The Role And Regulation Mechanism Of DNA Endonuclease Mus81 In The Metastasis Of Gastric Cancer

Part1 The expression of Mus81 in gastric cancer and its clinicopathological significance Objective: To study the expression of Mus81 in gastric cancer and clarify the correlation of Mus81 and clinicopathological significance.Methods: The RNA-sequence data from The Cancer Genome Altas(TCGA)were utilized to identify Mus81 expression in gastric cancer tissues.29 human specimens including gastric cancer tissues and paired adjacent normal tissues were obtained from the Department of Gastrointestinal Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology.Western blot and immunohistochemistry were used to determine the protein level of Mus81 in the surgical specimens.Then,the correlation of Mus81 and clinicopathological significance was analyzed.The relationship of Mus81 and overall survival was determined by Kaplan-Meier analysis.Furthermore,q RT-PCR and Western blot were respectively used to examine the m RNA and protein of Mus81 in human gastric cancer cell lines AGS,SGC7901,MGC803,BGC823,HGC27 and MKN45 and the immortalized human gastric mucosal cell line GES-1.Results: From the TCGA data,the expression of Mus81 was higher in 415 gastric cancer tissues than 34 normal tissues(P = 0.003).The Western blot data showed that Mus81 protein was elevated in gastric cancer tissues,compared with adjacent tissues(P = 0.0425).Additionally,immunohistochemistry staining revealed that Mus81 mainly located in the nucleus and was increased in gastric cancer tissues compared with normal tissues.According to the IHC scores,58.62% of the gastric cancer samples(17/29)were positive for Mus81 expression,while 27.58% of the normal tissues(8/29)were scored positive.Furthermore,Mus81 high expression was significantly correlated with lymph node metastases(P = 0.024),but not with gender(P = 0.720),age(P = 0.216),depth of invasion(P = 0.453),distant metastases(P = 0.527)and TNM stage(P = 0.527).Moreover,high expression of Mus81 positively correlated to the number of lymph node metastases(P = 0.0413).In addition,Kaplan-Meier analysis indicated that patients with Mus81 high expression had a significantly poorer overall survival(P = 1.6e-06).The Mus81 expression in gastric cancer lines was elevated both at the m RNA and protein levels.Conclusions: Mus81 is higher expressed in gastric cancer and correlated with lymph node metastasis in patients with gastric cancer.Part2 Mus81 promotes gastric cancer metastasis by regulating ZEB1 transcription.Objective: To study the effect of Mus81 on gastric cancer metastasis and its molecular mechanism.Methods: We checked the ability of migration in human gastric cancer cell lines AGS,SGC7901,MGC803,BGC823,HGC27 and MKN45 and the immortalized human gastric mucosal cell line GES-1 by Transwell assay,and analyzed the correlation between Mus81 expression and cells migration.Lentivirus was used to stably knock down Mus81 in SGC7901 and BGC823 cells and overexpress Mus81 in GES-1 and MGC803 cells.q RT-PCR was determined the Mus81 expression in knock down or overexpressed cells.The influence of Mus81 on cell proliferation was examined by MTT and colony formation assay.The cell cycle distribution was determined by PI staining and Flow cytometry.Cell migration was detected by wound healing and Transwell assay.q RT-PCR and Western blot were utilized to detect the EMT related molecule,including E-cadherin,N-cadherin,ZEB1,ZEB2,and Snail1.We designed the Ch IP primers according to ZEB1 promotor and performed Ch IP assays to investigate that Mus81 promotes ZEB1 transcription by binding to ZEB1 promoter.The correlation between Mus81 and ZEB1 in GC tissues was assessed by Pearson’s analysis.Results: The cells migration capability in AGS,SGC7901,MGC803,BGC823 and HGC27 is stronger than GES-1 cells and coincide with the Mus81 expression almost.Mus81 sh RNA significantly decreased the Mus81 expression in SGC7901 and BGC823 cells.The expression plasmid increased the Mus81 expression in GES-1 and MGC803.MTT,colony formation and cell cycle distribution assays revealed that Mus81 knockdown alone has limited effect on cell proliferation and cell cycle distribution(P>0.05).Wound healing and Transwell assays revealed that Mus81 knockdown decreased the migration ability ofSGC7901 and BGC823 cells and Mus81 overexpression enhanced the migration ability of GES-1 and MGC803.Mus81 knockdown increased E-cadherin expression and inhibited the expression of N-cadherin.Mus81 overexpression was associated with the opposite effect.Moreover,knockdown of Mus81 decreased the m RNA and protein expression of ZEB1 and did not affect the expression of ZEB2 and Snail1.From Ch IP,the binding between Mus81 and the promoter region of ZEB1 was observed.For Pearson’s analysis,Mus81 expression was positively correlated with the expression of ZEB1 in the 29 patients with gastric cancer(P = 0.0006,r = 0.5893).Conclusions: Mus81 promotes the migration of gastric cancer cells by regulating ZEB1 transcription.Part3 The molecular mechanism of BRD4 regulate Mus81 expression Objective: To study the mechanism by which BRD4 regulates gastric cancer metastasis through Mus81.Methods: To investigate the upstream molecule of Mus81,the small molecular inhibitors screening was carried out.BRD4 inhibiton by si RNA or AZD5153,cells migration was detected by wound healing and Transwell assay.q RT-PCR and Western blot were utilized to detect Mus81 m RNA and protein expression,respectively.Screening the intermediates mediating BRD4 regulation of Mus81 expression by bioinformatics analysis was performed.Identifying Sirt5 as the intermediate molecule and analyse the role of Sirt5 in GC cells migration and Mus81 expression.Furthermore,Western blot was used to examine the effect of BRD4 inhibition on the expression of Sirt5.Finally,the effect of AZD5153 and Mus81 on gastric cancer metastasis was analyzed by a nude mice model of lung metastasis.Results: The small molecular inhibitors screening results revealed AZD5153 significantly decreased Mus81 m RNA expression.BRD4 inhibition by si RNA or AZD5153 could decrease Mus81 m RNA and protein expression and inhibited GC cells migration.Moreover,BRD4 inhibition by AZD5153 had limited effect on cell migration in Mus81-depleted cells.Bioinformatics analysis showed BRD4 might regulate Mus81 in gastric cancer cells through Sirt5.Knockdown Sirt5 significantly decreased Mus81 expression and inhibited GC cells migration.Furthermore,knockdown Sirt5 could inhibit the ectopic expression of Mus81 and Mus81-induced cells migration.BRD4 inhibition suppressed Mus81 and ZEB1 expression.For nude mice model of lung metastasis,Mus81 knockdown inhibited the formation of lung metastasis nodule in vivo and AZD5153 could suppress GC cells metastasis by down-regulating Mus81 expression.Conclusions: BRD4 regulate Mus81 expression through Sirt5 in gastric cancer.AZD5153 suppress gastric cancer metastasis and BRD4 may be a potential target for the inhibition of gastric cancer metastasis.