The Mechanism Of ATXN3 Deubiquitination Regulating HDAC6 Promoting The Proliferation,Invasion And Metastasis Of Pancreatic Cancer

Objective: Pancreatic cancer is the seventh leading cause of cancer death worldwide,and the number of deaths is increasing year by year.Pancreatic ductal adenocarcinoma(PDAC)accounts for more than 90% of pancreatic cancer.The lack of blood supply and immunosuppressive microenvironment of pancreatic cancer hinder the penetration of drugs and reduce the effect of adjuvant chemotherapy.Therefore,it is of great significance to find new target drugs for molecular therapy of pancreatic cancer.Post-translational modification(PTM)of protein participates in many biological processes and maintains physiological homeostasis of human body.Ubiquitination is a well-known post-translational modification process of protein,which regulates biological processes such as cell apoptosis,immune response and tumorigenesis.Ataxin-3(ATXN3)is a member of the MJDs family in the de-ubiquitinase family.Most of the ubiquitination enzymes play a cancer promoting role in tumors.It has been reported that ATXN3 is involved in the occurrence and development of breast cancer,testicular cancer,lung cancer and other tumors,but the mechanism of action in pancreatic cancer is still unclear.Histone deacetylase 6(HDAC6)is a member of class II HDACs,which is expressed in many organs of human body.Previous studies have shown that HDAC6 is closely related to tumor,participates in the tumorigenesis process through a variety of ways,and can activate and affect the gene expression of some key molecules in the immune system.This topic focuses on the role of ATXN3 in the malignant biological behavior of pancreatic cancer and the regulation mechanism of HDAC6.Methods:1.From January 2019 to March 2020,a total of 40 cases of PDAC tissue and matched normal tissue adjacent to cancer were collected from the General Surgery Department of Shengjing Hospital affiliated to China Medical University.Real-time quantitative polymerase chain reaction(RT-q PCR)and western blot were used to detect the expression of ATXN3 m RNA and protein levels in PDAC tissues and paired normal tissues adjacent to cancer.Immunohistochemistry(IHC)was used to detect the expression of ATXN3 in 56 patients with PDAC.The clinical characteristics between ATXN3 and PDAC patients were analyzed based on the clinical data of patients.2.Compare the expression of ATXN3 m RNA in the normal human pancreatic duct epithelial cell line h TERT-HPNE and five PDAC cell lines MIAPa Ca2,Panc1,CFPAC1,SW1990,Bx PC3,select the cell lines with the highest and lowest expression,and construct the PDAC cell lines with stable over-expression and stable knockdown of ATXN3 through lentivirus as the follow-up experimental objects.The effect of ATXN3 gene on the proliferation of PDAC cells was detected by CCK-8 experiment and Ed U;The effect of ATXN3 gene on the invasion ability of PDAC cells was detected by Transwell test;The effect of ATXN3 on the migration ability of PDAC cells was detected by scratch test;The effect of ATXN3 gene on the apoptosis of PDAC cells was detected by Annexin V-FITC/PI experiment.3.Detect and analyze the protein HDAC6 that has an active relationship with ATXN3 through Bio GRID online database,and verify the targeted interaction relationship between ATXN3 and HDAC6 through immunofluorescence double staining test,immunoprecipitation test and ubiquitination test.Instantly silence HDAC6,detect the changes in the proliferation and invasion ability of PDAC cells through CCK-8 test and Transwell test,and verify the relationship between the two in PDAC cells.The tumorigenicity of ATXN3 in vivo was tested by subcutaneous tumorigenesis test in nude mice.The regulatory relationship between ATXN3 and HDAC6 was verified by Western blot and immunohistochemical staining.Results:1.RT-q PCR was used to detect the expression of ATXN3 m RNA in 40 PDAC tissue samples and 40 normal tissues adjacent to cancer.The results showed that the expression of ATXN3 m RNA in PDAC tissue was significantly higher than that in normal tissues adjacent to cancer.The expression of ATXN3 m RNA in PDAC tissue was 0.0112 ±0.0008,and the expression of ATXN3 m RNA in normal tissues adjacent to cancer was0.0078 ± 0.0005,P<0.01.Western blot was used to detect the expression of ATXN3 protein in 8 paired PDAC and paracancerous normal tissues.The expression of ATXN3 protein in PDAC tissue was significantly higher than that in paracancerous tissues.The relative expression of ATXN3 protein in PDAC tissue was 0.76 ± 0.385,and the relative expression of ATXN3 protein in paracancerous normal tissues was 0.31 ± 0.230,P<0.05.The analysis of clinical data of patients with IHC combination showed that the positive expression of ATXN3 was related to the size and stage of PDAC tumor(P<0.05),but not to the sex,age,histopathological grade and lymph node metastasis of patients(P>0.05).2.RT-q PCR results showed that the expression of ATXN3 m RNA in the normal pancreatic duct epithelial cell line h TERT-HPNE was(1.00 ± 0.08),the PDAC cell line with the highest expression of ATXN3 m RNA was SW1990(2.96 ± 0.26,P<0.05),and the cell line with the lowest expression was Panc1(1.08 ± 0.01,P>0.05).Panc1 cells were used to construct stable overexpression cell lines,which were divided into OE-NC,OE-ATXN3;SW1990 cells were used to construct stable knockdown cell lines,which were divided into sh-NC group and sh-ATXN3-1/2/3 group.The results of CCK-8experiment showed that the OD values of sh-ATXN3-1,sh-ATXN3-2 and sh-ATXN3-3groups at 48 h,72h and 96 h were significantly lower than those of sh-NC group(P<0.05),and the OD values of OE-ATXN3 group at 48 h,72h and 96 h were significantly higher than those of OE-NC group(P<0.05).Ed U test results showed that in SW1990 cells,the cell staining number of sh-ATXN3-1,sh2-ATXN3-2 and sh-ATXN3-3 was significantly lower than that of sh RNA-NC group,and in Panc1 cells,the cell staining number of OE-ATXN3 group was significantly higher than that of OE-NC group.The results of scratch test showed that the migration rate of sh-ATXN3-1,sh-ATXN3-2 and sh-ATXN3-3 groups were significantly lower than that of sh-NC group(P<0.05),while the migration rate of OE-ATXN3 group was significantly higher than that of OE-NC group(P<0.05).Transwell test results showed that the number of cells in sh-ATXN3-1group,sh-ATXN3-2 group and sh-ATXN3-3 group in SW1990 cells was significantly lower than that in sh-NC group(P<0.05),and the number of cells in OE-NC group in Panc1 cells was significantly lower than that in OE-ATXN3 group.The results of apoptosis experiment showed that the apoptosis rate of sh-ATXN3-1,sh-ATXN3-2 and sh-ATXN3-3 groups in SW1990 cells was significantly higher than that of sh-NC group(P<0.05),and the apoptosis rate of OE-NC group in Panc1 cells was significantly lower than that of OE-ATXN3 group.3.The proteins related to ATXN3 were detected and analyzed by Bio GRID online database.RT-q PCR was used to detect the expression of HDAC6 in PDAC tissues and normal tissues adjacent to cancer.The results showed that the expression of HDAC6 m RNA in PDAC tissues was higher than that in normal tissues adjacent to cancer(P<0.01).Western blot showed that the expression of HDAC6 in OE-ATXN3 group was higher than that in OE-NC group,and the expression of HDAC6 in sh-ATXN3 group was lower than that in sh-NC group.The expression of HDAC6 was positively correlated with the expression of ATXN3.The co-localization of ATXN3 and HDAC6 in SW1990and Panc1 cells was confirmed by immunofluorescence double staining;The interaction between ATXN3 and HDAC6 in SW1990 and Panc1 cells was confirmed by immunoprecipitation test.ATXN3 belongs to de-ubiquitination enzyme.Ubiquitin level was detected in the cells of ATXN3 overexpression and knockdown groups.The results showed that the ubiquitin expression level of HDAC6 protein in OE-ATXN3 group was lower than that in OE-NC group,and the ubiquitin expression level of HDAC6 protein in sh-ATXN3 group was higher than that in sh-NC group.Silencing HDAC6 was transiently transfected into the stable overexpression cell line Panc1 of ATXN3.The cell proliferation ability was detected by CCK-8 experiment.The OD values of OE-ATXN3+si RNA-NC group at 48 h,72h and 96 h were 1.080 ± 0.153,1.235 ± 0.158,1.385 ± 0.151,respectively;The OD values of OE-ATXN3+si RNA-HDAC6 group at48 h,72h and 96 h were 0.737 ± 0.082,0.929 ± 0.116 and 0.986 ± 0.104,respectively.The OD value of OE-ATXN3+si RNA-HDAC6 group at 48 h,72h and 96 h was significantly lower than that of OE-ATXN3+si RNA-NC group(P<0.05).The cell invasion ability was detected by Transwell test.The results showed that the number of invasive cells in OE-ATXN3+si RNA-NC group was 134.80 ± 16.10,the number of invasive cells in OE-ATXN3+si RNA-HDAC group was 68.20 ± 6.30,and the number of invasive cells in OE-ATXN3+si RNA-HDAC group was significantly lower than that in OE-ATXN3+si RNA-NC group(P<0.05).The cells in the overexpression and knockdown groups of ATXN3 were planted subcutaneously in nude mice.From the 12 th day to the21 st day,the tumor tissue volume in the OE-ATXN3 group was significantly larger than that in the OE-NC group(P<0.01),and the tumor tissue volume in the sh-ATXN3 group was significantly smaller than that in the sh-NC group(P<0.01).The overexpression of ATXN3 would promote the tumorigenicity of pancreatic cancer cells in vivo.The tumor tissues from mice were made into paraffin sections,and the expression of HDAC6 and PCNA was detected by immunohistochemistry.The results showed that HDAC6 was positive in OE-ATXN3 group and negative in sh-ATXN3 group.Western blot was used to detect the change of protein expression level of ATXN3 and HDAC6 in tumor tissue.The results showed that the expression of HDAC6 was positively correlated with ATXN3,and inhibition of ATXN3 expression would lead to the decrease of HDAC6 expression.Conclusions:1.The m RNA and protein levels of ATXN3 are highly expressed in PDAC tissues,which are closely related to the size and tumor stage of PDAC tumors.2.As the oncogenic gene of PDAC cells,ATXN3 can promote the proliferation,migration and invasion of PDAC cells and inhibit the apoptosis of PDAC cells.3.ATXN3 regulates the expression of HDAC6 through deubiquitination.ATXN3/HDAC6 promotes the proliferation,invasion and metastasis of PDAC.