Functional Study Of CK18 In Human Breast Cancer BT439 Cells

objective: To detect the expression of cytokeratin 18(CK18)in serum of healthy and breast cancer patients by Western Blot,and to screen out human breast cancer BT549 cells with high expression of CK18.Study on the biological function and mechanism of CK18 in BT549 cells.Methods: Detecting the expression of CK18 in serum of healthy and breast cancer patients by Western Blot and different expression of cytokeratin 18 in MDA-MB-231,BT549,MCF-7,MCF-10 A and 4T1 cell lines.The CK18 gene and the control vector targeted by the lentiviral vector were used to infect human breast BT549 cells with high expression of CK18,and a stable expression cell line was established.The silencing efficiency of CK18 gene was detected by WB.Furthermore,cell proliferation of was analyzed by CCK-8 assay and colony formation assay.Apoptosis rate was performed by flow cytometry(AnnexinV PE double staining).Cell cycle was detected by PI-FACS.Invasion and migration was measured by invasion chamber and Transwell assay.Furthermore,WB was used to analyze relative expression of Twist1,E-cadherin and vimentin proteins in invasion-related molecules,and investigated the potential molecular mechanism on invasion and migration.Result: The expression of CK18 was detected by WB in the serum of healthy and breast cancer patients.It was found that CK18 was positively expressed in 25 cases in healthy serum,and the positive rate was 96%.In 26 cases of breast cancer patients,19 cases was positive.The rate was 73%.The relative gray value of CK18/GAPDH was analyzed in serum of breast cancer patients and healthy.The higher CK18 expression in breast cancer patient.Detection of CK18 expression in different breast cancer cells.WB results showed that CK18 was expressed at high levels in human breast cancer BT549,MDA-MB-231 and mouse breast cancer 4T1 cell lines,while expression was low in human breast cancer MCF-7 and normal human mammary epithelial MCF-10 A cell lines.CK18 silencing stable expression of BT549 cell line was established by RNAi technology: uninfected BT549 cells were used as blank control group(Wt),infected empty vector virus was used as negative control group(shCon)and infect sub-viruses were experimental groups(CK18 shRNA-21,-22,-23 and-24).CK18 shRNA-23 group has the 73% silencing efficiency by WB,so CK18 shRNA-23 were selected as the next study model.The results of CCK-8 method and plate cloning assay: compared with the blank control group(Wt)and the negative control group(shCon),the proliferation of CK18-sh23 cells was inhibited,the ability colony formation was weakened(P< 0.05),suggesting that down-regulation of CK18 can inhibit the growth and proliferation of BT549 cells.The results of apoptosis FACS showed that the number of early apoptotic cells increased significantly in the experimental group,compared with the control group(7.30±0.82)% and the blank(6.50±0.50)%,the percentage of early apoptotic cells was increased significantly in the experimental group(14.77±1.17)%(P<0.05).The results of cell cycle that compared with negative control group(16.33±1.89)% and the blank(14.33±2.84)%,the proportion of G2 phase cells was higher in the experimental group(23.23±4.91)%.The cell scratch test found that compared with the blank group and the negative control group,the migration of the experimental group cells was significantly decreased.Further,Transwell assay confirmed that the number of transmembrane cells per field in the experimental group migration and invasion experiments were(43.33±2.31)and(30.33±0.578),which was significantly lower than the blank group(63.67±7.51)and(54.33±7.64)and the negative control group(62.67±3.79)and(50.33±4.93)respectively(P<0.05).WB results suggest that with the decrease of CK18 expression,Twist1 and Vimentin protein expression was down-regulated,while E-cadherin protein expression was up-regulated.Conclusion: 1.Compared with healthy serum,CK18 is highly expressed in breast cancer serum;2.The high expression of CK18 in BT549,MDA-MA-231,4T1 cell line,low expression in MCF-7,MCF-10 A cell line;3.Establishment and identification the efficiency of CK18 silenced;4.After CK18 gene silenced,the cell proliferation was inhibited,the clone,migration and invasion ability was decreased,the apoptosis rare was increased,and cell cycle was arrested.5.CK18 may participate in the invasion and metastasis through Twist1 pathway to induce EMT.