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.     

Lentivirus-mediated CCDC80 knock-out inhibits proliferation of ovarian cancer cells by decreasing Aib1 expression

AIM: To explore the role of coiled-coil domain-conaining protein 80 (CCDC80) gene deletion in the proliferation and apoptosis of human ovarian cancer ES-2 cells. METHODS: Lentivirus-mediated CCDC80 deletion in ovarian cancer cells was conducted by CRISPR/Cas9 method. Genomic sequencing was used to detect knock-out efficiency. The proliferation and colony formation of CCDC80 deletion cells were determined by cell growth curve and soft agar assay. The migration of CCDC80 deletion cells was measured by cell scratch assay. The apoptosis and cell cycle were analyzed by Annexin V/PI staining and flow cytometry. The protein levels of p-histone H3 and p-ERK1/2 were determined by Western blot. Nude mouse model was established to detect the tumorigenic capacity of CCDC80 deletion cells in vivo. RESULTS: Genomic sequencing results showed that CCDC80 was efficiently knocked out in ES-2 cells. CCDC80 deletion significantly repressed the proliferation, migration and colony formation of ES-2 cells (P<0.01).CCDC80 deletion increased the apoptosis rate and affected G¹ and S progression (P<0.01).CCDC80 deletion repressed the cell proliferation (P<0.01) in vivo. IHC results showed that CCDC80 deletion increased DNA damage and decreased cell proliferation. Western blot results showed that the protein level of p-histone H3 was decreased, while the protein level of p-ERK1/2 was increased in CCDC80 deletion cells (P<0.01). qPCR results showed that CCDC80 deletion significantly decreased Aib1 mRNA expression (P<0.01). CONCLUSION: Genetically CCDC80 deletion represses ES-2 cell proliferation, migration and colony formation, and promotes cell apoptosis by decreasing Aib1 expression.     

STC-1 mediates proliferation and apoptosis of gastric cancer cells through Bcl-2

AIM To investigate the effect of stanniocalcin-1 （STC-1） on the proliferation and apoptosis of gastric cancer AGS cells and the role of Bcl-2 in these processes. METHODS The AGS cells were transfected with the plasmids for STC-1 knockdown or over-expression. The cell proliferation was measured by MTT assay and colony formation assay. The migration ability was detected by scratch assay. Apoptosis was analyzed by Hoechst 33342 staining and flow cytometry with annexin V-FITC/PI double staining. The protein expression of Bcl-2, survivin, caspase-3 and cleared caspase-3 was determined by Western blot. The mRNA expression levels of STC-1 and Bcl-2 in 20 cases of clinical gastric cancer tissues and adjacent tissues were detected by RT-qPCR, and the correlation between them was analyzed by Pearson method. RESULTS After over-expression of STC-1, the proliferation and migration abilities of the AGS cells were increased, the expression of Bcl-2 and survivin was increased, while the expression of caspase-3 and cleared caspase-3 was decreased （P<0.05）. After knockdown of STC-1, the proliferation and migration abilities of the AGS cells were decreased, the expression of Bcl-2 and survivin was decreased, while the expression of caspase-3 and cleared caspase-3 was increased （P<0.05）. The mRNA expression levels of STC-1 and Bcl-2 in the gastric cancer tissues were higher than those in the adjacent tissues. Pearson correlation analysis showed that there was a positive correlation between STC-1 and Bcl-2 mRNA expression in the cancer tissues （r=0.308, P=0.011）. CONCLUSION STC-1 may regulate the biological function of gastric cancer cells by altering the expression level of Bcl-2.     

Effect of ARMCX1 on proliferation and apoptosis of cervical cancer SiHa cells

AIM: To observe the effect of Armadillo repeat-containing X-linked protein 1 (ARMCX1) on the proliferation and apoptosis of human cervical cancer SiHa cells by knock-down of ARMCX1 expression with small interfering RNA. METHODS: After knock-down of ARMCX1 expression, the cell cycle distribution and apoptosis of SiHa cells were detected by flow cytometry. The proliferation of SiHa cells was observed by plate colony formation assay after knock-down of ARMCX1 for 10 d. The protein levels of cell proliferation-and apoptosis-related molecules were determined by Western blot. RESULTS: After knock-down of ARMCX1 expression in the SiHa cells, the cell colony formation ability was significantly inhibited (P < 0.05), the cell cycle was arrested in S phase, and the protein levels of cyclin E and cell division cycle 25A (Cdc25A) in the SiHa cells were decreased. Meanwhile, knock-down of ARMCX1 expression promoted the apoptosis of SiHa cells, significantly reduced the protein expression of Bcl-2, and significantly increased the protein levels of Bax and active caspase-3 (P < 0.05). CONCLUSION: Knock-down of ARMCX1 expression inhibits the proliferation of SiHa cells and induces apoptosis.     

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 Rip2 overexpression on apoptosis in human pancreatic cancer cell line Panc-1

AIM: To observe the effect of receptor-interacting protein 2 (Rip2) overexpression on human pancreatic cancer cell line Panc-1. METHODS: pEGFP-C2 and pEGFP-Rip2 plasmids were respectively transfected into the Panc-1 cells using JetPRIME reagent. The cells were divided into control group, pEGFP-C2 group and pEGFP-Rip2 group. The apoptosis in the cells was detected 48 h after transfection by flow cytometry. Rip2 level and the expression of apoptosis-related proteins, Bax, cytoplasmic cytochrome c (Cyt-c) and Bcl-2, were analyzed by Western blot. The activity of caspase-3 was measured by colorimetric method. RESULTS: Rip2 protein expression significantly increased in the cells transfected with control and pEGFP-C2 plasmids. The apoptotic rate in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group, whereas no significant difference of apoptotic rate was observed between control group and pEGFP-C2 group. The protein expression of Bax and cytoplasmic Cyt-c was remarkably increased and the protein expression of Bcl-2 was obviously decreased in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group. The activity of caspase-3 in pEGFP-Rip2 group was obviously increased as compared with control group and pEGFP-C2 group. CONCLUSION: Overexpression of Rip2 is able to induce apoptosis in the Panc-1 cells, and the mechanism may be related to the up-regulation of Bax and cytoplasmic Cyt-c protein expression, down-regulation of Bcl-2 protein expression and enhancement of caspase-3 activity, thus activating intrinsic apoptotic pathway.     

Effect of biological clock gene Timeless silencing on apoptosis and invasion of ovarian cancer SKOV3 cells

AIM:To evaluate the effect of biological clock gene Timeless (TIM) silencing on the apoptosis and invasion ability of human ovarian cancer SKOV3 cells. METHODS:The protein expression of TIM in the ovarian cancer tissues and normal ovarian tissues was detected by immunohistochemistry, and the correlation between the protein expression of TIM in ovarian cancer tissues and the pathological features was analyzed. The ovarian cancer SKOV3 cells were transfected with PBS (blank control group), control siRNA (siRNA control group) or TIM siRNA (TIM siRNA group). The protein expression of TIM, Bcl-2, Bax, MMP-2, MMP-9, caspase-3 and caspase-9 was determined by Western blot. The apoptosis was detected by flow cytometry. The invasion ability was measured by Transwell chamber test. RESULTS:The positive expression rate of TIM in the ovarian cancer tissues (84.0%) was significantly higher than that in the normal ovarian tissues (10.0%; P<0.01). TIM expression was associated with ovarian cancer differentiation, depth of invasion, lymph node metastasis and TNM stage (P<0.05), but was not associated with age and pathological type (P>0.05). The protein expression levels of TIM, MMP-2, MMP-9 and Bcl-2 in TIM siRNA group were significantly decreased as compared with control group and siRNA control group (P<0.01), and the protein expression of Bax, caspase-3 and caspase-9 in TIM siRNA group was significantly increased as compared with blank control group and siRNA control group (P<0.01). No significant difference of the protein expression of TIM, MMP-2, MMP-9, Bcl-2, Bax, caspase-3 and caspase-9 between blank control group and siRNA control group was observed (P>0.05). The apoptotic rate in TIM siRNA group was significantly higher than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). The penetrated cell number in TIM siRNA group was significantly less than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). CONCLUSION:Silencing of TIM gene in ovarian cancer SKOV3 cells by siRNA promotes apoptosis, and inhibits cell invasion.     

Expression of miRNA-22 in ovarian cancer and effect of miRNA-22 over-expression on SKOV-3 ovarian cancer cell proliferation,migration and invasion

AIM: To examine the expression of miRNA-22 in the ovarian tissues and the effect of miRNA-22 over-expression on the proliferation, migration and invasion in SKOV-3 cells. METHODS: The expression levels of miRNA-22 in different ovarian tissues and SKOV-3 cells were determined by qPCR. miRNA-22 was over-expressed by transfection of miRNA-22 mimic. The cell viability was examined by CCK-8 assay. The cell migration was measured by wound healing test. The cell invasion was analyzed by Transwell assay. The protein expression levels of VEGF and P53 were determined by Western blot. RESULTS: Compared with the normal ovarian tissue, the expression level of miRNA-22 was remarkably decreased in the ovarian tumor tissues. After transfection with miRNA-22 mimic, the expression level of miRNA-22 in the SKOV-3 cells was significantly increased, while the cell viability, migration and invasion were obviously decreased. Moreover, the protein expression of VEGF and P53 was dramatically inhibited after over-expression of miRNA-22. CONCLUSION: The decreased miRNA-22 expression may be correlated with the development of ovarian can-cer. Over-expression of miRNA-22 decreases the cell viability, migration and invasion by reducing the protein expression of VEGF and P53.     

Effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45

AIM: To investigate the effects of CADM1 overexpression on proliferation and invasion of human gastric carcinoma cell line MKN-45. METHODS: The protein levels of CADM1 in 3 human gastric carcinoma cell lines were detected by Western blotting. Eukaryotic expression vector pcDNA-CADM1 was constructed and transfected into MKN-45 cells. The MKN-45 cells stably expressing CADM1 were selected by G418 and identified by Western blotting. Furthermore, CCK-8 assay and Boyden chamber were used to analyze the effects of CADM1 overexpression on the prolife ration and invasion of gastric carcinoma cells. Western blotting was also utilized to detect the levels of cell proliferation- and invasion-related proteins. RESULTS: Relative level of CADM1 protein in MKN-45 cells was significantly lower than that in MKN-28 cells and SGC-7901 cells. Additionally, eukaryotic expression vector pcDNA-CADM1 was successfully constructed and MKN-45 cells stably expressing CADM1 were obtained. Compared with non-treatment and pcDNA3.1 groups, the proliferation of MKN-45 cells was obviously inhibited in pcDNA-CADM1 group. The result of Boyden chamber showed that the migrated cell numbers in pcDNA-CADM1 group (52.35±3.89) were significantly lower than that in untreated group (101.53±6.89) and pcDNA3.1 group (98.77±7.03). Compared with non-treatment and pcDNA3.1 groups, the protein level of p21 was significantly up-regulated and protein expression of MMP-2 and MMP-9 was obviously down-regulated. CONCLUSION: Overexpression of CADM1 may markedly inhibit cell proliferation and reduce invasion ability, and thus may be a novel target for treating gastric carcinoma.     

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.     

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.     

Epigallocatechin gallate induces apoptosis of ovarian cancer cell line SKOV-3 via regulation of SIRT1-P53 pathways

AIM: To study the effect of epigallocatechin gallate (EGCG) on the growth and apoptosis of ova-rian cancer cell line SKOV-3 and its molecular mechanism. METHODS: SKOV-3 cells were treated with different concentrations of EGCG (0~50 μmol/L), SRT1720 (1 μmol/L) or EX527 (1 μmol/L) for 24 h. The cell activity was evaluated by CCK-8 assay. The apoptosis of SKOV-3 cells was analyzed by flow cytometry. The mRNA expression of Bcl-2 and Bax was detected by real-time PCR. SIRT1 deacetylase fluorometric assay kit was used to detect the activity of SIRT1. The protein levels of SIRT1 and acetylated P53 (Ac-P53) were determined by Western blot. RESULTS: EGCG or EX527 decreased the deacetylase activity and protein expression of SIRT1, and increased the level of Ac-P53 in a dose-dependent manner. Moreover, SRT1720 abrogated the effects of EGCG on the activity, apoptosis and SIRT1-P53 pathways in ovarian cancer cell line SKOV-3. CONCLUSION: EGCG inhibits the activity and induces apoptosis of ovarian cancer cell line SKOV-3 by regulating SIRT1-P53 pathways.     

Mitochondria associated endoplasmic reticulum membranes in cisplatin induced ovarian cancer cell apoptosis

AIM: To explore the structural change of mitochondria associated endoplasmic reticulum membranes (MAMs) in SKOV3 cells exposed to cisplatin. METHODS: The SKOV3 cells were treated with cisplatin at concentration of 6 mg/L. The protein levels of active caspase-3, as well as the colocalization of B-cell receptor-associated protein 31 (BAP31) and voltage-dependent anion channel protein 1 (VDAC1) in the SKOV3 cells were determined by the method of indirect immunofluorescence. The apoptotic rate of the SKOV3 cells was analyzed by flow cytometry. The structural change of MAMs was observed under transmission electron microscope. RESULTS: Under the confocal microscope, we found that cisplatin increased the protein levels of active caspase-3 as well as colocalization of BAP31 and VDAC1 in the SKOV3 cells. The results of flow cytometry demonstrated that cisplatin increased the apoptotic rate of the SKOV3 cells (P<0.05). The results of transmission electron microscopy showed that cisplatin induced increase in mitochondrial-associated membrane structures (P<0.05). CONCLUSION: Cisplatin induces SKOV3 cell apoptosis with increased MAMs contacts. MAMs may play a role in cisplatin induced SKOV3 cell apoptosis.     

Effect of gambogic acid on proliferation and apoptosis of human diffuse large B-cell lymphoma cell line OCI-LY-1

AIM: To investigate the effects of gambogic acid (GA) on the proliferation and apoptosis of human diffuse large B-cell lymphoma cells. METHODS: Diffuse large B-cell lymphoma cell line OCI-LY-19 was treated with GA. The cell viability and proliferation were evaluated by MTS assay. The morphological changes of the cells were observed under inverted microscope and fluorescence microscope with PI staining. The cell apoptosis was detected by flow cytometry. The changes of proteasome-related proteins Ubs, Bax, HSP90 and IκB-α, the apoptosis-related proteins PARP, pro-caspase-3 and caspase-8, and the proliferation-associated molecules ERK and STAT5 were detected by Western blotting. RESULTS: The proliferation of OCI-LY-19 cells was inhibited and the apoptosis was induced by GA. The expression of Ubs, Bax and HSP90 was up-regulated by GA treatment in a dose-dependent manner. The PARP protein was cleaved. The levels of apoptosis-related proteins pro-caspase-3 and pro-caspase-8, and proliferation-associated molecules p-Akt, p-ERK and p-STAT5 were down-regulated by GA treatment in a dose- and time-dependent manner. CONCLUSION: Gambogic acid down-regulates the activity of proteasome, inhibits cell proliferation and promotes cell apoptosis of diffuse large B cell lymphoma.     

Effects of shRNA targeting COL1A1 gene on proliferation and apoptosis of human breast cancer cell line MDA-MB-231

AIM: To investigate the effects of shRNA-mediated collagen type I alpha 1 (COL1A1) gene silencing on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231. METHODS: The specific recombinant vector pSilencer2.1-U6-COL1A1 was transiently transfected into human breast cancer cell line MDA-MB-231 with lipofectamine. RT-PCR and Western blotting were performed to detect the expression levels of COL1A1. MTT assay was employed to evaluate the effect of COL1A1 gene silencing on the cell proliferation. Flow cytometry was used to determine the apoptosis and cell cycle of transfected cells. The morphological characteristics of apoptosis were observed by Hoechst 33258 staining. RESULTS: Compared with mock group and scrambled group, the mRNA and protein levels of COL1A1 were reduced by pshRNA-COL1A1 transfection (P<0.05). The proliferation of MDA-MB-231 cells treated in shRNA-COL1A1 was significantly inhibited in a time-dependent way. The percentages of G₀/G₁ phase cells and early apoptotic rate were significantly higher in pshRNA-COL1A1 group than those in mock and scrambled group (P<0.05). The changes of apoptotic morphology such as cell shrinkage and nuclear condensation were also observed by staining with Hoechst 33258 under fluorescence microscope. CONCLUSION: Transfection of eukaryotic expression vector pshRNA-COL1A1 effectively inhibits the proliferation, induces apoptosis and arrests MDA-MB-231 cells in G0/G1 phase.     

Effect of quercetin on proliferation of human ovarian cancer SKOV-3 cells

AIM：To study the inhibitory effect of quercetin on the proliferation and apoptosis of human ovarian cancer SKOV-3 cells. METHODS：SKOV-3 cells were treated with different doses of quercetin. The inhibitory effect of quercetin on the proliferation was detected by MTT assay. The cell apoptosis was determined by immunocytochemistry. The cell cycle and apoptosis were assessed by flow cytometry. RESULTS：Quercetin inhibited the proliferation of ovarian cancer SKOV-3 cells in a time- and dose-dependent manner. Quercetin induced apoptosis of SKOV-3 cells. The proportion of S phase and the apoptotic rate were significantly increased, and the proportion of G2/M phase was reduced after treatment with quercetin. CONCLUSION：Quercetin inhibits the growth of ovarian cancer SKOV-3 cells in vitro, and promotes apoptosis through S phase arrest.     

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.     

High expression of BIRC5 enhances cell viability,inhibits apoptosis and is associated with poor prognosis in gastric cancer

AIM To investigate the expression of baculoviral inhibitor of apoptosis protein repeat-containing protein 5 （BIRC5） in gastric cancer tissue and its relationship with prognosis of gastric cancer patients, and to explore the effect of BIRC5 knock-down on the viability and apoptosis of gastric cancer cells. METHODS The expression of BIRC5 was detected by immunohistochemistry in 67 cases of gastric cancer tissues and paracancerous tissues for analyzing the relationships with clinicopathological characteristics. The mRNA and protein expression levels of BIRC5 in gastric carcinoma cell lines （AGS, MKN-1 and MGC-803） and normal gastric epithelial cell line GES-1 were detected by RT-qPCR and Western blot. The AGS cells were divided into blank group （no treatment）, Ctr-sh group （blank plasmid transfection） and BIRC5-sh group （BIRC5-shRNA plasmid transfection）. The interference efficiency of BIRC5-shRNA was evaluated by Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry, and the levels of apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. RESULTS BIRC5 was mainly expressed in cytoplasm, and the positive expression rate of BIRC5 in the gastric cancer tissues was higher than that in the adjacent tissues （P<0.01）. The positive rates of BIRC5 in the gastric cancer patients at TNM Ⅲ~Ⅳ stages and with lymph node metastasis were higher than those in the patients at TNM Ⅰ~Ⅱ stages and without lymph node metastasis, respectively （P<0.05）. The survival time of the patients with positive BIRC5 expression was shorter than that of the patients with negative BIRC5 expression （P=0.011 2）. The cell viability in BIRC5-sh group was lower than that in blank group and Ctr-sh group at time points of 48, 72 and 96 h. The apoptotic rate in BIRC5-sh group was increased compared with blank group and Ctr-sh group. The protein levels of cleaved caspase-3 and Bax in BIRC5-sh group were higher than those in blank group and Ctr-sh group, while the protein expression of Bcl-2 in BIRC5-sh group was lower than that in blank group and Ctr-sh group （P<0.05）. CONCLUSION High expression of BIRC5 in gastric cancer indicates poor prognosis. BIRC5 promotes the growth of gastric cancer cells and inhibits apoptosis.     

Knock-down of EZH2 expression promotes senescence of ovarian cancer cells

AIM: To investigate the molecular mechanism of down-regulated EZH2 expression promoting senescence of ovarian cancer cells. METHODS: Real- time PCR, Western blot and immunohistochemistry were used to detect the expression of EZH2 in ovarian cancer tissues, normal tissues, 4 ovarian cancer cell lines and IOSE80 cells. The ovarian cancer cells and IOSE80 cells were transfected with EZH2 siRNA (siEZH2) by Lipofectamine 2000 or treated with GSK126. Transfected IOSE80 cells were treated with ionizing radiation for 72 h, and negative control siRNA served as a control. The cell proliferation, apoptotic rate and senescence were detected by MTT assay, colony formation assay, flow cytometry and SA-β-Gal staining. The protein levels of EZH2, p53, p21, p16, caspase-3, cleaved caspase-3, PARP, cleaved PARP, H3K27me3, H3K27me2 and H3K27me1 were determined by Western blot. RESULTS: The EZH2 expression in the ovarian cancer tissues and ovarian cancer cells was significantly higher than that in the normal tissues and IOSE80 cells, respectively (P<0.01). siEZH2 significantly inhibited the proliferation of ovarian cancer cells, and promoted ionizing radiation-induced senescence. This effect was consistent with the cell phenotype after GSK126 treatment. Knock-down of EZH2 expression significantly inhibited the expression of H3K27me3, promoted the expression of p53, p21 and p16 (P<0.01), and had no effect on the protein levels of the key molecules in the apoptotic pathway. CONCLUSION: EZH2 is highly expressed in ovarian cancer tissues and ovarian cancer cells. Knock-down of EZH2 expression promotes the senescence of ovarian cancer cells via decrease in H3K27me3 level, thus inhibiting the proliferation of the cells.