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Objective To construct the lentiviral vector of human DNA methyltransferase 3A (DNMT3A) and to establish the stable breast caner MDA-MB-231 cell line. Methods The total RNA was extracted from MDA-MB-231 cells and cDNA containing DNMT3A was inverse transcribed. DNMT3A fragment containing EcoR I and BamH I sites was amplified by PCR and was inserted into pLVX-IRES-Hyg lentiviral vector. Then the recombinant expression vector pLVX-FLAG-DNMT3A was co-transfected into 293T cell line with packaging plasmids, and the culture supermatant containing the lentiviral particles was collected to infect MDA-MB-231 cells. After 48 h, 1 μg/μ puromycin was used to screen the infected cells, so as to obtain a cell line with stable expression. The protein and mRNA expression levels of DNMT3 A were detected by Western blotting and PCR. Results Bacteria colonies PCR, double restriction enzyme digestion, plasmid PCR identification, and DNA sequencing demonstrated the lentiviral vector pLVX-FLAG-DNMT3A was constructed. MDA-MB-231 cells were infected by the lentivirus, and the protein and mRNA expression of DNMT3A was significantly up-regulated following screening with puromycin. Conclusions The DNMT3A gene lentiviral vector pLVX-FLAG-DNMT3A is constructed successfully. The cell line stably expressing DNMT3A is screened in MDA-MB-231 cells, which provides an in vitro model for further study of molecular function and mechanism of DNMT3A in human breast cancer. [ABSTRACT FROM AUTHOR]

Objective To establish the human breast MDA-MB-231 cells stably expressing HMGA2 protein. Methods Total RNA of MDA-MB-231 cells in the logarithmic phase were extracted, and cDNA of HMGA2 gene was reversely tran­scribed. The recombinant pLVX-HMGA2 expression vector was acquired by inserting the target fragments which were ac­quired by PCR technology into the lentiviral expression plasmid pLVX-IRES-Puro. The forward and reverse primers, contai­ning EcoR1 and BamH1 restriction enzyme sites, were designed according to their CDS sequences. The recombinant lenti- virus carrying HMGA2 gene was obtained by co-transfecting HEK293T cells with the packaging plasmid，and the culture supernatant containing the lentiviral particles was collected. The recombinant plasmid was identified by bacterial colonies PCR，double restriction enzyme digestion，plasmid PCR，and sequencing comparison. MDA-MB-231 cells were divided in­to two groups. The cells in the experimental group were infected by the lentivirus carrying HMGA2 gene，simultaneously， the control group was infected by the lentivirus carrying only expression vector. Puromycin (1 pg/mL) was used to screen out the infected cells after 48 hours so as to obtain a cell line for stable expression. The mRNA and protein expression of HMGA2 was detected by Western blotting and real-time fluorescent quantitative PCR. Results The relative expression levels of HMGA2 mRNA in the observation group and the control group were 10 273.93 ±4 290. 77 and 1.18 ±0.81，re­spectively， and significant difference was found between these two groups (P < 0. 05 ). The relative expression levels of HMGA2 protein in the observation group and the control group were 1.570 ±0.080 and 0. 110 ±0.002，and the difference between the two groups was statistically significant ( P <0. 05 ) . Conclusion The breast cancer cell line MDA-MB-231 stably expressing HMGA2 is successfully established. [ABSTRACT FROM AUTHOR]

27-O-(E)-p-coumaric acyl ursolic acid( DY-17) from Ilex latifolia is a compound of the monomer. To investigate the DY-17 inducing apoptosis in the human breast cancer cell line, the MDA-MB-231 cells were used as research object in this experiment. The proliferation activity of the MDA-MB-231 cells stimulated with the different concentrations of DY-17 (20, 40 µmol · L(-1)) was detected at different time( 12, 24, 36, 48, 60,72 h) . We surveyed the DY-17 inducing apoptosis of the MDA-MB-231 cells with the fluorescent staining technology. The rate of MDA-MB-231 cells apoptosis and necrosis was determined by flow cell cytometry (FCC). Moreover, expression of JNK, phosphorylated JNK, Bax, PARP shear and caspase-3 shear related to JNK/SAPK pathways were investigated in every group ( control group, EGF group, EGF + DY-17 40 µmol · L(1) group and EGF + SP600125 group) with Western blot. The MTT results showed that, in the presence of DY-17, the proliferation activity of MDA-MB-231 cells decreased in a dose-dependent and time-dependent manner. The apoptosis and necrosis rates of MDA-MB-231 cells with DY-17(20, 40 µmol · L(-1)) groups was respectively 31.86%, 49.91% by flow cytometry and significantly increased compared with control group under Fluores- cence microscopy. Up-regulation of the JNK phosphorylation protein expression was observed in EGF group compared with control group. In addition, markedly decreased the expression of JNK phosphorylation protein were also surveyed in EGF + DY-17 40 µmol · L(-1) group compared with EGF group. The expression of Bax, shear PARP and shear caspase-3 protein in EGF + DY-17 40 µmol · L(-1) group were significantly increased in comparison with EGF group. The results showed DY-17 induced apoptosis of human breast cancer MDA-MB-231 cell line related to down-regulating JNK/SAPK signal pathways.

Objective: To construct the lentiviral expression vector of human TOX high mobility group box family member 3 (TOX3) gene and the MDA-MB-231 cell line which stably over-expresses TOX3 gene., Methods: TOX3 gene was synthesized by the gene synthesis method and amplified by PCR, and then cloned into pLVEF-1a/GFP-Puro vector to construct pLVEF-1a/GFP-Puro-TOX3 lentiviral vector. After restriction enzyme analysis and sequence identification, the lentiviral vector was packaged and the titer was detected. The human breast cancer MDA-MB-231 cells were transfected with the recombinant lentiviral vector and cultured selectively by puromycin to acquire stably transfected cells. MDA-MB-231 cells which expressed GFP were observed by fluorescence microcopy. And the expression levels of TOX3 mRNA and protein in transfected MDA-MB-231 cells were detected by real-time quantitative PCR(qRT-PCR) and Western blotting, respectively., Results: Restriction enzyme digestion and sequence analysis demonstrated that the lentiviral expression vectors of pLVEF-1a/GFP-Puro and pLVEF-1a/GFP-Puro-TOX3 were successfully constructed, and the viral titers were respectively 2×10(8) TU/mL and 1×10(8) TU/mL after lentiviral packaging. And after being transfected, more than 95% cells expressed GFP under a fluorescence microscope. The results of qRT-PCR and Western blotting showed that, when compared with the MDA-MB-231-NC negative control group, the expression of TOX3 mRNA and protein significantly increased in the MDA-MB-231-TOX3 group., Conclusion: The study successfully constructed lentiviral expression vector of TOX3 gene and obtained MDA-MB-231 cell line stably over-expressing TOX3 gene by transfection with the recombinant vector.

Objective: To establish a bioluminescent MDA-MB-231 cell line which can stably express luciferase and green fluorescent protein to allow bioluminescent imaging in nude mouse models bearing human triple-negative breast cancer xenografts., Methods: The lentivirus carrying luc2, eGFP and neo fusion genes were packaged in 293T cells via calcium phosphate co-precipitation. Human triple-negative breast cancer cell line MDA-MB-231 was infected by the lentivirus, and the positive cell clones were tested for eGFP and luc2 expressions by fluorescence microscopy and Xenogen IVIS200 bioluminescent imaging system, respectively. MTT assay, transwell invasion assay and wound healing assay were performed to evaluate the changes in the proliferation, invasion and migration abilities of the infected cells. The cells were then orthotopically implanted into the right second mammary fat pat of female BALB/c nude mice. The tumor growth was monitored by the in vivo imaging system every week, and the tumor tissues were harvested to evaluate the in vivo stability and tumorigenicity of the modified cells using cryosection and HE staining., Results: The lentivirus-infected MDA-MB-231cells could stably express luc2 and eGFP, and the luciferase activity reached 9689 phontons/s/per cell. No significant changes occurred in the biological activities of the lentivirus-infected MDA-MB-231 cells. We successfully established the nude mouse model bearing orthotopically implanted human triple-negative breast cancer cells., Conclusion: The modified MDA-MB-231 cell line can be detected sensitively at the primary implantation site and distant metastasis site in nude mice, which provides a convenient and sensitive platform for the research of metastatic mechanism and new antitumor drugs of human triple-negative breast cancer. The combination of eGFP and luc2 is superior to single reporter gene.

Objective: To study the anti-tumor effect of different proportions of elacridar (ELC) and doxorubicin (DOX), and determine the best combination ratio. Preparing a new type of acid-sensitive nanoparticles co-loaded with ELC and DOX using poly (benzyl malate) (PBM) as a carrier. Methods: PBM was prepared from L-aspartic acid by lactone ring opening method, then PEG was linked with acid sensitive hydrazone bond (Hz). The block polymer PBM-Hz-PEG was obtained and characterized by IR and 1H NMR. The nano micelles were prepared by dynamic dialysis. The particle size, polydispersity index (PDI), critical micelle concentration (CMC), drug loading capacity (DL) and entrapment efficiency (EE) of nano micelles were determined. Dynamic dialysis method was used to simulate the drug release performance in vitro for the drug loaded nano micelles. The cytotoxicity of the drug loaded nano micelles was evaluated by triple negative breast cancer MDA-MB-231 cell line in vitro. Results: ① ELC can enhance the sensitivity of DOX. They have the strongest antitumor effect when the molar ratio of ELC and DOX is 1:3. ② The results of IR and 1H NMR showed that the block copolymer PBM-Hz-PEG was successfully synthesized. ③ The particle size of the blank micelles was 69.67 依11.55 nm, the PDI was 0.245依0.026, and the CMC value was 3.9 滋g mL-1. The particle size of drug loaded nano micelles was slightly larger, ranging from 96.92 nm to 113.47 nm. The drug loading of ELC and DOX was consistent with the feed ratio. ④ The drug release rate curves and cytotoxicity tests in vitro of drug loaded micelles at pH 7.4 and pH 6.0 confirmed that the drug loaded micelles had good acid sensitivity. Conclusion: The combination of ELC and DOX has strong antitumor effect, and the PBM is an excellent carrier for both. The PBM-Hz-PEG nanomicelles have high drug loading rate, and their unique acid sensitivity can effectively reduce the toxicity of drugs on normal tissues and significantly increase the release of tumor tissues, which is expected to become a new intelligent drug delivery platform. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of microrna-221/222 (miR-221/222) on DOX resistance of breast cancer MDA-MB-231/doxorubicin (DOX) cells. Methods: miR-221/222 inhibitor (miR-221/222 inhibitor) was transfected into MDA-MB-231/DOX cells by liposome method, the blank control group and the negative control group were set up meanwhile. The transfection efficiency and expression level of miR-221/222 in MDA-MB-231 cell line and cell line MDA-MB-231/DOX cell line was detected by real time fluorescent quantitative PCR (qRT-PCR); the change of drug sensitivity of MDA-MB-231/DOX cells to dox after 48 hours of transfection was detected by CCK-8 tests; the apoptosis rate of MDA-MB-231/DOX cells was detected by flow cytometry (FCM); the upregulation of pro-apoptotic protein p53 up regulates apoptosis (PUMA), bcl-2 protein modifying factor (BMF) and cyclin kinase inhibitor p27 (p27kip1) in MDA-MB-231/DOX cells were detected by Western blotting (WB).Results: The expression level of miR-221/222 in MDA-MB-231/DOX cells was higher than that in MDA-MB-231 cells (P<0.05), the expression level of miR-221/222 in miR-221/222 inhibitor transfected by MDA-MB-231/DOX cells was lower than that in the blank control group and the negative control group (P<0.05) after 96 hours of treatment; compared with the blank control group, the expression level of miR-221/222 inhibitor transfected by MDA-MB-231/DOX cells was lower than that in the blank control group (P<0.05). The apoptotic rate of the cells increased significantly and the expression of apoptosis promoting protein puma, BMF and p27kip1 in the cells increased (P<0.05); the 50% inhibitory concentration (IC50) of drug resistance cells in the inhibitor group was significantly lower than that in the blank control group and the negative control group (P<0.05). Conclusion: miR-221/222 can increase the resistance of MDA-MB-231/DOX cells to DOX, [ABSTRACT FROM AUTHOR]

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Objective To investigate the effects of lysophosphatidic acid (LPA) on the migration and invasion of breast cancer MDA-MB-231 cells and the related mechanisms. Methods ① MDA-MB-231 cells were divided into three groups: transfection group, control sequence group, and blank group.Cells in the transfection group were cultured with 50 μmol/L LPA for 16 h after they were transfected with YAP-siRNA for 3 h. Cells in the control sequence group were cultured with 50 μmol/L LPA for 16 h after they were transfected with control sequence for 3 h. Cells in the blank group were normally cultured without transfection or LPA for 16 h. Transwell was performed to evaluate the cell migration and invasion.② MDA-MB-231 cells were cultured with 50 μmol/L LPA for 0, 10, 30, 60, 120, 240, and 480 min, and then we collected the cells and used Western blotting to evaluate the relative expression level of phosphorylated YAP (pYAP). MDA-MB-231 cells were cultured with 0, 1, 10, 20, and 50 μmol/L LPA for 120 min and then we collected the cells for detecting the pYAP expression level by using Western blotting.③ MDA-MB-231 cells were divided into seven groups: groups 1, 2, 3, 4, 5, 6, and 7, which were then added with cell culture medium only, SB203580 (MAPK inhibitor), LY294002 (PI3K inhibitor), MK2206 (Akt inhibitor), PD98059 (MEK inhibitor), C3 transferase (Rho inhibitor), and Y27632 (ROCK inhibitor), followed by 50 μmol/L LPA culture for 120 min. The pYAP expression was measured by Western blotting. Results ① The transwell cell migration test showed that the transmembrane cells in the transfection group, control sequence group, and blank group were 10.01±2.05, 12.13±2.44, and 7.06±2.54, respectively; the cell invasion test indicated that the transmembrane cells were 9.24±2.18, 11.54±2.09, and 6.75±1.48, respectively. The cell invasion or migration test showed that transmembrane cells were more in the control sequence group than those in the blank group (all P<0.05); both invasion and migration test showed that cell count in the transfection group was lower than that in the control sequence group (P<0.05). ② The pYAP expression levels in MDA-MB-231 cells cultured with 10 μmol/L LPA for 0, 10, 30, 60, 120, 240, and 480 min were 0.11±0.023, 0.08±0.002, 0.05±0.027, 0.04±0.012, 0.01±0.002, 0.03±0.004, and 0.05±0.007, respectively. The pYAP expression level in MDA-MB-231 cells cultured with 10 μmol/L LPA for 120 min was the lowest (all P<0.05). The pYAP expression levels in MDA-MB-231 cells cultured with 0, 1, 10, 20, and 50 μmol/L LPA were 0.23±0.024, 0.18±0.020, 0.09±0.022, 0.06±0.017, and 0.02±0.012, respectively. With the increase of LPA concentration, the relative expression of pYAP in MDA-MB-231 cells decreased gradually (all P<0.05). ③ The expression levels of pYAP in groups 1, 2, 3, 4, 5, 6, and 7 were 0.34±0.027, 0.32±0.022, 0.35±0.019, 0.33±0.021, 0.34±0.025, 0.74±0.029, and 0.65±0.022, respectively. No statistically significant difference in pYAP expression was detected among groups 1-5 (all P>0.05). However, the pYAP expression in groups 6 and 7 was respectively higher than that in groups 1-5 (all P<0.05). Conclusion LPA induces YAP dephosphorylation in MDA-MB-231 cell line through Rho and ROCK pathway, and thus promotes migration and invasion of MDA-MB-231 cells. [ABSTRACT FROM AUTHOR]

Objective To investigate whether epithelial-mesenchymal transition (EMT) is involved in the anti-invasion and anti-metastasis effects of curcumin on MDA-MB-231 breast cancer cell line and further analyze the underlying mechanisms. Methods MTT method was used to detect the anti-proliferative effect of curcumin on MDA-MB-231 cell line induced by lipopolysaccharide (LPS). The morphological changes were determined by optical and transmission electron microscopy, respectively. The expressions of Vimentin, E-cadherin, NF-κB and Snail were analyzed using RT-PCR and Western blotting. Results Curcumin could inhibit the occurrence of LPS-induced EMT of MDA-MB-231 breast cancer cell line, decrease the expression of LPS-induced EMT marker Vimentin and increase the expression of E-cadherin, resulting in the inhibition of in vitro cell motility and invasiveness. These actions were mediated by inactivating NF-κB-Snail signaling pathways. Conclusion Our data provide a new perspective of the anti-invasion mechanism of curcumin, indicating that the effect is in part due to its ability to inhibit the EMT process. [ABSTRACT FROM AUTHOR]

Objective: To investigate the role of vascular endothelial growth factor (VEGF) in regulating triple-negative breast cancer (TNBC) stem cells and the possible pathways involved in this regulatory mechanism., Methods: The Oncomine database, UALCAN database and Human Protein Atlas (HPA) database were used to analyze the expression of VEGF in breast cancer and its association with the molecular subtypes and prognosis of breast cancer. Sphere formation assay was carried out to examine the effects of hVEGF 165 on sphere formation ability of TNBC MDA-MB-231 cell line; Western blotting and RT-qPCR were performed to detect the expression of the tumor stem cell markers including CD44, c-Myc, Nanog, and ALDH1 and the activation of the related pathways., Results: Data from the online databases all showed a significant increase of VEGF expression in breast cancer tissues than in the adjacent tissues ( P < 0.0001), and its expression level was associated with the molecular subtypes of breast cancer. Specifically, the expression of VEGF was markedly higher in TNBC than in other subtypes of breast cancer. Survival analysis showed that breast cancer patients with a high VEGF expression had a significantly shortened overall survival ( P < 0.0001). In the cell experiments, the sphere formation ability of MDA-MB-231 cells was significantly enhanced after treatment with hVEGF 165 ( P =0.0029). Compared with the monolayer cells, MDA-MB-231 spheres showed significantly increased expressions of VEGF, NRP-1, CD44, Nanog and c-Myc. Treatment with hVEGF 165 resulted in significant time-dependent up-regulation of the expressions of CD44, c-Myc, Nanog and ALDH1 and down-regulation of CD24 expression in the cells. The results of Western blotting demonstrated that treatment with hVEGF 165 caused significant activation of the ERK/MAPK pathway in MDA-MB-231 cells., Conclusion: VEGF promotes cancer stemness of triple-negative breast cancer possibly through the ERK/MAPK pathway.

Objective: To study the effect of death decoy receptor 3 on the prognosis of breast cancer and the invasive function of breast cancer cells in vitro . Methods: Expression of DcR3 were assessed qualitatively by Q-PCR to analyze the correlation in 115 mammary tissue samples with a 10-year median follow-up. The expression of DcR3 was examined in MCF7 and MDA-MB-231 cell lines using immunocytochemical staining and RT-PCR. DcR3 knock-down cell sub-lines were constructed. The effects of reduced DcR3 expression were observed by establishing invasion and migration models. Results: Patients were divided into the good prognosis group ( n= 81) and the poor prognosis group ( n= 26). The expression of DcR3 in the poor prognosis group (133 350+49 646 copies/50 ng RNA)was significantly higher than that in the good prognosis group (5 393+1 428 copies/50 ng RNA, P= 0.020). DcR3 transcripts were found to be increased significantly in grade 2 cancers compared to well differentiated grade 1(82 844±34 068 copies/50 ng RNA, n= 39,) vs (5 371±3 500 copies/50 ng RNA, n= 20, P= 0.029).The DcR3 gene of MCF7 cell line and MDA-MB-231 cell line were successfully knocked out and verified that DcR3 knockout. And the invasion and migration of MCF7 cells were inhibited ( P= 0.009, P= 0.001). However, no significant difference was found in these two aspects of the MDA-MB-231 cell line ( P= 0.475, P= 0.102). Conclusion: DcR3 promotes the capacity of invasion of breast cancer cells and plays an important role in the metastasis of breast cancer. DcR3 detection is helpful to the judgment about prognosis of breast cancer.

Objective: To study if programmed death-ligand 1 (PL-L1) expression in breast cancer cell activates PD-L1/PD-1 pathway in dendritic cells to inhibit dendritic cell maturation., Methods: Human monocytes were induced to differentiate into immature dendritic cells using GM-CSF and IL-4, and further to mature dendritic cells using TNF-α. PD-L1-expressing breast cancer cell line MDA-MB-231 was co-cultured in contact with the dendritic cells to observe the effects of the breast cancer cells on the maturation of the dendritic cells. A PD-L1 blocking antibody was applied to the co-culture, and the changes in the inhibitory effect of the MDA-MB-231 cells on dendritic cell maturation was observed. TNF-α-induced dendritic cells were treated with a recombinant human PD-L1 protein to study the effect of PD-L1/PD-1 pathway activation on the maturation of dendritic cells. The expression of PD-L1 in MDA-MB-231 cells and the dendritic cell maturation marker HLA-DR and CD83 were analyzed using flow cytometry., Results: MDA-MB-231 cell line showed PD-L1 positivity on the cell membrane cells at a rate as high as (99.7∓0.15)%. In mature dendritic cells, the positivity rates for HLA-DR and CD83 were (88.8∓6.96)% and (18.36∓3.07)%, respectively, but in the co-culture system, the positivity rates of the dendritic cells were significantly decreased to (42.76∓10.52)% (P<0.01) and (9.93∓2.74)% (P<0.05), respectively, indicating that MDA-MB-231 cells inhibited the maturation of dendritic cells. Following treatment with a PD-L1 antibody isotype control, the percentages of HLA-DR- and CD83-positive cells in the co-culture were (45.17∓10.19)% and (10.15∓2.54)%, which were significantly increased to (63.46∓1.72)% and (16.46∓2.58)% after treatment with PD-L1 antibody, respectively (both P<0.05). Compared with the mature dendritic cell controls, the cells treated with the recombinant human PD-L1 protein exhibited significantly lowered percentages of HLA-DR-positive [from (84.23∓4.18)% to (2.56∓2.39)%, P<0.05] and CD83-positive cells [(87.26∓1.54)% to (60.67∓1.63)%, P<0.05]., Conclusion: The effect of PD-L1 antibody therapy on triple negative breast cancer can be partially mediated by blocking PD-L1 expression on breast cancer cell membrane, which attenuates the inhibition of dendritic cell maturation in the cancer microenvironment.

Objective: To investigate the influence of siRNA-mediated down-regulation of CD147 on growth, proliferation and movement of human breast cancer cell line MDA-MB-231., Methods: The protein expression of CD147, MMP-2 and TIMP-2 of the MDA-MB-231 cells were analyzed by ABC. Lentiviral expression vector of CD147 gene was constructed and transfected into MDA-MB-231 cells. RT-PCR and Western blot were used to detect the mRNA and protein level changes of CD147 genes to identify the optimal time point, followed by detection of changes of mRNA and protein expression of MMP-2 and TIMP-2 genes. CCK-8 reagent method and cell scratch test were used to detect the proliferation and migration change of MDA-MB-231 cells. The nude mouse model of breast cancer by hypodermic injection with MDA-MB-231 cells was established to document the effect of CD147 siRNA on the tumor transplants., Results: After transfection of lentiviral expression vector of CD147 gene, protein of CD147, MMP-2 and TIMP-2 were weakly or negative expressed, significantly weaker than those of control group (P < 0.01). After 72 hours of transfection, average down-regulation rate of CD147 and MMP-2 were 96.03% ± 0.84% and 96.03% ± 0.84%, respectively. Both CD147 mRNA and MMP-2 mRNA expression were down-regulated (P < 0.05), while TIMP-2 mRNA expression showed no significant deference (P > 0.05). No less than 2 days after transfection, cell growth of MDA-MB-231 cell line was found significantly inhibited (P < 0.05). After 24 hours of transection, average migration distance of MDA-MB-231 cell line and control group were (0.64 ± 0.12) mm and (4.69 ± 0.85) mm, respectively, which indicated a lower migrate speed. Down regulation of CD147 led to reduction of volume and mass of nude mouses. The growth of the carcinoma transplant was inhibited upon siRNA-mediated down-regulation of CD147 (P < 0.05), with an average tumor mass of (1.85 ± 0.98) g and both reduction of tumor size and tumor mass., Conclusions: CD147 may alter the MMP-2/TIMP-2 balance in MDA-MB-231 cells. CD147 gene silencing inhibits the proliferation and migration of MDA-MB-231 cells and the growth of carcinoma transplants in nude mice.

Objective: To prepare a recombinant lentiviral carring human lysosome-associated membrane protein type 2A (LAMP2A) gene shRNA, establish MDA-MB-231 cell line with a low expression of LAMP2A and observe the change in cell resistance to paclitaxel., Methods: Four shRNAs were designed according to the sequencing analysis of LAMP2A mRNA. The pGLV-EGFP-shRNA lentiviral vector was established by gene recombination technology and was confirmed by DNA sequencing. The lentiviral vector and the packaging vector were used to cotransfect the HEK293T cells to obtain the lentivirus against LAMP2A mRNA, and the titer of the virus was determined. The constructed shRNA lentivirus was applied to infect human breast cancer cell line MDA-MB-231, and then the cells were screened with puromycin for two weeks. The inhibitive efficacy of the shRNA on the LAMP2A protein was determined by Western blotting. After the breast cancer cell line with a low expression of LAMP2A was treated with three different concentrations of paclitaxel (1, 10, 100 nmol/L), MTT assay was performed to observe the difference in the proliferation ability between the control group and the low LAMP2A expression groups., Results: DNA sequencing revealed that the recombinant lentiviral plasmid was correctly constructed with the virus titer reaching 2×10(8); TU/mL. The LAMP2A protein expression in the obtained breast cancer cell line dropped drastically. After the treatment with paclitaxel at 10 and 100 nmol/L respectively, the drug resistance of cells with a low expression of LAMP2A was notably weaker than that of the control group (P<0.05)., Conclusion: The recombinant shRNA lentiviral vector against human LAMP2A gene was successfully constructed, and the breast cancer cell line MDA-MB-231 transfected with it stably expressed a low level of LAMP2A. It was proved that the down-regulation of LAMP2A could reduce the resistance of breast cancer cells to paclitaxel.

Objective: To investigate HIF-1α gene expression in human breast cancer cells and the role of HIF-1 in tumor metastasis and invasion., Methods: Human breast cancer cell lines were cultured under normoxic or hypoxic conditions. Western blot was used to evaluate the protein expression of HIF-1α, p-vimentin and α-tubulin. Small interfering RNA targeting HIF-1α was used to block the expression of HIF-1α in MDA-MB-231 and SK-BR-3 cell lines. Matrigel transwell and cell wound healing assays were used to detect the capability of cellular invasion and migration, respectively., Results: Under normoxic condition, all the cell lines tested showed a base-level of HIF-1α expression. The highest expression level of HIF-1α protein was obtained in the MDA-MB-231 cell line, which is also noticed to be highly invasive and migratory in behavior. HIF-1α siRNA was capable of blocking the protein expression of both HIF-1α and p-vimentin and in addition, the attenuated ability of invasion and migration., Conclusion: Since HIF-1 is able to promote invasion and metastasis of tumor cells, which may be considered as a target in anti-cancer therapy.

Objective: To construct a CXCR4 specific recombinant plasmid vector and study its inhibiting effect on invasion capacity in vitro of human breast cancer MDA-MB-231 cell line and its metastatic potential to the lung in nude mice., Methods: A CXCR4 specific recombinant plasmid vector was constructed and transfected into the cultured MDA-MB-231 cell line with lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of CXCR4, respectively. Invasion capability in vitro of the cells was evaluated by Boyden chamber. The cell proliferation capacity was detected by MTT method. The nude mouse model of lung metastasis was established by injection of MDA-MB-231 cells into the tail vein. The animals were sacrificed at 6 weeks after the tumor cells injection. Whole lung tissues were harvested, embedded in paraffin, sectioned serially, and the HE-stained paraffin sections were examined pathologically to evaluate the presence and number of metastatic tumors., Results: The CXCR4 mRNA expression rate was 29.5% +/- 3.8% in the CXCR4-shRNA group, significantly lower than that of the control group (69.7% +/- 2.6%, P < 0.01) and mock-control group (67.8% +/- 3.5%, P < 0.01). The CXCR4 protein expression rate was 15.4% +/- 1.1% in the CXCR4-shRNA group, significantly lower than that of the control group (39.0% +/- 2.4%, P < 0.01) and mock-control group (35.9% +/- 3.9%, P < 0.01). Silencing of CXCR4 by shRNA lead to a significant decrease in breast cancer cell invasion and proliferation capacity in vitro. Furthermore, tumor cells with CXCR4 shRNA permanent transfcetion had a much lower lung metastatic potential in nude mice than control cells and mock control cells in vivo., Conclusion: CXCR4 shRNA can inhibit the expression of CXCR4 and decrease the invasion and lung metastatic potential of human breast cancer cells.

Objective: To study the inhibitig effects of genistein on the growth of human breast cancer cell lines in vitro and its mechanisms. METHORD: Human breast cancer cell lines both MCF-7(positive estrogen receptor, ER+) and MDA-MB-231 (negative estrogen receptor, ER-) were cultured in vitro. The proliferation of cells was measured with MTT methord and the growth curve was drawn with cell count. The estrogen receptor in cells was show with immunohistochemistry., Result: Genistein inhibited proliferation of MCF-7 and MDA-MB-231 cell lines and inhibition was dependent on dose within some concentration range, IC50 being 32.5 mumol.L-1 and 46.8 mumol.L-1 respectively. Genisteins antiproliferation of MCF-7 was stimulated by ectogenesis estrogen but proliferation of MDA-MB-231 inhibited by geinstein was not related to estrogen. The positive signs of ER in cellular nuclei of MCF-7 cell line fed with genistein at concentration of 30 mumol.L-1 were significantly weaker than these not fed with genistein., Conclusion: Genistein obviously inhibits proliferation of both cell lines of MCF-7 and MDA-MB-231 in vitro and is dependent on dose. Genisteins antiproliferous effect on MCF-7 cell lines is stimulated by estrogen and this effect is related with ER, but genisteins inhibiting proliferation of MDA-MB-231 cell line is not through ER.