Abstract
目的
探讨癌旁组织中肝星状细胞（HSCs）分泌表达的circWDR25（hsa-circRNA-0004310）与肝细胞癌（HCC）患者行根治性切除术后的预后关系。
方法
肝星状细胞系LX-2分别与3种肝癌细胞系Hep3B、SMMC-7721、HCCLM3行transwell小室法共培养, 建立肿瘤细胞活化的HSCs, 收集HSCs上清分离外泌体, 提取RNA后检测circRNA基因表达谱; 收集2014年1月~2015年10月在重庆医科大学附属第一医院肝胆外科接受根治性切除手术的288例HCC患者的癌旁肝组织, 行原位杂交, 检测circWDR25与α-SMA的阳性表达量, 预后单因素分析和多因素分析分别采用Log-rank检验和Cox回归分析。
结果
基因表达谱发现肿瘤活化的LX-2所分离出的外泌体中circWDR25表达上调最显著; 癌旁circWDR25和HSCs呈正相关（r=0.156, P= 0.008）。术前AST>36 g/L、肿瘤多发、肿瘤>5 cm、HSC>70、circWDR25>190为影响HCC患者根治性切除术后总体生存率的独立危险因素; 术前AST>36、肿瘤多发、肿瘤>5 cm、有癌栓、HSC>70、circWDR25>190为肝癌患者无瘤生存的独立危险因素。
结论
癌旁circWDR25和HSCs是HCC患者根治性肝切除术后预后的影响因素, 二者在癌旁组织中的高表达与预后差密切相关。
Keywords: 肝细胞癌, 肝星状细胞, 外泌体, 环状RNA, circWDR25
Abstract
Objective
To study the association between the expression of peritumoral circWDR25 (hsa-circRNA-0004310) secreted by hepatic stellate cells (HSCs) and the prognosis of the patients with hepatocellular carcinoma (HCC) after curative resection.
Methods
HSCs cell line LX-2 was co-cultured separately with 3 liver cancer cell lines (Hep3B, SMMC-7721, and HCCLM3) in Transwell chambers to obtain tumor cell-activated HSCs. The supernatants of HSC cultures were collected to isolate the exosomes, from which total RNA was extracted to detect circRNA expression profile. We also collected specimens of paracancerous liver tissues from 288 HCC patients undergoing radical resection in our department from January, 2014 to October, 2015, and the expression levels of circWDR25 and α-SMA were detected with in situ hybridization. Log-rank test and Cox regression analysis were used for univariate and multivariate analysis of the factors affecting the patients' prognosis, respectively.
Results
Gene expression profiling revealed that the expression of circWDR25 was the most obviously up-regulated in the exosomes isolated from tumor-activated LX-2 cells. The expression of peritumoral circWDR25 was positively correlated with HSCs adjacent to the cancer loci (r=0.156, P=0.008). Multivariate analysis showed that a preoperative AST level >36 g/L, multiple tumors, a tumor diameter >5 cm, HSC>70, and circWDR25>190 were independent risk factors affecting the overall survival of HCC patients after radical resection; a preoperative AST level >36 g/L, multiple tumors, a tumor diameter >5 cm, presence of tumor thrombus, HSC>70, and circWDR25>190 were all independent risk factors for tumor-free survival in patients with liver cancer.
Conclusion
Peritumoral circWDR25 and HSCs are factors affecting the prognosis of HCC patients after radical hepatectomy, and their high expression in the adjacent tissues is closely related to a poor prognosis of the patients.
Keywords: hepatocellular carcinoma, hepatic stellate cells, exosomes, circRNA, circWDR25
肝细胞癌（HCC）是一种常见的消化系统恶性肿瘤，占原发性肝癌的90%，其发病率和死亡率分别位居世界第6位和第4位[1-3]。虽然外科手术已经明显改善了HCC患者的预后，但是由于其高死亡率，HCC患者的5年生存率仍然很低[4, 5]。肝星状细胞（HSCs）是位于肝窦内皮细胞与肝细胞之间的肝脏间质细胞，在细胞增殖、肝纤维化和肝脏肿瘤的发生发展过程中起着重要作用[6, 7]。环状RNA（circRNA）属于非编码RNA家族的成员，比线性RNA更加的保守、稳定，不易被降解[8, 9]，其特征是共价闭环结构，既没有5'-3'极性，也没有聚腺苷化的尾巴，它们是由单个前mRNA的后剪接产生的RNA转录本[10, 11]。CircRNA的异常表达在肝癌中也起着重要的作用[12-14]，且已有报道circRNA抑制肝癌的侵袭转移从而改善HCC患者的预后[15, 16]，但是肝星状细胞中的circRNA与HCC的关系未见报道。本研究中，我们利用基因表达谱分析了肿瘤活化HSCs所分泌的circRNA的差异表达水平，发现circWDR25上调最为显著。我们首次分析了癌旁组织中circWDR25与根治性切除后的HCC的预后关系，现将结果报告如下。
1. 资料和方法
1.1. 研究对象
288例接受根治性切除的HCC患者均来自我院肝胆外科。所有病例的入选标准如下：（1）年龄为18~79岁；（2）行R0根治性切除；（3）术前未进行任何抗癌治疗；（4）无肝外转移；（5）完整的临床病理资料及随访数据。排除标准：（1）肿瘤有肝外转移；（2）合并其他部位肿瘤；（3）合并血液或其他免疫系统疾病。术后对这些患者进行定期的电话、门诊等方式随访，每半年1次，记录患者生存状态和疾病进展情况。随访时间为手术日期到2021年1月31日，中位随访时间45.8月。所有患者均签署知情同意书。本研究经重庆医科大学伦理委员会批准。
1.2. 细胞和试剂
人肝星状细胞系LX-2（武汉普诺赛），人肝癌细胞系Hep3B（中科院上海细胞库），HCCLM3、SMMC-7721由重庆医科大学生命科学院馈赠；DMEM、PBS（Gibico），胎牛血清（PAN）；经地高辛标记的circWDR25及α-SMA寡核苷酸探针，探针合成序列分别为5'-CAA AGCCAAACCACCTTGAAAGTTTTATC-3'、5'-ATGG GAAAACAGCCCTGGGAGCATCGTC-3'，氯仿、异丙醇和无水乙醇（上海试一化学试剂有限公司）；0.4 μm transwell小室（Corning）。
1.3. Exo-free血清的制备
取300 mL胎牛血清装于超速离心管，热封仪将超速离心管封口，用超速离心机（Beckman optima XPN-100）在4℃、120 000 g条件下过夜离心18 h后，将超速离心管内的血清用0.22 μm滤菌器过滤后转移至无菌50 mL离心管管内，制备好的Exo-free血清于-20 ℃保存。
1.4. Transwell小室法共培养体系
HSCs细胞系LX-2按每孔1×104接种于24孔培养板中，将transwell小室（0.4 μm）放入培养板内，小室内分别接种1×104 Hep3B、SMMC-7721、HCCLM3，24 h后去除培养液后清洗贴壁生长的LX-23次后，用不含外泌体的完全培养基继续培养24 h后收集上清液。
1.5. 总RNA提取
获取HSCs培养上清，利用超速离心法分离出外泌体[17]，将-80 ℃保存的外泌体溶液冰上溶解，加入Trizol试剂1 mL，室温静置5 min充分裂解细胞；加入200 μL氯仿，剧烈震荡15 s，室温放置10 min，分离蛋白和RNA；4 ℃下12 000 g离心15 min，小心分离含RNA的无色相；加入400 μL异丙醇沉淀RNA，室温静置10 min；4 ℃下12 000 g离心15 min弃去上清，用75%无水乙醇清洗RNA沉淀；4 ℃下7500 g离心10 min弃去液体，室温干燥30 min；RNase-free水溶解RNA，测定RNA浓度和纯度，-80 ℃保存备用。
1.6. circRNA基因表达谱分析
利用试剂盒（Arraystar Human circRNA Array V2，8×15K, Arraystar）将分离出的HSCs外泌体的总RNA进行circRNA的基因表达谱分析，数据分析由上海康成生物科技有限公司完成。
1.7. 原位杂交（ISH）染色
将石蜡切片脱蜡，滴加0.3%的TritonX-100通透。分别滴加适量0.2 N盐酸和0.25%的胃蛋白酶后并用PBS洗涤。4%多聚甲醛固定后，杂交缓冲液于55 ℃孵育2 h。杂交缓冲液按1∶1000的比例稀释circWDR25、α-SMA探针，于85 ℃变性2 min，37 ℃平衡2 min。吸去多余的杂交缓冲液，滴加适量变性后的探针于组织切片上，于4 ℃孵育12~18 h。用2×SSC洗涤3次后，室温封闭15 min，弃去封闭液，滴加适量碱性磷酸酶标记的抗地高辛二抗（1∶800），4 ℃孵育过夜。PBS洗涤3次，滴加适量BCIP/NBT染色工作液染色，蒸馏水洗涤1~2次，梯度酒精脱水，二甲苯透亮，使用中性树胶封片。
1.8. 结果判读标准
染色强度评判标准：无着色，0分；弱着色，1分；中度着色，2分；强着色，3分。阳性细胞比例评判标准为：0~25%，1分；26~50%，2分；51~75%，3分；>75%，4分。两者分数相乘得到最终得分，得分大于中位数为高表达，小于中位数为低表达。
1.9. 统计学方法
采用SPSS 25.0统计软件分析数据。Kaplan-Meier法进行生存分析，生存率比较采用log-rank检验。多因素Cox比例风险回归模型分析肝细胞癌患者预后的影响因素。P < 0.05为差异有统计学意义。利用X-tile软件最小P值法[18]获得circWDR25与α-SMA阳性表达量的最佳界值分别为190、70。
2. 结果
2.1. circWDR25基因表达谱散点图：
circRNA基因表达谱（图 1）结果显示circWDR25表达上调最明显。circWDR25（图 2A）与α平滑肌肌动蛋白（α-SMA）（图 2B，活化肝星状细胞标志）主要分布于细胞质，多见于间质组织。
1.
肝癌细胞活化LX-2分泌的外泌体中的circRNA基因表达谱显示差异表达的circRNA散点图
Scatter plot of differential expression of circRNA shown by circRNA gene expression profile of the exosomes from LX-2 cells activated by hepatocellular carcinoma cells. A: Hep 3B. B: SMMC7721. C: HCCLM3.
2.
癌旁circWDR25（A）和α-SMA（B）的原位杂交表达
In situ hybridization for detecting the expression of circWDR25 (A) and α-SMA (B) in the adjacent tissues (Original magnification: ×200).
2.2. circWDR25与α-SMA的关系研究
计数每个高倍镜视野下的circWDR25与α-SMA阳性表达细胞数量，每张切片计数阳性表达最多的3个视野，并计算每个患者的阳性细胞平均数。相关分析发现癌旁circWDR25与α-SMA成正相关（r=0.156，P=0.008）。
2.3. HCC预后影响单因素分析
术前天冬氨酸氨基转移酶（AST）、肿瘤数目、肿瘤大小、有无癌栓、BCLC分期、HSC阳性表达量、circWDR25阳性表达量与肝细胞癌患者的累积生存率和无瘤生存率相关（表 1）。所有患者中位生存时间45.6月（1.5~74.3月），中位无瘤生存时间40.2月（1.0~ 74.2月）。
1.
288例不同临床病理特征的肝细胞癌患者累积生存率和累积无瘤生存率比较
Comparison of overall and tumor-free survival rates of 288 HCC patients with different clinicopathological characteristics
| Factors | Number | Overall survival rate | Tumo-free survial rate | P a | P b |
| a Comparison result of overall survival rate of each clinical index; b Comparison result of tumor- free survival rate of each clinical index; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; AFP: Alpha fetoprotein; BCLC: Barcelona Clinic Liver Cancer. According to "minimum P value" approach, the positive expression of circWDR25 was190, the positive expression of α-SMA was 70. | |||||
| Age (years) | 0.331 | 0.289 | |||
| ≤50 | 124 | 72.0% | 54.2% | ||
| > 50 | 164 | 65.4% | 44.9% | ||
| Gender | 0.744 | 0.339 | |||
| Male | 245 | 68.4% | 49.7% | ||
| Female | 43 | 66.7% | 44.2% | ||
| Cirrhosis | 0.235 | 0.346 | |||
| Yes | 250 | 69.5% | 49.7% | ||
| No | 38 | 59.6% | 43.6% | ||
| Preoperative ALT (U/L) | 0.336 | 0.548 | |||
| ≤40 | 156 | 71.0% | 51.1% | ||
| > 40 | 132 | 64.8% | 45.9% | ||
| Preoperative AST (U/L) | 0.028 | 0.047 | |||
| ≤36 | 173 | 74.2% | 54.7% | ||
| > 36 | 115 | 61.7% | 42.7% | ||
| PreoperativeAFP (μg/L) | 0.02 | < 0.001 | |||
| ≤20 | 114 | 76.4% | 62.0% | ||
| > 20 | 174 | 62.7% | 40.1% | ||
| Tumor number | < 0.001 | 0.006 | |||
| Single | 252 | 71, 5% | 51.8% | ||
| Multiple | 36 | 44.0% | 27.4% | ||
| Embolus | < 0.001 | < 0.001 | |||
| Yes | 88 | 54.0% | 29.2% | ||
| No | 200 | 74.2% | 57.4% | ||
| Tumor size (cm) | < 0.001 | < 0.001 | |||
| ≤5 | 197 | 79.2% | 57.8% | ||
| > 5 | 91 | 44.2% | 29.4% | ||
| BCLC stage | 0.001 | < 0.001 | |||
| 0-Ⅱ | 207 | 73.4% | 56.6% | ||
| Ⅲ | 81 | 54.6% | 29.0% | ||
| α-SMA | < 0.001 | < 0.001 | |||
| ≤70 | 62 | 92.5% | 85.8% | ||
| > 70 | 226 | 61.8% | 39.2% | ||
| CircWDR25 | 0.001 | < 0.001 | |||
| ≤190 | 197 | 73.9% | 57.4% | ||
| > 190 | 91 | 56.0% | 30.7% | ||
2.4. 多因素结果分析
将单因素分析结果中有统计学意义的因素纳入Cox回归模型进行多因素分析（表 2），结果显示：术前AST>36 g/L（HR=1.141 CI：1.015~1.282）、肿瘤多发（HR=2.825 CI：1.703~4.685）、肿瘤直径>5 cm（HR=3.435 CI：2.242~5.263）、α-SMA>70（HR=5.818 CI：2.115~16.005）、circWDR25>190（HR=1.918 CI：1.252~2.940）为HCC患者根治性肝切除术后生存的独立危险因素；同时，术前AST>36g/L（HR=1.136 CI：1.005~ 1.285）、肿瘤多发（HR=1.762 CI：1.132~2.744）、肿瘤直径>5 cm（HR=2.090 CI：1.475~2.961）、有癌栓（HR= 1.639 CI：1.146~2.344）α-SMA>70（HR=5.240 CI：2.539~ 10.816）、circWDR25>190（HR=1.732 CI：1.238~2.423）是肝癌患者无瘤生存的独立危险因素。
2.
肝细胞癌患者预后的多因素Cox回归分析
Multivariate Cox regression analysis of the prognosis of patients with HCC
| Variable | Overall survival rate | Tumor-free survival rate | |||
| HR (95% CI) | P | HR (95% CI) | P | ||
| aCompared with single tumor; HR: Hazard ratio; 95%CI; 95% confidence interval. | |||||
| Preoperative AST > 36 g/L | 1.141 (1.015-1.282) | 0.027 | 1.136 (1.005-1.285) | 0.041 | |
| Tumor number a | 2.825 (1.703-4.685) | < 0.001 | 1.762 (1.132-2.744) | 0.012 | |
| Tumor size (> 5 cm) | 3.435 (2.242-5.263) | < 0.001 | 2.090 (1.475-2.961) | < 0.001 | |
| Embolus | 1.044 (0.385-2.826) | 0.933 | 1.639 (1.146-2.344) | 0.007 | |
| α-SMA (> 70) | 5.818 (2.115-16.005) | 0.001 | 5.240 (2.539-10.816) | < 0.001 | |
| CircWDR25 (> 190) | 1.918 (1.252-2.940) | 0.003 | 1.732 (1.238-2.423) | 0.001 | |
2.5. CircWDR25与HCC患者根治性切除术后的预后关系
低circWDR25（≤190）组患者1、3、5年总体生存率为：93.9%、81.2%、73.9%，无瘤生存率为：81.2%、64.2%、57.4%；而高circWDR25（>190）组患者1、3、5年总体生存率为：75.7%、52.4%、46%，无瘤生存率为：69.2%、39.4%、25.1%。低circWDR25组1、3、5年总体生存率及无瘤生存率均高于高circWDR25组（P < 0.001，图 3）。
3.
circWDR25在肝细胞癌患者中的总体生存曲线
Overall survival curve of patients with HCC with different circWDR25 expression levels.
4.
circWDR25在肝细胞癌患者中的无瘤生存曲线
Tumor-free survival curve of patients with HCC with different circWDR25 expression levels.
3. 讨论
本研究首次证实癌旁肝星状细胞中的circWDR25表达水平与HCC患者术后的预后关系，基因表达谱分析比较发现，circWDR25在不同肝癌细胞刺激后的LX-2的外泌体中表达上调均最显著，癌旁肝组织中肝星状细胞与circWDR25的表达水平成正相关。HCC患者术后的预后关系分析提示，术前AST>36 g/L、肿瘤多发、肿瘤>5 cm、HSC>70、circWDR25>190为影响HCC患者根治性切除术后总体生存率的独立危险因素；术前AST>36、肿瘤多发、肿瘤>5 cm、有癌栓、HSC>70、circWDR25>190为HCC患者无瘤生存率的独立危险因素。同时发现低circWDR25组的总体生存率和无瘤生存率均高于circWDR25高表达组。
首先，肿瘤活化的HSCs可能通过外泌体源性的circWDR25参与HCC的发生发展过程。我们既往的多项研究也已经发现，HSCs能够通过多条信号通路（如TREM-1，IL-17，ERK）作用于肝癌细胞，从而促进HCC的恶性生物学行为[19]。同时，在肿瘤微环境中，活化的HSCs能够分泌白细胞介素-6、肿瘤坏死因子-α，参与肝癌细胞的“互动”，进而影响HCC的进程及预后[20, 21]。同时，HSCs能产生免疫调节细胞因子如（TGF-β）诱导调节性T细胞形成免疫抑制，从而促进肝癌细胞的免疫逃逸[22]。但是，HSCs和肝癌细胞的具体作用方式尚不清楚。最近的研究发现，外泌体在调节肿瘤微环境中肿瘤与正常细胞沟通中起着至关重要的作用[23, 24]，外泌体介导的circRNA转移被发现是癌症的一种新机制[25]。Huang等[26]报道了肝癌来源的外泌体环状RNA通过增强侵袭性和血管生成促进肝细胞癌的转移。本项研究为HSCs和肝癌细胞之间通过外泌体相互作用提供了新的实验室证据。
其次，circWDR25在HSCs和HCC之间的信息传递中起着关键作用。目前已经明确，circRNA可以作为微小RNA(miRNA)海绵、RNA结合蛋白海绵和翻译调节因子在基因表达调控中发挥着重要作用[27-29]。例如，circRNA-5692可以通过降低miR-328-5P的表达来提高DAB2IP的表达，从而抑制肝癌的发生发展[13]；circRNA Cdr1as通过海绵化miR-1270促进AFP的表达从而促进肝癌细胞的迁移、侵袭和增殖[30]。这项研究中，通过肝癌细胞刺激，并利用基因表达谱发现的HSCs中的circWDR25高度活化，且在癌旁组织中高表达，说明其参与了HSCs和HCC之间的“对话”，可能在HCC的发生发展中起着关键的作用，但具体的作用机制尚待进一步研究。
此外，癌旁circWDR25的高表达与HCC预后差密切相关，并与癌旁HSCs的表达成正相关，说明其是HSCs分泌的促瘤因子，能够促进肿瘤的恶性生物学行为。同时，癌旁组织中的circWDR25也可能作为评估HCC预后及手术筛选的一个参考指标。
综上所述，癌旁circWDR25和HSCs是HCC患者根治性肝切除术后预后的重要影响因素，二者在癌旁组织中的高表达与预后差密切相关。它们可能作为HCC根治性切除术后高复发风险患者的筛查和监测指标，并为患者术后的综合治疗提供参考依据。
Biography
刘磊，硕士，医师，E-mail: jorgelei@163.com
Funding Statement
国家自然科学基金（81703063）；重庆市科委基础科学与前沿技术研究项目（cstc2017jcyjBX0010, cstc2018jcyjAX0162）；重庆市教委科学技术研究项目（KJQN201800416）；重庆市科卫联合医学科研项目（2020GDRC013）
Supported by National Natural Science Foundation of China (81703063)
Contributor Information
刘 磊 (Lei LIU), Email: jorgelei@163.com.
廖 锐 (Rui LIAO), Email: liaorui99@163.com.
References
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