Construction Of Molecular Regulation Network Of Huaier In Breast Cancer And Functional Analysis Of Linc00339

BackgroundBreast cancer(BC)has become the most common life-threatening malignant diseases in women across the wold.In recent years,the incidence rate of breast cancer in China has also increased.Since the huge number of patients worldwide,a large amount of investment has been made in breast cancer research.Now,during the clinical practice,treatments has been developed into the systemic therapy,including surgery and adjuvant treatments,such as chemotherapy,radiotherapy and endocrine therapy.However,the side effects of conventional treatment are usually obvious and unbearable,which were the dilemma in the treatment of breast cancer.Traditional Chinese medicine has a long history in China,and was widely used as adjuvant therapy in many tumor treatment fields.Belonging to Hymenomycetes,Basidiomycotina,Trametes robiniophila Murr(Huaier)was first recorded in compendium of Materia Medica by Li Shizhen.In recent years,its anticancer activity has attracted the world's attention,and a large number of evidences show that Huaier has a significant effect on breast cancer.However,its pharmacological mechanism was very complicated and still remained largely unknown.In this project,we studied the function and clinical application value of Huaier in breast cancer through three parts.In the first part,using the bioinformatics analysis of the microarray,we successfully constructed the coexpression molecular network of Huaier for regulating breast cancer.In the second part,through further analysis of the coexpression molecular network,we found that linc00339 play an important role in the molecular network of Huaier in breast cancer.So we selected linc00339 for further study.We confirmed that linc00339 can affect the proliferation and metastasis of breast cancer by affecting the miR-4656/CSNK2B pathway both in vitro and vivo.In the third part,we analyzes the influence of Huaier on the survival time of breast cancer patients and serum tumor markers clinically.The data showed that Huaier can significantly improve the disease free survival time of breast cancer patients,and can reduce the levels of serum tumor markers of breast cancer patients.This further confirms that Huaier plays an important role in the adjuvant treatment of breast cancer.Part I Construction of Molecular Regulation Network of Huaier in Breast CancerObjective1.Analyze the effect of Huaier on molecular expression of breast cancer cells.2.Explore the function of differentially expressed genes after treatment of Huaier.3.Establish the molecular regulation network of Huaier on breast cancer.MethodsIn this part of the study,breast cancer cells treated and untreated by Huaier were collected and analyzed with Affymetric GeneChip Human Transcriptome Array 2.0.R package "limma" was applied to identify the differentially expressed genes including the data from lncRNA,miRNA,and mRNA.Heatmaps were used to show the expression levels of all the probes.Hierarchical clustering analysis was applied to present systematic variations between Huaier-treated group and control group.We used the GO(gene ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analyses to show the molecular processes after Huaier treatment.Weighted gene coexpression network analysis(WGCNA),an R package for weighted correlation network analysis,was applied to identify candidate therapeutic targets based on RNA sequencing and we constructed a molecular network of breast cancer regulated by Huaier.ResultsWe analyzed breast cancer cells before and after the Huaier treatment through the bioinformatics technology and we find that Huaier played an important part in the tumorigenesis and development of breast cancer.Various signaling pathways were influenced by the regulation of Huaier.Close relationship was found between Huaier,DNA repair and cell cycle,which further explained the anti-cancer activity of Huaier.In order to vividly show the changes of various molecules in breast cancer cells,mRNA,miRNA and IncRNA influenced by Huaier were exhibited on human chromosome by the Circos plot.By integrating the interaction between the key signal molecules associated with breast cancer,a molecular network of breast cancer regulated by Huaier was finally constructed.Conclusions1.There were a large number of differentially expressed lncRNA and mRNAbetween Huaier treated and untreated breast cancer cells.2.Huaier could regulate DNA repair,cell cycle and other tumor-related pathways.3.Based on the key differentially expressed genes,the molecular regulation networkof Huaier on breast cancer was established.Part ? Huaier inhibited progression and metastasis of breast cancer through Linc00339Objective1.Explore the key lncRNA in the regulation of Huaier on breast cancer progression and metastasis.2.Detect the expression of linc00339 in the Huaier treated and untreated breast cancer cells.3.Explore the effect of linc00339 on the proliferation and metastasis of breast cancer cells.4.Elucidate the molecular mechanism of linc00339 in the regulation of Huaier on breast cancer progression and metastasis.MethodsThrough analyzing the results in study of part I,we recongnized central role of linc00339 in the molecular network of breast cancer regulated by Huaier,and found that linc00339 had a close relationship with various factors.We assumed that linc00339 played an important part in the regulation effect of Huaier on breast tumor.Using real-time quantitative PCR,we verified that linc00339 was a direct target of Huaier.We also verified the inhibition effects of linc00339 on the chemosensitivity of breast cancer cells to Huaier by MTT assay.To further identify the downstream targets of linc00339,we used starBase to calculate the Spearman's correlation of linc00339 with its coexpressed mRNA.Then,miRNAs were also identified in silico analysis and the interaction was verified by luciferase assay and real-time quantitative PCR.ResultsWe validated the prediction that linc00339 played a key role in regulating the therapeutic sensitivity of Huaier in breast cancer treatment.The expression level of linc00339 in breast cancer cell lines was significantly decreased after Huaier treatment.Meanwhile,overexpression of linc00339 markedly decreased the cytotoxic effect of Huaier to breast cancer cells.Verified by website prediction and subsequent experiments,we found that linc00339 could decrease the expression of mir-4656 by working as a miRNA sponge(P<0.05)and up-regulate the expression of CSNK2B(P<0.05)to promote the proliferation of breast cancer.Huaier can significantly inhibit linc00339 in a time and dose-dependent manner(P<0.05).Then,the expression of mir-4656 was increased after the down-regulation of linc00339(P<0.05),which resulted in a significant decrease in the mRNA and protein levels of CSNK2B(P<0.05).In conclusion,linc00339 is the key factor in breast cancer to regulate the therapeutic sensitivity to Huaier treatment.And Huaier can inhibit the progress of breast cancer by regulating linc00339/miR-4656/CSNK2B signaling pathway.Conclusions1.Linc00339 is the key lncRNA in the regulation of Huaier on breast cancer progression and metastasis.2.The treatment of Huaier could significantly reduce the expression level of linc00339,and the high linc00339 expression is negatively correlated with the sensitivity of cells to Huaier.3.In vitro and in vivo experiments confirmed that linc00339 can significantly promote the proliferation and metastasis of breast cancer.4.Linc00339 could act as an oncogene through affecting the mir-4656/CSNK2B pathway.Part ? Efficacy of Huaier for Breast Cancer TreatmentObjective1.Analyze of Huaier on the survival time of breast cancer patients.2.Analyze of Huaier on serum tumor markers of breast cancer patients.3.Analysis of Huaier on the quality of life of breast cancer patientsMethodsOur study included 216 breast cancer patients treated with or without Huaier between January 2005 and October 2016 at Qilu Hospital,Shandong University,Jinan,China.Retrospective data obtained included demographics,clinicopathological characteristics,disease-free survival(DFS)and serum concentrations of tumor markers.DFS was the main outcome measure.ResultsA total of 216 patients were included.108 patients were involved into the control group and 108 patients were involved into the Huaier group.Baseline characteristics were well balanced between the study arms.The serum of breast cancer patients in Huaier treatment group and control group was tested.The results showed that Huaier could significantly reduce the serum CEA concentration,but had no significant effect on CA153 and CA125.Through Kaplan Meier analysis,we found that the disease-free survival time of patients in the treatment group was significantly longer than that in the control group(P=0.0002).In addition,we analyzed the effect of Huaier on the quality of life,and found that Huaier can improve sleep and(P=0.02)and anorexia(P=0.02),but there was no significant difference in irritability(P=0.99),memory loss(P=0.55),and fatigue(P=0.78).Therefore,Huaier granule can improve the quality of life of breast cancer patients.Through the above clinical data,it is confirmed that Huaier has an important clinical significance.Conclusions1.Huaier could reduce the concentration of serum tumor markers.2.Huaier could prolong the disease-free survival time of breast cancer patients3.Huaier can improve the quality of life of breast cancer patients.4.Huaier plays an important role in adjuvant treatment for breast cancer patients.