The Effect And Mechanism Of Coiled-Coil Protein CCDC85B On The Proliferation And Migration Of Gastric Cancer Cells

This study mainly explores the effect of coiled-coil domain containing protein CCDC85 B on the proliferation and migration of gastric cancer cells and the underlying mechanism.Gastric cancer is one of the most common gastrointestinal tumors.In the global cancer statistics in 2020,the incidence of gastric cancer ranks fifth and the mortality rate ranks fourth.Drinking,smoking,pickled food,and low intake of fruits and vegetables are all factors that induce gastric cancer.Helicobacter pylori infection is the main factor that induces gastric cancer.Due to its late diagnosis,early clinical symptoms are asymptomatic,the five-year survival rate of gastric cancer patients is less than 20%,and its treatment is mainly endoscopic resection and chemotherapy.Therefore,it is very important to explore the pathogenesis of gastric cancer and find its potential therapeutic targets.CCDC85B is a protein with two coiled-coil folding motifs.Because of its binding to hepatitis D antigen,it is also called DIPA which affects virus replication.Recent studies have shown that CCDC85 B exhibits opposite tumor suppressor and cancer-promoting effects depending on its subcellular location in tumors.For example,in non-small cell lung cancer,CCDC85 B is located in the cytoplasm,and its expression is higher than that of normal lung cells,which promotes the ability of tumor cell proliferation and migration.In osteosarcoma cells,CCDC85 B is located in the nucleus and plays a role of transcriptional inhibition.At present,the location and mechanism of CCDC85 B in gastric cancer cells are not clear,so we explored its role in the development of gastric cancer is very necessary,in order to provide a new molecular basis and theoretical research for the subsequent treatment of gastric cancer.The current research has achieved the following results:1.The location,expression and prognosis of CCDC85 B in gastric cancer cellsIn order to explore the role of CCDC85 B in gastric cancer cells,through multiple prognostic databases,we found that CCDC85 B gene is closely related to the prognosis of gastric cancer patients.If CCDC85 B is highly expressed,the patient’s prognosis is poor and the survival rate is low,which indicates that it may be a patient reference indicator for prognosis.Afterwards,through immunofluorescence experiments,we found that CCDC85 B is located in the nucleus and cytoplasm of gastric cancer cells,but it is more expressed in the cytoplasm.According to reading literature,we know that it is located in the cytoplasm may play the role of oncogene.Through IHC immunohistochemistry experiments on gastric cancer patients and normal adjacent tissue chips,it was also found that its expression was localized in the nucleus and cytoplasm,and its expression was higher in tumor tissues.Finally,we detected the expression of CCDC85 B in gastric cancer cell lines,and found that its expression in gastric cancer cell lines was more than that in normal gastric mucosal epithelial cells through fluorescence quantitative PCR and immunoblotting technology.Therefore,we speculate that CCDC85 B may act as a protooncogene in gastric cancer.2.CCDC85 B affects gastric cancer cell proliferation and migration abilityIn order to explore the specific effect of CCDC85 B on gastric cancer cells,we constructed a plasmid that interfered with CCDC85 B and overexpressed CCDC85 B,and performed experiments involving overexpression of CCDC85 B in MKN and HGC cell lines.Through MTT cell proliferation experiments,we found that after interference with CCDC85 B,compared with GFP interference in the control group,the proliferation speed of the cells in the CCDC85 B interference group was significantly slower and the cell proliferation ability was inhibited.The Ed U experiment also showed that the positive signals representing DNA synthesis in the interference CCDC85 B group were significantly less.Then we performed cell migration experiment,the experiment results show that after interference with CCDC85 B,the number of cells passing through the chamber is significantly reduced compared to the control group.The final plate cloning experiment also clearly shows that the cloning ability of cell is significantly inhibited after interference with CCDC85 B,in order to further verify that CCDC85 B can promote gastric cancer cell proliferation,migration,and clonal tumor formation ability,we overexpression CCDC85 B gene in MKN and HGC cells,the result of cellular phenomenon is completely opposite to interference phenomenon.After overexpression of CCDC85 B,whether it is cell proliferation,migration ability,as well as its cloning ability,have been significantly enhanced.These results all indicate that CCDC85 B acts as a proto-oncogene in gastric cancer cells,regulating the proliferation,migration,invasion and clonal formation of gastric cancer cells.3.CCDC85 B regulates the ubiquitination level of β-cateninIn order to explore the molecular mechanism behind the ability of CCDC85 B to regulate gastric cancer cell proliferation,migration,invasion,and clonal formation,we detected related cell cycle and migration and invasion proteins through Western blot experiments,and found that cadherin β-catenin,transcription factor c-MYC,cyclin CDK2 and Cyclin D1 were significantly down-regulated in gastric cancer cells after interference with CCDC85 B.Then we performed fluorescent quantitative PCR to detect the changes in m RNA levels and found that the quantitative level of β-catenin was almost unchanged,so we guessed CCDC85 B regulates β-catenin at the post-transcriptional level,and at the ubiquitination level to regulate the protein expression of β-catenin.In order to verify the conjecture,we immediately added drugs to the cells after interference with CCDC85 B,after adding the proteasome inhibitor MG132,western blot experiments showed that the expression of β-catenin protein in the interference CCDC85 B group recovered greatly.We added cycloheximide CHX to the cells overexpressing CCDC85 B,it was also found that the β-catenin half-life of the overexpression CCDC85 B group was significantly prolonged.Finally,we detected ubiquitination at the cellular level,and the results showed that after overexpression of CCDC85 B,the ubiquitination of β-catenin was significantly reduced,which further showed that CCDC85 B regulates the protein expression of β-catenin through the level of ubiquitination,and then affect the proliferation,migration and invasion of gastric cancer cells.4.CCDC85 B competes with β-catenin for binding to E3 ubiquitin ligase β-TrcpIn order to further explore how CCDC85 B affects the ubiquitination and degradation of β-catenin,we learned from the literature that CCDC85 B can bind to a variety of proteins by virtue of its coiled-coil structure,and rely on its binding protein to perform its function,so we passed the mass spectrometry data analysis of CCDC85 B detected β-catenin,a very important E3 ubiquitin ligase β-Trcp.In order to further explore whether they directly interact with each other,we constructed a prokaryotic inducible expression vector and induced expression by prokaryotic.Three proteins of CCDC85 B,β-Trcp andβ-catenin were expressed and purified.Through the GST-pull down experiment,we found that CCDC85 B directly binds to β-Trcp.Later,in order to explore whether CCDC85 B regulates the expression of β-Trcp,we found that interference CCDC85 B could not affect the protein expression of β-Trcp through Western blot detection.In order to explore the relationship between CCDC85 B and β-Trcp,we considered whether there is a competitive binding relationship.Through immunoprecipitation and Western blot experiments,we found that compared with the control group,the combination of β-catenin and β-Trcp in the gastric cancer cell group overexpressing CCDC85 B was significantly less,and the in vitro competitive binding experiment also showed that the combination of CCDC85 B protein with the increase of β-catenin,β-Trcp bound by β-catenin is gradually decreasing,which shows that CCDC85 B does prevent β-Trcp from binding β-catenin.The above results indicate that CCDC85 B affects the ubiquitination and degradation of β-catenin through competitive binding with β-Trcp.5.The specific binding site of CCDC85 B and β-TrcpThrough the above research,we have obtained the competitive binding relationship of CCDC85 B,β-Trcp,and β-catenin.In order to further determine the specific binding site of CCDC85 B and β-Trcp,we truncated CCDC85 B by amino acids,and then we found CCDC85 B binds to β-Trcp in the amino acid range of 40-100.Continue the amino acid truncation experiment.We locate the 80-100 amino acid range of CCDC85 B to combine with β-Trcp.We narrowed the binding range to the range of 80-85 by truncation,and finally we determined that the leucine at position 84 of CCDC85 B is the key site for binding to β-Trcp.After that,we explored the relevant protein data of the 84-site mutant and found that the ubiquitination and degradation of β-catenin protein of the mutant was significantly increased compared with that of the wild type.