lncrnasnhg10facilitateshepatocarcinogenesis …...coimmunoprecipitation cells were lysed in ip...

Genome and Epigenome

LncRNASNHG10FacilitatesHepatocarcinogenesisand Metastasis by Modulating Its HomologSCARNA13 via a Positive Feedback LoopTian Lan1, Kefei Yuan1,2, Xiaokai Yan1, Lin Xu1,2, Haotian Liao1, Xiangyong Hao1,3,Jinju Wang1, Hong Liu1, Xiangzheng Chen1,2, Kunlin Xie1, Jiaxin Li1,Mingheng Liao1, Jiwei Huang1, Yong Zeng1,2, and Hong Wu1,2

Abstract

Understanding the roles of noncoding RNAs (ncRNA) intumorigenesis and metastasis would establish novel avenuesto identify diagnostic and therapeutic targets. Here, weaimed to identify hepatocellular carcinoma (HCC)–specificncRNA and to investigate their roles in hepatocarcinogenesisand metastasis. RNA-seq of xenografts generated by lungmetastasis identified long noncoding RNA small nucleolarRNA host gene 10 (SNHG10) and its homolog SCARNA13as novel drivers for the development and metastasis of HCC.SNHG10 expression positively correlated with SCARNA13expression in 64 HCC cases, and high expression of SNHG10or SCARNA13 was associated with poor overall survival.As SCARNA13 showed significant rise and decline afteroverexpression and knockdown of SNHG10, respectively,we hypothesized that SNHG10 might act as an upstreamregulator of SCARNA13. SNHG10 and SCARNA13 coordi-nately contributed to the malignant phenotype of HCC cells,

where SNHG10 served as a sponge for miR-150-5p andinteracted with RPL4 mRNA to increase the expression andactivity of c-Myb. Reciprocally, upregulated and hyperacti-vated c-Myb enhanced SNHG10 and SCARNA13 expressionby regulating SNHG10 promoter activity, forming a positivefeedback loop and continuously stimulating SCARNA13expression. SCARNA13 mediated SNHG10-driven HCCcell proliferation, invasion, and migration and facilitatedthe cell cycle and epithelial–mesenchymal transition ofHCC cells by regulating SOX9. Overall, we identified acomplex circuitry underlying the concomitant upregulationof SNHG10 and its homolog SCARNA13 in HCC in theprocess of hepatocarcinogenesis and metastasis.

Significance: These findings unveil the role of a noncod-ing RNA in carcinogenesis and metastasis of hepatocellularcarcinoma.

IntroductionHepatocellular carcinoma (HCC) is the fourth most common

incident malignancy and third leading cause of cancer-relateddeath in China (1). Despite continuous progress in clinical detec-tion and treatment strategies, the prognosis of patients with HCCremains poor largely due to late diagnosis, a high postoperativerecurrence rate, andmetastasis (2). Because themolecularmechan-isms underlying the tumorigenesis and metastasis of HCC have

not yet been fully elucidated, identifying pivotal cancer-promotingmolecules would contribute to the understanding of HCC path-ogenesis and identification of potential therapeutic targets.

Long noncoding RNAs (lncRNA) belong to a class of RNAtranscripts over 200 nucleotides with no or low protein-codingpotential (3). Accumulating studies have shown that lncRNAsparticipate inmultiple biological regulatory processes, such as celldifferentiation, apoptosis, immune response, and carcinogene-sis (4). Small nucleolar RNAs (snoRNA), predominately found incell nucleolus, are a subgroupof ncRNAswith 60–300nucleotidesin length and guide the posttranscriptional modification of smallRNAs (5). Emerging evidence has demonstrated that abnormallyexpressed snoRNAs exert significant and comprehensive influ-ences on the carcinogenesis and progression in diverse humanmalignancies (6).

Recently, a complicated interaction between lncRNAs andsmall RNAs has been disclosed in which some lncRNAs canproduce or regulate small RNAs (7). For instance, lncRNAMIR17HG–derived miR-17�92 attenuates the TGFb signalingpathway to promote angiogenesis and tumor cell growth (8). Asa subclass of small RNAs, most snoRNAs are encoded in theintrons of small nucleolar RNA host genes (SNHG; ref. 6). Spe-cifically, the introns of primary RNA transcripts from SNHGs areprocessed into snoRNAs in the nucleus, while the exons arespliced into lncRNAs and transported to the cytoplasm, suggestingthat theremight beunderlying correlations between snoRNAs andthe homologous lncRNAs transcribed from SNHGs. However, the

1Department of Liver Surgery & Liver Transplantation, State Key Laboratory ofBiotherapy and Cancer Center, West China Hospital, Sichuan University andCollaborative Innovation Center of Biotherapy, Chengdu, China. 2Laboratory ofLiver Surgery, State Key Laboratory of Biotherapy and Cancer Center, WestChina Hospital, Sichuan University and Collaborative Innovation Center ofBiotherapy, Chengdu, China. 3Department of General Surgery, Gansu ProvincialHospital, Lanzhou, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

T. Lan and K. Yuan contributed equally to this article.

Corresponding Authors: Hong Wu, West China Hospital of Sichuan University,Chengdu, Sichuan 610041, China. Phone: 189-8060-1958; Fax: 028-85582944;E-mail: [email protected]; and Yong Zeng, [email protected]

Cancer Res 2019;79:3220–34

doi: 10.1158/0008-5472.CAN-18-4044

�2019 American Association for Cancer Research.

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particular relationships between these lncRNAs and homologoussnoRNAs as well as their specific roles in oncogenesis are poorlyunderstood.

In this study, by performing high-throughput RNA-sequencing(RNA-seq), we identified the contributions of the lncRNA smallnucleolar RNA host gene 10 (SNHG10) and its homologoussnoRNA, SCARNA13, in the development and progression ofHCC. Both SNHG10 and SCARNA13 were dramatically associ-ated with the malignant biological behaviors of HCC cells andpoor prognosis of patients with HCC. Specifically, SNHG10modulated the expression of SCARNA13 through themiR-150-5p/RPL4-c-Myb–positive feedback loop and SCARNA13 exertedits oncogenic activity by regulating SOX9 in HCC. Our find-ings characterized an epigenetic cause of hepatocarcinogenesisand metastasis with diagnostic and therapeutic implications.

Materials and MethodsEthical application

The protocols used in this study conformed to the ethicalguidelines of the 1975Declaration ofHelsinki andwere approvedby the Ethical Review Committees of Sichuan University(Chengdu, China).

Human tissuesHCC tissues and adjacent normal tissues were obtained fol-

lowing curative surgical resections from 64 patients with HCC atthe West China Hospital (Sichuan University, Chengdu, China).Ethical approvalwas granted from the Ethical ReviewCommitteesof Sichuan University (Chengdu, China), and written informedconsent was obtained from all the patients.

Cell lines and reagentsSNU-182, Huh-7, Hep3B, SK-Hep1, and SNU-387 cell lines

were purchased from the Shanghai Cell Bank Type Culture Col-lection Committee (CBTCCC, Shanghai, China). HEK293T cellline was purchased from the ATCC. The HCCLM3 cell line wasacquired from the State Key Laboratory of Biotherapy,West ChinaHospital (Chengdu, Sichuan, China). All cell lines were charac-terized by short-tandem repeat analysis, Mycoplasma testing, iso-zyme detection, and cell viability determination by third-partybiology services (Feiouer Biology Co., Ltd). Cells were cultured at37�C in a humidified incubator with 5% CO2 in DMEM(HyClone) supplemented with 10% FBS (PAN). ActinomycinD was obtained from Sigma-Aldrich.

Establishment of animal modelsThe animal studies were authorized by the Animal Ethic

Review Committees of the West China Hospital (Chengdu,Sichuan, China). Twenty male athymic BALB/c nude mice, ages4–5 weeks, were purchased from Beijing HFK Bioscience andwere fed under standard pathogen-free conditions. All surgicalprocedures were performed with sodium pentobarbital anes-thesia. Mice were subcutaneously injected with 200 mL of cellsuspension containing 2 � 106 cells in the right flanks. Tumorswere allowed to grow for 2 weeks, after which, pieces of thesubcutaneous tumors were excised and cut into 1-mm3 sec-tions, followed by upper-abdominal incisions on mice. Theright lobe of the liver was exposed, and part of the liver surfacewas moderately injured with scissors. A tumor section wasimplanted in the small incision on the liver surface. The liver

was returned to the peritoneal cavity; subsequently, theabdominal wall was strictly sutured. The tumors were permit-ted to grow for 5 weeks, followed by euthanization of themice. Next, the lung was carefully anatomized to expose thepulmonary metastatic focuses, the obvious ones of whichwere completely separated from the lung tissues. The largestone was subjected to primary culture to isolate xenograftedHCC cells, while the others were made into tissue sections andsubjected to hematoxylin and eosin (H&E) staining to deter-mine the pulmonary metastatic focuses by two pathologistsfrom the Department of Pathology, West China Hospital(Chengdu, Sichuan, China). All animal experiments werestrictly implemented in compliance with the NIH Guide forthe Care and Use of Laboratory Animals.

RNA FISHLocked nucleic acid-FISH (LNA-FISH) was performed to deter-

mine the subcellular location of SNHG10 and SCARNA13. LNAfluorescein–labeled probes against 18S rRNA, U6 snRNA,SNHG10, and SCARNA13 were designed and synthesized byRiboBio (RiboBio Biotechnology). FISH was conducted using theFluorescent in Situ Hybridization Kit (RiboBio Biotechnology),according to the manufacturer's protocol. Fluorescence signalswere scanned using the A1RþMP Confocal Laser MicroscopeSystem (Nikon).

RNA immunoprecipitation assaysRNA immunoprecipitation (RIP) assays were implemented

using the Magna RIP RNA-binding Protein ImmunoprecipitationKit (Millipore), according to the manufacturer's instructions.Anti-Ago2 antibody and normal IgG (Millipore) were used forimmunoprecipitation. The coprecipitated RNAs were purifiedwith phenol:chloroform:isoamyl alcohol and subsequentyanalyzed by qPCR to assess the enrichment of SNHG10 andmiR-150-5p to Ago2.

Chromatin immunoprecipitation assaysCells were cross-linked with 1% formaldehyde and quenched

in glycine solution. Chromatin immunoprecipitation (ChIP)assays were performed using the Pierce Magnetic ChIP Kit(Thermo Fisher Scientific), according to the manufacturer's pro-tocol. Anti–c-Myb antibody and normal IgG (Millipore) wereapplied for immunoprecipitation. ChIP-enriched DNA sampleswere quantified by qPCR to determine the c-Myb binding sites(MBS) of the SNHG10 promoter region. The data were shown asrelative enrichment normalized to control IgG. The sequences ofprimers used for ChIP-qPCR are presented in SupplementaryTable S1.

Chromatin isolation by RNA purification assaysTen oligonucleotide probes corresponding to the SNHG10

transcript were synthesized with biotin tags located at the 30 endby RiboBio (RiboBio Biotechnology). To eliminate nonspecificsignals, all probes were divided into two pools (even and oddprobe sets). The probe set targeting LacZ was used as a negativecontrol. Chromatin isolation by RNA purification (ChIRP) assayswere conducted using the EZ-Magna ChIRP RNA Interactome Kit(Millipore), according to the manufacturer's protocol. The pre-cipitated RNA was identified and quantified by qPCR to analyzethe enrichment of miR-150-5p to SNHG10. The sequences ofprobes used for ChIRP are listed in Supplementary Table S2.

Oncogenic Roles of SNHG10 and SCARNA13 in HCC

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CoimmunoprecipitationCells were lysed in IP lysis/wash buffer. The immune complex

was prepared using anti–c-Myb antibody, anti-RPL4 antibody, ornormal IgG (Millipore) and was subsequently captured using thePierce Classic IP Kit (Thermo Fisher Scientific), according to themanufacturer's instructions. The samples were separated on 10%SDS-PAGE gels and analyzed by Western blot.

Luciferase reporter assaysTo measure promoter activity, the cells were cotrans-

fected with pEZX-PL01-SNHG10, and c-Myb siRNAs or con-trol siRNA using Lipofectamine 2000 (Invitrogen). For30 UTR luciferase reporter assays, pmirGLO-SNHG10 orpmirGLO-SNHG10-mut(miR-150-5p) was cotransfected withmiR-150-5p mimics or miR-NC into HEK293T cells. Likewise,pmirGLO-SNHG10 or pmirGLO-SNHG10-mut(RPL4) wascotransfected with RPL4 siRNAs or control siRNA intoHEK293T cells. After 48 hours of transfection, Renilla andfirefly luciferase activities were detected by the Synergy MxMulti-Mode Microplate Reader (BioTek). Firefly luciferaseactivity was normalized to Renilla luciferase activity and waspresented as the relative luciferase activity.

Accession numbersThe Gene Expression Omnibus accession numbers for the

RNA-seq data of xenografted HCC cells, ChIRP-seq data ofSNHG10, and RNA-seq data for SCARNA13 knockdown areGSE120021, GSE119773, and GSE120095, respectively. Theaccession number for the quantitative proteomics data reportedin this article is Integrated Proteome Resources: IPX0001319000.

ReproducibilityEach experiment was performed in triplicate, and the data were

presented asmeans� SEM. All results were representative of threeseparate experiments.

Statistical analysisAll statistical analyses were performed using GraphPad Prism 7

Software (GraphPad Software) and SPSS version 17.0 Software(SPSS, Inc.). Regarding comparisons, Student t test, x2 test, theWilcoxon signed-rank test, and the Mann–Whitney test wereconducted as appropriate. Correlations were calculated by Pear-son correlation analysis. The median SNHG10 expression wasused as a cut-off value for grouping. The low SNHG10 group ineach of the 64 patients was defined as a value below the 50thpercentile. The high SNHG10 group in each of the 64 patientswas defined as a value above the 50th percentile. Likewise,patients were divided into the high and low SCARNA13 groupsaccording to the median SCARNA13 expression in HCCtissues. The survival curves were measured by the Kaplan–Meiermethod, and the differences were evaluated by the log-ranktest. The univariate and multivariate Cox proportional hazardsregression models were utilized to assess the independent fac-tors. Statistical significance was indicated by P values less than0.05 (�, P < 0.05; ��, P < 0.01; ��, P < 0.001).

Other detailed materials and methods are provided in theSupplementary Materials and Methods section. The sequencesof siRNA against specific target are presented in SupplementaryTable S3. The information of primary antibodies used is shown inSupplementary Table S4.

ResultsSNHG10 and SCARNA13 are elevated in pulmonary metastaticfocuses, as well as in HCC tissues, and are associated with thepoor prognosis of patients with HCC

To identify ncRNAs involved in HCC metastasis, we initiallygrafted HCC cells into nude mice and performed a screen of lungmetastasis based on orthotopic implantedmodels in vivo (Fig. 1A).Subsequently, RNA-seq was utilized to compare the expressionprofiles between xenografted HCC cells and parental HCC cells(Supplementary Fig. S1A) and demonstrated that 101 lncRNAswere differentially expressed by at least 2-fold change (Fig. 1B).Moreover, we found that six snoRNAs were upregulated and twosnoRNAs were downregulated in xenografts compared with thosein parental HCC cells (Fig. 1C).Moreover, the The Cancer GenomeAtlas (TCGA) Liver Hepatocellular Carcinoma (LIHC) data repos-itorywas analyzed to further investigate the roles of these candidatelncRNAs and snoRNAs in hepatocarcinogenesis. In total, 3,592lncRNAs and 113 snoRNAs were significantly dysregulated inHCC tissues compared with adjacent nontumor tissues from theTCGA LIHC dataset (Fig. 1B and C). With the intersection,24 lncRNAs (including SNHG10, DLGAP1-AS2, TMEM161B-AS1,LINC01004, and NNT-AS1) and 6 snoRNAs (SCARNA13,SNORD6, SNORD100, SNORD3A, SNORD94, and SNORA71C)were identified to possibly play crucial roles in both the tumori-genesis and metastasis of HCC. Intriguingly, among these abnor-mally expressed genes were SNHG10 and SCARNA13, which arethe different products from the same primary RNA transcript. Morespecifically, SCARNA13 isprocessed fromthe intronsof theprimaryRNA transcript from the SNHG10 gene, whereas the exons arespliced into the SNHG10 transcript (Fig. 1D), suggesting thatSNHG10 and SCARNA13 might synergistically contribute to thedevelopment and progression of HCC. Therefore, we focused onthis lncRNA SNHG10 and its homologous SCARNA13.

First, the protein-coding potential of SNHG10 and SCARNA13was analyzed to confirm whether they belonged to ncRNAs. Asexpected, the open reading frame (ORF) finder from the NationalCenter for Biotechnology Information (NCBI), Coding PotentialCalculator (CPC), and Coding Potential Assessment Tool (CPAT)revealed that neither SNHG10 nor SCARNA13 could encode anyprotein (Supplementary Fig. S1B–S1E).

Analysis of the TCGA LIHC data repository confirmed that theexpression levels of both SNHG10 and SCARNA13 were signif-icantly higher in HCC tissues than in adjacent normal tissues(Supplementary Fig. S1F and S1G), and SCARNA13 expressionwas statistically correlated with SNHG10 expression (Fig. 1E).qPCR analysis was performed to further validate their expressionlevels and correlation. Because SNHG10has twomajor transcriptsof 1,980nt and1,341nt in length (Supplementary Fig. S1H), bothshould be detected to determine SNHG10 expression. Comparedwith the long transcript (1,980 nt), the short transcript (1,341 nt)is spliced and lacks an intron. To distinguish these two transcripts,the reverse primer for the long transcript (1,980 nt) was designedin the intron region, while the reverse primer for the shorttranscript (1,341 nt) was designed across the exon-junction site.The forward primers of these two transcripts were identical.Importantly, PCR and Northern blot analysis identified the1,341 nt SNHG10, which does not overlap with the wholesequence of the SCARNA13 transcript as the steady and predom-inant transcript in HCC tissues and HCC cells. In contrast, the1,980 nt SNHG10 showed negligible expression (Supplementary

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Figure 1.

Identification of SNHG10 and SCARNA13, and their correlations with the poor prognosis of patients with HCC.A, Schematic model presenting the process toestablish lung metastasis screening mice models. The detailed information is provided in Materials and Methods (establishment of animal models). B and C, Theflow chart for selecting candidate lncRNAs and snoRNAs through the intersection of RNA-seq results and TCGA LIHC data repository. D, Genomic organization ofSNHG10 and SCARNA13 on human chromosome 14 (hsa chr14). E, Scatter plots of SNHG10 versus SCARNA13 expression in the TCGA LIHC data repository.Pearson correlation coefficients (r) and P values are shown. F and G, The expression of SNHG10 and SCARNA13 in 64 pairs of HCC tissues and adjacent normaltissues using qPCR. H, Scatter plots of SNHG10 versus SCARNA13 expression inWCH data repository. Pearson correlation coefficients (r) and P values are shown.I and J, Kaplan–Meier analyses of the correlations between SNHG10 or SCARNA13 expression level and overall survival of 64 patients with HCC. The medianexpression level was used as the cutoff. Values are expressed as the median with interquartile range. K, The expression of SNHG10 in five different HCC cell linesusing qPCR. Data are presented as mean� SEM. �� , P < 0.01.






Fig. S1I and S1J). Hence, the expression of SNHG10 was almostentirely determined by the levels of the 1,341 nt transcript.Although the transcript of 1,980 nt SNHG10 can barely bedetected, it overlaps the whole sequence of the SCARNA13transcript. Consequently, to accurately determine the expressionof SCARNA13 by qPCR, a specific stem-loop primer was designedto perform the unique reverse transcription of SCARNA13 (Sup-plementary Fig. S1K), after which, the product was verified bySanger sequencing (Supplementary Fig. S1L). Next, we examinedthe expression levels of SNHG10 and SCARNA13 in 64 pairs ofHCC tissues and their adjacent normal tissues from the WestChina Hospital (Chengdu, Sichuan, China) dataset. As expected,the levels of both SNHG10 and SCARNA13were elevated inHCCtissues compared with those in adjacent normal tissues (Fig. 1Fand G), and there was a statistically positive correlation betweenthem (Fig. 1H).

Furthermore, the relationship between the expression levels ofSNHG10 and SCARNA13 and the clinical characteristics wereanalyzed in 64 HCC tissues (Supplementary Table S5; Supple-mentary Table S6). Our results showed that high expression ofSNHG10was significantly associatedwith tumor size (P¼0.024),serum AFP (P ¼ 0.024), microvascular invasion (P ¼ 0.005),Edmondson's grade (P ¼ 0.001), TNM stage (P ¼ 0.011), andBCLC stage (P¼ 0.002). Likewise, high expression of SCARNA13was also statistically correlated with tumor size (P ¼ 0.045),serum AFP (P ¼ 0.012), microvascular invasion (P < 0.001),Edmondson's grade (P < 0.001), TNM stage (P ¼ 0.002), andBCLC stage (P < 0.001). More importantly, Kaplan–Meier analy-sis displayed that high expression of SNHG10 or SCARNA13was remarkably associated with poor overall survival (Fig. 1Iand J). Besides, SCARNA13, rather than SNHG10, could serveas an independent prognostic indicator for overall survivalaccording to univariate and multivariate Cox regression analysis(Supplementary Table S7; Supplementary Table S8).

Because SNHG10 expression was remarkably correlated withSCARNA13 expression, we asked whether there was a regulatoryrelationship between them. First, the expression of SNHG10 wasdetected in five different HCC cell lines. Compared withSNU-182, Hep3B, and Huh-7 cells, HCCLM3 and SNU-387 cellsdisplayed relatively high SNHG10 expression (Fig. 1K). Intrigu-ingly, we found that SCARNA13 showed a substantial increaseafter overexpression of SNHG10 in Huh-7 and Hep3B cells(Supplementary Fig. S2A–S2D), and knockdown of SNHG10resulted in significantly decreased expression of SCARNA13 inSNU-387 and HCCLM3 cells (Supplementary Fig. S2E–S2H).In contrast, depletion of SCARNA13 did not lead to any changein the expression of SNHG10 (Supplementary Fig. S2I–S2L).These results suggested that SNHG10 might act as the upstreamregulator of SCARNA13.

In addition, HCCLM3 cells infected with LV-shSNHG10exhibited an oval appearance (Supplementary Fig. S2M), where-as Huh-7 and Hep3B cells infected with LV-SNHG10 presenteda spindle-shaped appearance (Supplementary Fig. S2N), sug-gesting that SNHG10 might cause HCC cells to undergo theepithelial–mesenchymal transition (EMT).

SNHG10 facilitates the tumorigenesis and metastasis of HCCcells

To elucidate the oncogenic role of SNHG10 in hepatocarcino-genesis and metastasis, we examined the effects of SNHG10 oncell phenotypes. Depletion of SNHG10 significantly inhibited

SNU-387 and HCCLM3 cell cycle and proliferation, and inducedapoptosis (Fig. 2A and B; Supplementary Fig. S3A–S3F). More-over, the silencing of SNHG10 drastically weakened the invasiveand migratory abilities of SNU-387 and HCCLM3 cells (Fig. 2Cand D; Supplementary Fig. S3G and S3H).

To further investigate the tumorigenic effects of SNHG10 onHCC cells in vivo, SNU-387 and HCCLM3 cells were subcutane-ously injected into nude mice. Both the volumes and weights ofthe tumors in the LV-shSNHG10 group were remarkably lowerthan those in the LV-shCtrl group (Fig. 2E–G; SupplementaryFig. S4A–S4C), indicating that SNHG10 enhanced the tumorige-nicity of the HCC cells in vivo. Furthermore, the promoting effectsof SNHG10 on the metastasis of HCC cells were evaluated.We transplanted the indicated SNU-387 and HCCLM3 cells intothe livers of nude mice to construct orthotopic-implantedmodels for liver metastasis assays. The luciferase signal intensitiesof liver metastatic nodules were significantly declined in theLV-shSNHG10 group compared with those in the LV-shCtrlgroup (Fig. 2H; Supplementary Fig. S4D), demonstrating thatSNHG10 strengthened the intrahepatic metastatic ability of HCCcells. Eventually, SNU-387 and HCCLM3 cells were labeled withfirefly luciferase and were directly inoculated into the tail veins ofnude mice for lung metastasis assays. Apparently, the luciferasesignal intensities of mice in the LV-shSNHG10 group weremarkedly lower than those in the LV-shCtrl group (Fig. 2I; Sup-plementary Fig. S4E), suggesting that the lungmetastatic potentialof HCC cells could be promoted by SNHG10. H&E stainingrevealed that the metastatic foci in the LV-shSNHG10 group weredramatically decreased in tissue sections of the lungs and livers(Fig. 2J and K; Supplementary Fig. S4F and S4G).

Thereafter, we assessed the influences of overexpressing SNHG10on the HCC cell phenotype. Upregulation of SNHG10 significant-ly promoted the cell cycle, proliferation, and apoptotic resistanceof Huh-7, Hep3B, and SNU-182 (Supplementary Fig. S4H–S4M;Supplementary Fig. S5A–S5F). Moreover, overexpression ofSNHG10 drastically strengthened the invasive and migratoryabilities of Huh-7, Hep3B, and SNU-182 cells (SupplementaryFig. S5G–S5L). Regarding in vivo experiments, Huh-7, Hep3B, andSNU-182 cells infected with LV-SNHG10 were significantly asso-ciated with higher volumes and weights of the subcutaneoustumors, as well as with greater luciferase signal intensities of liverand lungmetastatic nodules (Supplementary Fig. S6A–S6O). Takentogether, these observations illuminated that SNHG10 functions asan oncogenic driver in the tumorigenesis and metastasis of HCC.

SCARNA13 boosts the tumorigenesis and metastasis of HCCcells

Because SCARNA13 and SNHG10 were concomitantly upre-gulated inHCC tissues, we evaluated the influences of SCARNA13on the malignant phenotype of HCC cells. Similar to SNHG10,knockdownof SCARNA13 tremendously suppressed the cell cycleand proliferation, and induced apoptosis of SNU-387 andHCCLM3 (Fig. 3A–H). Besides, downregulation of SCARNA13substantially impaired the invasive and migratory capabilities ofSNU-387 and HCCLM3 cells (Fig. 3I–L). These results identifySCARNA13 as an oncogenic promoter in HCC.

Transcription factor c-Myb upregulates the SNHG10 andSCARNA13 levels

Generally, the activation of oncogenes mediated by aberrantpromoter methylation levels is a key feature of cancer (9). Hence,

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Figure 2.

Inhibition of SNHG10 impairs the proliferation andmetastasis of HCC cells in vitro and in vivo. A and B, EdU immunofluorescence staining assays for SNU-387 andHCCLM3 cells transfected with SNHG10 siRNAs or the control. Scale bars, 100 mm. C andD, Transwell invasion assays for SNU-387 and HCCLM3 cells transfectedwith SNHG10 siRNAs or the control. Scale bars, 100 mm. E–G, Effects of SNHG10 knockdown in SNU-387 cells on tumor volumes and tumor weights in thesubcutaneous xenografts mice models. Scale bars, 5 mm; N¼ 5. H, Luciferase signal intensities of livers in each group 6 weeks after orthotopic implantation with1� 106 indicated SNU-387 cells. Scale bars, 5 mm. I, Luciferase signal intensities of mice in each group 6 weeks after tail intravenous injection with 5� 105

indicated SNU-387 cells. J and K, The metastatic foci derived from indicated SNU-387 cells in tissue sections of lungs and livers using H&E staining. Data arepresented as mean� SEM. �� , P < 0.01; ��, P < 0.001.






Figure 3.

Inhibition of SCARNA13 impairs the proliferation andmetastasis of HCC cells in vitro.A and B, CCK-8 assays for SNU-387 and HCCLM3 cells transfected withSCARNA13 ASOs or the control. C and D, Cell-cycle distribution was measured by propidium iodide staining in SNU-387 and HCCLM3 cells transfected withSCARNA13 ASOs or the control, followed by flow cytometric analysis. E and F, EdU immunofluorescence staining assays for SNU-387 and HCCLM3 cellstransfected with SCARNA13 ASOs or the control. Scale bars, 100 mm. G and H, Cell apoptosis was measured by FITC-Annexin V and propidium iodide staining inSNU-387 and HCCLM3 cells transfected with SCARNA13 ASOs or the control, followed by flow cytometric analysis. I and J, Transwell invasion assays for SNU-387and HCCLM3 cells transfected with SCARNA13 ASOs or the control. Scale bars, 100 mm. K and L,Wound-healing migration assays for SNU-387 and HCCLM3 cellstransfected with SCARNA13 ASOs or the control. Scale bars, 100 mm. Data are presented as mean� SEM. ns, not significant; �� , P < 0.01; �� , P < 0.001.

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to ascertain the underlying mechanisms for the elevated expres-sion of SNHG10 and SCARNA13 in HCC, we initially per-formed methylation analysis of the SNHG10 gene promoterusing whole-genome methylation data from the TCGA LIHCdataset. Because SNHG10 and SCARNA13 are processed fromthe exons and introns of primary RNA transcripts from theSNHG10 gene, respectively, the SCARNA13 gene promoter istheoretically equal to the SNHG10 gene promoter (6). Theanalysis results showed that neither SNHG10 nor SCARNA13expression was statistically associated with the methyla-tion levels of the SNHG10 gene promoter (SupplementaryFig. S7A and S7B), revealing that the dysregulation of SNHG10or SCARNA13 cannot be ascribed to the abnormal methylationlevels of the SNHG10 gene promoter.

In addition, the action of transcription factors (TF) in rec-ognizing and dynamically binding to degenerate sequencemotifs located at promoters plays a key role in transcrip-tion (10). Therefore, we speculated whether certain TFs wereresponsible for the aberrant expression of SNHG10 andSCARNA13. The intersection of JASPAR (11), PROMO (12),and LASAGNA (13) databases identified 9 TFs that possiblybound to the promoter region of the SNHG10 gene (Fig. 4A).Analysis of the TCGA LIHC dataset demonstrated that c-Mybexpression shows the highest correlation with SNHG10 andSCARNA13 expression among the 9 TFs (Fig. 4B and C; Sup-plementary Fig. S7C–S7P). Therefore, we deduced that c-Mybmight cause the elevated SNHG10 and SCARNA13 expression.As expected, knockdown of c-Myb significantly decreased theexpression of SNHG10 and SCARNA13 in SNU-387 andHCCLM3 cells (Fig. 4D and E; Supplementary Fig. S7Q–

S7S), identifying c-Myb as the upstream regulator of SNHG10and SCARNA13.

Subsequent bioinformatics analysis predicted five c-Myb bind-ing sites (MBS) in the promoter region of SNHG10 (Fig. 4F andG). Thereafter, ChIP assays confirmed the significantly highenrichment of c-Myb onMBS1 and MBS3 in the promoter regionof SNHG10 (Fig. 4H). To further determine the transcriptionalactivation of c-Myb on the SNHG10 gene promoter, cells werecotransfected with the SNHG10 promoter luciferase reporter(pEZX-PL01-SNHG10) and siRNAs targeting c-Myb. Depletionof c-Myb markedly reduced SNHG10 promoter activity inHEK293T cells (Fig. 4I). Collectively, these data indicated thatc-Myb can directly bind to the promoter region of SNHG10,leading to the upregulation of SNHG10 and SCARNA13 in HCCcells.

SNHG10 functions as a sponge for miR-150-5p to increasec-Myb expression

Theoretically, snoRNAs and the homologous lncRNAs arelocated in the nucleus and cytoplasm, respectively (6). RNAFISH was implemented to confirm the localization of SNHG10and SCARNA13, illustrating that SNHG10 is predominantlylocalized in the cytoplasm, whereas SCARNA13 is preferentiallysituated in the nucleus (Fig. 5A). Next, we investigated thespecific mechanism by which SNHG10 regulated the expressionof SCARNA13.

Emerging evidence has confirmed that cytoplasmic lncRNAscan serve as competing endogenous RNAs (ceRNA) to sequestermiRNAs, resulting in the release of corresponding miRNA-tar-geted mRNAs (14, 15). Accordingly, bioinformatics analysis ofmiRcode (16), lncRNASNP2 (17), and LncBase (18) suggested

that miR-150-5p can bind to the 309-315 nt site of SNHG10(Fig. 5B).

To verify whether SNHG10 and miR-150-5p were involved inthe RNA-induced silencing complex (RISC), RIP assays wereperformed utilizing the anti-Ago2 (the core component of theRISC) antibody. The results showed that both miR-150-5p andSNHG10 are drastically enriched in Ago2 immunoprecipitatescompared with those in the IgG pellet in SNU-387 and HCCLM3cells (Fig. 5C and D), suggesting that SNHG10 physically existedin Ago2-based miRNA-induced repression complex and is asso-ciated with miR-150-5p.

More importantly, ChIRP assays were conducted to deter-mine the direct interaction between SNHG10 and miR-150-5p.Ten oligonucleotide probes targeting SNHG10 were dividedinto an even set and an odd set to increase the specificityof ChIRP assays (19). The data validated the tremendousenrichment of miR-150-5p on SNHG10 in both even andodd probes pools relative to control LacZ probes set inSNU-387 and HCCLM3 cells (Fig. 5E and F). Moreover, trans-fection of miR-150-5p mimics significantly suppressed theluciferase activity of pmirGLO-SNHG10 that containedfull-length SNHG10 at the 30 UTR of Rluc. In contrast,pmirGLO-SNHG10-mut(miR-150-5p) presented no responseto miR-150-5p (Fig. 5G), confirming the sponging function ofSNHG10 to miR-150-5p.

Numerous studies have revealed that miR-150-5p interactswith the 30 untranslated region (UTR) of c-Myb mRNA andoverexpression of miR-150-5p downregulates c-Myb mRNAand protein levels (20, 21), identifying c-Myb as a directtarget of miR-150-5p. Strikingly, our previous data provedthat c-Myb could directly upregulate the expression ofSNHG10. Reciprocally, based on the sponging function ofSNHG10 to miR-150-5p and the confirmed inhibitory effectof miR-150-5p to c-Myb, we inferred that SNHG10-mediatedsequestration of miR-150-5p might be essential for the upre-gulation of c-Myb. To test this speculation, Huh-7 and Hep3Bcells with LV-SNHG10 were transfected with miR-150-5pmimics. The expression of c-Myb was increased upon upregu-lating SNHG10, whereas miR-150-5p overexpression entirelyabolished this effect (Fig. 5H). Conversely, SNU-387 andHCCLM3 cells with LV-shSNHG10 were transfected withmiR-150-5p inhibitors. The expression of c-Myb was declinedupon SNHG10 knockdown and was thoroughly rescued bythe miR-150-5p sponge (Fig. 5I). These findings demonstratedthat SNHG10 functions as a sponge for miR-150-5p to decreaseits suppressive effect on c-Myb, subsequently enhancing theexpression of c-Myb.

Because c-Myb could directly upregulate the expression ofSNHG10, it is reasonable to propose that SNHG10 and c-Mybmight form a positive feedback loop to sustain the elevatedexpression of SNHG10 in HCC. In addition, miR-150-5p didnot witness any statistical changes after silencing or overexpres-sing SNHG10 (Supplementary Fig. S8A and S8B), clarifyingthat SNHG10 could not exert any influences on the expressionof miR-150-5p in HCC. Nevertheless, because of the positivefeedback loop induced by c-Myb, the expression of SNHG10experienced a significant decline and growth after transfectionwith the miR-150-5p mimics and miR-150-5p inhibitors,respectively (Supplementary Fig. S8C–S8J). Together, thesedata further confirmed the positive feedback loop and ceRNAmodel involving SNHG10, miR-150-5p, and c-Myb.






SNHG10 modulates the expression of SCARNA13 through themiR-150-5p/RPL4-c-Myb–positive feedback loop

On the basis of the aforementioned positive feedback loop, aswell as the regulatory effect of c-Myb on SCARNA13, it can beinferred that SNHG10 might affect the expression of SCARNA13via the positive feedback loop mediated by c-Myb. Accordingly,SCARNA13 would accept the regulation of miR-150-5p and haveno influence onmiR-150-5p expression. Indeed, the expression of

SCARNA13 displayed statistical a decrease and increase aftertransfection with the miR-150-5p mimics and miR-150-5p inhi-bitors, respectively (Supplementary Fig. S8K–S8N). On the con-trary, the inhibition of SCARNA13 exerted no influence on theexpression of miR-150-5p (Supplementary Fig. S8O and S8P).Collectively, these results indicated SCARNA13 as the down-stream effector of the positive feedback loop consisting ofSNHG10, miR-150-5p, and c-Myb.

Figure 4.

C-Myb regulates the expression of SNHG10 and SCARNA13. A, Nine TFs were identified to possibly bind to SNHG10 gene promoter through the intersection ofJASPAR, PROMO, and LASAGNA databases. B and C, Scatter plots of SNHG10 or SCARNA13 and c-Myb expression in the TCGA LIHC data repository. Pearsoncorrelation coefficients (r) and P values are shown. D and E, The expression of SNHG10 and SCARNA13 in SNU-387 and HCCLM3 cells transfected with c-MybsiRNAs or the control. F and G, Schematic outlines of the predicted binding of c-Myb to SNHG10 gene promoter and the putative binding sites in the promoterregion. H, ChIP assays of the enrichment of c-Myb on MBSs in the promoter region of SNHG10 relative to IgG. I, Luciferase activity in HEK293T cells cotransfectedwith c-Myb siRNAs and luciferase reporter pEZX-PL01-SNHG10. Data are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. Dataare presented as mean� SEM. ns, not significant; � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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Figure 5.

SNHG10 functions as a sponge for miR-150-5p. A, RNA FISH assays for SNHG10 and SCARNA13. Nuclei were stained with DAPI. Scale bar, 10 mm. B, Predictedbinding sites between SNHG10 and miR-150-5p using bioinformatics analysis. C and D, RIP assays of the enrichment of Ago2 on SNHG10 and miR-150-5p relativeto IgG in SNU-387 and HCCLM3 cells. E and F, ChIRP assays of the enrichment of SNHG10 and miR-150-5p in both even and odd probes pools relative tocontrol LacZ probes set in SNU-387 and HCCLM3 cells. G, Luciferase activity in HEK293T cells cotransfected with miR-150-5p mimics and luciferase reporterpmirGLO-SNHG10 or pmirGLO-SNHG10-mut(miR-150-5p). Data are shown as the relative ratio of firefly luciferase activity to Renilla luciferase activity. H and I,Western blot analysis of c-Myb in indicated HCC cells with miR-150-5p mimics or inhibitors. Data are presented as mean � SEM. ns, not significant; �� , P < 0.01;�� , P < 0.001.






To clarify whether c-Myb was indispensable to the modulatoryeffect of SNHG10 on SCARNA13, rescue experiments wereconducted. Specifically, when the upregulation of c-Myb inLV-SNHG10 cells was completely reversed by c-Myb siRNA atthe RNA and protein levels, the expression of SCARNA13was stillstatistically higher than before (Fig. 6A; Supplementary Fig. S9Aand S9B), implying that SNHG10 might modulate SCARNA13not merely by regulating the expression of c-Myb. Nonetheless,these data could not exclude the possibility that miR-150-5p,rather than SNHG10, influenced SCARNA13 through othermechanisms. Thereafter, we performed further rescue experi-ments to test this hypothesis. When the expression of c-Mybremained stable at the RNA and protein level in LV-Control cellsafter cotransfection with the miR-150-5p inhibitors and c-MybsiRNA, the expression of SCARNA13 showed no significantchange (Fig. 6A; Supplementary Fig. S9A and S9B), validatingthat c-Myb completely mediated the regulatory effects ofmiR-150-5p on SCARNA13 expression. Therefore, it can be seenthat the expression level of c-Mybwas partially responsible for theupregulation of SCARNA13 caused by overexpressing SNHG10.

Apart from the expression level, the functional activity of c-Mybhas been proven to enormously stimulate the downstreamgenes (22). Recently, lncRNAs have been reported to bind tomRNA and improve the stability of mRNA, resulting in increasedprotein levels (15). Thus, we speculated that certain mRNAsbound by SNHG10 might regulate the functional activity ofc-Myb. To verify the hypothesis, ChIRP-seq was conducted topull down endogenous mRNAs bound by SNHG10 andsequence the retrieved RNA (Supplementary Fig. S9C). With theintersection of 407 gene transcripts found by ChIRP-seq and49 protein molecules proven to directly interact with c-Myb inthe BioGRID interaction database (23), two genes were screenedout, namely ribosomal protein L4 (RPL4) and damage-specificDNA binding protein 1 (DDB1; Fig. 6B). Compared with DDB1,RPL4 displayed significantly higher correlation with SNHG10 inthe TCGA LIHC dataset (Fig. 6C and D). More strikingly, aprevious study has described that RPL4 interacts with c-Myb andpositively regulates the transcriptional activity of c-Myb (24).Moreover, the expression of RPL4 showed significant reductionsat the RNA and protein levels after transfection with siRNAstargeting SNHG10 (Supplementary Fig. S9D–S9F). Thus, wededuced that SNHG10 might regulate the functional activity ofc-Myb by affecting the stability of RPL4 mRNA in HCC cells. Toconfirm this speculation, ChIRP-qPCR was initially implementedto verify the direct combination between SNHG10 and RPL4mRNA in SNU-387 and HCCLM3 cells, indicating that botheven and odd probes pools targeting SNHG10 presented signif-icantly higher enrichment of RPL4 mRNA than the control LacZprobes set (Fig. 6E and F). Subsequently, we identified sevenregions of highly complementary sequences between SNHG10and RPL4mRNA utilizing BLAST (http://blast.ncbi.nlm.nih.gov/;Supplementary Fig. S9G). Thereafter, these seven bindingsites were totally mutated in the full-length of SNHG10.SNU-387 and HCCLM3 cells were treated with actinomycinD to interrupt new RNA synthesis and the loss percentage ofRPL4 was measured within a 24-hour period. The findingsillustrated that the overexpression of SNHG10, rather than thatof SNHG10-mut(RPL4), prolongs the half-life of RPL4 mRNA(Fig. 6G and H).

Next, to evaluate the positive regulatory effect of RPL4 on thefunctional activity of c-Myb in HCC cells, luciferase reporters

containing c-Myb recognition elements (MRE) were constructed.Subsequently, the luciferase reporter assays revealed that RPL4could enhance the activity of MREs (Fig. 6I). Furthermore, coim-munoprecipitation assays were performed to determine the inter-action between RPL4 and c-Myb, validating the drastically higherenrichment of RPL4 in the anti–c-Myb group than in the IgGgroup (Fig. 6J). Reciprocally, the anti-RPL4 group exhibitedsubstantially increased enrichment of c-Myb compared with theIgG group (Fig. 6K). The effectiveness of siRNAs targeting RPL4 atthe RNA andprotein levels was verified in SNU-387 andHCCLM3cells (Supplementary Fig. S9H–S9J). Eventually, further rescueexperiments demonstrated that the expression of SCARNA13 didnot witness any statistical change in LV-SNHG10 cells aftercotransfection with c-Myb siRNA and RPL4 siRNA (Fig. 6L).Overall, our data demonstrated that SNHG10 promoted theexpression of c-Myb by, on one hand, absorbing miR-150-5pand, on the other hand, by enhancing the transcriptional activityof c-Myb through interacting with RPL4, consequently modulat-ing the expression of SCARNA13.

Eventually, we investigated whether this circuitry could func-tion under liver physiologic conditions using human normal liverepithelial cells THLE-2 and THLE-3. The results revealed that theexpression of SCARNA13 showed no statistical change after over-expressing SNHG10 in THLE-2 and THLE-3 cells (SupplementaryFig. S9K–S9N). Because c-Myb was responsible for the upregula-tion of SCARNA13 caused by overexpressing SNHG10, we sub-sequently investigated whether the expression of c-Myb could beaffected by SNHG10. Western blot analysis illustrated that upre-gulating SNHG10 exerted no influence on the expressionof c-Mybin THLE-2 and THLE-3 cells (Supplementary Fig. S9O). BecauseSNHG10 functioned as a sponge for miR-150-5p to decrease itssuppressive effect on c-Myb, subsequently enhancing the expres-sionof c-Myb inHCCcells, we speculated that SNHG10-mediatedsequestration of miR-150-5p might not operate due to the enor-mously high expression of miR-150-5p in THLE-2 and THLE-3cells. Indeed, the expression levels of miR-150-5p in THLE-2 andTHLE-3 cells were substantially higher than those in HCC cells(Supplementary Fig. S9P), indicating that miR-150-5p is indis-pensable to the activation of this circuitry in HCC cells.

SCARNA13 mediates SNHG10-driven HCC cell proliferation,invasion, and migration by regulating SOX9

On the basis of SNHG10 and SCARNA13 coordinately con-tributing to the malignant phenotype of HCC cells and SNHG10modulating the expression of SCARNA13, it can be deduced thatthe tumor-promoting effects of SNHG10 on HCC cells might bemediated by SCARNA13. Accordingly, SCARNA13 ASOs weretransfected into LV-SNHG10 cells. Knockdown of SCARNA13significantly rescued the influences of SNHG10 overexpressionon cell proliferation, invasion, and migration (Fig. 7A and B;Supplementary Fig. S10AandS10B). Thesefindings indicated thatSCARNA13mediates the tumor-promoting function of SNHG10.

To gain insights into the molecular mechanism underlying theoncogenic role of SCARNA13 in HCC, RNA-seq analysis wasimplemented for SCARNA13 knockdown (Supplementary Fig.S10C). Silencing of SCARNA13 downregulated the expression of188 genes and upregulated 275 genes. Analysis of the KEGGpathway showed that the cell cycle, TGFb, PI3K-Akt, and p53signaling pathways were influenced by inhibiting SCARNA13(Supplementary Fig. S10D). In addition, depletion of SCARNA13affected cell adhesion, cell proliferation, angiogenesis, and the

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http://blast.ncbi.nlm.nih.gov/


apoptotic process in GeneOntology analysis (Supplementary Fig.S10E), confirming the oncogenic activity of SCARNA13 in hepa-tocarcinogenesis and metastasis. To further identify candidatedownstream factors of SCARNA13, quantitative proteomic anal-ysis was conducted using the Tandem mass tag labeling method,revealing 182 proteins that were differentially expressed (Supple-mentary Fig. S10F). Eleven genes were screened out via taking theintersection of transcriptomics and proteomics (Fig. 7C). Amongthem was SOX9, whose expression showed the most significantdecrease at the protein level after SCARNA13 knockdown. Hence,

SOX9 was selected as the candidate downstream protein ofSCARNA13. Accordingly, inhibition of SCARNA13 resulted inthe remarkably decreased expression of SOX9 at the RNA andprotein levels in SNU-387 and HCCLM3 cells (Fig. 7D–F).Numerous studies have illustrated that SOX9 exerts enormousimpacts on the cell cycle and EMT of cancer cells (25–27).Therefore, the influences of SCARNA13 knockdown on theexpression of molecular markers of cell cycle and EMT weredetected. As expected, corresponding changes were observed forthe expression of these molecular markers. Particularly, the

Figure 6.

SNHG10 modulates SCARNA13expression through the miR-150-5p/RPL4-c-Myb–positive feedbackloop. A, The expression ofSCARNA13 in indicated HCC cellswith or without c-Myb siRNAand/or miR-150-5p inhibitors. B,RPL4 and DDB1 were screened outfrom the intersection of ChIRP-seqresults and the BioGRID interactiondatabase. C and D, Scatter plots ofSNHG10 versus RPL4 or DDB1expression in the TCGA LIHC datarepository. Pearson correlationcoefficients (r) and P values areshown. E and F, ChIRP assays of theenrichment of RPL4mRNA in botheven and odd probes pools relativeto control LacZ probes set in SNU-387 and HCCLM3 cells. G and H, TheRNA levels of RPL4mRNA at theindicated time points wereanalyzed by qPCR relative to time0 after blocking new RNA synthesiswith actinomycin D (2 mg/mL) inSNU-387 and HCCLM3 cells andnormalized to 18S rRNA. I,Luciferase activity in HEK293T cellscotransfected with RPL4 siRNAsand luciferase reporter containing12 MREs. Data are shown as therelative ratio of firefly luciferaseactivity to Renilla luciferase activity.J and K, Coimmunoprecipitationassays of the enrichment of RPL4on c-Myb and the enrichment ofc-Myb on RPL4 relative to IgG. L,The expression of SCARNA13 inindicated HCC cells with or withoutc-Myb siRNA and/or RPL4 siRNA.Data are presented as mean� SEM.ns, not significant; �, P < 0.05;�� , P < 0.01; �� , P < 0.001.






Figure 7.

SCARNA13 mediates SNHG10-driven HCC cell proliferation, invasion, and migration by regulating SOX9. A, EdU immunofluorescence staining assaysfor indicated Hep3B cells transfected with SCARNA13 ASOs or the control. Scale bars, 100 mm. B, Transwell invasion assays for indicated Hep3B cellstransfected with SCARNA13 ASOs or the control. Scale bars, 100 mm. C, Eleven downstream genes were identified by the intersection oftranscriptomics and proteomics. D–F, The expression of SOX9 at the RNA and protein level in SNU-387 and HCCLM3 cells transfected with SCARNA13ASOs or the control. G, Western blot analysis of molecular markers of cell cycle and EMT in SNU-387 and HCCLM3 cells transfected with SCARNA13ASOs or the control. H, Schematic model of the complex circuitry underlying concomitant upregulation of SNHG10 and SCARNA13 in HCC Cells. Dataare presented as mean � SEM. �� , P < 0.01; �� , P < 0.001.

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expression levels of CDK2, CDK3, Cyclin D1, Cyclin D3, ZEB1,Vimentin, N-cadherin, and Fibronectin presented statisticallydownward trends after suppressing SCARNA13 in SNU-387 andHCCLM3 cells. Conversely, transfection with SCARNA13 ASOsled to significantly increased expression of p21, p27, and E-cad-herin in HCC cells (Fig. 7G). Overall, these findings illuminatedthat the promoting effects of SNHG10 on tumorigenesis andmetastasis are mediated by SCARNA13, which promotes the cellcycle and EMT via upregulating SOX9 in HCC cells.

DiscussionHepatocarcinogenesis is regarded as a multistage process

involving genetic and epigenetic alterations, as well as extrinsicmicroenvironment factors, that ultimately result in themalignanttransformation of hepatocytes (28). Presently, HCC is frequentlydiagnosed at an advanced stage, because of insufficient progress inthe identification of ideal diagnostic biomarkers for HCC, pro-viding patients with limited therapies. More seriously, postoper-ative recurrence, mainly caused by intrahepatic metastasis, andextrahepatic metastasis, which most commonly occurs in thelungs, primarily explains the poor prognosis of patients withHCC (29). However, there has been little success in the exploi-tation of effective interventions against HCCmetastasis. To deter-mine the responsible regulators for hepatocarcinogenesis andmetastasis, we performed RNA-seq based on lung metastasisscreening mice models. By taking the intersection of sequencingdata and abnormally expressed genes in HCC tissues from theTCGA LIHC dataset, we identified lncRNA SNHG10 and itshomologous SCARNA13 as potential oncogenic drivers for hepa-tocarcinogenesis and metastasis.

Some 50 terminal oligopyrimidine (50 TOP) RNA transcriptsfrom SNHGs contain only short, poorly conserved ORFs and are,therefore, considered as lncRNAs from the perspective of thestructure and function (6). Recently, several SNHGs have beenidentified to be the critical factors for carcinogenesis and metas-tasis (30). For example, SNHG6 suppresses MAT1A expression byactivating themiR-1297/FUS pathway to regulate the global DNAmethylation levels, thus stimulating the phenotype of hepatomacells (31). SNHG15 maintains Slug stability in colon cancer cellsthrough interaction with the zinc finger domain of Slug, promot-ing colon cancer cellmigration (32). In this work, wefirst reportedthe cancer-promoting role of lncRNA SNHG10 in cancer. Specif-ically, overexpressionof SNHG10was observed in 64HCC tissuesfrom the WCH dataset, and was statistically associated with theclinical characteristics and prognosis of patients with HCC. Next,the phenotypic assays illustrated that SNHG10 exerted remark-able facilitating effects on cell proliferation, the cell cycle, apo-ptosis resistance, invasion, and metastasis in vitro and in vivo,determining the tumorigenic and metastasis-driving functions ofSNHG10 in HCC cells.

In addition, our data uncovered several dysregulated snoRNAsin the process of hepatocarcinogenesis and metastasis. As theprocessed product of primary RNA transcripts from SNHGs,snoRNAs play regulatory roles primarily by modifying rRNAs,acting as the precursors of miRNAs, and affecting RNA splicingvariants in the development and progression of cancer (6). Inaddition, small Cajal body–specific RNAs (scaRNA), located insmall membraneless subcompartments in the cell nucleus (Cajalbodies) rather than in the nucleolus, are a subset of snoRNAs (33).However, although scaRNAs structurally resemble snoRNAs, the

specific roles of scaRNAs in oncogenesis are not well studied. Inthis study, we provided the first evidence of scaRNA dysregulationin HCC. Precisely, SCARNA13 identified by our sequencing dataand the TCGA LIHC dataset showed elevated expression in 64HCC tissues from the WCH dataset. In addition, we found thatSCARNA13 is an independent prognostic indicator for the overallsurvival of patients with HCC. SCARNA13 dramatically contrib-uted to the malignant phenotypes of HCC cells, confirming thetumor-promoting function of SCARNA13 in HCC cells.

To our knowledge, regulatory relationships exist between miR-NAs and lncRNAs, particularly the transcripts from the host genesof miRNAs (7). For example, lncRNA MIR100HG–derived miR-100 and miR-125b were reported to upregulate MIR100HG byaffecting the transcription factor GATA6 (7). Therefore, we spec-ulated there might be similar modulatory correlation betweensnoRNAs and lncRNAs from SNHGs. Intriguingly, our resultsdemonstrated that SNHG10 exerts significant regulatory effectson the expression of SCARNA13. To be specific, SNHG10 servedas a sponge for miR-150-5p to abolish the suppressive effect ofmiR-150-5p on c-Myb, resulting in the elevated expression ofc-Myb. On the other hand, SNHG10 promoted the expression ofRPL4 by boosting the stability of RPL4 mRNA, leading to theimprovement of functional activity of c-Myb based on the directinteraction between RPL4 and c-Myb. Reciprocally, overexpressedand hyperactivated c-Myb enhanced the expression of SNHG10and SCARNA13 through directly binding to the promoter regionof SNHG10, thereby regulating its promoter activity and forminga positive feedback loop inHCC cells. Consequently, activation ofthis feedback loop continuously stimulated the expression ofSCARNA13 (Fig. 7H). Collectively, SNHG10 modulated itshomologous SCARNA13 via a positive feedback loop to facilitatethe development andprogression ofHCC.Ourfindings identifiedthe responsible lncRNAs and snoRNAs in hepatocarcinogenesisand metastasis, and presented novel implications for the molec-ular mechanisms of lncRNAs and snoRNAs in cancer research.More strikingly, we propose a complex regulatory relationshipbetween lncRNAs and its homologous snoRNAs.

In conclusion, we have identified a complex circuitry underly-ing the concomitant upregulation of SNHG10 and its homolo-gous SCARNA13 in HCC. SNHG10 regulates c-Myb by spongingmiR-150-5p and interacting with RPL4 mRNA, modulating theexpression of SCARNA13 and its downstream effector SOX9 inHCC cells. The investigation of SNHG10 and SCARNA13 pro-vides a luminous comprehension of hepatocarcinogenesis andmetastasis that may develop effective strategies for the diagnosisand treatment of HCC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Lan, K. Yuan, X. Hao, J. Wang, Y. Zeng, H. WuDevelopment of methodology: T. Lan, L. Xu, J. Wang, X. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Lan, L. Xu, H. Liao, X. Hao, H. Liu, J. Li, M. LiaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Lan, X. Yan, X. HaoWriting, review, and/or revision of the manuscript: T. Lan, K. Yuan, K. Xie,J. Huang, Y. ZengAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Liao, H. WuStudy supervision: K. Yuan, Y. Zeng, H. Wu






AcknowledgmentsWe thank Li Chai and Yan Wang from the Core Facility of West China

Hospital (Chengdu, Sichuan, China) for technical assistance and ShanghaiLu-Ming Biotech Co., Ltd. (Shanghai, China) for assistance with quantitativeproteomic analysis. This study was supported by grants from the NaturalScience Foundation of China (81872004, 81800564, 81770615, 81700555,81672882, and 81502441), National Key Technologies R&D Program(2018YFC1106803), the Science and Technology Support Program ofSichuan Province (2017SZ0003, 2018SZ0115), the Science and TechnologyProgram of Tibet Autonomous Region (XZ201801-GB-02), and the 1.3.5

project for disciplines of excellence, West China Hospital, Sichuan University(ZYJC18008).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 22, 2018; revised April 19, 2019; accepted May 13, 2019;published first May 17, 2019.

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2019;79:3220-3234. Published OnlineFirst May 17, 2019.Cancer Res Tian Lan, Kefei Yuan, Xiaokai Yan, et al. Loopby Modulating Its Homolog SCARNA13 via a Positive Feedback LncRNA SNHG10 Facilitates Hepatocarcinogenesis and Metastasis

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lncrnasnhg10facilitateshepatocarcinogenesis …...coimmunoprecipitation cells were lysed in ip...

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