The Deubiquitination Enzyme OTUD6B Stabilizes PVHL And Inhibites The Metastasis Of Renal Clear Cell Carcinoma

The deubiquitination enzyme OTUD6 B stabilizes p VHL and inhibites the metastasis of renal clear cell carcinomaBackground: Renal cell carcinoma is a kind of malignant tumor originating in the tubular epithelium of the kidney.According to the 2020 “World Cancer Report”,the incidence of kidney Cancer accounts for about 3% of the adult malignant tumor in the worldwide.the most common pathological type is clear cell Renal Cell Carcinoma,about 80% of Renal Cell Carcinoma.In recent decades,the incidence of kidney Cancer in most countries and regions all showed a trend of sustained growth.The cause of renal cancer is not clear,but its incidence is related to genetics,smoking,obesity,high blood pressure and so on.Hereditary renal carcinoma accounts for 2%~4% of the total,and most of them are inherited in the family by autosomal dominant inheritance.Hereditary renal carcinoma has been confirmed to include VHL syndrome and BHD syndrome.Moreover,VHL gene mutations have also been found in more than half of sporadic clear cell renal cell carcinomas,in addition to hereditary renal cell carcinomas.Previous studies in our laboratory have found that OTUD6 B,a member of the OTU-subfamily of deubiquitinated enzymes,is a tumor suppressor gene in HCC.OTUD6 B can interact with the VHL complex and stabilize PVHL in a way independent of enzyme activity,thereby antagonizing the VHL-HIF signaling pathway and inhibiting the metastasis of hepatocellular carcinoma.Although studies have shown that OTUD6 B can act as a positive regulator of VHL protein stability and act as a tumor suppressor in hepatocellular carcinoma,it is unclear whether OTUD6B has the same regulatory mechanism in clear cell renal cell carcinoma.In addition,more than half of the patients with clear cell renal cell carcinoma have VHL mutation,whether OTUD6 B can still play the function of stabilizing VHL protein in these patients and what is the relationship between OTUD6 B and the prognosis of patients,we conducted a further study on these.Aims To investigate whether deubiquitylating enzyme OTUD6 B can inhibit tumor development by stabilizing p VHL in clear cell renal cell carcinoma;To analyse the effects of OTUD6 B on proliferation,migration and EMT of cc RCC;To make sure whether OTUD6 B can continue to play a tumor suppressive role in VHL mutated cc RCC.Methods 1)Pathological information of patients with cc RCC and related expression data of OTUD6 B and VHL were obtained through TCGA database,and the correlation between the m RNA level of OTUD6 B and the protein level of VHL and tumor metastasis and prognosis was analyzed.2)Patients with cc RCC in the TCGA database were divided into the VHL mutation group and the wild group.The correlation between the m RNA level of OTUD6 B and the survival and tumor metastasis of the two groups was analyzed,and the VHL mutation group was further divided according to the type of mutation to analyze the correlation between OTUD6 B and the survival of different types of mutation groups.3)Three renal clear cell cancer cell lines（one VHL-WT ACHN cell line and two VHL-frameshift mutant A498 and 786 O cell lines）were selected for targeted transfection of OTUD6 B sh-RNA to down-regulate its levels.4)The above three cell lines were detected by cell migration and proliferation assay,and the effects of OTUD6 B knockdown on migration and proliferation of three renal clear cell cancer cells were observed.5)The changes of PVHL,hypoxia-inducing factor HIF-2α and related epithelial mesenchymal transformation（EMT）indexes after OTUD6 B knockdown were detected by Western blot.6)According to the VHL mutation database,more than ten kinds of VHL hot spot mutation plasmids were constructed to detect the regulation of OTUD6 B on point mutation VHL in 293 T cells,and several point mutation VHL were selected to further verify whether OTUD6 B interacts with these point mutation VHL and whether it can affect its ubiquitination level.Results 1)Analyzed the pathological information of patients with renal clear cell carcinoma in the TCGA database,it was found that the m RNA levels of OTUD6 B and VHL were correlated with tumor metastasis and patient prognosis.2)For patients with VHL mutated renal clear cell carcinoma,the m RNA level of OTUD6 B was still correlated with tumor metastasis and prognosis.For different types of VHL mutations,OTUD6 B is associated with the prognosis of patients with VHL missense mutations.3)Cell proliferation and migration assay results showed that knocking down OTUD6 B in three renal clear cell cancer cell lines resulted in increased migration of ACHN cell lines（P <0.05）,the migration ability of A498 and 786 O cell lines was not significantly changed,and the proliferation ability of the three cell lines was not affected.4)Western blot results showed that after OTUD6 B knockdown,the levels of PVHL and E-cadherin in ACHN cell lines decreased,while the levels of HIF-2α and Slug increased.In A498 and 786 O cell lines,the above indexes did not change significantly when OTUD6 B was knocked down.5)OTUD6B and hot VHL mutant plasmid were transfected in 293 T cells,and Western blot results showed that OTUD6 B still had a stabilizing effect on most point mutant VHL proteins,and reduced its ubiquitination degradation.Conclusion: In cc RCC,OTUD6 B can inhibit cell migration by stabilizing p VHL,down-regulating HIF-2α level,and for p VHL with partial point mutation,OTUD6 B can still exert tumor suppressive function through this effect.The deubiquitination enzyme OTUD6 B stabilizes p VHL and inhibites the metastasis of renal clear cell carcinomaMechanism of the deubiquitination enzyme OTUB2 involved in the growth and metastasis of hepatocellular carcinoma under hypoxiaBackground: Hepatocellular carcinoma（HCC）is the main histological subtype of liver cancer,accounting for more than 85% of primary liver cancer.Hepatitis B virus infection,hepatitis C virus infection,alcohol consumption,aflatoxin exposure,liver fluke infection,obesity and other major risk factors for liver cancer.China has the largest population of liver cancer in the world.Although the prevention and treatment of liver cancer have been continuously improved in recent years,the prevention and treatment of liver cancer is still not optimistic.As one of solid tumors,hepatocellular carcinoma often has a hypoxic microenvironment inside.As a key factor in tumor hypoxia response,hypoxia inducible factor（HIF）can regulate tumor angiogenesis,energy metabolism,cell proliferation,invasion and metastasis and other related genes.It leads to the increase of tumor malignancy and the change of tumor biological behavior.It has been mentioned in the past literature that the OTUs family of deubiquitinases may be regulated by hypoxia signals and their expression increases under hypoxia conditions.As a member of the OTUB2 family of deubiquitin enzymes,OTUB2 has been frequently reported in the literature in recent years,and has been involved in the occurrence and development of various tumors including thyroid cancer and endometrial cancer.However,its function in liver cancer remains to be further explored.Aims To explore the expression of OTUB2 in liver cancer and normal liver tissue,its influence on the prognosis of patients with HCC,and its role in the development of HCC,and to explore the regulation of OTUB2 by HIF in the culture of HCC cell lines in hypoxia environment.Methods 1)The m RNA expression level of OTUB2 in hepatocellular carcinoma and normal tissues was analyzed by TCGA database,and the relationship between the level of OTUB2 and the prognosis of patients was analyzed.2)Immunohistochemistry（IHC）was used to detect the expression of OTUB2 protein in 98 cases of hepatocellular carcinoma and 39 cases of adjacent normal tissues,and to analyze the relationship between the protein level and the pathological information of patients.3)The correlation between OTUB2 and m RNA levels of HIF-related downstream target genes was analyzed by TCGA data.4)The HIF and OTUB2 protein levels of SMMC7721 and LM3 liver cancer cell lines were detected by Western blot.5)After the addition of digoxin,the HIF and OTUB2 protein levels of SMMC7721 and LM3 liver cancer cell lines were detected by Western blot.Results 1)The m RNA levels of OTUB2 in 369 cases of hepatocellular carcinoma and 50 cases of normal liver tissue in TCGA database were analyzed.It was found that the expression of OTUB2 m RNA in cancer was significantly higher than that in normal tissue,and was correlated with the prognosis of patients.Patients with high expression of OTUB2 m RNA had worse prognosis.2)Immunohistochemical analysis showed that OTUB2 expression wassignificantly higher in HCC tissues compared with normal liver tissues（P <0.05）.The positive expression rate was 22.5%（9/40）in 40 normal liver tissues and 79.6%（78/98）in 98 hepatocellular carcinoma tissues,and the difference was statistically significant（P <0.001）.3)TCGA analysis showed that OTUB2 was positively correlated with the m RNA levels of HIF-related target genes,suggesting that OTUB2 might also be one of the HIF target genes.4)Western blot analysis showed that the HIF-2α and OTUB2 protein levels increased with the prolonging of hypoxia.5)After the addition of digoxin,HIF-2α protein level was inhibited by hypoxia culture,and OTUB2 protein level decreased with the prolongation of hypoxia.Conclusion Both m RNA and protein of OTUB2 are highly expressed in hepatocellular carcinoma and may be regulated by HIF-2α as a downstream target gene and participate in the progression of hepatocellular carcinoma in hypoxic environment.