The Molecular Mechanisms Of Huaier Inhibiting Breast Cancer Progression Through LncRNA-H19

BACKGROUND:Breast cancer is the most common cancer and the leading cause of cancer-related mortality among women worldwide.In China,new cases of female breast cancer account for about 12.2%of all the new cases in the whole world annually;the number of female patients who died of breast cancer accounts for approximate 9.6%of that in the world.Therefore,illustrating the biological and molecular mechanisms of how breast cancer takes place and develops to find new treatment modality is important for translational medicine research.In recent years,with the view of "breast cancer is a systemic disease" generally accepted,a comprehensive treatment system for breast cancer including surgical treatment,radiotherapy and chemotherapy,endocrine therapy,Chinese medicine therapy,biological therapy and targeted therapy gradually formed.With the progress made in clinical research and fundamental research,treatment for breast cancer has been improved in terms of both safety and treatment effect.However,in recent years the incidence and mortality of breast cancer are still on the rise.Besides,there are also a variety of problems in clinical treatment,such as unaffordable expense,drug resistance and systemic toxicity and side effects.Therefore,it is urgent to search for new biological agents with low toxicity,low drug resistance and sound curative effect.As an important part of integrated treatment in breast cancer,Traditional Chinese Medicine(TCM)has gained increasing attention around world in recent years.Huaier is a kind of medical fungi growing on trunks of trees,which has been used in China for 1600 years.Growing evidence has demonstrated that Huaier exerts promising anti-cancer effect in multiple cancer types,such as breast carcinoma,colon carcinoma,rectal carcinoma,hepatocarcinoma,gastric carcinoma,cervical carcinoma and ovarian carcinoma.Study on the molecular mechanism of antitumor effect of Huaier revealed that Huaier had regulating action on many important signaling pathways,such as p53 pathway,pAKT/mTOR/S6 pathway,JNK/P38 pathway,ER/pathway,NF-κB Wnt/beta-catenin pathway and PI3K/AKT pathway.Our previous study demonstrated that Huaier could inhibit cell proliferation,migration,invasion and tumor angiogenesis,induce cell apoptosis and autophagy,arrest cell cycle and modulate tumor specific immunity.However,the underlying mechanisms are not fully understood.Long noncoding RNA(lncRNA)is a class of RNA molecules composed of more than 200 nucleotides.Although lncRNAs do not have the protein encoding function,they can regulate gene expression through transcriptional regulation,transcriptional regulation,epigenetic modifications and so on,impacting the occurrence and development of many human diseases.In recent years,the important role of IncRNA in cancer development has been confirmed by a large number of studies,and laid the foundation for the application of IncRNA in clinical diagnosis and targeted therapy.For example,PCA3(also known as DD3),which is one of the earliest discovered IncRNA associated with cancer,has been used as biological markers for adjuvant diagnosis of prostate cancer in clinic for many years.Therefore,study of the regulatory role of IncRNA in cancer and its mechanism is of great significance.There’s no published paper about whether IncRNA mediates the antitumor function of Huaier,so we explore the new mechanism of anticancer effect of Huaier from the viewpoint of IncRNA,laying foundations for the clinical use of Huaier in breast cancer.OBJECTIVE1.Investigate differentially expressed genes of breast cancer cells using huaier treated or not through the method of gene chip analysis;2.Investigate the role and potential molecular mechanisms of IncRNA-H19 in the huaier related anticancer efficacy.METHODS1.Cell cultureIn this study,MDA-MB-231,MDA-MB-468 and MCF-7 breast cancer cell lines were cultured in DMEM medium containing 10%fetal bovine serum.They were cultured in 37℃,5%C02 environment and passaged once every 2-3 days.2.Analysis of differentially expressed genesIn this study,we extractd total RNA of breast cancer cells with or without Huaier treatment.The RNAs was reversed transcripted into cDNA,which was sent to the Affymetrix Company to conduct chip hybrid processing.We use the software provided by the company to analysethe original datathe company sent to us,the multiple method was adoptedto make the analysis of diffentially expressed genes between Huaier group and the control group.3.Functional analysis of genes(GO analysis):We conducted functional analysis in the GO database.We made function annotation for every differentially expressed genes analysed in the last step.And we select 20 GO functions with most significant changes between groups,according to the number of genes changed in each GO function and the p value based on Fisher test.Eventually,we mapped the distribution of GO functions.4.Pathway analysisUsing the specialized software provided by the company,we made annotation for the signaling pathways each diffenrentially expressed gene involvedin the KEGG signaling pathway database.Based on the number of genes that changed in each pathway and the p value of the significance based on the Fisher test,20 pathway with the most significant changes were selected and the pathway distribution was plotted.5.Network analysis of signaling pathwaysAccording to the differential genes shown in the above results and their involvement inthe pathway annotation,a network map of differential pathways was drawn using specialized analytical software.Among them,red represents pathway activation,blue represents pathway inhibition,and yellow represents there are both upregulation and downregulation of genes in the pathway.6.Real-time quantitative PCR(qPCR)We treated breast cancer cells with Trizol,and then the total RNA was extracted.We then usedthe reverse transcription kit to reversely transcribe RNA into cDNA or microRNA.Real-time quantitative PCR(qPCR)were conducted in the experiment,to detect the effects of Huaier on the expression level of IncRNA-H19 and miR-675-5p.7.Plasmid amplification and purificationWe cloned the IncRNA-H19 cDNA into the polyclonal site of the pcDNA3.1 vector and obtained the overexpression plasmid of IncRNA-H19.The plasmids were transformed into the susceptible Escherichia coli and coated on the LB culture board containing ampicillin,culturingfor 12-16h in 37℃ environment.Then we selected one bacteria colony in the LB culture board and added it into LB culture mediumcontaining ampicillin,culturingfor 12-16h in 37℃ environment.The plasmid was extracted by the endotoxin free maxi plasmid kit.8.Cell transfectionThe IncRNA-H19 overexpression plasmid,siRNA of IncRNA-H19,miR-675-5p mimics or miR-675-5p inhibitor was mixed with a certain amount of liposome 2000.And we added the mixture into the cells in according to the operating procedures to regulate the expression of corresponding genes.9.MTT experimentWe seededbreast cancer cells to 7 cell culture plates with 96 holes,and MTT was added to the cells after Huaier treatment for 0,1,2,3,4,5,6 days,respectively.Then the absorbance value at 490nm values was detected by microplate reader,to detect the overexpression or suppression of IncRNA-H19 or miR-675-5p on the sensitivity of breast cancer cellsto Huaier extract.10.Flow cytometryAfter transfection and Huaier treatment,cells were stained by AnnexinV-FITC/PI staining.The percentage of apoptotic cells was detected by flow cytometry to study the effect of overexpression or supression of IncRNA-H19 or miR-675-5p on cell apoptosis induced by Huaier.11.Western BlotAccording to the operation procedure,the cells were lysed and the total protein of the cells was extracted.Then we conducted SDS-PAGE electrophoresis,transmembrane,blocked by 5%evaporated milk,the first antibody incubation and the second antibody incubation.Finally we used chemiluminescencereagent to determine whether Huaier impacted breast cancer progression by regulating the expression of the downstream proteins of miR-675-5p.RESULTS1.Our results showed that after 8mg/ml Huaier treatment for 72h,there were significant changes in expression of 1116 lncRNAs.Of these,211 were up-regulated and 905 were down regulated.2.GO analysis showed that after Huaier stimulation,there were 129 GO functions changed.Of these,77 functions were upregulated and 52 functions were down regulated.;Pathway analysis showed that a total of 109 pathways had undergone significant changes,59 of which had been up-regulated and 50 had been down regulated;We conducted a molecular pathway map of Huaier effect and found that after the treatment of Huaier.3.Huaier extract reduced the expression of IncRNA-H19 and miR-675-5p.The overexpression of lncRNAH19 inhibited the cytotoxic effects of Huaier extract.4.Reduced lncRNA-H19 expression enhanced the function of Huaier extract,whereas downregulating miR-675-5p sensitived breast cancer cells to the effect of Huaier extract.5.Huaier extract increased the expression of CBL protein.CONCLUSION1.Huaier play the role of cancer suppression through action on multiple targets,such as inhibition of cell cycle progression,enhancement of apoptosis.2.Huaier could regulate the expression of lncRINA;Huaier extract reduces viability and induces apoptosis in breast cancer cells via lncRNA-H19-miR-675-5p-CBL axis regulation.SIGNIFICANCEThis study explored the new mechanisms of the anti-cancer effects of Huaier from the viewpoint of IncRNA,providing the rationale for the clinical use of Huaier.