MiRNA-129-1-3p In Reducing The Pirubicin-induced Cardiotoxicity By Rutin And The Sensitization Of Pirubicin Anti-breast Cancer Effects

As a common chemotherapy drug for a variety of solid tumors and malignant tumors of the hemolymph system,anthracycline pirarubicin(THP)has exact efficacy and strong anti-tumor effects.Breast cancer is one of the cancers treated with THP as the first-line drug.In the early diagnosis over the last 20 years,the mortality rate of breast cancer has been significantly reduced by surgery,chemoradiotherapy and targeted therapy.However,the improvement of survival rate has made people realize the importance of improving THP-induced cardiotoxicity in anti-cancer treatment.Clinical observations and studies showed that,one of the main toxic and side effects of THP is cardiotoxicity,which is often progressive and irreversible,significantly limiting its extensive and long-term use in clinical practices.Therefore,it is of great importance to find new drug targets to intervene in THP-induced cardiotoxicity.Rutin(RUT),a flavonoid,is one of the main active components of Sophora japonica,a traditional Chinese medicine listed in the Chinese Pharmacopoeia.It widely exists in Sophora japonica,Ginkgo biloba,jujube and other Chinese medicinal materials.RUT has pharmacological effects such as anti-free radical,anti-lipid peroxidation,anti-inflammatory,anti-cancer and cardiovascular protection.The research group has been carrying out research on the cardiovascular system protection of RUT for years.Rat experiment confirmed that RUT has protection against THP-induced cardiotoxicity.Besides,the microRNA(miRNA)expression profile of RUT's intervention of the THP-induced cardiotoxicity was constructed by miRNA chip technology.However,the specific mechanism of action of RUT has not been thoroughly explored.Besides,little is known about the role of miRNA in RUT's intervention in the pathogenesis and progression of the disease.In this study,it was found that miR-129-1-3p is a potential biomarker closely correlated with RUT's inhibition of cardiotoxicity and is highly conserved in different species on the basis of the analysis results of the miRNA expression profile of RUT's intervention of THP-induced myocardial damage and by miRbase database.Then,the target gene of miR-129-1-3p was searched by the Target Scan database.The KEGG enrichment analysis of the target gene showed that the Ca²⁺pathway in the results of the enrichment analysis was correlated with the mechanism of cardiotoxicity induced by anthracyclines.The target genes in the Ca²⁺pathway were selected and found that GRIN2D and miR-129-1-3p had the best targeting.Thus,it is speculated that RUT regulates the Ca²⁺pathway by directly targeting GRIN2D via miR-129-1-3p,thus playing a protective role in THP-induced cardiomyocyte injury.The dual-luciferase reporter gene assay verified that miR-129-1-3p can directly regulate GRIN2D.In addition,bibliographic retrieval showed that miR-129-1-3p plays an inhibitory role in a variety of tumors.Therefore,in this study,the reduction of the THP-induced cardiotoxicity by RUT and its sensitization and anti-breast cancer effects mediated by the Ca²⁺pathway regulated by miR-129-1-3p/GRIN2D were explored using cell and whole animal experiments,so as to systematically reveal the role of miR-129-1-3p/GRIN2D in the intervention of RUT in cardiotoxicity and the pathogenesis and progression of breast cancer,and clarify the possible mechanisms of RUT in the treatment of cardiotoxicity and breast cancer from the perspective of miRNA.(1)In cardiomyocyte(in vitro)experiments,RUT 100?M was used for pretreatment and THP 5?M was used to stimulate mouse myocardial HL-1 cells for24 h to simulate the improvement of the internal environment of the THP-induced HL-1 cell injury by RUT and explore the specific mechanism of RUT protection against THP-induced HL-1 cell injury at the cellular level.This was verified in HL-1cells by qPCR.The results showed that the miR-129-1-3p was down-regulated in the HL-1 cells after the treatment with THP and was up-regulated after RUT intervention.The trend of GRIN2D mRNA was contrary to that of GRIN2D mRNA,indicating that miR-129-1-3p/GRIN2D plays an important role in the protection of RUT against the THP-induced HL-1 cell injury.In order to study the role of miR-129-1-3p in the protection of RUT against the THP-induced cardiomyocyte injury,miR-129-1-3p mimics were transfected into HL-1 cell.It was confirmed by DCFH-DA,MAD,SOD,Fluo-3 AM,TUNEL and Western blot that,RUT and miR-129-1-3p can inhibit the THP-induced oxidative injury and calcium overload of cardiomyocytes,reduce the expression of Ca²⁺-associated pathway protein GRIN2D,calmodulin-1(Calm1),Ca²⁺/calmodulin-dependent protein kinase II?(Ca MK??)and phosphorylated Reynolds receptor 2(Ry R2-p S2814),increase the expression of ausculoplasmic endothelial reticulum calcium pump(SERCA2a)and sodium-calcium exchanger 1(NCX1),and reduce the apoptosis rate and inhibit the expression of apoptosis-associated proteins Bax/Bcl-2 and cleaved caspase-3.(2)In the breast cancer cell(in vitro)experiment,we cultured mouse mammary epithelial HC11 cells and mouse breast cancer 4T1 cells in vitro,and found by CCK-8test that,100?M RUT inhibits the proliferation of 4T1 cells and cooperates with THP.The detection of the expression of miR-129-1-3p in the two kinds of cells by qPCR showed that the expression levels of miR-129-1-3p in 4T1 cells were lower than those in HC11 cells.However,after RUT intervention,the expression levels of miR-129-1-3p in 4T1 cells increased,while the trend of GRIN2D mRNA was the opposite,indicating that miR-129-1-3p/GRIN2D also plays an important role in inhibiting the proliferation of mouse breast cancer cells by RUT.In order to study the inhibition of miR-129-1-3p on 4T1 cells,miR-129-1-3p mimics and miR-129-1-3p inhibitor were transfected into 4T1 cells,respectively.It was verified by plate cloning,wound-healing,Transwell,Fluo-3 AM and Western blot that,miR-129-1-3p mimics can inhibit the proliferation,migration and invasion of 4T1 cells and the expression of its relevant protein CD147,matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9)and vascular endothelial growth factor(VEGF)protein,inhibit the expression of Ca²⁺and GRIN2D,Calm1 and Ca MK??proteins,promote the expression of Bax/Bcl-2 and cleaved caspase-3 proteins,while miR-129-1-3p inhibitor has the opposite effects.(3)In animal(in vivo)experiment,a compound model of breast tumor bearing and THP-induced cardiotoxicity in mice was successfully built with BALB/c mice as the study subjects.After intragastric administration of RUT or tail intravenous injection of miR-129-1-3p agomiRs for 4 weeks,the cardiotoxicity of BALB/c mice was significantly reduced;electrocardiogram(ECG)and myocardial histopathological lesions were improved;the myocardial enzyme indexes tended to be normal;the expression of miRNA-129-1-3p in myocardial tissues increased,while that of GRIN2D mRNA decreased,reducing the levels of Ca²⁺;the expression of GRIN2D,Calm1,Ca MK??and Ry R2-p S2814 proteins decreased;the expression of SERCA2a and NCX1 proteins increased;the apoptosis of cardiomyocytes decreased;and the expression of Bax/Bcl-2 and cleaved caspase-3 proteins decreased.Besides,the growth and liver metastasis of breast tumor,and pulmonary inflammatory response in BALB/c mice were further controlled;the expression of miRNA-129-1-3p in tumor tissues further increased,while that of GRIN2D mRNA further decreased;the content of Ca²⁺and the expression of GRIN2D,Calm1,Ca MK??,CD147,MMP-2,MMP-9and VEGF were further inhibited;the apoptosis of cells and the expression of related proteins Bax/Bcl-2 and cleaved caspase-3 in tumor tissues were further promoted.To sum up,the following conclusions were drawn by in vivo and in vitro experiments in this study:(1)The in vitro and vivo THP-induced mouse HL-1 cell/myocardial damage experiment proved that RUT can reverse the THP-induced HL-1 cell/myocardial oxidative stress injury,the increase of mouse cardiac coefficient,serum myocardial enzyme damage,abnormal ECG and pathological changes of myocardial tissue,and protect against the THP-induced cardiotoxicity.(2)RUT can reverse the THP-induced down-regulation of miRNA-129-1-3p,the up-regulation of GRIN2D mRNA,calcium overload and apoptosis in mouse HL-1cell/myocardial tissues.This indicates that the mechanism of RUT of reversing the THP-induced mouse cardiotoxicity may be realized through regulating GRIN2D and Ca²⁺pathway by miR-129-1-3p and inhibiting myocardial apoptosis.(3)The in vitro and vivo experiments on the inhibition of breast cancer by THP confirmed that RUT can sensitize THP in inhibiting the proliferation,migration and invasion of 4T1 cells,inhibiting the growth and liver metastasis of the tumor and pulmonary inflammation response,and sensitize THP in inhibiting breast cancer.(4)RUT sensitizes THP in inhibiting the down-regulation of miRNA-129-1-3p and the up-regulation of GRIN2D mRNA in 4T1 cells/tumor tissues,further reduces calcium level and promotes apoptosis.This indicates that the mechanism of RUT inhibiting mouse breast cancer by sensitizing THP may be realized by regulating GRIN2D and Ca²⁺pathway by miR-129-1-3p and promoting the apoptosis of tumor cells.This study expanded the pharmacological effects of RUT,provided an important supplement to the biological functions of miR-129-1-3p/GRIN2D,proved that miR-129-1-3p may be a new target for treating the THP-induced cardiotoxicity and breast cancer,and can be used as the theoretical basis and experimental basis for the use of RUT as a novel drug for the treatment of cardiotoxicity caused by anthracyclines and breast cancer.