ZNF580 is up-regulated in S1P-induced migration and proliferation of endothelial cells

AIM: To investigate whether a novel human C2H2-type zinc finger protein ZNF580 is involved in the proliferation and migration of endothelial cells induced by sphingosine 1-phosphate (S1P). METHODS: The cDNA of EA.hy926 cells was analyzed by RT-PCR to determine the S1P receptor expression profile. The cells were incubated with S1P at different concentrations and for different time intervals. Total RNA and protein in treated EA.hy926 cells were analyzed by RT-PCR and Western blotting. SB203580, a chemical inhibitor of p38 MAPK, was used to determine whether p38 MAPK pathway had any effect on the up-regulation of ZNF580 expression by S1P. The plasmid pEGFP-ZNF580 or the synthetic ZNF580-siRNA was transfected into EA.hy926 cells with Lipofectamine 2000 for 48 h. Cell migration assay and MTT colorimetric assay were used to investigate the effects of ZNF580 on the motility and growth of endothelial cells. RESULTS: EA.hy926 endothelial cells expressed S1P1, S1P3 and S1P5 receptors. Furthermore, S1P up-regulated ZNF580 at mRNA and protein levels in a concentration- and time-dependent manner. The p38 MAPK pathway specific inhibitor SB203580 blocked the S1P-induced up-regulation of ZNF580 expression. Moreover, overexpression/silencing of ZNF580 in EA.hy926 cells led to enhancement/decrease of the migration and proliferation of the cells. CONCLUSION: S1P-induced migration and proliferation of endothelial cells are critical for angiogenesis. ZNF proteins usually play an essential role in altering gene expression and regulating the angiogenesis.     

Overexpression of N-terminal amino acids of p55γ regulatory subunit of PI3K inhibits cell adhesion of human gastric carcinoma MGC803 cells

AIM: To investigate the effect of the N-terminal 24-amino acid(N24) overexpression in p55γ re-gulatory subunit of phosphatidylinositiol 3-kinase(PI3K) on the cell adhesion of human gastric carcinoma cell MGC803.METHODS: MGC803 cells, which stably expressed GFP-N24 fusion protein and GFP alone, were generated by transfection with pEGFPN-24 plasmid and control plasmid pEGFP-C1, respectively. The morphological change of the cells was observed under inverted microscope. The expression of GFP-N24 fusion protein was detected by Western blot. The adhesion of the cells was determined by cell adhesion assay. The effects of GFP-N24 fusion protein on the expression of E-cadherin and β-catenin were analyzed by Western blot. The expression and secretion of matrix metalloproteinase 9(MMP9) and urokinase-type plasminogen activator(uPA) in the MGC803 cells were detected by gelatin zymography.RESULTS: MGC803/GFP-N24 cell line steadily expressed GFP-N24 fusion protein and MGC803/GFP cell line steadily expressing GFP were successfully established, but the expression of fusion protein GFP-N24 was lower than that of the control protein GFP. The morphological changes of the transfected cells from paving stone to fibroblast cell form after gene transfection, and the cytoplasm secretory granules were increased significantly. The cell adhesion to fibronectin and collagen decreased after GFP-N24 transfection. GFP-N24 fusion protein increased the expression of cell adhesion molecule E-cadherin and decreased the wnt signal pathway molecule β-catenin in the MGC803 cells. However, it did not affect the expression and secretion of tumor metastasis-related proteins MMP9 and uPA.CONCLUSION: Overexpression of N24p55γ inhibits cell adhesion by influencing the expression of E-cadherin and β-catenin in the MGC803 cells.     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Gene cloning,subcellular localization and expression in kidney tissue of human ASBT protein

AIM:To compare the cDNA sequences of ASBT (apical Na+/bile acid cotransporter) gene cloned from human kidney and intestine tissues, and to determine subcellular localization of ASBT in renal tubular epithelial cells and expression site of ASBT in human kidney tissue.METHODS:The total RNA was extracted from human kidney and intestine tissues. The full-length cDNA gene of ASBT was amplified by RT-PCR technique using primers with 8-peptide FLAG tag, sequenced and inserted into eukaryotic expression vector to construct ASBT protein expression vector, which was then transfected into swine renal tubular epithelial cell line LLC-PK1. The subcellular localization of ASBT protein was determined by immunofluorescence staining and laser confocal microscope. Immunohistochemical analysis was used to observe expression position of ASBT in kidney tissue.RESULTS:The results revealed that the sequence of kidney ASBT cDNA gene was identical to that of human intestine ASBT gene. Western blotting analysis indicated that ASBT protein was correctly expressed in LLC-PK1 cells. Confocal scanning analysis showed that ASBT protein was mainly localized at the cellular plasma membrane, consistent with the predicting result obtained by bioinformatics. The results of immunohistochemistry revealed that ASBT was expressed at brush border membrane of proximal renal tubular cells, but not expressed in distal tubule and renal interstitium.CONCLUSION:The gene sequence of kidney ASBT is the same as that of intestine ASBT and ASBT protein is expressed at the lumen (apical) membrane of proximal renal tubular epithelial cells.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.     

F-box domain is involved in regulating the proliferation of MCF-7 cells by FBXO39 protein

AIM: To investigate the effect of F-box domain on the regulation of MCF-7 cell proliferation by FBXO39 protein. METHODS: The effect of F-box domain on the localization of FBXO39 protein in the MCF-7 cells was investigated. MCF-7 cell cDNA library was used as the template resource. The full-length cDNA sequence of FBXO39 was amplified by PCR method and subcloned into eukaryotic expression vector pEGFP-C2. The pEGFP-FBXO39ΔF (F-box domain deletion mutation) plasmid was successfully constructed with the template resource of pEGFP-FBXO39 plasmid. The recombinant plasmids were transfected into the MCF-7 cells, and then the expression of FBXO39 and FBXO39ΔF were determined by Western blot. The cellular localization of FBXO39 and FBXO39ΔF were observed by confocal microscopy. The localization of endogenous FBXO39 in the MCF-7 cells was detected by immunofluorescence staining. In addition, MTT and EdU assays were used to measure the cell proliferation, flow cytometry was used to measure the cell cycle distribution, and immunohistochemical staining was used to observe the expression of FBXO39 in the breast cancer and para-carcinoma tissues. RESULTS: The eukaryotic expression vector pEGFP-FBXO39 and pEGFP-FBXO39ΔF were constructed successfully. F-box domain had no effect on the cell localization of FBXO39. FBXO39 promoted MCF-7 cell proliferation but FBXO39ΔF did not. FBXO39 was highly expressed in the breast cancer tissues. CONCLUSION: F-box domain had no effect on the cellular localization of FBXO39 protein. However, it plays an important role in the biological function of FBXO39. FBXO39 may be related to breast cancer tumorigenesis.     

Construction and identification of eukaryotic expression vector of bcl-2 siRNA

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.     

Effects of intracellular expression of Mycobacterium tuberculosis CFP10-ESAT6 fusion protein on proliferation and apoptosis of mouse macrophages

AIM：To establish Mycobacterium tuberculosis culture filtrate protein 10 (CFP10)-early secretory antigenic target 6 (ESAT6) eukaryotic expression vector and investigate the effect of intracellular expression of CFP10-ESAT6 fusion protein on the proliferation and apoptosis of mouse macrophages (RAW264.7 cells). METHODS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was constructed by inserting CFP10-ESAT6 fusion gene into eukaryotic expression vector pEGFP-N1, and then transfected into RAW264.7 cells to express CFP10-ESAT6 fusion protein. The viability of RAW264.7 cells was measured by MTT assay. Flow cytometry was applied to measure the apoptotic rate and Toll-like receptor 2 (TLR2) expression in RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein or staurosporine. RESULTS：The recombinant plasmid pEGFP-N1/CFP10-ESAT6 was successfully constructed and transfected into RAW264.7 cells. Compared with the control cells, intracellular expression of CFP10-ESAT6 fusion protein did not affect the viability of RAW264.7 cells, but could inhibit the apoptosis of RAW264.7 cells treated with Mycobacterium tuberculosis 19 kD lipoprotein. Moreover, CFP10-ESAT6-expressing macrophages had markedly lower expression of TLR2 on the surface. CONCLUSION：Intracellular expression of CFP10-ESAT6 fusion protein has no cytotoxicity on mouse macrophages, but can inhibit the apoptosis of the macrophages treated with Mycobacterium tuberculosis 19 kD lipoprotein through down-regulating the expression of TLR2.     

Effect of PRL-2 gene transfection on proliferation and cell cycle in hepatocellular cell line

AIM: To observe the changes of proliferation and cell cycle after PRL-2 gene effectively expressed in human hepatocellular cell line.METHODS: The PRL-2 vector was transfected into CL1 cell with lipofectamine reagent,the stable expression clones were screened by G418.The expression of PRL-2 mRNA was detected by real-time PCR.The expressive protein was identified by Western blotting.The subcellular localization was demonstrated by immunochemistry.The cell cycle was measured by flow cytometry.The population doubling time (TD) was analyzed by MTT assay.The expressions of cyclin A,cyclin D1,cyclin E,p16,p21Waf1 and p27Kip1 were detected by Western blotting.The p21Waf1 mRNA was determined by real- time PCR.RESULTS: The full length ORF of PRL-2 gene was inserted into the vector pcDNA3.1 (+),transfected into CL1 cells,and expressed successfully.Real-time PCR showed stable expression of PRL-2 mRNA.Western blotting confirmed the overexpression of PRL-2 protein.The subcellular localization of PRL-2 was in the plasmid.The proportion of cells in S-phase was increased.The population doubling time was reduced (P<0.01),a significant decrease was observed both in the mRNA and the protein expression of the p21Waf1 in comparison with untransfected or vector- transfected control cells (P<0.05).The expressions of cyclin D1,cyclin E,cyclinA,p16 and p27Kip1 were not appreciably different between the control and PRL-transfected cell lines.CONCLUSION: Eukaryocytic expression vector of PRL-2 has been successfully constructed,which shows stable and effective expression in CL1 cell line.PRL -2 increases cell proliferation by stimulating progression from G1 into S phase,which is primarily associated with decreased p21Waf1.     

Effects of hepatitis C virus core protein on cell cycle of HepG2 cells

AIM: To investigate the molecular mechanism of hepatitis C virus (HCV) chronic infection by studying the effect of its core protein on cell growth and the expression of cell cycle regulators such as cyclin D1 and pRb/p130 in HepG2 cells. METHODS: A eukaryotic expression vector that carried a gene encoding HCV-core-1b was constructed. The cDNA of HCV core protein was subcloned into pBabe-Flag-puro vector to generate pBabe-Flag-HCV-core-1b. The plasmid was transfected into Pheonix 293T packaging cells to produce retroviruses. The virus-containing supernatant collected from the cell culture was used to infect HepG2 cells and subsequently the cell line that stably expressed the core protein of HCV was obtained.The cell cycle was analyzed by flow cytometry. The expression of cyclin D1 and pRb/p130 was examined by Western blotting. RESULTS: The pBabe-Flag-HCV-core-1b vector was confirmed by DNA sequencing. The expression of HCV gene type 1b core protein was verified by Western blotting. The overexpression of HCV gene type 1b core protein impaired the cell cycle progression in G₀/G₁ phase and significantly reduced the levels of cyclin D1 and pRb/p130 in the cells. CONCLUSION: A eukaryotic expression plasmid that contains the cDNA of HCV core protein is successfully constructed, and a HepG2 cell line which stably expressed the core protein of HCV is also established. HCV gene type 1b core protein inhibits the cell cycle possibly through down-regulation of cyclin D1 and pRb/p130 proteins in the cells.     

Molecular cloning of ING4 gene and ING4-induced apoptosis in HeLa cells

AIM: To isolate a gene encoding mouse ING4, construct pcDNA3.0-ING4 recombinant eukaryotic expression plasmid and investigate its effects on HeLa cells in vitro. METHODS: The mouse ING4cDNA was amplified by RT-PCR from mouse liver. The eukaryotic expression vector pcDNA3.0-ING4 was constructed by DNA recombination technique. The recombinant plasmid pcDNA3.0-ING4 was identified by PCR, restriction enzyme digestion and DNA sequence analysis, then was transfected into HeLa cells by lipofectamine. The expression was determined by RT-PCR. Apoptosis was detected by fluorescence microscope with Hoechst33258 staining and laser scanning confocal microscope. Cell cycle distribution was measured with flow cytometry. RESULTS: RT-PCR product was about 750 bp specific fragment. Analysis by restricting enzyme digestion and PCR of pcDNA3.0-ING4 recombiant plasmid showed that results were about 750 bp, DNA sequencing revealed that ING4 cloning were successful. With Hoechst fluorescence staining, we found that the percentage of apoptotic rate in HeLa cells transfected with pcDNA3.0- ING4 (21.25%) was higher than that in HeLa cells transfected with pcDNA3.0 (8.91%,P<0.01). Apoptosis was also detected by laser scanning confocal microscope. Cell cycle analysis reavealed the cell number in S phase of HeLa cells transfected with pcDNA3.0- ING4 increased. CONCLUSION: The gene encoding mouse ING4 and construction of pcDNA3.0- ING4 eukaryotic expression vector were successfully obtained, ING4 could enhance apoptosis in HeLa cells.     

Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ eukaryotic expression vector and expression in NIH 3T3 cells

AIM: To construct a recombinant eukaryotic expression vector pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ and detect its expression in NIH 3T3 cells.METHODS: CD28-ζ cDNA was amplified from the plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/IgGFc/CD80 gene.The recombinant plasmids were transfected into NIH 3T3 cells,and resistant clones were obtained by G418 selection.The gene expression of the fusion protein was determined by RT-PCR and FACS.RESULTS: The recombinant eukaryotic vector was constructed successfully,determined by PCR and enzyme digestion analysis.The target gene was amplified from NIH 3T3 cells transfected with the vectors by RT-PCR.The FACS showed that recombinant protein was expressed in NIH 3T3 cells.CONCLUSION: Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ expression vector and its expression in NIH 3T3 cells lay the foundation for further research of generation of modified T lymphocytes to CD20 positive lymphoma.     

Mechanical stretch induces translocation of high-mobility group box 1 protein in THP-1 cells

AIM: To investigate the translocation of high-mobility group box 1 protein(HMGB1) in THP-1 cells induced by mechanical stretch. METHODS: The vector that expressed the fusion protein of HMGB1 and enhanced green fluorescent protein(EGFP) was constructed. The cDNA of HMGB1 and EGFP was subcloned into hemagglutinin(HA)-tagged vector pcDNA3-HA by two-step method. THP-1 cells were transfected with pcDNA3-HMGB1-EGFP and exposed to cyclic mechanical stretch at 20 % elongation using Flexercell 4000T cell stretching unit. The translocation of HMGB1 in THP-1 cells was observed under fluorescence microscope. RESULTS: The recombinant plasmid was verified by enzyme digestion. The green fluorescence accumulated in the nuclei of the cells, indicating that the fusion protein was highly expressed in THP-1 cells and localized in the nuclei. Eighteen hours after mechanical stretch, the green fluorescence was observed in the cytoplasm. At the same time, the green fluorescence was still localized in the nuclei of the control cells treated without mechanical stretch. CONCLUSION: The HMGB1-EGFP fusion protein is successfully and effectively expressed in THP-1 cells. Mechanical stretch induces the translocation of HMGB1 protein from nucleus to cytoplasm.     

Effect of human scavenger receptor SR-A I overexpression on uptaking ox-LDL and adhesion in 293T cells

AIM: To construct the recombinant eukaryotic expression plasmid pEGFP-C1-SR-A I for the high expression in 293T cells in order to identify functions of savenger receptor-A I (SR-A). METHODS: The primer was designed according to MSR1 cDNA and pEGFP-C1-SR-A I was constructed by standard molecular cloning technique and enzyme digestion. After sequencing, the plasmid was transfected into 293T cells by lipidosome method. The expression of scavenger receptor-A I was identified by RT-PCR and Western blotting. The foam cells were evaluated by the formation of lipid granules in the cells with oil red staining. Cell adhesion was analyzed by cell adhesion assay. RESULTS: 24 h after transfection, SR-A I mRNA was highly expressed and the high level of the protein was detected. The ratio of foam cell formation was doubled, the efficacy of cell adhesion was enhanced two times compared to the control group and the empty vector group. CONCLUSION: The recombinant eukaryotic expression plasmid has been constructed successfully with enhancing the function of uptake ox-LDL and adhesion in 293T cells by overexpression of SR-A I.     

Effect of estrogen receptor 2 on the cell proliferation in prostate cancer cell line PC-3M

AIM: To construct the recombination plasmid pcDNA3.1-hERβ with the human estrogen receptor 2 (ESR2) full length cDNA and transfect it into hormone-independent prostate cancer PC-3M cell line, and to study the effects of ESR2 on proliferation in transfected cells. METHODS: The complete cDNA of ESR2 was obtained from human ovary tissue by RT-PCR technique and cloned into eukaryotic expression vector pcDNA3.1 by using gene recombination technique to construct the pcDNA3.1-hERβ recombination plasmid. The plasmid was detected by endonuclease digestion and DNA sequencing and was transfected into PC-3M cells. MTT and FCAS assay were used to test the effects of ESR2 on the ability of proliferation in PC-3M cells. RT-PCR and Western blotting were used to detect the expressions of cyclinD1 and P21Cip1. RESULTS: The results of sequencing and endonuclease digestion demonstrated that the construction of pcDNA3.1-hERβ recombination plasmid was successful. The sequence analysis suggested that the ESR2 sequence detected by PCR was identical to that published in GenBank, and the product of endonuclease was as long as the complete human ESR2 gene. 48 h after transfected the pcDNA3.1-hERβ into PC-3M cells, the expression of ESR2 mRNA and protein levels increased significantly detected by RT-PCR and Western blotting. Compared to the cells transfected with vector as control, the PC-3M cells transfected with pcDNA3.1-hERβ showed that cell population decreased and proliferation activity degraded. FCAS showed that the cells in G0/G1 stage increased and in S stage or G2/M stage decreased. RT-PCR and Western blotting showed that the expression of cyclinD1 gene reduced and expression of P21Cip1 increased. CONCLUSION: The recombination of plasmid pcDNA3.1-hERβ is constructed and transfected into the PC-3M cells successfully. The activity of cell proliferation is inhibited after pcDNA3 transfection.1-hERβ. It is possible that ESR2 inhibits cell proliferation by the expression of proliferation related genes cyclinD1 and P21Cip1.