Effection Of Midkine On The Expression Of MMP-9of Lung Adenocarcinoma Cell Line A549An In Vitro

Objective:To observe the changes of MMP-9expression in the A549cells treated with midkine(MK). To explore whether there are any relati-onship between MK and MMP-9in the invasion and metastasis of lung cancer.Methods:(1) The cell proliferation of A549cells treated with MK measured by MTT assayGrowing cells were treated with midkine at a final concentration of0ng/ml,2ng/ml,10ng/ml,50ng/ml and250ng/ml, respectively. Culture medium was added in control group.Each concentration treatment was done in three copy wells.Fresh culture medium with different concenrtation of midkine or control medium was changed every two days.After exposure to various midkine for1～8d, respectively.MTT was added and the plates were incubated for an additional4h before absorbance at490nm was measured. All experiments were repeated for three times.(2) The expression levels of MMP-9mRNA of A549cell treated with MK performed by semi-quantitative RT-PCRA549cells were collected at after treated with0ng/ml,2ng/ml, lOng/ml,50ng/ml and250ng/ml midkine or culture medium for48h. The total RNAs were extracted from the cells and then semi-quantiative RT-PCR was performed to evaluate the MMP-9mRNA expression.35cycles of MMP-9were performed and the length of amplification was about320base pairs.The internal control was made by PCR with Primers specific for P-actin,35cycles of GAPDH were performed and the length of PCR product was about450base pairs. All experiments were repeated for three times.(3) The proteins expression levels of MMP-9of A549cell treated with MK performed by ImmunocytochemistryLogarithmic growth phase of the A549cells were collected,and resuspended at3×103cells/L with complete culture medium, suspension was added into24-well plate,each hole was1ml,attachment after8h and treated with midkine at a final concentration of0ng/ml,2ng/ml,10ng/ml,50ng/ml culture for1～8d.Each concentration treatment was done in three copy wells,fixed with4%formaldehyde, A549cells were incubated at4℃over night with MMP-9polyclonal rabbit antibodies(1:100).Then,the steps were in accordance with the immunohistochemistry kit and five random fields of vision were counted under light microscope.The antigen visualized with the immunoperoxidase-based system.Results:(1) The cell proliferation of A549cells treated with MK measured by MTT assayThe growth curve of different concentrations of MK intervene A549cells have the same trend. There is no difference of the first three days in the cell proliferation. Compared with control group, the differences were statistically significant(P<0.05).As the time goes,there is some difference of everyday,and the proliferation of A549cells reached a peak on the8th day. The results suggest that the the MK intervention effect on the proliferation of A549cells to a time dependent manner.Compare the difference among the groups after the intervention of midkine for seven days.There is no significant difference between2ng/ml,10ng/ml and the control.but there were significant difference compare50ng/ml with2ng/ml,10ng/ml,250ng/ml and the control,and the same to250ng/ml(P<0.05). The results of experiment show that50ng/ml is the best intervention concentration,and show that MK have a effect on the proliferation of A549cells with dose-effect relationship at a certain concentration range.Statistical analysis that the mean absorbance value (OD490) of the control and intervention group are0.732±0.0210.770±0.047、0.794±0.073.0.833±0.045,0.716±0.069.The results suggest that50ng/mL can be used as ideal interventions concentration.(2) The expression levels of MMP-9mRN A of A549cell treated with MK performed by semi-quantitative RT-PCRControl and treatment groups of A549cells are the results of statistical analysis that the mean MMP-9/GAPDH optical density ratio value of the control and intervention group are0.3883±0.0332、 0.2495±0.0072、0.2270±0.0119、0.1685±0.0028and0.1925±0.0077. Compared with the control group, the concentration of MK in the intervention group MMP-9mRNA expression were lower (P<0.01),and with the increase of MK,and the MMP-9mRNA expression showed a gradual increase in the downward trend, MK50ng/mL of MMP-9mRNA decreased is the lowest.50ng/ml with2ng/ml,10ng/ml,250ng/ml significant difference,and the same to250ng/ml(P<0.01). Although the250ng/ml compared with50ng/mL decline, but there is no statistically significant (P>0.05).(3) The proteins expression levels of MMP-9of A549cell treated with MK performed by ImmunocytochemistryCultrue the A549cells with different concentrations of MK for7days and the immunocytochemistry results shown by statistical analysis.The result show that the MMP-9protein expression of MK in the intervention groups is lower than the control group, but the decline is not obvious.It is to see that the MK intervention group and control group cells MMP-9protein expression was no statistically difference (P>0.05).Conclusions:1. MK can affect the proliferation of A549cells in a certain range of time and concentration.2. In a certain range,MK can inhibit the expression of MMP-9mRNA in A549cells with time-effect and dose-effect relationship.In addition,it may reduce or inhibit the invasion and metastasis of lung cancer.3. MK A549cells MMP-9protein expression was not obvious between the control group and intervention group.4. MK may promote the proliferation and down-regulation the MMP-9mRNA and involved in mediating invasion and metastasis in lung cancer.