摘要
目的观察CD36在肝细胞癌组织和细胞株中的表达水平,探讨CD36对人肝细胞癌细胞株增殖、迁移能力及人肝癌细胞异种移植裸鼠模型的影响。方法基于癌症基因组图谱(TCGA)数据库相关信息分析371份肝细胞癌及癌旁组织中CD36转录本表达水平差异。前瞻性收集2019年1月至2021年2月就诊于扬州大学附属医院行手术治疗的48例肝细胞癌患者癌组织及相应的癌旁组织,采用实时荧光定量聚合酶链反应(qRT-PCR)法检测组织中CD36 mRNA水平。采用蛋白质印迹法检测人肝癌细胞株Huh7、HCCLM3及人正常肝细胞株LO2中CD36蛋白水平。将载有CD36干扰序列的质粒和空质粒转染到Huh7细胞或HCCLM3细胞,分别为sh-CD36组和对照组;采用CCK-8法检测培养0、12、24、36、48、60 h各组细胞的增殖能力(以吸光度值表示),采用划痕愈合实验、Transwell实验检测各组细胞迁移能力。将sh-CD36组或对照组Huh7细胞注射于BALB/c裸鼠腋窝皮下,每组4只,构建人肝癌异种移植裸鼠模型;接种1周后每周测量肿瘤长径、短径并计算肿瘤体积,接种5周后处死裸鼠,收集肿瘤标本并称量质量;显微镜下观察肿瘤组织细胞形态,采用免疫组织化学法检测肿瘤组织中CD36、Ki-67蛋白表达情况。结果对TCGA数据库数据分析显示,肝癌组织中CD36转录本水平较癌旁组织高(4.2±1.8比3.2±1.5,t=2.28,P=0.035)。qRT-PCR法对48例肝细胞癌患者组织检测显示,肝癌组织中CD36 mRNA相对表达量高于癌旁组织(0.76±0.26比0.48±0.23,t=3.52,P<0.001)。蛋白质印迹法检测显示,Huh7、HCCLM3细胞中CD36蛋白水平均高于LO2细胞,分别为LO2细胞的(1.42±0.11)倍和(1.68±0.16)倍(均P<0.001)。在mRNA及蛋白水平上,sh-CD36组Huh7和HCCLM3细胞CD36均低于对应的对照组(均P<0.001)。CCK-8法检测显示,sh-CD36组Huh7细胞和HCCLM3细胞分别于培养36 h和24 h开始增殖能力均低于对应的对照组(均P<0.01)。划痕愈合实验显示,培养48 h的sh-CD36组Huh7细胞[(12±3)%比(30±5)%,t=4.01,P<0.001]和HCCLM3细胞划痕愈合率[(15±4)%比(29±5)%,t=4.16,P<0.001]均低于对应的对照组;Transwell实验显示,sh-CD36组Huh7细胞[(46±6)个/视野比(88±6)个/视野,t=5.56,P<0.001]及HCCLM3细胞24 h穿膜细胞数[(42±5)个/视野比(82±7)个/视野,t=5.34,P<0.001]均少于对应的对照组。皮下注射5周后,注射sh-CD36组Huh7细胞的裸鼠肿瘤体积[(682±268)mm3比(1375±512)mm3,t=4.73,P=0.006]和肿瘤质量[(432±95)mg/只比(871±109)mg/只,t=6.57,P<0.001]均低于注射对照组Huh7细胞的裸鼠;显微镜下观察,注射sh-CD36组Huh7细胞的裸鼠移植瘤标本中肿瘤细胞密度低于注射对照组Huh7细胞的裸鼠,CD36和Ki-67蛋白的表达水平亦均低。结论CD36在肝细胞癌患者癌组织及人肝癌Huh7、HCCLM3细胞株中表达均上调,其可能与肝癌细胞增殖和迁移相关。敲低CD36表达体外可明显抑制肝癌细胞增殖和迁移能力,对人肝癌细胞异种移植裸鼠模型肿瘤有抑制作用。
Objective To observe the expression of CD36 in hepatocellular carcinoma tissues and cell lines,and to investigate the effects of CD36 on the proliferation and migration abilities of human hepatocellular carcinoma cell lines and human hepatocellular carcinoma cell xenograft models in nude mice.Methods Differences in the expression levels of CD36 transcripts in 371 hepatocellular carcinoma and paracancerous tissues were analyzed based on information from The Cancer Genome Atlas(TCGA)database.Cancer tissues and corresponding paracancerous tissues of 48 hepatocellular carcinoma patients who were diagnosed and underwent surgical treatment at the Affiliated Hospital of Yangzhou University from January 2019 to February 2021 were prospectively collected,and the levels of CD36 mRNA in the tissues were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)method.Western blotting was used to detect CD36 protein levels in human hepatocellular carcinoma cell lines Huh7 and HCCLM3 and human normal liver cell line LO2.Plasmids containing CD36 interfering sequences and empty plasmids were transfected into Huh7 cells or HCCLM3 cells for sh-CD36 group and control group,respectively.The CCK-8 assay was used to detect the proliferation ability(expressed as absorbance value)of cells in each group at 0,12,24,36,48 and 60 h of culture,and the scratch healing assay and Transwell assay were used to detect the migration ability of cells in each group.The Huh7 cells of sh-CD36 group or control group were injected into the axillary subcutis of BALB/c nude mice,with 4 mice in each group,to construct nude mice models of human hepatocellular carcinoma xenografts;the long and short diameters of tumor were measured weekly after 1 week of inoculation,and the tumor volume was calculated.The nude mice were put to death after 5 weeks of inoculation,and the tumor specimens were collected and weighed;the tumor cell morphology was observed under the microscope,and the expressions of CD36 and Ki-67 proteins in the tumor tissues was detected by immunohistochemistry(IHC).Results Analysis of the data from the TCGA database showed that the level of CD36 transcripts was higher in hepatocellular carcinoma tissues compared with that in paracancerous tissues(4.2±1.8 vs.3.2±1.5,t=2.28,P=0.035).Tissues detection using qRT-PCR in 48 patients with hepatocellular carcinoma showed that the relative expression of CD36 mRNA in hepatocellular carcinoma tissues was higher than that in paracancerous tissues(0.76±0.26 vs.0.48±0.23,t=3.52,P<0.001).Western blotting assay showed that CD36 protein level in Huh7 and HCCLM3 cells was higher than that in LO2 cells,which were(1.42±0.11)times and(1.68±0.16)times higher than LO2 cells,respectively(both P<0.001).At the mRNA and protein levels,the CD36 of Huh7 and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group(both P<0.001).CCK-8 assay showed that the proliferative ability of Huh7 cells and HCCLM3 cells in the sh-CD36 group was lower than that in the corresponding control group after 36 and 24 h of culture(both P<0.01).Scratch healing assay showed that the scratch healing rates of Huh7 cells[(12±3)%vs.(30±5)%,t=4.01,P<0.001]and HCCLM3 cells[(15±4)%vs.(29±5)%,t=4.16,P<0.001]in the sh-CD36 group were lower than those in the corresponding control group at 48 h of culture;Transwell assay showed that the number of Huh7 cells[(46±6)cells/field of view vs.(88±6)cells/field of view,t=5.56,P<0.001]and HCCLM3 cells[(42±5)cells/field of view vs.(82±7)cells/field of view,t=5.34,P<0.001]penetrating into the membrane in 24 h in the sh-CD36 group was less than that in the corresponding control group.Five weeks after subcutaneous injection,the tumor volume[(682±268)mm3vs.(1375±512)mm3,t=4.73,P=0.006]and tumor mass[(432±95)mg/mouse vs.(871±109)mg/mouse,t=6.57,P<0.001]of nude mice injected with Huh7 cells of the sh-CD36 group were lower than those of nude mice injected with Huh7 cells of the control group;under the microscope,the density of tumor cells in transplanted tumor specimens of nude mice injected with Huh7 cells of the sh-CD36 group was lower than that in nude mice injected with Huh7 cells of the control group,and the expression levels of both CD36 and Ki-67 proteins were also low.Conclusions CD36 expression is up-regulated in cancer tissues of hepatocellular carcinoma patients and human hepatocellular carcinoma cell lines Huh7 and HCCLM3,and it may associate with cell proliferation and migration of hepatocellular carcinoma.Knockdown of CD36 expression significantly inhibits the proliferation and migration abilities of hepatocellular carcinoma cells in vitro,and inhibits the tumors of human hepatocellular carcinoma cell xenograft models in nude mice.
作者
张立洪
吴巍
胥广才
张培建
陶立德
Zhang Lihong;Wu Wei;Xu Guangcai;Zhang Peijian;Tao Lide(Department of Hepatobiliary Surgery,Affiliated Hospital of Yangzhou University,Yangzhou 225012,China)
出处
《肿瘤研究与临床》
CAS
2024年第2期98-104,共7页
Cancer Research and Clinic
基金
国家自然科学基金(82001964)。
关键词
癌
肝细胞
抗原
CD36
细胞增殖
细胞运动
Carcinoma,hepatocellular
Antigens,CD36
Cell proliferation
Cell movement