LPS Cooperates With TGF-β1to Promote The Invasive Migration Of Breast Cancer Cell

Objective: To investigate the biological effect of both LPS and TGF-β1on thesubtype of breast cancer—luminal-A—MCF-7and its underlying molecularmechanism. Methods:(1) In vitro, four groups are established: control group, grouptreated with LPS alone, group treated with TGF-β1alone and group treated with bothLPS and TGF-β1.(2) Using inverted microscopy to observe the morphology ofMCF-7cells after they are incubated with cytokines.(3) Using Rhodamine staining toobserve the cytoskeletons of MCF-7cells.(4) Using transwell method to judgeMCF-7cells’ capability of invasion and migration.(5) Through Gelatin zymographyto detect expression level of matrix metalloproteinase.(6) With real-time PCR toidentify epithelial-mesenchymal transition (EMT) by analyzing EMT related genesincluding E-cadherin, Vimentin, Snail-2.(7) Performing western blot assay to detectSmad signal pathway.Results:(1) MCF-7cells in the group treated with both LPS and TGF-β1haveobtained an elongated, mesenchymal phenotype, while most cells in both controlgroup and LPS group have a cobblestone-like appearance. And only a small part ofcells in TGF-β1group have an elongated, mesenchymal phenotype.(2) The results ofRhodamine staining show that the cytoskeletons of MCF-7cells in LPS combinedwith TGF-β1group change much, with obvious assemble of actin. Under thefluorescence microscope, we find that the percentage of typical mesenchymal cells inall the cells of control group and LPS group is1%, and the percentage of typicalmesenchymal cells in all the cells of TGF-β1group is2%. Particularly, the percentagein LPS combined with TGF-β1group is4%, and compared with other groups, theresult have statistical significance (P<0.01).(3) The results of transwell method showthat under microscope, the average number of cells passing through transwell in control group and LPS group is only1, and in TGF-β1group the average number isthree, particularly, the average number in LPS combined with TGF-β1group is six.Compared with other groups, the results of LPS combined with TGF-β1group havestatistical significance (P<0.01).(4) The results of gelatin zymography assay showthat MCF-7cells in control group and LPS group secrete very little activated MMP-9.Besides, TGF-β1group secretes twice activated MMP-9as much as secretion ofcontrol group or LPS group. Particularly, LPS combined with TGF-β1group secretesactivated MMP-9twice than TGF-β1group, and the results have statisticalsignificance.(5) The results of using real-time PCR to detect EMT-related genesshow that little change of E-cadherin, Vimentin and Snail-2in both LPS group andcontrol group is found; that in TGF-β1group, the expression of E-cadherin decreasesin a transient way and the expression of Vimentin increases in a transient way, onlySnail has a continuous-up expression; in the group treated with both LPS and TGF-β1,E-cadherin has decreased persistently, while Vimentin and Snail-2has increasedpersistently.(6) The results of using real-time PCR to detect receptors of TGF-β1show that little change of TGF-βRI expression is found in control group and LPSgroup. TGF-β1group has twice expression level of TGF-βRI, particularly, LPScombined with TGF-β1group has four times up-regulation.(7) The results of westernblot show that phosphorylation of Smad2in TGF-β1group reaches the highest levelat the time of half an hour, and then decreases as time goes by. By the end of threehours, the phosphorylation level returns to the initial level. However, when we test thephosphorylation of Smad2in the seventh day of stimulation, the results show thatonly LPS combined with TGF-β1group still express high level of phosphorylatedSmad2.Conclusions: LPS cooperating with TGF-β1can promote migration and invasionof MCF-7cells by activating EMT related genes such as Vimentin, Snail-2, enhancingthe expression level of TGF-βRI and maintaining the phosphorylation of Smad2.