cbm-prepress-cbm--1--1-cbm567-cbm--1-cbm567

Cancer Biomarkers -1(2015)1–101

DOI 10.3233/CBM-150567IOS Press

Long non-coding RNA HNF1A-AS is

upregulated and promotes cell proliferation and metastasis in nasopharyngeal carcinoma

Kun Zhuang a ,b ,1,Qiang Wu b ,1,Chun-Shun Jin a ,?,Hui-Jun Yuan b and Jin-Zhang Cheng a

Department of Otolaryngology,Head and Neck Surgery,the Second Hospital of Jilin University,Changchun,Jilin,China b

Department of Otolaryngology,Head and Neck Surgery,the First Af?liated Hospital of Harbin Medical University,Harbin,Heilongjiang,China

Abstract.

BACKGROUND:Nasopharyngeal carcinoma (NPC)is a common head and neck cancer with an incidence of 10–30cases per 100,000in southern China.Although primary treatment includes radiation therapy,prognosis is still unsatisfactory.OBJECTIVE:In this study,we examined the role of HNF1A-AS in NPC progression in vitro and in vivo .

METHODS:Relative levels of long non-coding RNA (LncRNA),HNF1A-AS,were evaluated in tumor tissues from 20patients with NPC as well as from cultured NPC cell lines.Lentivirus-mediated HNF1A-AS knockdown was conducted in NPC cell lines,CNE-2and SUNE-1.Cell migration and invasion abilities were estimated in vitro by colony-formation,wound-healing,and transwell assays.Cell cycle analysis was used to further examine the role of HNF1A-AS in cell proliferation.The tumor size of 24male mice with or without HNF1A-AS knockdown was monitored once a week.The underlying mechanism of HNF1A-AS-mediated cell proliferation was studied by western blot analysis.

RESULTS:Lentivirus-mediated HNF1A-AS knockdown suppressed cell proliferation and migration abilities.In mice injected with CNE-2and SUNE-1,depletion of HNF1A-AS caused inhibition of tumor growth,whereas cell cycle analysis showed that HNF1A-AS-knockdown resulted in cell accumulation in the G0/G1phase.Moreover,HNF1A-AS was found to be associated with epithelial to mesenchymal transition.

CONCLUSIONS:Overall,our results suggest that LncRNA,HNF1A-AS potentially regulates NPC tumorigenesis.This could help in development of new strategies for NPC diagnosis and treatment.

Keywords:Long non-coding RNA,HNF1A-AS,nasopharyngeal carcinoma,proliferation,metastasis,RNAi

1.Introduction

Nasopharyngeal carcinoma (NPC)is very common 2in certain well-de?ned populations such as Southeast 3Asia and the Middle East/North Africa,although it is 4relatively rare in other populations [1].Despite various

1Kun

Zhuang and Qiang Wu contributed equally to this work.

?Corresponding author:Chun-Shun Jin,Department of Otolaryn-gology,Head and Neck Surgery,the Second Hospital,Jilin Univer-sity,NO.218Ziqiang Street,Changchun,Jilin 130041,China.Tel.:+86043188796636;E-mail:JIDAJCS@https://www.360docs.net/doc/24218388.html,.

advances in chemoradiotherapy,no notable progress 6has been achieved in the 5-year survival rate of pa-7tients with NPC [2],mostly because NPC is a highly 8metastatic and invasion-prone form of epithelial can-9cer.It has also been shown to be related with sex and 10age distribution [3,4],racial/ethnic pattern [5],secular 11trends [6],diet [7],occupational exposures [8,9],and 12genetic variation [10,11].However,the speci?c mech-13anism underlying NPC tumorigenesis is far from clear.14Therefore,there is an urgent need to identify molecu-15lar biomarkers to establish novel strategies for targeted 16therapy against NPC.

ISSN 1574-0153/15/$35.00c

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Despite >90%of the eukaryotic genome being tran-18scribed,only 1%to 2%encodes proteins,which in-19dicates a large proportion of non-coding RNAs (ncR-20NAs)present in eukaryotes [12,13].These are cat-21egorized as housekeeping or regulatory (small and 22long)ncRNAs [14,15].Long ncRNAs (LncRNAs)are 23>200nucleotides in length,with generally none or 24very few open reading frames (ORFs).These function 25as a scaffold for the RNA-directed DNA methylation 26pathway [16,17].Advances in technology have helped 27identify thousands of LncRNAs and their activities.28For example,Wang et al.demonstrated that LncRNAs 29regulate transcription directly by various mechanisms 30such as interaction with RNA-binding proteins [18].31However,speci?c functions of LncRNAs have not 32been fully understood to date.

33The LncRNA,HNF1A-AS has recently begun to re-34ceive attention and has been reported to regulate cell 35proliferation and migration in human oesophageal ade-36nocarcinoma [19]and lung adenocarcinoma [20]cells.37In this study,we investigated the role of HNF1A-AS 38in NPC in vivo and in vitro .We found that HNF1A-39AS was overexpressed in both,clinical NPC tissues 40and cultured NPC cell lines.Furthermore,HNF1A-AS 41knockdown decreased tumor growth in a mouse model 42and inhibited cell migration by accumulating cells in 43G0/G1phase in CNE-2and SUNE-1cells.44

2.Materials and methods 45

2.1.Clinical NPC tissues

Tumor tissue and matched,adjacent non-cancerous 47tissues were obtained from 20patients with NPC who 48were admitted to the Second Hospital,Jilin University.49All patients provided informed consent.50

2.2.Cell culture

Cell lines including 5-8F,6-10B,HONE-1,CNE-522,SUNE-1,C666-1,and NP69were obtained from 53the American Type Culture Collection (ATCC;NY ,54USA)and cultured in Dulbecco’s modi?ed Eagle’s 55medium (DMEM;Gibco,Los Angeles,CA,USA)sup-56plemented with 10%fetal bovine serum (FBS;Gibco)57and antibiotics (penicillin and streptomycin)in a hu-58midi?ed incubator with 5%CO 2at 37?C.For im-59munoblotting and wound-healing assays,cells were 60seeded into 6-well plates.For cell-based functional as-61says,cells were cultured in 96-well plates.For migra-62tion and invasion analysis,cells were incubated in 24-63well plates.

2.3.Lentivirus-mediated HNF1A-AS knockdown 65

Lentivirus vector encoding increased green ?uores-66cent protein (GFP)cloned with DNA inserts express-67ing small hairpin RNA (shRNA)targeting the HNF1A-68AS sequence (5 -CACCTGCATTCAAACTCGGAC 69TGTT-3 )was used.Packaging,puri?cation,and de-70termination of HNF1A-AS knockdown viral titer were 71performed as previously described [21,22].Recombi-72nant lentivirus was generated from HEK 293T cells as 73per manufacturer instructions.

2.4.Quantitative reverse transcription polymerase

75chain reaction (qRT-PCR)76

For total RNA extraction,cells were washed twice 77with phosphate-buffered saline (PBS)and processed 78with Trizol reagent (TaKaRa,Dalian,China)as per the 79standard protocol.Subsequently,1μg RNA from each 80sample was reverse-transcribed into cDNA using the 81PrimeScript Real Time Master Mix (Perfect Real Time 82kit;TaKaRa),followed by real-time polymerase chain 83reaction (PCR)using the SYBR Premix Ex Taq Kit 84(TaKaRa)and the following primers:85HNF1A-AS:

5 -TCAAGAAATGGTGGCTAT-3 (forward)875 -GCTCTGAGACTGGCTGAA-3 (reverse);

Glyceraldehyde 3-phosphate dehydrogenase 89(GAPDH):

5 -GTGGACATCCGCAAAGAC-3 (forward)915 -AAAGGGTGTAACGCAACTA-3 (reverse).92

2.5.Immunoblotting analysis

Cells were lysed with a lysis buffer containing 94the protein extraction reagents RIPA (Beyotime,Nan-95tong,China),protease inhibitor cocktail,and phenyl-96methylsulfonyl?uoride.The concentration and quality 97of each protein sample were determined using protein 98assay kits (Bio-Rad,California,USA).Sodium dode-99cyl sulfate polyacrylamide gel electrophoresis (SDS-100PAGE)was performed with 40μg protein loaded into 101each well on 12%polyacrylamide gel.Proteins were 102transferred onto 0.22-μm nitrocellulose membranes 103(Bio-Rad)and incubated with speci?c antibodies.En-104hanced chemiluminescence substrate was used to vi-105sualize the bands and protein expression was quan-106ti?ed by Quantity One software (Bio-Rad).GAPDH 107was included as an internal control.Primary antibod-108ies against CdC25C,cyclin B1,Snail,GAPDH,and

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secondary antibodies were purchased from Santa Cruz 110Biotechnology (Santa Cruz,USA).Antibodies against 111N-cadherin,E-cadherin,and vimentin were obtained 112from Abcam (Cambridge,UK).

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2.6.Cell proliferation and colony formation assays 114

Cell proliferation assay was performed using the 115MTT kit (Sigma-Aldrich,St.Louis,MO,USA)ac-116cording to the manufacturer protocol.Brie?y,cells 117were cultured with or without lentivirus-mediated 118HNF1A-AS knockdown for 5days and 100μl Cell 119Titer-Glo solution was added to lyse the cells.After 120incubation at room temperature for 10min,the lumi-121nescence in each well was measured at the absorbance 122of 450nm.For colony formation assay,CNE-2and 123SUNE-1cells were suspended in Top Agar (0.45%No-124ble agar)in 6-well plates containing Base Agar (0.9%125Noble agar)in triplicate.After incubation at 37?C for 1263weeks,colonies were ?xed with chilled methanol and 127stained with 0.1%crystal violet.Visible colonies were 128manually counted in triplicate.129

2.7.Cell cycle assay

130

For cell cycle evaluation,scrambled and HNF1A-131AS shRNA-treated CNE-2and SUNE-1cells were 132trypsinized and ?xed in chilled methanol.After RNase 133treatment,cells were incubated with protease inhibitor 134for 30min and ?uorescence intensity was determined 135using the FC500?ow cytometer (Beckman,USA)?t-136ted with an FL-1H ?lter.Moreover,Mod?t 2.0soft-137ware was used to quantify the percentage of cells in 138G0/G1,S,and G2/M phases.

139

2.8.Wound healing assay

140

After shRNA-mediated stable knockdown of HNF-1411A-AS,CNE-2and SUNE-1cells were seeded into 1426-well plates containing 1ml DMEM medium and 143then incubated until approximately 90%con?uence.144The plates were scratched linearly with 100-μl pipette 145tips,washed thoroughly,and allowed to recover at 14637?C.After 24h,cells were washed with PBS and 147photographed under a light microscope (magni?cation:148×40).Migration areas were determined for uncovered 149wound areas using an image-analysis program (Image-150Pro Plus 4.5,Media Cybernetics,MD).

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2.9.Cell migration and invasion assays

152

CNE-2or SUNE-1cells (3×104)in serum-free 153medium were seeded into the upper chambers for mi-154gration (Millipore,Massachusetts,USA)and Matrigel 155invasion assays (Sigma-Aldrich),24h after transfec-156tion.The lower chambers were ?lled with 200μl 157DMEM containing 10%FBS.After 8h of incubation 158at 37?C,cells that migrated to the lower chamber were 159?xed in paraformaldehyde,stained with 0.1%crystal 160violet (Sigma-Aldrich),and photographed.A total of 1615random ?elds were counted manually under a mi-162croscope (Nikon).Triplicate independent experiments 163were performed for each cell line.

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2.10.Xenograft tumor model

165

For the tumor formation assay,24male,athymic 166BALB/c nude mice (6weeks old)were purchased from 167SLRC Laboratory Animal Co.(Shanghai,China)and 168randomly divided into 4groups.HNE-2and SUNE-1691cells with or without stable HNF-1A-AS knock-170down were cultured in 6-well plates and resuspended 171at 1×108cells/ml after washing with PBS.Subse-172quently,100μl cell suspension was subcutaneously 173injected into each mouse.Tumor volumes were mea-174sured once a week,beginning from day 7after the tu-175mor cells were injected.All mice were sacri?ced at 4176weeks post injection and the subcutaneous growth of 177each tumor was measured and photographed.Animal 178care procedures and the study protocol were approved 179by the Model Animal Research Center of the Sec-180ond Hospital,Jilin University and performed strictly 181in accordance with Institutional Animal Care and Use 182Guidelines.Every effort was made to minimize suffer-183ing of the animals.

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2.11.Statistical analysis

185

The results are shown as means ±standard deriva-186tion (SD).Student’s t -test was used to evaluate the sig-187ni?cance of inter-group differences.Values with p <1880.05were considered statistically signi?cant.189

3.Results

190

3.1.HNF1A-AS is upregulated in both,clinical NPC

191tissues and cultured NPC cell lines 192

We ?rst detected the expression of LncRNA,193HNF1A-AS in https://www.360docs.net/doc/24218388.html,pared with matched,non-

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4K.Zhuang et al./Long non-coding RNA HNF1A-AS is upregulated and promotes cell proliferation and metastasis in NPC

Fig.1.HNF1A-AS is overexpressed in both,clinical NPC tissues and cultured NPC cells.(A)qRT-PCR analysis of the 20clinical NPC cases as well as their adjacent,non-cancerous tissues.Relative HNF1A-AS mRNA levels in tumor tissues were signi?cantly higher than those in the adjacent tissues.???p <0.001,tumor versus (vs )adjacent.(B)qRT-PCR analysis of HNF1A-AS expression in 5NPC cell lines and 1control epithelial cell line,NP69.HNF1A-AS was observed to be upregulated in these NPC cell lines,of which CNE-2and SUNE-1exhibited highest HNF1A-AS expression.?p <0.05;??P <0.01;NPC cell lines vs NP69.

cancerous tissues,clinical NPC tissues from 20pa-195tients showed >3-fold higher expression of HNF1A-196AS on average (Fig.1A).NP69,an immortalized na-197sopharyngeal epithelial cell line,was used as a non-198cancerous control,while other cell lines including 5-1998F,6-10B,HONE-1,CNE-2,SUNE-1,and C666-2200are human NPC cells cultured in vitro .We determined 201the relative mRNA levels of HNF1A-AS in different 202NPC cell lines.Figure 1B reveals that HNF1A-AS ex-203pression in all NPC cell lines was signi?cantly in-204creased compared with that in NP69cells.Notably,205the HNF1A-AS level in SUNE-1cells was increased 2068-fold and even higher in CNE-2cells.Meanwhile,207HNF1A-AS expression was elevated 3-,5.7-,2.2-,and 2084-fold in 5-8F,6-10B,HONE-1,and C666-2cells,re-209spectively.Therefore,CNE-2and SUNE-1cells were

210

chosen as representative NPC cells for subsequent 211analysis.These data suggest that HNF1A-AS is in-212creased in human NPCs.

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3.2.shRNA-mediated HNF1A-AS knockdown

214successfully decreased HNF1A-AS expression in 215CNE-2and SUNE-1cells 216

Next,effects of HNF1A-AS in human NPC were 217explored.We initially infected the NPC cells,CNE-2182and SUNE-1,with lentivirus stably expressing ei-219ther HNF1A-AS-speci?c or scrambled shRNA.Fig-220ure 2A shows the high infection ef?ciency of NPC 221cell lines,CNE-2and SUNE-1(approximately 95%).222Subsequently,RT-PCR revealed the relative mRNA 223levels of HNF1A-AS in both cell lines.In CNE-2224cells,HNF1A-AS level was decreased by 76%by 225shHNF1A-AS lentivirus as compared to the scrambled 226shRNA (Fig.2B).In SUNE-1cells,total HNF1A-AS 227mRNA level fell to 14%of the control (Fig.2C).These 228results suggest that lentivirus-mediated knockdown de-229creases HNF1A-AS expression with high infection ef-230?ciency.

231

3.3.HNF1A-AS promotes cell proliferation and

232colony formation in vitro 233

Next,we examined the role of HNF1A-AS in hu-234man NPCs.No notable differences between HNF1A-235AS shRNA-treated and control groups were observed 236in the ?rst 3days for CNE-2cells and 2days for 237SUNE-1cells.However,the proliferation rate was sup-238pressed by 32.5%on the 4th day and by 39%on the 2395th day,in CNE-2cells (Fig.3A),as compared to 24033.3%on the 3rd day for SUNE-1cells,followed by 241further decrease with time,due to the inhibition effect 242of HNF1A-AS (Fig.3B).Colony-formation assay con-243?rmed that HNF1A-AS knockdown signi?cantly de-244creased the number of colonies in both NPC cell lines 245(Fig.3C).Quanti?cation of the colonies showed that 246about 70colonies were formed in case of scrambled 247shRNA-treated CNE-2cells,while only 21colonies 248were observed in case of speci?c shRNA-treated cells.249Meanwhile,in SUNE-1cells,the number of colonies 250decreased by 68%in shRNA-treated group compared 251with that in the control group (Fig.3D).These data 252imply that HNF1A-AS promotes cell proliferation in 253NPC cell lines.

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Fig.2.Lentivirus-mediated HNF1A-AS knockdown signi?cantly decreased the expression of HNF1A-AS.(A)NPC cell lines,CNE-2and SUNE-1were transfected with either scrambled or HNF1A-AS shRNA.Fluorescence microscopy indicated high infection ef?ciency (>98%)for both cell lines.qRT-PCR analysis showed that mRNA levels of HNF1A-AS were remarkably decreased in CNE-2(B)and SUNE-1(C)cells.??p <0.01,???p <0.001,shRNA vs scramble.GAPDH was included as an inner control.(Colours are visible in the online version of the article;https://www.360docs.net/doc/24218388.html,/10.3233/CBM-150567)

3.4.HNF1A-AS knockdown causes cell cycle arrest in

255G0/G1phase in NPC cell lines

256

The effect of HNF1A-AS on cell cycle distribu-257tion in CNE-2and SUNE-1cells was determined by 258cell cycle analysis.After 24-h incubation,cells were 259stained with propidium iodide and subjected to ?ow 260cytometry.In CNE-2cells,HNF1A-AS knockdown in-261creased the cell number in G0/G1phase by approxi-262mate 1.5-fold,but decreased the percentage of cells in 263S and G2/M phases by 65%and 66.7%,respectively 264(Fig.4C).Likewise,>25.7%cells in S and G2/M 265phases shifted to G0/G1phase when SUNE-1cells 266were treated with lentivirus expressing speci?c shRNA 267against HNF1A-AS (Fig.4D).These data reveal that 268HNF1A-AS knockdown arrested cell cycle progres-269sion by accumulating cells predominantly in G0/G1270phase in both,CNE-2and SUNE-1cell lines.

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3.5.HNF1A-AS knockdown inhibits tumor growth in

272mice 273

We then examined the bioactivity of HNF1A-AS in 274vivo via intraperitoneal injection of CNE-2and SUNE-

275

1NPC cells with or without speci?c shRNA against 276HNF1A-AS.Weekly measurement of tumor volumes 277revealed a large extent of difference in tumor sizes 278between HNF1A-AS-knockdown and control groups 279from the 3rd week in both,cell line-injected mice.280Speci?cally,the tumor sizes in CNE-2-injected mice 281were reduced by 58.8%and 64%at the 3rd and 4th 282week,respectively (Fig.5A).Similar results were ob-283tained for the SUNE-1injected mice,where difference 284between HNF1A-AS-knockdown and control groups 285was 54%at the 3rd week and 69.7%at the 4th week.286These data implicate that HNF1A-AS promotes tumor 287growth in NPC.

288

3.6.HNF1A-AS promotes cell migration and invasion

289in vitro 290

We also explored the effects of HNF1A-AS on 291metastasis with in vitro wound healing assay as well as 292migration and Matrigel invasion assays.As shown in 293Figs 6A and 6B,CNE-2cells in the scrambled group 294?lled 67%of the scratched wound as compared to only 29544%when HNF1A-AS was knocked down.Similarly,

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A C

Fig.3.HNF1A-AS Knockdown inhibits cell proliferation and colonies formation in vitro .MTT assay revealed that proliferation rate was signi?-cantly inhibited on the 4th day for CNE-2cells (A)and on the 3rd day for SUNE-1cells (B)after HNF1A-AS knockdown and further decreased with time.(C)Colony-formation assay showed HNF1A-AS shRNA other than scrambled infection blocked the formation of colonies in CNE-2and SUNE-1cell lines.(D)Quanti?cation of colonies indicated that knockdown of HNF1A-AS inhibited cell proliferation for both cell lines.??p <0.01,shRNA vs scrambled.

Fig.4.HNF1A-AS knockdown caused cell cycle arrest in G0/G1phase in NPC cell lines.(A-B)Cells were starved for 24h and then treated with scrambled or HNF1A-AS-speci?c shRNA for another 24h.Cell cycle analysis revealed that knockdown of HNF1A-AS caused most of the cells to remain in G0/G1phase.Cell distributions in CNE-2cells (C)and SUNE-1cells (D)after distinct treatments.Data are shown as the means ±SD from 3independent experiments.??p <0.01,?p <0.05,shRNA vs scrambled.(Colours are visible in the online version of the article;https://www.360docs.net/doc/24218388.html,/10.3233/CBM-150567)

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Fig.5.HNF1A-AS knockdown suppressed tumor growth in NPC mouse model.A nude mouse model of NPC was established and the tumor volume in each mouse was measured once a week.Generally,mice in the scrambled group exhibited a larger tumor size compared with the HNF1A-AS shRNA-treated group from the 3rd week after inoculation.The difference in tumor size between the 2groups became more signi?-cant with time,for both CNE-2cell (A)and SUNE-1(B)injection.?p <0.05,shRNA vs scrambled.(Colours are visible in the online version of the article;https://www.360docs.net/doc/24218388.html,/10.3233/CBM-150567)

metastasis ability was also inhibited in SUNE-1cells 297treated with HNF1A-AS shRNA.As for transwell as-298says,numerous cells migrated through the membrane 299of the chamber and notable disparities were detected 300between scrambled and HNF1A-AS shRNA-treated 301groups.Brie?y,for the migration assay,cells numbers 302migrating to the lower chamber decreased by 66.7%303for CNE-2cells and 62.5%for SUNE-1cells (Fig.6D).304In the invasion assays,the differences were 69.6%305and 62.5%,respectively (Fig.6E).These results sug-306gest that HNF1A-AS increases cell metastasis in vitro ,307which was also validated by subsequent analyses.

308

3.7.Knockdown of HNF1A-AS reverses the process of

309epithelial to mesenchymal transition (EMT)310

EMT is a critical process in epithelial cells,known 311to be involved in NPC tumorigenesis [23–25].There-312fore,we explored the potential mechanism by which 313HNF1A-AS regulates NPC metastasis and examined 314the potential relationship between HNF1A-AS and 315EMT.As shown in Fig.7,the expression of EMT 316biomarkers including CdC25C,cyclin B1,Snail,N-317cadherin,and vimentin were decreased when HNF1A-318AS was knocked down in both,CNE-2and SUNE-1319cell lines,whereas cell-to-cell adhesion molecule,E-320Cadherin showed an increase,which conforms to the 321process of EMT.These data suggest that HNF1A-AS 322is positively related with the process of EMT in NPC.

323

4.Discussion

324

Nasopharyngeal carcinoma (NPC)is an interesting 325form of cancer because of its de?ned racial and ge-326ographic distribution [1,26].Almost 84,400incident 327NPC cases and 51,600NPC-related deaths were re-328ported in 2008by the International Agency for Re-329search on Cancer [27].Due to anatomic constraints and 330high radiosensitivity of NPC,early-stage NPC is gen-331erally treated by radiotherapy,while standard treatment 332of advanced NPC is chemoradiotherapy [28].

333Although HNF1A-AS is reported to be upregulated 334in lung adenocarcinoma and oesophageal adenocarci-335noma [19,20],the speci?c mechanism is still far from 336clear.Moreover,the role of HNF1A-AS in other tu-337mors remains to be explored.In this study,we con-338?rmed that HNF1A-AS overexpression in NPC is re-339lated to tumor growth and cell migration as well as 340contributes to the process of EMT.However,further 341studies are required to explain the detailed underlying 342mechanism of HNF1A-AS-mediated regulation of tu-343morigenesis.

344EMT is an important process,where epithelial cells 345lose their morphology and obtain a mesenchymal phe-346notype,genetically characterized by the decline of ep-347ithelial proteins such as Zo-1and E-cadherin,and the 348overexpression of mesenchymal proteins such as N-349cadherin,vimentin,and ?bronectin [29,30].Further-

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Fig.6.HNF1A-AS promotes the abilities of cell migration and invasion in vitro .(A-B)Scratched wound healing assay revealed that in both,CNE-2and SUNE-1cells,cell migration ability was suppressed when HNF1A-AS was knocked down.??p <0.01,shRNA vs scrambled.(C)Representative images of migration and Matrigel invasion assay for both cell lines.(D-E)Quanti?cation of the transwell assay results revealed that the number of cells that migrated decreased signi?cantly for CNE-2and SUNE-1cells.??p <0.01,shRNA vs scrambled.(Colours are visible in the online version of the article;https://www.360docs.net/doc/24218388.html,/10.3233/CBM-150567)

Fig.7.HNF1A-AS knockdown reversed the EMT process.Expres-sion of EMT markers,including CdC25C,cyclin B1,Snail,N-cad-herin,and vimentin were signi?cantly suppressed when HNF1A-AS was knocked down in both,CNE-2and SUNE-1cell lines,while the expression of E-cadherin was increased.GAPDH was included as an inner control.

more,Snail and Slug are 2critical members of the 351zinc-?nger family and exert a central transcriptional 352function in the regulation of EMT via direct bind-353ing to speci?c E-boxes on the promoter region of E-354cadherin [31].It is widely recognized that EMT par-355ticipates in tumor cell metastasis and growth.Further-356more,aggressive cancer cells often manifest with loss 357of epithelial features and acquisition of mesenchymal 358phenotype [32].We demonstrated that when HNF1A-359AS was knocked down using speci?c shRNA,expres-360sion of mesenchymal proteins such as N-cadherin and 361vimentin was upregulated,while the level of epithelial 362protein,E-cadherin was decreased in both cell lines.363These ?ndings suggest that HNF1A-AS is positively 364associated with EMT.

365Thus,we found that HNF1A-AS was overexpressed 366in both,clinical NPC tissues and cultured NPC cells,367demonstrated by RT-PCR.Lentivirus-mediated 368HNF1A-AS knockdown showed that HNF1A-AS pro-369moted cell proliferation and migration in CNE-2and 370SUNE-1NPC cells.Xenograft mouse model bear-371ing NPC revealed that HNF1A-AS knockdown inhib-

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ited tumor growth,while cell cycle analysis showed 373that speci?c shRNA-treated CNE-2and SUNE-1cells 374accumulated in the G0/G1phase.HNF1A-AS thus,375shows high potential as a new prognostic marker and 376therapeutic target for treatment of NPC.

377

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