Study On Mechanism Of Ring Finger Protein14 Promoting Proliferation And Invasion Of Hepatoma Cells By Down-Regulating Hepatocyte Nuclear Factor-1?

Malignant tumors progress rapidly with a poor prognosis,which is one of the main culprits threatening the health of contemporary human beings.Hepatocellular carcinoma(HCC),as one of the most common malignant tumors of the digestive system,ranks fifth in the incidence and second in mortality globally,while it ranks fourth in the incidence and second in mortality in China.The number of yearly new cases of HCC in China took up more than half of the global yearly new cases.HCC is a severe threat to Chinese people' life and health.The onset of hepatocellular carcinoma is occult,and it progresses rapidly,with a high recurrence rate and poor prognosis.The overall survival rate within 1 year of HCC patients is less than 50% and less than 10% within 5 years.The current understanding of the pathogenesis of hepatocellular carcinoma remains unclear.Therefore,extending the research on the molecular mechanism of hepatocellular carcinoma carcinogenesis is of great significance for the clinical treatment and prognosis of HCC patients.In recent years,more and more studies have shown that epigenetic regulatory disorders are crucial in the occurrence and development of tumors.As an important post-translational epigenetic modification,ubiquitination modification is highly involved in many cellular biological processes,and its dysregulation can promote the occurrence of many malignant tumors.Ring finger protein 14(RNF14),as a member of RBR(RING-between-RING)subfamily of E3 ubiquitin ligase,has a RING finger domain,which can cause ubiquitination and degradation of itself and its substrates.RNF14 plays an important role in the development of many gastrointestinal tumors.It has been reported that RNF14 can accelerate the progression of Barrett's esophagus to esophageal adenocarcinoma;RNF14 can cooperate with the TCF / LEF to target promoter and then stabilizing the recruitment of ?-catenin,thereby positively regulating the transduction of Wnt signals in colon cancer cells,which is very important for the survival of colon cancer cells;Another study have reported that RNF14 can promote the progression of cell cycle from G1 phase to S phase,promoting DNA replication and synthesis and regulating the proliferation of tumor cells;In addition,studies have shown that RNF14 also plays an important role in the development of prostate cancer and breast cancer.However,the effect of RNF14 on the occurrence and development of HCC remains unclear,while there is no corresponding research to prove that RNF14 can participate in the regulation of malignant biological behaviors of tumors through its E3 ubiquitin ligase-dependent manner.Hepatocyte nuclear factors(HNFs)are predominantly expressed in the liver,and can regulate the expression of a variety of liver-specific genes,which are essential for liver development,hepatocyte differentiation and regeneration.The main members of HNFs family include HNF1,HNF3,HNF4 and so on.HNF1?,as a member of this family,is closely related to the occurrence of hepatocellular carcinoma.It is reported that HNF1? can inhibit the proliferation of hepatoma cells in vitro and in vivo,suggesting that HNF1? is a tumor suppressor of hepatocellular carcinoma.In molecular mechanism,the stability of HNF1? protein is poor,with a half-life of only 4-6 hours,suggesting that it might be regulated by ubiquitin-proteasome system.When searching for the potential substrates of RNF14 through the uniprot protein database(http://www.uniprot.org/),we found that there is a mutual binding between HNF1? and RNF14,suggesting that RNF14 might be the E3 Ubiquitin ligase of HNF1?.Therefore,this article focuses on the regulatory effects of RNF14 on the proliferation,invasion and metastasis of hepatoma cells,as well as the exact mechanism by which RNF14 recognizes and binds HNF1? to promote its degradation.It is of certain value to further clarify the molecular mechanism of the occurrence and development of HCC and improve the clinical treatment effect and outcomes of HCC patients.Materials and Methods:1.Regulatory role of RNF14 in the occurrence and development of liver cancerIn this part of the research,the expression of RNF14 in hepatocellular carcinoma tissues and adjacent tissues,in normal human hepatocytes and hepatoma cells were compared through database query and Western blot experiments;Through transwell migration and invasion experiments,the effects of RNF14 on the invasion and metastasis of hepatoma cells were analyzed;Through CCK8 proliferation experiment,colony formation experiment and subcutaneous tumor formation experiment in mice,the effect of RNF14 on the proliferation of hepatoma cells were studied.2.RNF14 regulates HNF1? and its mechanismIn This part of the research,with a half-life experiment and MG132 to block the ubiquitin proteasome system,the possible degradation pathways of HNF1? were analyzed;Through immunoprecipitation experiment,the mutual binding effects between RNF14 and HNF1? were studied;to study exogenous Through overexpression and knockout of RNF14,the effects of RNF14 on HNF1? expression were studied;And through Ni-NTA pull-down experiment,we investigate whether RNF14 can affect the ubiquitination level of HNF1?.The main results and conclusions of this research are as follows:1.RNF14 is highly expressed in liver cancer tissues and hepatoma cells;(1)The expression of RNF14 mRNA in liver cancer tissues is significantly higher than in adjacent tissues;(2)Compared with normal human hepatocytes L02,the expression of RNF14 in hepatoma cells(Hep3B,HepG2,SMMC-7721,BEL-7404,Huh7,PLC)were significantly increased.2.RNF14 promotes the proliferation of hepatoma cells(1)RNF14 enhances the proliferation ability of hepatoma cells(Huh7,BEL-7404);(2)RNF14 enhances the colony-forming ability of hepatoma cell BEL-7404;(3)Knockdown of RNF14 significantly inhibit the subcutaneous tumorigenic ability of hepatoma cell BEL-7404.3.RNF14 promotes the migration and invasion of hepatoma cells(1)RNF14 promotes the migration ability of liver cancer cells(Huh7,BEL-404);(2)RNF14 promotes the invasion ability of liver cancer cells(Huh7,BEL-7404).4.HNF1? has a ubiquitin-proteasome degradation pathway(1)HNF1? has a relatively short half-life of 4-6 hours;(2)HNF1? is regulated by the ubiquitin-proteasome system.5.RNF14 may degrade HNF1? through ubiquitin-proteasome pathway(1)The immunoprecipitation experiment proves that there is a mutual binding between HNF1? and RNF14;(2)Overexpression of RNF14 inhibits the expression of HNF1?;(3)Down-regulating RNF14 promotes HNF1? expression;(4)RNF14 increases the ubiquitination level of HNF1?.In Conclusion,the main results of this research are as follows:(1)RNF14 is highly expressed in liver cancer tissues and liver cancer cells,and can promote the migration,invasion and value-added capabilities of liver cancer cells;(2)RNF14 can recognize and bind HNF1?,a tumor suppressor of liver cancer,and can inhibit the expression of HNF1?,suggesting that RNF14 may exert its cancer-promoting effect by down-regulating HNF1?;(3)There is a ubiquitin-proteasome degradation pathway in HNF1?,and RNF14 can up-regulate the ubiquitination level of HNF1?,suggesting that HNF1? may be a ubiquitination substrate of RNF14,and RNF14 may exert its cancer-promoting effect by degrading HNF1? through ubiquitination..This study proposed for the first time that RNF14 can promote the proliferation,invasion and metastasis of liver cancer cells,indicating that RNF14 plays an important role in promoting the development of liver cancer.It was found that HNF1? may be a ubiquitinated substrate for RNF14,and RNF14 may be passed through ubiquitin.The degradation of HNF1? promotes the proliferation,invasion and metastasis of liver cancer cells,and improves the understanding of RNF14,an E3 ubiquitin ligase molecule that has important functions in tumors.It is urgent to further study the specific ubiquitination regulation mechanism of HNF1? by RNF14,provide new directions and research ideas for exploring the molecular mechanism of liver cancer,and provide new choices and directions of apparent targeted therapy for the treatment of patients with liver cancer in the future.