The Research On SUMO1 And UBA2 Abnormally High Expressions And Their Resistance To Oxaliplatin In Colorectal Cancer

Colorectal cancer(CRC)is the third most common cancer in men and the second in women worldwide.China is the country with the largest number of new cases and deaths every year.However,the mechanism of occurrence,development in CRC still remains unclear.SUMOylation is a post-translational modification characterized by covalent and reversible binding of small ubiquitin-like modifier(SUMO)to a target protein.SUMO proteins are critically involved in the modulation of nuclear organization and cell viability.Their expression is significantly increased in processes associated with carcinogenesis and chemotherapy resistance.However,little is known about the role of SUMOylation in CRC.In our study,we undertook RT-PCR,Western blot and immunohistochemistry to test SUMO1 and SUMO2 expression in CRC.We found the SUMO1 expressions in tumor tissues were more significant than in SUMO2 expression,which was in accordance with the results form ONCOMINE analysis.SUMO1 played a more important role in the process of carcinogenesis.Subsequently,we conducted lentivirus vectors inhibiting or overexpressing SUMO1 to infect colon cancer cells and made immunohistochemistry for SUMO1 by tissue microarray.The results were shown that SUMO1 overexpression could significantly enhance the malignant abilities of proliferation,invasion and metastasis in tumor cells,accelerate cell cycle,and promote resistance to oxaliplatin.UBA2 was one of the heterodimer of SUMO1 activating enzyme subunit 1.UBA2 was significantly up-regulated in cancerous tissues in comparison with adjacent normal mucosa and UBA2 might be an independent prognostic factor for CRC.UBA2 was a key molecule of SUMO1 promoting carcinogenesis of colorectal cancer and oxaliplatin resistance.UBA2 knockdown could significantly inhibit the malignant biological behavior of colon cancer cells,promote cancer cell apoptosis and G1 phase arrest,and increase the sensitivity of oxaliplatin.In order to elucidate the mechanism of SUMO1 and UBA2 promoting carcinogenesis and oxaliplatin resistance,RNA-seq was used to sequence the colon cancer cells with UBA2 knockdown and oxaliplatin and analyzed their related differential genes and enrichment pathway.We discovered that CDKN1 A was probably the target gene of UBA2.SUMO1 might accelerate the transformation of cancer cells cycle to promote the development of CRC,inhibit cancer cells apoptosis to be resistant to oxaliplatin by indirectly regulating CDKN1 A.Part Ⅰ.Expression of SUMOs in CRCObjective:To examine the expression of SUMO1 and SUMO2 in normal mucosa and tumor tissues and then identify their intensity difference in CRC.Methods:RT-PCR,Western blot and immunohistochemistry were used to examine the expression of SUMO1 and SUMO2 in normal mucosa and tumor tissues,combined with Oncomine online software.Results:(1)SUMO1 and SUMO2 expression was up-regulated in cancerous tissues compared with the corresponding non-cancerous mucosa at the RNA and protein levels.(2)The expression of m RNA and protein in SUMO1 was significantly higher than that in SUMO2(3)Oncomine data mining software analyzed SUMO1 and SUMO2 expression in 215 CRC cases from TCGA database.The expression of SUMO1 was significantly higher than SUMO2,which was in accordance with our results.Conclusions:The high expression of SUMOs in CRC suggested that SUMOs may be closely related with the occurrence and development of CRC.The expression of SUMO1 was significantly higher than SUMO2,indicating that SUMO1 played a more important role than SUMO2 in the process of CRC.Part Ⅱ.The research on role of SUMO1 in CRC and its resistance to oxaliplatinObjective:To further investigate the expression and function of SUMO1 in CRC,and to explore its mechanism of resistance to oxaliplatinMethods:We constructed lentivirus vectors inhibiting or overexpressing SUMO1 to infect colon cancer cells,conducted immunohistochemistry for SUMO1 by tissue microarray and made nude mice tumorigenicity compare tumor growth after SUMO1 overexpression.Results:(1)SUMO1 knockdown could weaken colon cancer cells abilities of proliferation,migration and invasion;vice versa,SUMO1 overexpression could enhance colon cancer cells abilities of proliferation,migration and invasion.(2)SUMO1 knockdown had little effect on the apoptosis,but it could lead to G1 phase block.(3)Up-expression of SUMO1 was significantly correlated with T stage,N stage,M stage,AJCC stage.Those patients with positive HOXA13 expression had an obviously lower overall survival(OS)and disease-free survival(DFS)rate than patients with negative SUMO1 expression.(4)The colon cancer cells with SUMO1 overexpression were resistant to oxaliplatin.(5)SAE1,UBA2 and UBC9 were significantly enhanced in colon cancer cells with SUMO1 overexpression,especially UBA2 and UBC9.The UBA2 expression was increased by dose-dependent of concentration of oxaliplatin.(6)The IC50 value of oxaliplatin was significantly reduced after colon cancer cells with SUMO1 overexpression were knockdown UBA2.(7)SUMO1 overexpression promoted tumor growth and resistance to oxaliplatin in nude mice.Conclusions : SUMO1 overexpression could significantly enhance the malignant abilities of proliferation,invasion and metastasis in tumor cells,accelerate cell cycle,and promote resistance to oxaliplatin.UBA2 was one of the heterodimer of SUMO1 activating enzyme subunit 1.UBA2 was a key molecule of SUMO1 promoting carcinogenesis of colorectal cancer and oxaliplatin resistance.Part Ⅲ.The research on role of UBA2 in CRC and its resistance to oxaliplatinObjective:To further investigate the expression and function of UBA2 in CRC,and to explore its mechanism of resistance to oxaliplatinMethods:We conducted immunohistochemistry for UBA2 by tissue microarray,constructed lentivirus vectors inhibiting UBA2 to infect colon cancer cells,and made next generation sequencing to compare differential genes and analyze enrichment pathway for UBA2.Results:(1)UBA2 expression was significantly up-regulated in cancerous tissues compared with the corresponding non-cancerous mucosa;The UBA2 expression from the normal intestinal mucosa to low-grade intraepithelial neoplasia to high-grade intraepithelial neoplasia to adenocarcinoma showed a trend of gradual enhancement.(2)Up-expression of UBA2 was significantly correlated with tumor size,T stage,N stage,M stage,AJCC stage.Those patients with positive UBA2 expression had an obviously lower overall survival(OS)and disease-free survival(DFS)rate than patients with negative UBA2;UBA2 was a significant independent prognostic factor and could serve as a putative marker for diagnosis and prognosis of CRC.(3)UBA2 knockdown could significantly inhibit the malignant biological behavior of colon cancer cells,promote cancer cell apoptosis and G1 phase arrest,and increase the sensitivity of oxaliplatin.(4)UBA2 knockdown inhibited tumor growth and sensitivity to oxaliplatin in nude mice(5)RNA-seq was used to sequence the colon cancer cells with UBA2 knockdown and oxaliplatin and analyzed their related differential genes and enrichment pathway.Conclusions: We preliminary discovered that CDKN1 A was probably the target gene of UBA2.UBA2 was a key molecule of SUMO1 promoting carcinogenesis of colorectal cancer and oxaliplatin resistance.SUMO1 might accelerate the transformation of cancer cells cycle to promote the development of CRC,inhibit cancer cells apoptosis to be resistant to oxaliplatin by indirectly regulating CDKN1 A.