Effect Of CCNL1and TIMP1Genes On Cells Proliferation And Related Genes Expression In Human Breast Cancer Cells

Breast cancer is a common malignancy of women's health, cancer occurrenceand development is a multi-factor and multi-mutation accumulation process, Causeof cancer is unknown. The tissue inhibitors of metalloproteinases1(TIMP1) is notonly a matrix metalloproteinase inhibitor,but also as an anti-tumor markers toinfluence breast cancer prognosis. Many scholars have found that TIMP1involved intumor cell proliferation, apoptosis and tumor angiogenesis, and along with poorprognosis. Cyclin L1(CCNL1) was identified as a new nuclear protein recently,isbelong to cyclin L1proteins.CCNL1contains the C-terminal arginine and serinerich(RS)of the domain, play an important role in the first mRNA splicing. In headand neck squamous cell carcinoma (HNSCC) and CCNL1cancer occur in situ playsa key role,and as an indicator of occult advanced tumor stage.Therefore,CCNL1isconsidered a candidated gene biomarkers of head and neck squamous cellcarcinoma.In order to study the development and progression of breast cancer geneexpression changes,we randomly selected10patients with breast cancer and nearbreast cancer tissue,and differences in screening by gene chip technology of breastcancer gene expression in breast cancer and near breast cancer tissue.The303differentially expressed genes and112unknown genes analysis by microarryresearch. The analysis and verification of the difference was significant for33screened genes.We found that cell proliferation of CCNL1and TIMP1gene expression wassignificantly increased in these33selected differentially expressed genes. So wespeculated that some mechanism may exist between CCNL1and TIMP1andregulation of human breast cancer cells proliferation,and the existence of these twogenes may influence gene expression relationship with each other for the study ofthese two genes function. we first conducted a prokaryotic expression of its study byRT-PCR and cloned CCNL1and TIMP1genes which length were624bp and1581bp. CCNL1and TIMP1gene connected with the prokaryotic expression vector pET28awas successfully constructed E.coli expression vector pET28a-TIMP1andpET28a-CCNL1,express and purify the target protein by Western blot methoddetected CCNL1and TIMP1protein which size is28.5KD and59.6KD after identifythe correct species. To lay the foundation for future animal experiments.To further study the CCNL1and TIMP1gene to the breast cancer cell growthand the relationship between of CCNL1and TIMP1gene expression in breast cancercells. we put the KOZAK sequences connected with CCNL1and TIMP1gene5'endwhich were able to promote gene expression in eukaryotic. And these two geneswere connected with the eukaryotic expression vector pcDNA3.1to built CCNL1and TIMP1gene eukaryotic expression plasmid (pc-CCNL1-K,pc-CCNL1,pc-TIMP1-K and pc-TIMP1) successfully.while we constructed a full-length RNAinterference plasmid according to CCNL1and TIMP1genes. This gene eukaryoticexpression vector was transfected into MBA-MD-231and MCF-7cells and detectedthe CCNL1and TIMP1gene expression in MBA-MD-231and MCF-7cells. Theresults show that,KOZAK sequence can significantly improve the CCNL1andTIMP1gene expression in human breast cancer cell (MBA-MD-231andMCF-7).We build CCNL1and TIMP1genes interference plasmid and results showthat CCNL1RNAi-1173and TIMP1RNAi-229were best on the interference effectat the transcriptional level.These two genes over-expression plasmids were transfected into MBA-MD-231and MCF-7cells and detected CCNL1and TIMP1gene expression.The results showthat CCNL1overexpression can lead to increasing of TIMP1gene expression andTIMP1overexpression can cause CCNL1gene expression.These CCNL1andTIMP1genes interference plasmids transfected into MBA-MD-231and MCF-7cellsand detect the CCNL1and TIMP1gene expression. The results show that,CCNL1gene expression by the interference caused of TIMP1gene expression increasedsignificantly and TIMP1gene expression interfered with the increased geneexpression of CCNL1effect was not significant. For the further study of the interaction between two genes, The TIMP1gene over-expression plasmid andCCNL1interference plasmid were cotransfected the breast cancer cells. The resultshowed a significant increasing in TIMP1expression, TIMP1and CCNL1cooverexpression also showed significant expression of TIMP1and CCNL1.We use TIMP1and CCNL1gene interference plasmids and over-expressionplasmids were transfected into MBA-MD-231and MCF-7cells because of theTIMP1and CCNL1gene for cell growth regulation and detected cell growth byMTT. The results show that,TIMP1in breast cancer cells has a positive regulatoryrole and CCNL1was negative in breast cancer cells.The performance of a strongpromotion of cell proliferation when TIMP1overexpression and CCNL1interference together were transfected in breast cancer cells. The performanceshowed more pronounced inhibition of cell proliferation when CCNL1overexpression and TIMP1interference together were transfected breast cancercells.Cell proliferation is not significant changed when TIMP1and CCNL1overexpressed together were transfected in breast cancer cells. Cell proliferation alsono obvious changed when TIMP1and CCNL1interference together were transfectedin breast cancer cells.Description that TIMP1and CCNL1while raising in breastcancer cells were the rate of cell proliferation to maintain the internal adjustmentmechanism to maintain cell balance needs.We have reserch the TIMP1and CCNL1gene-related genetic changes anddetected CCNL1, MMP1, MMP2, CCNL2and P53gene expression changeed whenTIMP1and CCNL1gene over-expressed by Western blot. The results show that,TIMP1gene overexpression can cause of increasing expression of P53and MMP2,while CCNL1overexpression also affected the expression of P53and MMP2.TIMP1and CCNL1gene over-expression did not have significant impact of MMP1and CCNL2expression.The gene of Breast cancer and near breast cancer tissue changes proved by thepreliminary study, this study also found there was close connection between the geneTIMP1and CCNL1, and CCNL1genes on breast cancer cells growth was play a negative regulatory role. This new information will has several important functionson the study of the mechanism of breast cancer.