Study On The Mechanism Of Shear Stress Regulating Breast Tumor Cells Migration In Suspension Mechanical State

Hematogenous metastasis(HM)of tumor cells is one of the main ways to cause distant metastasis of tumor and the derived target organ failure is the main cause of tumor death.The tumor cells experienced HM acquired elevated migration ability,in addition to biochemical factors,the microenvironment mechanical stimulation of tumor cells in HM is also an important factor enhancing tumor metastasis.The migration ability of tumor cells was always enhanced after HM.It was confirmed by preliminary studies of our laboratory that suspension mechanical state(Sus)participated in the migration process of breast tumor cells(BTCs)by regulating cyclooxygenase-2(COX-2)and the migration of BTCs was also regulated by shear stress(SS)through extracellular signal-regulated kinase,respectively.However,it is now not fully understood that the effect of the combination of Sus and SS(Co SS),that is SS under Sus,on tumor migration ability and the relevant biomechanical mechanism.Therefore,from the perspective of biomechanics,this study combines the two single factors of Sus and SS in HM to deeply explore the influence of SS under Sus on metastasis of BTCs and its possible mechanobiological mechanism.The main research contents and results are as follows:(1)Co SS regulates the migration of BTCs by nuclear lamina protein A/C(Lamin A/C)and large tumor suppressor(LATS)through yes-associated protein(YAP)MDA-MB-231 and SK-BR-3 cells were traditionally cultured adherently and cultured on low-adhesion culture plate with suspended cells loaded with SS by coaxial cylindrical rotational viscometer.The effect of Co SS on the migration of BTCs was examined.It is revealed that although the migration ability,invasion ability,aggregation of cytoskeleton(F-actin)and accumulation of Lamin A/C protein were weaker than those in Sus,they were still up-regulated significantly compared with these in adhesion state(Adh).It is suggested that Co SS promotes BTCs migration,in which Lamin A/C may be involved.Lentiviruses were transfected into MDA-MB-231 cells to ensure the different expression of LMNA gene.It was found that the knockdown of LMNA inhibited the migration of BTCs under Co SS.In line with it,the results of animal model of tumor metastasis revealed that the knockdown of LMNA inhibited the migration of BTCs in vivo.In order to explore the possible mechanobiological mechanism of Lamin A/C in regulating the migration of BTCs under the Co SS,the regulation of Lamin A/C on mechanotransduction signaling factor YAP and YAP on the migration of BTCs were examined.In Sus,YAP was mainly distributed in the cytoplasm of MDA-MB-231 and SK-BR-3 cells but entered the nucleus after suspended cells being loaded with SS partially,which reflecting the response of YAP to Sus and Co SS.In MDA-MB-231 cells,knockdown of LMNA inhibited YAP downstream genes in Sus,while it was promoted under Co SS.This trend was opposite in MDA-MB-231 cells overexpressing LMNA.It was suggested that the high expression of Lamin A/C in Sus made the cell obtain enhanced ability of binding YAP to nucleus,while restricted YAP from entering the nucleus in Co SS.Lentiviruses were transfected into MDA-MB-231 cells to ensure the different expression of YAP gene.It was demonstrated that knockdown of YAP decreased but the overexpression of YAP increased the migration of BTCs.In order to explore whether LATS,a classical regulator of YAP,participate in YAP dependent BTCs migration regulation under Co SS,the activation of LATS and its influence on BTCs migration were tested.The results illustrated that the protein expression of p LATS increased in Co SS than that of Adh,though the expression level was lower than that of Sus.MDA-MB-231 cells were transfected with lentivirus to achieve stable knockdown of LATS gene.It was found that knockdown of LATS decreased the migration of BTCs.(2)Co SS enhances transforming growth factor-β(TGF-β)involved EMT of BTCs through YAP by microRNA-29bAs EMT was supposed to be one of the key factors for the enhancement of BTCs migration,the mechanobiological mechanism of Co SS promoting the migration of BTCs was intend to be studied in depth from the perspective of regulation of YAP on EMT.The EMT of BTCs under Co SS was detected.The results showed that the nuclear localization of YAP in epithelial BTCs could also be regulated by Co SS and EMT occurred reversibly in MCF-7 and SK-BR-3 cells.To verify the effect of nuclear localization change of YAP under Co SS on EMT of BTCs,the overexpression and knockdown of YAP in MCF-7 cells were carried out by lentivirus transfection,respectively.It was found that the overexpression of YAP promoted the occurrence of EMT but knockdown of YAP inhibited it.To find out the regulatory mechanism of YAP on EMT under Co SS post-transcriptionally,the microRNA sequencing results of the suspended and adherent MDA-MB-231 cells aquired by our group before were analyzed.Gene expression detection not only verified the sequencing results,but also confirmed that the expression level of microRNAs under Co SS was located between that in Adh and Sus in MCF-7cells.Among them,although microRNA-29 b regulating EMT can not regulate YAP reversely,it is a downstream target gene of YAP,which is determined to be the key microRNA for the following study.At the same time,the overexpression of microRNA-29 b inhibited the occurrence of EMT but knockdown of microRNA-29 b promoted.In addition,the expression of TGF-β regulated negatively by microRNA-29 b was increased under Co SS,which indicated that TGF-β involved in EMT regulation of BTCs by YAP/microRNA-29 b.Moreover,it was further confirmed that knockdown of TGF-β by small interfering RNA(si RNA)inhibited EMT and cell migration but TGF-βpretreatment promoted.In this study,it was proved that the migration of BTCs was promoted by Lamin A/C through YAP,and the mechanobiological mechanism behind was that YAP mediated BTCs EMT through microRNA-29 b post-transcriptionally.The result of this study is helpful to understand the vital role of the effect of SS under Sus in the HM on BTCs migration and provide a substantial theoretical basis for the development of new drugs and clinical prognosis targeting Lamin A/C,YAP and microRNA-29 b.