兰州大学机构库 >第二临床医学院
基于肝癌细胞研究PA-MSHA抑制PD-L1及sPD-L1的机制
Alternative TitleResearch on the Mechanism of PA-MSHA Inhibiting PD-L1 and sPD-L1 in Hepatoma Carcinoma Cell
魏航之
Subtype博士
Thesis Advisor张有成
2023-08-29
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name医学博士
Degree Discipline外科学
Keyword肝细胞癌 HCC PA-MSHA PA-MSHA β-catenin β-catenin 甘露糖基 mannosyl PD-L1 PD-L1 sPD-L1 sPD-L1
Abstract

【背景】近年来肝细胞癌(HCC)的发病率逐步上升,成为导致癌症相关死亡的第四大恶性肿瘤。只有不到30%的患者可以被早期确诊并有手术机会。对于晚期HCC,当前疗效最好的分子靶向药物(MTA)和免疫检查点抑制剂(ICIs)的预后并不令人满意。因此探索HCC耐药机制及找寻新的有潜力的MTA和ICI迫在眉睫,开发这种恶性肿瘤的创新治疗方法的临床意义至关重要。当前铜绿假单胞菌-甘露糖敏感血凝素(PA-MSHA)基于其增强免疫、调节肿瘤免疫应答的作用被相关部门批准用于临床辅助治疗感染,辅助治疗胃癌、结肠癌、乳腺癌等癌症。PA-MSHA通过与细胞膜上的甘露糖基特异性结合而凝集红细胞等,PA-MSHA亦通过甘露糖基特异性结合影响肝癌、胃癌等类型肿瘤细胞膜受体蛋白发挥作用。虽然已有较多研究对PA-MSHA的抗肿瘤机制有深入研究,目前还未有报道表明PA-MSHA对PD-L1有抑制作用。因此本研究基于HCC细胞探索PA-MSHA对PD-L1、sPD-L1的作用及机制,为PA-MSHA可能作为ICIs或ICIs的辅助用药奠定理论基础。

【目的】本研究以PA-MSHA对PD-L1的显著抑制作用为研究中心,验证相关分子在HCC中的表达;验证PA-MSHA对HCC细胞生物学行为的影响;探索PA-MSHA抑制PD-L1的可能机制;探索PA-MSHA抑制sPD-L1对体外培养的外周血单个核细胞(PBMC)PD-1表达及凋亡的影响。为PA-MSHA治疗HCC提供新思路,为PA-MSHA的抗肿瘤研究提供新方向。

【方法】(1)免疫荧光(IF)、免疫组化(IHC)检测HCC组织β-catenin、甘露糖苷乙酰氨基葡萄糖转移酶1(MGAT1)、α-甘露糖苷酶II、甘露糖基、PD-L1的表达情况,分析与临床病理特征的关系。(2)通过光学显微镜、透射电镜、CCK-8实验、平板克隆形成以及transwell小室实验评估PA-MSHA对肝癌细胞生物学功能及细胞结构的影响。western blot检测PA-MSHA干预后肝癌细胞紧密连接蛋白Claudin-1的变化。构建Huh7细胞的裸鼠皮下种植瘤模型,评估PA-MSHA对肿瘤组织生长的抑制情况及本研究相关分子表达的影响。(3)通过 qRT-PCR 、western blot检测PA-MSHA干预后肝癌细胞β-catenin、c-Myc、PD-L1表达变化。采用质粒和小干扰RNA(siRNA)构建β-catenin过表达和下调模型,PA-MSHA干预后western blot检测β-catenin、c-Myc、PD-L1、凋亡相关蛋白、MGAT1表达。(4)qRT-PCR检测PA-MSHA干预后肝癌细胞MGAT各亚型、α-甘露糖苷酶各亚型表达的变化,筛选可能作用的靶点;western blot检测MGAT1、α-甘露糖苷酶II的表达;IF检测甘露糖基变化。通过质粒构建肝癌细胞MGAT1过表达模型,PA-MSHA干预后western blot检测PD-L1表达。(5)酶联免疫吸附测定(ELISA)检测PA-MSHA对肝癌细胞sPD-L1的影响。构建sPD-L1干预体外培养的PBMC模型,western blot检测PBMC PD-1和凋亡蛋白表达。GEPIA分析HCC样本中sPD-L1与患者预后的关系,绘制Kaplan-Meier生存曲线。

【结果】(1)PD-L1、β-catenin、c-Myc、甘露糖苷酶II、甘露糖基在HCC组织中高表达,MGAT1低表达。患者年龄增加,MGAT1表达增加且甘露糖基减低。HCC组织中PD-L1和MGAT1表达负相关。MGAT1与HCC组织中中甲胎蛋白(AFP)表达相反,也和肝癌患者血液中AFP水平相反。HCC中β-catenin与癌旁组织中β-catenin中度相关。(2)CCK-8、平板克隆形成和transwell小室实验结果表明,PA-MSHA可显著抑制HCC细胞的增殖、迁移和侵袭能力,PA-MSHA干预增加HCC细胞Claudin-1的表达。电镜观察细胞结构,PA-MSHA干预导致细胞质空泡、染色质边缘聚集、内质网扩张、线粒体嵴断裂等典型凋亡特征。(3)PA-MSHA干预HCC细胞后β-catenin的抑制基因AXIN2、APC上调,CTNNB1(β-catenin)、c-Myc、PD-L1基因表达下调。PA-MSHA干预后细胞和移植瘤组织中β-catenin、c-Myc蛋白减少,PD-L1显著减少。PA-MSHA干预β-catenin过表达和下调模型表明PA-MSHA通过影响β-catenin部分抑制PD-L1表达。PA-MSHA通过抑制β-catenin促进HCC细胞凋亡。(4)PA-MSHA干预HCC细胞后qRT-PCR检测的基因有MGAT1、MGAT3、MGAT4C、MGAT5、MAN1B1、MAN1C1、MAN2A1,其中MGAT1、MAN2A1相对表达量是对照组两倍以上。PA-MSHA干预Huh7细胞移植瘤后MGAT1、MGAT3、MAN2A1基因表达增加。PA-MSHA干预HCC细胞后MGAT1、甘露糖苷酶II蛋白表达增加,western blot检测表明PA-MSHA可通过影响β-catenin增加MGAT1,最后减低PD-L1。PA-MSHA干预后IF、IHC检测裸鼠种植瘤组织,显示β-catenin,c-Myc,甘露糖基减低,MGAT1增加。(5)经PA-MSHA干预后两种肝癌细胞培养基中sPD-L1浓度均明显降低。未经PA-MSHA干预的Huh7细胞其上清对PBMC PD-1的抑制最显著,PA-MSHA干预后的Huh7细胞上清对PBMC PD-1没有显著抑制,PA-MSHA干预的Huh7细胞上清对PBMC无明显促凋亡作用。GEPIA分析sPD-L1高表达组的HCC患者总生存期(OS)显著低于sPD-L1低表达组。

【结论】(1)PA-MSHA显著抑制HCC细胞PD-L1的表达,其中部分抑制作用可能是通过PA-MSHA抑制β-catenin,减低PD-L1基因和蛋白的表达引起。PA-MSHA可通过抑制β-catenin促进HCC细胞凋亡。(2)PA-MSHA可能通过抑制β-catenin继而降低甘露糖基的表达,影响HCC细胞PD-L1的N-糖基化过程,最后影响PD-L1蛋白的表达量。(3)PA-MSHA显著抑制HCC细胞sPD-L1表达。PA-MSHA通过抑制sPD-L1,减轻HCC细胞产生sPD-L1对PBMC PD-1的抑制,减轻sPD-L1引起的PBMC凋亡,PA-MSHA有作为ICIs或ICIs辅助用药的理论基础。

【背景】近年来肝细胞癌(HCC)的发病率逐步上升,成为导致癌症相关死亡的第四大恶性肿瘤。只有不到30%的患者可以被早期确诊并有手术机会。对于晚期HCC,当前疗效最好的分子靶向药物(MTA)和免疫检查点抑制剂(ICIs)的预后并不令人满意。因此探索HCC耐药机制及找寻新的有潜力的MTA和ICI迫在眉睫,开发这种恶性肿瘤的创新治疗方法的临床意义至关重要。当前铜绿假单胞菌-甘露糖敏感血凝素(PA-MSHA)基于其增强免疫、调节肿瘤免疫应答的作用被相关部门批准用于临床辅助治疗感染,辅助治疗胃癌、结肠癌、乳腺癌等癌症。PA-MSHA通过与细胞膜上的甘露糖基特异性结合而凝集红细胞等,PA-MSHA亦通过甘露糖基特异性结合影响肝癌、胃癌等类型肿瘤细胞膜受体蛋白发挥作用。虽然已有较多研究对PA-MSHA的抗肿瘤机制有深入研究,目前还未有报道表明PA-MSHA对PD-L1有抑制作用。因此本研究基于HCC细胞探索PA-MSHA对PD-L1、sPD-L1的作用及机制,为PA-MSHA可能作为ICIs或ICIs的辅助用药奠定理论基础。

【目的】本研究以PA-MSHA对PD-L1的显著抑制作用为研究中心,验证相关分子在HCC中的表达;验证PA-MSHA对HCC细胞生物学行为的影响;探索PA-MSHA抑制PD-L1的可能机制;探索PA-MSHA抑制sPD-L1对体外培养的外周血单个核细胞(PBMC)PD-1表达及凋亡的影响。为PA-MSHA治疗HCC提供新思路,为PA-MSHA的抗肿瘤研究提供新方向。

【方法】(1)免疫荧光(IF)、免疫组化(IHC)检测HCC组织β-catenin、甘露糖苷乙酰氨基葡萄糖转移酶1(MGAT1)、α-甘露糖苷酶II、甘露糖基、PD-L1的表达情况,分析与临床病理特征的关系。(2)通过光学显微镜、透射电镜、CCK-8实验、平板克隆形成以及transwell小室实验评估PA-MSHA对肝癌细胞生物学功能及细胞结构的影响。western blot检测PA-MSHA干预后肝癌细胞紧密连接蛋白Claudin-1的变化。构建Huh7细胞的裸鼠皮下种植瘤模型,评估PA-MSHA对肿瘤组织生长的抑制情况及本研究相关分子表达的影响。(3)通过 qRT-PCR 、western blot检测PA-MSHA干预后肝癌细胞β-catenin、c-Myc、PD-L1表达变化。采用质粒和小干扰RNA(siRNA)构建β-catenin过表达和下调模型,PA-MSHA干预后western blot检测β-catenin、c-Myc、PD-L1、凋亡相关蛋白、MGAT1表达。(4)qRT-PCR检测PA-MSHA干预后肝癌细胞MGAT各亚型、α-甘露糖苷酶各亚型表达的变化,筛选可能作用的靶点;western blot检测MGAT1、α-甘露糖苷酶II的表达;IF检测甘露糖基变化。通过质粒构建肝癌细胞MGAT1过表达模型,PA-MSHA干预后western blot检测PD-L1表达。(5)酶联免疫吸附测定(ELISA)检测PA-MSHA对肝癌细胞sPD-L1的影响。构建sPD-L1干预体外培养的PBMC模型,western blot检测PBMC PD-1和凋亡蛋白表达。GEPIA分析HCC样本中sPD-L1与患者预后的关系,绘制Kaplan-Meier生存曲线。

【结果】(1)PD-L1、β-catenin、c-Myc、甘露糖苷酶II、甘露糖基在HCC组织中高表达,MGAT1低表达。患者年龄增加,MGAT1表达增加且甘露糖基减低。HCC组织中PD-L1和MGAT1表达负相关。MGAT1与HCC组织中中甲胎蛋白(AFP)表达相反,也和肝癌患者血液中AFP水平相反。HCC中β-catenin与癌旁组织中β-catenin中度相关。(2)CCK-8、平板克隆形成和transwell小室实验结果表明,PA-MSHA可显著抑制HCC细胞的增殖、迁移和侵袭能力,PA-MSHA干预增加HCC细胞Claudin-1的表达。电镜观察细胞结构,PA-MSHA干预导致细胞质空泡、染色质边缘聚集、内质网扩张、线粒体嵴断裂等典型凋亡特征。(3)PA-MSHA干预HCC细胞后β-catenin的抑制基因AXIN2、APC上调,CTNNB1(β-catenin)、c-Myc、PD-L1基因表达下调。PA-MSHA干预后细胞和移植瘤组织中β-catenin、c-Myc蛋白减少,PD-L1显著减少。PA-MSHA干预β-catenin过表达和下调模型表明PA-MSHA通过影响β-catenin部分抑制PD-L1表达。PA-MSHA通过抑制β-catenin促进HCC细胞凋亡。(4)PA-MSHA干预HCC细胞后qRT-PCR检测的基因有MGAT1、MGAT3、MGAT4C、MGAT5、MAN1B1、MAN1C1、MAN2A1,其中MGAT1、MAN2A1相对表达量是对照组两倍以上。PA-MSHA干预Huh7细胞移植瘤后MGAT1、MGAT3、MAN2A1基因表达增加。PA-MSHA干预HCC细胞后MGAT1、甘露糖苷酶II蛋白表达增加,western blot检测表明PA-MSHA可通过影响β-catenin增加MGAT1,最后减低PD-L1。PA-MSHA干预后IF、IHC检测裸鼠种植瘤组织,显示β-catenin,c-Myc,甘露糖基减低,MGAT1增加。(5)经PA-MSHA干预后两种肝癌细胞培养基中sPD-L1浓度均明显降低。未经PA-MSHA干预的Huh7细胞其上清对PBMC PD-1的抑制最显著,PA-MSHA干预后的Huh7细胞上清对PBMC PD-1没有显著抑制,PA-MSHA干预的Huh7细胞上清对PBMC无明显促凋亡作用。GEPIA分析sPD-L1高表达组的HCC患者总生存期(OS)显著低于sPD-L1低表达组。

【结论】(1)PA-MSHA显著抑制HCC细胞PD-L1的表达,其中部分抑制作用可能是通过PA-MSHA抑制β-catenin,减低PD-L1基因和蛋白的表达引起。PA-MSHA可通过抑制β-catenin促进HCC细胞凋亡。(2)PA-MSHA可能通过抑制β-catenin继而降低甘露糖基的表达,影响HCC细胞PD-L1的N-糖基化过程,最后影响PD-L1蛋白的表达量。(3)PA-MSHA显著抑制HCC细胞sPD-L1表达。PA-MSHA通过抑制sPD-L1,减轻HCC细胞产生sPD-L1对PBMC PD-1的抑制,减轻sPD-L1引起的PBMC凋亡,PA-MSHA有作为ICIs或ICIs辅助用药的理论基础。

Other Abstract

Background: In recent years, the incidence rate of hepatocellular carcinoma (HCC) has gradually increased, becoming the fourth largest malignant tumor leading to cancer related deaths. Less than 30% of patients can be diagnosed early and have the opportunity for surgery. For advanced HCC, the current best molecular targeted agent (MTA) and immune checkpoint inhibitors (ICIs) have unsatisfactory prognosis. Therefore, it is urgent to explore the mechanism of HCC drug resistance and to find new potential MTA and ICIs, and it is of great clinical significance to develop innovative therapeutic methods for this malignant tumor. Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA) is currently approved by relevant departments for clinical adjuvant treatment of infection, adjuvant treatment of stomach carcinoma, colon carcinoma, breast carcinoma and other cancers based on its role of enhancing immunity and regulating tumor immune response. PA-MSHA agglutinates red blood cells by specifically binding to mannosyl the cell membrane, and PA-MSHA also affects some membrane receptor proteins of HCC, stomach carcinoma and other types of tumors by specifically binding to mannosyl. Although there have been many in-depth studies on the anti-tumor mechanism of PA-MSHA, no reports indicating that PA-MSHA has an inhibitory effect on PD-L1. Therefore, this research explored the effect and mechanism of PA-MSHA on PD-L1 and sPD-L1 based on HCC cells, laying a theoretical foundation for PA-MSHA to be used as ICIs or ICIs adjunct.

Objective: This research focused on the significant inhibitory effect of PA-MSHA on PD-L1 and verified the expression of related molecules in HCC; To verify the effect of PA-MSHA on the cell biological behavior of HCC cells; To explore the possible mechanism of PA-MSHA inhibiting PD-L1; To explore the effect of PA-MSHA inhibition of sPD-L1 on PD-1 expression and apoptosis of peripheral blood mononuclear cells (PBMC) in vitro. It provides a new idea for the treatment of HCC by PA-MSHA and a new direction for the anti-tumor research of PA-MSHA.

Methods:(1)The expressions of β-catenin, mannoside acetylglucosamine transferase 1 (MGAT1), α-mannosidase II, mannosyl, and PD-L1 in HCC were detected by immunofluorescence detection (IF) and immunohistochemical detection (IHC), and their relationship with clinicopathological characteristics were analyzed. (2) The effects of PA-MSHA on the biological function of hepatocellular carcinoma cells were evaluated by optical microscopy, transmission electron microscopy, CCK-8 assay, plate clone formation assay and transwell chamber assay. Western blot was used to detect the change of tight junction protein Claudin-1 in hepatocellular carcinoma cells after PA-MSHA intervention. A subcutaneous implantation tumor model of nude mice with Huh7 cells was constructed to evaluate the inhibition of PA-MSHA on tumor tissue growth and the influence of related molecular expression in this research. (3) The expression changes of β-catenin, c-Myc and PD-L1 in hepatoma cells after PA-MSHA intervention were detected by qRT-PCR and western blot. Plasmid and small interfering RNA (siRNA) were used to construct the overexpression and down-regulation models of β-catenin. After PA-MSHA intervention, the expressions of β-catenin, c-Myc, PD-L1, apoptosis-related proteins and MGAT1 were detected by western blot. (4) qRT-PCR was used to detect the expression changes of MGAT subtypes and α-mannosidase subtypes in hepatoma cells after PA-MSHA intervention, and screen possible targets. The expression of MGAT1 and α-mannosidase II were detected by western blot. Mannosyl changes were detected IF. MGAT1 overexpression model of hepatoma cells was constructed by plasmid, and PD-L1 expression was detected by western blot after PA-MSHA intervention. (5) The effect of PA-MSHA on sPD-L1 was detected by enzyme-linked immunosorbent assay (ELISA). The PBMC model of sPD-L1 intervention in vitro was constructed, and the expression of PBMC PD-1 and apoptotic protein was detected by western blot. GEPIA analyzed the relationship between sPD-L1 and patient prognosis in HCC samples, and plotted Kaplan-Meier survival curve.

Results:(1) PD-L1, β-catenin, c-Myc, mannosidase II and mannosyl were highly expressed in HCC tissues, while MGAT1 was low. MGAT1 expression may increase and mannosyl may decrease with the increase of age. There was a negative correlation between PD-L1 and MGAT1 expression in HCC tissues. MGAT1 is opposite to the expression of alpha-fetoprotein (AFP) in HCC tissues and to the level of AFP in the blood of HCC patients. β-catenin in HCC was moderately associated with β-catenin in paracancer tissue. (2) The results of CCK-8, plate cloning formation and transwell chamber experiments showed that PA-MSHA could significantly inhibit the proliferation, migration and invasion ability of HCC cells, and PA-MSHA intervention increased the expression of Claudin-1 in HCC cells. PA-MSHA intervention led to cytoplasmic vacuolation, chromatin edge aggregation, endoplasmic reticulum expansion, mitochondrial ridge breakage and other typical apoptotic characteristics. (3)After PA-MSHA intervention in HCC cells, β-catenin suppressor genes AXIN2 and APC were up-regulated, and CTNNB1 (β-catenin), c-Myc and PD-L1 gene expressions were down-regulated. After PA-MSHA intervention, β-catenin and c-Myc proteins decreased in cells and transplanted tumor tissues, and PD-L1 decreased significantly. PA-MSHA interferes with β-catenin overexpression and down-regulation models, suggesting that PA-MSHA partially inhibits PD-L1 expression by affecting β-catenin. PA-MSHA promotes apoptosis of HCC cell by inhibiting β-catenin. (4) After PA-MSHA intervention in HCC cells, the genes detected by qRT-PCR included MGAT1, MGAT3, MGAT4C, MGAT5, MAN1B1, MAN1C1 and MAN2A1, among which the relative expression levels of MGAT1 and MAN2A1 were more than twice that of the control group. The expression of MGAT1, MGAT3, and MAN2A1 genes increased after PA-MSHA intervention in transplantation tumors. The expression of MGAT1 and mannosidase II protein increased after intervention of PA-MSHA on HCC cells. western blot analysis showed that PA-MSHA could increase MGAT1 by affecting β-catenin, and finally reduce PD-L1. After PA-MSHA intervention, IF and IHC detection of implanted tumor tissue of nude mice showed that β-catenin, c-Myc, mannosyl decreased and MGAT1 increased. (5) After PA-MSHA intervention, the concentration of sPD-L1 in the culture medium of two kinds HCC cells decreased significantly. The supernatant of Huh7 cells without PA-MSHA intervention had the most significant inhibition on PBMC PD-1, the supernatant of Huh7 cells after PA-MSHA intervention had no significant inhibition on PBMC PD-1, and the supernatant of Huh7 cells after PA-MSHA intervention had no obvious pro-apoptotic effect on PBMC. GEPIA analysis showed that the overall survival (OS) of HCC patients in the group with high sPD-L1 expression was significantly lower than that in the group with low sPD-L1 expression.

Conclusions: (1)PA-MSHA significantly inhibits the expression of PD-L1 in HCC cells, and part of the inhibition may be caused by PA-MSHA inhibiting β-catenin and reducing the expression of PD-L1 gene and protein. PA-MSHA can promote HCC cell apoptosis by inhibiting β-catenin. (2)PA-MSHA may inhibit β-catenin and then reduce the expression of mannosyl, affect the N-glycosylation process of PD-L1 in HCC cells, and finally affect the expression level of PD-L1 protein. (3) PA-MSHA significantly inhibits sPD-L1 expression in HCC cells. By inhibiting sPD-L1, PA-MSHA alleviates the inhibition of sPD-L1 on PBMC PD-1 produced by HCC cells and alleviates the apoptosis of PBMC caused by sPD-L1. PA-MSHA has a theoretical basis to be used as ICIs or ICIs adjuvant.

Subject Area消化道肿瘤
MOST Discipline Catalogue医学 - 临床医学 - 外科学(含:普外、骨外、泌尿外、胸心外、神外、整形、烧伤、野战外)
URL查看原文
Language中文
Other Code262010_120200901591
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/539790
Collection第二临床医学院
Affiliation
兰州大学第二临床医学院
Recommended Citation
GB/T 7714
魏航之. 基于肝癌细胞研究PA-MSHA抑制PD-L1及sPD-L1的机制[D]. 兰州. 兰州大学,2023.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[魏航之]'s Articles
Baidu academic
Similar articles in Baidu academic
[魏航之]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[魏航之]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.