of lung carcinoma cells. METHODS: Recombinant plasmids harboring 19-nt-long small interfering RNA (siRNA) were constructed and tested to selectively downregulate hHBrk1 gene in human lung cancer 95D cell line in vitro by stable transfection with Lipofectamine 2000. The mRNA level of the cells transfected with siRNA plasmids were monitored by Northern blotting and RT-PCR. Growth curve and flow cytometry were applied to determine the cell proliferation and cell cycle. Ability of cell migration was measured by Trans-well system. RESULTS: hHBrk1 gene was silenced by targeting siRNA, and stable silencing cell model was constructed. No difference in proliferation and clone formation between hHBrk1 silencing cells and control cells was observed. The ability of migration was decreased in hHBrk1 silencing cells as compared with control cells. CONCLUSION: hHBrk1 may play an important role in migration of the lung cancer cells.     

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.     

NAC-1 -specific siRNA enhances paelitaxel-induced apoptosis of ovarian cancer cell line HO8910

AIM: To observe the effect of NAC-1 -specific siRNA alone, or in combination with paelitaxel on proliferation and apoptosis of human ovarian cancer cell line HO8910. METHODS: Ovarian cancer cells were treated with NAC-1 siRNA alone or in combination with paelitaxel. The level of NAC-1 mRNA was assessed by real-time quantitative PCR. Western blotting analysis was used to detect NAC-1 protein and the activation of epidermal growth factor receptor(EGFR) downstream signals,Akt and ERK. The cell proliferation rate was measured by MTT assay, and the cell cycle and apoptosis were determined by flow cytometry. RESULTS: After treated with NAC-1 -specific siRNA for 48 h, the expression of NAC-1 at mRNA and protein levels in HO8910 cells decreased by 71.1% and 80.5%, respectively. The cells in G₁ phase increased. The protein levels of p-Akt and p-ERK were decreased by 43.7% and 49.8%, respectively. After treated with NAC-1 -specific siRNA for 72 h, the proliferation inhibitory rate of the cells was increased to 45.6% as compared with the cells treated with negative siRNA. Apoptotic rate of the cells treated with NAC-1 siRNA (0.5 μmol/L combined with 2 μmol/L of paelitaxel) for 72 h was (30.93±4.57)%,higher than that of the cells treated with paelitaxel alone[(23.85±3.65)%]. CONCLUSION: NAC-1 siRNA suppresses NAC-1 gene expression and EGFR downstream signaling activation, inhibits cell proliferation and enhances the responsiveness of ovarian cancer cells to paelitaxel. The combination treatment produces synergistic inhibition.     

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.     

Expression of SCD-1 in cervical carcinoma and effect of its down-regulation on proliferation and apoptosis of cervical carcinoma cells

AIM:To examine the expression of stearoyl-CoA desaturase-1 (SCD-1) in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki, and to investigate the effect of down-regulation of SCD-1 on the proliferation and apoptosis of cervical carcinoma cells. METHODS:The expression of SCD-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, and cervical carcinoma cell lines HeLa, SiHa and CaSki. SCD-1 siRNA and control siRNA were utilized to transfect CaSki cells by Lipofectamine 2000, and SCD-1 protein level was determined by Western blotting after transfection. Furthermore, CCK-8 and flow cytometry were utilized to investigate the changes of cell proliferation and apoptosis after transfection with SCD-1 siRNA in CaSki cells. Subsequently, the activities of caspase-3 and caspase-9 were analyzed by Caspase-Glo3/7 and 9 detection kit after transfection with SCD-1 siRNA in CaSki cells. Finally, the protein expression of Bcl-2 and Bax was detected by Western blotting. RESULTS:The protein expression of SCD-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues, and the protein expression of SCD-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which CaSki cells displayed the highest SCD-1 protein level. In addition, the protein expression of SCD-1 in SCD-1 siRNA group was significantly lower than that in untreated group and control siRNA group. Compared with untreated group and control siRNA group, the proliferation of CaSki cells was markedly inhibited in SCD-1 siRNA group. Early apoptotic rate in SCD-1 siRNA group was evidently higher than that in untreated group and control siRNA group. The activities of caspase-3 and caspase-9, and the level of Bax protein were significantly elevated, and the protein level of Bcl-2 was obviously reduced after transfection with SCD-1 siRNA in CaSki cells. CONCLUSION: SCD-1 may play an important role in the occurrence and development of cervical carcinoma, and its down-regulation, which mediates cell proliferation inhibition and apoptosis, may be tightly associated with the activities of caspase-3 and caspase-9, and the protein expression of Bcl-2 and Bax.     

Effect of microtubule-destabilizing protein stathmin gene silencing on proliferation and apoptosis of nasopharyngeal carcinoma cell line 5-8F

AIM: To investigate the effects of stathmin gene silencing on nasopharyngeal carcinoma cell line 5-8F. METHODS: Double-strand siRNA targeting to stathmin gene was obtained by chemical synthesis and annealing, and was sub-cloned into the vector pGenesil-1.1. The plasmid was introduced into 5-8F cells by liposome-mediated transfection. The gene expression of stathmin, and the proliferation, morphology and apoptosis of the cells were analyzed by Western blotting, MTT assay and flow cytometry. RESULTS: The cell suppression rate in stathmin gene silencing group was (53.01?1.12)%, significantly higher than that in transfection reagent group and in negative control group. The cell apoptotic rate in stathmin gene silencing group was (8.75?0.67)%, also significantly higher than that in transfection reagent group and in negative control group (P<0.05). CONCLUSION: Silencing of stathmin gene in nasopharyngeal carcinoma cells inhibits the cell proliferation and induces cell apoptosis.     

Effects of resveratrol on proliferation of ARPE-19 cells

AIM:To investigate the effects of resveratrol (Res) on the proliferation of ARPE-19 cells and to explore the possible mechanisms. METHODS:After ARPE-19 cells were treated with Res at concentrations of 0, 50, 100, 150, 200 and 300 μmol/L for 24 h, 48 h and 72 h, the effects of Res on the proliferation of the cells were tested by CCK-8 assay. The ARPE-19 cells were treated with Res at concentrations of 0, 100, 150 and 200 μmol/L for 48 h. The effects of Res on the cell cycle and apoptosis were detected by flow cytometry with Annexin V-FITC/PI staining. The protein expression of proliferating cell nuclear antigen (PCNA) was detected by immunofluorescent assay. The mRNA expression of PCNA, P21 and P27 was determined by real-time PCR. RESULTS: The results of CCK-8 assay showed that Res inhibited the proliferation of ARPE-19 cells in a time- and dose-dependent manner. The treatment with Res for 48 h resulted in an arrest of cell cycle at S phase without increasing cell apoptosis. Res inhibited the protein expression of PCNA in ARPE-19 cells. The results of real-time PCR showed that Res increased the mRNA expression of P21 and P27, and decreased the mRNA expression of PCNA. CONCLUSION: Res inhibits the proliferation of ARPE-19 cells and induces the cell cycle arrest at S phase. The mechanism may be related to up-regulation of P21 and P27, and down-regulation of PCNA.     

Effect of PTEN gene overexpression on cell cycle and tumor invasion in a human ovarian carcinoma SKOV3 cell line

AIM:To investigate the role of phosphatase and tensin homology deleted on chromosome(PTEN) gene in the cell cycle and invasion ability of human ovarian carcinoma SKOV3 cell line in vitro. METHODS:Human ovarian carcinoma SKOV3 cells were transfected with a eukaryotic expression plasmid vector containing PTEN gene in vitro,and then the positive cell clones were selected and amplified. MTT method was used to observe the inhibitory rate,flow cytometry was used to detect the cycle of transfected PTEN cells and apoptosis level. Western blotting analysis was used to determine PTEN gene expression. The invasiveness of transfected cells were measured quantitatively by Matrigel invasion assays (Transwell chamber). RESULTS:The expression of PTEN mRNA in SKOV3 cells increased after transfection with PTEN gene. Flow cytometry showed that the percentage of cells in S phase increased,but that in G₂/M phase decreased. Invasiveness of SKOV3 was significantly decreased. CONCLUSION:The transfection of PTEN gene into SKOV3 cells can inhibit human ovarian carcinoma SKOV3 cell proliferation,invasion and induce SKOV3 cell apoptosis.     

Inhibition of human hepatocellular carcinoma cell proliferation by galactose receptor mediated c-myc antisense oligonucleotide

AIM: To evaluate the inhibitory effect of galactose (Gal)-polyethyleneimine (PEI)-c-myc antisense oligodeoxynucleotide (ASODN) complex on proliferation of human hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma cell line Bel-7402 was treated with Gal-PEI-ASODN complex. Cell proliferation was tested by trypan blue dye at different time points and with various concentrations of ASODN treatment. Cell morphology was observed under inverted microscope, cell hypodiploid percentage was analyzed by flow cytometry and cell ultrastructure was observed through electron microscopy. RESULTS: Compared with ASODN group (20 μmol/L) from 0 h to 96 h, Gal-PEI-ASODN complex (with ASODN 0.75 μmol/L) significantly suppressed Bel-7402 cells proliferation, the ASODN concentration within Gal-PEI-ASODN complex and time course acquired were significantly lower and shorter, respectively. Incubated with pure ASODN at different concentrations for 72 hours, cell proliferation was inhibited and IC₅₀ was 20.9 μmol/L; while mediated with galactose receptor for 48 hours, ASODN significantly inhibited cell proliferation and IC₅₀ was only 0.294 μmol/L, the inhibitory efficacy of ASODN enhanced 70.9 folds. While Bel-7402 cells were incubated with Gal-PEI-ASODN complex for 48 hours, cell hypodiploid percentage was much higher than ASODN groups and cell apoptosis was seen under electron microscopy. CONCLUSIONS: Galactose receptor mediated ASODN delivery may significantly increase proliferation inhibition efficacy on Bel-7402 cells.     

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.     

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.     

Up-regulation of microRNA-187* inhibits proliferation of human colon cancer cell line HCT116

AIM:To investigate the expression of microRNA-187* (miR-187*) in human colon cancer cell lines and normal colon tissues, and to determine the effects of miR-187* up-regulation on the proliferation and cell cycle of human colon cancer cell line HCT116. METHODS:The expression profiling of miRNAs in 3 colorectal adenocarcinoma samples and their matched normal tissue samples was performed using miRNA microarray chip. Total RNA was isolated from 8 colon cancer cell lines and 10 normal colon tissues. The miR-187* level was detected by Taqman real-time RT-PCR. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1), the possible target of miR-187*, was also detected. Synthetic miR-187* mimics were transfected into HCT116 cell line by LipofectamineTM 2000. The mRNA expression of miR-187* and BMI-1 in HCT116 cell line was measured by real-time RT-PCR. Cell growth and cell cycle were assayed by MTS method and flow cytometry. RESULTS:miR-187* was found to be differentially expressed between colorectal adenocarcinoma and normal tissues. The expression of miR-187* in 8 colon cancer cell lines was down-regulated, while BMI-1 mRNA was up-regulated. Compared with blank control group, miR-187* expression was remarkably increased after transfection with miR-187* mimics, and ectopic expression of miR-187* significantly inhibited the mRNA expression of BMI-1. The cell growth was inhibited in miR-187* mimics group, and proliferating cell nuclear antigen (PCNA) mRNA expression was decreased. The cells at G2/M phase in miR-187* mimics group were significantly increased. CONCLUSION: miR-187* is down-regulated in human colon cancer cell lines. Up-regulation of miR-187* not only inhibits the proliferation but also influence the cell cycle of HCT116 cells, which might act as a tumor suppressor in colorectal cancer by inhibiting the expression of BMI-1.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Effect of abnormal expression of miR-141 on malignant biological behaviors of human hepatocarcinoma cells

AIM: To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells. METHODS: The RNA from SMMC-7721 cells and HL-7702 cells was extracted. SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine. After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. The changes of migration ability were investigated by Transwell invasion assay. RESULTS: The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells transfected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P < 0.05). The percentages of G₁ phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migration ability was inhibited (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P < 0.05). When miR-141 was down-regulated, the percentages of G₁ phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P < 0.05). CONCLUSION: miR-141 is down-regulated in human hepatocarcinoma cell line. Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells. miR-141 may function as a tumor suppressor gene during HCC development.     

Knockdown of RRM2 gene by siRNA inhibits human breast cancer cell proliferation,migration and tumor growth in nude mice

AIM: To explore the effect of ribonucleotide reductase M2 (RRM2) gene knockdown by siRNA on the proliferation and migration of human breast cancer MCF-7 cells and the tumor growth in BALB/c nude mice. METHODS: The mRNA and protein expression leves of RRM2 in human breast cancer cell line MCF-7 and human normal breast cell line MCF-10A were determined by real-time PCR and Western blotting. siRNA-RRM2 was constructed and transfected into MCF-7 cells at different time points and different concentrations. The silencing efficiency of RRM2 gene was detected by real-time PCR. The cell proliferation was measured by CCK-8 assay. The migration was observed using Transwell cell migration system. The effect of siRNA-RRM2 on the tumor growth was determined in nude mice. RESULTS: The mRNA and protein levels of RRM2 were higher in MCF-7 cells than those in MCF-10A cells. siRNA-RRM2 down-regulated the expression of RRM2 in MCF-7 cells in a time-and concentration-dependent manner. The results of CCK-8 assay showed that siRNA-RRM2 inhibited the proliferation ability of MCF-7 cells, but not that of MCF-10A cells. The results of Transwell assay indicated that siRNA-RRM2 inhibited the migration ability of MCF-7 cells. siRNA-RRM2 also inhibited the tumor growth in nude mice. CONCLUSION: RRM2 overexpression is associated with the breast cancer proliferation and migration. Suppression of RRM2 function is a potential therapeutic strategy for treating breast cancer.