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 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.     

Effect of miR-375 on viability,cell cycle and apoptosis of colon cancer HCT116 cells

AIM: To investigate the effect of microRNA-375 (miR-375) on the viability, cell cycle and apoptosis of HCT116 cells.METHODS: The expression of miR-375 in different colorectal cancer cell lines was detected by real-time PCR. The miR-375 mimics was transfected into HCT116 cells by Lipofectamine^TM 2000. The mRNA expression of miR-375 and AEG-1 was detected by real-time PCR. The HCT116 cell viability was detected by MTT assay. The changes of apoptosis and cell cycle distribution were analyzed by flow cytometry.RESULTS: Real-time PCR showed that miR-375 expression was the lowest in HCT116 among 4 colorectal cancer cell lines. The expression level of miR-375 significantly increased in miR-375 mimics group compared with that in the negative control group. The high expression level of miR-375 significantly inhibited the mRNA expression of AEG-1. After transfection with miR-375 mimics, the cell viability was inhibited, the apoptotic rate was increased, the proportion of G₁-stage cells was increased, and the proportion of S-stage cells was decreased.CONCLUSION: miR-375 inhibits the viability, mediates the cell cycle arrest and promotes the apoptosis of colon cancer HCT116 cells. miR-375 may act as a tumor suppressor in colorectal cancer by inhibiting AEG-1.     

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.     

Growth inhibition of colorectal cancer HCT116 cells by lincRNA-p21 through STAT3 signaling pathway

AIM: To study the effect of long intergenic non-coding RNA-p21 (lincRNA-p21) on the growth inhibition of colorectal cancer HCT116 cells via STAT3 signaling pathway. METHODS: The human colorectal cancer cell line HCT116 was used to construct the cells with over-expression of lincRNA-p21 by transfection of pcDNA-lincRNA-p21, and negative control cells were also set up. After transfection, the expression level of lincRNA-p21 was detected by RT-qPCR. The cell viability and proliferation were examined by MTT assay and plate colony formation assay, respectively. The protein levels of STAT3 and phosphorylated STAT3 (p-STAT3) were determined by Western blot. After STAT3 signaling pathway activator SD19 was used to treat the colorectal cancer HCT116 cells with over-expression of lincRNA-p21, Western blot was used to detect the protein levels of STAT3 and p-STAT3, MTT assay was used to measure the viability of the cells, and flow cytometry analysis was used to determine the cell apoptosis. RESULTS: Compared with control group and pcDNA group, the expression of lincRNA-p21 in pcDNA-lincRNA-p21 group was significantly up-regulated, the cell proliferation was inhibited, and the protein levels of STAT3 and p-STAT3 were significantly decreased (P<0.05). After treatment with STAT3 activator SD19, the protein levels of STAT3 and p-STAT3 in pcDNA-lincRNA-p21+SD19 group were higher than those in pcDNA-lincRNA-p21 group, the cell viability was increased, and the apoptotic rate was decreased significantly (P<0.05). CONCLUSION: Over-expression of lincRNA-p21 inhibits the growth of colorectal cancer HCT116 cells. STAT3 signaling pathway activator abolishes the growth inhibitory effect of lincRNA-p21 over-expression. lincRNA-p21 inhibits the growth of colorectal cancer cells by inhibiting the activation of STAT3 signaling.     

Effect of DEC1 gene over-expression on proliferation and invasion abilities of human esophageal cancer ECA109 cells

AIM: To investigate the effect of DEC1 gene over-expression on the proliferation and invasion abilities of human esophageal cancer ECA109 cells.METHODS: ECA109 cells were transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group). The mRNA and protein levels of DEC1, cyclin D1 and MMP-9 were evaluated by real-time PCR and Western blot, respectively. The effects of DEC1 over-expression on the proliferation and invasion abilities of the ECA109 cells were evaluated by CCK-8 assay, colony formation assay and Transwell test respectively.RESULTS: The DEC1 expression level in ECA109 cells in DEC1 group was significantly higher than that in vector group (P<0.01), but the levels of MMP9 and cyclin D1 expression were opposite (P<0.01). However, both the proliferation and invasion abilities of ECA109 cells in DEC1 groups decreased significantly as compared with those in vector group (P<0.05).CONCLUSION: The over-expression of DEC1 significantly inhibits the proliferation and invasion of ECA109 cells, which may be involved in the expression of cyclin D1 and MMP9.     

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.     

Effect of CIP2A over-expression on proliferation and apoptosis of gastric epithelial cells

AIM: To investigate the effects of cancerous inhibitor of protein phosphatase 2A (CIP2A) over-expression on the proliferation and apoptosis of gastric epithelial cells.METHODS: The mRNA expression of CIP2A and cyclin D1 in the tissues of normal gastric mucosa and gastric polyps was detected by RT-qPCR. The GES-1 cells were divided into control group, Ad-emp group and Ad-CIP2A group. The cell proliferation ability was detected by MTT assay and BrdU assay, and the cell apoptosis was analyzed by flow cytometry. The expression of apoptosis-related molecules was determined by Western blot and RT-qPCR. The levels of inflammatory cytokines were measured by ELISA after GES-1 cells were infected with Ad-emp and Ad-CIP2A. Furthermore, the protein levels of p-Rb, E2F1 and cyclin D1 in the GES-1 cells was determined by Western blot after transfected with CIP2A siRNA.RESULTS: The expression of CIP2A and cyclin D1 in adenomatous gastric polyps tissues was significantly higher than that in normal gastric mucosa tissues, and no significant change of that between hyperplastic gastric polyps tissues and normal gastric mucosa was observed. After transfected with CIP2A, the proliferation ability of GES-1 cells was increased, the cell apoptosis was inhibited, the concentrations of IL-1β and IL-10 was up-regulated and the protein levels of p-Rb, E2F1 and cyclin D1 were increased, while the protein levels of p-Rb, E2F1 and cyclin D1 were significantly decreased after transfected with CIP2A siRNA.CONCLUSION: CIP2A promotes the proliferation and inhibits the apoptosis of GES-1 cells by activating Rb/E2F1.     

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.     

FOXQ1 gene mediate angiogenesis and proliferation induced by Shh in SW480 cells

AIM: To explore the effect of FOXQ1 gene silencing on angiogenesis and proliferation ability of colon cancer cells induced by Sonic hedgehog(Shh). METHODS: Lentivirus expressing different FOXQ1-shRNA or negative cantrol(NC)-shRNA was used to infect the SW480 cells. The best silencing condition was screened and used in the following experiments. The SW480 cells were divided into interfered group(FOXQ1-shRNA) and control group(NC-shRNA). The MTT assay was used to observe the doubling time and cell activity. Tube formation assay was performed to detect the ability of angiogenesis. Meanwhile, the expression of vascular endothelial growth factor(VEGF)-A, matrix metalloproteinase(MMP) 2 and cyclin D1 at mRNA and protein levels was determined by real-time PCR and Western blot. After induction of the cells by recombinant Shh proteins, the changes of angiogenesis and proliferation ability in each group were detected. At the same time, the transformation of related gene was examined. RESULTS: Compared with control group, the angiogenic ability in interfered group was decreased, and no obvious difference of proliferation ability was observed. The expression of VEGF-A and MMP2 was declined significantly, and the expression of cyclin D1 was not obviously changed. Recombinant Shh proteins improved the expression of FOXQ1 gene. Compared with NC-shRNA group, after induction, the angiogenic ability of FOXQ1-shRNA group was decreased, and the proliferation ability was not obviously changed. CONCLUSION: FOXQ1 gene mediates the angiogenic ability but does not affect the proliferation ability of SW480 cells. Meanwhile, it may be regulated by shh pathway.     

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.     

Effects of SphK1 and FAK on epithelial-mesenchymal transition in colon cancer HCT116 cells

AIM: To investigate the effects of sphingosine kinase l(SphK1) and focal adhesion kinase(FAK) on the epithelial-mesenchymal transition(EMT) of human colon cancer HCT116 cells. METHODS: Human colon cancer HCT116 cells were divided into 3 groups. N, N-dimethylsphingosine(DMS) was used to suppress the activity of SphK1. PF573228 was used to suppress the activation of FAK. The cells treated with equal volume of culture medium severed as control group. The cell viability was measured by MTT assay. The protein expression of SphK1, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and matrix metalloproteinase(MMP) 2 was analyzed by Western blot. The mRNA expression of SphK1, sphingosine-1-phosphate(S1P), FAK, E-cadherin and vimentin was detected by real-time PCR. The ability of tumor cell migration was measured by wound-healing assay. RESULTS: The cell viability of HCT116 cells was suppressed by DMS and PF573228 in dose and time dependent manners. DMS significantly suppressed the expression of SphK1, FAK, N-cadherin, vimentin and MMP2, meanwhile enhanced the expression of E-cadherin. PF573228 reduced the expression of FAK, SphK1, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin(P<0.01). In addition, the migration ability of HCT116 cells was significantly decreased by treating with DMS and PF573228(P<0.01). Compared with control group, the mRNA expression of FAK, SphK1, S1P and vimentin was decreased, while the expression of E-cadherin was increased significantly in PF573228 group and DMS group(P<0.05). CONCLUSION: SphK1 and FAK signaling pathways may play an important role in the occurrence of EMT in the colon cancer HCT116 cells.     

Effect of 3 isoforms of Ikaros on proliferation of human ovarian cancer SKOV3 cells

AIM: To investigate the effect of Ikaros isoforms on the proliferation of human ovarian cancer SKOV3 cells. METHODS: Three isoforms of Ikaros, IK1, IK2 and IK6, were transfected into ovarian cancer SKOV3 cells. CCK-8 assay and cell counting were used to detect the effects of Ikaros isoforms on the proliferation of SKOV3 cells. The cell cycle was analyzed by flow cytometry. The cell cycle-related proteins were detected by Western blot. RESULTS: IK1 and IK2 expression inhibited SKOV3 cells proliferation. Flow cytometry analysis indicated that IK1 and IK2 induced SKOV3 cell cycle arrest at the G₁ phase. IK6 isoform exerted no obvious effect on the proliferation or cell cycle of SKOV3 cells. Compared with control EV group, IK1 group and IK2 group showed a dramatic elevation in the expression of the cell cycle inhibitor p21, along with a substantial decrease in the expression of the cell cycle inducers cyclin D1 and cyclin D2, which did not change in IK6 group. CONCLUSION: IK1 and IK2 significantly inhibit the proliferation of ovarian cancer SKOV3 cells and induce cell cycle arrest at G₁ phase by regulation of cell cycle-related proteins cyclin D1, cyclin D2 and p21, while IK6 isoform exerts no obvious effect on the proliferation and cell cycle of SKOV3 cells.     

Emodin inhibits proliferation and blocks cell cycle of human breast cancer MCF-7 cells

AIM：To investigate the effects of emodin on the proliferation of human breast cancer MCF-7 cells and its mechanisms. METHODS：MTT assay was used to observe the viability of MCF-7 cells. The cell cycle distribution and apoptosis of MCF-7 cells was analyzed by flow cytometry. The membrane surface morphology and three-dimensional ultrastructure of MCF-7 cells were observed under atomic force microscope (AFM). RESULTS：MTT assay showed that emodin could inhibit MCF-7 cell proliferation in a dose-dependent manner. Flow cytometric analysis demonstrated that emodin induced cell cycle arrest at G0/G1 phase. Annexin V/PI double staining confirmed that emodin had no effect on cell apoptosis. AFM images revealed that the cell nuclear area was full and the surface of cell membrane was flat and smooth in control group. Compared with control group, the cell nuclear area collapsed and shrank in emodin group at 48 h. The cell membrane ultrastructure showed that the particles in emo-din group had an intensive distribution. The height of cell nuclear area was decreased, and the surface average roughness (Ra) and root mean square roughness (Rq) were elevated in emo-din group compared with control group. CONCLUSION: Emodin has cytotoxicity on MCF-7 cells via cell cycle arrest at G0/G1 phase and ultrastructural changes.