The Function And Mechanism Of Long Noncoding RNA AURKAPS1 In Liver Cancer

Liver cancer is the fourth most commonly diagnosed cancer and the second most frequent cause of cancer death in men worldwide?There are three subtypes of primary liver cancers,including hepatocellular carcinoma(HCC)which represents the major histological subtype,accounting for 85% of cases of primary liver cancer;Intrahepatic cholangiocarcinoma(ICC)is the second most frequent type of liver cancer,and its incidence has been increasing;Combined HCC–cholangiocell?Lar carcinoma(HCC-CCA),which contains both hepatocellular and biliary epithelial cells.Long ncRNAs are greater than 200 nucleotides.LncRNA has several origin,including long Intergenic lncRNAs(lincRNAs),long intronic noncoding RNA,antisense RNAs,promoter associated long RNA,enhancer RNAs,pseudogene,3'untranslated region and long stress induced noncoding transcripted variants.Similar to protein-coding genes,lncRNAs are also polyadenylated and are transcribed by RNA polymerase II,with independent gene promoters,binding sites of transcription factor,epigenetic modification,exons and introns.Studies have demonstrated that lncRNAs can reg?Late chromatin remodeling and altered gene expression by interact with polycomb repressivecomplex(PRC);they can also promote the expression of target genes of some specific miRNAs by acting as compete RNA(ceRNA).Recently,researchers find the expression of more than 4% lncRNAs are altered in hepatitis virus infection.For example,low expression of human ortholog RNA of Dreh(hDERH)is associated with poor survival of HCC.HOTAIR(Homeobox antisense intergenic RNA)as a most studied lncRNA,is upregulated in various cancers and correlated with the recurrence and lymph node metastasis.All these findings infer that lncRNA is a potential risk index,diagnosis markers and new therapy target.Aurora kinase-A(AURKA),as a crucial serine/threonine kinase,plays a key role in the processes of mitosis and meiosis.AURKA promotes the separation of sister chromatids through phosphorylating microtubule-associated proteins,motor proteins which reguLate polymeric microtubules and promote spindle formation.In tumor cells,AURKA participates in the process of DNA damage mediated by P53 or BRCA1.Besides,overexpression of AURKA can enhance tumor resistance to chemotherapeutic drugs.Some studies suggest that high expression of AURKA can regulate the process of epithelial-mesenchymal transition(EMT),promoting tumor recurrence and metastasis.AURKAPS1,as a lncRNA,is the pseudogenes of AURKA,and locates in the intron region of RAB3GAP2(RAB3 GTPase activating non-catalytic protein subunit 2)on chromosome 1.However,the expression and function of AURKAPS1 in tumors has not been reported.In this study,the expression of AURKAPS1 in hepatocellular carcinoma is detected by Real-time PCR.Moreover the cell biology function and the molecular mechanism of AURKAPS1 is explored by constructing overexpressing cancer cell lines.In this study,we focus our attention on lncRNA AURKAPS1.To explore the expression,function and molecular mechanism of AURKAPS1 in liver cancer,this study is divided into three parts.Part ?:The expression of lncRNA AURKAPS1 in liver cancer tissues and the correlation analysis of clinicopathological characters Methods 1.Analysis of the genome localization and homology blast of AURKAPS1 sequence using UCSC genome database.2.The sequence blast between AURKA and AURKAPS1 using Blast software.3.The expression of lncRNA AURKAPS1 in 124 pairs of hepatocellular carcinoma and matched normal tissues are detected by Real-time qPCR assay.4.SPSS17.0 statistical analysis software is used to analyze all data.Single sample t-test analysis is used to analyze the expression of AURKAPS1,ANOVA is used to assess the relationship between mRNA expression and clinicopathological Characters.Results 1.AURKAPS1 locates in the first intron of the RAB3GAP2 gene which is on chromosome 1,and it is not conserved between vertebrates.2.AURKAPS1 gene lacks 359-560 coding region sequence compared with AURKA gene sequence,has a 25 bp difference in the 3' extremity,and some nucleotide mutation and loss..3.The mRNA expression of AURKAPS1 in HCC tumor tissues was significantly higher(p<0.05),and its expression is associated with tumor size and TNM stage(p <0.05),but not with gender,age,history of hepatitis,lymph node metastasis(p>0.05).Part ? The function of lncRNA AURKAPS1 on tumor proliferation and metastasis Methods 1.AURKAPS1 overexpressing liver cancer cell lines are constructed by infected with lentivirus-coated AURKAPS1 plasmid.2.The effect of AURKAPS1 on tumor proliferation in vitro is examined by MTT,flow cytometry sorting analysis and clone formation assays.3.The effect of AURKAPS1 on tumor proliferation in vivo is examined by subcutaneous tumor-burdened assay.4.The effect of AURKAPS1 on cancer movement,migration and invasion in vitro is examined by Wound healing and Transwell assays.5.The effect of AURKAPS1 on cancer metastasis in vivo is examined by tail intravenous injection tumor-burdened assay.Results 1.Overexpression of AURKAPS1 promotes the cell cycle of liver cancer cells from G1 phase to G2/S phase,and cellular proliferation.2.AURKAPS1 overexpression or control cells are inoculated subcutaneously in nude mice,the mice were sacrificed after 4 weeks,and then tumor tissue blocks are stripped and weighed.The results showed that overexpression AURKAPS1 promotes the proliferation of HCC cells in vivo.3.Results of wound healing assay and Transwell assay reveal that restoring the expression of AURKAPS1 promotes cell movement,migration and invasion.4.The Intravenously injected nude mice show overexpression AURKAPS1 promotes tumor cell metastasis.Part ? The mechanism of lncRNA AURKAPS1 in liver cancer Methods 1.The potential miRNAs that can bind with AURKAPS1 are analyzed with DIANA TOOLS software.2.Functioal analysis of potential miRNAs is performed with STARBASE software.3.The protein expression of potential downstream genes RAC1 and ERK1/2 in AURKAPS1 overexpressing cells are measured by Western Blot assay.4.Constructing the luciferase plasmid of RAC1 3'UTR and examing the regulation of RAC1 by miRNAs.5.Constructing the luciferase plasmid of AURKAPS1 and examing the reg?Lation of AURKAPS1 by miRNAs.6.The effect of AURKAPS1 on the regulation of RAC1 by miRNAs is examined by Western blot assay.7.The effect of AURKAPS1 on cellular membrane ruffles is examined by Confocal laser scanning microscopy.8.The expression of RAC1 in HCC tissues is detected by Immunohistochemistry (IHC)assay.Results 1.Several miRNAS including miR-218,miR-134,mi R-636,miR-192,miR-182 and so on that can bind with AURKAPS1.2.Functional analysis show that AURKAPS1 may regulate genes such as RAC1?ERK.3.Overexpression of AURKAPS1 can increase the protein expression of RAC1 and promote the activation of ERK.4.Luciferase results display that miR-182,miR-155 and miR-142 can target both AURKAPS1 and RAC1.5.Overexpression of AURKAPS1 promotes the formation of cellular membrane ruffles.6.IHC reveals that RAC1 is significantly upregulated in HCC tissues.Conclusion 1.The expression of AURKAPS1 is significantly higher in HCC tissues;higher level of AURKAPS1 is associated with tumor size and TNM stage.2.AURKAPS1 promotes the proliferation of liver cancer cells in vivo and in vitro.3.AURKAPS1 enhances the ability of cellular movement,migration,invasion and metastasis.4.MiR-142?miR-155?miR-182?mi R-194?miR-218 can regulates the expression of RAC1? 5.AURKAPS1 can increases the protein expression of RAC1,promotes the activation of ERK,and enhance the formation of membrane ruffles by binding with miR-182,mi R-155 and mi R-142 competively.