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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]

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]