Abstract
目的
研究miR-424通过调控ATG14表达影响自噬和肝癌细胞增殖。
方法
收集肝癌患者因手术切除的肝癌组织及其癌旁组织，采用qRT-PCR法检测肝癌组织中miR-424-5p，ATG14的表达。培养人肝癌细胞系HepG2、SMMC-7721、Huh-7、MHCC97H、HCCLM3和人正常肝细胞LO2，qRT-PCR法检测上述各细胞系的miR-424-5p表达，选取表达量较高的Huh-7和表达量较低的HCCLM3肝癌细胞为研究对象，Huh-7细胞实验分组（n=3）：干扰miR-424-5p表达阴性对照组（in-NC组），干扰miR-424-5p表达组（in-miR-424-5p组）；HCCLM3细胞实验分组（n=3）：过表达miR-424-5p组（mi-miR-424-5p组），另设过表达miR-424-5p阴性对照组（mi-NC组）；过表达miR-424-5p阴性对照+过表达ATG14阴性对照组（mi-NC+NC组）；过表达miR-424-5p阴性对照+过表达ATG14组（mi-NC+ ATG14组）；过表达miR-424-5p+过表达ATG14阴性对照组（mi-miR-424-5p+NC组）；过表达miR-424-5p+过表达ATG14组（mi-miR-424-5p+ATG14组）。qRT-PCR法及Western blot法验证各组miR-424-5p和ATG14的转染情况。采用MTT法检测各组细胞的细胞增殖，流式细胞术检测各组细胞的细胞凋亡水平，Western blot检测各组细胞的ATG14和自噬相关蛋白LC3、Beclin1和P62的表达水平，双荧光素酶报告基因实验检测miR-424-5p和ATG14之间的相互作用。
结果
在肝癌组织和细胞中ATG14高表达，miR-424-5p低表达。与相应对照组相比，过表达miR-424-5p抑制肝癌细胞增殖和促进凋亡（P < 0.05）；干扰miR-424-5p表达及过表达ATG14促进肝癌细胞增殖和抑制凋亡（P < 0.05）；miR-424可能通过靶向ATG14发挥抑癌的作用。干扰miR-424-5p表达及过表达ATG14均能提高肝癌细胞自噬体标志蛋白LC3-ΙΙ/LC3-Ι、Beclin1的表达水平（P < 0.05），降低自噬受体蛋白P62的表达水平（P < 0.05）；过表达miR-424-5p则降低肝癌细胞自噬体标志蛋白LC3-ΙΙ/ LC3-Ι、Beclin1的表达水平（P < 0.05），提高自噬受体蛋白P62的表达水平（P < 0.05）。
结论
miR-424调控ATG14表达影响自噬，从而抑制肝癌细胞增殖。
Keywords: 肝癌, miR-424-5p, ATG14, 自噬, 细胞增殖, 细胞凋亡
Abstract
Objective
To determine whether miR-424 affects cancer cell proliferation and autophagy through ATG14 in hepatocellular carcinoma (HCC) cells.
Methods
We detected miR-424-5p and ATG14 expression levels in surgical specimens of HCC and adjacent tissues and in different HCC cell lines (HepG2, SMMC-7721, Huh-7, MHCC97H, and HCCLM3) and normal human hepatocyte LO2 cells using qRT-PCR and Western blotting. In the cell transfection experiments, we observed the effects of miR-424-5p knockdown in Huh-7 cells and the effects of overexpression miR-424-5p and ATG14 in HCCLM3 cells on the proliferation, cell cycle, apoptosis and expression levels of autophagy-related proteins (LC3, Beclin1 and p62). Dual luciferase reporter assay was used to verify the possible interaction between miR-424-5p and ATG14.
Results
In HCC tissues and cells, ATG14 was highly expressed and miR-424-5p expression was downregulated. In HCC cells, overexpression of miR-424-5p obviously suppressed cell proliferation and promoted cell apoptosis (P < 0.05), while inhibiting miR-424-5p or overexpressing ATG14 significantly promoted cell proliferation and inhibited cell apoptosis (P < 0.05). Dual luciferase reporter assay indicated that miR-424-5p inhibits HCC cells by targeting ATG14. In addition, inhibition of miR-424-5p and overexpression of ATG14 both enhanced the expressions of LC3-ΙΙ/LC3-Ι and Beclin1 and decreased p62 expression (P < 0.05), but miR-424-5p overexpression reduced the expressions of LC3-ΙΙ/LC3-Ι and Beclin1 and increased p62 expression (P < 0.05).
Conclusion
MiR-424 inhibits HCC cell autophagy and proliferation through regulating ATG14.
Keywords: hepatocellular carcinoma, miR-424-5p, ATG14, autophagy, proliferation, apoptosis
肝细胞癌（HCC）是原发性肝癌的主要组织学形式，其发病率占肝癌的70%~90%[1]。截至2018年，肝癌在全世界上常见的癌症中排第6，2018年肝癌相关死亡人数约为78.1万[2]。HCC的高转移率和复发率导致HCC的5年生存率很低[3]。因此研究HCC发生及其生殖调控机制对于肝癌的防治具有十分重要的意义。
自噬是一个高度保守的分解代谢过程，通过退化受损的细胞器和蛋白质来维持细胞内稳态[4]。同时在HCC中发挥重要作用[5]。近年来，有关自噬与肝癌的研究越来越多[6, 7]。自噬相关基因（ATGs）是启动自噬的关键基因，ATG14作为PI3K复合物的特异亚基，被认为是肝癌细胞自噬的关键调节因子[8]。这提示ATG14介导的肝癌细胞自噬可作为治疗肝癌的潜在靶点。
研究发现miRNA的表达与多种恶性肿瘤相关，可影响肿瘤细胞的增殖、凋亡、分化、侵袭和转移[9, 10]。且有文献报道，miR-424-5p在肝癌的发生发展中具有重要作用[11]，但其是否通过细胞自噬的作用肝癌细胞尚未被研究。
本研究以肝癌组织中低表达的miR-424为主要研究靶点，构建miR-424过表达和敲降的肝癌细胞模型，观察miR-424对ATG14表达、自噬和肝癌细胞增殖的影响，从而为研究抑制肝癌细胞的生长、肝癌的有效防治奠定理论基础。
1. 材料和方法
1.1. 方法
1.1.1. 临床资料
在中南大学湘雅三医院进行部分肝切除术的HCC患者中，收集36例肝癌患者因手术切除的肝癌组织及癌旁组织，所有样本的获得及后续的实验研究均通过我院伦理审查委员会审查和患者或家属的知情同意。所有样本离体后迅速放入液氮中保存，用于后续检测miR-424-5p，ATG14的表达。患者入组要求：所有患者在手术前均未接受过放化疗、射频治疗或生物治疗等治疗措施，术后病理学诊断为原发性HCC。
1.1.2. 术后随访及生存分析
采用电话和门诊复诊方式随访，患者死于肝癌为终点事件，死于其他疾病、失访或至随访结束时患者仍存活为截尾事件。随访指标为患者术后临床检验指标和患者生存情况，Kaplan-Meier法绘制生存曲线。
1.1.3. 细胞培养
人肝癌细胞系HepG2、SMMC-7721、Huh-7、MHCC97H、HCCLM3和人正常肝细胞LO2购买中国科学院上海细胞生物研究所。所有细胞系在含10%胎牛血清的DMEM培养基（Gibco, Grand Island, NY, USA）中培养，培养基中添加1 %青霉素/链霉素，细胞置于37 ℃、5% CO2条件下培养。
1.1.4. 细胞转染
miR-424-5p mimic和miR-424-5p inhibitor（50 mg）及其阴性对照，ATG14过表达载体质粒（pcDNA3.1-ATG14，2 μg）及其阴性对照均购买自吉玛基因公司（上海）。选取对数生长期的Huh-7、HCCLM3肝癌细胞，按照2×105/孔密度接种于24孔培养板中，待细胞密度达50 %~70 %时，更换为不含胎牛血清的RPMI 1640培养基。Huh-7细胞实验分组（n=3）：干扰miR-424-5p表达阴性对照组（in-NC组）：转染miR-424-5p抑制剂的阴性对照；干扰miR-424-5p表达组（in-miR-424-5p组）：转染miR-424-5p抑制剂；HCCLM3细胞实验分组（n=3）实验分组：过表达miR-424-5p阴性对照组（mi-NC组）：转染miR-424-5p模拟剂的阴性对照；过表达miR-424-5p组（mi-miR-424-5p组）：转染miR-424-5p模拟剂、；in-NC组、in-miR-424-5p组、过表达miR-424-5p阴性对照+过表达ATG14阴性对照组（mi-NC+NC组）：转染miR-424-5p模拟剂的阴性对照和过表达ATG14阴性对照质粒；、过表达miR-424-5p阴性对照+过表达ATG14组（mi-NC+ATG14组）：转染miR-424-5p模拟剂的阴性对照和过表达ATG14质粒；、过表达miR-424-5p+过表达ATG14阴性对照组（mi-miR-424-5p+NC组）：转染miR-424-5p模拟剂和过表达ATG14阴性对照质粒；、过表达miR-424-5p+过表达ATG14组（mi-miR-424-5p+ATG14组）：转染miR-424-5p模拟剂和过表达ATG14质粒。转染使用Lipofectamine 2000试剂（Invitrogen, Carlsbad, CA, USA），按说明书的操作进行。
1.1.5. qRT-PCR
使用TRIZOL（Invitrogen）提取总RNA。mRNA利用逆转录试剂盒（TaKaRa）进行逆转录，mi RNA逆转录使用mi Script逆转录试剂盒（Qiagen）合成。所有操作按试剂盒的使用说明进行。使用LightCycler 480（Roche）荧光定量PCR仪检测基因的表达，反应条件按荧光定量PCR试剂盒（SYBR Green Mix, Roche）的操作说明进行。热循环参数为：95 ℃ 10 s，然后95 ℃ 5 s，60 ℃ 10 s，72 ℃ 10 s，共45个循环；最后72 ℃延伸5 min。定量PCR每个反应设置3个重复。miRNA内参用U6，mRNA内参用GAPDH。数据分析采用2-ΔΔCt法，ΔΔCt=实验组（Ct目标基因-Ct内参）-对照组（Ct目标基因-Ct内参）。各基因及其内参的扩增引物序列详见表 1。
1.
引物序列
Primer sequences
| Name of primer | Sequences |
| miR-424-5p-F | CAGCAGCAATTCATGT |
| miR-424-5p-R | TGGTGTCGTGGAGTCG |
| U6-F | CTCTCGCTTCGGCAGCACA |
| U6-R | ACGCTTCACGAATTTGCGT |
| ATG14-F | CGTCTACTTCGACGGCCGCGA |
| ATG14-R | CTCTTGGTGCCGTTGTGCTCG |
| GAPDH-F | AGTCCACTG GCGTCTTCA |
| GAPDH-R | GAGTC CTTCCACGATACCAA |
1.1.6. MTT法检测细胞增殖情况
将各组分别培养24、48、72 h后，每孔加MTT液（5 mg/ mL，Merck KGaA）20 μL，37 ℃，5%CO2培养箱孵育4 h，终止培养，弃去培养液。每孔再加入DMSO，剂量为150 μL，轻匀摇动10 min，促进结晶溶解。在酶联免疫检测仪上450 nm波长下测定各孔的吸光度值（A450 nm）；将吸光度值设置为纵坐标，时间设置为横坐标后绘制MTT曲线图。每组的吸光度值重复测量3次取平均值。
1.1.7. 流式细胞术检测细胞凋亡水平
将细胞制成单细胞悬液，2000 r/min离心收集，PBS洗2次后，重悬于结合缓冲液中，在含有约105细胞的195 μL细胞悬液中加入5 μLAnnexin-V-FITC染色液和PI染色液，混合均匀，避光孵育10 min后使用流式细胞仪（BD Biosciences）检测凋亡率。
1.1.8. Western blot检测相关蛋白表达
使用RIPA裂解液（碧云天）裂解细胞，获得蛋白样品。利用BCA试剂盒（碧云天）测量蛋白浓度后，取相应体积的蛋白加入上样缓冲液（碧云天）混匀，沸水浴加热3 min，使蛋白变性。80 V电泳30 min，待溴酚蓝进入分离胶后改用120 V，电泳1~2 h。转膜在冰浴中进行，转膜电流为300 mA，时间为60 min。转膜后把膜放入洗涤液中漂洗1~2 min，再将膜放入封闭液中室温封闭60 min，或者4 ℃封闭过夜。室温下，摇床上孵育一抗（GAPDH（5174S，1∶1000）、LC3-Ⅰ/LC3-Ⅱ（12741S，1∶1000）、Beclin1（3495S，1∶1000）、p62（88588S，1∶1000）、购于Cell Signaling, Boston, USA和ATG14（ab241298，1∶ 1000）购于abcam，CA，USA）1 h，洗涤液洗涤3次，每次10 min。转入二抗（辣根过氧化物酶标记的山羊抗兔IgG，1∶5000，北京康维世纪生物科技有限公司）中，室温下孵育1 h，洗涤3次，每次10 min。在膜上滴加显影液后，利用化学发光成像系统（Bio-rad）进行检测。
1.1.9. 双荧光素酶报告基因实验
通过StarBase工具（<a href="http://starbase.sysu.edu.cn/" target="_blank">http://starbase.sysu.edu.cn/</a>）预测miR-424-5p与ATG14结合的位点。进入StarBase首页，点击miRNA-mRNA工具栏，随后在target该栏输入ATG14，确定后，点击“Quick Search”，随后在Search这栏输入miR-424-5p，结果表明miR-424-5p与ATG14存在结合位点。根据预测的结果，分别设计和合成结合位点的野生序列和突变序列（wt-ATG14和mut-ATG14）。分别将结合位点的野生序列和突变序列插入荧光素酶报告基因载体（pGL3-Basic）中，再分别与miR-424-5p mimic（50 nmol/L）或阴性对照（mimic NC）共转染HCCLM3细胞。转染后使用Promega GLOMAX检测仪器（Promega）检测各组Firefly荧光素酶活性和Renilla荧光素酶活性，Renilla荧光素酶活性作为内参，Firefly荧光素酶与Renilla荧光素酶活性的比值为荧光素酶的相对活性。
1.2. 统计学分析
用GraphPad prism7软件进行统计学分析，所有数据用均数±标准差表示。两组之间的比较采用t检验，多组之间的比较采用单因素方差分析，相关性采用Pearson相关分析，Kaplan-Meier用于生存分析。P < 0.05为差异具有统计学意义。
2. 结果
2.1. miR-424-5p在肝癌组织或细胞中低表达情况
2.1.1. miR-424-5p在肝癌组织中低表达
对36个肝癌组织和相匹配的癌旁样本中miR-424-5p的检测发现：miR-424-5p在肝癌组织中的表达显著下调（图 1A，P= 0.002）；进一步根据肝癌组织miR-424-5p平均相对表达量，将患者分为高、低两表达组，低表达miR-424-5p与肿瘤大小，HBV感染，AFP含量，TNM分级有关，和年龄、性别无关（表 2）。随访通过生存分析比较了两组患者五年生存率的差异，与miR-424-5p高表达的患者的生存率相比，miR-424-5p低表达的患者在五年内的生存率显著降低（图 1B，P=0.04）。
1.
肝癌组织及癌旁组织中miR-424-5p的表达水平
Quantification of miR-424-5p expressions in hepatocellular carcinoma tissues and adjacent tissues. A: miR-424-5p expression in hepatocellular carcinoma tissues and adjacent tissues. B: Kaplan-Meier survival analysis; **P < 0.01.
2.
miR-424-5p的表达与HCC患者临床特征的关系
Association of miR-424-5p expression with clinicopathological characteristics of the patients with hepatocellular carcinoma
| Characteristics | n=36 | miR-424-5p | P | |
| High(18) | Low (18) | |||
| HBV: Hepatitis B; APF: Alpha fetoprotein. Chi-square test was used for statistical analysis. | ||||
| Age (year) | 0.738 | |||
| ≥60 | 20 | 9 | 11 | |
| < 60 | 16 | 9 | 7 | |
| Gender | 0.724 | |||
| Male | 24 | 13 | 11 | |
| Female | 12 | 5 | 7 | |
| Tumor size (cm) | 0.044 | |||
| ≥5 | 17 | 5 | 12 | |
| < 5 | 19 | 13 | 6 | |
| HBV infection | 0.012 | |||
| YES | 24 | 8 | 16 | |
| NO | 12 | 10 | 2 | |
| AFP (ng/mL) | 0.035 | |||
| ≥20 | 23 | 8 | 15 | |
| < 20 | 13 | 10 | 3 | |
| TNM classification | 0.045 | |||
| Ⅰ-Ⅱ | 31 | 18 | 13 | |
| Ⅲ-Ⅳ | 5 | 0 | 5 | |
2.1.2. miR-424-5p在肝癌细胞中低表达
qRT-PCR法检测了5个肝癌细胞系（HepG2、SMMC-7721、Huh-7、MHCC97H和HCCLM3）和人正常肝细胞（LO2）中miR-424-5p的表达，结果显示，miR-424-5p在5个肝癌细胞系中的表达均显著低于LO2细胞（P=0.02，P < 0.001），其中在Huh-7细胞中表达量最高，在HCCLM3细胞表达量最低，选择Huh-7和HCCLM3细胞进行后续实验（图 2）。
2.
miR-424-5p在不同肝癌细胞和正常肝细胞的表达比较
miR-424-5p expression in hepatocellular carcinoma cell lines and normal hepatocytes; *P < 0.05, ***P < 0.001.
2.2. miR-424-5p抑制自噬和肝癌生长
2.2.1. qRT-PCR检测
qRT-PCR检测Huh-7细胞和HCCLM3细胞中miR-424-5p表达水平，结果显示：在HCCLM3细胞中mi-miR-424-5p组miR-424-5p的表达量均显著高于mi-NC组（P < 0.001）；在Huh-7细胞中inmiR-424-5p组miR-424-5p的表达量均显著低于in-NC组（P < 0.001，图 3）。
3.
转染miR-424-5p mimic/inhibitor肝癌细胞中miR-424-5p的表达水平
Expression of miR-424-5p after the transfection with miR-424-5p mimic or inhibitor. A: qRTPCR for detecting the expression of miR-424-5p after the transfection with miR-424-5p mimic; B: qRT-PCR for detecting the expression of miR-424-5p after the transfection with miR-424-5p inhibitor. ***P < 0.001.
2.2.2. MTT法检测
采用MTT法检测细胞增殖活力，结果显示在HCCLM3细胞中转染miR-424-5p mimic后，与相应阴性对照组相比，mi-miR-424-5p组细胞增殖显著被抑制（P=0.02），在Huh-7细胞中转染miR-424-5p inhibitor后，与相应阴性对照组相比，mi-miR-424-5p组细胞增殖均明显增强（P=0.02，图 4）。
4.
MTT法检测不同miR-424-5p表达水平的HCCLM3细胞和Huh-7细胞的的增殖情况
MTT assay for assessing proliferation of MCF-7 and MDA-MB-231 cells with miR-4443 overexpression or knockdown. A: Proliferation of HCCLM3 cells. B: Proliferation of Huh-7 cells. *P < 0.05.
2.2.3. 流式细胞术检测
采用流式细胞术检测细胞凋亡发现mi-miR-424-5p组中HCCLM3细胞凋亡水平显著高于mi-NC组（P=0.02），in-miR-424-5p组中Huh-7细胞凋亡水平显著低于in-NC组（P=0.008，图 5）。
5.
流式细胞术检测不同miR-424-5p表达水平的HCCLM3和Huh-7细胞的凋亡率
Flow cytometric analysis of apoptosis of HCCLM3 (A) and Huh-7 (B) cells after miR-424-5p overexpression or knockdown. *P < 0.05, **P < 0.01.
2.2.4. Western blot检测
HCCLM3细胞：与mi-NC组比较：mi-miR-424-5p组自噬体标志蛋白LC3-ΙΙ/LC3-Ι、Beclin1的表达水平显著降低（P=0.14，P=0.008），自噬受体蛋白P62的含量显著升高（P=0.004）。Huh-7细胞：与in-NC比较，in-miR-424-5p组自噬体标志蛋白LC3-ΙΙ/LC3-Ι、Beclin1的表达水平显著升高（P=0.006，P=0.007），自噬受体蛋白P62的含量显著降低（P=0.004，图 6）。
6.
Western blot检测不同miR-424-5p表达水平的HCCLM3和Huh-7细胞中自噬相关蛋白LC3Ⅱ/LC3Ⅰ、Beclin1和P62的表达水平
Western blot analysis of the expressions of autophagy-related proteins LC3Ⅱ/LC3Ⅰ, Beclin1 and P62 in HCCLM3 (A) and Huh-7 (B) cells after miR-424-5p overexpression or knockdown. *P < 0.05, **P < 0.01.
2.3. ATG14是miR-424-5p在HCC中的下游靶点
2.3.1. ATG14在肝癌组织中高表达
通过生物信息学Starbase（<a href="http://starbase.sysu.edu.cn" target="_blank">http://starbase.sysu.edu.cn</a>）预测到ATG14可以与miR-424-5p结合，结合位点及突变序列(<xref ref-type="fig" rid="Figure7">图 7A</xref>)。评估36个肝癌组织和相匹配的癌旁样本中ATG14的水平，ATG14在肝癌中表达显著上调（<xref ref-type="fig" rid="Figure7">图 7B</xref>，<italic>P</italic>=0.005），进一步分析发现，ATG14与miR-424-5p水平呈负相关（<xref ref-type="fig" rid="Figure7">图 7C</xref>）。
7.
ATG14在肝癌组织中高表达
TG14 is overexpressed in hepatocellular tissues. A: StarBase was used to predict the binding of miR-424-5p with ATG14. B: qRTPCR analysis of the expression of ATG14 in hepatocellular carcinoma tissues and matched adjacent tissues. C: Spearman correlation analysis showing a negative correlation between miR-424-5p andATG14. **P < 0.01.
2.3.2. qRT-PCR及Western blot检测
qRT-PCR及Western blot法检测过表达和沉默miR-424-5p细胞中的ATG14表达，过表达miR-424-5p的HCCLM3细胞中ATG14表达明显下降（P=0.001，P=0.008），而in-miR-424-5p组中Huh-7细胞中ATG14表达明显升高（P= 0.001，P=0.006，图 8）。
8.
转染miR-424-5p inhibitor/mimic肝癌细胞中ATG14的表达水平
Expression of ATG14 in Huh-7 and HCCLM3 cell lines after transfection with miR-424-5p inhibitor or mimic. qRTPCR (A, C) and Western blotting (B, D) were used to detect the expression ofATG14 in the cells. **P < 0.01.
2.3.3. 双荧光素酶报告实验
双荧光素酶报告基因实验结果显示，mut-ATG14组与miR-424-5p共转染后荧光素酶活性没有变化，而wt-ATG14组与miR-424-5p mimic共转染后荧光素酶活性显著降低，wt-ATG14组与miR-424-5p inhibitor共转染后荧光素酶活性显著升高（P=0.03，P=0.02，图 9）。
9.
双荧光素酶报告基因活性检测
Dual luciferase reporter assay for determine the targeting relationship between miR-424-5p and ATG14. *P < 0.05.
2.4. miR-424-5p通过ATG14抑制自噬和肝癌生长
2.4.1. qRT-PCR及Western blot检测
与mi-NC+NC组相比，mi-NC+ATG14组细胞中ATG14的表达水平明显增加（P=0.003，P=0.006），而mi-miR-424-5p+NC组细胞中ATG14的表达水平明显降低（P=0.001，P=0.009），而mi-miR-424-5p+ATG14组较mi-miR-424-5p+NC组细胞中ATG14的表达水平明显增加（P=0.003，P= 0.007，图 10）。
10.
各组HCCLM3细胞中ATG14的表达水平
Expression ofATG14 in HCCLM3 cells in each group detected by qRT-PCR (A) and Western blotting (B). **P < 0.01.
2.4.2. MTT法检测
与mi-NC+NC组比较：mi-NC+ ATG14组细胞增殖明显增强（P=0.02），mi-miR-424-5p+NC组细胞增殖明显被抑制（P=0.03）；mi-miR-424-5p+ATG14组较mi-miR-424-5p+NC组增殖明显增强（P=0.03，图 11）。
11.
MTT法检测各组肝癌细胞的增殖情况
Cancer cell proliferation detected by MTT assay. *P < 0.05.
2.4.3. 流式细胞术检测
与mi-NC+NC组相比，mi-NC+ATG14组细胞凋亡明显减少（P=0.02），而mi-miR-424-5p+NC组细胞凋亡明显增加（P=0.008），而mi-miR-424-5p+ATG14组较mi-miR-424-5p+NC组凋亡明显减少（P=0.02，图 12）。
12.
流式细胞术检测各组肝癌细胞凋亡情况
Flow cytometric analysis of HCC cell apoptosis in each group. *P < 0.05, **P < 0.01.
2.4.4. Western blot检测
与mi-NC+NC组相比，miNC+ATG14组自噬体标志蛋白LC3-ΙΙ/LC3-Ι，Beclin1的表达水平显著升高（P=0.008，P=0.006），而自噬受体蛋白P62的含量显著降低（P=0.009），而mi-miR-424-5p+NC组自噬体标志蛋白LC3-ΙΙ/LC3-Ι，Beclin1的表达水平显著降低（P < 0.001，P=0.003），而自噬受体蛋白P62的含量显著升高（P=0.008），而mi-miR-424-5p+ ATG14组较mi-miR-424-5p+NC组自噬体标志蛋白LC3-ΙΙ/LC3-Ι，Beclin1的表达水平显著升高(P < 0.001，P=0.002)，而自噬受体蛋白P62的含量显著降低（P= 0.006，图 13）。
13.
Western blot检测自噬相关蛋白LC3II/LC3I、Beclin1和P62的表达水平
Expression of LC3II/LC3I, Beclin1 and P62 in HCC cells detected by Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001.
3. 讨论
miRNAs是一类具有调控功能的非编码RNA，长度在20~25个核苷酸，通过与信使RNA（mRNA）的3'非翻译区（3'UTR）结合，在转录和转录后水平上调控基因表达，在肿瘤中可以作为一个癌基因后抑癌基因发挥作用。许多miRNAs已被证明可以调节肿瘤的增殖能力，例如miR-7[12]、miR-19b-1-5p[13]、miR-206[14]。尽管有研究已经证明了miR-424-5p在HCC中的表达异常[15, 16]，且miR-424-5p与各种肿瘤的发生发展密切相关，比如口腔癌[17]、非小细胞肺癌[18]、直肠癌[19]等。且目前有报道表明miR-424-5p可以影响细胞周期，从而抑制肝癌细胞的增殖[20]，且miR-424-5p会影响肝癌细胞的活力，迁移侵袭与血管形成[21-23, 11]。但miR-424-5p如何影响自噬及其影响HCC发生发展中的机制尚不清楚。
在本研究中，肝癌患者癌组织中miR-424-5p表达显著下调，且miR-424-5p的表达与其预后密切相关。这意味着miR-424-5p显著参与HCC的进展。同时miR-424-5p在五个肝癌细胞系（HepG2、SMMC-7721、Huh-7、MHCC97H和HCCLM3）中的表达均显著低于LO2细胞。在HCCLM3和Huh-7肝癌细胞分别转染miR-424-5p mimic或miR-424-5p inhibitor，qRT-PCR法验证转染成功可供后续使用。另外本研究发现，miR-424-5p过表达显著抑制肝癌细胞增殖，促进细胞凋亡，而miR-424-5p低表达发挥了相反的作用。提示miR-424-5p可对肝癌发挥抑癌作用。
细胞自噬是一种“自我进食”现象，其通过将细胞内受损的细胞器和错误折叠的蛋白质靶向溶酶体降解从而维持细胞稳态。它可以通过维持氧化代谢或促进糖酵解来增强实体瘤缺氧区域的肿瘤细胞存活率，进而对肝癌的进展起促进作用。考虑到自噬在癌症中的作用[24]，我们推测miR-424-5p也可能影响肝癌细胞自噬。Beclin-1是编码调节自噬的蛋白质，充当PI3K复合物的核心亚基，在自噬中起核心作用，其表达水平可反映自噬[25]的发生。微管相关蛋白1，轻链3（LC3）是自噬小体膜上的标记蛋白，以LC3-Ⅰ和LC3-Ⅱ两种形式产生，介导微管和细胞骨架成分之间的物理相互作用。在自噬过程中，LC3-Ⅰ转化为LC3-Ⅱ并被招募为自噬小体，这是自噬小体形成的关键步骤。此时，P62作为LC3-Ⅱ的底物，与LC3-Ⅱ形成复合物，最终被自噬溶酶体降解[26]。因此，Beclin-1、LC3、p62是自噬最关键的蛋白，是常用的自噬标记物。在我们的研究中，在HCC细胞中，LC3-Ⅱ/LC3-Ⅰ和Beclin1水平与miR-424-5p表达呈负相关，而p62的表达随着miR-424-5p表达的增加而增加。由此可见，miR-424-5p抑制了肝癌细胞自噬。
哺乳动物自噬相关基因14（ATG14）又称为Barkor/Atg14（L），人源性ATG14由492氨基酸构成，其N端为半胱氨酸残基富集区域，哺乳动物中高度保守的半胱氨酸残基形成卷曲螺旋结构，该结构对磷脂酰肌醇3激酶（PI3K）复合物靶向内质网必不可少。ATG14是PI3K复合物中的一个特定亚基，它将PI3K复合物靶向自噬体形成的可能位点，从而对复合物进行分选，使PI3K复合物在自噬中发挥特定作用。ATG14在各种肿瘤细胞如胃癌、卵巢癌中通常会激活自噬[8, 27-29]，此外据报道，ATG14的下调显著抑制了肝癌细胞的增殖和迁移能力[30]。基于以上研究结果，我们进一步研究ATG14的上游调控因子。我们通过生物学在线预测软件预测到ATG14与miR-424-5p存在结合位点，据此可猜测miR-424-5p可能通过调控ATG14的表达影响自噬在肝癌细胞增殖过程中发挥调控作用。在本研究中，ATG14在HCC组织及细胞中被上调，此外，ATG14在HCC细胞中的表达与miR-424-5p的表达呈负相关。且通过qRT-PCR及Western blot证实miR-424-5p可以抑制肝癌细胞内源性ATG14的表达，这可能与肝癌细胞自噬和增殖密切相关。
本结果表明，与相应对照组相比，ATG14过表达促进肝癌细胞增殖，抑制细胞凋亡。ATG14过表达部分抑制了过表达miR-424-5p抑制的肝癌细胞增殖。miR-424-5p对肝癌细胞的凋亡有促进作用，而这种作用被过表达ATG14所抵消。更重要的是，过表达ATG14可促进miR-424-5p过表达抑制的肝癌细胞自噬。
综上所述，本研究证实了miR-424-5p靶向ATG14表达在HCC组织和细胞系中抑制自噬，从而抑制肝癌细胞增殖的可能作用机制，这可能是影响肝癌发生发展的一条重要通路，提示肝癌的发生、发展与miR-424-5p表达水平密切相关，miR-424-5p的表达水平的检测可以作为肝癌诊断的参考指标，对于临床上肝癌早期诊断和早期治疗提供了新的思考和方向。
Biography
赵梓丹，在读硕士研究生，E-mail: zidan7041@163.com
Contributor Information
赵 梓丹 (Zidan ZHAO), Email: zidan7041@163.com.
冯 超 (Chao FENG), Email: fengchao7626@163.com.
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