The Function Of Chemokine Receptor CCR7 On Metastasis Of Breast Cancer And Its Regulative Mechanism

Objective: RNA interference technology was used to inhibit the expression of CCR7 and investigate its effect on metastasis in breast cancer cell lines. Then, protein spectrum was performed for detecting differentially expressed proteins induced by interference of CCR7 to investigate molecules that involved in the regulative role of CCR7. These studys could be useful for investigating the mechanism of CCR in the metastasis of breast cancer and meaning for finding new diagnose markers and therapeutic target.Methods :1) Breast cancer cell lines MDA-MB-231、4T1-luc、MDA-MB-361、T47D、MDA-MB-468、MCF-7、MCF10A、184B5、184AI、HCC1806、HCC1937、HCC1580 and MCF10A were routinely cultured. Then q-PCR and western blot were used to detect the expression of CCR7 in different cell lines to determine the cell line that expressed higher CCR7. 2) Screened breast cell lines were transfected into CCR7-siRNA. Then, after total RNA and protein were extracted from CCR7-siRNA group and control group, respectively, q-PCR and western blot were performed for detecting the interfering effect. 3) Scratch and transwell experiments were used to investigate the effect of CCR7 on metastasus in breast cancer cell. 4) Protein spectrum was performed for screening differentially expressed proteins, and further q-PCR and Western blot were used to determine the key proteins that involved in the regulative role of CCR7.Results: 1. The mRNA expression levels of CCR7 were higher in T47D,HCC1500, SK-RB-3, BT474 and MCF7 cell lines compared with others. The protein expression levels of CCR7 were highest in 4T1-Luc, T74D and MCF7 cell lines,higher in MB231, MB468 and HCC1937 cell lines. While, the protein expression levels of CCR7 were fewer in 184B5, 184A1 and MCF10A cell lines. So, MCF7 and T47D cell lines were usded to further study. 2. After transfecting CCR7 siRNA into T47D and MCF7 cell lines, q-PCR was performed for detecting the expression of CCR7. The results showed that CCR7-siRNA 1# and CCR7-siRNA2# could effectively inhiting the mRNA expression of CC7 in T47D and MCF7 cell lines. The results of western blot were further determine the above results. 3. The results of scratch and transwell experiments showed that cells in shCCR7C and shCCR7D group exhibited weaker migration ability compared with cells in shLacZ group. 4. We found that EPHA2、PVR、SQSTMI、ANXA6、PTRF、ALDH3A2 and EPCAM were differentially expressed proteins using protein spectrum. The results of q-PCR and Western blot showed that ALDH3A2 and PTRF might involved in the regulative role of CCR7.Conclusion: (1) when the CCR7 in different amount of expression of the breast cancer cell lines is different, in high degree malignant cells expressing quantity is higher, may be when the CCR7 expression level of high and low has certain correlation with breast cancer metastasis of malignant degree.(2) the removal of breast cancer cells after the low CCR7 was knocked down, causing ALDH3A2 and PTRF to decrease.(3) interfering with the migration of CCR7 to inhibit breast cancer cells may be achieved by lowering the expression of ALDH3A2 and PTRF, which requires further study.