| CORRELATION BETWEEN ESTROGEN RECEPTOR AND RSK4 GENE EXPRESSION AND METHYLATION OF RSK4 GENE PROMOTER IN BREAST CANCERObjective: To analyze the expression of ER and RSK4 in breast cancer tissues and adjacent tissues.To compare the expression of RSK4 mRNA in ER positive/ER negative breast cancer tissues and breast cancer cells.The relationship between the promoter methylation degree of RSK4 gene and the expression of the RSK4 gene was assessed.Method: The expression of ER and RSK4 protein in breast cancer tissues and adjacent tissues was detected by immunohistochemistry.The expression of RSK4 mRNA in breast cancer tissues and adjacent tissues was detected by qRT-PCR.The expression of RSK4 protein in ER positive and ER negative breast cancer cells was detected by Western blot.The promoter methylation degree of RSK4 gene in breast cancer tissues,adjacent tissues and breast cancer cells was detected by Bisulfite Genomic Sequence(BSP).Results :(1)The expression of RSK4 protein in breast cancer tissues(2.889±1.167)was lower than that in adjacent tissues(4.111±0.782),and the difference was statistically significant(P=0.01<0.05);RSK4 mRNA in breast cancer tissues(0.546±0.320)was lower than that in adjacent tissues(1.000±0.000),and the difference was statistically significant(P=0.003<0.05).(2)The expression of ER protein in breast cancer tissues(4.556±0.726)was higher than that in adjacent tissues(2.556±0.726),and the difference was statistically significant(P=0.001<0.05).(3)The expression of RSK4 mRNA in ER-positive breast cancer tissues(0.672±0.293)was significantly higher than that in ER-negative breast cancer tissues(0.408±0.200),and the difference was statistically significant(P=0.037<0.05).(4)The expression of RSK4 protein in ER-positive breast cancer cells MCF-7(1.507±0.054)was significantly higher than that of ER-negative breast cancer cells MDA-MB-231(1.400±0.061)and MDA-MB-453(0.868±0.063).The differences were statistically significant(P=0.037<0.05,P=0.000<0.05).(5)The promoter methylation degree of RSK4 gene in breast cancer tissues(66.2%)was higher than that in adjacent tissues(31.2%);the promoter methylation degree of RSK4 gene in ER-positive breast cancer tissues(45.0%)was lower than that in ER-negative breast cancer tissues(56.9%);the promoter methylation degree of RSK4 gene in ER-positive breast cancer cells MCF-7(42.8%)was lower than that in ER-negative breast cancer cells MDA-MB-231(52.8%)And MDA-MB-453(71.8%).Conclusion:Compared with adjacent tissues,the expression level of RSK4 gene in breast cancer tissues was lower;the expression level of RSK4 gene in ER+ breast cancer tissues and cells was higher than that in ER-breast cancer tissues and cells,respectively;the higher the expression of the RSK4 gene,the lower the degree of promoter methylation.FUNCTIONAL EFFECTS OF RSK4 GENE OVEREXPRESSION ON ER-POSITIVE MCF-7 BREAST CANCER CELLSObjective: To evaluate the effect of RSK4 gene on the cell function of ER-positive MCF-7 breast cancer cells.Method: The RSK4 gene lentiviral expression vector(p Lenti-RSK4-EGFP)carrying the RSK4 gene coding sequence was co-transfected into HEK293 T cells together with the packaging plasmid.After 48 hours,the virus solution containing RSK4 lentivirus was harvested from HEK293 T cell culture.MCF-7cells were infected with p Lenti-RSK4-EGFP virus as an experimental group(RSK4-OE group),and MCF-7 cells were infected with blank vector Lenti-EGFP virus as a negative control group(NC group),untreated MCF-7cells as a blank control group(blank group).(1)The expression of RSK4 m RNA and RSK4 protein in three groups were detected by q RT-PCR and Western blot.(2)CCK-8 was used to detect the growth vigor of the three groups of cells for1-5 days and to plot the growth curve.(3)The ability of three groups of cell clones to be detected was tested using a plate cloning assay.(4)Transwell was used to detect the migration ability of three groups of cells.(5)Flow cytometry was used to detect the apoptosis levels of the three groups.(6)The effect of three groups of cell supernatants on the lumen formation of huvec cells was examined by lumen formation assay.Results:(1)The expression of RSK4 mRNA in the experimental group(314040.000±170851.800)was higher than that in the negative control group(3.366±2.807)(P=0.008<0.05)and the blank control group(1.000±0.000)(P=0.008<0.05).The expression of RSK4 protein in the experimental group(1.067±0.056)was higher than that in the negative control group(0.710±0.022)(P=0.000<0.05)and the blank control group(0.813±0.090)(P=0.002<0.05).The differences were statistically significant.(2)The absorbance at OD450 nm of the experimental group on day 2,3,4,and 5 was significantly lower than that of the negative control group(P=0.000,P=0.000,P=0.000,P=0.000)and the blank control group(P = 0.000,P = 0.000,P = 0.000,P = 0.000),and the difference was statistically significant.(3)The clonal ability of the experimental group was significantly lower than that of the negative control group(P=0.001<0.05)and the blank control group(P=0.001<0.05).(4)The migration ability of the experimental group was significantly lower than that of the negative control group(P=0.000<0.05)and the blank control group(P=0.000<0.05).(5)The degree of apoptosis in the experimental group was significantly higher than that in the negative control group(P=0.000<0.05)and the blank control group(P=0.000<0.05).(6)The number of lumens induced by the supernatant of the experimental group was significantly lower than that of the negative control group(P=0.001<0.05)and the blank control group(P=0.001<0.05).The differences were statistically significant..Conclusion : Overexpression of the RSK4 gene attenuates the proliferation,cloning,migration,and induction of huvec cell lumen formation in ER+ MCF-7 breast cancer cell line,and promotes apoptosis of this cell line.EFFECT OF ESTROGEN(E2)ON RSK4 GENE EXPRESSION AND RSK4 GENE PROMOTER METHYLATION IN ER-POSITIVE MCF-7 BREAST CANCER CELLSObjective: To investigate whether the estrogen/estrogen receptor signal(E2/ER signal)has a methylation regulation mechanism for the RSK4 gene.Method: Estrogen E2 at different concentrations(1 n M,5 n M,10 n M)was used to intervene in three groups of MCF-7 cells,and MCF-7 cells treated with estrogen were used as experimental groups(1 n M group,5 n M group,10 n M group).MCF-7 cells without estrogen intervention were used as a control group.(1)After 48 hours of estrogen intervention,the expression of ER and RSK4 protein in four groups of cells were detected by Western blot.(2)The methylation of the RSK4 gene promoter in four groups of cells was detected by BSP method.Results:(1)The expression of ER protein in 1 n M group,5 n M group and10 n M group were higher than that in control group(P=0.000,P=0.000,P=0.000).and the difference was statistically significant.The expression of ER protein in5 n M group cells was higher than that in 1 n M group(P=0.000),and the expression of ER protein in 10 n M group was higher than that in 1 n M group(P=0.000).The difference was statistically significant.There was no significant difference between 5 n M group and 10 n M group(P=0.446).(2)The expression of RSK4 protein in 1 n M group,5 n M group and 10 n M group were lower than that in control group(P=0.021,P=0.000,P=0.000).The difference was statistically significant.The expression of RSK4 protein in 5 n M group and 10 n M group were lower than that in 1 n M group(P=0.000,P=0.000),and the expression of RSK4 protein in 10 n M group was lower than that in 5 n M group(P=0.001).(3)The expression of ER protein was negatively correlated with the expression of RSK4 protein(r =-0.914,P = 0.000).(4)The degree of methylation of the RSK4 gene promoter in the experimental group(53.8% in the1 n M group,71.2% in the 5 n M group,75.3% in the 10 n M group)was higher than that in the control group(36.9%).With increasing estrogen concentration,the degree of methylation of the RSK4 gene promoter in MCF-7 cells is gradually increasing.Conclusion:The E2/ER signal has methylation regulation on the RSK4gene;there was a negative correlation between the expression of ER protein and the expression of RSK4 protein. |
PDF Full Text Request