| Part One: Correlation between PLAC1 expression and prognosis of the patients with breast cancerObjective: To elucidate the correlation between the expression of PLAC1 in breast cancer tissues and the clinical parameters,and the relationship between the expression of PLAC1 and the prognosis of the patients with breast cancer.Methods: The expression of PLAC1 was detected by immunohistochemistry(IHC)in 250 breast cancer tissues;Chi-square Test be used to analyze the relationship between PLAC1 expression and the clinical parameters;COX regression analysis and Kaplan-Meier curve were used to analyze whether the expression of PLAC1 has prognostic significance for breast cancer.Results: The results of immunohistochemistry showed that there was obvious correlation between the expression of PLAC1 and clinical stage(P=0.035),lymph node metastasis status(P=0.001),and estrogen receptor status(P=0.023)in 250 cases of breast cancer tissues.PLAC1 expression was negatively associated with patients’ age(P=0.097),tumor size(P=0.082),tumor grade(P=0.772),and HER2 status(P=0.62).COX regression analysis showed that tumor stage(P=0.002,HR=1.937,CI=1.267-2.962 95%),HER2 status(P<0.001,HR=0.345,95% CI=0.217-0.549)and hormone receptor status(P<0.001,HR=0.231,CI=0.149-0.359 95%)are the independent risk factors of overall survival(OS);Tumor size(P=0.004,HR=0.516,CI=0.330-0.809 95%),tumor stage(P<0.001,HR=2.199,CI= 95%(1.445-3.345),HER2 status(P=0.003,HR=0.513,95% CI=0.332-0.793),hormone receptor status(P<0.001,HR=0.301,CI=0.194-0.468 95%)and PLAC1 expression(P<0.001,HR=2.261,CI=11.480-3.454 95%)are the independent risk factors of metastasis-free survival(MFS);In breast cancer patients with stage of T1-T2,the expression of PLAC1 is independent risk factor of OS(P=0.002,HR=2.547,95% CI=1.422-4.563)and MFS(P=0.001,HR=2.738,95% CI=1.549-4.804).The Kaplan-Meier curve analysis revealed that high expression of PLAC1 in patients with breast cancer correlation with shorter metastasis-free survival(log-rank,test,P<0.001);in breast cancer patients with stage of T1-T2,the overexpression of PLAC1 has a significant correlation with shorter survival time(log-rank test,P=0.012)and shorter metastasis free survival(log-rank test,P<0.001).Conclusion: PLAC1 expression was positively associated with the clinical stage,lymph node metastasis and hormone receptor status.The overexpression of PLAC1 in tumor tissue indicates that patients have a high risk of recurrence and metastasis;PLAC1 is not only independent risk factors for tumor metastasis,and is an independent risk factor for the overall survival in patients with T1-T2 stage.The overexpression of PLAC1 is associated with shorter survival time and earlier metastasis possibility.Part Two: PLAC1 promotes the migration and invasion of breast cancer cells in vitro and in vivoObjective: To elucidate the effect of PLAC1 expression in vitro and in vivo on the migration and invasion of human breast cancer cells.Methods: RT-PCR and Western blot were used to detect the expression of PLAC1 in breast cancer cell lines,and MDA-MB-231 and MCF-7 cells lines were selected as tool cells for further research.In human breast cancer MDA-MB-231 and MCF-7 cell lines,stable cell lines with overexpression or knockdown of PLAC1 were constructed.The expression of PLAC1 in MDA-MB-231 and MCF-7 stable transfected cell lines was verified by Western Blot;Transwell migration assay was used to study the effect of PLAC1 on migration ability of breast cancer cells;Transwell invasive assay was used to study the effect of PLAC1 on invasive ability of breast cancer cells.PLAC1 overexpressing and contrast MDA-MB-231 cells were injected into the tail vein of nude mice to study the effect of PLAC1 on invasion and metastasis of human breast cancer cells.PLAC1 overexpression and empty vector of MDA-MB-231 cells wereinjected into the tail vein of nude mice to study the effect of PLAC1 on invasion and metastasis of breast cancer cells.H&E staining was used to detect the pathological changes of liver and lung of nude mice.Results: In human breast cancer MDA-MB-231-PLAC1 and MCF-7-PLAC1 stable transfected cell lines,the expression of PLAC1 was significantly higher than that in the empty vector group.In MDA-MB-231-sh RNA and MCF-7-sh RNA stable transfected cell lines,the expression of PLAC1 was significantly lower than that in the empty vector group.Transwell migration test showed that the number of MDA-MB-231 or MCF-7 cells passing through the basement membrane in PLAC1 overexpression group significantly inceased compared with the empty vectorl group(p<0.05),and the number of cells passing through the basement membrane in the PLAC1 knockdown of MDA-MB-231 and MCF-7 cells significantly decreased compared with the empty vectorl group(p<0.05).Transwell invasive test showed that the number of MDA-MB-231 or MCF-7 cells passing through the basement membrane in PLAC1 overexpression group significantly inceased compared with the empty vectorl group(p<0.05),and the number of cells passing through the basement membrane in the PLAC1 knockdown of MDA-MB-231 and MCF-7 cells significantly decreased compared with the empty vectorl group(p<0.05).In vivo,the number of tumor metastasis of lung and liver in nude mice was observed by the naked eye and microscope showed that PLAC1 overexpression was significantly higher than that the control group(p<0.05).Conclusion: PLAC1 overexpression can significantly enhance the migration and invasion ability of human breast cancer cells in vitro and in vivo.Knockdown of the PLAC1 protein can significantly inhibit the migration and invasion ability of breast cancer cells in vitro.Part There: PLAC1 Promotes Invasion and Metastasis of Breast Cancer through Furin/NICD/PTEN Signaling PathwayObjective: Our previous study shows that PLAC1 can enhance the invasion and metastasis of breast cancer cells in vivo and in vitro,but the specific mechanism is not clear.The main purpose of this part is to clarify the possible molecular mechanism of PLAC1 to promote tumor cells invasion and metastasis.Methods: Immunoprecipitation and proteome analysis were used to detect the proteins combined with endogenous PLAC1 in MCF-7 cells,and selected the possible protein Furin that coact with PLAC1 to promote the invasion and metastasis of breast cancer cells.Gene microarray was used to analysis the change of downstream of PLAC1,and identified the possible regulatory mechanism of PLAC1 in MDA-MB-231 cells.CO-IP and immunofluorescence colocalization further confirm that PLAC1 and Furin are directly combined with each other.The PLAC1 overexpression and the empty vector group of MDA-MB231 and MCF-7 cells were transfected with Furin si RNA or control respectively to determine whether PLAC1 promote the migration and invasion of breast cancer cells through the interaction with Furin.Western Blot was used to detect the changes of pathway related factors after overexpression of PLAC1,and the changes of related molecules after rescue Furin knockdown.The PLAC1 overexpression and the empty vector group of MDA-MB231 and MCF-7 cells were transfected with PTEN plasmid or control respectively to determine whether PLAC1 promote the migration and invasion of breast cancer cells through regulating PTEN expression.IHC was used to detect the expression of related factors in nude mice liver and lung tissues.Results: The results of immunoprecipitation and proteome analysis showed that there was a binding relationship between Furin and PLAC1.CO-IP and immunofluorescence colocalization further clarified that Furin and PLAC1 were binding proteins;gene microarray results showed that increased expression of PLAC1 can significantly activate PI3K/AKT signaling pathway and inhibit the expression of the key regulatory factor PTEN;In PLAC1 overexpress of breast cancer cell lines MDA-MB231 and MCF-7,western Blot results demonstrated that PLAC1 could regulate the expression of NICD,Hes1,PTEN and PI3K/AKT pathway related proteins,MMP2 and MMP9.In rescue experiment,Furin si RNA or control were transfected in PLAC1 overexpression and the empty vector group of MDA-MB231 and MCF-7 cells,and the transwell migration and invasion test showed that the number of MDA-MB-231 and MCF-7 cells passing through the basement membrane significantly decreased in Furin knockdown compared with control(p<0.05).Also in the PLAC1 over expression of MDA-MB231 and MCF-7 cells and the control group were transfected with PTEN plasmid or control,and the transwell migration and invasion test showed that the number of MDA-MB-231 and MCF-7 cells passing through the basement membrane significantly decreased in PTEN overexpression compared with control(p<0.05).Western Blot results showed that the knockdown of Furin significantly reversed the changes in NICD,Hes1,PTEN and PI3K/AKT pathway related proteins and their downstream MMP2 and MMP9 induced by PLAC1 overexpression.IHC was used to detecte the expression of NICD,PTEN,MMP2 and MMP9 in nude mouse liver and lung tissues,which results showed that PLAC1 could regulate the NICD and PI3K/AKT related proteins expression in vivo.Conclusion: PLAC1 regulates the expression of NICD and Hes1 by interaction with Furin protein,which affects the expression of PTEN and PI3K/AKT signaling pathway to promote the invasion and metastasis of breast cancer. |
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