Expression of TSTA3 in esophageal squamous-cell carcinoma and its significance

AIM To investigate the expression of TSTA3 in esophageal squamous cell carcinoma (ESCC) and the effects of TSTA3 on the proliferation, invasion and migration abilities of human ESCC cell lines KYSE150 and KYSE450. METHODS The expression of TSTA3 was detected by immunohistochemistry in 45 cases of ESCC and matched adjacent tissues. The mRNA expression levels of TSTA3 in ESCC cells were detected by qPCR. Over-expression of TSTA3 in KYSE150 cells and KYSE450 cells was carried out by lentivirus infection. The effects of TSTA3 on the viability and colony formation ability of ESCC cells were examined by MTT assay and colony formation assay. Transwell assays were performed to detect the effects of TSTA3 on migration and invasion abilities of ESCC cells. The effect of TSTA3 on core fucosylation modification of ESCC cells was analyzed by flow cytometry. RESULTS The expression of TSTA3 in the ESCC tissues was significantly higher than that in matched adjacent tissues (P<0.01). Over-expression of TSTA3 had no effect on the proliferation of ESCC cells (P>0.05), but promoted the migration and invasion of ESCC cells (P<0.01). Moreover, over-expression of TSTA3 increased the core fucosylation modification level of ESCC cells (P<0.01). CONCLUSION TSTA3 may promote the development and progression of ESCC via regualting fucosylation modification level, and serve as a biomarker for early diagnosis and treatment of ESCC.     

Effect of BMP9 over-expression on migration and invasion of human lung squamous-cell carcinoma NCI-H520 cells

AIM: To investigate the effect of bone morphogenetic proteins 9(BMP9) on the migration and invasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism. METHODS: The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial (HBE) cells was detected by RT-PCR and Western blot. The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expression of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot. The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays. The mRNA and protein levels of the migration-related factor matrix metalloproteinase 2(MMP2) were detected by RT-PCR and Western blot. The level of phosphorylated Smad1/5(p-Smad1/5) was detected by Western blot. Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9. The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays. RESULTS: The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells. After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly decreased, and the mRNA and protein levels of MMP2 were decreased. Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability. CONCLUSION: Over-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells. The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.     

Effect of long non-coding RNA MEG3 on invasion and migration of colorectal cancer cells

AIM: To investigate the expression of long non-coding RNA maternally expressed gene 3(MEG3) in colorectal cancer(CRC) cells, and to observe the effect of MEG3 on the invasion and migration of CRC cells. METHODS: The levels of MEG3 in human normal colon cell NCM460 and CRC cells SW48 and LoVo were detected by real- time PCR. MEG3 was over-expressed by plasmid transfection, and the effects of MEG3 on the invasion and migration of SW48 and LoVo cells were analyzed by Transwell assay and wound healing assay. The expression of matrix metalloproteinase(MMP) family proteins was determined by Western blotting. RESULTS: The level of MEG3 was down-regulated in CRC cells compared with normal colon cell NCM460. The invasion and migration of CRC cells were reduced after MEG3 over-expression. Transwell invasion and migration assays showed that the numbers of transmembrane SW48 and LoVo cells were smaller in MEG3 over-expression group than control group(CONCLUSION: The expression of MEG3 is down-regulated in CRC cells. Over-expression of MEG3 inhibits the invasion and migration of CRC cells. TIMP-2, MMP-2 and MMP-9 might play an important role in this regulation.     

Progress on genomic aberrations in esophageal squamous-cell carcinoma

Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies with poor prognosis in China. Since most clinical confirmation of the patients with ESCC is diagnosed at an advanced stage or with lymph node metastasis, the treatment effect was very poor. With the applications of high-throughput microarray and whole-genome sequencing or whole-exome sequencing, several novel cancer-related genes have been identified and revealed, and these genes may become new biomarkers or therapeutic targets. This review is to summarize the research progress in ESCC genome reported recently.     

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.     

Effects of miRNA-25 on proliferation of human esophageal squamous-cell carcinoma cell line TE1

AIM: To investigate the effects and mechanisms of microRNA-25(miRNA-25) on the proliferation of human esophageal squamous-cell carcinoma cell line TE1. METHODS: The abundance of miRNA-25 in different tissues was measured by RT-PCR. After silencing or over-expression of miRNA-25 with mimics or inhibitor in TE1 cells, the cell proliferation, cell cycle distribution and the expression of cyclin E1 and cyclin-dependent kinase 2(CDK2) at mRNA and protein levels were measured by CCK-8 assay, BrdU detection, flow cytometry, RT-PCR and Western blot, respectively. RESULTS: miRNA-25 was prominent in esophageal mucosal tissue and highly expressed in TE1 cells (P<0.05). Over-expression of miRNA-25 increased TE1 cell proliferation, promoted the cell cycle progression and enhanced the entrance of the cells into S phase (P<0.05). Inverse results were obtained after down-regulation of miRNA-25(P<0.05). Furthermore, the expression of cyclin E1 and CDK2 at mRNA and protein levels was significantly increased after over-expression of miRNA-25, but decreased after down-regulation of miRNA-25(P<0.05). CONCLUSION: miRNA-25 enhances cell cycle transition by increasing the expression of cyclin E1 and CDK2, thus accelerating TE1 cell proliferation. This study provides a novel mechanism by which miRNA-25 increases the proliferation of human esophageal squamous-cell carcinoma cell line TE1, suggesting that down-regulation of miRNA-25 may be a potential new therapeutic strategy for treating esophageal squamous-cell carcinoma.     

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.     

Long non-coding RNA TTTY15 expression in osteosarcoma and its effect on viability and invasion ability of osteosarcoma cells

AIM:To observe the expression of long noncoding RNA TTTY15 in osteosarcoma tissues and cell lines and to explore its effect on the viability and invasion ability of osteosarcoma cell lines. METHODS:qPCR was used to detect the expression of TTTY15 in 11 cases of osteosarcoma and its adjacent tissues. The mRNA levels of TTTY15 in osteosarcoma cell lines (143B, Saos2, MG-63, U2OS and HOS) and human osteoblast cell line hFOB1.19 were also tested. TTTY15 was down-regulated after transfected with small interfering RNA in MG-63 cells, the cell line with the highest level of TTTY15. The effect of TTTY15 knockdown on the viability of MG-63 cells was measured by CCK-8 assay. The cell cycle distribution was analyzed by flow cytometry. The effect of TTTY15 knockdown on the cell invasion ability was detected by Transwell assay. The levels of miR-216b-5p and FOXM1 mRNA were detected by qPCR, and the changes of the related proteins were determined by Western blot. RESULTS:Compared with the adjacent tissues, the expression of TTTY15 increased in the osteosarcoma tissues (P<0.01). Compared with the human osteoblast cell line, the expression of TTTY15 increased in the osteosarcoma cell lines (P<0.05), and the level of TTTY15 in the MG-63 cells was the highest (P<0.01). After knockdown of TTTY15 expression in the MG-63 cells, the cell viability was decreased (P<0.05), cell cycle progression was inhibited (P<0.01), and the cell invasion ability was decreased (P<0.01). The expression of miR-216b-5p was increased (P<0.01) and the expression of FOXM1 mRNA was decreased (P<0.01). The protein expression of FOXM1, CDK4, cyclin D1, MMP-2 and N-cadherin was decreased, while the protein expression of E-cadherin was increased (P<0.05). CONCLUSION:The expression of TTTY15 is increased in the osteosarcoma tissues and cell lines. The low expression of TTTY15 inhibits the cell viability and invasion ability of osteosarcoma cells. The possible mechanism is that the knockdown of TTTY15 expression results in the increase in miR-216b-5p expression and the down-regulation of FOXM1 expression.     

Effects of siRNA-mediated nestin silencing on invasion and migration of human esophageal cancer ECA109 cells

AIM: To investigate the effect of small interference RNA(siRNA)-mediated silencing of nestin gene on the invasion and migration of human esophageal cancer ECA109 cells and the possible mechanism. METHODS: The esophageal cell line ECA109 was transfected with siRNA targeting nestin and the cell invasion and migration abilities were observed. The expression of nestin, MMP2, MMP9, VEGF, and total and nuclear β-catenin proteins in the transfec-ted cells were determined by real-time PCR and Western blot. RESULTS: Compared with control group, the expression of nestin at mRNA and protein levels was significantly down-regulated in the ECA109 cells transfected with nestin-siRNA, so was the expression of MMP2, MMP9, VEGF, and total and nuclear β-catenin proteins. The levels of invasion and migration capacities of ECA109 cells transfected with nestin-siRNA were lower than those in the cells transfected with control-siRNA. CONCLUSION: Knockdown of nestin expression significantly inhibits the invasion and migration of the esophageal cancer cells, which may act via suppressing β-catenin translocation to the nucleus and influencing the expression of MMP2, MMP9 and VEGF.     

Reduced expression of SIRT2 in serous ovarian carcinoma promotes cell proliferation,migration and invasion

AIM: To compare the expression of SIRT2 in ovarian surface epithelial (OSE) cell line and serous ovarian carcinoma (SOC) cell lines, and to investigate the effects of SIRT2 on the cell proliferation, migration and invasion. METHODS: The expression levels of SIRT2 in the OSE cell line and the SOC cell lines were determined by Western blot. The SIRT2 siRNAs and overexpression construct were designed and verified. Transient transfection of SIRT2 siRNAs or overexpression construct was performed, and the effect of SIRT2 on the cell proliferation, migration and invasion was evaluated. RESULTS: SIRT2 levels in the 5 strains of SOC cell lines were significantly lower than that in the OSE cell line. SIRT2 knockdown in HOSEpiC cells significantly enhanced the ability of cell colony formation and accelerated the cell growth rate. On the contrary, overexpression of SIRT2 in HO8910 cells dramatically repressed the number of cell colonies and cell activity. SIRT2 significantly changed the ability of ovarian cell migration. Knockdown of SIRT2 facilitated the cell invasion. CONCLUSION: The expression of SIRT2 in the SOC cells is significantly down-regulated. In the OSE cells, SIRT2 acts as a tumor suppressor and mediates the inhibition of cell proliferation, migration and invasion.     

Effect of wogonin on OSCC cell growth and invasion

AIM: To investigate the molecular mechanism of wogonin on the growth and invasion of oral squamous-cell carcinoma (OSCC) cell line SCC-4. METHODS: After treatment with various doses (0, 20, 40, 60, 80 and 100 mg/L) of wogonin for the indicated time, MTT assay was used to evaluate cell viability. The cell apoptosis was detected by flow cytometry with Annexin V and propidium iodide (Annexin V/PI) double staining. The cell invasion ability was analyzed by Transwell assay. The activation of Wnt/β-catenin signaling molecules was assessed by Western blot. RESULTS: Wogonin inhibited the viability and invasion of SCC-4 cells but promoted cell apoptosis in a dose-and time-dependent manner. Wogonin treatment obviously decreased the activation of β-catenin. Moreover, the expression of downstream targets cyclin D1, matrix metalloproteinase-2 (MMP-2) and MMP-9 were obviously down-regulated, accompanied by the up-regulation of anti-apoptotic protein Bcl-2. Wnt/β-catenin activator LiCl remarkably attenuated the inhibitory effect of wogonin on the activation of Wnt/β-catenin signaling molecules. Importantly, the inhibition of cell growth and invasion ability by wogonin treatment was dramatically attenuated after LiCl exposure. CONCLUSION: Wogonin blocks SCC-4 cell growth and invasion mainly by regulating Wnt/β-catenin signaling, indicating that it is a potential suppressor for OSCC and may be a potential target for the development of anti-OSCC therapy.     

Long non-coding RNA HOTAIR modulates proliferation and apoptosis of acute lymphoblastic leukemia cells

AIM: To explore the influence of long non-coding RNA HOTAIR on the proliferation and apoptosis of acute lymphoblastic leukemia cells via the regulation of glucocorticoid receptor (GR).METHODS: The expression le-vels of HOTAIR and GR mRNA in human bone marrow stromal cell line HS-5 and human acute lymphoblastic leukemia cell lines MOLT-4, CCRF-CEM and CEM-C1 were examined by RT-qPCR. HOTAIR was knocked down by siRNA in acute lymphoblastic leukemia cells. CCK-8 assay was used to assess the cell viability, and the effect of si-HOTAIR on the proli-feration of CEM-C1 cells was evaluated by BrdU method. The effect of si-HOTAIR on apoptosis of CEM-C1 cells was examined by Hoechst 33342 staining and Caspase-Glo^® 3/7 assay. Western blot was utilized to examine the protein level of GR.RESULTS: The expression level of HOTAIR in acute lymphoblastic leukemia cells was significantly increased as compared with normal human bone marrow stromal cells (P<0.01). The viability and proliferation of acute lymphoblastic cells was inhibited, the apoptosis was induced, and the anti-proliferation effect of dexamethasone on CEM-C1 cells was enhanced after knockdown of HOTAIR expression (P<0.01). The expression of GR was up-regulated at both mRNA and protein levels (P<0.01).CONCLUSION: Long non-coding RNA HOTAIR may modulate the viability, proliferation and apoptosis of acute lymphoblastic leukemia cells via a GR regulatory way.     

Effects of oridonin on invasion and migration of human hepatocellular carcinoma MHCC-97H cells

AIM: To investigate the effects of oridonin on the invasion and migration of hepatocelluar carcinoma cells. METHODS: Human hepatocelluar carcinoma MHCC-97H cells were cultured and treated with 5, 10 or 20 μmol/L oridonin. The migration ability was measured by wound healing assay. The invasion ability was examined by Transwell invasion assay. The adhesion capabilities were evaluated by adhesion assay. The protein levels of LIM kinase-1 (LIMK-1), cofilin and phosphorylated cofilin (p-cofilin) were determined by Western blot. RESULTS: Oridonin inhibited the migration, invasion and adhesion abilities of MHCC-97H cells in a dose-dependent manner (P<0.05). After oridonin treatment, the expression of cofilin had no obvious change, but the protein levels of LIMK-1 and p-cofilin decreased significantly. CONCLUSION: Oridonin inhibits the invasion and migration of MHCC-97H cells. The mechanism may be related with the regulatory effect of oridonin on LIMK-1/cofilin signal transduction pathway.     

Calreticulin promotes migration and invasion of nasopharyngeal carcinoma cells by inducing cell EMT

AIM:To observe the expression of calreticulin (CRT) in nasopharyngeal carcinoma tissues, analyze the significance of clinical pathology and the influence on epithelial-mesencymal transition (EMT) of CNE2 cells. METHODS:The expression of calreticulin was detected by immunohistochemistry in 52 nasopharyngeal carcinoma and 57 nasopharyngeal benign tissues, and the significance of clinical pathology was evaluated. The calreticulin gene-specific small interfering RNA was constructed, and then was transfected into the NPC cell line CNE2 using the cationic liposome method. The effect of CRT on the morphological changes of the CNE2 cells was observed under light microscope. The effect of CRT on the cell migration and invasion abilities of the CNE2 cells was detected by Transwell migration and invasion assays. The expression of EMT-related proteins E-cadherin, vimentin, transforming growth factor (TGF)-β and matrix metalloproteinase (MMP)-9 in the CNE2 cells was determined by Western blot. RESULTS:The positive expression rate of CRT in the benign lesion tissues was 19.29% (11/57), which was significantly increased in the nasopharyngeal carcinoma tissues as 82.69% (43/52). The expression rate of CRT was positively correlated with the stage of nasopharyngeal carcinoma and lymph node metastasis (P<0.05). Knockdown of CRT expression made the CNE2 cells showing a spindle shape to a flat, cobblestone-like epithelial state change, arranged more compact, and the migration and invasion abilities were significantly decreased (P<0.05). Knockdown of CRT expression resulted in significant increase in the protein expression of E-cadhe-rin, and the decreases in the protein expression of vimentin, TGF-β and MMP-9 in the CNE2 cells (P<0.05). CONCLUSION:Calreticulin expression in nasopharyngeal carcinoma is significantly higher and positively correlated with nasopharyngeal carcinoma stage and lymph node metastasis. Calreticulin promotes cell migration and invasion of nasopharyngeal carcinoma CNE2 cells by inducing EMT.     

Effect of sirtuin 6 on proliferation of hepatocellular carcinoma cells

AIM: To illuminate the effect of sirtuin 6 (SIRT6) on the proliferation of hepatocellular carcinoma (HCC) cells. METHODS: The mRNA expression of SIRT6 in the peripheral blood from 200 cases of HCC patients and 50 cases of healthy people was detected by real-time quantitative PCR (RT-qPCR). The mRNA expression levels of SIRT6 in the peripheral blood from 200 cases of HCC patients were combined with multiple clinicopathologic parameters for statistical analysis. The protein expression of SIRT6 in primary hepatocytes, immortalized hepatocytes and 4 hepatoma cell lines were determined by Western blotting. The silencing of SIRT6 was conducted by transfection of vector expressing short hairpin RNA targeting on SIRT6, and the protein level of SIRT6 was measured by Western blotting. The viability of HCC cells was tested by MTS assay. DNA synthesis was analyzed by Cell-Light^TM EdU Apollo® 488 In Vitro Imaging Kit. The abilities of colony formation and anchorage-dependent growth were measured by colony formation assay and soft agar assay, respectively. RESULTS: The mRNA expression of SIRT6 in the peripheral blood of HCC patients was significantly higher than that in the healthy people, and its expression was highly associated with tumor size, tumor grade and vascular invasion. SIRT6 expression in 4 hepatoma cell lines was significantly higher than that in the others. SIRT6 silencing led to a significant decrease in the cell viability as tested by MTS assay. EdU staining revealed that SIRT6 silencing reduced DNA synthesis. SIRT6 silencing reduced the ability of colony formation and anchorage-dependent growth as determined by colony formation assay and soft agar assay, respectively. CONCLUSION: Sirtuin 6 promotes the proliferation and malignant transformation of HCC cells.     

Long non-coding RNA PVT1 regulates effect of miR-551 on migration and invasion abilities of ovarian cancer through Wnt signaling pathway

AIM: To investigate the expression of long non-coding RNA PVT1 in ovarian cancer and the role of PVT1 in migration and invasion abilities of ovarian cancer cells.METHODS: The expression of PVT1 in ovarian cancer tissue, normal ovarian tissue and different ovarian cancer cell lines was detected by qPCR. Transwell assay was used to detect the invasion ability of ovarian cancer cells after PVT1 silencing. The migration ability of the ovarian cancer cells after PVT1 silencing was detected by scratch test. The interaction between PVT1 and microRNA (miR)-551 was analyzed by dual-luciferase reporter assay. The effect of miR-551-inhibitor on the invasion and migration abilities of ovarian cancer cells after PVT1 silencing was detected by Transwell assay and scratch test. The expression of Wnt signaling pathway-related proteins was determined by Western blot after PVT1 silencing. The effects of PVT1 silencing on tumor weight and volume of ovarian cancer were examined by subcutaneous tumor transplantation in nude mice.RESULTS: The expression of PVT1 in ovarian cancer tissue was significantly higher than that in normal ovarian tissue (P<0.05). The expression level of PVT1 in ovarian cancer cell line ES-2 was the highest. PVT1 silencing inhibited the invasion and migration abilities of the ovarian cancer cells. After PVT1 silencing, miR-551-inhibitor promoted the invasion and migration abilities of the ovarian cancer cells. The expression of Wnt signaling pathway-related proteins was decreased after PVT1 silencing (P<0.05). Compared with negative control group, the tumor volume and weight in PVT1-siRNA group were significantly decreased (P<0.05).CONCLUSION: PVT1 plays an important role in the development of ovarian cancer. PVT1 regulates the invasion and migration abilities of ovarian cancer cells through Wnt signaling pathway.     

Effects of luteolin on invasion,migration and adhesion of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the effects of luteolin on invasion, migration and adhesion of human hepatocelluar carcinoma HepG2 cells.METHODS: HepG2 cells were cultured and treated with luteolin at 10, 20 and 40 μmol/L respectively. The invasion capability was examined by cell invasion assay. The migration ability was examined by wound healing assay. The adhesion capability was measured by adhesion assay. The protein levels of E-cadherin, N-cadherin, vimentin and Snai1 were determined by Western blot analysis.RESULTS: Luteolin inhibited the invasion, migration and adhesion ability of HepG2 cells in vitro in a dose-dependent manner. After treatment with luteolin, the expression of E-cadherin was increased significantly and the expression of N-cadherin, vimentin and Snai1 were decreased significantly.CONCLUSION: Luteolin inhibits the invasion, migration and adhesion ability of human hepatocelluar carcinoma HepG2 cells. The mechanism may be related to the regulatory effects of luteolin on epithelial-mesenchymal transition.     

Effect of ZNF281 on proliferation of hepatocellular carcinoma cells

AIM:To investigate the role of zinc finger protein 281 (ZNF281) in the proliferation of hepatocellular carcinoma (HCC) cells. METHODS:The mRNA expression levels of ZNF281 in peripheral blood mononuclear cells from 80 cases of healthy people and 206 cases of HCC patients were determined by real-time PCR. Statistical analysis were used to illustrate the relationship between the mRNA expression levels of ZNF281 in the peripheral blood mononuclear cells and the clinicopathologic parameters of HCC patients. Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of ZNF281 in hepatoma cell lines and immortalized hepatocytes. The silencing of ZNF281 was conducted by transfection of small interfering RNA targeting ZNF281, and then the proliferation of HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was tested by Cell-Light^TM EdU Apollo^®488 In Vitro Imaging Kit. The ability of colony formation of the HCC cells was measured by colony formation assay, and the ability of anchorage-indepen-dent growth was detected by soft agar test. RESULTS:The mRNA expression level of ZNF281 in the peripheral blood mononuclear cells from HCC patients was significantly increased compared with the healthy people, and the high expression level was positively correlated with tumor size, tumor stage and tumor vascular invasion. Concurrently, the expression level of ZNF281 in hepatoma cell lines was significantly higher than that in immortalized hepatocytes. More importantly, the silencing of ZNF281 inhibited the proliferation, DNA synthesis, colony formation and anchorage-independent growth of the HCC cells. CONCLUSION:ZNF281 promotes the proliferation of HCC cells.     

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.