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·論著·

LncRNA-PVT1對(duì)人胰腺癌細(xì)胞株HPAF-Ⅱ增殖和凋亡的影響*

彭娟菲#黃鳳婷莊燕妍陳文穎張世能&

中山大學(xué)孫逸仙紀(jì)念醫(yī)院消化內(nèi)科(510120)

#Email: pengjfei0305@163.com

&本文通信作者，Email: shinengz@163.net

背景：近年來(lái)，長(zhǎng)非編碼RNA(lncRNAs)在腫瘤發(fā)生、發(fā)展中的作用日益受到重視。研究發(fā)現(xiàn)lncRNA-PVT1在多種人類惡性腫瘤中表達(dá)上調(diào)并發(fā)揮促癌作用。目的：探討PVT1在人胰腺癌細(xì)胞中的表達(dá)及其對(duì)HPAF-Ⅱ細(xì)胞增殖和凋亡的影響。方法：以脂質(zhì)體轉(zhuǎn)染技術(shù)將siRNA-PVT1轉(zhuǎn)染入HPAF-Ⅱ細(xì)胞；以real-time PCR檢測(cè)PVT1 mRNA表達(dá)，MTS實(shí)驗(yàn)和平板克隆形成實(shí)驗(yàn)檢測(cè)細(xì)胞增殖能力，流式細(xì)胞分析檢測(cè)細(xì)胞周期和細(xì)胞凋亡，蛋白質(zhì)印跡法檢測(cè)凋亡相關(guān)蛋白和原癌基因蛋白c-Myc表達(dá)。結(jié)果：以人永生化正常胰腺導(dǎo)管上皮細(xì)胞株H6c7作為對(duì)照，各人胰腺癌細(xì)胞株中以HPAF-Ⅱ細(xì)胞PVT1 mRNA表達(dá)升高最為明顯。與轉(zhuǎn)染陰性對(duì)照siRNA和未予轉(zhuǎn)染siRNA的細(xì)胞相比，轉(zhuǎn)染siRNA-PVT1的HPAF-Ⅱ細(xì)胞PVT1 mRNA表達(dá)顯著降低，細(xì)胞增殖能力減弱，發(fā)生細(xì)胞周期G1期阻滯并出現(xiàn)凋亡峰，細(xì)胞中凋亡相關(guān)蛋白cleaved-caspase-3、cleaved-PARP表達(dá)上調(diào)，Bcl-2/Bax比值降低，c-Myc蛋白表達(dá)下調(diào)。結(jié)論：LncRNA-PVT1在人胰腺癌細(xì)胞株HPAF-Ⅱ中呈高表達(dá)，其可能通過(guò)調(diào)控c-Myc表達(dá)影響HPAF-Ⅱ細(xì)胞的增殖和凋亡。

關(guān)鍵詞胰腺腫瘤；長(zhǎng)非編碼RNA；PVT1；原癌基因蛋白質(zhì)c-myc；細(xì)胞增殖；細(xì)胞凋亡

Effect of LncRNA-PVT1 on Proliferation and Apoptosis of Human Pancreatic Cancer Cell Line HPAF-Ⅱ

PENGJuanfei,HUANGFengting,ZHUANGYanyan,CHENWenying,ZHANGShineng.

DepartmentofGastroenterology,SunYat-senMemorialHospital,SunYat-senUniversity,Guangzhou(510120)

Correspondence to: ZHANG Shineng, Email: shinengz@163.net

Background: Recent studies have shown that long non-coding RNAs (lncRNAs) play important roles in carcinogenesis and cancer biology and the related context has attracted more and more attentions. PVT1, which encodes a lncRNA, is reported to be up-regulated and exhibit pro-oncogenic activity in a wide variety of human cancers. Aims: To investigate the expression of PVT1 in human pancreatic cancer cells and its effect on proliferation and apoptosis of HPAF-Ⅱ cells. Methods: One target siRNA against PVT1 was synthesized and transfected into HPAF-Ⅱ cells by using lipofactamine technique. PVT1 mRNA expression was detected by real-time PCR; capability of cell proliferation was examined by MTS and colony formation assays; cell cycle progression and apoptosis were measured by flow cytometry; and Western blotting was performed to determine the expressions of apoptosis-related proteins and proto-oncogene protein c-Myc. Results: The mRNA expression of PVT1 in several human pancreatic cancer cell lines, especially HPAF-Ⅱ cells was significantly higher than that in H6c7, a human immortalization normal pancreatic ductal epithelial cell line. Compared with HPAF-Ⅱ cells transfected with negative control siRNA or without transfection, silencing of PVT1 by siRNA-PVT1 resulted in remarkable reduction in cell proliferation, cell cycle G1 phase arrest, and notable apoptosis; meanwhile, the expressions of apoptosis-related proteins (cleaved-caspase-3 and cleaved-PARP) were up-regulated, the ratio for Bcl-2/Bax was decreased, and the expression of c-Myc protein was down-regulated. Conclusions: LncRNA-PVT1 is highly expressed in human pancreatic cancer cell line HPAF-Ⅱ. It may affect the proliferation and apoptosis of HPAF-Ⅱ cells partially through regulating c-Myc expression.

Key wordsPancreatic Neoplasms;Long Non-Coding RNA;PVT1;Proto-Oncogene Proteins c-myc;

Cell Proliferation;Apoptosis

胰腺癌早期診斷率低，根治性手術(shù)率低，預(yù)后極差，死亡率高[1]，探討胰腺癌的發(fā)生、發(fā)展機(jī)制有利于提高其臨床診治水平。近年來(lái)，長(zhǎng)非編碼RNA(long non-coding RNAs, lncRNAs)在腫瘤發(fā)生、發(fā)展中的作用日益受到重視。LncRNAs是一類轉(zhuǎn)錄本長(zhǎng)度超過(guò)200 nt 的RNA分子，不編碼蛋白質(zhì)，但可通過(guò)表觀遺傳學(xué)以及轉(zhuǎn)錄和轉(zhuǎn)錄后調(diào)控層面調(diào)控基因表達(dá)[2]。LncRNA-PVT1(plasmacytoma variant translocation 1)基因定位于染色體8q24[3]，研究發(fā)現(xiàn)其參與了Burkitt淋巴瘤[4]、多發(fā)性骨髓瘤[5]、卵巢癌和乳腺癌[6]、結(jié)直腸癌[7]等多種惡性腫瘤的發(fā)生、發(fā)展，并與預(yù)后不良相關(guān)。已知PVT1能調(diào)節(jié)人胰腺癌細(xì)胞對(duì)吉西他濱的化療敏感性[8]，但PVT1在胰腺癌細(xì)胞中的表達(dá)情況及其與胰腺癌細(xì)胞生物學(xué)特性的關(guān)系則尚未見(jiàn)相關(guān)文獻(xiàn)報(bào)道。本研究檢測(cè)了PVT1在人胰腺癌細(xì)胞中的表達(dá)情況，并初步探討其對(duì)HPAF-Ⅱ細(xì)胞增殖和凋亡的影響，以期為進(jìn)一步明確PVT1在胰腺癌發(fā)生、發(fā)展中的作用奠定基礎(chǔ)。

材料與方法

一、細(xì)胞株和主要試劑

人胰腺癌細(xì)胞株P(guān)ANC-1、SW1990、MIAPaca-2、BXPC-3、DAN-G、HPAF-Ⅱ購(gòu)自ATCC公司；人永生化正常胰腺導(dǎo)管上皮細(xì)胞株H6c7由加拿大安大略癌癥研究所Ming-Sound Tsao教授惠贈(zèng)，體外培養(yǎng)傳代。RPMI 1640培養(yǎng)基、K-SFM無(wú)血清培養(yǎng)基、Opti-MEM?減血清培養(yǎng)基、胎牛血清(FBS)、重組表皮生長(zhǎng)因子(rEGF)、牛垂體提取物(BPE)、牛血清白蛋白(BSA)、Lipofectamine?RNAiMAX轉(zhuǎn)染試劑、RIPA細(xì)胞裂解液(Thermo Fisher Scientific Inc.)；siRNA-PVT1(si-PVT1)、siRNA-Negative Control(si-NC)、siRNA-Negative Control-FAM(si-NC-FAM)由蘇州吉瑪基因藥物科技有限公司設(shè)計(jì)、合成(表1)；RNAiso Plus總RNA提取試劑、PrimeScriptTMRT Master Mix(Perfect Real Time)、SYBR?Premix Ex TaqTM(Tli RNaseH Plus)(TaKaRa Bio Inc.)；MTS試劑(Promega Corporation)；Annexin V-FITC/PI細(xì)胞凋亡檢測(cè)試劑盒(Bender)；細(xì)胞周期檢測(cè)試劑盒(碧云天生物技術(shù))；caspase-3、PARP、Bcl-2、Bax、GAPDH抗體和相應(yīng)二抗(Cell Signaling Technology, Inc.)；c-Myc抗體(Santa Cruz Biotechnology, Inc.)；PVDF膜、ImmobilonTMWestern Chemiluminescent ECL超敏發(fā)光液(Merck Millipore Corporation)；細(xì)胞培養(yǎng)耗材(Corning Incorporated)。

表1　siRNAs序列

二、方法

1. 細(xì)胞培養(yǎng)：HPAF-Ⅱ細(xì)胞置于含10% FBS的RPMI 1640培養(yǎng)基(含100 U/mL青霉素和100 μg/mL鏈霉素)中，于37 ℃、5% CO2、飽和濕度條件下培養(yǎng)，每2～3 d換液或傳代1次，0.02% EDTA和0.25%胰蛋白酶1∶1混合消化，完全培養(yǎng)基中和。H6c7細(xì)胞置于K-SFM無(wú)血清培養(yǎng)基(含100 U/mL青霉素、100 μg/mL鏈霉素、0.2 μg/L rEGF和 20 mg/L BPE)，于37 ℃、5% CO2、飽和濕度條件下培養(yǎng)，0.05%胰蛋白酶消化，0.4% BSA中和。

2. 實(shí)驗(yàn)分組和瞬時(shí)轉(zhuǎn)染：實(shí)驗(yàn)設(shè)4個(gè)組別：si-PVT1組、si-NC組和si-NC-FAM組分別以Lipo-fectamine?RNAiMAX轉(zhuǎn)染試劑將si-PVT1、si-NC和si-NC-FAM(終濃度120 nmol/L)轉(zhuǎn)染入HPAF-Ⅱ細(xì)胞，si-Blank Cell組(si-BC組)HPAF-Ⅱ細(xì)胞常規(guī)培養(yǎng)，不予轉(zhuǎn)染siRNA。轉(zhuǎn)染方法：HPAF-Ⅱ細(xì)胞以2.5×105/孔常規(guī)培養(yǎng)于6孔板，待融合度達(dá)60%～70%時(shí)，將培養(yǎng)基換成Opti-MEM?減血清培養(yǎng)基，嚴(yán)格按試劑說(shuō)明書操作進(jìn)行轉(zhuǎn)染。

3. Real-time PCR：以RNAiso Plus試劑提取各人胰腺癌細(xì)胞株總RNA，瓊脂糖凝膠電泳法檢測(cè)RNA完整性，以PrimeScriptTMRT Master Mix(Perfect Real Time)逆轉(zhuǎn)錄合成cDNA，操作均嚴(yán)格按試劑說(shuō)明書進(jìn)行。采用NCBI/Primer-BLAST軟件設(shè)計(jì)PCR引物，由上海TaKaRa公司合成：β-actin(內(nèi)參)上游5’-GTT GCG TTA CAC CCT TTC TTG AC-3’，下游5’-CTC GGC CAC ATT GTG AAC TTT G-3’；PVT1上游5’-TTG GCA CAT ACA GCC ATC AT-3’，下游5’-GCA GTA AAA GGG GAA CAC CA-3’。PCR擴(kuò)增采用SYBR?Green Ⅰ方法，2-ΔΔCt法計(jì)算PVT1 mRNA相對(duì)表達(dá)量。實(shí)驗(yàn)獨(dú)立重復(fù)3次，每次每一樣本設(shè)3個(gè)復(fù)孔。

4. MTS實(shí)驗(yàn)：各組HPAF-Ⅱ 細(xì)胞以4×103/孔(100 μL)接種于96孔板，待融合度達(dá)50%～70%時(shí)進(jìn)行轉(zhuǎn)染；分別于轉(zhuǎn)染后24 h、48 h、72 h、96 h加入10 μL MTS，37 ℃、5% CO2、飽和濕度條件下培養(yǎng)3.5 h，于酶聯(lián)免疫檢測(cè)儀波長(zhǎng)492 nm處測(cè)定吸光度(A)值，以未接種細(xì)胞、僅加入培養(yǎng)基和MTS的空白孔調(diào)零。實(shí)驗(yàn)獨(dú)立重復(fù)3次，每組設(shè)5個(gè)復(fù)孔。

5. 平板克隆形成實(shí)驗(yàn)：各組HPAF-Ⅱ細(xì)胞轉(zhuǎn)染后 72 h，以4×103/孔接種于6孔板，常規(guī)培養(yǎng)9 d后，棄培養(yǎng)基，PBS洗2次，4%多聚甲醛溶液固定15 min，0.1%結(jié)晶紫染色20 min，以PBS洗去殘余染料，待自然干燥后拍照。實(shí)驗(yàn)獨(dú)立重復(fù)3次。

6. 流式細(xì)胞分析：各組HPAF-Ⅱ細(xì)胞轉(zhuǎn)染后72 h，以PBS重懸為(1～5)×109/L的細(xì)胞懸液，取0.5 mL 立即用于細(xì)胞凋亡檢測(cè)，0.5 mL用于細(xì)胞周期檢測(cè)，操作均嚴(yán)格按試劑盒說(shuō)明書進(jìn)行。實(shí)驗(yàn)獨(dú)立重復(fù)3次。

7. 蛋白質(zhì)印跡法：各組HPAF-Ⅱ細(xì)胞轉(zhuǎn)染后72 h，RIPA細(xì)胞裂解液裂解、提取細(xì)胞總蛋白，BCA法測(cè)定蛋白濃度。每組取總蛋白50 μg上樣，恒壓濃縮膠60 V、分離膠100 V電泳約90 min，4 ℃ 200 mA 轉(zhuǎn)膜120 min，漂洗轉(zhuǎn)印后的PVDF膜，室溫3次×10 min，5% BSA室溫封閉1.5 h，加入一抗4 ℃ 孵育過(guò)夜，1×TBST洗膜3次×10 min，加入二抗室溫孵育2 h，1×TBST洗膜3次×10 min，ECL發(fā)光液顯色。實(shí)驗(yàn)獨(dú)立重復(fù)3次。

三、統(tǒng)計(jì)學(xué)分析

結(jié)果

一、PVT1在人胰腺癌細(xì)胞中的表達(dá)

以人永生化正常胰腺導(dǎo)管上皮細(xì)胞株H6c7作為對(duì)照，real-time PCR檢測(cè)顯示人胰腺癌細(xì)胞株P(guān)ANC-1、SW1990、BXPC-3、DAN-G、HPAF-Ⅱ中的PVT1 mRNA表達(dá)明顯升高，以HPAF-Ⅱ細(xì)胞升高最為顯著(P<0.01)，人胰腺癌細(xì)胞株MIAPaca-2中的PVT1 mRNA表達(dá)則明顯降低(圖1A)，故選擇HPAF-Ⅱ細(xì)胞為研究對(duì)象進(jìn)行后續(xù)實(shí)驗(yàn)。

二、siRNA轉(zhuǎn)染效率

si-PVT1、si-NC以及與兩者相同大小的si-NC-FAM轉(zhuǎn)染入HPAF-Ⅱ細(xì)胞后，熒光顯微鏡觀察顯示，si-NC-FAM組帶綠色熒光的HPAF-Ⅱ細(xì)胞(圖1C)占細(xì)胞總數(shù)(圖1B)的百分率>90%；real-time PCR檢測(cè)顯示，si-PVT1組HPAF-Ⅱ細(xì)胞中的PVT1 mRNA表達(dá)顯著低于si-NC組和si-BC組(P<0.05)，si-NC與si-BC兩組間差異則無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)(圖1D)。

三、抑制PVT1對(duì)HPAF-Ⅱ細(xì)胞增殖和凋亡的影響

MTS實(shí)驗(yàn)顯示，轉(zhuǎn)染后48 h起，si-PVT1組HPAF-Ⅱ細(xì)胞A值開(kāi)始顯著低于si-NC組和si-BC組(P<0.01)(圖2A)；平板克隆形成實(shí)驗(yàn)顯示，轉(zhuǎn)染后72 h，si-PVT1組細(xì)胞形成的克隆數(shù)顯著低于si-NC組和si-BC組(P<0.01)，si-NC與si-BC兩組間差異則無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)(圖2B)。

圖1　人胰腺癌細(xì)胞PVT1表達(dá)(A)以及siRNA轉(zhuǎn)染效率(B、C、D)

流式細(xì)胞分析顯示，轉(zhuǎn)染后72 h，si-PVT1組HPAF-Ⅱ細(xì)胞出現(xiàn)凋亡峰，凋亡率顯著高于si-NC組和si-BC組(P<0.05)(圖2C)；蛋白質(zhì)印跡法檢測(cè)進(jìn)一步證實(shí)，與si-NC組相比，si-PVT1組凋亡相關(guān)蛋白cleaved-caspase-3(P=0.000 8)、cleaved-PARP(P=0.000 4)表達(dá)顯著上調(diào)，Bcl-2/Bax比值降低(P=0.049 5)(圖2D)。

上述發(fā)現(xiàn)提示抑制PVT1可抑制HPAF-Ⅱ細(xì)胞增殖，誘導(dǎo)細(xì)胞凋亡。

四、抑制PVT1對(duì)HPAF-Ⅱ 細(xì)胞細(xì)胞周期的影響

流式細(xì)胞分析顯示，轉(zhuǎn)染72 h后，si-PVT1組G1期細(xì)胞比率較si-NC組和si-BC組顯著增高(P<0.01)，G2期細(xì)胞比率顯著降低(P<0.01)，各組間S期細(xì)胞比率無(wú)明顯差異(P>0.05)(圖2E、表2)，結(jié)合MTS實(shí)驗(yàn)結(jié)果，推測(cè)抑制PVT1可引起HPAF-Ⅱ細(xì)胞細(xì)胞周期G1期至G2期轉(zhuǎn)換阻滯。

五、PVT1調(diào)控HPAF-Ⅱ細(xì)胞中的c-Myc表達(dá)

蛋白質(zhì)印跡法檢測(cè)顯示，轉(zhuǎn)染72 h后，si-PVT1組HPAF-Ⅱ 細(xì)胞中的c-Myc表達(dá)較si-NC組顯著降低(P=0.012 3)，si-NC與si-BC兩組間差異則無(wú)統(tǒng)計(jì)學(xué)意義(P=0.803 2)(圖3)，提示PVT1對(duì)c-Myc表達(dá)具有調(diào)控作用，抑制PVT1可下調(diào)c-Myc表達(dá)。

圖2　抑制PVT1對(duì)HPAF-Ⅱ細(xì)胞增殖(A、B)、凋亡(C、D)和細(xì)胞周期(E)的影響

圖3　抑制PVT1對(duì)HPAF-Ⅱ細(xì)胞中c-Myc表達(dá)的影響

組別G1期G2期S期si-BC29.51±1.5636.19±1.4434.30±0.64si-NC27.11±1.4037.16±1.3735.73±1.53si-PVT153.05±3.17**9.98±0.60**36.97±3.53

**與si-NC組比較，P<0.01

討論

既往諸多研究發(fā)現(xiàn)胰腺癌組織和細(xì)胞中存在lncRNAs表達(dá)異常，如高表達(dá)的HOTAIR[9]、H19[10]、HOTTIP[11]、MALAT-1[12]、HULC[13]、AF339813[14]和低表達(dá)的gas5[15]、ENST00000480739[16]，這些異常表達(dá)的lncRNAs可通過(guò)特定機(jī)制調(diào)節(jié)胰腺癌細(xì)胞的分化、增殖、凋亡、遷移、侵襲等生物學(xué)行為，從而影響胰腺癌的發(fā)生、發(fā)展進(jìn)程，如H19可通過(guò)拮抗miRNA let-7增強(qiáng)其靶mRNA HMGA2介導(dǎo)的上皮-間質(zhì)轉(zhuǎn)換(EMT)，從而促進(jìn)胰腺導(dǎo)管腺癌細(xì)胞侵襲和遷移[10]，而HOTTIP可通過(guò)調(diào)節(jié)多個(gè)HOX基因家族成員表達(dá)而影響胰腺癌細(xì)胞的增殖、凋亡和遷移[11]。近年研究發(fā)現(xiàn)lncRNA-PVT1在多種人類惡性腫瘤中表達(dá)上調(diào)并發(fā)揮促癌作用。You等[8]的研究顯示，使人胰腺癌細(xì)胞株ASPC-1中的PVT1功能失活可增強(qiáng)細(xì)胞對(duì)吉西他濱的化療敏感性。Huang等[17]發(fā)現(xiàn)，與癌旁非癌組織相比，PVT1在胰腺導(dǎo)管腺癌組織中呈顯著高表達(dá)，并與腫瘤臨床分期和預(yù)后不良相關(guān)。但PVT1在人胰腺癌細(xì)胞中的表達(dá)情況及其可能作用機(jī)制尚未見(jiàn)相關(guān)文獻(xiàn)報(bào)道。

本研究采用real-time PCR技術(shù)檢測(cè)了PVT1在人永生化正常胰腺導(dǎo)管上皮細(xì)胞株H6c7和多種人胰腺癌細(xì)胞株中的表達(dá)情況，結(jié)果顯示與H6c7細(xì)胞相比，PVT1在人胰腺癌細(xì)胞株HPAF-Ⅱ、DAN-G、SW1990、BXPC-3、PANC-1中呈高表達(dá)，在人胰腺癌細(xì)胞株MIAPaca-2中則呈低表達(dá)，以HPAF-Ⅱ細(xì)胞表達(dá)升高最為明顯。以脂質(zhì)體轉(zhuǎn)染技術(shù)將siRNA-PVT1轉(zhuǎn)染入HPAF-Ⅱ細(xì)胞后，其PVT1 mRNA表達(dá)顯著降低，發(fā)生細(xì)胞周期G1期阻滯，細(xì)胞增殖明顯受到抑制并出現(xiàn)凋亡峰，凋亡相關(guān)蛋白cleaved-caspase-3、cleaved-PARP表達(dá)上調(diào)，Bcl-2/Bax比值降低，提示沉默PVT1表達(dá)可抑制HPAF-Ⅱ細(xì)胞增殖，誘導(dǎo)細(xì)胞凋亡。

PVT1基因與原癌基因Myc共同定位于染色體8q24，位于Myc基因下游50 kb，并延伸入Myc末端著絲粒200 kb，兩者在腫瘤細(xì)胞中常共同擴(kuò)增[18]。PVT1基因啟動(dòng)子區(qū)近轉(zhuǎn)錄起始點(diǎn)區(qū)域(-155/-95)存在Myc結(jié)合位點(diǎn)(E-box CACGCG)，Myc可與此位點(diǎn)結(jié)合而調(diào)節(jié)PVT1轉(zhuǎn)錄，即PVT1是Myc的靶基因之一[19]。此外，研究發(fā)現(xiàn)PVT1亦可調(diào)節(jié)Myc蛋白表達(dá)。在Myc與PVT1共同擴(kuò)增的乳腺癌細(xì)胞株中，抑制PVT1表達(dá)并不影響c-Myc mRNA水平，但可使c-Myc蛋白表達(dá)下調(diào)，并促進(jìn)細(xì)胞凋亡；在Myc陽(yáng)性結(jié)腸癌細(xì)胞株HCT116中敲除PVT1基因可致Myc蛋白表達(dá)下調(diào)，細(xì)胞增殖能力減弱，在小鼠中形成腫瘤的能力消失[3]。本研究發(fā)現(xiàn)以siRNA沉默HPAF-Ⅱ細(xì)胞中的PVT1表達(dá)可顯著降低c-Myc蛋白表達(dá)，提示PVT1可能系通過(guò)調(diào)節(jié)c-Myc表達(dá)在胰腺癌細(xì)胞中發(fā)揮作用。

綜上所述，lncRNA-PVT1在人胰腺癌細(xì)胞株HPAF-Ⅱ中呈高表達(dá)，可促進(jìn)HPAF-Ⅱ細(xì)胞增殖，調(diào)節(jié)細(xì)胞周期分布，并抑制細(xì)胞凋亡，上述作用可能是通過(guò)調(diào)控c-Myc表達(dá)實(shí)現(xiàn)的。后續(xù)擬進(jìn)一步開(kāi)展體內(nèi)實(shí)驗(yàn)以驗(yàn)證本研究結(jié)果，并著重探討PVT1的具體作用機(jī)制。

參考文獻(xiàn)

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(2015-10-14收稿；2015-10-23修回)

*基金項(xiàng)目：國(guó)家自然科學(xué)基金面上項(xiàng)目(81572348)；廣東省自然科學(xué)基金(2015A030313115)；廣州市科技計(jì)劃項(xiàng)目科學(xué)研究專項(xiàng)(201510010206)

DOI:10.3969/j.issn.1008-7125.2016.03.002