Epigenetic Mechanism Studies Of De-ubiquitinase USP22 In Gastric Cancer And L-GSH Capped Gold Nanoclusters In Reprogramming Gastric Cancer Cells

Gastric cancer(GC)is a worldwide common malignancy with high mortality and low cure rates.There is an urgent need to clarify the mechanism of GC and develop new treatment strategies.Epigenetic mechanism refers to changes in gene expression due to external factors or changes in the environment without altering the genetic DNA sequence.In this study,we focus on investigating the epigenetic mechanism of gastric cancer stem cells(CSCs)stemness sustaining and the the epigenetic modification of GC cells caused by low cytotoxic nanomaterials.The main results are as follows:1.Gastric CSCs have been identified and demonstrated to be associated with the initiation and metastasis of GC.And a large number of de-ubiquitinases have been reported to regulate the CSC-related factors and signaling pathways to maintain CSC stemness.Increased expression of ubiquitin-specific protease 22(USP22)has been associated with several poor prognosis of cancers,including gastric cancer.However,the role of USP22 in GC remains unclear.Using lentivirus-mediated shRNA technology,we constructed the stable USP22 knockdown GC cell lines.Results show that USP22 knockdown significantly inhibited the formation and maintenance of gastric CSCs,accompanied by a variety of CSC markers down-regulated;preclinical tumor xenografts experiment in mice showed that knockdown of USP22 significantly inhibited GC initiation and progression.Further bioinformatics analysis of the TCGA database showed that the core member of the Poly-comb group protein(PcG),BMI1,was significantly associated with poor prognosis in patients with gastric cancer.Previous studies have reported that BMI1 protein regulates self-renewal of stem cell and development of cell lineages.Similar to the effect of knock down of USP22,BMI1 silencing inhibits the formation of gastric cancer stem cells.The addition of TAT-BMI1 protein into the cultured cells in vitro could rescue the impaired ablility of gastric CSCs formation and self-renewal caused by USP22 knockdown and could increase the expression of CSC stemness markers,which proves that BMI1 protein is an important downstream signaling molecule of USP22.Furthermore,immunofluorescence staining showed that USP22 and BMI1 were co-located in the nucleus.Co-IP results showed that USP22 interacted with BMI1.Western blot analysis of BMI1 in cells caused by the way of overexpressing and silencing of USP22 revealed that USP22 directly stablize BMI1,in vitro ubiquitination immunoprecipitation test further proved that USP22 is a deubiquitination enzyme of BMI1.2.L-glutathione(L-GSH)-capped gold nanoclusters(AuNCs@L-GSH)have been widely used in GC targeting and imaging due to their low cytotoxicity,good water solubility and bio-compatibility.However,there is little research associated with the epigenetic effects of AuNCs@L-GSH.Therefore,it is very necessary to study its epigenetic effects on the cells.HEK293 FT cells and GC cell line MGC803 cells were treated with low cytotoxic AuNCs@L-GSH.The dot blot results showed that AuNCs@L-GSH could significantly decrease 5-hydroxymethylcytosine(5hmC)levels,in a dose-dependent manner.Epigenetic marker 5hmC was the "sixth base" involved in gene transcriptional regulation.Further studies showed that AuNCs@L-GSH can not only down-regulate TET1 and TET2 mRNA expression,but also induce intracellular ROS production,and ROS levels can be further enhanced by induction of TET protein aggregation,thereby reducing enzyme activity of conversion of the 5-methylcytosine(5mC)into 5hmC.Studies on gene expression profiles and bioinformatics analysis have shown that changes in TET-5hmC signaling will affect multiple important downstream targets and then regulate multiple physiological processes.In conclusion,our studies demonstrate that USP22 is a deubiquitination enzyme of BMI1,USP22-BMI1 signal axis is involved in the regulation of the stemness of gastric CSCs and the progression of GC,low cytotoxic AuNCs@L-GSH nanomaterials can directly affect TET and 5hmC,which lay foundation for further clarification of the function of USP22 and AuNCs@L-GSH.