Preliminary Study On The Epigenetic Mechanism Of Agrocybe Cylindracea Polysaccharide Inhibiting The Proliferation Of Colorectal Cancer Cells

Colorectal cancer(CRC)is a global cancer of the digestive system.Epidemiological studies show that the incidence and fatality rate of CRC rank among the top three in the world,posing a great threat to the health of all mankind.Agrocybe cylindracea,as a common edible fungus in daily life,is not only nutritious but also has medicinal and health value.Polysaccharide is a bioactive component with high content,which has broad research value and application prospect.In this study,Agrocybe cylindracea polysaccharide(ACP)was used as the research object to detect the inhibitory effect of ACP on the proliferation of different colorectal cancer cells through cell experiments in vitro.RNA-seq and Ch IP-seq technologies were used to explore the potential molecular mechanism of ACP-induced lysosomal dysfunction in colorectal cancer HCT-116 cells and then triggered the apoptosis program from the perspective of epigenetics.The main research contents and conclusions are as follows:1.Exploring the effect of ACP on the proliferation and gene expression of colorectal cancer cells.The inhibitory effect of ACP on the proliferation of colorectal cancer cells was detected by cell clone formation assay and CCK8 assay,and a cell type was selected to explore the mechanism.The results showed that ACP significantly inhibited the proliferation ability of three types of colorectal cancer cells(HCT-116,HT29,and CT26)in a concentration and time dependent manner,with HCT-116 cells being the most significantly inhibited.Importantly,ACP had no significant toxicity to normal intestinal epithelial cells IEC-6.Bioinformatics analysis of RNA-seq results showed that a total of 2783 differentially expressed genes were identified in HCT-116 cells treated with 400 μg/m L ACP.Through GO and KEGG enrichment analysis,it was found that apoptosis,lysosome,mitochondrial depolarization and cytochrome c release pathways were significantly up-regulated in HCT-116 cells after ACP treatment,while energy metabolism and TCA cycle pathways were significantly down regulated.In addition,GSEA enrichment analysis showed that ACP up-regulated apoptosis-related pathways and down-regulated cell cycle pathway.2.Exploring the effect of ACP on lysosomal function of HCT-116 cells and its mechanism.Lyso-Tracker Red fluorescent probe and Lyso-Sensor Green fluorescent dye were used to analyze the changes in the number of lysosomes and intracellular acid value of HCT-116 cells after ACP treatment,respectively.Ch IP-seq experiment was used to explore the epigenetic mechanism of ACP affecting the function of lysosomes in HCT-116 cells.The results showed that ACP significantly induced excessive lysosomal accumulation and elevated intracellular acid values,which resulted in lysosomal dysfunction.Next,Western Blot data showed that ACP significantly increased the expression of H3K27 ac protein and histone acetyltransferase CBP protein.Therefore,Ch IP-seq experiments were performed to determine the effect of ACP on gene expression at the binding site of H3K27 ac modification.Combined RNA-seq data and Ch IP-seq data analysis showed that some H3K27 ac regulated lysosome-related upregulated genes,including CTSD,EBP and CLTB,were found in the ACP-treated group.Furthermore,the expression and activity of cathepsin D(CTSD)in ACP-treated group were enhanced by immunofluorescence assay and cathepsin D activity detection kit,which again illustrated that ACP induced lysosomal dysfunction in HCT-116 cells.In addition,immunofluorescence results showed that ACP not only enhanced the expression of nuclear transcription factor EB(TFEB),but also promoted the translocation of TFEB to the nucleus.In summary,ACP promoted H3K27 ac expression by up-regulating CBP,which in turn promoted the binding of the transcription factor TFEB,activated the transcription of lysosome-related genes such as CTSD,and promoted lysosomal excessive accumulation and dysfunction.In addition,we speculated that ACP-induced lysosomal dysfunction leaded to cathepsin D leakage,triggering mitochondrial apoptosis in HCT-116 cells.3.Exploring the effect and mechanism of ACP on mitochondrial apoptosis in HCT-116 cells.The number of apoptotic cells was detected by acridine orangeethidium bromide(AO-EB)staining and Annexin V-FITC/PI double staining.The mitochondrial membrane potential and apoptosis-related proteins were detected by Mito Tracker Red fluorescent probe and Western Blot,respectively.HCT-116 cells were pretreated with cathepsin D activity inhibitor(PepA)to explore the role of cathepsin D in mitochondrial apoptosis pathway.It was found that the number of apoptotic cells was significantly increased in the 300 μg/m L and 400 μg/m L ACPtreated groups,indicating that ACP significantly induced apoptosis.In addition,ACP significantly down-regulated the expression of anti-apoptotic protein Bcl-2 and upregulated the expression of pro-apoptotic protein Bax,resulting in the decrease of mitochondrial membrane potential and the release of pro-apoptotic factors into the cytoplasm,which promoted the expression of Cleaved Caspase-9 and Cleaved Caspase-3.Interestingly,pretreatment with PepA could attenuate the inhibitory effect of ACP on the proliferation of HCT-116 cells and the induction of mitochondrial apoptosis.In addition,the results of mitochondrial stress testing experiment showed that ACP downregulated ATP production,basal respiration value,maximum respiration value,non-mitochondrial respiration value and mitochondrial spare respiratory energy,and induced mitochondrial dysfunction.In conclusion,ACP induced mitochondrial apoptosis in HCT-116 cells via cathepsin D.To sum up,ACP significantly activated the transcription of lysosomal genes(including CTSD)through H3K27 ac modification,thereby promoting lysosomal excessive accumulation and dysfunction.Meanwhile,ACP-induced lysosomal dysfunction released cathepsin D,triggering mitochondrial apoptosis in HCT-116 cells to inhibit proliferation.In addition,ACP induced mitochondrial dysfunction.The results of this study provided data support for the development of ACP as an anticancer drug,which is conducive to exploring the economic value of Agrocybe cylindracea.