Abstract
目的
建立索拉菲尼诱导的肝细胞癌(hepatocellular carcinoma, HCC)耐药细胞株,筛选出耐药细胞株中的高表达基因,进一步探讨肝细胞癌中与索拉菲尼耐药的相关基因.
方法
选取人肝癌细胞株PLC及Huh7,采用药物间歇诱导法分别建立索拉菲尼诱导的耐药细胞株,运用高通量测序技术(RNA-Seq)测定两株耐药细胞株中高表达基因,运用生物信息学数据库分析高表达基因与肝癌临床生物学指标相关性,筛选出肝癌耐药机制中可能参与的基因.
结果
通过基因测序得到两株肝癌耐药细胞株中的高表达基因,在设定的限定条件下进行进一步筛选, 限定条件为两株耐药细胞中共同表达的基因且表达差异倍数大于4倍,差异具有统计学意义(P<0.05),发现两株耐药细胞共同表达前12位基因符合限定条件,进一步运用Ualcan数据库分析12个基因与肝癌组织表达水平,分期,分级及生存的关系,筛选出符合条件的基因CBX4.
结论
成功构建了索拉菲尼诱导的肝癌耐药细胞株,并筛选出与索拉菲尼耐药相关基因CBX4,为进一步耐药机制探讨提供依据.
Keywords: 肝细胞癌, 索拉菲尼耐药, TCGA, CBX4
Abstract
Objective
To establish the drug-resistant cell lines of hepatocellular carcinoma (HCC) induced by sorafenib, and to screen out the high expression genes in drug-resistant cell lines of HCC induced by sorafenib, then to explore the genes related to sorafenib resistance in hepatocellular carcinoma.
Methods
The human PLC and Huh7 cell lines were obtained, then the PLC and Huh7 drug-resistant cell lines were induced with sorafenib by using intermittent induction in vitro. CCK8 assay was used to detect the IC50 value of sorafenib for evaluation of drug sensitivity of hepatocellular carcinoma cell lines in PLC and Huh7. All the up regulated genes in PLC and Huh7 drug-resistant cell lines induced by sorafenib were screened out using high-throughput cDNA sequencing (RNA-Seq), Ualcan database was used to analyze the correlations between the up regulated genes in PLC and Huh7 drug-resistant cell lines induced and four clinical biological characteristics of hepatocellular carcinoma, including the gene expressions between normal samples and tumor samples, tumor stage, tumor grade, and patient overall survival, to find the genes that might be involved in the mechanism of sorafenib resistance of hepatocellular carcinoma.
Results
All the up regulated genes detected by the using high-throughput cDNA sequencing (RNA-Seq) in PLC and Huh7 drug-resistant cell lines were further screened out by following conditions:(1) genes co-expressed in PLC and Huh7 drug-resistant cells induced by sorafenib, (2) the fold change was more than 4 times and the difference was statistically significant (P <0.05), the top 12 up regulated genes in PLC and Huh7 drug-resistant cell lines were found, which were TPSG1, CBX4, CLC, CLEC18C, LGI4, F2RL1, S100A6, HABP2, C15ORF48, ZG16, FOLH1, and EPCAM. Compared with the correlations between the twelve genes and the clinical biological characteristics by Ualcan database, the potentially significant gene CBX4 was screened out.
Conclusion
The human PLC and Huh7 drug-resistant cell lines of hepatocellular carcinoma induced by sorafenib were successfully established. CBX4, the gene related to sorafenib resistance in hepatocellular carcinoma, was screened out by the high-throughput cDNA sequencing (RNA-Seq) and further analysis using Ualcan database, which is providing a powerful basis for further research on the mechanism of sorafenib resistance of hepatocellular carcinoma.
Keywords: Hepatocellular carcinoma, Sorafenib resistance, TCGA, CBX4
原发性肝癌(hepatocellular carcinoma, HCC)是常见的消化系统恶性肿瘤,全球范围内每年新发病例数超过85万,病死人数高达70万,肝癌患者病死率在所有癌症中位居第二(占所有癌症病死率8.2%)[1], 近年来其发病率和病死率均呈上升趋势.肝癌治疗主要包括手术切除,介入消融疗法,肝动脉化疗栓塞术以及化疗等综合治疗,但是肝癌早期诊断困难,肝切除术后复发,转移率高,对常规化疗容易产生耐药,使得患者的预后水平整体较差,仅有10% 的HCC 患者生存期超过5 年[2,3].
索拉菲尼是一种口服多激酶抑制剂,通过抑制酪氨酸激酶和下游的丝氨酸/苏氨酸激酶活性,从而发挥抑制肿瘤血管生成和促进肿瘤细胞凋亡等作用[4,5].多个Ⅲ期临床试验的结果显示,索拉菲尼可显著延长晚期患者的生存期,并且在不同种族,不同状态都有明确的生存获益,因此,2007 年被美国FDA 批准用于HCC 的治疗[6].目前,索拉菲尼已成为晚期不可进行肝切除或具有转移性HCC患者的标准一线治疗方案[7],然而,索拉菲尼容易产生耐药,显著影响其疗效,且耐药机制不明.
本研究旨在采用间歇诱导法,使用索拉菲尼诱导人肝癌细胞系PLC和Huh7 在体外建立一种分子靶向药物的耐药模型,通过基因测序获得耐药细胞株中的所有异常高表达的基因,进一步通过生物信息学分析,利用Ualcan 数据库中肝癌临床病理特征与基因表达之间的相关性,筛选出索拉菲尼诱导的耐药细胞模型中的参与基因,探讨索拉菲尼可能的耐药机制,为索拉菲尼耐药的肝癌治疗提供理论依据[8].
1. 材料与方法
1.1. 材料
索拉菲尼购自美国Selleck 公司,人肝细胞系PLC 和Huh7 由本实验室保存; DMEM,胎牛血清,胰酶及相关培养试剂购自美国Gibco 公司; Cell Counting Kit-8 试剂盒(CCK-8 试剂盒) 购自日本DOJINDO 公司.
1.2. 方法
1.2.1 细胞培养 人肝癌细胞系PLC 和Huh7 培养于含10%(体积分数)胎牛血清的DMEM(含青链霉素) 中,在37 ℃,5%(体积分数)CO2的培养箱中孵育,待细胞成长度达到70%~80%进行胰酶消化传代.
1.2.2 PLC和Huh7耐药细胞株的建立 采用药物间歇诱导法筛选耐药细胞株,取对数生长期PLC 和Huh7 细胞接种至含10%(体积分数)FBS 的DMEM培养液中,在PLC及Huh7细胞培养基中添加终浓度为1 μmol /L的索拉菲尼,隔日换液,根据密度传代,培养2周后,逐渐提升药物浓度,最终以PLC 与Huh7 各自的IC50值药物浓度6.4 μmol /L 和7.6 μmol /L 作为筛选浓度,作用72 h 后弃去含药培养液,加入新鲜的DMEM 培养液,筛选4 周后培养皿中仅存活少量耐药细胞,将存活的耐药细胞消化并转移至规格适宜的培养皿中,以避免细胞密度过低影响生长状态;继续保持索拉菲尼药物浓度进行持续培养,并在细胞汇合度到达70%~80% 时进行传代.经过3 个月的培养,获得对索拉菲尼具有耐药性的细胞株,并将其维持培养在含1 μmol /L 的索拉菲尼的完全培养液中.
1.2.3 CCK8 法检测索拉菲尼的IC50值 取生长良好的对数期细胞按每孔1×104 细胞100 μL 接种于96 孔板培养.实验组加入不同浓度的索拉菲尼,每次处理3 个复孔,另设不加索拉菲尼的对照组以及不加细胞的空白组.37 ℃,5% CO2培养箱中培养,适时换液,待细胞覆盖率达到75% 时加入药物索拉菲尼.加药后48 h 换液,再分别每孔加入CCK8 试剂(5 g/L)置于37 ℃, 5% CO2培养箱内继续培养1 h 后,酶联免疫分析仪检测490 nm 处光密度值.按以下公式计算在索拉菲尼的作用下,肿瘤细胞的生存率.细胞生存率=[加药组D490-空白组D490]/[对照组D490-空白组D490]×100%,并计算出其IC50值.
1.2.4 高通量cDNA测序(high-throughput cDNA sequencing, RNA-Seq) RNA-Seq实验由Novogene(北京)公司,按照Illumina标准程序进行测序.使用TRIZOL试剂(Invitrogen公司,美国)从PLC及Huh7原代细胞和耐药细胞中提取总RNA,使用DNaseⅠ处理,去除基因组DNA污染.用带有oligo(dT)的磁珠富集样品中的mRNA,随后将mRNA打成短片段并以其为模板合成双链cDNA,后者经纯化,末端修复,加A尾,连接测序接头,选择合适的片段大小进行PCR扩增,按照Illumina标准的步骤创建mRNA-Seq文库,并在Illumina HiSeq 2000平台上进行测序.
1.2.5 Ualcan 数据库 Ualcan(http://ualcan.path.uab.edu/index.html) 数据库使用来自31 种癌症类型的癌症基因组图谱(the Cancer Genome Atlas,TCGA) 3 级RNA-seq 和临床数据,根据个体肿瘤组织和正常样本基因表达水平,癌症分期,肿瘤分级,种族,体质量分析,以及各种肿瘤亚组中查询基因的相对表达或其他临床病理特征; 估计基因表达水平和临床病理特征对患者生存的影响; 确定个体癌症类型中的顶部过度表达和表达不足(上调和下调) 基因[9].运用Ualcan 数据库分析经测序筛选基因在肝癌组织与正常组织中表达的差异.
1.3. 统计学分析
应用SPSS 20.0统计软件,计量资料以x±s表示,两组间比较采用独立样本t检测, P<0.05认为差异有统计学意义.
2. 结果
2.1. 索拉菲尼诱导的肝癌细胞株IC50值测定
随着药物浓度的增加, Huh7和PLC的细胞死亡率随之升高(图1).同样经索拉菲尼诱导48 h后,耐药细胞株的IC50值明显增高,其中Huh7细胞株的IC50值为(1.54±0.18) μmol /L,耐药细胞株的IC50值上升为(50.90±1.70) μmol /L;PLC 的细胞株的IC50值为(2.56±0.37) μmol /L,耐药细胞株上升为(56.90±1.80) μmol /L.
1.
CCK8 法检测索拉菲尼对PLC和Huh7 细胞增殖抑制作用
The inhibitory effect of sorafenib on the proliferation of PLC and Huh7 cells by CCK8 assay
PLC and Huh7 drug-resistant cell lines were established after 12 weeks using intermittent induction with sorafenib. Cell proliferation assay was performed to calculate cell mortality based on sorafenib treatment. sr, sorafenib resistant.
2.2. 基因测序筛选耐药细胞株的基因表达
成功建立PLC及Huh7耐药细胞株后,分别提取PLC及Huh7原代细胞株及耐药细胞株的mRNA,进行基因测序,比较两种细胞中差异倍数大于2倍的基因,PLC中有442个高表达基因,Huh7中有260个高表达基因,再设定限定条件进一步筛选基因,限定条件为在两种耐药细胞株中均有表达和差异倍数>4倍(P<0.05),筛选出前12位高表达基因,分别是TPSG1,CBX4,CLC,CLEC18C,LGI4,F2RL1,S100A6,HABP2,C15ORF48,ZG16,FOLH1,EPCAM(图2).进一步运用Ualcan数据库分析这些基因在肿瘤组织与正常组织中的表达,临床病理特征以及进行生存分析.
2.
基因测序PLC及Huh7耐药细胞株中的基因表达
Gene expression in PLC and Huh7 drug-resistant cell lines by High-throughput cDNA sequencing
The up regulated genes in PLC and Huh7 drug-resistant cell lines were detected by RNA-Seq, the top twelve genes which fold change were more than 4 times.
2.2.1 肿瘤组织与正常组织的基因表达差异分析 12个基因中,TPSG1,CBX4,CLEC18C,LGI4,F2RL1,S100A6,C15ORF48共7个基因在肿瘤组织中表达均升高,差异有统计学意义.CLC,HABP2,ZG16,FOLH1,EPCAM共5个基因在正常组织中的表达高于肿瘤组织(图3).
3.
肝细胞癌肿瘤组织与正常组织的基因表达差异
Gene expression differences based on sample types in hepatocellular carcinoma
Seven genes are screened out from all the twelve genes which gene expression in tumor sample are higher than in normal tissues (P<0.05).
2.2.2 肿瘤分期与基因表达差异分析 在肿瘤组织高表达的7个基因中,Ⅳ期患者高表达且具有统计学差异的基因为CBX4和S100A6,其余5个基因TPSG1,CLEC18C,LGI4,F2RL1,C15ORF48在Ⅳ期患者中高表达,但是差异无统计学意义(图4).
4.
肝细胞癌肿瘤分期与基因表达差异
Gene expression differences based on tumor stage in hepatocellular carcinoma
Two genes, CBX4 and S100A6, are screened out from the seven genes which gene expression in stage Ⅳ are higher than in normal tissues (P<0.05).
2.2.3 肿瘤组织学分级与基因表达差异分析 在肿瘤组织高表达的7个基因中,低分化肝癌中高表达且具有统计学差异的基因为CBX4和C15ORF48,其余5个基因TPSG1,CLEC18C,LGI4,F2RL1,S100A6在低分化肿瘤中虽然表达增高,但是差异无统计学意义(图5).
5.
肝细胞癌肿瘤组织学分级与基因表达差异
Gene expression differences based on tumor grade in hepatocellular carcinoma
Two genes, CBX4 and C15ORF48, are screened out from the seven genes which gene expression in grade IV are higher than in normal tissues (P <0.05).
2.2.4 淋巴结转移与基因表达差异分析 在肿瘤组织高表达的7个基因中,淋巴结转移患者均高表达该基因,且差异具有统计学意义(图6).
6.
肝细胞癌淋巴结转移与基因表达差异
Gene expression differences based on nodal metastasis in hepatocellular carcinoma
Gene expression of all the seven genes with nodal metastasis are higher than in normal tissues (P<0.05).
2.2.5 生存分析与基因表达差异关系 在肿瘤组织高表达的7个基因中,低表达该基因使得患者生存期延长且差异具有统计学意义的基因为CBX4,LGI4,S100A6,C15ORF48(图7).索拉菲尼耐药细胞株中的基因表达与临床病理特征见表1,将基因表达与组织学分级,分期以及生存时间相关性用分值表示,筛选得分最高的基因为CBX4.
7.
肝细胞癌生存分析与基因表达差异
Gene expression differences based on patient survival in hepatocellular carcinoma
The overall survival of patients were prolonged with lower gene expression of these four genes, which are CBX4, LGI4, S100A6 and C15ORF48(P <0.05).
1.
索拉菲尼耐药细胞株中的靶基因表达差异分析(TCGA数据库)
Screening the target genes in sorafenib resistant cell lines using TCGA dataset analysis
| Gene name | Gene expression | Tissues | OS | Metastasis | Grade | Scores | |||||
| in Huh7 | in PLC | T/N>1 | P<0.05 | L/H>1 | P<0.05 | M1/M0>1 | P<0.05 | G4/N>1 | P<0.05 | ||
| The scores were carried out through identifying the genes expression from four parts: 1, tumor and normal tissues; 2, overall survival; 3, tumor stage; 4, tumor grade. For example, if the level of a gene expression in tumor higher than in normal tissues, it should be scored 1 point, but then the reverse would be scored 0. The similar as followed: low expression of a gene capturing long term survival is scored 1 point; high expression of a gene in HCC with metastasis is scored 1 point; high expression of a gene in HCC with high grade is scored 1 point; statistical significance (P<0.05) of each of above four elements is scored 1 point. Maximum score is 8 points. Gene of full score is CBX4. T, tumor; N, normal; L, low expression; H, high expression; OS, overall survival; G,grade. | |||||||||||
| TPSG1 | 11.82 | 4.00 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| CBX4 | 10.56 | 5.42 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 8 |
| CLC | 9.86 | 7.00 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| CLEC18C | 8.74 | 4.04 | 1 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | 4 |
| LGI4 | 5.74 | 5.90 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 0 | 6 |
| F2RL1 | 5.40 | 4.46 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| S100A6 | 5.02 | 5.00 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 7 |
| HABP2 | 4.84 | 7.06 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| C15ORF48 | 4.38 | 4.46 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 6 |
| ZG16 | 4.36 | 4.42 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
| FOLH1 | 4.30 | 4.02 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 |
| EPCAM | 4.16 | 4.72 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
3. 讨论
原发性肝癌是全球第五大常见恶性肿瘤,我国是肝癌高发国家,统计数据约占全球统计数据的55%.由于肝癌起病隐匿,大部分患者一经诊断往往已处于肝癌晚期阶段,索拉菲尼作为晚期肝癌标准一线治疗方案,却因其疗效维持时间短和患者易出现耐药,限制了它在临床中的应用.目前,一旦出现索拉非尼耐药,晚期肝癌患者没有更佳的治疗方案可供选择,因此,索拉非尼耐药机制的研究显得尤为迫切和重要.
国内外研究表明[5,7],在肝细胞癌中索拉菲尼的耐药机制可能包括肝细胞癌的自分泌及旁分泌途径,肿瘤微环境中的基质细胞,免疫细胞及细胞外基质等,也可通过细胞因子,缺氧,自噬等多种因素.运用传统的研究方法从基因库中筛选出耐药基因需要耗费大量时间和精力,结合现代生物信息学手段从已建立的癌症患者数据库中的数据与相关研究基因进行分析,可以快速筛选出目的基因.
TCGA (The Cancer Genome Atlas) 数据库收录癌症患者临床样本及基因相关信息,将统计学和生物信息学知识综合运用到癌症患者的数据分析中发挥其预测作用.TCGA 汇集了全球范围内高通量测序,基因芯片检测数据,包括RNA-Seq 数据[10],miRNA 芯片数据[11],CMV 数据等大量生物医学信息,与传统医学研究缺乏信息学基础相比,TCGA 数据库存储了海量基因组和临床数据供我们分析使用,为基础医学和转化医学构建了桥梁,也为癌症研究及治疗提供了新思路.本实验建立了两种索拉菲尼诱导的人肝癌耐药细胞株,通过高通量cDNA基因测序测定出耐药细胞株中差异表达基因几百个,进一步将两株耐药细胞株中共同表达的前12位基因,通过借助Ualcan数据库具体分析耐药细胞株中基因表达与肝癌临床病理特征之间的相关性,能够快速筛选出可能与肝癌索拉菲尼耐药相关靶基因.
综上所述,本研究利用生物信息学数据库最终筛选出的靶基因为CBX4,CBX4是表观遗传调控复合物多梳蛋白家族(polycomb group,PcG) 的重要成员之一,PcG蛋白可以通过修饰染色质对靶基因进行转录抑制,在调控细胞分化,衰老,死亡和肿瘤发生中发挥着重要作用[12].CBX4主要通过其SUMO E3 酶的作用调控多种重要蛋白,在调控细胞周期,维持胚胎干细胞多潜能性和自我更新能力,肿瘤发生中发挥重要作用[13].多项研究表明,CBX4异常表达与肝癌,乳腺癌,骨肉瘤等恶性肿瘤发生密切相关[14,15,16],这些结果提示在肝细胞癌中,CBX4可能是索拉菲尼耐药机制的重要参与蛋白.本课题组将进一步在耐药细胞株中过表达和沉默CBX4,通过体外细胞层面及体内动物实验验证CBX4的作用,并进一步寻找作用CBX4蛋白的调节基因,探讨肝细胞癌中索拉菲尼耐药的分子机制,为寻找逆转耐药靶点的研究提供理论依据.
Funding Statement
国家自然科学基金（81872025）; 北京市自然科学基金（7182030）; 北京大学临床肿瘤学院科学研究基金(10-29)（10-29）
Supported by the National Natural Science Foundation of China(81872025); the Beijing Natural Science Foundation(7182030); Peking University School of Oncology Science Foundation(10-29)
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