The Expression And Mechanism Of Ring Finger And WD Repeat Domain 3 In Hepatocellular Carcinoma

HCC is one of the most common malignant tumours and exhibits high invasiveness and a high mortality rate,meanwhile,the incidence of HCC has increased rapidly.HCC ranked the sixth among the most common malignancies and the third in the malignant tumor causes death in worldwide.In our country,because there are a large number of hepatitis B patients,HCC ranks the second in the incidence of malignant tumors.In spite of this,along with the unbalanced development of regional economic development and the backwardness of health consciousness in underdeveloped areas,the actual morbidity and mortality rates of HCC may higher than reported data.Despite advances in medical research,early diagnosis of HCC remains difficult,and most patients are diagnosed with middle-or late-stage disease.For patients diagnosed in the early stage of the disease,only a few treatments can be used,and long-term survival after treatment has not improved much in recent years.With the development of biomedicine,more basic studies have been carried out on HCC,but the underlying mechanism remains unclear,and promising interventions are needed.Multiple signal transduction pathways are involved in the cellular response to stress.The ubiquitin-proteasome system(UPS)plays an important role in processes including DNA damage repair,transcription regulation,cell cycle arrest and apoptosis.In DNA damage repair pathway,ataxia telangiectasia mutated(ATM)and ATM and Rad-3 related(ATR)are two checkpoint kinases,Several recent studies have focused on DNA damage repair and the role of the protein RING finger and WD repeat domain 3(RFWD3)in the ATM/ATR pathways.RFWD3 contains a RING finger domain and displays in vitro E3 ubiquitin ligase activity;it has an SQ-rich region in the N-terminus,a coiled-coil domain and a WD40 domain in the C-terminus,and sequence comparison revealed that the residues flanking serine 46 and serine 63 of RFWD3 are conserved.RFWD3 primarily described as an ATM and ATR phosphorylation substrate thus on multiple lysine residues in response to DNA damage.RFWD3 accumulates at stalled replication forks as part of the DNA damage response.At these sites,RFWD3 co-localizes with RPA and binds via its WD40 domain at the C-terminus to replication protein A2(RPA2).It functions in replication checkpoint signalling upon replication stress and promotes replication fork restart after homologous recombination(HR).There is little published information regarding the relationship between HCC and RFWD3.DNA damage repair plays an important role in tumor progression,it is noteworthy that all known E3 ligases function as negative regulators of p53 by promoting its degradation,which is inhibited in response to DNA damage.However,RFWD3 is regarded as a tumour suppressor gene,as recent functional and mechanism studies have indicated that it maintains the stability of p53.In our preliminary qRT-PCR experiments,we found that HCC tissues showed higher RFWD3 gene expression than adjacent normal tissues,prompting us to conduct further experiments.In the present study,we designed the use of the human hepatocellular carcinoma cell lines to simulate the HCC model and to revalidate it in nude mice to prove the role of RFWD3 in HCC.First RFWD3 gene expression was analyzed by qRT-PCR,WB and IHC in HCC and adjacent tissues,in addition,the correlation index and prognosis of HCC patients were analyzed with clinical data.Second the lentivirus was used to silencing the expression of RFWD3 to obtain the stably transfected cells in the HCC cell lines BEL-7404 and HCC-LM3.After checking silence efficiency,celigo/cell cycle/apoptosis and MTT test were used to verify the proliferation and apoptosis of the cells,cell migration and invasion were assessment by scratch wound and transwell assay.Third transfected cells were implanted subcutaneously and tail vein injection in nude mice to observe tumor growth and metastasis.Finally use protein antibody microarray,gene microarray and Ingenuity Pathway Analysis(IPA)to analyze the molecular regulation network.Based on the above conclusions,demonstrated the feasibility of RFWD3 protein as a potential target for molecular target therapy of liver cancer and provides theoretical basis for further basis and clinical trials.Part 1.The expression of RFWD3 in HCC and its correlation with clinical indicators and prognosisAim:Explore the expression of RFWD3 in HCC tissue samples,analyzed the relationship between the relevant indicators and prognosis of HCC patients with clinical data.Methods:1.The expression of RFWD3 in 57 normal liver tissues and 57 HCC tissues were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).2.The expression of RFWD3 in 16 normal liver tissues and 16 HCC tissues were detected by western blot(WB).3.The expression of RFWD3 in 32 normal liver tissues and 32 HCC tissues were detected by immunohistochemical(IHC).Furthermore,the relationship between the clinical data and the relevant indicators of HCC patients was analyzed.4.In order to expand the samples size and reduce the sampling error to ensure the accuracy and reliability of the calculated results,we also used the data of the GEPIA database for RFWD3 expression and related survival analysis.Results:1.qRT-PCR,WB and IHC detection found that the expression of RFWD3 in HCC was significantly increased compared with that of adjacent tissues(P<0.05).2.analyzed the clinical data and IHC,the high expression of RFWD3 was significantly related to tumor volume and TNM staging(P<0.05).3.Survival analysis found that the high expression of RFWD3 had a shorter total survival period,suggesting that RFWD3 may play an important role in promoting the progression of liver cancer(P<0.05).Part 2.The effect of Knockdown of RFWD3 on HCC cell phenotypeAim:Knock down RFWD3 expression with lentivirus in HCC cells,explore the effect of cell phenotype in vitro experiments.Methods:1.Verify the expression of RFWD3 in HCC cell line BEL-7404,HCC-LM3,BEL-7402 and SMMC-7721.2.Knock down RFWD3 expression with lentivirus in HCC cells(Control group:shCtrl;Silent group:shRNAi).3.Using qRT-PCR,WB and fluorescence imaging system to verify the silence efficiency of shRNAi.4.Use MTT to detect the effect of shRNAi on cell proliferation ability.5.Use flow cytometry to detects the effect of shRNAi on its cell cycle status.6.Use flow cytometry to detects the effect of shRNAi on its cell apoptosis status.7.The effect of shRNAi on migration ability was detected by cell scratch healing experiment.8.Use transwell to detects the effect of shRNAi on its migration and invasion ability.Results:1.qRT-PCR showed that RFWD3 was stable in all four HCC cell lines,the highest expression were in BEL-7404 and HCC-LM3 cell lines(AcT>6).2.The mRNA and protein expression levels of RFWD3 were significantly decreased after lentivirus transfection(P<0.05).3.After shRNAi,the cell cycle was blocked in the G2/M period,the apoptosis rate increased,and the proliferation ability was inhibited(P<0.05).4.After shRNAi,the ability of migration and invasion decreased significantly(P<0.05).Part 3.The effect of Knockdown of RFWD3 on HCC cell xenograft model in nude miceAim:Knock down RFWD3 expression with lentivirus in HCC cells,explore the effect of the biological behavior in vivo experiments.Methods:1.The model of hypodermic HCC in nude mice was constructed using shRNAi cells.2.The HCC lung metastasis model in nude mice was constructed by shRNAi cells.3.The effect of biological behavior was detected by imaging and HE staining.Results:1.The tumor volume of the shRNAi group decreased significantly in the subcutaneous nude mice models(P<0.05).2.The metastasis and pulmonary sizes of the shRNAi group was significantly reduced in the HCC lung metastasis nude mice model(P<0.05).3.In the model of HCC lung metastasis,HE staining confirmed that the number and volume of lung tumor metastases in shRNAi group were significantly reduced(P<0.05).Part 4.The molecular biological mechanism of RFWD3 in HCC.Aim:Using gene chip and antibody microarray technology,exploring the possible molecular biological mechanisms of RFWD3 in HCC to demonstrate the possibility of RFWD3 protein as a potential target for molecular target therapy of HCC and provide theoretical basis for further foundation and clinical trials.Methods:1.Protein antibody microarray was used to detect the expression of related proteins in the Stress and Apoptosis signaling pathway after shRNAi.2.Gene expression microarray was used to detect the change of gene expression profile after shRNAi.3.Using IPA to analyze the related network control mechanism.Results:1.The Stress and Apoptosis protein antibody chip found the protein expression levels of IkBa(Total),Survivin and a-Tubulin were significantly lowered.The protein phosphorylation level of P44/42 MAPK(ERK1/2),HSP27,p53,SAPK/JNK,Chkl,Chk2,IkBa(Ser32/36),eIF2a and TAK1 were significantly lowered(P<0.01).2.After screening,the gene expression profile chip data showed that there were 454 mRNA expression changes significantly(above 2-fold,P<0.05).3.Genes involved in cell death,viability and survival,cell movement and invasion,cellular assembly and organization,DNA replication,recombination and repair,and cellular function and maintenance were significantly enriched.Classical pathway analysis next re-vealed that Wnt/?-catenin and TGF-? signalling pathways were altered following RFWD3 silencing(P<0.05).Conclusions1.RFWD3 gene expression was increased in liver cancer,and was closely related to tumor size and TNM staging.RFWD3 low expression of HCC patients had a higher survival time than RFWD3 expression patients.2.After the silence expression of RFWD3,in vitro experiments confirmed that the cell cycle was blocked in the G2/M phase,the apoptosis rate increased,and the proliferation was inhibited,while the invasion and metastasis ability decreased significantly in HCC cells.3.After the silence expression of RFWD3,it was confirmed that the growth,invasion and metastasis of HCC cells were significantly decreased.4.RFWD3 mediated HCC tumor development by regulating the MAPK,Wnt/?-catenin and TGF-? signalling pathways.