| Objective:Breast cancer is a malignant tumor derived from epithelial cells in the lobular unit of breast terminal ducts.It has become the most common malignant tumor and the leading cause of cancer death in women worldwide.And we are researching the mechanism of breast cancer progression and looking for a molecular target for early diagnosis and treatment.Recent evidence indicated that malignant human breast epithelial cells expressed GP5 protein.But its expression level and function were not clear.The aim of this study was to reveal the influence of abnormal expression of GP5 in breast cancer tissues and the associations between the methylation modification and GP5 protein expression,and to explore the molecular mechanismof proliferation,invasion and migration,and to provide reference for clinical diagnosis and treatment of breast cancer.Methods:1.The total RNA was extracted from the cancer tissues and adjacent tissues of 41 breast cancer patients with pathological examination confirmed by the pathology department of the Second Affiliated Hospital of Kunming Medical University.The mRNA expression of GP5 in two tissues was detected by QPCR,and the protein expression of GP5 was detdced by immunohistochemical staining.2.The mRNA expression of GP5 in tree cells was tested by QPCR after RNA was extracted from breast cancer cell line MDA-MB-231,MCF-7 and human normal breast epithelial cell line MCF-10A.3.The DNA samples were extracted from cancer tissues and adjacent tissues of 41 breast cancer patients,19 ductal carcinoma in situ,and 15 lymph node metastase.The methylation of GP5 promoter region was analyzed by methylation sequencing.4.Lentivirus infected breast cancer cell MDA-MB-231 to over-express and knock down GP5?Then we used CCK-8,flow cytometry and scratch experiments to observe whether GP5 overexpression and knockdown affect the biological behavior of this tumor cell.5.The transcriptome of breast cancer cells with GP5 knocking down and control cells was analysised by high-throughput sequencing technology.The relevant signaling pathway was verified by RT-PCR and western blot.Results:1.GP5 mRNA levels were significantly increased in breast cancer tissues(8.195±10.903)compared with corresponding normal tissues(1.480±3.541)(P<0.0001),and GP5 protein expression in tumor tissues(30.468±16.806)was also significantly higher than corresponding normal tissue(4.421±3.286)(P<0.001):2 MDA-MB-231 mRNA levels(2.139±0.114)and MCF-7 mRNA levels(6.746 ±0.517)were significantly higher than human normal breast epithelial cell MCF-10A mRNA levels(1.010±0.096)(P<0.01);3.The methylation of GP5 promoter region was observed more frequently in ductal carcinoma in situ(0.425±0.169),invasive breast cancer(0.419±0.215),and lymph node metastasis tissue(0.394±0.207)compared with corresponding normal tissue samples(0.265±0.099)(P<0.05),The methylation of primary breast cancer tissues(0,549±0.152)was also more frequently compared with lymph node metastases tissues(0.414±0.1 34)(P<0.001),and the results showed that site methylation specificity and special methylation haplotypes existed in different kind of tissues;4.The high expression of GP5 was significantly associated with higher nuclear grade,TNM stage and Her-2 negativity(P<0.05),but not with age,menopause,tumor size,vascular invasion,nerve invasion,nipple invasion,ER,PR,Ki-67 and lymph node metastasis.The high expression of GP5 was significantly correlated with a worse prognosis in RFS;5.After lentivirus infecting breast cancer cells,the OD values was significantly increased by up-regulating GP5 expression compared with control group at each observation time in CCK-8 experiments(P<0.05),and downregulated GP5 expression was lower than that of control group(P<0.05);The results showed that the cell cycle of experimental group was not significantly different from control group.Apoptosis test results showed that the apoptosis rate of GP5 overexpression group was higher compared with control group,but the difference was not statistically significant;scratch experiments showed that the cell migration rate was significantly increased by up-regulating GP5 expression compared with control group at 24h and 48h(P<0.001),and the cell migration rate was significantly decreased by down-regulating GP5 expression at 24h and 48h(P<0.001);6.High-throughput transcriptome sequencing results showed that there were 141 specific genes,including 128 genes up-regulated and 13 genes down-regulated.The PI3K mRNA levels(3.053±0.033),AKT mRNA levels(2.335±0.178)were significantly increased by up-regulating GP5 expression compared with control group(0.985±0.015),(0.915±0.085),P<0.001,and significantly decreased by down-regulating GP5 expression(P<0.05).The expression levels of N-Cadherin,Vimentin,MMP2,and MMP9 proteins were significantly increased by up-regulating GP5 expression compared with control group(P<0.05),and significantly decreased by down-regulating GP5 expression(P<0.05).Conclusion:1.GP5 is highly expressed in breast cancer tissues and may play a important role as a cancer-promoting gene in breast cancer.The high expression of GP5 was significantly associated with higher nuclear grade,TNM stage and Her-2 negativity.2.Abnormal methylation of GP5 promoter region may be not the dominant factor of regulating its protein expression.3.GP5 can promote the proliferation,invasion,and metastasis of breast cancer cells.4.GP5 may promote the proliferation,invasion,and metastasis by activating PI3K/AKT signaling pathway to upregulate MET,and is expected to become a potential target for clinic diagnosis and treatment. |
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