| BackgroundHepatoblastoma(HB) is the most common liver tumor with an estimated incidence of 1 per 100,000 children,originating from liver progenitor cells that acquire malignant transformation during embryogenesis.Certain inherited conditions,as well as congenital anomalies and low birth weight,are believed to increase the risk of HB,however the etiology of HB remains elusive due to the extreme rarity of this disease.Somatic mutations in genes encoding components of the Wnt/β-catenin are prevalent in sporadic HB cases.Recently,the interplay of Wnt/β-catenin and Myc signaling has been shown to play a critical role in poorly differentiated aggressive HB.Overexpression of mutant β-catenin in mice induces HBs that express high levels of c-Myc.These findings suggest Myc as a potential therapeutic target for HB.However,MYC does not harbor any cavities into which small molecules can easily bind,indicating Myc oncoproteins are undruggable.In addition,small molecule drugs in combination with cytotoxic chemotherapy have not been developed for HB patients.Since the event-free survival rate is less than 50% in children with high-risk HB and pediatric liver transplantation is not easily accessible,current major research efforts are toward identifying new regulators of HB.Micro RNAs(mi RNAs)are small,endogenous,non-coding RNAs that regulate gene expression by specifically binding to 3’-untranslated region(3’-UTR)of target gene m RNAs.Mi RNAs are involved in many important processes,including development,proliferation,differentiation,apoptosis,and stress response.Because mi RNAs regulate diverse physiological and developmental processes,their own expression and processing must be tightly controlled.Due to the rarity of HB and limited availability of fresh-frozen tumor samples,analyzing mi RNA expression in HB was mostly achieved from formalin fixed paraffin embedded samples.Several mi RNAs have been reported aberrantly expressed in HB,including mi R-21 and mi R-221.Despite these studies,how mi RNAs control the development of HB remains little known.LIN28 B is a highly conversed RNA binding protein,which can regulate cell fate to control embryonic development.It sustains the proliferative and metabolic capacities of pluripotent stem cells,and facilitates the transition from naive to primed pluripotency.Aberrantly expressed LIN28 B plays an oncogenic role in various human cancers.Aurora kinase A(AURKA) is a serine-threonine mitotic kinases,which plays an important role in regulating mitosis,cell division,and cell cycle progression.Overexpression of AURKA promotes cell cycle progression via disrupting cell cycle checkpoints,leading to aneuploidy and genomic instability;the latter is a hallmark of malignant transformation.Emerging results suggest that inhibiting AURKA may be a novel strategy for reducing the stability of Myc protein that has not been previously targetable.Ras-related nuclear protein(RAN)is a well known nuclear trafficking protein,which is a member of Ras family.RAN is overexpressed in diverse malignancies,and promotes AURKA phosphorylation.Recently,a study demonstrated that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis,providing a pathway may be amenable to therapeutic targeting.In this study,we aimed at understanding the function of LIN28 B and AURKA in HB.We demonstrate that both genes are direct targets of mi R-26a-5p.LIN28 B and AURKA are upregulated in HB and knockdown of both genes by si RNAs or by mi R-26a-5p inhibits HB tumor cell proliferation and foci formation.Our results suggest that mi R-26a-5p plays an important regulatory role in HB,providing new insights into developing treatment strategies for HB.MethodHB formalin fixed paraffin embedded(FFPE)samples Patient HB FFPE samples were collected retrospectively from the First Hospital of Jilin University,China.The study was approved by the institutional review board at the First Hospital of Jilin University.Cell culture Human hepatocyte cell line HC-04,human hepatoblastoma cell line Hep G2 and HUH6 were cultured in DMEM medium supplemented with 10% calf serum.Hep G2 was generously provided by Dr.Yuzuru Shiio and Dr.Hongbing Wang.HUH6,was a generous gift from Dr.Thomas Pietschmann(22).Hep293 TT cells;which was a generous gift from Dr.Yuzuru Shiio,were cultured in RPMI 1640 supplemented with 10% fetal calf serum,25 m M HEPES,and 1m M sodium pyruvate.Oligonucleotides,plasmids and si RNAs Mi R-26a-5p mimics,mi R-29a-3p mimics,and negative control oligonucleotides(mi R-CON)were purchased from Sigma(#HMI0415,#HMC0434,#HMC0002).Small interfering pool RNA of Lin28 B,RAN and control RNA(si Lin28 B,si RAN,si Con,5 si RNAs per pool)were purchased from Santa Cruz(#SC-105614,#SC-36382,#SC-37007).LIN28 B and AURKA plasmids were purchased from addgene(#51375 and #20427).Luciferase reporter gene plasmids including wild-type AURKA 3’UTR,wild-type LIN28 B 3’UTR and their seed mutants were synthesized and subcloned into pmir GLO vector,which were confirmed by sequencing.Cell transfection Cells were seeded in 6-well plate the day before experiment and transfected using RNAi MAX Transfection Reagent(Thermo Fisher Scientific,#13778075),with micro RNA mimics(mi R-26a-5p or mi R-29a-3p)/negative control at final concentration a total of 50 n M;or with si RNAs(si Lin28 B or si RAN)/ negative control at final concentration of 50 μM.After 48 hours,cells were harvested for RNA and protein isolation,respectively.3’UTR luciferase reporter assays HUH6 and Hep G2 cells were seeded in 24-well plates at a density of 5×104 cells/well.After 24 hr,wild-type AURKA 3’UTR and LIN28 B 3’UTR or their seed mutant luciferase reporter vectors with mi R-26 mimic or mi R-29 mimic were transfected into the cells using RNAi MAX transfection reagent.48 hours after transfection,the Dual-luciferase reporter assay system(Promega,Madison,USA)was applied to determine changes in relative luciferase units as described previously.All assays were done in triplicates,and all values were normalized for transfection efficiency against Renilla luciferase activities.Cell proliferation assay MTS assay was performed to measure the proliferative ability of HUH6,Hep293 TT and Hep G2 cells,as described previously.In brief,a density of 5 × 103 cells per well were seeded into 96-well plates containing complete DMEM or 1640(100 μl)in triplicate for each condition and were maintained in an incubator at 37 °C and 5% CO2.MTS solution(20 μl)was added to each well and incubated for 2 hr.Then,absorbance at 490 n M(A490)was read on a microplate reader(168–1000 Model 680,Bio-Rad Laboratories),and proliferation curves were plotted.Colony formation assay was conducted as previously described.Quantitative real-time PCR(q RT-PCR)Total RNA was extracted from tissues or cells with TRIzol reagent(Thermo Fisher Scientific,USA,#15596018).Real-time quantification of m RNA levels of the genes of interest was performed using Brilliant II SYBR Green QPCR Master Mix(BIO-RAD,Germany,#1725274)as described previously.Normalized Ct values were subjected to statistical analysis,and the fold difference was calculated by the ΔΔ Ct method as described previously.Human β-ACTIN and U6 m RNAs were also measured as internal controls for normalization.Western blotting Tissue or cell lysate were harvested by radioimmune precipitation assay(RIPA)lysis buffer with protease inhibitors(Thermo Fisher Scientific,#78410).30 μg proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes according to standard procedures.Membranes were blocked,immunostained with primary antibodies and finally detected by horseradish peroxidase-conjugated corresponding secondary antibody.Signals was visualized with Super Signal West Pico Chemiluminescent Substrate(Thermo Fisher Scientific,#34080)according to the manufacturer’s protocol.Equal loading of protein was verified with β-actin.RNA extraction from formalin fixed paraffin embedded(FFPE)samples Total RNAs from 16 FFPE tissues blocks were isolated using commercially available mi RNeasy FFPE Kit,according to the respective manufacturers’ instructions(QIANGEN,Germany,#217504).ResultsThe expression of LIN28 B,RAN and AURKA is upregulated in human HB livers Hepatoblastoma is resulted from the failure of cells to differentiate properly into their final cell types during fetal or postnatal development.Recently,LIN28 B was identified as an oncogenic driver in high-risk neuroblastoma.LIN28B-RAN-AURKA signaling was further revealed to drive neuroblastoma oncogenesis.We were interested in determining whether LIN28,RAN and AURKA act as oncogenes in hepatoblastoma.We first examined their expression in human tissue biopsies from HB patients by q PCR and Western blot.The m RNA levels of LIN28 B and RAN were significantly higher in HB than in normal livers,whereas AURKA m RNAs showed greater variations among the samples.Interestingly,LIN28 B protein exhibited larger variations among the samples,whereas RAN and AURKA proteins were notably upregulated in HB vs non-tumors.The inconsistency between m RNA and protein expression of LIN28 B and AURKA in HB suggests that both transcriptional and translational mechanisms may be involved.A molecule link exists between LIN28 B,RAN and AURKAWe next assessed the expression levels of LIN28 B,RAN and AURKA in a panel of human HB cell lines vs normal hepatocyte cell line HC-04.All three genes were highly expressed in HUH6,Hep G2,Hep293 TT cells,but with a low expression in HC-04.To further investigate the molecule link between LIN28 B,RAN,and AURKA,we knocked-down LIN28 B using si LIN28 B and examined m RNA expression of RAN and AURKA in all three HB cells.LIN28 B depletion lead to decreased RAN m RNA expression in HUH6,Hep G2,and Hep293 TT cells,whereas it decreased AURKA m RNA expression in HUH6,Hep G2,but not in Hep293 TT cells.Interestingly,knocking-down of RAN by si RNA significantly decreased AURKA m RNA but also had a modest effect in reducing LIN28 B m RNA in all three HB cells.At the protein levels,si LIN28 B decreased RAN protein expression in HUH6 and Hep G2,but not in Hep293 TT cells,whereas RAN-si RNA decreased AURKA protein in all three lines but had a moderate effect on LIN28 B protein expression.Taken together,the results suggest that LIN28 B may act upstream of RAN and AURKA,whereas RAN may be an activator of AURKA expression but may also regulate LIN28 B in a feedback fashion.In addition,post-transcriptional regulation may be a mechanism contributing to the differential expression pattern between m RNA and protein.Knockdown of LIN28 B and RAN suppresses HB cell proliferation and colony formationTo determine the functional roles of LIN28 B and RAN in HB,we depleted LIN28 B and RAN using their respective si RNAs in HUH6,Hep G2,and Hep293 TT cells.Knockdown of LIN28 B or RAN markedly diminished HB cell proliferation.In addition,si LIN28 B and si RAN notably suppressed the colony formation of HB cells.The results suggest an oncogenic effect of LIN28 B and RAN in HB.LIN28B and AURKA are direct target genes of mi R-26a-5p in HBThe prediction of mi RNA target regions by Target Scan(http://www.targetscan.org/)indicated that both LIN28 B 3’UTR and AURKA 3’UTR contain a binding site for mi R-26a-5p.Both target sequences are conserved in several different species.To further investigate whether LIN28 B and AURKA were directly regulated by mi R-26a-5p,luciferase reporter assays were performed in HUH6 and Hep G2 cells.Co-transfection of mi R-26a-5p mimics markedly decreased luciferase reporter activities of LIN28 B 3’UTR and AURKA 3’UTR,which were relieved when the mi R-26a-5p seed regions were mutated.Mi R-26a-5p was significantly suppressed in HB cell lines HUH6,Hep G2 and Hep293 TT,compared to HC-04 cells).In addition,Western blot showed decreased LIN28 B and AURKA protein expression after transfection with mi R-26a-5p mimics in HB cells.Interestingly,RAN protein was also decreased by mi R-26a-5p mimics in HUH6 and Hep G2 cells.We examined the micro RNA target prediction databases Targetscan and Micro RNA.org,but found no mi R-26a-5p binding sites within RAN 3’UTR,arguing that RAN is not a canonical mi R-26a-5p target.The results suggest that RAN down-regulation likely resulted from LIN28 B knockdown.Downregulation of mi R-26a-5p is associated with poor patient outcomes in HBTo establish the clinical relevance of mi R-26a-5p in HB,we determined its expression in 16 paraffin HB tissues and compared with normal children liver specimens.Overall,the expression level of mi R-26a-5p was much lower in HB tissues vs normal tissues.As shown in Table 1,mi R-26a-5p expression in stage one disease was compatible with normal livers,however its level decreased drastically in stage four disease.In addition,the expression level of mi R-26a-5p in patients with poor outcomes was the lowest than other stages(p<0.01).Mi R-26a-5p inhibits HB cell proliferation and colony formationTo further investigate the potential function of mi R-26a-5p in HB,we transfected mi R-26a-5p mimics in Hep293 TT,HUH6 and Hep G2 cells.mi R-26a-5p mimics,but not the control mimics nor mi R-29a-3p mimics,drastically attenuated the proliferation of these cells as compared with the control cells.Because mi R-26a-5p showed a similar effect on Hep293 TT,HUH6 and Hep G2 cells,we next examined the ability of mi R-26a-5p to inhibit foci formation using Hep293 TT cells.Overexpression of mi R-26a-5p mimics also dramatically decreased colony formation.On the contrary,mi R-26a-5p inhibitor increased colony formation.In addition,the inhibitory effect of mi R-26a-5p on Hep293 TT proliferation was rescued by mi R-26a-5p inhibitor.The results suggest a tumor suppressor function of mi R-26a-5p in HB.LIN28B and AURKA block the inhibitory effect of mi R-26a-5p o HB cell growthBecause LIN28 B and AURKA are downstream targets of mi R-26a-5p,we conducted rescue experiments.As expected,re-expression of LIN28 B and AURKA blocked the effect of mi R-26a-5p on the repression of colony formation and cellular growth of Hep293 TT cells,suggesting that the effect of mi R-26a-5p is mediated through both genes.Conclusion(1)LIN28B、RAN、AURKA are highly expressed in hepatoblastoma.(2)LIN28B-RAN-AURKA signaling network promotes hepatoblastoma tumorigenesis.Knowdown LIN28 B or RAN,the ability of cell proliferation and conloy formation is decreased.(3)Micro RNA-26-5p functions as a new inhibitor of hepatoblastoma by repressing LIN28 B and AURKA expression.Micro RNA-26-5p gover hepatoblastoma tumorigenesis through LIN28B-RAN-AURKA signaling network. |
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