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

刘淑岩; 杨其昌; 沈屹; 周虹; 钱金锋

doi:10.12449/JCH240721

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

南通市第一人民医院病理科，江苏南通 226014

基金项目:

南通大学研究生创新计划项目 (YKC16070)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：刘淑岩负责课题设计，资料分析，撰写论文；杨其昌、沈屹、周虹参与收集数据，修改论文；钱金锋负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
钱金锋， qjinfeng@126.com （ORCID： 0009-0003-5024-7423）

计量
- 文章访问数: 230
- HTML全文浏览量: 112
- PDF下载量: 36
- 被引次数: 0
出版历程
- 收稿日期: 2023-11-09
- 录用日期: 2023-12-15
- 出版日期: 2024-07-25

Role and prognostic value of matrix metalloproteinase-7 in the migration and immune cell infiltration of hepatocellular carcinoma

Department of Pathology，The First People’s Hospital of Nantong，Nantong，Jiangsu 226014，China

Research funding:

The Post-graduation Innovation Project Program of Nantong University (YKC16070)

More Information

Corresponding author: QIAN Jinfeng， qjinfeng@126.com （ORCID： 0009-0003-5024-7423）

摘要

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。
- 肝肿瘤 /
- 基质金属蛋白酶7 /
- 细胞运动
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.
- Liver Neoplasms /
- Matrix Metalloproteinase 7 /
- Cell Movement

HTML全文

图 1 RT-qPCR及Western Blot实验筛选最有效的MMP7_siRNA

注： a，RT-qPCR方法检测MMP7 mRNA表达水平；b，Western Blot检测MMP7蛋白表达水平。与NC_siRNA组比较，*P<0.05。

Figure 1. Utilized RT-qPCR and Western Blot assays to identify the MMP7_siRNA with the highest efficacy

下载: 全尺寸图片幻灯片

图 2 pMMP7转染MHCC97H后MMP7表达水平的变化

注： a，pMMP7转染MHCC97H的转染效率（×100）；b，pMMP7及Vector抽提质粒效果；c，RT-qPCR检测pMMP7转染MHCC97H后MMP7 mRNA表达水平；d，Western Blot检测pMMP7转染MHCC97H后MMP7蛋白表达水平。

Figure 2. The expression level of MMP7 after transfection with pMMP7 into MHCC97H

下载: 全尺寸图片幻灯片

图 3 扫描电镜观察MHCC97H伪足的变化（×3 000）

Figure 3. The changes of pseudopodia were observed by scanning electron microscopy （×3 000）

下载: 全尺寸图片幻灯片

图 4 Transwell小室检测MHCC97H迁移能力的变化（×200）

Figure 4. The change of migration ability of liver cancer cell line （MHCC97H） was detected by Transwell chamber（×200）

下载: 全尺寸图片幻灯片

图 5 在TCGA-HCC中MMP7表达与免疫细胞及免疫浸润评分的关系

注： a，TCGA-HCC中，MMP7与免疫细胞浸润的相关性；b，在TCGA-HCC中，MMP7与免疫浸润评分的相关性。

Figure 5. Relationship between MMP7 expression and immune cells and immunoinfiltration in TCGA-HCC

下载: 全尺寸图片幻灯片

图 6 MMP7与肝癌患者预后生存期的相关性

注： a，TCGA-HCC中MMP7的表达与OS的关系；b，TCGA-HCC中MMP7表达与DSS的关系。

Figure 6. Correlation between MMP7 and prognosis of patients with liver cancer

下载: 全尺寸图片幻灯片

表 1 siRNA 序列

Table 1. Sequences of siRNA

基因名称	序列（5′-3′）
MMP7_siRNA1	正义链	GGAAAGAGAAGUAAUUCAAdTdT
MMP7_siRNA1	反义链	UUGAAUUACUUCUCUUUCCdTdT
MMP7_siRNA2	正义链	GACGGAUGGUAGCAGUCUAdTdT
MMP7_siRNA2	反义链	UAGACUGCUACCAUCCGUCdTdT
MMP7_siRNA3	正义链	CGAUUAGUGUCAAAGGCUUdTdT
MMP7_siRNA3	反义链	AAGCCUUUGACACUAAUCGdTdT
MMP7_siRNA4	正义链	GCAUUUCAGGAAAGUUGUAdTdT
MMP7_siRNA4	反义链	UACAACUUUCCUGAAAUGCdTdT
NC_siRNA	正义链	UUCUCCGAACGUGUCACGUdTdT
NC_siRNA	反义链	ACGUGACACGUUCGGAGAAdTdT

下载: 导出CSV

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
MMP7	R：CAACATCTGGCACTCCACATC	179
GAPDH	F：GAAGGTGAAGGTCGGAGTC	226
GAPDH	R：GAAGATGGTGATGGGATTTC	226

下载: 导出CSV

参考文献(43)

[1]	SUNG H, FERLAY J, SIEGEL RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71( 3): 209- 249. DOI: 10.3322/caac.21660.
[2]	CRISTESCU R, MOGG R, AYERS M, et al. Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy[J]. Science, 2018, 362( 6411): eaar3593. DOI: 10.1126/science.aar3593.
[3]	THOMPSON SM, CALLSTROM MR, JONDAL DE, et al. Heat stress-induced PI3K/mTORC2-dependent AKT signaling is a central mediator of hepatocellular carcinoma survival to thermal ablation induced heat stress[J]. PLoS One, 2016, 11( 9): e0162634. DOI: 10.1371/journal.pone.0162634.
[4]	MASUZAKI R, YOSHIDA H, TATEISHI R, et al. Hepatocellular carcinoma in viral hepatitis: Improving standard therapy[J]. Best Pract Res Clin Gastroenterol, 2008, 22( 6): 1137- 1151. DOI: 10.1016/j.bpg.2008.11.005.
[5]	HERNANDEZ-GEA V, TOFFANIN S, FRIEDMAN SL, et al. Role of the microenvironment in the pathogenesis and treatment of hepatocellular carcinoma[J]. Gastroenterology, 2013, 144( 3): 512- 527. DOI: 10.1053/j.gastro.2013.01.002.
[6]	CHEN JF, LU XL, ZHANG XY, et al. Expression and clinical significance of matrix metalloproteinase 7 and heparan sulfate proteoglycan 2 in pancreatic cancer[J]. J Clin Hepatol, 2023, 39( 9): 2169- 2174. DOI: 10.3969/j.issn.1001-5256.2023.09.020. 陈金凤, 陆小柳, 张晓韵, 等. 基质金属蛋白酶7(MMP7)和硫酸乙酰肝素糖蛋白2(HSPG2)在胰腺癌中的表达及临床意义[J]. 临床肝胆病杂志, 2023, 39( 9): 2169- 2174. DOI: 10.3969/j.issn.1001-5256.2023.09.020.
[7]	CHEN L, KE XY. MMP7 as a potential biomarker of colon cancer and its prognostic value by bioinformatics analysis[J]. Medicine, 2021, 100( 9): e24953. DOI: 10.1097/MD.0000000000024953.
[8]	LI XF, AIERKEN ALD, SHEN L. IPO5 promotes malignant progression of esophageal cancer through activating MMP7[J]. Eur Rev Med Pharmacol Sci, 2020, 24( 8): 4246- 4254. DOI: 10.26355/eurrev_202004_21004.
[9]	GOBIN E, BAGWELL K, WAGNER J, et al. A pan-cancer perspective of matrix metalloproteases(MMP) gene expression profile and their diagnostic/prognostic potential[J]. BMC Cancer, 2019, 19( 1): 581. DOI: 10.1186/s12885-019-5768-0.
[10]	DING XX, ZHU QG, ZHANG SM, et al. Precision medicine for hepatocellular carcinoma: Driver mutations and targeted therapy[J]. Oncotarget, 2017, 8( 33): 55715- 55730. DOI: 10.18632/oncotarget.18382.
[11]	LI TW, FAN JY, WANG BB, et al. TIMER: A web server for comprehensive analysis of tumor-infiltrating immune cells[J]. Cancer Res, 2017, 77( 21): e108- e110. DOI: 10.1158/0008-5472.CAN-17-0307.
[12]	XU WX, ZHANG J, HUA YT, et al. An integrative pan-cancer analysis revealing LCN2 as an oncogenic immune protein in tumor microenvironment[J]. Front Oncol, 2020, 10: 605097. DOI: 10.3389/fonc.2020.605097.
[13]	GTEX CONSORTIUM. The genotype-tissue expression(GTEx) project[J]. Nat Genet, 2013, 45( 6): 580- 585. DOI: 10.1038/ng.2653.
[14]	SHEN W, SONG Z, ZHONG X, et al. Sangerbox: A comprehensive, interaction-friendly clinical bioinformatics analysis platform[J]. Imeta, 2022, 1( 3): e36. https://doi.org/10.1002/imt2.36 DOI: 10.1002/imt2.36
[15]	AJUCARMELPRECILLA A, PANDI J, DHANDAPANI R, et al. In silico identification of hub genes as observing biomarkers for gastric cancer metastasis[J]. Evid Based Complement Alternat Med, 2022, 2022: 6316158. DOI: 10.1155/2022/6316158.
[16]	RAJENDRAN L, IVANICS T, CLAASEN MP, et al. The management of post-transplantation recurrence of hepatocellular carcinoma[J]. Clin Mol Hepatol, 2022, 28( 1): 1- 16. DOI: 10.3350/cmh.2021.0217.
[17]	YANG JD, HAINAUT P, GORES GJ, et al. A global view of hepatocellular carcinoma: Trends, risk, prevention and management[J]. Nat Rev Gastroenterol Hepatol, 2019, 16( 10): 589- 604. DOI: 10.1038/s41575-019-0186-y.
[18]	LEOWATTANA W, LEOWATTANA T, LEOWATTANA P. Systemic treatment for unresectable hepatocellular carcinoma[J]. World J Gastroenterol, 2023, 29( 10): 1551- 1568. DOI: 10.3748/wjg.v29.i10.1551.
[19]	ZAVADIL J, ROHAN T, JURÁČEK J, et al. Biomarkers as prognostic and predictive factors in patients with hepatocellular carcinoma undergoing radiological oncological interventions[J]. Klin Onkol, 2023, 36( 2): 104- 111. DOI: 10.48095/ccko2023104.
[20]	ZHANG JF, ZHANG J. Research progress on diagnosis and treatments for microvascular invasion of hepatocellular carcinoma[J/CD]. Chin J Hepat Surg(Electronic Edition), 2022, 11( 1): 104- 108. DOI: 10.3877/cma.j.issn.2095-3232.2022.01.022. 张剑锋, 张剑. 肝细胞癌微血管浸润诊治研究进展[J/CD]. 中华肝脏外科手术学电子杂志, 2022, 11( 1): 104- 108. DOI: 10.3877/cma.j.issn.2095-3232.2022.01.022.
[21]	SZARVAS T, CSIZMARIK A, VÁRADI M, et al. The prognostic value of serum MMP-7 levels in prostate cancer patients who received docetaxel, abiraterone, or enzalutamide therapy[J]. Urol Oncol, 2021, 39( 5): 296. DOI: 10.1016/j.urolonc.2020.09.005.
[22]	TREGUNNA R. Serum MMP7 levels could guide metastatic therapy for prostate cancer[J]. Nat Rev Urol, 2020, 17( 12): 658. DOI: 10.1038/s41585-020-00396-3.
[23]	DU F, FENG WB, CHEN S, et al. Sex determining region Y-box 12(SOX12) promotes gastric cancer metastasis by upregulating MMP7 and IGF1[J]. Cancer Lett, 2019, 452: 103- 118. DOI: 10.1016/j.canlet.2019.03.035.
[24]	JAFFAR J, WONG M, FISHBEIN GA, et al. Matrix metalloproteinase-7 is increased in lung bases but not apices in idiopathic pulmonary fibrosis[J]. ERJ Open Res, 2022, 8( 4): 00191- 02022. DOI: 10.1183/23120541.00191-2022.
[25]	SANG HY, WU S, CHEN XF, et al. FAM46B suppresses proliferation, migration and invasion of non-small cell lung cancer via β-catenin/MMP7 signaling[J]. Transl Cancer Res, 2019, 8( 4): 1497- 1505. DOI: 10.21037/tcr.2019.07.27.
[26]	RONG WQ, ZHANG Y, YANG L, et al. Post-surgical resection prognostic value of combined OPN, MMP7, and PSG9 plasma biomarkers in hepatocellular carcinoma[J]. Front Med, 2019, 13( 2): 250- 258. DOI: 10.1007/s11684-018-0632-1.
[27]	YE LT, KRIEGL L, REITER FP, et al. Prognostic significance and functional relevance of olfactomedin 4 in early-stage hepatocellular carcinoma[J]. Clin Transl Gastroenterol, 2020, 11( 1): e00124. DOI: 10.14309/ctg.0000000000000124.
[28]	HE XX, SHI LL, QIU MJ, et al. Molecularly targeted anti-cancer drugs inhibit the invasion and metastasis of hepatocellular carcinoma by regulating the expression of MMP and TIMP gene families[J]. Biochem Biophys Res Commun, 2018, 504( 4): 878- 884. DOI: 10.1016/j.bbrc.2018.08.203.
[29]	LIAO LJ, DUAN DY, LIU YF, et al. LHPP inhibits hepatocellular carcinoma cell growth and metastasis[J]. Cell Cycle, 2020, 19( 14): 1846- 1854. DOI: 10.1080/15384101.2020.1783472.
[30]	JEGGO PA, PEARL LH, CARR AM. DNA repair, genome stability and cancer: A historical perspective[J]. Nat Rev Cancer, 2016, 16( 1): 35- 42. DOI: 10.1038/nrc.2015.4.
[31]	CHATTERJEE A, RODGER EJ, ECCLES MR. Epigenetic drivers of tumourigenesis and cancer metastasis[J]. Semin Cancer Biol, 2018, 51: 149- 159. DOI: 10.1016/j.semcancer.2017.08.004.
[32]	CHEN F, FAN YM, CAO PX, et al. Pan-cancer analysis of the prognostic and immunological role of HSF1: A potential target for survival and immunotherapy[J]. Oxid Med Cell Longev, 2021, 2021: 5551036. DOI: 10.1155/2021/5551036.
[33]	XU ZH, HE CY, FANG XD. Research progress in relationship between mitochondrial inner membrane mitochondrial protein and tumor[J]. J Jilin Univ(Med Edit), 2023, 49( 1): 231- 236. DOI: 10.13481/j.1671-587X.20230131. 徐仲航, 何成彦, 房学东. 线粒体内膜蛋白与肿瘤关系的研究进展[J]. 吉林大学学报(医学版), 2023, 49( 1): 231- 236. DOI: 10.13481/j.1671-587X.20230131.
[34]	LI TW, FU JX, ZENG ZX, et al. TIMER2.0 for analysis of tumor-infiltrating immune cells[J]. Nucleic Acids Res, 2020, 48( W1): W509- W514. DOI: 10.1093/nar/gkaa407.
[35]	ANGELOVA M, CHAROENTONG P, HACKL H, et al. Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy[J]. Genome Biol, 2015, 16( 1): 64. DOI: 10.1186/s13059-015-0620-6.
[36]	LEE N, ZAKKA LR, MIHM MC Jr, et al. Tumour-infiltrating lymphocytes in melanoma prognosis andcancerimmunotherapy[J]. Pathology, 2016, 48( 2): 177- 187. DOI: 10.1016/j.pathol.2015.12.006.
[37]	DENKERT C, VON MINCKWITZ G, DARB-ESFAHANI S, et al. Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: A pooled analysis of 3771 patients treated with neoadjuvant therapy[J]. Lancet Oncol, 2018, 19( 1): 40- 50. DOI: 10.1016/S1470-2045(17)30904-X.
[38]	GALON J, BRUNI D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies[J]. Nat Rev Drug Discov, 2019, 18( 3): 197- 218. DOI: 10.1038/s41573-018-0007-y.
[39]	ZHAO YD, SCHAAFSMA E, GORLOV IP, et al. A leukocyte infiltration score defined by a gene signature predicts melanoma patient prognosis[J]. Mol Cancer Res, 2019, 17( 1): 109- 119. DOI: 10.1158/1541-7786.MCR-18-0173.
[40]	MCGRANAHAN N, SWANTON C. Clonal heterogeneity and tumor evolution: Past, present, and the future[J]. Cell, 2017, 168( 4): 613- 628. DOI: 10.1016/j.cell.2017.01.018.
[41]	WEI GG, ZHANG HL, ZHAO HP, et al. Emerging immune checkpoints in the tumor microenvironment: Implications for cancer immunotherapy[J]. Cancer Lett, 2021, 511: 68- 76. DOI: 10.1016/j.canlet.2021.04.021.
[42]	ZHANG YY, ZHANG ZM. The history and advances in cancer immunotherapy: Understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications[J]. Cell Mol Immunol, 2020, 17( 8): 807- 821. DOI: 10.1038/s41423-020-0488-6.
[43]	MURCIANO-GOROFF YR, WARNER AB, WOLCHOK JD. The future of cancer immunotherapy: Microenvironment-targeting combinations[J]. Cell Res, 2020, 30( 6): 507- 519. DOI: 10.1038/s41422-020-0337-2.