The Role And Mechanism Of MiR34a In Combination With Adriamycin And The Pharmacodynamic Evaluation Of The Co-delivery Nano-system Against The Progression Of Drug-resistant Breast Cancer

BackgroundMultidrug resistance of tumors has become the most serious problem that hinders the progress and prognosis of tumor chemotherapy.Doxorubicin(Dox)is an anthracycline anti-tumor drug that inhibits the growth of tumor cells by targeting Top 2A.Dox is a clinically first-line chemotherapeutic drug for the treatment of breast cancer,but its single-use inhibitory effect is not complete,especially in advanced breast cancer.After breast cancer cells develop resistance,they are more prone to invade and migrate,which makes clinical treatment more difficult and leading to a poor prognostic.Therefore,how to effectively reverse the resistance of breast cancer to Dox and improve the clinical symptoms of breast cancer patients has become a top priority.MicroRNA(miR)is a type of small non-coding RNA with a length of about 18-25 nucleotides,which mainly functions by binding to the target gene.The imbalance of miR expression is related to many diseases.Recent studies have shown that miR also plays a key role in the chemotherapy resistance of cancer.miR34a has been confirmed to participate in the occurrence and development of various tumors through multiple targets such as Bcl-2,CD44,C-MET,SIRT1,CDK,MYC or through multiple signaling pathways such as PI3K/Akt,Ras/Raf/Mek,Notch,et al.However,due to the numerous targets of miR,the mechanisms regulating drug resistance are still unclear.Based on the above research background,we speculate that the combination of miR34a and Dox may increase the sensitivity of drug-resistant breast cancer cells to Dox.Objective:The first research purpose is to explore whether miR34a can reverse breast cancer resistance to Dox,and whether the combination of miR34a and Dox can synergistically inhibit the progression of Dox-resistant breast cancer and elucidate the mechanism of miR34a in reversing drug resistance.miR34a with a negatively charge is easily degraded and unstable,which makes it difficult to get into the cell membrane,which greatly limits its application in vivo.So,the second research purpose is to solve the shortcomings of miR34a by constructing a nano-drug system simultaneously delivering miR34a and Dox in order to reverse breast cancer resistance.Specifically,it is divided into two parts:1)To study the mechanism of miR34a reversing breast cancer resistance to Dox;2)To study whether chitosan(CS)can carry miR34a into breast cancer cells,and whether a co-loaded Dox and miR34a nano-drug carrier system based on hyaluronic acid(HA)-targeted conjugated linoleic acid(CLA)modified chitosan could be constructed and the effect in vivo and in vitro was evaluated.Part I Study on the mechanism of miR34a reversing Dox resistance in breast cancerResearch purposes:Whether the combined administration of miR34a and Dox can synergistically inhibit the progression of drug-resistant breast cancer and its molecular mechanism was explored.Research methods:In this study,human breast cancer MCF-7 cells and Dox-resistant human breast cancer MCF-7/A cells were used as the experimental subjects in vitro,and BALB/c-nu nude mice were used as the experimental subjects in vivo.1)miR over-expression technology was employed to detect the effect of miR34a on the proliferation,invasion,metastasis and adhesion of MCF-7/A cells.2)The effect of miR34a in reversing the resistance of MCF-7/A cells to Dox and its molecular mechanism was investigated.The multiple of resistance of MCF-7/A cells and MCF-7 cells to Dox was detected;The synergic indexes when miR34a and Dox was co-administration and Dox was used alone were calculated.The best combined administration was screened to study the effects on the proliferation,apoptosis,invasion and metastasis of MCF-7/A cells.MTT method was used to detect the effects of the two on the proliferation of MCF-7/A cells,and Hoechst staining and Annexin V/P1 double staining method were used to detect the effect on cell apoptosis.The scratch and Transwell method were used to detect the effect of the combination on the invasion and metastasis of MCF-7/A cells.Western blotting was used to detect the protein expression changes in key proliferation/apoptosis proteins(Bcl-2,Bax,Cleaved PARP),migration-related proteins(E-cadherin,N-cadherin,CD44),and drug resistance-related proteins(P-gp,MRP,GST-?,BCRP,Top2A)when combination with miR34a and Dox.3)To explore the key target of miR34a affecting the biological effects of Dox-resistant breast cancer cells:miRDB target prediction software was employed to predict the key target of miR34a affecting drug resistance.miR34a transfection was used to over-express the target gene.Western blotting and RT-PCR method was used to detect whether miR34a had a regulatory effect on the target gene,and the plasmid over-expression method or siRNA interference method were used to detect the effect of the target gene on the proliferation,invasion and metastasis of Dox resistant and Dox sensitive cells.Western blotting was used to detect the influence of the target gene on the expression levels of invasion and metastasis related proteins such as E-cadherin and N-cadherin.The dual luciferase reporter gene detection system was used to verify the relationship between miR34a and the target gene.4)To detect the effect of miR34a and Dox co-administration on Notch/NF-?B and Ras/Raf/Mek/Erk signaling pathways,and Notch inhibitor(DAPT),NF-?B inhibitor(PDTC),Ras inhibitor(salirasib)and Mek inhibitor(Pimasertib)were introduced to verify whether the combination of miR34a and Dox can regulate the above-mentioned target genes through the above two pathways.5)A subcutaneous MCF-7/A tumor-bearing model of BALB/c-nu nude mice was established to study whether the combination of miR34a and Dox can synergistically inhibit the progression of breast cancer.Western blotting,immunofluorescence and immunohistochemical were employed to verify the related protein changes of Bcl-2,Bax,Snail,and so on.Experimental results:1)Compared with MCF-7 cells,Dox-resistant MCF-7/A cells are more prone to invade,migrate and adhere by the scratch and Transwell test.The level of E-cadherin protein in MCF-7/A cells were significantly down-regulated,while the protein expressions of N-cadherin and CD44 protein were significantly up-regulated in MCF-7/A cells by Western blotting.2)Compared with MCF-7 cells,the gene expression of miR34a in Dox-resistant MCF-7/A cells was significantly reduced.After MCF-7/A cells were transfected with 50nM,100nM and 150nM miR34a mimics,the expression of miR34a significantly increased determined by RT-PCR.MTT results showed that miR34a inhibited the proliferation,invasion and metastasis of MCF-7/A cells and the adhesion of HUVEC cells in a dose-dependent manner.Western blotting results showed that miR34a can significantly increased the protein expression of E-cadherin,and reduced the protein expression of N-cadherin and CD44 in MCF-7/A cells.3)The drug resistance index of MCF-7/A to MCF-7 cells was about 24.98.The combination of 25 nM,50 nM and 100 nM miR34a with different concentrations of Dox caused a decrease of the IC50 of Dox from 106.17 to 41.35,29.28 and 14.08 ?M,and the synergy index are 2.57,3.63 and 7.54,respectively.The combination of 10?M Dox and 100nM miR34a can significantly inhibit the proliferation of MCF-7/A cells and promote apoptosis,which may be related with the decrease of the ratio of Bcl-2/Bax and the increase of the protein expression of Cleaved PARP.4)The combination of 10 ?M Dox and 100 nM miR34a has no significant effect on the expressions of classical resistance proteins such as P-gp,GST-? and MRP,but can significantly reduce the protein expressions of BCRP and Top2A.Meanwhile,the combination could inhibit the efflux function of P-gp.5)The combination of 7 ?M Dox and 100 nM miR34a can significantly inhibit the invasion and metastasis of MCF-7/A cells and up-regulate the protein expression of E-cadherin and down-regulate the protein expression of N-cadherin.6)7 ?M Dox and 100 nM miR34a can significantly reduce the intracellular expression of Snail by IF and Western blotting.Transfection of miR34a in vitro lead to a decrease of Snail at the protein and mRNA levels.The miRDB target prediction website shows that Snail and miR34a have base complementary pairing binding sequences.The dual luciferase reporter gene detection system showed that only miR34a and wild-type Snail co-transfected group could reduce the relative ratio of luciferase,indicating that miR34a can directly target Snail.7)Small interfering RNA technology was used to interfere(knock down)the expression of Snail(Si-Snail)in MCF-7/A cells.Its effects on cell proliferation,invasion and metastasis,and adhesion was determined.The successful interference of Snail was verified by Western blotting.Si-Snail had no significant effect on the proliferation of MCF-7/A cells by MTT assay,but significantly inhibited the invasion,metastasis and adhesion of MCF-7/A cells by scratch test and Transwell assay.Si-Snail significantly down-regulated the expression of N-cadherin and CD44,and up-regulated the expression level of E-cadherin by Western blotting.Meanwhile,Rescue experiment showed Snail interference combined transfection of miR34a did not further inhibit the invasion and metastasis of MCF-7/A cells.On the contrary,the plasmid over-expression of Snail in MCF-7 cells was employed to detect the effects on cell proliferation,invasion and metastasis,and adhesion.The successful up-regulation of Snail was verified by Western blotting and RT-PCR.Snail over-expression has no significant effect on the proliferation of MCF-7 cells by MTT assay,but it significantly promoted the invasion,metastasis and adhesion with HUVECs cells of MCF-7 cells by scratch test and Transwell assay.Over-expression of Snail could significantly down-regulate the expression level of E-cadherin and up-regulate N-cadherin and CD44 by Western blotting.8)Western blotting experiments showed that the combination of Dox and miR34a inhibited the protein expression of Notch,NF-?B,pan-Ras,k-Ras,Raf,p-Mek and p-Erk.Notch inhibitor DAPT,NF-?B inhibitor PDTC,Ras inhibitor salirasib and Mek inhibitor pimasertib were introduced to the experiments,and results showed Dox and miR34a could dose-dependently inhibit the protein expression of Notch,NF-?B,pan-Ras,p-Mek and Snail.9)Combination of Dox and miR34a could significantly inhibit the progression of transplanted MCF-7/A breast cancer in nude mice,and reduced the protein expression of Bcl-2/Bax and Snail and increased the protein expression of PARP By Western blotting.IF and IH results showed that the protein expression of Snail in tumor tissues for the Dox and miR34a combined group was significantly reduced.Summary:miR34a can significantly increase the sensitivity of Dox in drug-resistant breast cancer.The combination of Dox and miR34a can significantly inhibit the proliferation,invasion and metastasis of breast cancer cells in vivo and in vitro,and induce apoptosis.The cooperative anti-tumor mechanism of the two may be achieved by regulating Snail directly or through Notch/NF-?B and Ras/Raf/Mek/Erk signalling pathway indirectly.Part ? Reversing drug resistance of breast cancer by CD44 targeted chitosan nano-system co-delivery of miR34a and DoxResearch purposes:In the part I,we have verified that the combination of miR34a and Dox can synergistically inhibit the progress of drug-resistant breast cancer.However,as a miR,miR34a can be easily degraded and cannot enter the cell membrane due to its negatively charge properties,causing it difficult to be used in clinic.In order to solve these shortcomings,the part ? of the research aimed to construct a targeted co-delivery vector for miR34a and Dox co-administration and the effects on proliferation,migration,invasion and adhesion were investigated,and the mechanisms in vitro and in vivo were explored.Research methods:1)CS/miR34a NPs were prepared by ion gel method,and the morphology,particle size and potential of CS/miR34a NPs were detected by TEM,particle size analyzer.qRT-PCR was used to detect the cellular level of miR34a,fluorescence microscope and flow cytometer was employed to detect whether CS/miR34a NPs can enter cells and the effects on cell proliferation,invasion and metastasis of MDA-MB-231 cells was evaluated.2)CLA-CS was synthesized,and 1H-NMR and FT-IR were used to verify the successful synthesis of CLA-CS,and the modification rate of CLA was calculated.Self-assembly method was used to prepare CLA-CS blank nanoparticles(NPs).Dox and miR34a were loaded into the NPs and HA were adsorbed on the surface of drug-loaded NPs to obtain a co-delivery NPs called CCmDH NPs.3)TEM was used to observe the morphology of CCmDH NPs,and the particle size and potential were determined.Ultraviolet spectrophotometer was used to detect the encapsulation rate and drug loading of Dox.The in vitro release of Dox from CCmDH NPs was examined.Agarose gel electrophoresis was used to detect the loading of miR34a by CCmDH NPs,and to detect whether CCmDH NPs could resist degradation against RNase and serum.4)Fluorescence microscope and flow cytometry were used to detect whether CCmDH NPs can be taken up by MCF-7/A cells.Western blotting was used to detect the protein expression of CD44 in MCF-7/A and MCF-7 cells,and qRT-PCR was used to detect whether CCmDH NPs could increase the level of miR34a in MCF-7/A cells.5)MTT assay was used to detect whether CLA-CS is toxic to normal cell HUVEC.The effects of free Dox and CCmDH NPs on the proliferation of MCF-7/A cells at the same Dox dose were examined by MTT,and the effects of free Dox and CCmDH NPs on the apoptosis of MCF-7/A cells were employed by Hoechst staining.Western blotting was used to detect the protein expression changes of cleaved PARP and Bcl-2.The scratch and Transwell method were used to detect the migration and invasion of MCF-7/A cells in the free Dox and CCmDH NPs group.And Western blotting was used to detect protein changes of metastasis-related proteins such as E-cadherin,N-cadherin,MMP2,CD44 and Snail.6)IVIS Kinetic in vivo imaging system was employed to detect the targeting ability of CCmDH NPs in vivo.After constructing the transplanted MCF-7/A tumors in nude mice,normal saline,miR34a,Dox,CCmD NPs and CCmDH NPs was administrated to evaluate the anti-tumor effect of each group,and the tumor inhibition rate was calculated.Western blotting was used to detect the protein changes of Bcl-2,Bax,Snail,E-cadherin and N-cadherin in tumor tissues after different treatment.IH and IF were used to detect the changes of the key target protein in the tumor tissue.At last the effects of CCmDH NPs on related signaling pathway of CCmDH NPs were evaluated.Experimental results:1)The prepared CS/miR34a NPs has an average particle size of about 135 nm and a surface potential of about 34 mV.The CS/miR34a NPs have a good stability which can resist the degradation of serum and RNase.And CS/miR34a NPs can be taken up by MDA-MB-231 cells,leading to a significant increase of miR34a in the cells.And CS/miR34a NPs can inhibit the proliferation,invasion and migration of MDA-MB-231 cells.2)CLA-CS was successfully synthesized verified by 1H-NMR and FT-IR,and the substitution degree of CLA on CS was about 9.03%.CCmDH NPs are spherical without adhesion by TEM.Dox NPs had a particle size of about 323.33 nm and a potential of 35.7 mV by DLS.After miR34a and HA are adsorbed,the particle size reduced to 240.1 nm and 180 nm,and the potential reduced to 35.3 mV and 16.5 mV.CCmDH NPs could release Dox more easily in acidic medium(pH5.0 PBS)than physiological medium(pH7.4 PBS)and has certain slow-release characteristics within 96 h.3)CCmDH NPs could effectively protect miR34a from the degradation of serum and RNase enzyme by agarose gel electrophoresis.4)CCmDH NPs can be uptaken up by MCF-7/A cells detected by Fluorescence microscope.Compared with MCF-7 cells,the level of miR34a in MCF-7/A cells was significantly reduced and CCmDH NPs can increase the expression of miR34a in MCF-7/A cells,indicating that the constructed CCmDH NPs can effectively transfer miR34a into MCF-7/A cells.5)The synthesized CLA-CS has no obvious toxic effect on the proliferation of normal cell HUVECs.CCmDH NPs can inhibit the proliferation of MCF-7/A in a dose-dependent and time-dependent manner.CCmDH NPs group could significantly induce apoptosis by Hoechst experiment.And the protein expression of Cleaved PARP in the CCmDH NPs group was significantly increased,while the expression of Bcl-2 protein was significantly decreased.6)The blank NPs group and the miR34a group have almost no effect on cell migration and invasion.For miR34a NPs and CCmDH NPs groups,cell migration was obviously inhibited,and CCmDH NPs had a stronger inhibitory effect.The expression of E-cadherin protein in the CCmDH NPs group was significantly increased,while the expressions of N-cadherin,MMP2,CD44 and Snail protein were significantly reduced by Western blotting.7)In vivo imaging results of small animals showed that CCmDH NPs not only have tumor targeting properties but also have certain slow-release properties.8)The tumor inhibition rate of the CCmDH NPs group was about 73.7%,while the tumor inhibition rates in the Dox,miR34a or CCmD NPs groups were 55.3%,42.5%,and 33.9%,respectively.The protein level of Bcl-2,N-cadherin and Snail in the CCmDH NPs group was significantly reduced,and the level of Bax,E-cadherin was significantly increased by Western blotting.IH and IF indicated that Snail protein was highly expressed in tumor control group,while CCmDH NPs group could significantly reduced the level of Snail.9)CCmDH NPs can significantly inhibit the expressions of K-Ras,NF-?B and Notch in the Notch/NF-?B and Ras/Raf/Mek/Erk pathways.Summary:CS/miR34a NPs can significantly inhibit the proliferation,invasion and metastasis of breast cancer cells.The prepared CCmDH NPs are spherical,with a particle size of about 180 nm and a potential of about 16 mV.CCmDH NPs can resist the degradation of serum and RNase and can be uptaken by MCF-7/A cells,causing an increase of miR34a level in MCF-7/A cells.CCmDH NPs can promote cell apoptosis by regulating the expressions of Bcl-2/Bax and PARP,and inhibit cell invasion and metastasis by regulating the expressions of E/N-cadherin,CD44,Snail,and MMP2 both in vivo and in vitro.It has a targeting effect and a certain slow-release effect.It can significantly inhibit the growth of MCF-7/A tumors in nude mice,and can inhibit Notch/NF-?B and Ras/Raf/Mek/Erk signaling pathways.Conclusions:1)miR34a can significantly increase the sensitivity of drug-resistant breast cancer cells to Dox.The combination of Dox and miR34a can significantly inhibit the proliferation,invasion and metastasis and induce apoptosis of drug-resistant breast cancer cells in vitro and in vivo.The underlying mechanism may be achieved by regulating Snail directly or through Notch/NF-?B and Ras/Raf/Mek/Erk signaling pathways indirectly.2)The prepared nano drug delivery system based on CLA-CS and HA(CCmDH NPs)can simultaneously load Dox and miR34a with a good stability,and can protect miR34a from degradation of serum and RNase.CCmDH NPs can significantly promote the cell uptake of Dox and miR34a.CCmDH NPs can inhibit proliferation,invasion and metastasis and induce apoptosis of MCF-7/A cells through regulating the protein level of Bax,PARP,Bcl-2,E-cadherin,N-cadherin,CD44,Snail,MMP2,etc.In addition,CCmDH NPs can significantly inhibit the progression of Dox-resistant breast cancer,which may be related to the regulation of Notch/NF-?B and Ras/Raf/Mek/Erk signaling pathways.