Role And Mechanism Of ROS/miR-200c/ZEB1 Axis In UTMD Inhibition Of Breast Cancer EMT

Background:Breast cancer is one of the most common malignant tumors in women,and its inci-dence is increasing year by year.Recurrence and metastasis of breast cancer are the main causes of patients' poor prognosis and death.Epithelial-mesenchymal transition(EMT)is a key regulator of aggressive invasion and metastasis.During the EMT pro-cess,the epithelial cells lose the apical-basal polarity and cell-cell adhesion and get mesenchymal characteristics.The major EMT markers comprise epithelial markers,such as E-cadherin,and mesenchymal markers,such as Vimentin,N-cadherin,and fi-bronectin.More than 95%of breast cancer cells are derived from epithelial cells,and studies have confirmed that metastasis of breast cancer,progression of ductal carci-noma in situ to invasive breast cancer,changes in tumor immune microenvironment,and chemotherapy drug resistance are all closely related to the EMT process.Therefore,the inhibition of EMT process is one of the most important problems in clinical treat-ment of breast cancer.Ultrasound-targeted microbubble destruction(UTMD)is one of the hotspots of ul-trasound research in recent years,and has shown a great potential in the diagnosis and treatment of tumors.Ultrasound combined with micro/nano contrast agent delivery sys-tem is a promising non-invasive targeted drug delivery/gene technology,which has many advantages such as high drug load,targeting,modifiable,increased efficacy and reduced adverse reactions.In tumor therapy,UTMD can not only serve as the carrier of anti-tumor drugs and genes,but also play an important role in inducing tumor cell apoptosis,increasing chemotherapy sensitization and reducing toxic and side effects,reversing multi-drug resistance of tumor cells.However,the mechanism of UTMD is still unclear.The effect of UTMD in inhibiting EMT has not been systematically stud-ied.Therefore,the first step of this study was to verify the role of UTMD in inhibiting breast cancer EMT.Studies have confirmed that sonoporation is the main mechanism for UTMD to achieve targeted drug release,but it is insufficient to explain the multiple biological effects produced by UTMD.Endocytosis is one of the mechanisms of UTMD-mediated drug/gene targeted delivery.Under the treatment of UTMD,a certain amount of hydro gen peroxide(H2O2)is produced to make the calcium ion flow,and the calcium-de-pendent potassium ion channel is opened,thus stimulates the endocytosis.H2O2 is the main component of Reactive oxygen species(ROS).Sonodynamic therapy(SDT),as a new tumor treatment technology,has been proved by a large number of studies that ROS production is the main mechanism of its anti-tumor effect.SDT takes advantage of the powerful penetrating ability of ultrasound to penetrate deep into the tissue,acti-vates the sonodynamic agent,and produces ROS to kill tumor cells.Therefore,we pro-posed the following hypothesis:is ROS production also an important mechanism of UTMD?The production of ROS is an important factor in aging and disease.A lot of studies have elucidated the molecular mechanism of ROS/miRNA axis and its role in tumor-igenesis.Studies have confirmed that ROS and miR-200c form positive feedback loops.ROS induces upregulation of miR-200c,and miR-200c also influences the pro-duction of ROS by regulating the expression of target genes.miR-200c was found to be significantly decreased in various malignant tumors such as breast cancer,ovarian cancer and endometrial cancer,which can up-regulated the expression of its target fac-tors ZEB1 and ZEB2 to enhance the migration and invasion ability of tumors.miR-200c is one of the main regulatory factors of EMT.In some malignant tumors,miR-200c and ZEB1 regulate the occurrence and development of tumors by forming a two-way negative feedback loop.The down-regulation of miR-200c expression and up-reg-ulation of ZEB1 expression in MDA-MB-231 and BT549 cells of triple negative breast cancer were closely related to the poor prognosis of breast cancer.PART ?A preliminary study on the inhibiting effect of UTMD in the breast cancer MDA-MB-231 cells EMT processObjectives:(1)The experiments in vitro confirmed that UTMD can inhibit breast cancer EMT.(2)To preliminarily verify that UTMD can improve intracellular ROS level.Methods:Human breast MDA-MB-231 cells were treated with MB,US and UTMD,respec-tively.The generation of oxidative stress and the levels of miR-200c,ZEB1 and Vi-mentin were evaluated quantitatively and qualitatively by the measurement of intracel-lular ROS,qRT-PCR,western blot assay and transwell assay.Results:1.The inhibitory effect of UTMD treatment on EMT of MDA231 cells(1)The western Blot results showed that ZEB1 and Vimentin expression levels of MDA-MB-231 cells of the US group and UTMD group were significantly decreased(P<0.05),Compared with the US group,the expression levels of the two proteins in UTMD group were more significantly decreased(P<0.05).(2)The transwell assay support the conclusion from the point of the tumor cells mi-gratory ability.It proved that UTMD can suppress migration in MDA-MB-231 cells,which indicating that the UTMD can be potentially used in EMT inhibition.2.The intracellular ROS generation induced by UTMDThe intracellular ROS levels were analyzed qualitatively and quantitatively by fluo-rescence microscopy and flow cytometry,respectively.Fluorescence microscopy results showed that,compared with the control group,the ROS fluorescence in cells of the US and UTMD groups was significantly increased,which confirmed that the intracellular ROS level was increased.Under the treatment of UTMD,the intracellular ROS fluorescence of MDA-MB-231 cells in UTMD+NAC group was not obvious.Flow cytometry results were as follows:control:0.72±0.21%;MB:1.27±0.42%;US:1 1.02±0.75%;UTMD:29.04±0.67%;UTMD+NAC:2.67±0.22%.Compared with the control group,the intracellular ROS levels of the US and UTMD groups were signifi-cantly increased(P<0.05 and P<0.001).The ROS level of UTMD group was higher than that of US group(P<0.05).There were no statistically significant differ-ences between the UTMD+NAC group and the control group(P>0.05).Conclusions:1.The results of the first part of the study showed that under the treatment of UTMD,the expression levels of EMT-related proteins Vimentin and ZEB1 were significantly decreased,and the number of cell migration was reduced,which proved that UTMD could inhibit the EMT process of breast cancer for the first time.2.We confirmed that the intracellular ROS level was increased under the treatment of UTMD,and the antioxidant NAC could effectively reduce the intracellular ROS level.Therefore,we speculated that the production of ROS might be an important mechanism of UTMD,which was further confirmed in the following experiments.PART ?UTMD modulates the EMT process through the regulation of the ROS/miR-200c/ZEB1 axisObjectives:We explored the mechanism of UTMD in inhibiting breast cancer EMT,and inves-tigated that the generation of ROS induced by UTMD inhibited EMT through the reg-ulation of miR-200c/ZEB1 axis.By adding ROS inhibitors and silencing miR-200c expression,the role of ROS/miR-200c/ZEB 1 axis in UTMD inhibition of breast cancer MDA-MB-231 cells EMT was verified.Methods:In order to prove the role of UTMD induced oxidative stress and miR-200c in the EMT inhibition,The ROS scavenger NAC and miR-200c-3p inhibitor were used before UTMD treatment.The effects of NAC and miR-200c-3p inhibitor on the levels of miR-200c,ZEB1 and Vimentin expression under UTMD treatment were evaluated by the measurement of qRT-PCR,western blot assay and transwell assay.Results:1.The expression of miR-200c modulate by UTMDThe qRT-PCR results shown that,compared with the control group,the expression of miR-200c was up-regulated to 1.64 folds in the US group and 2.56 folds in the UTMD group(P<0.05).The expression of miR-200c in UTMD group was higher than that in US group(P<0.05).The cells treated with MB alone did not show any signifi-cant changes in miR-200c expression.2.UTMD inhibits the EMT process through the regulation of the ROS/miR-200c/ZEB1 axis(1)Inhibition of intracellular ROS generation under the treatment of UTMD reduces the expression of miR-200cCompared with UTMD group,the expression levels of miR-200c in UTMD+NAC group and UTMD+miR-200c inhibitor group were significantly decreased(P<0.05).the expression of miR-200c in UTMD+NAC and UTMD+ miR-200c inhibitor group have no statistically significant difference to the control group(P>0.05).(2)Under the treatment of UTMD,the down-regulation of the miR-200c expression can up-regulate the expression of EMT-related proteins expressionThe western Blot results showed that ZEB1 and Vimentin expressions of MDA-MB-231 cells in the UTMD+NAC group and the UTMD+miR-200c inhibitor group were significantly increased compared to the UTMD group(P<0.05).ZEB1 and Vimentin expressions in UTMD+NAC group and UTMD+miR-200c inhibitor group were not significantly different from those in control group(P>0.05).(3)Under the treatment of UTMD,the down-regulation of the miR-200c expression can enhance the cell migration ability.The transwell assay results revealed that a significant decrease of cell migration was observed in the UTMD group(P<0.05 vs.control,UTMD+NAC,UTMD+miR inhib-itor),and no significant difference was found among the control,UTMD+NAC,and UTMD+miR inhibitor groups(all P>0.05).Conclusions:UTMD up-regulated miR-200c expression.The role of ROS and miR-200c in the inhibition of breast cancer EMT induced by UTMD was verified by adding NAC and miR-200c-3p inhibitor,respectively.The results showed that under the treatment of UTMD,the inhibition of ROS or down-regulation of miR-200c expression could up-regulated the expresssion of EMT-related proteins Vimentin and ZEB1,and increased the number of migratory cells.These results confirmed that UTMD inhibited breast cancer EMT through ROS/miR-200c/ZEB1 axis.PART ?TGF-?1 induces EMT model of breast cancer MCF-7 cells to verify the effect of UTMD in inhibiting breast cancer EMTObjectives:(1)TGF-?1 induced EMT model of breast cancer MCF-7 cells.(2)To detect the effect of UTMD on the EMT process of breast cancer MCF-7 cells.(3)To verify the role of ROS/miR-200c/ZEB1 axis in UTMD's inhibition of EMT in breast cancer MCF-7 cells.Methods:MCF-7 cells were stimulated by TGF-?1.The morphological changes of MCF-7 cells were observed by microscope.The expression of Vimentin and E-cadherin in MCF-7 cells was detected by western blot assay.Human breast MCF-7 cells were treated with MB,US and UTMD,respectively.The expression of Vimentin,ZEB1 and E-cadherin were evaluated quantitatively by west-ern blot assay.The numbers of migratory cells were evaluated by transwell assay.In order to prove the role of UTMD induced oxidative stress and miR-200c in the EMT inhibition,The ROS scavenger NAC and miR-200c-3p inhibitor were used before UTMD treatment.The effects of NAC and miR-200c-3p inhibitor on the levels of Vi-mentin,ZEB1 and E-cadherin expression and the numbers of migratory cells under UTMD treatment were evaluated by the measurement of western blot assay and transwell assay.Results:1.TGF-?1 can induce EMT model of breast cancer MCF-7 cellsMicroscopically,the cells were elongated and spindle shaped under the stimulation of TGF-?1.Western Blot results showed that compared with the control group,E-cad-herin expression was decreased and Vimentin expression was increased in both 10ng/mL and 20ng/mL TGF-?1 treatment groups after 48h treatment.Compared with the 20ng/mL TGF-?1 group,the decrease of E-cadherin expression and the increase of Vimentin expression were more obvious in the 10ng/mL TGF-?1 group than in the 20ng/mL TGF-?1 group.Therefore,the concentration of TGF-?1 in subsequent exper-iments was determined to be 10ng/mL.2.UTMD inhibits the EMT process of breast cancer MCF-7 cellsCompared with the control group,the expression of ZEB1 and Vimentin in MCF-7 cells in US group and UTMD group was decreased,the expression of E-cadherin was increased,and the number of migratory cells was decreased(P<0.05).The UTMD group had more significant changes in all indexes(vs.US group,P<0.05).There were no significant differences in the expression of three proteins and the number of migra-tory cells between MB group and control group(all P>0.05).3.UTMD regulates the EMT of breast cancer MCF-7 cells through the ROS/miR-200c/ZEB1 axisCompared With the UTMD group,the expression of ZEB1 and Vimentin in MCF-7 cells in the UTMD+NAC group and the UTMD+miR-200c inhibitor group were in-creased,the expression of E-cadherin was decreased,and the number of migratory cells was increased(all P<0.05).The expression levels of the above three proteins and the number of migrated cells in UTMD+NAC group and UTMD+miR-200c inhibitor group were not significantly different from those in the control group(all P>0.05).Conclusions:1.10ng/mL TGF-?1 can successfully induce EMT model of breast cancer MCF-7 cells.2.Under the effect of UTMD,the expression of Vimentin and ZEB1 in breast cancer MCF-7 cells decreased,the expression of E-cadherin protein increased,and the number of migratory cells decreased,which confirmed that UTMD can inhibit the EMT process of breast cancer MCF-7 cells.3.The role of ROS and miR-200c in UTMD inhibition of EMT in breast cancer MCF-7 cells was verified by adding NAC and miR-200c inhibitor,respectively.We speculated that UTMD could inhibit the EMT of breast cancer MCF-7 cells through the ROS/miR-200c/ZEB1 axis.In conclusions,this study confirmed that UTMD can inhibit the process of breast cancer EMT through in vitro studies on MDA-MB-231 cells and MCF-7 cells.These results indicated that UTMD could induce the production of intracellular ROS and in hibit EMT by regulating the ROS/miR-200c/ZEB1 axis,which may be the potential molecular mechanism of UTMD inhibiting EMT.This new mechanism provides a new strategy for UTMD to be used in cancer therapy.