長(zhǎng)鏈非編碼RNA HIF1A-AS1對(duì)缺氧誘導(dǎo)的胰腺癌PANC1細(xì)胞自噬的調(diào)節(jié)作用

許豐 徐月梅 夏菲珍 姜玉華 張波 李先鵬

·論著·

長(zhǎng)鏈非編碼RNA HIF1A-AS1對(duì)缺氧誘導(dǎo)的胰腺癌PANC1細(xì)胞自噬的調(diào)節(jié)作用

許豐 徐月梅 夏菲珍 姜玉華 張波 李先鵬

目的 觀察長(zhǎng)鏈非編碼RNA HIF1A-AS1對(duì)缺氧誘導(dǎo)的胰腺癌PANC1細(xì)胞自噬的調(diào)節(jié)作用。方法 應(yīng)用三氣培養(yǎng)箱及缺氧混合氣體(94%N2、5% CO2、1% O2)缺氧培養(yǎng)胰腺癌PANC1細(xì)胞3、6、12、24、36、48 h，實(shí)時(shí)熒光定量PCR法檢測(cè)HIF1A-AS1的表達(dá)。通過(guò)攜帶HIF1A-AS1過(guò)表達(dá)的重組腺病毒感染PANC1細(xì)胞和通過(guò)脂質(zhì)體將靶向HIF1A-AS1的siRNA轉(zhuǎn)染PANC1細(xì)胞分別獲得過(guò)表達(dá)、低表達(dá)HIF1A-AS1的PANC1細(xì)胞株，以常規(guī)培養(yǎng)的PANC1細(xì)胞作為對(duì)照。對(duì)照組、過(guò)表達(dá)組、低表達(dá)組細(xì)胞分別缺氧培養(yǎng)24 h，采用實(shí)時(shí)熒光定量PCR法檢測(cè)各組PANC1細(xì)胞HIF1A-AS1的表達(dá)，流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率，蛋白質(zhì)印跡法檢測(cè)自噬相關(guān)蛋白Beclin 1的表達(dá)。結(jié)果 缺氧培養(yǎng)的PANC1細(xì)胞HIF1A-AS1的表達(dá)隨缺氧時(shí)間的延長(zhǎng)而增加，36 h時(shí)達(dá)峰值，從6 h開(kāi)始均顯著高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。以對(duì)照組表達(dá)量為1，過(guò)表達(dá)組、低表達(dá)組細(xì)胞HIF1A-AS1的表達(dá)量分別為4.49±0.53、0.49±0.07，過(guò)表達(dá)組高于對(duì)照組，低表達(dá)組低于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。對(duì)照組、過(guò)表達(dá)組、低表達(dá)組PANC1細(xì)胞經(jīng)缺氧培養(yǎng)24 h后的細(xì)胞凋亡率分別為(8.27±1.28)%、(6.56±1.49)%、(19.9±2.34)%，過(guò)表達(dá)組低于對(duì)照組，低表達(dá)組高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)；Beclin 1表達(dá)量分別為1.05±0.11、1.29±0.19、0.38±0.18，過(guò)表達(dá)組高于對(duì)照組，低表達(dá)組低于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。結(jié)論 HIF1A-AS1可能通過(guò)促進(jìn)缺氧誘導(dǎo)胰腺癌PANC1細(xì)胞的自噬，參與胰腺癌的發(fā)病過(guò)程。

胰腺； 細(xì)胞系，腫瘤； 長(zhǎng)鏈非編碼RNA； HIF1A-AS1； 自噬； 缺氧

Fund program: Natural Science Foundation of Zhejiang Province(LY16H160004)

缺氧微環(huán)境作為胰腺癌等實(shí)體腫瘤的重要特征之一，可誘導(dǎo)腫瘤細(xì)胞中多種耐藥基因的表達(dá)，導(dǎo)致化療耐藥[1]。自噬是真核細(xì)胞通過(guò)清除損傷細(xì)胞器或變性蛋白質(zhì)維持細(xì)胞穩(wěn)態(tài)的降解機(jī)制，在腫瘤形成、轉(zhuǎn)移及化療耐藥中起重要作用，但其調(diào)控機(jī)制尚未闡明[2-3]。長(zhǎng)鏈非編碼RNA(long non-coding RNA, lncRNA)是最新發(fā)現(xiàn)的一類在表觀遺傳、轉(zhuǎn)錄水平、翻譯水平都具有調(diào)節(jié)作用的非編碼RNA分子[4-5]，可通過(guò)調(diào)節(jié)腫瘤細(xì)胞的自噬水平參與腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移進(jìn)程[6-7]。存在于缺氧誘導(dǎo)因子1α(hypoxia-inducible factor-1α，HIF-1α)反義鏈上的lncRNA HIF1A-AS1參與肝癌、非小細(xì)胞肺癌等腫瘤的發(fā)生和發(fā)展[8-9]。本研究在缺氧環(huán)境中培養(yǎng)胰腺癌PANC1細(xì)胞，觀察細(xì)胞HIF1A-AS1表達(dá)對(duì)細(xì)胞自噬活性的影響，探討其可能的調(diào)控機(jī)制。

材料與方法

一、PANC1細(xì)胞HIF1A-AS1表達(dá)的檢測(cè)

胰腺癌PANC1細(xì)胞株購(gòu)自ATCC公司，常規(guī)培養(yǎng)傳代。應(yīng)用三氣培養(yǎng)箱及缺氧混合氣體(94% N2、5% CO2、1% O2)培養(yǎng)細(xì)胞3、6、12、24、36、48 h，以常規(guī)培養(yǎng)細(xì)胞作為對(duì)照組。應(yīng)用Trizol提取各組細(xì)胞總RNA，先利用隨機(jī)引物逆轉(zhuǎn)錄成cDNA，再采用實(shí)時(shí)熒光定量PCR法檢測(cè)HIF1A-AS1表達(dá)。HIF1A-AS1引物正義序列為5′-TTCGGTACTTTACGCACCCT-3′，反義序列為5′-TTTTCCTCCTTTTCGCCAGC-3′；內(nèi)參β-actin引物正義序列為5′-TGGCATCCACGAAACTACCT-3′，反義序列為5′-CGTACAGGTCTTTGCGGATG-3′。反應(yīng)條件：95℃ 15 s，58℃ 10 s，72℃ 20 s，40個(gè)循環(huán)。由儀器自帶軟件獲取Ct值，應(yīng)用公式2-△△Ct計(jì)算其相對(duì)表達(dá)量，以對(duì)照組表達(dá)量計(jì)為1。

二、HIF1A-AS1過(guò)表達(dá)及低表達(dá)PANC1細(xì)胞株的構(gòu)建及鑒定

HIF1A-AS1過(guò)表達(dá)的重組腺病毒由上海百力格生物技術(shù)公司合成，感染PANC1細(xì)胞，建立HIF1A-AS1過(guò)表達(dá)PANC1細(xì)胞株(過(guò)表達(dá)組)。靶向HIF1A-AS1的siRNA由上海吉瑪制藥公司設(shè)計(jì)并合成，采用脂質(zhì)體法轉(zhuǎn)染PANC1細(xì)胞，建立HIF1A-AS1低表達(dá)細(xì)胞株(低表達(dá)組)。收集兩組細(xì)胞，抽提細(xì)胞總RNA，應(yīng)用實(shí)時(shí)熒光定量PCR法檢測(cè)過(guò)表達(dá)、低表達(dá)細(xì)胞HIF1A-AS1表達(dá)，鑒定高、低表達(dá)細(xì)胞株構(gòu)建是否成功。

三、PANC1細(xì)胞凋亡的檢測(cè)

取對(duì)照組、過(guò)表達(dá)組、低表達(dá)組對(duì)數(shù)生長(zhǎng)期PANC1細(xì)胞，在缺氧環(huán)境中培養(yǎng)24 h。收集各組細(xì)胞，用預(yù)冷PBS洗滌2次。加入500 μl的Binding Buffer懸浮細(xì)胞，依次加入5 μl的Annexin V-FITC、5 μl的PI，混勻，置室溫避光反應(yīng)5～15 min，上流式細(xì)胞儀檢測(cè)各組細(xì)胞凋亡率。

四、PANC1細(xì)胞自噬標(biāo)志蛋白Beclin 1表達(dá)的檢測(cè)

取對(duì)照組、過(guò)表達(dá)組、低表達(dá)組對(duì)數(shù)生長(zhǎng)期PANC1細(xì)胞，在缺氧環(huán)境中培養(yǎng)24 h，收集各組細(xì)胞，用細(xì)胞裂解液置冰上裂解30 min，離心5 min去除細(xì)胞碎片，收集上清液，應(yīng)用BCA法測(cè)定蛋白濃度后取30 μg蛋白樣品行蛋白質(zhì)印跡法檢測(cè)細(xì)胞Beclin 1蛋白表達(dá)，以β-actin為內(nèi)參?？笲eclin 1一抗1∶1 000稀釋，辣根過(guò)氧化物酶標(biāo)記的二抗 1∶2 000稀釋，最后ECL發(fā)光，X線片曝光、顯影、定影。通過(guò)Image J軟件獲取條帶灰度值，以目的條帶與內(nèi)參條帶的灰度值比表示蛋白相對(duì)表達(dá)量。

五、統(tǒng)計(jì)學(xué)處理

結(jié) 果

一、缺氧培養(yǎng)對(duì)PANC1細(xì)胞HIF1A-AS1表達(dá)的影響

以對(duì)照組表達(dá)量為1，缺氧培養(yǎng)3、6、12、24、36、48 h的PANC1細(xì)胞的HIF1A-AS1表達(dá)量分別為1.37±0.21、1.96±0.27、2.22±0.16、3.46±0.54、4.26±0.27和3.04±0.23。HIF1A-AS1的表達(dá)隨缺氧時(shí)間的延長(zhǎng)而增加，在36 h時(shí)達(dá)峰值。缺氧6 h以上各組HIF1A-AS1的表達(dá)量均顯著高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。

二、過(guò)表達(dá)及低表達(dá)HIF1A-AS1細(xì)胞鑒定

以對(duì)照組表達(dá)量為1，過(guò)表達(dá)組、低表達(dá)組細(xì)胞HIF1A-AS1的表達(dá)量分別為4.49±0.53、0.49±0.07。過(guò)表達(dá)組顯著高于對(duì)照組，低表達(dá)組顯著低于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。

三、HIF1A-AS1對(duì)缺氧培養(yǎng)的PANC1細(xì)胞凋亡的影響

對(duì)照組、過(guò)表達(dá)組、低表達(dá)組PANC1細(xì)胞經(jīng)缺氧培養(yǎng)24 h后的細(xì)胞凋亡率分別為(8.27±1.28)%、(6.56±1.49)%、(19.9±2.34)%，過(guò)表達(dá)組顯著低于對(duì)照組，低表達(dá)組顯著高于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01，圖1)。

圖1 缺氧培養(yǎng)24 h后對(duì)照組(1A)、過(guò)表達(dá)組(1B)、低表達(dá)組(1C)PANC1細(xì)胞的凋亡率

四、HIF1A-AS1對(duì)缺氧培養(yǎng)的PANC1細(xì)胞自噬標(biāo)志蛋白Beclin 1表達(dá)的影響

常氧培養(yǎng)24 h后，對(duì)照組、過(guò)表達(dá)組、低表達(dá)組PANC1細(xì)胞Beclin 1的表達(dá)量分別為0.15±0.08、0.31±0.15、0.13±0.11，過(guò)表達(dá)組明顯高于對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義(P<0.01)，而低表達(dá)組與對(duì)照組的差異無(wú)統(tǒng)計(jì)學(xué)意義。缺氧培養(yǎng)24 h后，對(duì)照組、過(guò)表達(dá)組、低表達(dá)組細(xì)胞的Beclin 1表達(dá)量分別為1.05±0.11、1.29±0.19、0.38±0.18，過(guò)表達(dá)組顯著高于對(duì)照組，低表達(dá)組顯著低于對(duì)照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01，圖2)。

圖2 對(duì)照組(1)、低表達(dá)組(2)、過(guò)表達(dá)組(3)常氧(2A)及缺氧(2B)培養(yǎng)24 h后PANC1細(xì)胞Beclin 1蛋白表達(dá)

討 論

腫瘤細(xì)胞的高代謝和快速增殖狀態(tài)引起腫瘤組織局部微環(huán)境的氧含量持續(xù)降低，造成腫瘤內(nèi)部的缺氧微環(huán)境。缺氧微環(huán)境引起缺氧相關(guān)轉(zhuǎn)錄因子的表達(dá)，進(jìn)而誘導(dǎo)多種信號(hào)通路激活，不僅提高其惡性程度及轉(zhuǎn)移能力，還抑制針對(duì)腫瘤細(xì)胞的免疫反應(yīng)，促使腫瘤細(xì)胞自身發(fā)生改變以逃避免疫細(xì)胞的攻擊[10-12]。因此，缺氧微環(huán)境下腫瘤細(xì)胞基因表達(dá)調(diào)控機(jī)制研究將為其靶向治療提供理論和實(shí)驗(yàn)依據(jù)。

自噬是一種溶酶體依賴的真核細(xì)胞自身降解機(jī)制，在細(xì)胞功能維持穩(wěn)定方面具有重要作用[13]。近年來(lái)的研究發(fā)現(xiàn)自噬與腫瘤發(fā)展和耐藥機(jī)制密切相關(guān)。自噬的“雙刃劍”作用，對(duì)耐藥的腫瘤細(xì)胞具有雙面作用。適量的自噬能夠促使耐藥細(xì)胞生存，而過(guò)度自噬能促使耐藥腫瘤細(xì)胞死亡[14]。研究發(fā)現(xiàn)胰腺癌細(xì)胞在缺氧微環(huán)境下通過(guò)氧化應(yīng)激誘導(dǎo)黏蛋白-4(MUC4)降解，從而促進(jìn)細(xì)胞自噬、增強(qiáng)細(xì)胞存活[15]。同時(shí)，自噬過(guò)程中溶酶體通過(guò)調(diào)控基因轉(zhuǎn)錄誘導(dǎo)胰腺癌細(xì)胞代謝形式發(fā)生改變[16]。但胰腺癌細(xì)胞自噬調(diào)控的具體作用機(jī)制還在初步研究階段。

研究發(fā)現(xiàn)，lncRNA作為具有重要調(diào)控作用的一類非編碼RNA，在多種腫瘤的發(fā)生、發(fā)展和轉(zhuǎn)移的過(guò)程中表達(dá)明顯異常，提示其可能參與腫瘤的發(fā)病過(guò)程[17]。HIF1A-AS1位于人的14號(hào)染色體HIF1α的反義鏈上，其成熟體長(zhǎng)度為652 nt。近期有研究顯示在胸主動(dòng)脈瘤組織中HIF1A-AS1表達(dá)明顯增加，HIF1A-AS1可能通過(guò)促進(jìn)平滑肌細(xì)胞凋亡，調(diào)控胸主動(dòng)脈瘤的發(fā)生發(fā)展進(jìn)程[18]。本研究結(jié)果顯示HIF1A-AS1的水平與缺氧誘導(dǎo)的胰腺癌細(xì)胞自噬活性明顯相關(guān)，抑制HIF1A-AS1能夠顯著降低缺氧誘導(dǎo)的胰腺癌細(xì)胞自噬水平，但其具體調(diào)控機(jī)制還需深入探索。

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(本文編輯：冀凱宏)

Regulatory role of long-chain non-coding RNA HIF1A-AS1 on hypoxia-induced the autophagy of pancreatic cancer PANC1 cells

XuFeng,XuYuemei,XiaFeizhen,JiangYuhua,ZhangBo,LiXianpeng.

DepartmentofGastroenterology,YinzhouHospital,MedicalSchool,NingboUniversity,Ningbo315040,China

LiXianpeng,Email：849840290@qq.com

Objective To observe the regulatory role of long non-coding RNA HIF1A-AS1 on the autophagy of pancreatic cancer PANC1 cells induced by hypoxia. Methods The pancreatic cancer PANC1 cells were cultured in a three-gas incubator filled with hypoxic gas mixture (94% N2，5% CO2，1% O2) for 3, 6, 12, 24, 36 and 48 h. HIF1A-AS1 overexpression and low expression PANC1 cells were obtained by the infection of recombinant adenovirus carrying HIF1A-AS1 and the transfection of HIF1A-AS1 targeting siRNA by liposome, and routinely cultured PANC1 cells served as control. The expression of HIF1A-AS1 of PANC1 cells was detected by real-time quantitative PCR after being cultured in hypoxia-induced condition for 24 h. The apoptosis rate was detected by flow cytometry. The autophagy related proteins Beclin 1 were detected by western blot. Results The expression of HIF1A-AS1 in hypoxic cells was increased as the hypoxic time increased since 6 h and peaked at 36 h, which was significantly higher than that in control group (P<0.01). HIF1A-AS1 relative expression in HIF1A-AS1 overexpression and low expression PANC1 cells was 4.49±0.53 and 0.49±0.07, which were normalized to that of control group with the relative expression of 1. Control group had lower HIF1A-AS1 expression than HIF1A-AS1 overexpression PANC1 cells but higher HIF1A-AS1 in HIF1A-AS1 low expression PANC1 cells, and the differences were statistically significant (P<0.01).The cell apoptosis rate of control, HIF1A-AS1 overexpression and low expression PANC1 cells was (8.27±1.28)%, (6.56±1.49)% and (19.9±2.34)% after 24 h hypoxic culture. Control group had higher HIF1A-AS1 expression than HIF1A-AS1 overexpression PANC1 cells but lower HIF1A-AS1 in HIF1A-AS1 low expression PANC1 cells, and the differences were statistically significant (P<0.01). The expression of Beclin 1 protein was protein 1.05±0.11, 1.29±0.19 and 0.38±0.18, respectively. Control group had lower Beclin 1 expression than HIF1A-AS1 overexpression PANC1 cells but higher Beclin 1 in HIF1A-AS1 low expression PANC1 cells, and the differences were statistically significant (P<0.01). Conclusions HIF1A-AS1 can promote autophagy of pancreatic cancer PANC1 cells induced by hypoxia and participate in the pathogenesis and metastasis of pancreatic cancer.

Pancreas; Cell line, tumor; Long non-coding RNA; HIF1A-AS1; Autophagy; Anoxia

10.3760/cma.j.issn.1674-1935.2017.03.008

315040 寧波，寧波大學(xué)醫(yī)學(xué)院附屬鄞州醫(yī)院消化科

李先鵬，Email: 849840290@qq.com

浙江省自然科學(xué)基金(LY16H160004)

2017-01-04)