Long noncoding RNA AWPPH promotes hepatocellular carcinoma progression through YBX1 and serves as a prognostic biomarker

    Long noncoding RNA AWPPH promotes hepatocellular carcinoma progression through YBX1 and serves as a prognostic biomarker Xiaodong Zhao, Yanbo Liu, Shuo Yu PII: DOI: Reference:

S0925-4439(17)30126-6 doi:10.1016/j.bbadis.2017.04.014 BBADIS 64745

To appear in:

BBA - Molecular Basis of Disease

Received date: Revised date: Accepted date:

31 January 2017 8 April 2017 16 April 2017

Please cite this article as: Xiaodong Zhao, Yanbo Liu, Shuo Yu, Long noncoding RNA AWPPH promotes hepatocellular carcinoma progression through YBX1 and serves as a prognostic biomarker, BBA - Molecular Basis of Disease (2017), doi:10.1016/j.bbadis.2017.04.014

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title: Long noncoding RNA AWPPH promotes hepatocellular carcinoma progression through

†

IP

T

YBX1 and serves as a prognostic biomarker

†

Department of Oncological Surgery, The Second Hospital of Hebei Medical University, No. 215

Peace West Road, Shijiazhuang 050000, China

Equal contributors

TE

D

Correspondence: [email protected], Tel: +86-0311-66003910, Fax: +86-0311-66003910.

CE P

*

AC

†

The First Department of Surgery, Feixiang Central Hospital, Handan 057550, China

MA

2

NU

1

SC R

Xiaodong Zhao1 , Yanbo Liu2 , Shuo Yu1*

1

ACCEPTED MANUSCRIPT Abstract Long noncoding RNAs (lncRNAs) have been shown to play important roles in various cancers.

IP

T

However, the clinical significances and biological roles of lncRNAs in hepatocellular carcinoma

SC R

(HCC) remain largely unknown. In this study, using online-available data sets and quantitative real-time PCR, we identified a novel lncRNA termed lncRNA-AWPPH, which is highly expressed in HCC tissues. Its upregulation is correlated with encapsulation incomplete, microvascular invasion,

NU

advanced TNM stage and BCLC stage. Cox proportional hazards regression analysis revealed that high

MA

lncRNA-AWPPH expression is an independent prognostic factor for poor recurrence-free and overall survival. Functional experiments showed that overexpression of lncRNA-AWPPH promotes HCC cell

D

proliferation and migration in vitro, and tumor growth and metastasis in vivo. Conversely, depletion of

TE

lncRNA-AWPPH has opposite effects on HCC. Mechanistically, lncRNA-AWPPH interacts with

CE P

YBX1, promotes YBX1-mediated activation of SNAIL1 translation, and upregulates SNAIL1 expression. Furthermore, lncRNA-AWPPH promotes YBX1-mediated activation of PIK3CA

AC

transcription, upregulates PIK3CA expression, and activates PI3K/AKT pathway. Depletion of YBX1 abolishes the effects of lncRNA-AWPPH on SNAIL1 and PIK3CA, and also the biological roles of lncRNA-AWPPH on HCC cells. In conclusion, this study identifies a novel lncRNA termed lncRNA-AWPPH which is highly expressed in HCC, indicates poor prognosis of HCC patients, and promotes HCC cell proliferation, migration, and in vivo tumor growth and metastasis via a novel regulatory mechanism of interacting with YBX1.

Keywords: long noncoding RNA; hepatocellular carcinoma; proliferation; metastasis; YBX1

2

ACCEPTED MANUSCRIPT 1. Introduction Liver cancer is the sixth most common form of cancer with 782,000 cases reported in 2012 worldwide,

IP

T

and its incidence is continuing to rise [1, 2]. Dismayingly, the prognosis of liver cancer patients is very

SC R

poor with 16.6% of liver cancer patients surviving for five years after being diagnosed in United States [3, 4]. Hepatocellular carcinoma (HCC) is the major histological type of liver cancer [5]. Despite there have been advances in the medical treatments for HCC, including surgery, transplant and radiation,

NU

effective treatment for postsurgical recurrence and metastasis is still lacking, which accounts for the

MA

poor outcomes of HCC patients [6-8]. Accurately identifying patients with high risk of recurrence and metastasis would allow appropriate management for improving prognosis. However, until now

D

researchers do not completely understand the molecular mechanisms underlying the recurrence and

TE

metastasis of HCC [9, 10]. Therefore, further understanding the critical molecular mechanisms and

CE P

searching for reliable biomarkers for predicating recurrence and survival would be urgent. Recent progressions have revealed a class of long noncoding RNA (lncRNA) with critical roles in

AC

various pathophysiological processes [11-13]. lncRNA has limited protein coding potential with longer than 200 nucleotides in length [14]. Accumulating evidences demonstrate that lncRNAs play important roles in tumor initiation, progression, metastasis, drug-resistance, recurrence and et al. [15-18]. Furthermore, aberrant expressions of lncRNAs have been found in many tumors, supporting the critical roles of lncRNAs in tumors [19-21]. Although several lncRNAs have been shown to play various roles in HCC, including lncRNA-HEIH, lncRNA-ATB, DANCR, GIHCG, and et al. [22-25], the overall number of lncRNAs is much larger than mRNAs [26], and so whether other lncRNAs also function as critical regulators in HCC and indicate patients’ prognosis need further investigation. In this study, using online-available data sets, we identified a novel lncRNA which is upregulated in

3

ACCEPTED MANUSCRIPT HCC tissues and associated with poor prognosis of HCC patients. We further validated the expression of this lncRNA in enlarged clinical samples and analyzed its prognostic values. In vitro and in vivo

AC

CE P

TE

D

MA

NU

SC R

molecular mechanisms exerting by this lncRNA were also explored.

IP

T

functional experiments were performed to investigate its biological roles. Furthermore, the potential

4

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Patients and tissue samples

IP

T

Eighty-eight pairs of HCC tissues and adjacent noncancerous hepatic tissues, and 20 portal vein tumor

SC R

thrombus (PVTT) tissues were obtained from patients undergoing curative hepatectomy at the Second Hospital of Hebei Medical University. None of the patients received preoperative treatment. Tissue samples were immediately frozen in liquid nitrogen after surgery and stored at -80˚C until use. All

NU

resected tissues were confirmed by histopathological examination. Tumor clinical stage was defined

MA

according to the Barcelona Clinic Liver Cancer (BCLC) staging classification. This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University and written

D

informed consent was obtained from all patients before the initiation of this study.

TE

2.2. Cell culture

CE P

Human immortalized, nontransformed liver cell line QSG-7701, and HCC cell lines SMMC-7721, HCCLM3, Huh7, and HepG2 were obtained from Cell Bank, Chinese Academy Sciences (Shanghai,

AC

China). QSG-7701 and SMMC-7721 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA), HCCLM3 and Huh7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco), and while HepG2 cells were cultured in Eagle's Minimum Essential Medium (Gibco). All the cells were maintained with 10% fetal bovine serum (Gibco) supplemented at 37°C in an atmosphere containing 5% CO2. 2.3. RNA isolation and quantitative real-time PCR (qRT-PCR) Total RNA from human tissues and cultured cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Then 2 µg of RNA were reverse-transcribed into complementary DNA (cDNA) using the M-MLV Reverse Transcriptase

5

ACCEPTED MANUSCRIPT (Invitrogen). qRT-PCR was performed on Applied Biosystems 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR® Premix Ex Taq™ II (Takara, Dalian, China). The

IP

T

expressions of lncRNAs and mRNAs were normalized to GAPDH. The gene-specific primers

SC R

sequences used were as follows: for lncRNA-AWPPH: 5'-CTGGATGGTCGCTGCTTTTTA-3' (forward) and 5'-AGGGGGATGAGTCGTGATTT-3' (reverse); for lncRNA-HEIH: 5'-CCTCTTGTGCCCCTTTCTT-3' (forward) and 5'-ATGGCTTCTCGCATCCTAT-3' (reverse); for

NU

TP53TG1: 5'-CTTTCCTTTAATCTTCGGAGGC-3' (forward) and

MA

5'-TGCCAGCTCTCAGAGTCCTT-3' (reverse); for SNAIL1: 5'-TGCGTCTGCGGAACCTG-3' (forward) and 5'-GGACTCTTGGTGCTTGTGGA-3' (reverse); for PIK3CA:

D

5'-TTCTGTCTCCTCTAAACCC-3' (forward) and 5'-TATCTTGCCGTAAATCATCC-3' (reverse); for

TE

GAPDH: 5'-GGAGCGAGATCCCTCCAAAAT-3' (forward) and

CE P

5'-GGCTGTTGTCATACTTCTCATGG-3' (reverse); for β-actin: 5'-GGGAAATCGTGCGTGACATTAAG-3' (forward) and 5'-TGTGTTGGCGTACAGGTCTTTG-3'

AC

(reverse); for U6: 5'-GCTTCGGCAGCACATATACTAAAAT-3' (forward) and 5'-CGCTTCACGAATTTGCGTGTCAT-3' (reverse). The relative expressions of lncRNAs and mRNAs were calculated using the comparative Ct method. 2.4. 5' and 3' Rapid Amplification of cDNA Ends (RACE) To determine the transcriptional initiation and termination sites of lncRNA-AWPPH, 5'-RACE and 3'-RACE analyses were performed using a 5'/3' RACE Kit (Roche, Mannheim, Germany) following the manufacturer’s protocol. The primers used for the RACE analyses were as follows: SP1, 5'-CAGACAAATGGGAAACCGAC-3'; SP2, 5'-GAGGGGGATGAGTCGTGAT-3'; SP3, 5'-ATCTGAAGACAGGCACGGGC-3'; SP5, 5'-AGCACCTCTACCTGTTGCCCG-3'.

6

ACCEPTED MANUSCRIPT 2.5. Northern blot Northern blot was performed using the NorthernMax® Kit (Ambion, Grand Island, NY, USA)

IP

T

following the manufacturer’s protocol. Briefly, a total of 10 µg indicated RNA was separated by

SC R

formaldehyde gel electrophoresis and transferred to Biodyne Nylon membrane (Pall, NY, USA). PCR was performed to obtain biotin-16-dUTP (Roche)-labeled lncRNA-AWPPH cDNA probe. The primers used were as follows: 5'-CATCATTGGGAATGGAGGGA-3' (forward) and

NU

5'-CACAGTGGGCAGTTGGAAC-3' (reverse). After 1 hour of prehybridization in ULTRAhyb-Oligo

MA

buffer, the membrane was hybridized at 68°C for 12 hours in ULTRAhyb-Oligo buffer containing the denatured probe. After being washed, the membrane was detected using an Odyssey infrared scanner

D

(Li-Cor, Lincoln, NE, USA).

TE

2.6. Cytoplasmic, nuclear, and polyadenylated RNA isolation

CE P

Cytoplasmic and nuclear RNA were isolated and purified using the Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA, USA) following the manufacturer’s protocol. Polyadenylated

AC

RNA was captured and purified using the GenEluteTM mRNA Miniprep Kit (Sigma-Aldrich, Saint Louis, MO, USA) following the manufacturer’s protocol. 2.7. Vectors and stable cell lines construction Full length of lncRNA-AWPPH was PCR amplified from cDNA using the Q5® Hot Start High-Fidelity DNA Polymerase (NEB, Ipswich, MA, USA) following the manufacturer’s protocol. The primers used were as follows: 5'-CCCAAGCTTTATGAAGAGAATGTCGGG-3' (forward) and 5'-CGGGATCCCTGTTTTCTTTAGTTTTGCTT-3' (reverse). Then the PCR product was subcloned into the Hind III and BamH I sites of pcDNA3.1 (Invitrogen), named pcDNA3.1-AWPPH. For construction of lncRNA-AWPPH stably overexpressed cells, pcDNA3.1-AWPPH or pcDNA3.1 was

7

ACCEPTED MANUSCRIPT transfected into SMMC-7721 cells, and selected with neomycin (800 µg/ml) for four weeks.

IP

fragment was subcloned into pSPT19 (Roche), named pSPT19-AWPPH.

T

pcDNA3.1-AWPPH was double digested with Hind III and BamH I, and the lncRNA-AWPPH

SC R

Two independent cDNA oligonucleotides specifically targeting lncRNA-AWPPH (shAWPPH-1 and shAWPPH-2) were synthesized by GenePharma (Shanghai, China) and inserted into the shRNA expression vector pGPH1/Neo. One cDNA oligonucleotide specifically targeting YBX1 (shYBX1)

NU

were synthesized by GenePharma and inserted into the shRNA expression vector pGPH1/Hygro. The

MA

shRNAs target sites were as follows: for shAWPPH-1: GGTCTGGTCGGTTTCCCATTT; for shAWPPH-2: GGAATGCAGCTGAAAGATTCC; for shYBX1: GGTTCCCACCTTACTACATGC.

D

shAWPPH-1, shAWPPH-2, or scrambled shControl was transfected into HCCLM3 cells, and selected

TE

with neomycin (1000 µg/ml) for four weeks. shYBX1 or scrambled shControl was transfected into

CE P

SMMC-7721 cells, and selected with Hygromycin B (400 µg/ml) for four weeks. All the transfections were carried out using Lipofectamine 3000 (Invitrogen) following the

AC

manufacturer’s protocol.

2.8. Cell proliferation assay Glo cell viability assays and ethynyl deoxyuridine (EdU) incorporation assays were performed to assess cell proliferation. Glo cell viability assays were carried out using the Cell Titer-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Briefly, a total of 3,000 indicated HCC cells were plated in 96-well plates. The luminescence values at each time point were collected and plotted into the cell proliferation curves. EdU incorporation assays were carried out using an EdU kit (Roche) according to the manufacturer’s protocol. The results were acquired using Zeiss AxioPhot Fluorescence Microscope (Carl Zeiss,

8

ACCEPTED MANUSCRIPT Oberkochen, Germany) and quantified using Image-Pro plus 6.0 software. 2.9. Wound healing assays

IP

T

Indicated HCC cells were plated in 6-well plates and cultured at 37°C. Once cells were attached

SC R

completely, they were scraped to form a wound in the middle of the plates. After scraping the cells, the medium was replaced with serum-free medium. After incubation for 72 hours, the fraction of cell coverage across the line was measured.

NU

2.10. Cell migration assay

MA

40,000 indicated HCC cells suspended in serum-free medium were seeded in the upper chambers of a 24-well transwell insert (Millipore, Bedford, MA, USA). 1 µg/ml cell proliferation inhibitor Mitomycin

D

C was added to the upper chambers. Medium supplemented with 10% serum was added to the lower

TE

chambers. After 48 hours’ incubation, cells remaining on the upper surface of inserts were scraped off

CE P

and migrated cells on the lower surface were fixed with methanol, stained with crystal violet, and counted under a microscope.

AC

2.11. Animal studies

All animal studies were approved by the Ethics Committee of the Second Hospital of Hebei Medical University. 5×106 luciferase-labeled indicated HCC cells were injected subcutaneously into the axillary fossa of male athymic BALB/c nude mice. The tumor volume was measured every week after injection with a caliper, and was calculated as a×b2/2 (a, long axes; b, short axes). At indicated time after inoculation, the mice were monitored using the IVIS@ Lumina II system (PerkinElmer, Hopkinton, MA, USA) 10 min after intraperitoneal injection of luciferin (PerkinElmer). For liver orthotopic xenograft assays, subcutaneous tumors were cut into 1 mm3 sections. An upper-abdominal incision was made while the mice were anesthetized. The liver was exposed and part

9

ACCEPTED MANUSCRIPT of the liver surface was mechanically injured with scissors. A tumor piece was fixed in the liver tissue, and then the liver was returned to the peritoneal cavity and the abdominal wall was closed. At indicated

IP

T

time after inoculation, the mice were monitored using the IVIS@ Lumina II system (PerkinElmer) 10

SC R

min after intraperitoneal injection of luciferin.

For lung metastasis assays, 2×106 luciferase-labeled indicated HCC cells were injected into the tail vein of nude mice. At indicated time after inoculation, the mice were monitored using the IVIS@ Lumina II

NU

system (PerkinElmer) 10 min after intraperitoneal injection of luciferin.

MA

2.12. Immunohistochemistry assay

Immunohistochemistry for Ki67 was carried out on paraffin sections using a Ki67 specific antibody

D

(Cell Signaling Technology, Boston, MA, USA) and a horseradish peroxidase-conjugated IgG

CE P

2.13. RNA pull-down assay

TE

(Invitrogen). Then the proteins in situ were visualized with 3, 3-diaminobenzidine.

lncRNA-AWPPH and its antisense RNA were in vitro transcribed from pSPT19-AWPPH, and

AC

biotin-labeled using Biotin RNA Labeling Mix (Roche) and T7/SP6 RNA polymerase (Roche) following the manufacturer’s protocols. 50 pmol of purified biotinylated lncRNA-AWPPH was incubated with 1 mg protein extracts from SMMC-7721 cells for 1 hour at 4 °C. Then the complexes were mixed with Dynabeads Myone Streptavidin T1 beads (Invitrogen) for additional 1 hour and washed with PBS. The proteins binding to the beads were boiled in SDS buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then the gels were silver-stained and the specific bands were excised and analyzed by mass spectrometry or directly detected by western blot. 2.14. Western blot

10

ACCEPTED MANUSCRIPT Proteins were extracted from cells using RIPA Lysis Buffer and PMSF (Beyotime Co., Jiangsu, China) following the manufacturer’s protocols. Then the proteins were separated by SDS-PAGE, followed by

IP

T

being transferred to a nitrocellulose membrane. After being blocked with bovine serum albumin, the

SC R

blots were incubated with antibodies for YBX1 (Millipore), ANXA1 (Abcam, Hong Kong, China), PGK1 (Abcam), SNAIL1 (Abcam), PI3K p110α (Cell Signaling Technology), AKT p-S473 (Cell Signaling Technology), AKT (Cell Signaling Technology) or GAPDH (Abcam). Then the blots were

NU

incubated with IRdye 700-conjugated goat anti-mouse IgG or IRdye 800-conjugated goat anti-rabbit

2.15. RNA immunoprecipitation (RIP)

MA

IgG, and were detected using an Odyssey infrared scanner (Li-Cor).

D

RIP assays were carried out using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation

TE

Kit (Millipore) and YBX1 specific antibody (Millipore) following the manufacturer’s protocol.

CE P

Retrieved RNAs were reverse-transcribed into cDNA and detected by qRT-PCR as above described. 2.16. Polysome fractionation

AC

Polysome fractionation was performed using sucrose density gradient centrifugation as previously described [27]. Indicated HCC cells were lyzed using Nonidet P-40 lysis buffer (20 mM HEPES-KOH pH 7.6, 100 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.25% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml pepstatin, and 10 µg/ml cycloheximide). Whole-cell lyses were centrifuged at 13,000 rpm for 20 min at 4 °C to clear nuclei and cell debris. Supernatants were layered onto 15–55%(w/v) sucrose gradients and centrifuged at 39,000 rpm for 4 hours at 4°C. After centrifugation, fractions were pumped out by upward displacement, followed by RNA extraction, reverse-transcription into cDNA, and detection by qRT-PCR as above described. 2.17. Chromatin immunoprecipitation (ChIP)

11

ACCEPTED MANUSCRIPT ChIP assays were carried out using the EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore) and YBX1 specific antibody (Millipore) following the manufacturer’s protocol. Retrieved

IP

T

DNAs were detected by qRT-PCR as above described. The primer sequences used were as follows:

SC R

for PIK3CA promoter: 5'-GAAGAGCAGCCCCAACTGTA-3' (forward) and 5'-GAGGGGCAGAGCCTACAATC-3' (reverse). 2.18. Statistical analysis

NU

All statistical analyses were carried out using SPSS 18.0 software. As indicated in figure legends,

MA

Mann-Whitney test, Log-rank test,Wilcoxon signed-rank test, Pearson chi-square test, Cox proportional hazards regression, and Student’s t test were performed. P < 0.05 was considered as statistically

AC

CE P

TE

D

significant.

12

ACCEPTED MANUSCRIPT 3. Results 3.1. lncRNA-AWPPH is highly expressed in HCC and an independent prognostic factor for HCC

IP

T

patients

SC R

To identify lncRNAs involved in the progression and prognosis of HCC, we analyzed online-available data sets, including GSE54238 (lncRNAs microarray data of 30 noncancerous hepatic tissues and 23 HCC tissues) and GSE40144 (lncRNAs microarray data of 59 HCC tissues with disease-free survival

NU

and overall survival information). Among the differently expressed lncRNAs, we noted that lncRNA

MA

NR_024373 was highly expressed in HCC tissues compared with noncancerous hepatic tissues from GSE54238 (Fig. 1A). Furthermore, Kaplan-Meier survival analysis revealed that high NR_024373

D

expression was correlated with poor disease-free survival and overall survival from GSE40144 (Fig.

CE P

(lncRNA-AWPPH).

TE

1B and C). Hereafter we named NR_024373as lncRNA associated with poor prognosis of HCC

We further measured the expression pattern of lncRNA-AWPPH in our own cohort, including 88 pairs

AC

of HCC tissues and adjacent noncancerous hepatic tissues, and 20 PVTT tissues. PVTT is the primary route for intrahepatic metastasis in HCC patients. As shown in Fig. 1D, lncRNA-AWPPH was highly expressed in HCC tissues compared with noncancerous hepatic tissues, and was further upregulated in PVTT tissues. We also measured lncRNA-AWPPH expression in human immortalized, nontransformed liver cell line QSG-7701, and HCC cell lines SMMC-7721, HCCLM3, Huh7, and HepG2. As shown in Figure S1A, lncRNA-AWPPH was highly expressed in the HCC cell lines compared with immortalized, nontransformed liver cell line. To analyze the correlations between lncRNA-AWPPH expression and clinicopathological characteristics in the 88 HCC patients, Pearson chi-square test was performed, and the results showed that high lncRNA-AWPPH expression was

13

ACCEPTED MANUSCRIPT correlated with encapsulation incomplete, microvascular invasion, advanced TNM stage, and advanced BCLC stage (Table 1). Kaplan-Meier survival analysis and log-rank test was performed to detect the

IP

T

association between lncRNA-AWPPH expression and these 88 HCC patients’ prognoses. As shown in

SC R

Fig. 1E and F, high lncRNA-AWPPH expression was correlated with poor recurrence-free survival and overall survival. Cox proportional hazards regression analysis further revealed that high lncRNA-AWPPH expression in HCC tissues was an independent prognostic factor for poor

NU

recurrence-free survival (hazard ratio, 2.579; 95% confidence interval, 1.425 to 4.668; P = 0.002) and

MA

overall survival (hazard ratio, 3.509; 95% confidence interval, 1.574 to 7.820; P = 0.002) (Table 2). The full-length sequence of lncRNA-AWPPH was determined using 5' and 3' RACE analyses and

D

presented in Figure S1B. Northern blot analysis of lncRNA-AWPPH in QSG-7701 and HCCLM3 cells

TE

confirmed the expected size (Figure S1C). lncRNA-AWPPH was poly (A)-positive and located both in

CE P

the nucleus and cytoplasm (Figure S1D and E). Collectively, these data demonstrate that lncRNA-AWPPH is highly expressed and associates with

AC

multiple aggressive characteristics of HCC. Moreover, these data implied that lncRNA-AWPPH could serve as a prognostic biomarker for HCC patients. 3.2. lncRNA-AWPPH promotes HCC cell proliferation and migration in vitro To explore the biological roles of lncRNA-AWPPH in HCC, we stably overexpressed lncRNA-AWPPH in SMMC-7721 cells (Fig. 2A). Glo cell viability assays showed that enhanced expression of lncRNA-AWPPH significantly increased SMMC-7721 cell viability (Fig. 2B). EdU incorporation assays further revealed that ectopic expression of lncRNA-AWPPH promoted SMMC-7721 cell proliferation (Fig. 2C). Furthermore, we stably depleted lncRNA-AWPPH in HCCLM3 cells using two independent lncRNA-AWPPH specific shRNAs (Fig. 2D). Glo cell viability

14

ACCEPTED MANUSCRIPT assays revealed that depletion of lncRNA-AWPPH significantly decreased HCCLM3 cell viability (Fig. 2E). EdU incorporation assays further revealed that depletion of lncRNA-AWPPH significantly

IP

T

inhibited HCCLM3 cell proliferation (Fig. 2F). We further explored the effects of lncRNA-AWPPH on

SC R

HCC cells migration. Wound healing assays showed that enhanced expression of lncRNA-AWPPH increased SMMC-7721 cells migratory speed (Fig. 2G). Transwell assays showed that enhanced expression of lncRNA-AWPPH promoted SMMC-7721 cells migration (Fig. 2H). Furthermore, wound

NU

healing assays and transwell assays both revealed that depletion of lncRNA-AWPPH significantly

MA

inhibited HCCLM3 cells migration (Fig. 2I and J). These results demonstrate that lncRNA-AWPPH promotes HCC cells proliferation and migration in vitro.

D

3.3. lncRNA-AWPPH promotes HCC tumor growth and metastasis in vivo

TE

To further confirm the biological roles of lncRNA-AWPPH in HCC, we labeled lncRNA-AWPPH

CE P

stably overexpressed and control SMMC-7721 cells with firefly luciferase, and then injected these cells subcutaneously into athymic nude mice. Subcutaneous tumor growth was measured every 7 days, and

AC

the mice were sacrificed at 21th day after injection, when the tumors were excised and weighed. As shown in Fig. 3A and B, ectopic expression of lncRNA-AWPPH significantly enhanced tumor growth in vivo. The photon flux of subcutaneous tumor also revealed that ectopic expression of lncRNA-AWPPH promoted subcutaneous tumor growth in vivo (Fig. 3C). Ki67 staining of subcutaneous tumor further revealed that ectopic expression of lncRNA-AWPPH promoted SMMC-7721 cell proliferation in vivo (Fig. 3D). In addition, we established orthotopic tumor models using firefly luciferase labeled lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells. As shown in Fig. 3E, ectopic expression of lncRNA-AWPPH significantly enhanced hepatic orthotopic xenograft growth.

15

ACCEPTED MANUSCRIPT lncRNA-AWPPH stably depleted and control HCCLM3 cells were also labeled with firefly luciferase, and then injected subcutaneously into athymic nude mice. As shown in Fig. 3F-H, depletion of

IP

T

lncRNA-AWPPH significantly inhibited subcutaneous tumor growth in vivo. Ki67 staining of

SC R

subcutaneous tumor further revealed that depletion of lncRNA-AWPPH inhibited HCCLM3 cell proliferation in vivo (Fig. 3I). In addition, depletion of lncRNA-AWPPH also inhibited hepatic orthotopic xenograft growth (Fig. 3J).

NU

To explore the effects of lncRNA-AWPPH on HCC metastasis, we injected luciferase labeled

MA

lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells directly into tail veins of nude mice. As shown in Fig. 3K, ectopic expression of lncRNA-AWPPH significantly promoted lung

D

metastasis of SMMC-7721 cells. Furthermore, luciferase labeled lncRNA-AWPPH stably depleted and

TE

control HCCLM3 cells were also injected directly into tail veins of nude mice. The results showed that

CE P

depletion of lncRNA-AWPPH significantly inhibited lung metastasis of HCCLM3 cells (Fig. 3L). Collectively, these data demonstrate that lncRNA-AWPPH promotes HCC tumor growth and

AC

metastasis in vivo.

3.4. lncRNA-AWPPH binds to YBX1 protein Recent studies have demonstrated that many lncRNAs exert their functions through physically associating with proteins [22, 28, 29]. To investigate whether lncRNA-AWPPH functions in such a manner, we performed RNA pull-down assays to identify proteins bound to lncRNA-AWPPH. lncRNA-AWPPH specifically bound protein band was excised and subjected to mass spectrometry (Fig. 4A and Table 3). The RNA and DNA binding protein Y-box binding protein 1 (YBX1) was the primary protein identified by mass spectrometry. Independent RNA pull-down assays followed by western blot verified the specific binding of YBX1, but not ANXA1 and PGK1, to lncRNA-AWPPH

16

ACCEPTED MANUSCRIPT (Fig. 4B). In addition, RIP assays with an YBX1 specific antibody revealed that lncRNA-AWPPH, but not lncRNA-HEIH and GAPDH mRNA, bound to YBX1 (Fig. 4C). TP53TG1 was used as a positive

IP

T

control [30]. These data demonstrate that lncRNA-AWPPH could bind to YBX1 protein. However, the

SC R

protein level of YBX1 was not changed in lncRNA-AWPPH overexpressed SMMC-7721 cells and lncRNA-AWPPH depleted HCCLM3 cells (Fig. 4D and E). These results imply that the binding between lncRNA-AWPPH and YBX1 does not change the protein level of YBX1.

NU

3.5. lncRNA-AWPPH activates SNAIL1 translation via YBX1

MA

Previous report has revealed that YBX1 promoted breast cancer invasion and metastasis through binding to and activating SNAIL1 mRNA translation [31]. To investigate whether the association

D

between lncRNA-AWPPH and YBX1 affects the effects of YBX1 on SNAIL1, we first performed RIP

TE

assays with YBX1 specific antibody in lncRNA-AWPPH stably overexpressed SMMC-7721 cells and

CE P

lncRNA-AWPPH stably depleted HCCLM3 cells. As shown in Fig. 5A and B, YBX1 bound to not only lncRNA-AWPPH, but also SNAIL1 mRNA in HCC cells. Furthermore, ectopic expression of

AC

lncRNA-AWPPH enhanced the binding between YBX1 protein and SNAIL1 mRNA (Fig. 5A). Conversely, depletion of lncRNA-AWPPH inhibited the binding between YBX1 protein and SNAIL1 mRNA (Fig. 5B). Then we compared the total and polysome-bound (translationally active) SNAIL1 mRNA levels in lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells, lncRNA-AWPPH stably depleted and control HCCLM3 cells. As shown in Fig. 5C and D, neither overexpression nor depletion of lncRNA-AWPPH affected SNAIL1 total mRNA levels. But overexpression of lncRNA-AWPPH significantly increased the proportion of SNAIL1 mRNA in polysomal fractions, and while depletion of lncRNA-AWPPH significantly decreased the proportion of SNAIL1 mRNA in polysomal fractions (Fig. 5C and D). These results suggest that lncRNA-AWPPH

17

ACCEPTED MANUSCRIPT activates SNAIL1 translation. Western blot analysis further confirmed that overexpression of lncRNA-AWPPH upregulated SNAIL1 protein level, and while depletion of lncRNA-AWPPH

IP

T

decreased SNAIL1 protein level (Fig. 5E and F).

SC R

To investigate whether the effects of lncRNA-AWPPH on SNAIL1 mRNA translation are dependent on YBX1, we stably depleted YBX1 in SMMC-7721 cells using YBX1 specific shRNAs (Fig. 5G), which did not regulate lncRNA-AWPPH expression (Fig. 5H). The total and polysome-bound (translationally

NU

active) SNAIL1 mRNA levels in lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells

MA

with YBX1 depletion were measured by qRT-PCR. The results showed that depletion of YBX1 abolished the upregulation of SNAIL1 mRNA in polysomal fractions caused by lncRNA-AWPPH

D

overexpression (Fig. 5I, compared with Fig. 5C). Western blot analysis further showed that depletion of

TE

YBX1 abolished the upregulation of SNAIL1 protein level caused by lncRNA-AWPPH overexpression

CE P

(Fig. 5J, compared with Fig. 5E). Collectively, these data demonstrate that lncRNA-AWPPH actives SNAIL1 translation via YBX1 in HCC cells.

AC

3.6. lncRNA-AWPPH activates PI3K/AKT pathway via YBX1 Except the roles of cytoplasmic YBX1 on translational activation of SNAIL1 and tumor metastasis, nuclear YBX1 is known to bind to PIK3CA promoter, activate its transcription, and promote tumorigenesis [30, 32]. Due to lncRNA-AWPPH locates both in the nucleus and cytoplasm, we next investigated whether the nuclear lncRNA-AWPPH regulates the effects of YBX1 on PIK3CA. ChIP assays with YBX1 specific antibody in lncRNA-AWPPH stably overexpressed SMMC-7721 cells and lncRNA-AWPPH stably depleted HCCLM3 cells were performed. As shown in Fig. 6A and B, YBX1 not only bound to lncRNA-AWPPH, but also bound to PIK3CA promoter in HCC cells. Furthermore, ectopic expression of lncRNA-AWPPH enhanced the binding of YBX1 protein to the PIK3CA

18

ACCEPTED MANUSCRIPT promoter (Fig. 6A). Conversely, depletion of lncRNA-AWPPH inhibited the binding of YBX1 protein to the PIK3CA promoter (Fig. 6B). qRT-PCR results showed that overexpression of lncRNA-AWPPH

IP

T

increased PIK3CA mRNA level, and while depletion of lncRNA-AWPPH decreased PIK3CA mRNA

SC R

level (Fig. 6C and D). Western blot analysis revealed that overexpression of lncRNA-AWPPH upregulated PI3K protein level and increased phosphorylation of AKT (Fig. 6E). Conversely, depletion of lncRNA-AWPPH decreased PI3K protein level and phosphorylation of AKT (Fig. 6F). To

NU

investigate whether the effects of lncRNA-AWPPH on PIK3CA transcription are dependent on YBX1,

MA

we measured PIK3CA mRNA level, PI3K protein level, and AKT phosphorylation level in lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells with YBX1 depletion. As shown

D

in Fig. 6G and H, depletion of YBX1 abolished the upregulation of PIK3CA mRNA level, PI3K protein

TE

level, and AKT phosphorylation level caused by lncRNA-AWPPH overexpression (compared with Fig.

CE P

6C and E). Collectively, these data demonstrate that lncRNA-AWPPH actives PIK3CA transcription and PI3K/AKT pathway via YBX1 in HCC cells.

AC

3.7. Depletion of YBX1 abolishes the effects of lncRNA-AWPPH on HCC cells growth and metastasis in vitro and in vivo

YBX1 is known to activate SNAIL1 translation and PIK3CA transcription, and further promote tumor growth and metastasis. In our above results, we have found that lncRNA-AWPPH also activated SNAIL1 translation and PIK3CA transcription via binding to YBX1. To further investigate whether the effects of lncRNA-AWPPH on HCC cells growth and metastasis are dependent on YBX1, we measured the proliferation of lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells with YBX1 depletion using Glo cell viability assays and EdU incorporation assays. As shown in Fig. 7A and B, depletion of YBX1 abolished the pro-proliferative effects of lncRNA-AWPPH on SMMC-7721 cells

19

ACCEPTED MANUSCRIPT (compared with Fig. 2B and C). Transwell assays and wound healing assays showed that depletion of YBX1 abolished the pro-migratory effects of lncRNA-AWPPH on SMMC-7721 cells (Fig. 7C and D,

IP

T

compared with Fig. 2G and H). Subcutaneous tumor growth assays showed that depletion of YBX1

SC R

abolished the pro-growth effects of lncRNA-AWPPH on subcutaneous xenograft (Fig. 7E-G, compared with Fig. 3A-C). Liver orthotopic tumor models showed that depletion of YBX1 abolished the pro-growth effects of lncRNA-AWPPH on liver orthotopic xenograft (Fig. 7H, compared with Fig. 3E).

NU

Tail veins injection models showed that depletion of YBX1 abolished the increasing of lung metastasis

MA

caused by lncRNA-AWPPH overexpression (Fig. 7I, compared with Fig. 3K). Collectively, these results showed that depletion of YBX1 abolishes the effects of lncRNA-AWPPH on HCC cells growth

AC

CE P

TE

D

and metastasis in vitro and in vivo.

20

ACCEPTED MANUSCRIPT 4. Discussion The disappointing outcomes of HCC patients are largely due to frequent recurrence and metastasis [33,

IP

T

34]. Reliable predicting the risk of recurrence and providing the optimal medical management would

SC R

have great benefits for improving HCC patients’ outcomes [35]. Unfortunately, the molecular mechanisms underlying the recurrence and metastasis are still largely unknown and so the molecular biomarkers defining the risk of recurrence are less effective [33, 36]. The aberrant expression of

NU

lncRNAs in tumors is one of the mainly observed molecular phenotypes of cancers [24, 37, 38]. To

MA

investigate whether the aberrant expressed lncRNAs could be biomarkers indicating recurrence and metastasis, we searched online-available data sets and identified a novel lncRNA termed

D

lncRNA-AWPPH, which is upregulated in HCC tissues and further upregulated in liver metastatic

TE

PVTT tissues. The increased expression of lncRNA-AWPPH is correlated with encapsulation

CE P

incomplete, microvascular invasion, advanced TNM stage and BCLC stage. Furthermore, Kaplan-Meier survival analysis and Cox proportional hazards regression analysis verified that

AC

lncRNA-AWPPH is not only correlated with poor recurrence-free survival and overall survival, but also an independent prognostic factor for recurrence-free survival and overall survival. Our results demonstrate that lncRNA-AWPPH could serve as a prognostic biomarker for HCC. Some of the aberrantly expressed lncRNAs are potential diver events of tumor initiation and progression, whereas others may be just bystander events accompanying the tumor initiation and progression [39]. Thus, it is important to distinguish the driver events from the bystander events. In this study, to investigate whether lncRNA-AWPPH have a role in HCC, we performed in vitro and in vivo functional experiments. Glo cell viability and EdU incorporation assays verified that lncRNA-AWPPH promotes HCC cell proliferation. Wound healing assays and transwell assays verified that

21

ACCEPTED MANUSCRIPT lncRNA-AWPPH promotes HCC cell migration. Subcutaneous tumor growth assays and liver orthotopic xenograft assays verified that lncRNA-AWPPH promotes HCC tumor growth in vivo. Tail

SC R

demonstrate that lncRNA-AWPPH functions as an oncogene in HCC.

IP

T

veins injection assays showed that lncRNA-AWPPH promotes HCC lung metastasis. These results

Another evidence supporting the functional roles of lncRNA in cancers is the involvement of lncRNA in critical carcinogenic or metastatic signaling pathway [40, 41]. Elucidating the exact action

NU

mechanisms of lncRNA is also the most difficult aspects in lncRNA research. Combining RNA

MA

pull-down assays and mass spectrometry analysis, we identified YBX1 as a specific lncRNA-AWPPH interacting protein. YBX1 is a highly conserved and multifunctional protein, which binds both DNA

D

and RNA, participates in transcription regulation, RNA processing and translation regulation, and plays

TE

pro-oncogenic roles in tumor progression, metastasis, and drug resistance in various cancers [42-46].

CE P

YBX1 has been reported to directly activate SNAIL1 mRNA translation, induce epithelial-mesenchymal transition, and enhance metastatic potential of breast cancer cells [31]. YBX1

AC

has also been reported to bind to the promoter of PIK3CA, stimulate its transcription, and activate PI3K/AKT pathway in breast cancer and colon cancer cells [30, 32]. In the present study, we found that through interacting with YBX1, lncRNA-AWPPH enhances the binding between YBX1 and SNAIL1 mRNA, upregulates the proportion of SNAIL1 mRNA in polysomal fractions, and promotes SNAIL1 mRNA translation in HCC cells. Through interacting with YBX1, lncRNA-AWPPH also enhances the binding between YBX1 and PIK3CA promoter, promotes PIK3CA transcription, and activates PI3K/AKT pathway in HCC cells. The effects of lncRNA-AWPPH on SNAIL1 and PIK3CA are dependent on YBX1 as depletion of YBX1 abolished these effects of lncRNA-AWPPH. The interaction between lncRNA-AWPPH and YBX1 did not change YBX1 protein level, but changed the function of

22

ACCEPTED MANUSCRIPT YBX1 protein. These data implied that the specific binding between lncRNA-AWPPH and YBX1 may changed the structure of YBX1, and then changed the affinity of YBX1 to other DNAs and RNAs,

IP

T

which need further investigation. Furthermore, the biological roles of lncRNA-AWPPH on HCC cell

SC R

proliferation, migration, and in vivo tumor growth and metastasis are also dependent on YBX1, as depletion of YBX1 abolished the pro-oncogenic roles of lncRNA-AWPPH. These results demonstrate that the specific binding between lncRNA-AWPPH and YBX1 could enhance the effects of YBX1 on

NU

SNAIL1 mRNA translation and PIK3CA transcription, and also the oncogenic roles of YBX1 in HCC.

MA

Whether other reported targets of YBX1 could also be regulated by lncRNA-AWPPH need further investigation. Anyway, our results confirmed that the important roles of lncRNA-AWPPH in HCC are

D

dependent on YBX1.

TE

In conclusion, we identify a novel lncRNA lncRNA-AWPPH which is highly expressed in HCC,

CE P

indicates poor prognosis of HCC patients, and promotes HCC cell proliferation, migration, and in vivo tumor growth and metastasis via binding to YBX1. Our data suggest that lncRNA-AWPPH would be a

AC

prognostic biomarker and a potential therapeutic target for HCC.

23

ACCEPTED MANUSCRIPT Abbreviations lncRNA: long noncoding RNA; HCC: hepatocellular carcinoma; PVTT: portal vein tumor thrombus;

IP

T

BCLC: Barcelona Clinic Liver Cancer; qRT-PCR: quantitative real-time PCR; cDNA: complementary

SC R

DNA; RACE: rapid amplification of cDNA ends; EdU: ethynyl deoxyuridine; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RIP: RNA immunoprecipitation; ChIP: chromatin immunoprecipitation; lncRNA-AWPPH: lncRNA associated with poor prognosis of HCC; YBX1:

MA

NU

Y-box binding protein 1.

Conflict of interest

CE P

Authors' contributions

TE

D

The authors declare that they have no competing interests.

SY and XZ conceived and designed the experiments. XZ and YL performed the experiments. SY, XZ

AC

and YL analyzed the data and wrote the paper. All authors read and approved the final manuscript.

Authors' information 1

Department of Oncological Surgery, The Second Hospital of Hebei Medical University, No. 215

Peace West Road, Shijiazhuang 050000, China. 2The First Department of Surgery, Feixiang Central Hospital, Handan 057550, China.

24

ACCEPTED MANUSCRIPT References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, 2012, CA: a cancer journal for clinicians, 65 (2015) 87-108.

T

[2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA: a cancer journal for clinicians, 66 [3] L. Laursen, A preventable cancer, Nature, 516 (2014) S2-3.

IP

(2016) 7-30.

[4] R.L. Siegel, K.D. Miller, A. Jemal, Cancer Statistics, 2017, CA: a cancer journal for clinicians, 67

SC R

(2017) 7-30.

[5] F. Ahmed, J.F. Perz, S. Kwong, P.M. Jamison, C. Friedman, B.P. Bell, National trends and disparities in the incidence of hepatocellular carcinoma, 1998-2003, Preventing chronic disease, 5 (2008) A74.

NU

[6] V. Brower, Sorafenib plus cisplatin for hepatocellular carcinoma, The Lancet. Oncology, 17 (2016) e424.

[7] A. Forner, J.M. Llovet, J. Bruix, Hepatocellular carcinoma, Lancet, 379 (2012) 1245-1255.

MA

[8] Y.F. Zhang, M. Shi, R.P. Guo, A novel risk score (MCCT) to guide decision for treatment of advanced hepatocellular carcinoma with transarterial chemoembolisation: a derivation and validation study, Lancet, 388 Suppl 1 (2016) S35.

[9] K.J. Yong, C. Gao, J.S. Lim, B. Yan, H. Yang, T. Dimitrov, A. Kawasaki, C.W. Ong, K.F. Wong, S.

D

Lee, S. Ravikumar, S. Srivastava, X. Tian, R.T. Poon, S.T. Fan, J.M. Luk, Y.Y. Dan, M. Salto-Tellez, L.

TE

Chai, D.G. Tenen, Oncofetal gene SALL4 in aggressive hepatocellular carcinoma, The New England journal of medicine, 368 (2013) 2266-2276. [10] M. Sherman, Recurrence of hepatocellular carcinoma, The New England journal of medicine, 359

CE P

(2008) 2045-2047.

[11] C.P. Ponting, P.L. Oliver, W. Reik, Evolution and functions of long noncoding RNAs, Cell, 136 (2009) 629-641.

[12] T.R. Mercer, M.E. Dinger, J.S. Mattick, Long non-coding RNAs: insights into functions, Nature

AC

reviews. Genetics, 10 (2009) 155-159. [13] X. Liu, Z.D. Xiao, L. Han, J. Zhang, S.W. Lee, W. Wang, H. Lee, L. Zhuang, J. Chen, H.K. Lin, J. Wang, H. Liang, B. Gan, LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress, Nature cell biology, 18 (2016) 431-442. [14] J.T. Kung, D. Colognori, J.T. Lee, Long noncoding RNAs: past, present, and future, Genetics, 193 (2013) 651-669. [15] T. Hirano, R. Yoshikawa, H. Harada, Y. Harada, A. Ishida, T. Yamazaki, Long noncoding RNA, CCDC26, controls myeloid leukemia cell growth through regulation of KIT expression, Molecular cancer, 14 (2015) 90. [16] E. Raveh, I.J. Matouk, M. Gilon, A. Hochberg, The H19 Long non-coding RNA in cancer initiation, progression and metastasis - a proposed unifying theory, Molecular cancer, 14 (2015) 184. [17] F. Chen, J. Mo, L. Zhang, Long noncoding RNA BCAR4 promotes osteosarcoma progression through activating GLI2-dependent gene transcription, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 37 (2016) 13403-13412. [18] X.T. Zhu, J.H. Yuan, T.T. Zhu, Y.Y. Li, X.Y. Cheng, Long noncoding RNA glypican 3 (GPC3) antisense transcript 1 promotes hepatocellular carcinoma progression via epigenetically activating GPC3, The FEBS journal, 283 (2016) 3739-3754.

25

ACCEPTED MANUSCRIPT [19] L.J. Ding, Y. Li, S.D. Wang, X.S. Wang, F. Fang, W.Y. Wang, P. Lv, D.H. Zhao, F. Wei, L. Qi, Long Noncoding RNA lncCAMTA1 Promotes Proliferation and Cancer Stem Cell-Like Properties of Liver Cancer by Inhibiting CAMTA1, International journal of molecular sciences, 17 (2016). [20] C. Liu, J. Lin, Long noncoding RNA ZEB1-AS1 acts as an oncogene in osteosarcoma by

T

epigenetically activating ZEB1, American journal of translational research, 8 (2016) 4095-4105. [21] A. Lin, C. Li, Z. Xing, Q. Hu, K. Liang, L. Han, C. Wang, D.H. Hawke, S. Wang, Y. Zhang, Y. Wei,

IP

G. Ma, P.K. Park, J. Zhou, Y. Zhou, Z. Hu, Y. Zhou, J.R. Marks, H. Liang, M.C. Hung, C. Lin, L. Yang, The LINK-A lncRNA activates normoxic HIF1alpha signalling in triple-negative breast cancer, Nature

SC R

cell biology, 18 (2016) 213-224.

[22] F. Yang, L. Zhang, X.S. Huo, J.H. Yuan, D. Xu, S.X. Yuan, N. Zhu, W.P. Zhou, G.S. Yang, Y.Z. Wang, J.L. Shang, C.F. Gao, F.R. Zhang, F. Wang, S.H. Sun, Long noncoding RNA high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans,

NU

Hepatology, 54 (2011) 1679-1689.

[23] J.H. Yuan, F. Yang, F. Wang, J.Z. Ma, Y.J. Guo, Q.F. Tao, F. Liu, W. Pan, T.T. Wang, C.C. Zhou, S.B. Wang, Y.Z. Wang, Y. Yang, N. Yang, W.P. Zhou, G.S. Yang, S.H. Sun, A long noncoding RNA

MA

activated by TGF-beta promotes the invasion-metastasis cascade in hepatocellular carcinoma, Cancer cell, 25 (2014) 666-681.

[24] S.X. Yuan, J. Wang, F. Yang, Q.F. Tao, J. Zhang, L.L. Wang, Y. Yang, H. Liu, Z.G. Wang, Q.G. Xu, J. Fan, L. Liu, S.H. Sun, W.P. Zhou, Long noncoding RNA DANCR increases stemness features of

D

hepatocellular carcinoma by derepression of CTNNB1, Hepatology, 63 (2016) 499-511.

TE

[25] C.J. Sui, Y.M. Zhou, W.F. Shen, B.H. Dai, J.J. Lu, M.F. Zhang, J.M. Yang, Long noncoding RNA GIHCG promotes hepatocellular carcinoma progression through epigenetically regulating miR-200b/a/429, Journal of molecular medicine, 94 (2016) 1281-1296.

CE P

[26] M.K. Iyer, Y.S. Niknafs, R. Malik, U. Singhal, A. Sahu, Y. Hosono, T.R. Barrette, J.R. Prensner, J.R. Evans, S. Zhao, A. Poliakov, X. Cao, S.M. Dhanasekaran, Y.M. Wu, D.R. Robinson, D.G. Beer, F.Y. Feng, H.K. Iyer, A.M. Chinnaiyan, The landscape of long noncoding RNAs in the human transcriptome, Nature genetics, 47 (2015) 199-208.

AC

[27] A.M. El-Naggar, C.J. Veinotte, H. Cheng, T.G. Grunewald, G.L. Negri, S.P. Somasekharan, D.P. Corkery, F. Tirode, J. Mathers, D. Khan, A.H. Kyle, J.H. Baker, N.E. LePard, S. McKinney, S. Hajee, M. Bosiljcic, G. Leprivier, C.E. Tognon, A.I. Minchinton, K.L. Bennewith, O. Delattre, Y. Wang, G. Dellaire, J.N. Berman, P.H. Sorensen, Translational Activation of HIF1alpha by YB-1 Promotes Sarcoma Metastasis, Cancer cell, 27 (2015) 682-697. [28] R. Kogo, T. Shimamura, K. Mimori, K. Kawahara, S. Imoto, T. Sudo, F. Tanaka, K. Shibata, A. Suzuki, S. Komune, S. Miyano, M. Mori, Long noncoding RNA HOTAIR regulates polycomb-dependent chromatin modification and is associated with poor prognosis in colorectal cancers, Cancer research, 71 (2011) 6320-6326. [29] S. Zhang, G. Zhang, J. Liu, Long noncoding RNA PVT1 promotes cervical cancer progression through epigenetically silencing miR-200b, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, 124 (2016) 649-658. [30] A. Diaz-Lagares, A.B. Crujeiras, P. Lopez-Serra, M. Soler, F. Setien, A. Goyal, J. Sandoval, Y. Hashimoto, A. Martinez-Cardus, A. Gomez, H. Heyn, C. Moutinho, J. Espada, A. Vidal, M. Paules, M. Galan, N. Sala, Y. Akiyama, M. Martinez-Iniesta, L. Farre, A. Villanueva, M. Gross, S. Diederichs, S. Guil, M. Esteller, Epigenetic inactivation of the p53-induced long noncoding RNA TP53 target 1 in human cancer, Proceedings of the National Academy of Sciences of the United States of America, 113

26

ACCEPTED MANUSCRIPT (2016) E7535-E7544. [31] V. Evdokimova, C. Tognon, T. Ng, P. Ruzanov, N. Melnyk, D. Fink, A. Sorokin, L.P. Ovchinnikov, E. Davicioni, T.J. Triche, P.H. Sorensen, Translational activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes an epithelial-mesenchymal transition, Cancer cell, 15

T

(2009) 402-415. [32] A. Astanehe, M.R. Finkbeiner, P. Hojabrpour, K. To, A. Fotovati, A. Shadeo, A.L. Stratford, W.L.

IP

Lam, I.M. Berquin, V. Duronio, S.E. Dunn, The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1, Oncogene, 28 (2009) 2406-2418.

SC R

[33] Q.H. Ye, W.W. Zhu, J.B. Zhang, Y. Qin, M. Lu, G.L. Lin, L. Guo, B. Zhang, Z.H. Lin, S. Roessler, M. Forgues, H.L. Jia, L. Lu, X.F. Zhang, B.F. Lian, L. Xie, Q.Z. Dong, Z.Y. Tang, X.W. Wang, L.X. Qin, GOLM1 Modulates EGFR/RTK Cell-Surface Recycling to Drive Hepatocellular Carcinoma Metastasis, Cancer cell, 30 (2016) 444-458.

NU

[34] J. Ding, S. Huang, S. Wu, Y. Zhao, L. Liang, M. Yan, C. Ge, J. Yao, T. Chen, D. Wan, H. Wang, J. Gu, M. Yao, J. Li, H. Tu, X. He, Gain of miR-151 on chromosome 8q24.3 facilitates tumour cell migration and spreading through downregulating RhoGDIA, Nature cell biology, 12 (2010) 390-399.

MA

[35] A. Budhu, M. Forgues, Q.H. Ye, H.L. Jia, P. He, K.A. Zanetti, U.S. Kammula, Y. Chen, L.X. Qin, Z.Y. Tang, X.W. Wang, Prediction of venous metastases, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment, Cancer cell, 10 (2006) 99-111.

D

[36] X. Zhao, T. Wang, B. Liu, Z. Wu, S. Yu, T. Wang, Significant association between upstream

TE

transcription factor 1 rs2516839 polymorphism and hepatocellular carcinoma risk: a case-control study, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 36 (2015) 2551-2558.

CE P

[37] C.R. Goding, Targeting the lncRNA SAMMSON Reveals Metabolic Vulnerability in Melanoma, Cancer cell, 29 (2016) 619-621.

[38] G.K. Pandey, S. Mitra, S. Subhash, F. Hertwig, M. Kanduri, K. Mishra, S. Fransson, A. Ganeshram, T. Mondal, S. Bandaru, M. Ostensson, L.M. Akyurek, J. Abrahamsson, S. Pfeifer, E.

AC

Larsson, L. Shi, Z. Peng, M. Fischer, T. Martinsson, F. Hedborg, P. Kogner, C. Kanduri, The risk-associated long noncoding RNA NBAT-1 controls neuroblastoma progression by regulating cell proliferation and neuronal differentiation, Cancer cell, 26 (2014) 722-737. [39] Z.Z. Chen, L. Huang, Y.H. Wu, W.J. Zhai, P.P. Zhu, Y.F. Gao, LncSox4 promotes the self-renewal of liver tumour-initiating cells through Stat3-mediated Sox4 expression, Nature communications, 7 (2016) 12598. [40] A.R. Bassett, A. Akhtar, D.P. Barlow, A.P. Bird, N. Brockdorff, D. Duboule, A. Ephrussi, A.C. Ferguson-Smith, T.R. Gingeras, W. Haerty, D.R. Higgs, E.A. Miska, C.P. Ponting, Considerations when investigating lncRNA function in vivo, eLife, 3 (2014) e03058. [41] R.A. Gupta, N. Shah, K.C. Wang, J. Kim, H.M. Horlings, D.J. Wong, M.C. Tsai, T. Hung, P. Argani, J.L. Rinn, Y. Wang, P. Brzoska, B. Kong, R. Li, R.B. West, M.J. van de Vijver, S. Sukumar, H.Y. Chang, Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis, Nature, 464 (2010) 1071-1076. [42] D. Li, X. Liu, J. Zhou, J. Hu, D. Zhang, J. Liu, Y. Qiao, Q. Zhan, LncRNA HULC modulates the phosphorylation of YB-1 through serving as a scaffold of ERK and YB-1 to enhance hepatocarcinogenesis, Hepatology, (2016). [43] J. Zheng, W. Dong, J. Zhang, G. Li, H. Gong, YB-1, a new biomarker of glioma progression, is

27

ACCEPTED MANUSCRIPT associated with the prognosis of glioma patients, Acta biochimica et biophysica Sinica, 48 (2016) 318-325. [44] M. Shiota, N. Fujimoto, K. Imada, A. Yokomizo, M. Itsumi, A. Takeuchi, H. Kuruma, J. Inokuchi, K. Tatsugami, T. Uchiumi, Y. Oda, S. Naito, Potential Role for YB-1 in Castration-Resistant Prostate

T

Cancer and Resistance to Enzalutamide Through the Androgen Receptor V7, Journal of the National Cancer Institute, 108 (2016).

IP

[45] C. Pagano, O. di Martino, G. Ruggiero, A.M. Guarino, N. Mueller, R. Siauciunaite, M. Reischl, N.S. Foulkes, D. Vallone, V. Calabro, The tumor-associated YB-1 protein: new player in the circadian

SC R

control of cell proliferation, Oncotarget, (2016).

[46] S.P. Tsofack, C. Garand, C. Sereduk, D. Chow, M. Aziz, D. Guay, H.H. Yin, M. Lebel, NONO and RALY proteins are required for YB-1 oxaliplatin induced resistance in colon adenocarcinoma cell lines,

AC

CE P

TE

D

MA

NU

Molecular cancer, 10 (2011) 145.

28

ACCEPTED MANUSCRIPT Figure legends Fig. 1. lncRNA-AWPPH expression level in clinical tissues samples and its correlation with HCC

IP

T

patients’ prognosis. (A) lncRNA NR_024373 expression intensities in 23 HCC tissues and 30

SC R

noncancerous hepatic tissues from GSE54238. Data are shown as median with interquartile range. P < 0.0001 by Mann-Whitney test. (B and C) Kaplan–Meier survival estimates of the correlations between lncRNA-AWPPH expression and disease-free (B) and overall survival (C) of 59 HCC patients from

NU

GSE40144. The median expression level was used as the cut-off. P = 0.0038 by Log-rank test for (B).

MA

P = 0.0058 by Log-rank test for (C). (D) lncRNA-AWPPH expression level in 88 pairs of HCC tissues and adjacent noncancerous hepatic tissues, and 20 PVTT tissues. Data are shown as median with

D

interquartile range. For comparison of noncancerous tissues and HCC tissues, P < 0.0001 by Wilcoxon

TE

signed-rank test. For comparison of HCC tissues and PVTT tissues, P < 0.0001 by Mann-Whitney test.

CE P

(E and F) Kaplan–Meier survival estimates of the correlations between lncRNA-AWPPH expression and recurrence-free (E) and overall survival (F) of 88 HCC patients from (D). The median expression

AC

level was used as the cut-off. P = 0.0066 by Log-rank test for (E). P = 0.0035 by Log-rank test for (F). Fig. 2 The effects of lncRNA-AWPPH on HCC cell proliferation and migration in vitro. (A) lncRNA-AWPPH expression level in lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells. (B) The effects of lncRNA-AWPPH overexpression on SMMC-7721 cell proliferation were determined by Glo cell viability assays, and the relative cell viability to 0 hour is shown. (C) The effects of lncRNA-AWPPH overexpression on SMMC-7721 cell proliferation were determined by EdU incorporation assays. Representative images are shown. Scale bars = 200 µm. (D) lncRNA-AWPPH expression level in lncRNA-AWPPH stably depleted and control HCCLM3 cells. (E) The effects of lncRNA-AWPPH depletion on HCCLM3 cell proliferation were determined by Glo cell viability

29

ACCEPTED MANUSCRIPT assays, and the relative cell viability to 0 hour is shown. (F) The effects of lncRNA-AWPPH depletion on HCCLM3 cell proliferation were determined by EdU incorporation assays. Representative images

IP

T

are shown. Scale bars = 200 µm. (G) The effects of lncRNA-AWPPH overexpression on SMMC-7721

SC R

cell migration were determined by wound healing assays. Representative images are shown. (H) The effects of lncRNA-AWPPH overexpression on SMMC-7721 cell migration were determined by transwell assays. Representative images are shown. Scale bars = 100 µm. (I) The effects of

NU

lncRNA-AWPPH depletion on HCCLM3 cell migration were determined by wound healing assays.

MA

Representative images are shown. (J) The effects of lncRNA-AWPPH depletion on HCCLM3 cell migration were determined by transwell assays. Representative images are shown. Scale bars = 100 µm.

TE

***P < 0.001 by Student’s t test.

D

Results are shown as mean ± SD based on at least three independent biological repeats. **P < 0.01,

CE P

Fig. 3 The effects of lncRNA-AWPPH on HCC tumor growth and metastasis in vivo. (A) lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells were injected subcutaneously

AC

into nude mice. Subcutaneous tumor volumes measured every 7 days were shown. (B) Subcutaneous tumor weights were measured at 21th day after injection with indicated SMMC-7721 cells. (C) Luciferase signal intensities of subcutaneous tumors at 21th day after injection with indicated SMMC-7721 cell. (D) Ki67 immunohistochemistry staining of tumors derived from (C). Representative images are shown. Scale bars = 50 µm. (E) Luciferase signal intensities of hepatic orthotopic xenograft at 21th day after inoculation with tumors derived from lncRNA-AWPPH stably overexpressed or control SMMC-7721 cells. (F) LncRNA-AWPPH stably depleted and control HCCLM3 cells were injected subcutaneously into nude mice. Subcutaneous tumor volumes measured every 7 days were shown. (G) Subcutaneous tumor weights were measured at 28th day after injection

30

ACCEPTED MANUSCRIPT with indicated HCCLM3 cells. (H) Luciferase signal intensities of subcutaneous tumors at 28th day after injection with indicated HCCLM3 cell. (I) Ki67 immunohistochemistry staining of tumors derived

IP

T

from (H). Representative images are shown. Scale bars = 50 µm. (J) Luciferase signal intensities of

SC R

hepatic orthotopic xenograft at 28th day after inoculation with tumors derived from lncRNA-AWPPH stably depleted or control HCCLM3 cells. (K) Luciferase signal intensities of lung metastases at 28th day after tail veins injection with lncRNA-AWPPH stably overexpressed or control SMMC-7721 cells.

NU

(L) Luciferase signal intensities of lung metastases at 35th day after tail veins injection with

MA

lncRNA-AWPPH stably depleted or control HCCLM3 cells. For all panels, n = 6 mice in each group. Results are shown as mean ± SD.**P < 0.01 by Mann-Whitney test.

D

Fig.4 lncRNA-AWPPH binds to the YBX1 protein. (A) SDS-PAGE of proteins from SMMC-7721

TE

cells pulled down by lncRNA-AWPPH or its antisense RNA. The arrow indicates the band specific to

CE P

lncRNA-AWPPH excised for mass spectrometry. (B) Proteins pulled down by lncRNA-AWPPH or its antisense RNA were detected by western blot withYBX1, ANXA1, or PGK1 specific antibody. (C) RIP

AC

assays were carried out in SMMC-7721 cells using an YBX1 specific antibody or IgG to retrieve endogenous RNAs bound toYBX1. Enriched RNAs were measured by qRT-PCR and accounted as percentage of input. Results are shown as mean ± SD based on three independent biological repeats. *P < 0.05, **P < 0.01 by Student’s t test. (D) The protein levels of YBX1 in lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells. (E) The protein levels of YBX1 in lncRNA-AWPPH stably depleted and control HCCLM3 cells. Representative images are shown for all western blot analyses. Fig. 5 lncRNA-AWPPH accelerates SNAIL1 translation via YBX1. (A and B) RIP assays were carried out in lncRNA-AWPPH stably overexpressed and control SMMC-7721cells (A) or lncRNA-AWPPH

31

ACCEPTED MANUSCRIPT stably depleted and control HCCLM3 cells (B) using an YBX1 specific antibody or IgG to retrieve endogenous RNAs bound toYBX1. The retrieved RNAs were measured by qRT-PCR with SNAIL1

IP

T

mRNA or lncRNA-AWPPH specific primers. Enrichment was accounted as percentage of input. (C and

SC R

D) The total or polysomal fractionated SNAIL1 mRNAs in lncRNA-AWPPH stably overexpressed and control SMMC-7721cells (C) or lncRNA-AWPPH stably depleted and control HCCLM3 cells (D) were measured by qRT-PCR. (E and F) SNAIL1 protein levels in lncRNA-AWPPH stably overexpressed and

NU

control SMMC-7721cells (E) or lncRNA-AWPPH stably depleted and control HCCLM3 cells (F) were

MA

measured by western blot. (G) YBX1 protein levels in YBX1 stably depleted and control SMMC-7721 cells were measured by western blot. (H) LncRNA-AWPPH expression levels in YBX1 stably depleted

D

and control SMMC-7721 cells were measured by qRT-PCR. (I) The total or polysomal fractionated

TE

SNAIL1 mRNAs in lncRNA-AWPPH stably overexpressed and control SMMC-7721cells with stable

CE P

YBX1 depletion were measured by qRT-PCR. (J) SNAIL1protein levels in lncRNA-AWPPH stably overexpressed and control SMMC-7721cells with stable YBX1 depletion were measured by western

AC

blot. Results are shown as mean ± SD based on three independent biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test. Fig. 6 lncRNA-AWPPH activates PI3K/AKT pathway via YBX1. (A and B) ChIP and RIP assays were carried out in lncRNA-AWPPH stably overexpressed and control SMMC-7721cells (A) or lncRNA-AWPPH stably depleted and control HCCLM3 cells (B) using an YBX1 specific antibody or IgG to retrieve endogenous DNAs or RNAs bound toYBX1. The retrieved DNAs were measured by qPCR with primers specific to PIK3CA promoter. The retrieved RNAs were measured by qRT-PCR with lncRNA-AWPPH specific primers. Enrichment was accounted as percentage of input. (C and D) PIK3CA mRNA levels in lncRNA-AWPPH stably overexpressed and control SMMC-7721cells (C) or

32

ACCEPTED MANUSCRIPT lncRNA-AWPPH stably depleted and control HCCLM3 cells (D) were measured by qRT-PCR. (E and F) PI3K protein levels and AKT phosphorylation levels in lncRNA-AWPPH stably overexpressed and

IP

T

control SMMC-7721cells (E) or lncRNA-AWPPH stably depleted and control HCCLM3 cells (F) were

SC R

measured by western blot. (G) PIK3CA mRNA levels in lncRNA-AWPPH stably overexpressed and control SMMC-7721cells with stable YBX1 depletion were measured by qRT-PCR. (H) PI3K protein levels and AKT phosphorylation levels in lncRNA-AWPPH stably overexpressed and control

NU

SMMC-7721 cells with stable YBX1 depletion were measured by western blot. Results are shown as

MA

mean ± SD based on three independent biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

D

Fig. 7 Depletion of YBX1 abolishes the effects of lncRNA-AWPPH on HCC cells growth and

TE

metastasis. (A) The proliferation of lncRNA-AWPPH stably overexpressed and control SMMC-7721

CE P

cells with stable YBX1 depletion was detected by Glo cell viability assays, and the relative cell viability to 0 hour is shown. (B) The proliferation of lncRNA-AWPPH stably overexpressed and

AC

control SMMC-7721cells with stable YBX1 depletion was detected by EdU incorporation assays. Representative images are shown. Scale bars = 200 µm. (C) The migration of lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells with stable YBX1 depletion was detected by transwell assays. Representative images are shown. Scale bars = 100 µm. (D) The migration of lncRNA-AWPPH stably overexpressed and control SMMC-7721cells with stable YBX1 depletion was detected by wound healing assays. Representative images are shown. For (A-D), results are shown as mean ± SD based on three independent biological repeats. P > 0.05 by Student’s t test. (E) lncRNA-AWPPH stably overexpressed and control SMMC-7721 cells with stable YBX1 depletion were injected subcutaneously into nude mice. Subcutaneous tumor volumes measured every 7 days were shown. (F)

33

ACCEPTED MANUSCRIPT Subcutaneous tumor weights were measured at 28th day after injection with indicated SMMC-7721 cells. (G) Luciferase signal intensities of subcutaneous tumors at 28th day after injection with indicated

IP

T

SMMC-7721 cell. (H) Luciferase signal intensities of hepatic orthotopic xenograft at 28th day after

SC R

inoculation with tumors derived from lncRNA-AWPPH stably overexpressed or control SMMC-7721 cells with stable YBX1 depletion. (I) Luciferase signal intensities of lung metastases at 35th day after tail veins injection with lncRNA-AWPPH stably overexpressed or control SMMC-7721 cells with

NU

stable YBX1 depletion. For (E-I), n = 6 mice in each group. Results are shown as mean ± SD. P > 0.05

AC

CE P

TE

D

MA

by Mann-Whitney test.

34

ACCEPTED MANUSCRIPT Table 1

lncRNA-AWPPHa P-valueb High

44

44

Gender

0.502

40

38

Female

4

6

Age 22

≤50

22

D

AFP (µg/l)

CE P

HBsAg

TE

>20

23

26

31

18

13

35

35

Negative

9

9

Liver cirrhosis

AC

0.265

1.000

Positive

0.632

With

31

33

Without

13

11

Tumor size (cm)

0.667

≥5

24

26

<5

20

18

Tumor number

0.437

Multiple

8

11

Single

36

33

Pathological satellite Present

0.831

21

MA

>50

NU

Male

≤20

SC R

All cases

Low

IP

Variable

T

Correlations between lncRNA-AWPPH expression and clinicopathological characteristics

0.118 12

19

35

ACCEPTED MANUSCRIPT Absent

32

25

Encapsulation 23

13

No

21

31

IP

Complete

T

0.030

Microvascular invasion Present

18

28

Absent

26

16

Differentiation

0.334

7

III-IV

37

I

20

11

24

33

D

II-III

TE

BCLC stage

0.045

0.033

17

8

27

36

CE P

B-C

40

MA

TNM stage

4

NU

II

A

SC R

0.033

Median expression level of AWPPH was used as the cutoff.

b

P-value was acquired by Pearson chi-square test.

AC

a

AFP, alpha-fetoprotein; HBsAg, hepatitis B surface antigen.

36

ACCEPTED MANUSCRIPT Table 2 Multivariate analysis of several variables for RFS and OS

IP

Hazard ratio (95%

Variable

T

Recurrence-free survival

Overall Survival

Hazard ratio (95%

CI) 2.579 (1.425-4.668)

Gender (male vs. female)

1.008 (0.391-2.599)

Age (>50 vs. ≤50 years)

0.705 (0.360-1.383)

AFP (>20 vs. ≤20 µg/l)

P-value

CI)

0.002

3.509 (1.574-7.820)

0.002

0.986

0.867 (0.311-2.416)

0.784

0.310

0.661 (0.295-1.478)

0.313

1.240 (0.589-2.611)

0.570

2.698 (0.909-8.009)

0.074

HBsAg (positive vs. negative)

1.751 (0.765-4.007)

0.185

1.817 (0.618-5.341)

0.277

Liver cirrhosis (with vs. without)

1.524 (0.683-3.400)

0.303

3.595 (1.182-10.931)

0.024

Tumor size (≥5 cm versus <5 cm)

3.955 (1.789-8.742)

0.001

9.034 (2.937-27.785)

0.000

0.880

0.439 (0.137-1.414)

0.168

2.601 (1.075-6.290)

0.034

3.604 (1.259-10.317)

0.017

1.375 (0.671-2.821)

0.384

1.746 (0.686-4.442)

0.242

1.543 (0.593-4.017)

0.374

1.234 (0.390-3.900)

0.721

Differentiation (III-IV vs. II)

1.091 (0.341-3.485)

0.883

1.823 (0.347-9.559)

0.478

TNM stage (I vs. II-III)

0.500 (0.158-1.581)

0.238

0.548 (0.127-2.366)

0.421

BCLC stage (A vs. B-C)

0.800 (0.330-1.942)

0.622

1.099 (0.329-3.673)

0.878

MA

D

Tumor number (multiple vs.

1.074 (0.423-2.729)

TE

single)

NU

lncRNA-AWPPH (high vs. low)

SC R

P-value

Pathological satellite (positive vs.

CE P

negative)

Encapsulation (no vs. complete)

Microvascular invasion (positive

AC

vs. negative)

P-value was acquired by Cox proportional hazards regression. CI, confidence interval

37

ACCEPTED MANUSCRIPT Table 3 Mass spectrometry analysis of the proteins pulled down by lncRNA-AWPPH in HCCLM3 cells

No. of

(Da)

peptide

1

35902.68

9

HUMAN Nuclease-sensitive element-binding protein 1 (YBX1)

2

38918.07

2

HUMAN Annexin A1 (ANXA1)

3

44985.28

2

HUMAN Phosphoglycerate kinase 1 (PGK1)

4

22336.31

2

HUMAN Histone H1.3 (HIST1H1D)

5

23715.31

1

HUMAN Non-POU domain-containing, octamer-binding protein (NONO)

6

38692.23

1

HUMAN Docking protein 6 (DOK6)

T

Protein mass

Sequence header

AC

CE P

TE

D

MA

NU

SC R

IP

Hits

38

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Fig. 1

39

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 2

40

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Fig. 3

41

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Fig. 4

42

AC

Fig. 5

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

43

AC

Fig. 6

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

44

Fig. 7

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

45

ACCEPTED MANUSCRIPT Highlights A novel lncRNA termed lncRNA-AWPPH is highly expressed in HCC tissues.

IP

T

High lncRNA-AWPPH expression is an independent prognostic factor for poor recurrence-free and

SC R

overall survival.

lncRNA-AWPPH promotes HCC cell proliferation and migration in vitro, and tumor growth and metastasisin vivo.

NU

lncRNA-AWPPH interacts with YBX1, promotes YBX1-mediated activation of SNAIL1 translation,

MA

and upregulates SNAIL1 expression.

lncRNA-AWPPH promotes YBX1-mediated activation of PIK3CA transcription, upregulates PIK3CA

AC

CE P

TE

D

expression, and activates PI3K/AKT pathway.

46