ABSTRACT
Objective To construct the silencing vector of FOS like antigen 1 (FOSL1) gene, and study the effects of FOSL1 on cell proliferation, cell invasiveness and the methylation level of PRDM10 gene in breast cancer cell line MDA-MB-231. Methods The FOSL1 silencing vector of gene pLVX-shRNA-FOSL1-shRNA was purchased. The FOSL1 silencing vector and the empty vector were separately transfected into MDA-MB-231, which were regarded as transfection group and empty group, respectively. Untransfected MDA-MB-231 was used as control group. FOSL1 was verified by PCR in MDA-MB-231. The cell proliferation ability and cell invasion ability of MDA-MB-231 were detected by MTT and Transwell assay, respectively. MSP was used to detect the methylation status of PRDM10 gene. The mRNA and protein expression levels of PRDM10 gene were detected by Q-PCR and Western-blot assay. Results MTT results showed that the optical density (OD) values were significantly lower in transfection group compared with those of control group at 24 h, 48 h and 72 h (all P<0.05), and the same as those compared with empty group at 48 h and 72 h (both P<0.05). Compared with empty group and control group, Transwell assays showed that the cell invasive abilities of MDA-MB-231 were decreased in transfection group (both P<0.05), and MSP assay showed that the methylation of PRDM10 gene was decreased in MDA-MB-231, and Q-PCR and Western-blot tests showed that the expressions of PRDM10 gene were increased in mRNA level and in protein level. Conclusion Silencing of FOSL1 gene inhibits the proliferation and invasion of MDA-MB-231 cells, which might be related to the demethylation of PRDM10 gene in the cells.
ABSTRACT
Objective To investigate the effect of the over-expressed CTCF on apoptosis factors Bax and Bcl-2 in human breast cancer cell line MDA-MB-231. Methods Reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expressions of CTCF,Bax and Bcl-2 in MDA-MB-231. The overexpression vector of CTCF/pEGFP-N1 was constructed. The overexpression plasmid CTCF/pEGFP-N1 and the empty vector plasmid pEGFP-N1 were transfected into breast cancer cell line MDA-MB-231 by lentivirus transfection, and the MDA-MB-231 cells were divided into CTCF group and control group. After successfully transfection of MDA-MB-231 identified by RT-PCR, real time quantitative PCR (Q-PCR) was used to detect the mRNA levels of Bax and Bcl-2 in MDA-MB-231 of the CTCF group and the control group. The protein levels of Bax and Bcl-2 were detected by Western blot assay and enzyme-linked immunosorbent assay (ELISA). Results The expression of CTCF was not found in MDA-MB-231, and expressions of Bax and Bcl-2 were found in MDA-MB-231. Results of Q-PCR showed that the mRNA levels of Bax were 4.63±1.08 and 2.27±0.16 in CTCF group and control group, respectively, and they were statistically significant (t=27.50, P<0.05). The mRNA levels of Bcl-2 were 1.39±0.14 and 3.56 ± 0.97 in CTCF group and control group, and there was significant difference between two groups(t=39.00, P<0.05). Results of Western blot assay showed that the protein level of Bax was higher in CTCF group compared with that of control group. The protein level of Bcl-2 was lower in CTCF group compared with that of control group. Results of ELISA showed that the protein levels of Bax were 15.25±2.17 and 6.24±1.78 in CTCF group and control group, respectively, and there was significant difference between the two groups (t=26.84, P<0.05). The protein levels of Bcl-2 were 4.59 ± 0.97 and 10.68 ± 1.93, and there was significant difference between the two groups (t=21.72, P<0.05). Conclusion The over-expressed CTCF can promote the expression of apoptotic factors and inhibit the expression of anti-apoptotic factors in breast cancer cells.
ABSTRACT
Objective To investigate the effect of the over-expressed CTCF on apoptosis factors Bax and Bcl-2 in human breast cancer cell line MDA-MB-231. Methods Reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expressions of CTCF,Bax and Bcl-2 in MDA-MB-231. The overexpression vector of CTCF/pEGFP-N1 was constructed. The overexpression plasmid CTCF/pEGFP-N1 and the empty vector plasmid pEGFP-N1 were transfected into breast cancer cell line MDA-MB-231 by lentivirus transfection, and the MDA-MB-231 cells were divided into CTCF group and control group. After successfully transfection of MDA-MB-231 identified by RT-PCR, real time quantitative PCR (Q-PCR) was used to detect the mRNA levels of Bax and Bcl-2 in MDA-MB-231 of the CTCF group and the control group. The protein levels of Bax and Bcl-2 were detected by Western blot assay and enzyme-linked immunosorbent assay (ELISA). Results The expression of CTCF was not found in MDA-MB-231, and expressions of Bax and Bcl-2 were found in MDA-MB-231. Results of Q-PCR showed that the mRNA levels of Bax were 4.63±1.08 and 2.27±0.16 in CTCF group and control group, respectively, and they were statistically significant (t=27.50, P<0.05). The mRNA levels of Bcl-2 were 1.39±0.14 and 3.56 ± 0.97 in CTCF group and control group, and there was significant difference between two groups(t=39.00, P<0.05). Results of Western blot assay showed that the protein level of Bax was higher in CTCF group compared with that of control group. The protein level of Bcl-2 was lower in CTCF group compared with that of control group. Results of ELISA showed that the protein levels of Bax were 15.25±2.17 and 6.24±1.78 in CTCF group and control group, respectively, and there was significant difference between the two groups (t=26.84, P<0.05). The protein levels of Bcl-2 were 4.59 ± 0.97 and 10.68 ± 1.93, and there was significant difference between the two groups (t=21.72, P<0.05). Conclusion The over-expressed CTCF can promote the expression of apoptotic factors and inhibit the expression of anti-apoptotic factors in breast cancer cells.