| Objective:1.To investigate the effect of PLCD1 on the migration and invasion of human breast cancer cells;2.To analyze the regulatory effect of PLCD1 on ERK/β-catenin/MMP7 signaling pathway;3.To explore the potential mechanism of PLCD1 affecting ERK/β-catenin/MMP7 signaling pathway.Methods:1.The online softwares(Oncomine,Kaplan-Meier Plotter,BioGRID3.4,bc-GenExMiner)were used to analyze the expression of PLCD1 and KIF3 A in human breast cancer,the prognostic value of PLCD1 in human breast cancer,and the correlation between PLCD1 and KIF3A;2.Real-time PCR was used to detect the expression of PLCD1 in human breast cancer tissues and paired cancer adjacent tissues;RT-PCR was used to detect the expression of PLCD1 in human breast cancer cell lines;3.PLCD1 plasmid was used to express PLCD1;siPLCD1 and siKIF3 A were used to knock down the expression of PLCD1 and KIF3Arespectively;U0126 was used to inhibit the expression of pERK1/2;4.Wound-healing assayand Transwell assay were used to detect the migration and invasion ability of human breast cancer cells;5.Western blot was used to detect the expression of PLCD1,KIF3 A,ERK1/2,GSK-3β,β-catenin,MMP7;6.IHC was used to detect the expression of PLCD1,ERK1/2,β-catenin and MMP7;7.ELISA was used to detect the expression of MMP7 protein in the cell cultured supernatants.Results:1.Compared with the matched adjacent tissues,the expression of PLCD1 in human breast cancer tissues is relatively lower;compared with normal breast tissue,the expression of PLCD1 in breast cancer tissue is lower;compared with negative lymph node metastasis of breast cancer,the expression of PLCD1 in positive lymph node metastasis of breast cancer is relatively lower;compared with the breast cancer patients with low expression of PLCD1,the breast cancer patients with high expression of PLCD1 haslonger distant metastasis free survival and overall survival;2.PLCD1 was expressed in human breast cancer cell lines BT-549,SK-BR-3 and normal human breast tissues,but was weakly expressed in T47 D,and was absent in MDA-MB-231,MDA-MB-468,MCF-7 and ZR-75-1 cells;3.In MDA-MB-231 cells,the 24 hours migration distancein control group and PLCD1-expressedgroup was 28.8±1.0 and 21.06±1.1 respectively(p<0.01),the number of cell migration was 274±23 and 113±16(p<0.01),the number of cell invasion was 137±12 and 62±10(p<0.01);in MCF-7 cells,the 48 hours migration distance in control group and PLCD1-expressedgroup was 17.6±0.9 and 9.55±1.4(p<0.01);in BT-549 cells,the 24 hours migration distance in blank control group,negative control group,siPLCD1-01 and siPLCD1-02 groupswas 18.8±1.2,17.9±1.5,29.6±2.4 and 29.6±2.5(p<0.01),the number of cell migration was165±9,166±12,323±27 and 286±31(p<0.01),the number of cell invasion was 71±4,75±12,123±12 and107±20(p<0.01);4.In MDA-MB-231 and MCF-7 cells,the protein level of p ERK1/2,pGSK-3β,active-β-catenin and MMP7 were lower in PLCD1-expressed group compared with control group,the expression of MMP7 protein in the cell cultured supernatants was also lower;in BT-549 cells,the protein level of p ERK1/2,pGSK-3β,active-β-catenin and MMP7 were higher in siPLCD1 groups compared with control group,the expression of MMP7 protein in the cell cultured supernatants was also higher;in the nude mice model,IHC assay showed that in the PLCD1-expressed tumor,the protein level of pERK1/2,active-β-catenin and MMP7 were lower;in MDA-MB-231 and MCF-7 cells,specific inhibition of pERK1/2,the expression of pGSK-3β,active-β-catenin and MMP7 were decreased;5.Compared with normal breast tissues,the expression of KIF3 A is higher in human breast cancer tissues and has correlation with the expression of PLCD1(r=-0.09,p<0.0001,n=5164);in BT-549 cells,knockdown of PLCD1 increased the expression of KIF3 A,knockdown of KIF3 A did not affect the expression of PLCD1;6.In MDA-MB-231 cells,the 24 hours migration distance in blank control group,negative control group and siKIF3A-01 and siKIF3A-02 groups was 31.0±1.2,31.2±1.1,17.9±0.9 and 17.2±0.5(p<0.01),the number of cell migration was 167±9,164±9,76±11 and 95±12(p<0.01),the number of cell invasion was 125±12,124±9,67±8 and 88±8(p<0.01);In BT-549 cells,the 24 hours migration distance in blank control group,negative control group and siKIF3A-01 and siKIF3A-02 groups was27.3±1.0,26.9±0.8,8.0±0.9 and 13.6±1.3(p<0.01),the number of cell migration was 269±15,260±15,122±16 and 119±16(p<0.01),the number of cell invasion was 174±10,173±5,76±6 and 76±8(p<0.01);7.In MDA-MB-231 and BT-549 cells,the levels of pERK1/2,pGSK-3β,active-β-catenin and MMP7 was decreased,and the MMP7 protein in cell cultured supernatants was also decreased when KIF3 A was knocked down;8.When PLCD1 and KIF3 A were knocked down simultaneously,the 16 hours migration distance in fivegroups was 13.6±1.3,13.4±1.0,23.7±0.8,23.6±1.2 and 17.3±1.2 respectively(p<0.01),the number of cell migration was 153±13,155±7,227±9,230±17 and 145±13(p<0.01),the number of cell invasion was 81±7,80±5,163±11,168±12 and 85±6(p<0.01);9.when PLCD1 and KIF3 A were simultaneously knocked down in BT-549 cells,the expression of pERK1/2,pGSK-3β,active-β-catenin and MMP7 was decreased and the MMP7 protein in the cell cultured supernatants was also decreased compared with the knockdown of PLCD1 alone.Conclusion:1.PLCD1 suppresses the migration and invasion of human breast cancer cells;2.PLCD1 plays a role of tumor suppressor gene by modulating KIF3A-mediated ERK1/2/β-catenin/MMP7 signaling pathway. |
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