Abstract
目的
研究miR-607异常表达在肝细胞癌（HCC）中的临床意义及对肝癌细胞生长转移的影响。
方法
荧光定量PCR检测45对HCC及癌旁组织中miR-607表达，分析miR-607表达与患者临床病理特征间的相关性。通过转染miR-607 mimics提高HCC细胞（Huh-7和HCCLM3）内miR-607的表达水平。采用CCK-8、流式细胞仪、划痕愈合实验及transwell小室模型分别检测过表达miR-607对HCC细胞增殖、凋亡、迁移和侵袭的影响。双荧光素酶报告基因系统检测miR-607是否与潜在靶点TRPC5的mRNA 3'-非翻译区直接结合。Western blot检测过表达miR-607对HCC细胞内TRPC5、CCND1、MMP2表达及Akt磷酸化的影响。
结果
MiR-607在HCC组织及HCC细胞中的表达水平均降低（P < 0.05）；miR-607低表达与肿瘤直径>5 cm、血管侵犯及较晚TNM分期（Ⅲ+Ⅳ）密切相关（P < 0.05）。过表达miR-607抑制HCC细胞增殖、迁移、侵袭并诱导细胞凋亡（P < 0.05）。双荧光素酶报告基因系统证实miR-607与TRPC5 mRNA 3'-非翻译区直接结合（P < 0.05）。过表达miR-607抑制HCC细胞内TRPC5、CCND1及MMP2表达并下调Akt的磷酸化水平（P < 0.05）。
结论
miR-607低表达与肝细胞癌恶性临床特征密切相关，miR-607可能通过下调TRPC5表达进而抑制Akt通路激活来发挥抗HCC生长转移作用。
Keywords: miR-607, 肝细胞癌, TRPC5, Akt通路, 转移
Abstract
Objective
To investigate the clinical implications of abnormal expression of miR-607 in hepatocellular carcinoma (HCC) and its influence on HCC cell proliferation and migration.
Methods
The expression of miR-607 in 45 pairs of HCC and adjacent tissues were detected with real-time PCR, and the correlation between miR-607 expression and clinicopathological features of the patients was analyzed. The effects of transfection with miR-607 mimics on proliferation, apoptosis, migration and invasion of two HCC cell lines (Huh-7 and HCCLM3) were evaluated using CCK-8 assay, flow cytometry, wound-healing assay and Transwell assay. A dual-luciferase reporter system was used to detect the direct binding between miR-607 and 3'-UTR of TRPC5 mRNA. Western blotting was used to measure the expressions of TRPC5, CCND1, MMP2 and phosphorylated Akt in the HCC cells.
Results
The expression of miR-607 was significantly decreased in HCC tissues (P=0.029) and HCC cell lines (P < 0.05). In HCC patients, a low expression of miR-607 was associated with a larger tumor size (>5 cm, P=0.031), vascular invasion (P=0.027) and advanced TNM stages (Ⅲ + Ⅳ, P=0.015). In the two HCC cell line, overexpression of miR-607 significantly inhibited cell proliferation, migration, and invasion and enhanced cell apoptosis (P < 0.05). The results of dualluciferase reporter assay confirmed that TRPC5 was a direct target of miR- 607 in HCC cells. Overexpression of miR-607 significantly inhibited the expressions of TRPC5, CCND1, and MMP2 and suppressed Akt phosphorylation in HCC cells (P < 0.05).
Conclusion
A low expression of miR-607 in HCC is associated with poor clinicopathological phenotypes of HCC. Overexpression of miR-607 inhibits HCC growth and metastasis possibly by down- regulating TRPC5, which causes Akt signaling inactivation.
Keywords: miR-607, hepatocellular carcinoma, TRPC5, Akt signal pathway, metastasis
肝细胞癌（HCC）具有生长速度快、易发生脉管侵犯和肝内外转移的特点，目前已成为我国致死率最高的恶性肿瘤[1]。MicroRNA（miRNA, miR）是一种长度小于22个碱基的单链非编码RNA，miRNAs的异常表达在HCC发生发展中发挥关键作用[2]。MiR-607位于人类10号染色体，最早在抗埃博拉病毒相关miRNA中[3]被发现，随后miR-607被发现与神经精神疾病[4, 5]及多种癌症[6, 7]的进展密切相关。胰腺癌中，血清miR-607低表达患者出现淋巴结转移、肝脏转移和周围神经侵犯比例更高、预后更差[8]，体外过表达miR-607能够抑制胰腺癌细胞增殖和迁移[9]，提示miR-607在胰腺癌中具备液体活检标志物价值并能发挥抗癌作用。目前，仅通过测序发现HCC患者血清外泌体miR-607表达水平降低[10]，但HCC组织中miR-607的表达和具体生物学功能尚缺乏研究。因此，本研究拟通过探讨miR-607在HCC中异常表达的临床意义和调节HCC生长转移的功能机制，以期明确miR-607在HCC疾病进展中的作用。
1. 资料和方法
1.1. 资料
收集2019年1月1日~2021年1月1日于安康市人民医院普通外科行手术切除的HCC组织及对应癌旁组织标本45例，包括男性29例，女性16例，年龄50~77岁，中位年龄63岁。所有患者术前均未接受过靶向、介入、放化疗等辅助治疗并且均签署知情同意书，实验方案由西安交通大学第一附属医院伦理委员会审核批准（批号：2021伦审科字第347号）。MicroRNA模拟物（mimics，miR-607及阴性对照）、microRNA PCR扩增引物试剂盒（miR-607及U6）、野生型及突变型TRPC5 mRNA 3'-非翻译区（3'-UTR）双荧光素酶报告基因系统由广州锐博生物科技有限公司提供。LO2、HepG2、Hep3B、Huh-7及HCCLM3细胞由西安交通大学第一附属医院肝胆外科实验室保存。DMEM液体培养基及胎牛血清（Gibco）。RNA提取试剂Trizol、转染试剂LipofectamineTM 3000及RT-PCR试剂盒（Invitrogen）。SYBR Green实时荧光定量PCR试剂盒（BIO-RAD）。TRPC5、Akt、Ser473磷酸化Akt、CCND1、MMP2及GAPDH一抗（proteintech）。CCK-8试剂盒、流式细胞仪凋亡检测试剂盒（上海生工生物有限公司）。TRPC5引物（上游5'-GTTCTAGGTTTCATTTGGGGTGAGA- 3'；下游5'-ACATTTCCCATTCCTCCCTTGG-3）、' GA PDH引物（上游5'-CCAGGGCTGCTTTTAACTCT-3'；下游5'-GGACTCCACGACGTACTCA-3）' 购自上海生工生物有限公司。Transwell小室（CORNING），人工基质胶（B&D）。
1.2. 方法
1.2.1. 细胞培养与传代
正常人永生化肝细胞LO2及肝癌细胞HepG2、Hep3B、Huh-7及HCCLM3细胞株培养于含10%胎牛血清的DMEM培养基中，置于细胞培养箱中，在37 ℃、5% CO2及饱和湿度环境下进行培养传代。稳定传代2~3代后，取对数生长期细胞进行后续实验。
1.2.2. qRT-PCR检测miR-607及TRPC5 mRNA表达
使用Trizol试剂按操作指南提取临床标本组织或体外培养细胞中的总RNA。定量后，每份组织样品或细胞样品取等量RNA配制20 μL的逆转录反应体系合成cDNA。cDNA稀释10倍后取5 μL配制实时荧光定量PCR体系，按如下条件进行PCR反应：95 ℃预变性30 s，1循环；95 ℃变性5 s，60 ℃退火延伸30 s，40循环。以U6 RNA为miR-607内参，以GAPDH为TRPC5的内参，采用2-△△Ct法计算miR-607及TRPC5 mRNA的相对表达量。
1.2.3. miR-607 mimics细胞转染
HCC细胞以过夜培养达50%~70%之密度接种于6孔板中。miR-607 mimics转染分组：阴性对照组（miR-ctrl）加入100 pmoL/孔的阴性对照mimics及5 μL转染试剂，miR-607组（miR-607）加入100 pmoL/孔的miR-607 mimics及5 μL转染试剂；以无血清DMEM培养基调整终体积为2 mL/孔。按后续实验要求确认转染时间。
1.2.4. CCK-8检测细胞增殖
分别收集转染24、48 h及72 h后的HCC细胞，制备密度为1×105/mL的无血清DMEM基细胞悬液，以200 μL/孔接种于96孔板。加入10 μL/孔CCK-8 solution试剂后放置于细胞培养箱内避光培养2 h。于酶标仪上测定各孔450 nm的吸光度A450 nm。每个样本独立重复实验3次。
1.2.5. 流式细胞仪检测凋亡
取转染72 h后的HCC细胞，用1×Binding Buffer制备密度为1×106/mL的细胞悬液，每管加入500 μL细胞悬液、5 μLAnnexin V-FITC和10 μLPropidium Iodide。混匀后，室温下避光反应10 min，进行流式细胞仪检测。每组样本独立重复实验3次。
1.2.6. 划痕愈合实验检测细胞迁移
取转染24 h后的HCC细胞，以过夜培养达80%~90%之密度接种于6孔板中。过夜培养后用无菌200 μL移液器枪头沿培养孔中线划过制作初始划痕。以无血清DMEM培养基清洗培养孔以洗净悬浮细胞。每孔再次加入无血清DMEM培养基2 mL，将6孔板置于培养箱中继续培养48 h。在显微镜下拍照记录划痕剩余距离，计算细胞迁移情况。
1.2.7. Transwell小室模型检测细胞侵袭
用无血清DMEM培养基按1:8比例稀释50 mg/L的基质胶100 μL/孔覆盖transwell小室上室面，于37 ℃细胞培养箱内风干1 h制成侵袭小室模型。取转染48 h后的HCC细胞用无血清DMEM培养基重悬，以2×105/mL的密度加入100 μL/孔的细胞悬液。24孔细胞培养板内加入750 μL/孔含10%胎牛血清的DMEM培养基，建立细胞侵袭检测模型。培养完成后，PBS清洗小室、4%多聚甲醛固定，并用0.1%的结晶紫染色10 min。擦除上室面未成功穿过小室膜的细胞，显微镜下观察并计数下室面细胞数目。
1.2.8. 生物信息学分析
使用Targetscan网站（<a href="https://www.targetscan.org/" target="_blank">https://www.targetscan.org/</a>）确定miR-607与潜在靶基因TRPC5 mRNA 3'-UTR区的结合位点。
1.2.9. 双荧光素酶报告基因系统
Huh-7及HCCLM3细胞分别以1×105/孔种于24孔板中过夜培养，每种细胞均设置4个分组Wt-TRPC5质粒+ miR- ctrl mimics；Mut-TRPC5质粒+miR-ctrl mimics；Wt-TRPC5质粒+ miR-607 mimics；Mut-TRPC5质粒+miR-607 mimics。采用LipofectamineTM 3000将混合物转染入特定细胞中培养48 h后，按双荧光素酶检测试剂说明书进行荧光强度测定。
1.2.10. Western blot
提取转染48 h后的HCC细胞总蛋白并调齐所有样品的蛋白浓度。采用垂直电泳法，利用4%~10% SDS-PAGE凝胶按相对分子质量分离总蛋白。采用湿转法，按100 V转印2 h之条件将分离后的蛋白转移至NC膜上，5% BSA室温封闭NC膜1 h。裁取目的蛋白所在的位置条带，按1:1000比例配制TRPC5、Akt、Ser473磷酸化Akt、CCND1、MMP2及GAPDH一抗，4 ℃孵育过夜。PBS洗去未结合一抗后采用1:5000稀释的HRP标记山羊抗兔二抗室温孵育NC膜1 h。于暗室内，ECL法曝光目的蛋白，以条带灰度强度计算蛋白相对表达量。
1.3. 统计学分析
采用SPSS22.0统计软件进行分析，连续性资料以均数±标准差表示，两组间比较采用两独立样本t检验。非连续性资料结果采用Pearson卡方检验，以P < 0.05表示差异具有统计学意义。
2. 结果
2.1. miR-607在肝细胞癌中表达及临床意义
分析45对HCC及癌旁组织的miR-607表达，HCC组织中miR-607的平均表达水平低于对应癌旁组织（t=2.214，P=0.029，图 1A）。以HCC组织中miR-607平均表达量为截断值，将45例肝细胞癌患者分为miR-607高表达组（n=16）和miR-607低表达组（n=29）。miR-607低表达与肿瘤直径>5 cm（χ2=4.640，P=0.031）、血管侵犯（χ2=4.865，P=0.027）及较晚的TNM分期（Ⅲ + Ⅳ，χ2=5.940，P=0.015）密切相关（表 1）。在体外培养细胞中，与正常人永生化肝细胞LO2相比，所检测HCC细胞系中miR-607的表达水平均降低（HepG2：P=0.044，Hep3B：P=0.041，Huh-7：P=0.037，HCCLM3：P=0.031；图 1B）。选择Huh-7及HCCLM3细胞进行mimics转染以开展下一步实验，结果显示，与miR-ctrl组相比，转染miR-607 mimics提高了两种HCC细胞内miR-607的表达水平（Huh-7：P < 0.001，图 1C；HCCLM3：P=0.003，图 1D）。
1.
miR-607在HCC中的表达以及在Huh-7和HCCLM3细胞中过表达miR-607
Expression of miR-607 in HCC and overexpression of miR-607 in Huh-7 and HCCLM3 cells. A: Down-regulated expression of miR-607 in HCC tissues (*P < 0.05 vs tumor-adjacent tissues). B: miR-607 expression is lower in the 4 HCC cell lines (HepG2, Hep3B, Huh-7, and HCCLM3) than in LO2 cells (*P < 0.05 vs LO2). C, D: Transfection of miR-607 mimics increases miR-607 expression in Huh-7 (C) and HCCLM3 (D) cells (*P < 0.05 vs miR-ctrl group).
1.
miR-607异常表达在肝细胞癌中的临床意义
Correlation between miR-607 expression and clinicopathological features of HCC patients
| Clinical features | miR-607 expression | χ2 | P | ||
| Low (n=29) | High (n=16) | ||||
| Gender | Male | 19 | 10 | 0.041 | 0.840 |
| Female | 10 | 6 | |||
| Age (year) | < 60 | 8 | 9 | 3.604 | 0.058 |
| ≥60 | 21 | 7 | |||
| HBsAg | Negative | 7 | 6 | 0.896 | 0.344 |
| Positive | 22 | 10 | |||
| Liver cirrhosis | Absent | 9 | 9 | 2.732 | 0.098 |
| Present | 20 | 7 | |||
| Tumor size (cm) | ≤5 | 7 | 9 | 4.640 | 0.031 |
| > 5 | 22 | 7 | |||
| Vascular invasion | No | 10 | 11 | 4.865 | 0.027 |
| Yes | 19 | 5 | |||
| Edmondson-steiner grading | Ⅰ+Ⅱ | 11 | 10 | 2.501 | 0.114 |
| Ⅲ+Ⅳ | 18 | 6 | |||
| Serum AFP level (ng/mL) | < 400 | 13 | 11 | 2.371 | 0.124 |
| ≥400 | 16 | 5 | |||
| TNM stage | Ⅰ+Ⅱ | 9 | 11 | 5.940 | 0.015 |
| Ⅲ+Ⅳ | 20 | 5 | |||
2.2. 过表达miR-607抑制HCC细胞增殖并诱导凋亡
与miR-ctrl组相比，miR-607组Huh-7细胞的增殖活力在转染48 h后明显下降（P=0.037），并且在转染72 h后仍可检测出差异（P=0.023，图 2A）。同时，转染72 h后通过流式细胞仪分析发现，miR-607组细胞凋亡比例高于转染miR-ctrl组（P=0.005，图 2B）。过表达miR-607可抑制HCCLM3的增殖活力（48 h：P=0.045，72 h：P=0.025；图 2C）并促进细胞凋亡发生（P=0.004，图 2D）。
2.
过表达miR-607抑制HCC细胞增殖并促进凋亡
Overexpression of miR-607 inhibits proliferation and promotes apoptosis in HCC cells. A, C: Overexpression of miR-607 inhibits proliferation of Huh-7 (A) and HCCLM3 (C) cells. B, D: Overexpression of miR-607 promotes apoptosis of Huh-7 (B) and HCCLM3 (D) cells. *P < 0.05 vs miR-ctrl group.
2.3. 过表达miR-607抑制HCC细胞迁移和侵袭
细胞划痕愈合试验发现，与miR-ctrl组相较，miR- 607组中Huh-7细胞的迁移距离缩短（P=0.011，图 3A）。Transwell侵袭小室结果显示，miR-ctrl组Huh-7细胞穿膜数量少于miR-607组Huh-7细胞穿膜数量（P=0.005，图 3B）。过表达miR-607具有抑制HCCLM3细胞迁移（P=0.023，图 3C）和侵袭（P=0.003，图 3D）的效应。
3.
过表达miR-607抑制HCC细胞迁移和侵袭
Overexpression of miR-607 inhibits migration and invasion of HCC cells. A, C: Overexpression of miR-607 inhibits migration of Huh-7 (A) and HCCLM3 (C) cells. B, D: Overexpression of miR-607 inhibits invasion of Huh-7 (B) and HCCLM3 (D) cells. *P < 0.05 vs miR-ctrl group.
2.4. miR-607靶向TRPC5抑制Akt通路激活
在Starbase、miRDB、TargetScan数据库进行生物信息学检索发现，经典瞬时受体电位通道5（TRPC5）可能是miR-607的潜在靶点之一（图 4A）。采用双荧光素酶报告基因系统检测证实，miR-607能够与HCC细胞内TRPC5 mRNA的野生型3'-UTR区直接结合（Huh-7：P=0.004，HCCLM3：P=0.011；图 4B）。PCR及Western blot检测显示，过表达miR-607下调HCC细胞内TRPC5 mRNA（Huh-7：P < 0.001，HCCLM3：P=0.002；图 4C）及蛋白质（Huh-7：P=0.001，HCCLM3：P=0.013；图 4D）的表达水平。
4.
miR-607靶向TRPC5抑制Akt通路激活
miR-607 inhibits Akt pathway activation through targeting TRPC5. A: miR-607 was predicted to bind to TRPC5 mRNA 3'-UTR region by bioinformatics analysis. B: Overexpression of miR-607 inhibits luciferase activity of wild-type (Wt) but not mutant-type (Mut) 3'-UTR vectors of TRPC5 in HCC cells. C: Overexpression of miR-607 decreases TRPC5 mRNA expression in HCC cells. D, E: Total expressions of TRPC5, CCND1, MMP2 and phosphorylated Akt are decreased in Huh7 (D) and HCCLM3 (E) cells. *P < 0.05 vs miR-ctrl group.
与miR-ctrl组相比，过表达miR-607的HCC细胞内Akt的磷酸化水平下调（Huh-7：P=0.034，HCCLM3：P=0.041）；同时，增殖相关分子CCND1（Huh-7：P=0.026，HCCLM3：P=0.029）及侵袭相关分子MMP2（Huh-7：P=0.004，HCCLM3：P=0.001）的表达水平降低（图 4D、E）。
3. 讨论
MicroRNA异常表达与肿瘤生长转移[11, 12]和患者预后[13]密切相关，其中miR-607在非小细胞肺癌中表达下调并提示预后不良[14]；慢性淋巴细胞白血病中miR-607通过下调WNT信号通路受体FZD3表达发挥细胞周期阻滞和凋亡诱导作用[15]。本研究发现，miR-607在HCC组织中的表达水平显著下调，miR-607低表达与肿瘤体积增大、血管侵犯和较晚的临床分期等肿瘤生长转移特征密切相关；体外实验表明miR-607可以显著抑制HCC细胞的增殖、迁移、侵袭，并诱导细胞凋亡的发生。肿瘤中MicroRNA的表达下调大多与长链非编码RNA、环状RNA等其他内源性RNA的分子海绵的吸附效应有关[16]。宫颈癌中miR-607被lncRNATP73-AS1吸附降解，导致其对下游靶点CCND2的抑制作用减弱，引起肿瘤发生凋亡抵抗和侵袭转移[17]。在HCC中发现lncRNATP73-AS1表达显著升高[18]，并促进HCC增殖侵袭[19]和放疗抵抗[20]，上述结果从侧面揭示了本研究所发现的miR-607低表达原因。
TRPC5是一种以同源四聚体形式锚定于细胞膜上的Ca2+离子通道[21]，开放后可介导细胞Ca2+内流[22]。据报道，TRPC5作为促癌因子在肺癌[23]、胃癌[24]中表达升高并与肿瘤生长转移和患者不良预后相关。生物信息学分析推断TRPC5可能是miR-607的潜在靶点之一。我们的研究通过双荧光素酶报告基因证实miR-607可与TRPC5的mRNA 3'-UTR区直接结合，PCR及Western blot结果也表明miR-607能下调TRPC5表达，说明TRPC5是miR-607的靶基因之一。
在肺鳞癌中，miR-607通过下调钙激活核苷酸酶1表达抑制肺癌细胞增殖侵袭[25]，提示Ca2+信号参与了miR-607的抑癌过程。研究证实，Ca2+信号异常活动是TRPC5发挥促癌作用的主要机制。结肠癌中TRPC5介导的Ca2+内流可激活HIF-1α/Twist轴促进上皮间质转化发生[26]。乳腺癌细胞中TRPC5的通过Ca2+依赖转录因子NFATC3诱导多药耐药蛋白1表达将细胞内阿霉素泵至胞外，诱导原发性耐药形成[27]。在HCC中，通道受体异常表达引起的Ca2+内流能激活PI3K/Akt通路来促进HCC生长和转移[28]。据此线索，本研究发现过表达miR-607能够削弱Akt的磷酸化并下调CCND1、MMP2等Akt通路下游效应分子的表达，初步揭示了miR-607抗肿瘤的潜在生物学机制。由于目前尚未能在裸鼠荷瘤模型中进一步验证miR-607的抗癌活性及体内作用机制，因此尽管本研究在体外分子过程方面进行了较为深入的探索，但其结果仍有一定的局限性，需要在未来的工作中进一步完善。
综上所述，HCC中低表达miR-607与肿瘤恶性临床特征密切相关。MiR-607通过下调TRPC5表达进而抑制Akt通路激活来发挥抗HCC生长转移作用。调控miR-607表达具有靶向治疗肝细胞癌潜力。
Biography
李超，博士，助理研究员，博士后，E-mail: lca03326@btch.edu.cn
Funding Statement
国家自然科学基金（81773128）；陕西省自然科学基础研究计划（2021JQ-397）；北京市医院管理中心“青苗”计划（QML20210903）
Supported by National Natural Science Foundation of China (81773128)
Contributor Information
李 超 (Chao LI), Email: lca03326@btch.edu.cn.
姜 业臻 (Yezhen JIANG), Email: jyzbhh@163.com.
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