Investigation Of The Effect Of HDAC3 On Proliferation And Lipid Metabolism Of Gastric Cancer And Its Molecular Mechanism

Gastric cancer,a common malignant tumour worldwide originating from the gastric mucosal epithelium.It has the second highest incidence and the third leading,Gastric cancer is a multifactorial comprehensive disease which can be caused by environmental and genetic factors.In recent years,although surgical resection,radiotherapy and molecular targeted therapy in gastric cancer treatment have progressed,the prognosis is still poor and 5-year survival rates is low,therefore,it is urgent to explore the complex pathogenesis of gastric cancer.Histone deacetylase(HDAC)is a class of proteases.Histone acetylation plays an important role in chromatin remodeling and gene expression regulation.Histone acetylation activates gene expression.The deacetylated histones inhibit the expression of specific genes,especially the expression of some tumor suppressor genes.HDAC3 is a member of the HDAC family.Current research indicates that HDAC3 plays an important role in various cancers such as liver cancer,colorectal cancer,and neuroblastoma.However,there are few reports on the research of HDAC3 gene in gastric cancer,especially the interaction between HDAC3 and lipid metabolism in gastric cancer.Therefore,this topic studies the effect of HDAC3 on the proliferation,migration and lipid metabolism of gastric cancer cells and its further investigation on the regulation mechanism of gastric cancer cells,hoping to provide new ideas and a certain theoretical basis for the treatment of gastric cancer.Our main research results are as follows: 1.The effect of histone deacetylase inhibitor TSA on the proliferation of gastric cancerWe consulted the literature and the MTT experiment to obtain the appropriate concentration and treatment time for TSA treatment of MKN-45 and SGC-7901 cells.Subsequently,MKN-45 and SGC-7901 cells were treated with appropriate concentration,and tested by MTT and BrdU experiments.The results showed that TSA significantly inhibited the number of MKN-45 and SGC-7901 cells and the positive rate of BrdU.It was found that the number of MKN-45 and SGC-7901 cells blocked in G0/G1 phase was significantly higher than the control group after TSA treatment through flow cytometry analysis.Western Blotting experiment detected the down-regulation of G0/G1 related proteins,which further confirmed TSA induced cell cycle arrest at G1 phase.Then the cell flow cytometry experiment was used to detect the apoptosis,it was found that the number of apoptotic cells increased significantly after TSA treatment,the Western Blot experiment also verified that TSA induced apoptosis of gastric cancer cells.In order to further determine the ability of TSA to inhibit the proliferation of gastric cancer cells,a soft agar experiment was carried out.The results showed that TSA significantly repress the clone formation ability of gastric cancer cells in vitro.In order to further explore the specific molecular mechanism of TSA inhibiting the proliferation and apoptosis of gastric cancer cells,we detected the key proteins of PI3K/AKT pathway that is closely related to cell proliferation,migration,invasion and apoptosis through Western Blotting experiments.TSA enhanced H3 acetylation and inhibited the activation of PI3K/AKT.The above results indicate that TSA inhibits the proliferation ability of gastric cancer cells and induces apoptosis by inhibiting the PI3 K / Akt pathway.2.Effects of HDAC3 on cell proliferation,migration and invasion and tumorigenic ability of gastric cancer cellsBased on the effect of histone deacetylase inhibitor TSA on gastric cancer proliferation and apoptosis,we readed the literature: when HDAC3 is down-regulated and TSA treatment can significantly increase the expression of FGF21 which is an important regulator of liver lipid metabolism in cells.TSA can reduce the expression of HDAC3 and increase the expression of KAT2 A to improve the in vitro development ability of bovine SCNT embryos.TSA increases the expression of Dleu2 / miR-15 a / 16-1 by HDAC3 in lung small cell carcinoma and inhibits cell proliferation.It is found that the role of TSA in tumors is closely related to histone deacetylase HDAC3 via these documents.So we have studied and explored the role of HDAC3 in gastric cancer.We analyzed the prognosis database of TCGA gastric cancer patients and found that HDAC3 was negatively correlated with the prognosis of gastric cancer patients.Then we used lentiviral-mediated shRNA interference technology to down-regulate the expression of HDAC3,it was found that down-regulating the expression of HDAC3 significantly inhibited the proliferation of gastric cancer cells through MTT and BrdU experiments.Next,we examined the effect of HDAC3 on the cycle of gastric cancer cells.It was found that down-regulating the expression of HDAC3 caused the cycle arrest in the G0/G1 phase,and Western blotting detected the expression of cycle-related proteins proved it.The transwell experiment results showed that the migration and invasion ability of gastric cancer cells was significantly weakened after down-regulating the expression of HDAC3.At the same time,Western blotting detection of the expression of proteins related to migration and invasion also confirmed this.We restored the expression of HDAC3 when HDAC3 expression was down-regulated,and we found that the inhibition of migration ability of gastric cancer cells was restored.It was found that the down-regulation of HDAC3 expression inhibited the cloning formation ability in vitro through soft agar cloning experiments,and the experimental results were restored after the restoration of HDAC3 expression.Next,we performed a subcutaneous tumor formation experiment in nude mice,the results showed that down-regulated HDAC3 expression significantly suppressed the tumorigenicity of cells in vivo.Finally,H&E staining and immunohistochemistry experiments were performed on the tumor tissues from mice,it was found that the tumor mass was significantly smaller than that in the control group,and the expression of HDAC3 and Ki67 was also significantly reduced in the experimental group.PI3K / AKT is a common important signal pathway related to tumorigenesis and development in human tumors.We tried to detect PI3K/AKT by Western blotting and found that down-regulation of HDAC3 expression significantly reduced the expression of key proteins in the PI3K/AKT pathway,while the expression of related proteins in PI3K/AKT pathway was significantly increased after the HDAC3 expression was restored.The above results showed that inhibition of HDAC3 can inhibit gastric cancer cell proliferation,migration and invasion by inhibiting the PI3K/AKT pathway.3.Histone deacetylase HDAC3 regulates lipid metabolism and ROS-induced apoptosis of gastric cancerLipids have many important biological functions accompanied with multiple complex metabolic pathways in the body.Lipids have a variety of important biological functions.Abnormal lipid metabolism can cause many human diseases.Recent studies have shown that abnormal lipid metabolism may be an important factor in the development of cancer.we explored the effect of HDAC3 on gastric cancer lipid metabolism.First,we examined the effect of down-regulation of HDAC3 in MKN-45 and SGC-7901 cells on fatty acid and cholesterol pathways,it was found that mRNA and protein levels of key genes in fatty acid and cholesterol pathways were significantly up-regulated.However,restoring HDAC3 expression also restored the RNA and protein levels of these key genes in fatty acid and cholesterol pathways.Therefore,we used cholesterol detection reagents and found that the cholesterol content in MKN-45 and SGC-7901 cells that down-regulated HDAC3 increased significantly.This indicates that HDAC3 is involved in the metabolism of fatty acids and cholesterol in gastric cancer cells.Cholesterol and fatty acids are essential lipids involved in many biological processes,but excessive lipids can lead to the production of reactive oxygen species(ROS)which further induces lipotoxicity and leads to cell death.Reactive oxygen species(ROS)detection experiments showed that reactive oxygen species(ROS)are activated and produced in large quantities after down-regulation of HDAC3 expression in gastric cancer cells.It was found that the mRNA levels of several important antioxidant genes involved in oxidative stress were obviously activated and enhanced by qRT-PCR experiment.Based on the occurrence of oxidative stress,the study detected apoptosis and found that the number of apoptosis of gastric cancer cells increased significantly after down-regulation of HDAC3,and the expression of apoptotic protein was further verified that by Western blotting.In the study,the molecular mechanism of HDAC3 regulating lipid metabolism was studied.The regulation of cholesterol biosynthesis is achieved through a simple feedback suppression system.The main regulator of this process is the sterol response element binding proteins(SREBF1 and SREBF2),HMGCR is also a key rate-limiting enzyme for cholesterol biosynthesis.Finally,it was found that HDAC3 can interact with SREBP2 and HMGCR through immunoprecipitation experiments.The above results indicate that down-regulation of HDAC3 induces the activation of the cholesterol synthesis pathway of gastric cancer cells,and thus the cholesterol and free cholesterol levels are significantly increased to induce ROS production,ROS induces oxidative stress and apoptosis.In summary,down-regulation of HDAC3 not only inhibits the proliferation and migration of gastric cancer cells but also up-regulates the cholesterol synthesis pathway leading to abnormal lipid metabolism causing lipid toxicity to induce apoptosis of gastric cancer cells.