Effect Of Silencing OTUD1 Gene On Proliferation And Migration Of Human Gallbladder Carcinoma Cells

1.Background and 0bjectiveGallbladder cancer is the most common malignant tumor in biliary system diseases.Its incidence rate ranks 5th in the incidence of human digestive system tumors,and the incidence is very hidden.It is often diagnosed along with gallstones,sometimes called "accidental gallbladder cancer." "Because of the absence of special symptoms and signs in the early stage of gallbladder cancer,routine physical examination is difficult to find.Most of them have missed the best opportunity for diagnosis and treatment at the time of discovery,and naturally progress to the end stage.Most of the prognosis is poor,and the 5-year survival rate is less than 5%.In clinical work,how to detect and diagnose gallbladder cancer early and determine further treatment plans is of great significance.Nowadays,people’s living standards have improved and their health has been paid more and more attention.With the various tests and examinations such as ultrasound,CT and MRI,the early diagnosis rate of gallbladder cancer can be improved.The most common risk factor for gallbladder cancer is gallstones.The incidence of gallstones increases with age,mostly cholesterol gallstones,especially in women with obesity.Patients with cholelithiasis are often associated with chronic inflammation of the gallbladder and bacterial infection of the biliary system,which secrete large amounts of toxins.Studies have shown that biliary bacterial infection combined with chronic inflammation can induce epithelial metaplasia and atypical hyperplasia of the biliary wall,and these changes are closely related to the occurrence of gallbladder cancer.High expression of estrogen receptors in gallbladder epithelial cells may also be a risk factor for gallstone formation.Estrogen receptor activation is a reductase of Mars,a rate-limiting enzyme that increases cholesterol synthesis,promotes cholesterol synthesis,increases cholesterol levels in the gallbladder,and forms gallbladder.Increased likelihood of cholesterol gallstones,which in turn increases the risk factors for gallbladder cancer.Globally,gallbladder cancer has ethnic differences,Asians have a high incidence,Indians and Maori have a high incidence,and European incidence is low.The theoretical molecular weight of the OTUD1 domain is 51.063 kDa,with a pI value of 5.68,belonging to the OTUB subfamily of the ovarian tumor family.OTUD1 is a cysteine protease containing an OTU domain that produces deubiquitination of ubiquitinated proteins in vitro.In 2012,OTUD1 was first reported to be located on chromosome 10p12.Mevissen et al.reported the potential specificity of OTUD1 for the linkage specificity of K63 diubiquitin and its weak specificity for K33 ubiquitin-binding proteins.However,the specific substrates and functions of OTUD1 in cells are still unknown.Studies have shown that the absence of OTUD1 allows breast cancer cells to undergo epithelial cell mesenchylysis to obtain cancer stem cell characteristics,which in turn can spread to distant organs such as bone and lung.The researchers subsequently identified OTUD1 as a potent negative regulator of the transforming growth factor beta(TGF-beta)signaling pathway,while the TGF-beta signaling pathway is a strong inducer of EMT and cancer cell stemness.The ability of the OTUD1 gene to regulate the TGF-β signaling pathway is mainly achieved by its deubiquitination,especially the de-polyubiquitination of SMAD7.SMAD7 is a negative regulator of TGF-β signaling.Polyubiquitinated SMAD7 is a substrate for the expression product of OTUD1 gene,and its protein level is stabilized by deubiquitination in vivo or in vitro.The rate of presentation of SMAD7 protein is reduced and it is more stable in cells.Have to exist.The study also found that OTUD1 also promotes the degradation of the TGF-Β pathway type I receptor(TβRI)on the cell membrane.The activity and stability of p53 are regulated by post-translational modifications,including phosphorylation,acetylation,methylation,glycosylation and ubiquitination.Ubiquitination is the most famous post-translational modification of this,which regulates the stability of p53 by E3 ubiquitin ligase.The end of E3 ubiquitin ligase-mediated p53 ubiquitination is a decrease in the stability of p53 protein and an increased tendency to degrade.Studies have found that OTUD can maintain p53 point stability at the cellular level,thereby enhancing the anti-cancer gene function of p53 gene,maintaining the integrity of the genome and preventing the malignant transformation of cells.2.MethodsBy consulting the medical record system of our hospital,we selected the gallbladder cancer specimens diagnosed by postoperative pathological diagnosis,collected the corresponding paraffin sections and performed immunohistochemical staining of OTUD1 gene in the pathology department of our hospital to understand the gene in the specimens of clinical gallbladder cancer cases.Express the situation.GBC-SD cells were transfected with OTUD1 small interfering RNA(siRNA),real-time PCR was used to detect the gene silencing efficiency after siRNA transfection,western blot was used to detect the expression of OUTD1 protein in cells,and CCK-8 method was used to investigate gene silencing.The effect of proliferative viability,cell scratch assay to determine the ability of cells to migrate in vitro.3.ResultThe OTUD1 protein was positively expressed in 16 specimens.The positive cytoplasm was positive in the positive control group and not in the negative control group.After immunohistochemical staining of OTUD1 gene translation products in adherent cells of gallbladder carcinoma,OTUD1 protein was positively expressed in each sample and localized in the cytoplasm.The difference of 2-ΔΔCt between the three groups of q-PCR experiments was statistically significant.The LSD-t test was continued.The results showed that the transfection group was significantly different from the blank control group and NC transfection group(P 0.000).There was no significant difference between the blank control group and the NC transfection group(P=0.051).The western-blot experiment calculated the ratio of the gray value of the OTUD1 protein to the gray value of the internal reference.The results showed that the gray ratio of the transfection group was statistically different from the blank control group and the NC transfection group(P 0.000).There was no significant difference in the gray ratio between the blank control group and the NC transfection group(P=0.554).There was a statistically significant difference in the healing rate between the three groups in the cell scratch test.The LSD-t test was continued.The results showed that the transfection group was statistically different from the blank control group and the NC transfection group(P values were 0.028,0.018,respectively).There was no significant difference between the blank control group and the NC transfection group(P=0.733).CCK-8 experiment calculated the difference between the absorbance of different groups of cells and the absorbance of zero-adjusted cells.The difference in absorbance difference between the three groups was statistically significant.Continued LSD-t test showed that the cell viability of the transfected group was different from the blank.The difference between the control group and the NC transfection group was statistically significant(P value was 0.000).There was no significant difference between the blank control group and the NC transfection group(P=0.522),and the cell viability was(6.36±3.2)×100%.4.ConclusionsGallbladder cancer cells have a certain OTUD1 positive rate.After silencing OTUD1 gene,gallbladder cancer cells have stronger proliferation ability and migration ability in vitro.