Effect Of Prl-3 Antibody Combined With Pemetrexed On Proliferation, Migration, Apoptosis And The Expression Of Prl-3, P-ERK And VEGF Proteins Of Human Lung Adenocarcinoma A549 Cells

Objective:To investigate the effects of PRL-3 antibody combined pemetrexed on proliferation,migration,apoptosis and the expression of PRL-3,p-ERK and VEGF proteins in human lung adenocarcinoma A549 cells,and explored its possible mechanism.Methods:?1?Cell culture and experimental grouping:A549 cells were cultured in 1640 medium?containing 10%fetal bovine serum?,and in a humidified incubator with saturated humidity?37?and 5%CO₂?,and the logarithmic growth phase cells were used for the experiment.Experimental groups:control group?no drug intervention group?,PRL-3 antibody group?IC50 concentration?,pemetrexed group?IC50 concentration?and PRL-3antibody?IC50 concentration?+pemetrexed group?IC50 concentration?.?2?A549 cells were treated with different concentrations of PRL-3 antibody?0,2.5,5,10,20,40,80,160,320ng/mL?.The CCK-8 test was used to measure the absorbance?OD?of A549 cells and calculate the inhibition rate of cell proliferation and IC50 concentration of PRL-3 antibody?164ng/ml?.The half inhibition concentration of pemetrexed?180ng/ml?was obtained according to the references,and IC50 was used in subsequent experiments.?3?A549 cells in control group and experimental group were treated at different time?24h,48h,72h?.The CCK-8 test was used to measure the absorbance?OD?of A549 cells and calculate the inhibition rate of cell proliferation.The difference between each group was compared by statistical method.The best time was chosen as the intervention time of follow-up experimental drugs.?4?Transwell chamber method was used to detect the cell migration ability:The experimental group was the same as the previous design,A549 cells in each group were treated for72 hours,Transwell chamber method was used to detect the cell migration ability of each group,and statistical method was used to compare the two groups.?5?Flow cytometry was used to detect the apoptosis rate:The experimental group was the same as the previous design.A549 cells in each group were treated for 72 hours,the apoptosis rate of each group was detected by flow cytometry,and the two groups were compared.?6?Western bolt was used to detect the protein expression of PRL-3,p-ERK and VEGF:The experimental group was the same as the previous design,A549 cells in each group were cultured for 72 hours,and the protein expression levels of PRL-3,p-ERK and VEGF were detected by Western bolt method,and the two groups were compared.?7?The correlation of PRL-3,P-ERK and VEGF protein was analyzed by linear correlation analysis.Results:?1?Results of CCK-8 method:IC50 of PRL-3 antibody was164ng/ml;compared with the control group,the inhibition of PRL-3 antibody on A549 cell proliferation increased with the increase of concentration?P<0.05?,but the inhibition of low concentration PRL-3 antibody group?2.5ng/ml?on A549 cell proliferation was not significant?P>0.05?;compared with the control group,the inhibitory effect of the combination group on A549 cell proliferation was better than that of the single drug group?P<0.05?,and the inhibition increased with time?P<0.05?,and the 72 hours was the best intervention time.?2?Results of Transwell chamber method:A549 cells in each group were treated for 72 hours,the number of migrating cells in the control group,PRL-3antibody group,pemetrexed group and PRL-3 antibody plus pemetrexed group were respectively?146.433±2.136?,?101.033±1.155?,?101.233±0.503?,?65.233±1.858?;the number of migration cells in each experimental group was significantly lower than that in the control group?P<0.05?,and the number of migration cells in the combination group was significantly lower than that in the PRL-3 antibody and pemetrexed group?P<0.05?.?3?Results of flow cytometry:A549 cells in each group were treated for 72 hours,the apoptosis rate of A549 cells in control group,PRL-3 antibody group,pemetrexed group and PRL-3 antibody plus pemetrexed group was?5.133±0.304?%,?29.790±1.637?%,?28.807±1.588?%,?47.297±0.869?%;the apoptosis rate of each experimental group was significantly higher than that of the control group?P<0.05?,and the apoptosis rate of the combination group was significantly higher than that of PRL-3 antibody group and pemetrexed group?P<0.05?.?4?Western bolt test results:A549 cells in each group were treated for 72 hours,the expression levels of PRL-3 protein in the control group,PRL-3 antibody group,pemetrexed group and PRL-3 antibody+pemetrexed group were 0.711±0.047?0.307±0.050?0.364±0.062?0.106±0.030,the difference is statistically significant?F=80.560,P=0.000?;The expression levels of p-ERK protein were 1.237±0.127?0.481±0.051?0.520±0.051?0.153±0.036,the difference is statistically significant?F=111.059,P=0.000?.The expression levels of VEGF protein were0.843±0.066?0.324±0.009?0.365±0.013?0.104±0.023,The difference is statistically significant?F=227.208,P=0.000?.The expression levels of PRL-3,VEGF and p-ERK proteins in each experimental group were significantly lower than those in the control group?P<0.05?,and the expression levels of PRL-3,VEGF and p-ERK in the combination group were significantly lower than those in the PRL-3 antibody and pemetrexed group?P<0.05?.?5?PRL-3 protein and p-ERK protein expression level showed a positive correlation,PRL-3 protein and VEGF protein expression level showed a positive correlation,VEGF protein and p-ERK protein expression level showed a positive correlation.Conclusions:PRL-3 antibody combined with pemetrexed can inhibit the proliferation,migration and induce apoptosis of A549 cells,which is better than the monotheraphy group.The mechanism may be related to the inhibition of the expressions of PRL-3,P-ERK and VEGF protein.