The Relevant Research Of Between Tight Junction Protein Occludin And The Biological Characteristics Of Human Breast Cancer Cells

Objective：(1)To determine the expression of occludin gene in different human breastcancer cell lines MCF-7、MDA-MB-231、SKBR-3. To study the relationship between thechange of occludin and malignancy multiplication in different human breast cancer cell linesMCF-7、MDA-MB-231、SKBR-3.(2) To construct plasmid vector pcDNA3.1(+)-occludin, then,occludin gene was transfected into human breast cell lines MDA-MB-231and SKBR-3which is relatively hyp-expression occludin gene. To investigate how occludin geneaffect the cell proliferation、apoptosis and migrate in MDA-MB-231and SKBR-3.Methods:(1)The mRNA and protein expression of occludin in human breast cell linesMCF-7、MDA-MB-231、SKBR-3were examined by RT-PCR, Western-blot andimmunohistochemistry. Cell multiplication was determined by MTT. The cell cycleand Apoptosis was measured by flow cytometry (FCM). the migration of cells wasdetected by Transwell; the adherence was detected by Fibronectin protein.(2) PCRamplication occludin gene,which was used to construct plasmid vector pcDNA3.1(+)–occludin.(3)A recombinate pcDNA3.1(+)–occluding and an empty pcDNA3.1(+) weretransfered into MDA-MB-231and SKBR-3cells by useing LipofectaminTM2000.After transfection, RT-PCR, immunohistochemistry and Western-blot methods wereused to analyze expression of occludin mRNA and protein in MDA-MB-231andSKBR-3cells before and after transfection.(4) Transwell test, Fibronectin protein, FCMtest were used to search for the effects of occludin expression on transfected cellsproliferation, migration, adherence, apoptosis.Results:(1)Compared with MCF-7cell, the expression of occludin mRNA and proteinwere lower in MDA-MB-231and SKBR-3cells(P<0.05), while the speed of maliganatmultiplication and synthesis period were higher(P<0.01).(2)The plasmid vectorpcDNA3.1(+)-occludin was constructed successfully. Gene sequencing was not found basic group deficiency and mutation.(3)The occludin gene was transfected successfullyto MDA-MB-231and SKBR-3by using LipofectamineTM-2000, the subclone cell linesMDA-MB-231/pcDNA3.1(+)-occludin and SKBR-3/pcDNA3.1(+)-occludin, whichhighly expressed occludin were successfully selected.(4) Compared withMDA-MB-231and MDA-MB-231/pcDNA3.1(+) cells, the results of FCM、Fibronectinprotein and traswell in MDA-MB-231/pcDNA3.1(+)-occludin and SKBR-3/pcDNA3.1(+)-occludin cells indicated:growth velocity obviously steped down(P<0.01),the percentage of G1phase obviously increased and cell number in S phase weresharply decreased(P<0.01), the percentage of cell apoptosis were obviouslyincreased(P<0.01), penetrating membrance cells were decreased(P<0.01). the adhesioncells were fewer (P<0.01).Conclusions:(1) occludin and human breast cancer cellsmalignant multiplication has certain negative relevance.(2) Exogenous occludinexpresion may inhibit growth、migration、adherence and promote apoptosis in humanbreast cells MDA-MB-231and SKBR-3.