基质金属蛋白酶7在肝癌细胞迁移及免疫细胞浸润中的作用与预后价值
DOI: 10.12449/JCH240721
Role and prognostic value of matrix metalloproteinase-7 in the migration and immune cell infiltration of hepatocellular carcinoma
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摘要:
目的 评估基质金属蛋白酶7(MMP7)在肝癌细胞迁移及免疫细胞浸润中的作用及预后价值。 方法 分别构建下调或上调靶基因MMP7的MMP7_siRNA、pMMP7转染肝癌细胞株(MHCC97H)。采用RT-qPCR、Western Blot分别检测细胞中靶基因mRNA及蛋白的表达水平。扫描电镜和Transwell小室实验分别观察细胞伪足和迁移能力的变化,并采用生物信息学的方法,在TCGA、TIMER数据库分析MMP7与免疫细胞及肝癌患者免疫浸润评分之间的相关性,并进一步研究MMP7与肝癌患者预后的相关性。相关性分析采用Spearman方法。Sanger Box在线工具评估MMP7在肝癌总体生存期、疾病特异性生存期方面的意义。Kaplan-Meier法绘制生存曲线,Log-rank检验评估不同组样本之间的预后差异。 结果 构建的MMP7_siRNA、pMMP7转染MHCC97H细胞后均能有效下调或上调靶基因MMP7的表达,在MHCC97H细胞中MMP7被干扰后细胞伪足明显减少,并且变短,但是过表达MMP7之后细胞表面丝状伪足数量明显增多,并且伪足长度变长,放射状排列。Transwell小室结果发现,MMP7_siRNA2能够显著降低细胞迁移能力(P<0.05),而转染pMMP7后,细胞迁移能力显著增加(P<0.05)。MMP7的表达与B淋巴细胞(r=0.37)、CD4+T淋巴细胞(r=0.40)、中性粒细胞(r=0.49)、巨噬细胞(r=0.49)、树突状细胞(r=0.47)显著相关(P值均<0.05)。在TCGA数据库中,基于总体生存期最佳截断值将肝癌患者分成MMP7高表达组(n=267)和MMP7低表达组(n=146),结果发现,MMP7高表达组的总体生存期明显低于MMP7低表达组(P<0.05);基于疾病特异性生存期最佳截断值将肝癌患者分成MMP7高表达组(n=257)和MMP7低表达组(n=145),结果发现,MMP7高表达组的疾病特异性生存期也低于MMP7低表达组(P<0.05)。 结论 MMP7促进了肝癌细胞的迁移,并在免疫细胞浸润中发挥主要作用,MMP7的表达也与肝癌的预后具有明显相关性。 Abstract:Objective To investigate the role and prognostic value of matrix metalloproteinase-7 (MMP7) in the migration and immune cell infiltration of hepatocellular carcinoma. Methods MMP7_siRNA for downregulating the target gene MMP7 and pMMP7 for upregulating MMP7 were constructed and were used to transfect hepatocellular carcinoma cell line (MHCC97H). RT-qPCR and Western Blot were used to measure the mRNA and protein expression levels of the target gene in cells. Scanning electron microscopy and Transwell assay were used to observe the changes in cell pseudopodia and migration ability, and bioinformatics methods were used to investigate the correlation of MMP7 with immune cells and immune infiltration score in TCGA and TIMER databases in patients with hepatocellular carcinoma, as well as the association between MMP7 and the prognosis of patients with hepatocellular carcinoma. The Spearman method was used for correlation analysis. Sanger Box online tool was used to assess the value of MMP7 in the overall survival curve and disease-specific survival of hepatocellular carcinoma. The Kaplan-Meier method was used to plot survival curves, and the Log-rank test was used for comparison of prognosis between different samples. Results After MHCC97H cells were transfected with MMP7_siRNA or pMMP7, there was a significant reduction or increase in the expression of the target gene MMP7; after downregulation of MMP7, there were significant reductions in the number and length of the pseudopodia, while after MMP7 overexpression, there were significant increases in the number and length of filopodia with radial arrangement. The Transwell chamber assay showed that MMP7_siRNA2 significantly reduced the migration ability of cells (P<0.05), and there was a significant increase in migration ability after pMMP7 transfection. The expression of MMP7 was significantly correlated with B lymphocytes (r=0.37, P<0.05), CD4+ T lymphocytes (r=0.40, P<0.05), neutrophils (r=0.49, P<0.05), macrophages (r=0.49, P<0.05), and dendritic cells (r=0.47, P<0.05). In the TCGA database, the patients with hepatocellular carcinoma were divided into MMP7 high expression group with 267 patients and MMP7 low expression group with 146 patients based on overall survival, and the results showed that the MMP7 high expression group had a significantly shorter overall survival time than the MMP7 low expression group (P<0.05); based on the disease-specific survival time, the patients were divided into MMP7 high expression group with 257 patients and MMP7 low expression group with 145 patients, and the analysis showed that the MMP7 high expression group also had a significantly shorter disease-specific survival time than the MMP7 low expression group (P<0.05). Conclusion MMP7 promotes the migration of hepatocellular carcinoma cells and plays a major role in immune cell infiltration, and the expression of MMP7 is also significantly associated with the prognosis of hepatocellular carcinoma. -
Key words:
- Liver Neoplasms /
- Matrix Metalloproteinase 7 /
- Cell Movement
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2020年全球新增癌症病例1 930万例,癌症死亡人数近1 000万例,其中肝癌是癌症死亡的第三大原因,成为威胁人类健康的主要因素[1]。肝癌的治疗方法主要包括传统的手术切除、放疗、化疗,以及最近越来越受到关注的免疫检查点阻断治疗,但是癌症的高复发及高转移风险使得癌症患者的生存期不能有效延长[2-5],因此探寻新的分子用于肝癌的免疫治疗具有重要意义,对癌症患者的预后生存也具有重要价值。基质金属蛋白酶7(matrix metalloproteinase-7,MMP7)是基质金属蛋白酶家族中的主要成员之一,由267个氨基酸组成,具有蛋白水解酶活性,能够促进许多生长因子的释放,如表皮生长因子受体、肝素结合表皮生长因子受体、胞外结构域的细胞表面分子脱落[6],主要参与细胞外基质降解的过程如:胚胎发育、组织重塑等,在另外一些疾病如关节炎、癌症转移的过程中也发挥重要作用[7-8]。研究[9]发现,MMP7对多种恶性肿瘤的发生及转移过程具有重要意义。本文主要分析MMP7在肝癌中的表达及在肝癌迁移浸润中的作用,并探讨其与肝癌预后的关系,从而为肝癌的临床治疗和预后提供新思路。
1. 材料与方法
1.1 细胞培养和抗体
人肝癌细胞株(MHCC97H)购自上海中山医院肝癌研究所,保存于南通百奥迈科生物技术有限公司,将MHCC97H在含10%胎牛血清的DMEM培养基中生长,并在5% CO2、37 ℃恒温培养箱中进行培养。兔抗人MMP7抗体购自杭州联科生物公司,鼠抗人GD购自美国Abcam公司,本研究中所用的二抗羊抗鼠IgG-HRP和羊抗兔IgG-HRP均购自美国Abcam公司。
1.2 siRNA及过表达质粒设计与合成和细胞转染
MMP7_ siRNA、pMMP7的设计与合成均由南通百奥迈科生物技术公司完成(表1),在肝癌细胞中,分别取2.5 μL稀释好的siRNA或2.5 μL稀释好过表达质粒加入到含10%胎牛血清的50∶1 DMEM血清基础培养基中,混匀,室温孵育5 min。然后由LipofectamineTM 2000转染入肝癌细胞株,另外将构建的pMMP7转染肝癌细胞株(MHCC97H)后,利用荧光显微镜分别在明场和荧光下(×100)对比观察,计算转染率。
表 1 siRNA 序列Table 1. Sequences of siRNA基因名称 序列(5′-3′) MMP7_siRNA1 正义链 GGAAAGAGAAGUAAUUCAAdTdT 反义链 UUGAAUUACUUCUCUUUCCdTdT MMP7_siRNA2 正义链 GACGGAUGGUAGCAGUCUAdTdT 反义链 UAGACUGCUACCAUCCGUCdTdT MMP7_siRNA3 正义链 CGAUUAGUGUCAAAGGCUUdTdT 反义链 AAGCCUUUGACACUAAUCGdTdT MMP7_siRNA4 正义链 GCAUUUCAGGAAAGUUGUAdTdT 反义链 UACAACUUUCCUGAAAUGCdTdT NC_siRNA 正义链 UUCUCCGAACGUGUCACGUdTdT 反义链 ACGUGACACGUUCGGAGAAdTdT 1.3 RT-qPCR法检测基因mRNA的表达水平
首先用RISOTM RNA提取试剂提取相应的RNA,之后用RT-qPCR方法检测MMP7及管家基因APDH mRNA表达水平,甘油醛-3-磷酸脱氢酶(GAPDH)的表达水平为内参,引物序列见表2。之后反应结束分析RT-qPCR的扩增曲线,同时观察熔解曲线,按每个反应的Ct值,用2-ΔΔCt[10]分析实验结果。
表 2 RT-qPCR 引物序列Table 2. Sequences of RT-qPCR primers基因名称 序列(5′-3′) 长度(bp) MMP7 F:ACAGGCTCAGGACTATCTCAAG 179 R:CAACATCTGGCACTCCACATC GAPDH F:GAAGGTGAAGGTCGGAGTC 226 R:GAAGATGGTGATGGGATTTC 1.4 Western Blot法
将MHCC97H细胞转染后利用SDS蛋白裂解液裂解细胞后提取总蛋白,测定蛋白浓度后,进行凝胶电泳。待转膜结束,取出PVDF膜,加入5%脱脂奶粉溶液进行封闭室温2 h。之后进行一抗(MMP7抗体一抗工作浓度为1∶500,GAPDH一抗工作浓度为1∶400)即孵育4 ℃过夜,次日恢复室温后,加入相应二抗即经辣根过氧化物酶标记的羊抗兔/鼠IgG抗体(1∶500),室温孵育PVDF膜2 h,二抗孵育结束后,室温,TBST清洗10 min×3次,准备荧光剂ECL:200 μL A+200 μL B,PVDF膜正面朝上,均匀滴加ECL显影液,显影仪显影:将PVDF膜放至显影仪内,均匀滴加显影液,关闭显影仪,软件直接读取蛋白印迹条带,之后用凝胶图像处理系统(Image J)分析目的条带的分子量和净光密度值。
1.5 Transwell小室实验
将MHCC97H细胞转染48 h后0.25%胰酶消化细胞,DMEM基础培养基重悬细胞并调整细胞密度为1×106个/mL,且小室在实验前用基础培养基进行孵育至少1 h。在Transwell上室中加入已经稀释好的细胞悬液100 µL,下室加入600 µL培养液:DMEM完全培养基与条件培养基1∶1混合。置于37 ℃、5% CO2条件下细胞培养箱孵育24 h,经4%的多聚甲醛固定细胞15 min后,0.5%结晶紫溶液染色15 min后,PBS清洗5 min×3次,将小室倒扣于载玻片上,在显微镜下观察下室的细胞,分别计数穿膜细胞并拍照。
1.6 电镜样品前期制备及扫描电镜观察
将灭菌后的小圆玻片放入24孔板中,分别接种细胞,进行转染后吸除培养基,加入500 mL PBS轻轻漂洗3遍,弃PBS后,加入2.5%的戊二醛,在4 ℃条件下固定细胞24 h;细胞处理后,由南通大学扫描电镜室利用扫描电镜进行采图处理,之后在扫描电镜下观察细胞形态及伪足变化。
1.7 MMP7与免疫细胞及免疫浸润评分
在TIMER数据库[11]分析MMP7与B淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞、中性粒细胞、巨噬细胞、树突状细胞的相关性。计算每个肝癌患者的基质、免疫和评估评分[12]。
1.8 统计学方法
使用SPSS 20.0统计软件进行数据分析。相关性分析采用Spearman方法。分析TCGA数据库[13]中肝癌患者的生存数据,并利用Sanger Box在线工具评估MMP7在肝癌中总体生存期(OS)、疾病特异性生存期(DSS)方面的意义[14-15]。R软件包maxstat分别计算OS、DSS中ENSG00000137673(MMP7)的最佳截断值,设置最小分组样本数大于25%,最大分组样本数小于75%,基于此将肝癌患者分为MMP7高表达组和低表达组,计算危险比(HR)和P值,进一步使用R软件包survival的survfit函数分析两组的预后差异,Kaplan-Meier法绘制生存曲线,Log-rank检验评估不同组样本之间的预后差异。P<0.05为差异有统计学意义。
2. 结果
2.1 筛选MMP7_siRNA
将构建的4种MMP7_siRNA和阴性对照组NC_siRNA转入MHCC97H。RT-qPCR和Western Blot结果表明,与NC-siRNA组相比,MMP7_siRNA2下调最明显(P<0.05)(图1),用于后续实验。
2.2 检测pMMP7转染MHCC97H后MMP7的表达
将构建的pMMP7转染MHCC97H,荧光显微镜观察发现转染效率达到80%,并且琼脂糖凝胶电泳表明所抽提的质粒大小与标准相符。RT-qPCR及Western Blot实验结果发现,pMMP7组中MMP7 mRNA及蛋白水平的表达均明显增高(P值均<0.05)(图2)。
2.3 观察MMP7对MHCC97H细胞伪足的影响
在MHCC97H中分别转染MMP7_siRNA2和pMMP7后,经扫描电镜显示,与阴性对照组相比,MMP7_siRNA2组中细胞伪足明显减少,并且变短。pMMP7组中细胞表面丝状伪足数量明显增多,并且伪足长度变长,放射状排列(图3)。
2.4 MMP7对细胞迁移的影响
MMP7_siRNA2及pMMP7分别转染MHCC97H,Transwell小室结果发现,MMP7_siRNA2能够显著降低细胞迁移能力(P<0.05),而转染pMMP7后,细胞迁移能力显著增加(P<0.05)(图4)。
2.5 在肝细胞癌(HCC)中MMP7表达与免疫细胞及免疫浸润的关系
对TIMER数据库分析发现,TCGA-HCC中MMP7的表达与B淋巴细胞(r=0.37)、CD4+T淋巴细胞(r=0.40)、中性粒细胞(r=0.49)、巨噬细胞(r=0.49)、树突状细胞(r=0.47)显著相关(P值均<0.05)(图5a)。进一步分析发现,在TCGA-HCC中根据MMP7基因表达分别计算每个肝癌患者stromal评分、Immune评分和ESTIMATE评分,结果都表明在肝癌中MMP7与免疫浸润呈显著正相关(P值均<0.01)(图5b)。
2.6 MMP7与肝癌患者预后生存期的关系分析
在TCGA数据库中,首先评估了MM7对肝癌患者OS的影响,通过计算获得MMP7的最佳截断值3.76,将肝癌患者分成MMP7高表达组(267例,64.6%)和MMP7低表达组(146例,35.4%),MMP7高表达组的OS明显低于MMP7低表达组(w=17.35,P<0.05)(图6a)。其次,分析MM7对肝癌患者DSS的影响,通过计算获得MMP7最佳截断值3.76,将肝癌患者分成MMP7高表达组(257例,63.9%)和MMP7低表达组(145例,36.1%),MMP7高表达组的DSS也低于MMP7低表达组(w=9.61,P<0.05)(图6b)。
3. 讨论
肝癌是成人最常见的恶性肿瘤,也是全球范围内危害公共健康的问题之一,5年相对生存率为18.4%,总生存率也很差。局部、区域和转移患者的5年生存率分别为33%、10%和2%[16-17]。由于缺乏症状,患者发现不适时往往已经到了中晚期[18-20]。因此,寻找与肝癌转移及预后相关的靶点对于肝癌患者的预后生存具有重要意义。
MMP7是MMP家族的主要成员,具有蛋白水解酶的特性。由于缺乏与HPX的同源序列,MMP7不需要特异识别底物的结构就可以降解几乎所有的细胞外基质成分[6],从而促进肿瘤细胞的转移。在前列腺癌、结肠癌患者的预后评估中MMP7的高表达具有较高的预后价值[21-22],在胃癌和肺癌的浸润及转移过程中MMP7高度参与上皮间质转换并促进癌症进展[23-25]。Rong等[26]研究发现在肝癌血浆中MMP7的高表达水平与肝癌较低的OS有明显相关性。Ye等[27]研究发现,Olfactomedin 4通过下调MMP7表达降低了肝癌细胞的迁移及侵袭能力。He等[28]研究也发现,分子靶向抗癌药物通过调节MMP家族成员MMP7、MMP9等的表达来抑制肝癌细胞的侵袭和转移。
在本研究中首先分析了MMP7在肝癌细胞株MHCC97H迁移中的作用。利用RT-qPCR及Western Blot方法筛选出最有效的MMP7_siRNA即MMP7_siRNA2,并将MMP7_siRNA2及构建的MMP7过表达质粒(pMMP7),分别转入MHCC97H细胞,结果表明在肝癌细胞株MHCC97H中,MMP7_siRNA2与pMMP7均能够有效下调或者上调MMP7的表达。扫描电镜观察发现,MMP7_siRNA2组中细胞伪足明显减少,并且变短;pMMP7组中细胞表面丝状伪足数量明显增多,并且伪足长度变长,放射状排列。Transwell小室迁移实验发现MMP7_siRNA2细胞系肝癌细胞株MHCC97H的迁移能力减弱,而pMMP7细胞系能够明显促进肝癌细胞的迁移。Liao等[29]也发现磷酸组氨酸无机焦磷酸酶能够通过作用于MMP7、MMP9、CCNB1的表达来抑制HCC细胞生长和转移,这也与本实验结果相一致。
肿瘤的发生是一个多步骤、多层次的过程,与癌基因的激活、肿瘤抑制基因的抑制作用、表观遗传改变修饰和肿瘤微环境的变化等多种因素相关[30-33]。肿瘤微环境是由肿瘤细胞、多种免疫细胞(包括淋巴细胞、自然杀伤细胞、巨噬细胞和树突状细胞)以及细胞外基质、血管和其他物质组成的复杂结构。肿瘤进展在很大程度上受到肿瘤微环境中免疫细胞的组成和丰度的影响,不同类型的免疫细胞在肿瘤微环境中起着关键的调节作用,并且有助于肿瘤的异质性[34-40]。越来越多的证据[41-43]表明,癌细胞与肿瘤微环境各种成分之间的相互作用有助于肿瘤的免疫逃逸,并最终导致肿瘤的增殖、复发和转移。因此利用生物信息学技术,分析TIMER数据库中MMP7与肿瘤免疫细胞的关系,结果表明TCGA-HCC中MMP7的表达与B淋巴细胞、CD4+T淋巴细胞、中性粒细胞、巨噬细胞、树突状细胞5种免疫细胞呈显著正相关(P值均<0.05),表明MMP7可能通过免疫细胞浸润参与HCC的发生和发展。之后进一步分析发现在TCGA-HCC中根据MMP7基因表达分别计算每个肝癌患者stromal评分、Immune评分和ESTIMATE评分,这三种评分结果都表明在肝癌中MMP7与免疫浸润呈显著正相关(P值均<0.01)。因此推测MMP7可能通过免疫细胞浸润调控肝癌的发生和发展。通过进一步分析TCGA数据库中MMP7与肝癌预后的关系,结果表明MMP7高表达时肝癌患者的OS和DSS明显降低,提示MMP7可以作为评估肝癌患者预后的有效指标。
综上所述,通过分子生物学实验和生物信息学技术分析,本研究揭示了MMP7能够促进肝癌细胞的迁移,其表达与B淋巴细胞、CD4+T淋巴细胞、中性粒细胞、巨噬细胞、树突状细胞的浸润水平相关,在肝癌细胞系中MMP7与肝癌患者的免疫浸润显著相关。除此之外,MMP7高表达与肝癌不良预后有关,可作为肝癌新的潜在预后标志物和靶向治疗的潜在靶点。但由于实验条件有限,并未进行MMP7相关肝癌体内实验,因此未来应实施体内实验进一步验证MMP7的功能并探讨其促进肝癌发生发展的潜在分子机制。
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表 1 siRNA 序列
Table 1. Sequences of siRNA
基因名称 序列(5′-3′) MMP7_siRNA1 正义链 GGAAAGAGAAGUAAUUCAAdTdT 反义链 UUGAAUUACUUCUCUUUCCdTdT MMP7_siRNA2 正义链 GACGGAUGGUAGCAGUCUAdTdT 反义链 UAGACUGCUACCAUCCGUCdTdT MMP7_siRNA3 正义链 CGAUUAGUGUCAAAGGCUUdTdT 反义链 AAGCCUUUGACACUAAUCGdTdT MMP7_siRNA4 正义链 GCAUUUCAGGAAAGUUGUAdTdT 反义链 UACAACUUUCCUGAAAUGCdTdT NC_siRNA 正义链 UUCUCCGAACGUGUCACGUdTdT 反义链 ACGUGACACGUUCGGAGAAdTdT 表 2 RT-qPCR 引物序列
Table 2. Sequences of RT-qPCR primers
基因名称 序列(5′-3′) 长度(bp) MMP7 F:ACAGGCTCAGGACTATCTCAAG 179 R:CAACATCTGGCACTCCACATC GAPDH F:GAAGGTGAAGGTCGGAGTC 226 R:GAAGATGGTGATGGGATTTC -
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