The Molecular Mechanism Of Proliferation And Metastasis Regulated By CRNDE In Breast Cancer Cells

Objective Breast cancer is one of the most common malignant tumors in women of the world,accounting for 32% of the female malignant tumors in China.Although modern technology can make detection and treatment of breast cancer early,the fatality rate of breast cancer is still high,the main cause of this result is the metastatasis of cancer cells.However,the mechanism of metastasis in breast cells is still unknown.So,find out the mechanism of the proliferation and metastasis of breast cancer is the point to the treatment of breast cancer.Recent studies have shown that lnc RNA participate in the mechanism of tumor metastasis,and has becaome a hot spot in tumor study.Long noncoding RNA(lnc RNA),Colorectal Neoplasia Differentially Expressed,CRNDE was found in colorectal tumor in 2011,which plays an important role at metastasis,proliferation and invasion of colorectal tumor.Through our present experiments also showed that the expression of lnc RNA CRNDE is low in high metastatic of breast cancer cells compared with low metastatic,this mean the expression level of lnc RNA CRNDE might have a relationship with the metastasis of breast cancer cells.So this study is to explore the relationship between lnc RNA CRNDE and the mechanism of regulating proliferation and metastasis in breast cancer cells.Methods 1.Breast cancer cells MDA-MB-231 and MCF-7 were cultivated in vitro.According to the different transfected plasmid,the cells were divided into two groups: the experimental group and the control group.Then the microscope examination of GFP fluorescence ensure the plasmid transfection into cells.2.After transferation,the m RNA expression of EMT marker: E-cadherin and Vimentin;transcription factor marker: Snail and E2F-1;CDK2,CDK4,Cyclin D,Cyclin E,and protooncogene P21 in the cells were detected by q RT-PCR.3.After transferation,the protein expression of Rb,E2F-1,E-cadherin and Vimentin in the cells were detected by western blot.4.After transferation,the ability of transfer and invasion were detected by Woundhealing assay and transwell assay.5.After transferation,the ratio of G1/G0 and the ability of proliferation were detected by flow cytometry analysis and clones formation.Result 1.The expression of CRNDE was low in high metastatic MDA-MB-231 cells compared with low metastatic MCF-7 cells.2.After transfection,the wound healing experiments detected that the migration ability of MCF-7 and MBA-MD-231 have increased compared with the control group.3.The invasion ability of MCF-7 cells was significantly higher than control group.4.After transfection cells with Si RNA-CRNDE and Si RNA-control.The expression of E-cadherin and Snail in MCF-7 cells was significantly lower than that in control group.And that the expression of Vimentin in MCF-7 cells was significantly higher than that in control group.We also found that the expression of E-cadherin in MDA-MB-231 cells was down-regulated after CRNDE knockdown,but the changes of Vimentin and Snail were not obvious at m RNA level.5.After transfection,the changes of protein levels in Vimentin and E-cadherin were consistent with those at the m RNA level.6.After transfection,compared with the control group,the expression of cyclin D in MCF-7 cells increased significantly.And the expression of cyclin E in MDA-MB-231 cells was up-regulated.7.After transfection 48 h,compared with control group,the m RNA levels of CDK2 in MCF-7 and MDA-MB-231 cells were increased.8.After transfection,the m RNA expression of E2F-1 and P21 was up-regulated in MCF-7 cells by q RT-PCR assay.The expression of Rb and E2F-1 was also up-regulated by Western blot.9.After transfection,flow cytometry assay showed that the ratio of G1/G0 was not significant changed,and cell clone formation assay showed that lnc RNA CRNDE have no significant effect in cell proliferation compared the control group.Conclusion 1.The lnc RNA CRNDE is negatively correlated with the metastasis of human breast cancer cells.It is low in high metastatic breast cancer and is high in low metastatic breast cancer.2.Lnc RNA CRNDE might inhibit EMT passway through snail mediated,and inhibit the migration ability and invasive ability of MCF-7 and MDA-MB-231.3.CRNDE was no statistically significant effect on cell proliferation.4.In breast cancer cells,the expression of lnc RNA CRNDE might be used as a new indicator to determine the ability of cell metastasis.