Study On The Mechanism Of Inhibitory Effect Of Inonotus Obliquus Polysaccharide On Breast Cancer Immune Infiltration

As a large medicinal and edible fungus,Inonotus Obliquus is popular in Europe,especially in Russia,which exhibited a series of medicinal activity.With a long period of folk exploration,Inonotus Obliquus polysaccharide(IOP)has been proved beneficial to the common chronic diseases,such as diabetes,heart disease,etc.,especially cancer,IOP exhibited a wide range of anticancer activities,including liver cancer,lung cancer,colorectal cancer,etc.However,most of the study was conducted in vitro,and limited research focus on molecular mechanism and in vivo.The annual incidence rate of breast cancer is increasing all around world.Nearly 2 million women diagnosed with breast cancer every year(date from 2013),and caused 600 thousand patients die.Compared with other cancers,breast cancer exhibited the highest incidence rate/mortality rate in female cancer.With the accumulation of the BC molecular mechanism data,targeted therapy has become to a key method for BC treatment.Regarding the dominate function of immune response in the occurrence and prognosis of breast cancer,the improve of immune ability,through the tumor immune response regulation pathway,can be regarded as an effect treatment to the BC patients.In the study of tumor immune microenvironment,some researches on relatively stable cell infiltration mode will significantly affect the diagnosis and survival outcome of tumor.Such studies based on histological data and high-throughput data analysis are generally referred to as immune infiltration-related studies.It can analyze the information from key genes to cell level and to survival information level of clinical samples in a coherent way,and can screen and evaluate the research samples with statistical analysis method.The present study focused on the effect of IOP to the immune infiltration of breast cancer,which conducted a research evaluated the bioactivity of IOP to the BC rat model by cancer cell line 4T-1 in vivo.The study is meaningful to reveal the inhibitive mechanism of cancer,the preventing and treating of breast cancer,and the immunotherapy of clinical breast cancer.In this study,rat breast cancer cell line 4T-1 was considered as the research materials.Firstly,the immune-related molecular targets to BC was screened by bioinformatics;then,transcriptome was used to investigate the mechanism of IOP alleviating 4T-1 BC mouse model,and evaluated the changing of particular immune-related molecular targets;finally,PCR and WB were used to analyze the immune-related molecular targets changing above in mouse tumor tissue.In this study,we intend to determine the effect of IOP to the immune infiltration of BC.The main results are as follows:1.The polysaccharide content of wild Inonotus Obliquus was high.The mixture of crude polysaccharide was 13.6g by hot water extraction from 50 g Inonotus Obliquus power and 3.2g polysaccharide by impurity removal.After ion-exchange column elution and separation,six main components of IOP1-IOP6 were obtained.The total recovery rate of polysaccharide was 89.2%.According to the results obtained from the MTT experiment,it can be known that the IOP can inhibit human breast cancer cell MDA-MB-231 and mouse breast cancer cells 4T-1.The inhibitory effect of IOP-5 polysaccharide was the best.The IC50 of human breast cancer cell MDA-MB-231 was 294 ± 55.7 ?g·m L-1 and IC50 of mouse breast cancer cell 4T-1 was 214.5 ± 32.1 ?g·m L-1.2.We enrolled 1094 BC patient data and obtained 1399 survival related immune related DEGs from the database.In this study,multivariate Cox analysis was used to establish a risk assessment model of TAIG based on the DEGs above.According to the results obtained,a higher TAIG level means a lower clinical survival,a higher level of AJCC-TNM stage and a higher pathological stage of advanced breast cancer.Meanwhile,In the high TAIG group,the patients exhibited a significantly decreased of the immune infiltration density of CD8+T cells,memory rest CD4 + T cells,M0 macrophages and M2 macrophages,where CDHGA2,SPIB,ADRB1,FLT3 and NFKBIE can be regarded as the critical immune related molecular markers among the 17 immune related genes which utilized in TAIG scoring system.3.According to the transcriptome results,IOP-5 can inhibit the growth of breast tumor in model mice,effectively.Which decreased the growth rate of tumor,caused a lower final BC tumor weight in vivo,and its antitumor activity was positively correlated with the concentration.In the high dose group,we screened 3135 differentially expressed genes,which including 192 immune-related DEGs.According to the TAIG index established in this study,we screened five molecular markers which were close correlation with immune infiltration,including: NFKBI(up regulation),FLT3,ADRB1,SPIB and IFNE(down regulation).The above molecular markers can be regarded as the core molecular markers of IOP-5 that inhibit the immune infiltration of breast cancer.4.IOP-5 can significantly inhibit the proliferation of rat breast cancer cell line 4T-1 in a concentration dependent.The BC immune invasion mechanism of IOP-5 is up regulated the expression of NFKBI,thus inhibited NFk B signal pathway.With the decrease of FLT3,ADRB1,NFKBI and IFNE expression,the immune microenvironment of BC was changing,thus inhibited the metastasis of BC tumor.The result indicated that,IOP-5 could improve the BC immune infiltration situation in a concentration dependent manner.