The Relationship Between Matriptase, HAI-1 Expression And The Invasion, Migration And Cisplatin Efficacy Assessment Of Endometrial Cancer Cells

Objective:To detect the expression of Matriptase and HAI-1 in three endometrial cancer cells. Explore the relationship of Matriptase expression and invasion and migration of endometrial cancer cells using RNAi to disturb Matriptase gene expression. The three endometrial cancer cell were divided into low, medium and high concentration groups depending the treatment of different concentration of cisplatin.After the treatment, investgate the correlation of the expression of Matriptase and HAI-1 and the capacity of invasion and migration, the ratio of the opoptosis.Methods: The endometrial cancer cells were cultured in vitro. Then, detect the three endometrial cancer cells m RNA and protein expression level of Matriptase and HAI-1 by Real-time fluorescence quantitative PCR and Western blot respectly. We successfully constructed Matriptase lentivirus si RNA plasmid expression vectors andapply the transient transfection technique in order to suppress the expression of Matriptase of HEC-1A and RL952 which express high level of Matriptase. Then, the ability of invasion and migration were compared before and after transfection using scratch assay and transwell chamber assay separately. Detect the m RNA and protein expression level of Matriptase and HAI-1 after the treatment of different concentration of cisplatin by Real-time fluorescence quantitative PCR and Western blot respectly. Applying the scratch assay and transwell chamber assay to test the capacity of invasion and migration. The flow cytometry was used tu monitoring the impact of cisplatin on the proliferation of cancer cells.Results:1. The relative expression of Matriptase m RNA in HEC-1A, HEC-1B and RL952 cells were(0.2123 ± 0.021),(6.95E-4 ± 1.2E-4) and(0.1778 ± 0.013) respectively,while the relative expression of HAI-1m RNA were(0.2827 ± 0.049),(0.0263 ±0.0043) and(0.2420 ± 0.032). The protein expression of Matriptase and HAI-1was positive in HEC-1A and RL952, but they both were not expressed or were below detectable levels in HEC-1B, which were consistent with the m RNA level.2. We constructed the Knock down(KD),Control(CON) and Negative control(NC)group using the Matriptase gene lentiviral vector(Ma-Si RNA-89873), the normal medium and lentiviral vectors NO53 respectively. The m RNA expression of Matriptase in KD group was significantly down-regulated(HEC-1A: 0.2157 ±0.0124 vs 0.0358 ± 0.0111; RL952: 0.1849 ± 0.0053 vs 0.0341 ± 0.0017) and the inhibition rate were 83.4% and 81.5% respectively. The m RNA expression of HAI-1 in KD group were up slightly compared with the CON group(HEC-1A:0.3142 ± 0.0277 vs 0.2900 ± 0.0292; RL952: 0.2829 ± 0.0021 vs 0.2611 ± 0.0192),but the difference were not statistically significant(P> 0.05). The changement of Matriptase and HAI-1 protein levels were consistent with the m RNA level.3. The cell scratch assay results showed that the migration distances of HEC-1A CON, KD and the NC group were(206.67 ± 28.38)μm,(79.2 ± 6.82) μm and(197.96 ± 16.83) μm respectively, while the migration distances of RL952 CON,KD and NC group were respectively(184.57 ± 21.97) μm,(76.8 ± 5.48) μm and(175.34 ± 14.73) μ m; P(CON vs KD) <0.001, P(CON vs NC)> 0.05.Person correlation analysis indicated Matriptase m RNA expression levels were positively correlated with the migration distance(r HEC-1A=0.97,r RL952=0.982).4. The transwell chamber assay showed that the invasion cells of KD group were significantly reduced compared with the CON group(HEC-1A: 139.25 ± 12.3112 vs 48.6 ± 4.8496; RL952: 150 ± 7.0710 vs 53.3 ± 5.6376), there were statistical differences significance(P <0.05), the invasion cells of HEC-1A and RL952 NC groups were(132 ± 8.3120) and(145 ± 6.0711) respectively, the differences were not statistically significant(P> 0.05). Person correlation analysis indicated Matriptase m RNA expression level was positively correlated with the invasion cells(r HEC-1A=0.975,r RL952=0.994).5. The Real-time q PCR results showed the Matriptase m RNA expression of low concentrations of cisplatin group were increased(P <0.05), while they were decreased in the high concentration of cisplatin group(P <0.05) after different concentration of cisplatin treatment.The HAI-1 m RNA expression levels showed a downward trend after the first rise in the HEC-1A and RL952 cells treatment group. The HAI-1 m RNA expression levels showed a decline trend in HEC-1B cells treatment group. The protein expression levels of Matriptase and HAI-1protein were coincide with m RNA expression level in the experimental groups.6. The scratch assay results showed the same changment of migration distance after different concentrations of cisplatin treatment in the three endometrial cancer that the migration distance were significantly widened in the low concentrations of cisplatin treatment group(P <0.05) while there were shortened in the high concentration of cisplatin group(P <0.05).7. The transwell chamber assay results showed the same changment of invasion cells after different concentrations of cisplatin treatment in the three endometrial cancer that the invasion cells were significantly increased in the low concentrations of cisplatin treatment group(P <0.05) while there were decreased in the high concentration of cisplatin group(P <0.05).8. The flow cytometry results showed that the apoptosis rate of HEC-1A treatment group(0, 2, 10, 50 mg/L DDP) were 84.4%, 82.8%, 75.0% and 41.4%; the apoptosis rate of HEC-1B treatment group(0, 2, 10, 50 mg/L DDP)were 76.9%,73.5%, 65.6% and 31.8%; the apoptosis rate of RL952 treatment group(0, 1, 2, 5mg/L DDP)were 89.4%, 86.3%, 65.8% and 31.1%. one-way ANOVA indicated that the differences were significant(FHEC-1A=34.548, PHEC-1A < 0.001;FHEC-1B=64.141, PHEC-1B<0.001; FRL952= 115.043, P RL952<0.001), the P values were less than 0.05 expect PHEC-1A(0 mg/L vs 2 mg/L)=0.418, PHEC-1B(0 mg/L vs2 mg/L)=0.412 and P RL952(0 mg/L vs 1 mg/L)=0.418.Conclusion:1. The endometrial cancers’ capacity of invasion and migration was limited while gene expression level of Matriptase was specifically down-regulated by applying the RNAi technology. It implied that the Matriptase expression level was closely related with the invasion and migration of endometrial cancer cells.2. The expression of Matriptase and HAI-1 are closely related to the invasion and migration capacity of endometrial cancer cell. Futher more, they are associated with the proportion of apoptosis induced by cisplatin which can be expected to become targets of cisplatin efficacy assessment.