Studies On Mechanisms Of RAI16 And SNORD113-1 In Tumorigenesis Of Hepatocellular Carcinoma

Background and Aim:Primary hepatocellular carcinoma represents the sixth most common cancer worldwide and the third leading cause of cancer-related death(Data of WHO GLOBOCAN project,2012).Hepatocellular carcinoma(HCC)accounts for 80%~90% of primary hepatocellular carcinoma.Despite the major advances achieved in the diagnostic workup and treatment of HCC,5-year survival rates after resection for early-stage HCC ranges between 17% and 53%,and recurrence rates can be as high as 70%.Now,it is one of the important hotspots to study HCC biomarkers and therapy targets.HCC biomarkers include serum and tissue markers.At present,many HCC tissue markers have been reported,mainly including GPC3,HSP70,TAG72,Ki-67,HepPar1,HERC5 and so on.HCC serum markers include AFP,AFP-L3,GP73,APO-J,DKK-1,HCR2,Midkine,de-?-carboxy prothrombin,GPC3,?-1-fucosidase,HGF,VEGF,TGF-?,EGFR,Wnt,angiopoietin-1/2,NOTCH and the like.While,AFP is the only one widely used in HCC clinical diagnosis as serum marker.However,AFP is negative in about 1/3 of HCC patients.Therefore,further identification of biomarkers involved in the development of HCC,not only helps to understand the molecular mechanism of HCC,but also helps to find early diagnosis markers and potential therapy targets for HCC.In this study,we selected RAI16 and SNORD113-1,which were dysregulated in HCC tissues,to study their roles and mechanisms in tumorigenesis of HCC.1.Study on mechanisms of RAI16 promoting HCC tumorigenesisRAI16(Retinoic acid-induced protein 16)belongs to retinoic acid-induced protein family,the molecular weight of which is about 82 kDa.Homology analysis shows that RAI is conserved in human,rat,mouse,rabbit and zebrafish,which indicates that RAI might play an important role in basic cell function.A number of studies have shown that RAI can regulate the development of multiple diseases.RAI16 is a relatively new protein.Its structure and function are not clear.Our previous study reported that RAI16 could enhance tumorigenesis in HCC as the target of miR-483-5p.And,RAI16 might serve as a useful therapeutic agent for HCC gene therapy and tumor marker for HCC diagnosis.Further,the cells over-expressing RAI16 showed resistant to apoptosis to some extent.However,the mechanism of anti-apoptosis of RAI16 is still unclear.This study was aimed to explain RAI16-mediated anti-apoptotic effects in HCC.Methods:1.Proteomics(Immunoprecipitation and MS/MS)was used to find the proteins interacted with RAI16.2.Different lengths of RAI16,PKA-RII?,HSP70,14-3-3? expression plasmids,were constructed respectively.And,mapping study and immunoprecipitation were used to verify the directly interacted surfaces between RAI16 and PKA-RII?,HSP70,or 14-3-3?.3.RAI16 and PKA-RII? proteins were purified.ELISA and sHt31 peptide were used to verify the interaction between RAI16 and PKA-RII?.4.The Scansite Motif Scanner was used to predict the phosphorylation sites of RAI16 and HSP70.5.Immunoprecipitation,Western Blotting,single amino acid mutation,phosphorylation assay and autoradiography,were applied to detect the phosphorylation of HSP70 and RAI16.6.Apoptosis of cells was detected by flow cytometry.Results:1.Endogenous and exogenous RAI16 interacts directly with PKA-RII?;RAI16 Aa352-370 binds with PKA-RII?D/D domain directly,and presents an amphipathic helix,which is the AKB domain.RAI16 acts as a novel AKAP.2.Endogenous and exogenous RAI16 interacts directly with HSP70;RAI16 central portion(Aa251-500)binds with HSP70 C terminal(Aa441-641).And RAI16 anchors PKA to HSP70.Then,PKA phosphorylates HSP70 Ser486,which plays an important role in antiapoptosis.3.Endogenous and exogenous RAI16 interacts directly with 14-3-3?;RAI16 central portion(Aa251-500)binds with 14-3-3? central portion(Aa81-160).4.Ser325 of RAI16 can be phosphorylated by PKA.The phosphorylation of RAI16 promotes the recruitment of 14-3-3?.And,the interaction of RAI16 and 14-3-3? is dependent on the RAI16/PKA-RII complex.5.After binding with RAI16,14-3-3? inhibits the phosphorylation of HSP70 by PKA.Conclusion:This study identifies RAI16 as a novel AKAP firstly,which anchors PKA to specific subcellular compartment and mediates specific phosphorylation.RAI16 mediates PKA phosphorylation of HSP70 and the phosphorylation of RAI16 by the anchored PKA promotes the recruitment of 14-3-3?,which,in turn,inhibits RAI16 mediated PKA phosphorylation of HSP70.These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC.2.Study on roles and mechanisms of SNORD113-1 in HCC tumorigenesisSnoRNAs are a group of non-coding RNAs,the length of which is in the range of 60~300 nucleotides.SnoRNAs are predominantly found in the nucleolus and functions as guide RNAs for post-transcriptional modification of rRNAs and some spliceosomal RNAs.The length of SNORD113-1(small nucleolar RNA 113-1)is 70 nucleotides.SNORD113-1 structurally belongs to C/D box snoRNA.Its gene is located in the Dlk1-Dio3 region of chromosome 14.While,the specific expression of SNORD113-1 in HCC tumor tissues,and the roles of SNORD113-1 in the development and progression of HCC,have not been reported.Methods:1.The expression of SNORD113-1 in HCC tumor tissues and adjacent tissues was detected by micro-array or qRT-PCR.2.According to the clinical pathological information of HCC patients,the statistical analysis and prognosis were analyzed to study the predictive value of SNORD113-1 on the survival time of HCC patients and the correlation with other clinical pathological features.3.The promoter region of SNORD113-1 was identified by dual luciferase reporter assay system.4.The methylation level of CpG islands was detected by sodium sulfite sequencing.5.SNORD113-1 was overexpressed or knocked down by using expression plasmids or small interfering RNAs.Cell Titer-Blue,Colony formation assay,Flow Cytometry,Transwell units,Matrigel invasion units were used to detect the effect of SNORD113-1 on HCC cell proliferation,cell cycle,apoptosis,migration and invasion.6.The effect of SNORD113-1 on the proliferation of HCC in vivo was studied in xenograft nude mice.7.Cancer pathway reporter assays and Western Blotting were used to study the effect of SNORD113-1 in MAPK/ERK,TGF-? and other signal pathways.Results:1.SNORD113-1 is significantly downregulated in 77.6%(87/112)of HCC samples,compared with that of adjacent tissues.2.The mean relapse-free survival in patients with low SNORD113-1 expression(n = 87)is 68.4 months,whereas the mean relapse-free survival in those with high SNORD113-1 expression(n = 25)is 99.8 months.The downregulation of SNORD113-1 is associated with the poor prognosis of HCC patients.3.The gene of SNORD113-1 locates in Human 14 th chromosome.The F1(82370922~82371234)fragment of Human 14 th chromosome is predicted and verified that has strong promoter activity.So F1 is the putative promoter of SNORD113-1.4.44.4%(12/27)CpGs are hypermethylated in tumors compared with normal pituitaries.Thus,methylation in the putative promoter region of SNORD113-1gene is higher in HCC tumor tissues than in those from adjacent non-tumor tissues.5.Overexpression of SNORD113-1 can inhibit the proliferation of HCC cells.And,more cells remain in the S phase.Knockdown of SNORD113-1 promotes the proliferation of HCC cells,and more cells remain in the G2-M phase.But there is no significant effect on cell apoptosis,migration and invasion.6.In a xenograft nude mouse model,tumors generated by injection with p3.1-SNORD113-1 transfected HepG2 cells are significant smaller compared with the empty vector transfected or non-transfected HepG2 cells groups.However,tumors generated from SNORD113-1 siRNA transfected HepG2 cells are significant larger after injection.SNORD113-1 can suppress HCC tumorigenesis in vivo.7.Knockdown of SNORD113-1 promotes the phosphorylation of MEK,ERK1/2 and SMAD2/3;Overexpression of SNORD113-1 inhibits the phosphorylation of MEK,ERK1/2 and SMAD2/3 in MAPK / ERK and TGF-? signaling pathways.Conclusion:In the present study,we demonstrated that the CpG island methylation of SNORD113-1 putative promoter region results in downregulation of SNORD113-1 in HCC tissues.And,low expression of SNORD113-1 promotes cell proliferation,then enhances HCC tumorigenesis.Mechanism studies shows that SNORD113-1 inhibits HCC tumorigenesis by regulating the phosphorylation of MEK,ERK1/2 in MAPK/ERK signaling pathway and SMAD2/3 in TGF-? signaling pathway.3.SummaryIn summary,the work uses Human transcriptome array and identifies RAI16 and SNORD113-1 dysregulated in HCC.Further study suggests that RAI16 promotes HCC tumorigenesis,while SNORD113-1 suppresses HCC tumorigenesis.(1)RAI16 is identified as a novel AKAP firstly.RAI16 mediates PKA phosphorylation of HSP70,then involves in the anti-apoptosis in HCC.RAI16 binds with PKA and 14-3-3? simultaneously,forming a multi-signal molecular complex,then mediates the signaling transduction coordinately.(2)SNORD113-1 acts as a HCC suppressor,and suppresses HCC tumorigenesis in MAPK/ERK and TGF-? pathways-dependent mechanisms.In the present paper,we study the anti-apoptotic mechanisms of RAI16 in HCC,and the roles and mechanisms of SNORD113-1 in HCC tumorigenesis,which further deepening our understanding of HCC,and providing a basis for identifying potential diagnosis markers and potential therapy targets for HCC.