WO2024000613A1 - Mogat2在制备肝细胞癌诊断和预后判断产品中的应用 - Google Patents

Mogat2在制备肝细胞癌诊断和预后判断产品中的应用 Download PDF

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WO2024000613A1
WO2024000613A1 PCT/CN2022/104290 CN2022104290W WO2024000613A1 WO 2024000613 A1 WO2024000613 A1 WO 2024000613A1 CN 2022104290 W CN2022104290 W CN 2022104290W WO 2024000613 A1 WO2024000613 A1 WO 2024000613A1
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mogat2
hepatocellular carcinoma
tissue
prognosis
diagnosis
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王燕
姜纯一
何鑫
靳钦
张曙
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南通大学附属医院
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

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  • the present invention relates to the field of molecular markers, in particular to the application of MOGAT2 (monoacylglycerol acyltransferase 2) in the preparation of products for diagnosis and prognosis of hepatocellular carcinoma.
  • MOGAT2 monoacylglycerol acyltransferase 2
  • liver cancer is the sixth most common human malignant tumor in the world (841,80 cases/year) and the fourth leading cause of cancer-related deaths (781,631 cases/year).
  • Hepatocellular carcinoma (HCC) originates from liver cells and accounts for the vast majority (approximately 75-85%).
  • HCC Hepatocellular carcinoma
  • Abnormal lipid metabolism is closely related to hepatocellular carcinoma, which is different from normal liver tissue and other tumors in terms of lipid metabolism.
  • metabolism-related genes to predict the diagnosis of hepatocellular carcinoma and the prognosis of liver cancer patients. . Therefore, there is an urgent need to establish a new metabolism-related biomarker and therapeutic target that can reliably predict the prognosis of HCC to supplement and improve current screening strategies for HCC diagnosis and prognosis.
  • MOGAT2 is a member of the MOGAT gene family. It is a membrane-bound acyltransferase belonging to the diacylglycerol acyltransferase (DGAT) gene family. It plays an important role in the monoacylglycerol pathway for the synthesis of triacylglycerol and is found in the human small intestine and liver. Highly expressive. Recent studies have shown that MOGAT2 is an attractive target for the treatment of obesity, diabetes, and non-alcoholic steatohepatitis. However, the specific expression of MOGAT2 in hepatocellular carcinoma and its value in diagnosis and prognosis have not yet been studied.
  • DGAT diacylglycerol acyltransferase
  • the purpose of the present invention is to provide the application of MOGAT2 in the preparation of hepatocellular carcinoma diagnosis and prognosis judgment products, so as to solve the problems existing in the above-mentioned existing technologies, to quickly diagnose hepatocellular carcinoma and judge the prognosis of hepatocellular carcinoma patients, to supplement and improve liver function. Shortcomings of current screening strategies for cell carcinoma diagnosis and prognosis.
  • the present invention provides the following solutions:
  • the present invention provides the application of MOGAT2 lipid metabolism-related biomarkers in the preparation of products for diagnosis and/or prognosis of hepatocellular carcinoma.
  • the product includes reagents and/or kits for detecting the expression level of MOGAT2.
  • the present invention also provides a reagent for detecting the expression level of MOGAT2 for use in preparing a kit for predicting the prognosis of patients with hepatocellular carcinoma.
  • the kit includes primers for amplifying MOGAT2 and primers for amplifying the internal reference ⁇ -actin gene.
  • the primers for amplifying MOGAT2 are:
  • Upstream primer 5′-TCTCTTCGATCTTCCCCGGTA-3′,
  • Downstream primer 5′-GATGCCCAGCAAGTTTCCG-3′;
  • the primers for amplifying the internal reference ⁇ -actin gene are:
  • Upstream primer 5′-CAGCAAGGACTGGTCTTTCTAT-3′,
  • Downstream primer 5′-GCGGCATCTTCAAACTC-3′.
  • the present invention obtains MOGAT2, a new molecular marker that assists in predicting the prognosis of hepatocellular carcinoma through screening.
  • MOGAT2 is lowly expressed in hepatocellular carcinoma tissue and highly expressed in adjacent normal liver tissue.
  • MOGAT2 By detecting the molecular marker MOGAT2 in patient tissue, The expression level of MOGAT2 can quickly diagnose and predict the prognosis of patients with hepatocellular carcinoma. Therefore, the molecular marker MOGAT2 provided by the present invention can supplement and improve the deficiencies of current screening strategies in the diagnosis and prognosis of hepatocellular carcinoma.
  • Figure 1 shows the GEPIA and UALCAN database analysis of MOGAT2 mRNA expression levels in hepatocellular carcinoma tissue
  • LIHC in the figure represents hepatocellular carcinoma
  • Normal represents normal liver tissue
  • Primary tumor represents primary tumors
  • Figure 2 shows the correlation between MOGAT2 expression and postoperative survival of patients with hepatocellular carcinoma and other tumors analyzed by UALCAN and GEPIA databases;
  • LIHC in the figure represents hepatocellular carcinoma
  • BLCA represents bladder urothelial carcinoma
  • HNSC head and neck squamous cell carcinoma.
  • READ stands for rectal adenocarcinoma;
  • Figure 3 shows the GEPIA database analysis of the correlation between MOGAT2 expression in hepatocellular carcinoma tissues and some hepatocellular carcinoma-related genes (AFP, GPC3, HSPA4 and TP53);
  • Figure 4 is the GEPIA database analysis of the correlation between MOGAT2 expression and proliferation-related factors (MKI67, MYC) in hepatocellular carcinoma tissues;
  • Figure 5 is a graph comparing the expression levels of MOGAT2 mRNA in 40 pairs of fresh hepatocellular carcinoma and adjacent tissues;
  • A is the use of qRT-PCR to detect the level of MOGAT2 mRNA in hepatocellular carcinoma and adjacent tissues, and ⁇ -actin is the internal reference.
  • B is the ROC curve of A; in the figure, Adjacent normal tissue represents para-cancerous tissue, and HCC represents hepatocellular carcinoma tissue;
  • Figure 6 is a graph comparing the protein expression levels of MOGAT2 in 40 pairs of fresh hepatocellular carcinoma and adjacent tissues;
  • A is the use of Western blot method to detect the protein level of MOGAT2 in hepatocellular carcinoma and adjacent tissues, GAPDH is the internal reference, and
  • B is Quantitative histogram of A; in the figure, Adjacent normal tissue and AN represent para-cancerous tissue, HCC and H represent hepatocellular carcinoma tissue;
  • Figure 7 shows the results of tissue chip combined with IHC observation of MOGAT2 expression in 363 pairs of hepatocellular carcinoma and para-cancerous paraffin tissues;
  • B is the ROC curve of A ;
  • Adjacent normal represents the paracancerous tissue
  • HCC represents the hepatocellular carcinoma tissue;
  • Figure 8 is a Kaplan-Meier plotter showing the relationship between MOGAT2 expression and the five-year survival rate of patients with hepatocellular carcinoma (P ⁇ 0.05, the difference is statistically significant).
  • BCA protein assay kit Biosharp; MOGAT2 polyclonal antibody (for Westemblot test): Invitrogen Company; Horseradish peroxidase-labeled goat anti-rabbit secondary antibody (for Westemblot test): American CST Company; ECL luminescence kit: American Thermofisher company.
  • Two-step immunohistochemical detection kit rabbit anti-human MOGAT2 polyclonal antibody (for immunohistochemical testing): Invitrogen Company; horseradish peroxidase-labeled goat anti-rabbit secondary antibody (for immunohistochemical testing) : CST Company of the United States; 0.01mol/L citric acid buffer (pH6.0): Beijing Zhongshan Biotechnology Co., Ltd.; DAB staining solution kit: Fuzhou Maixin Biotechnology, China; xylene, neutral gum, etc. are provided by the Pathology Department .
  • MOGAT2 is lowly expressed in hepatocellular carcinoma tissues.
  • MOGAT2 is related to the expression of MOGAT2 in patients with hepatocellular carcinoma and other tumors, such as It is positively correlated with postoperative survival of patients with bladder urothelial cancer, head and neck squamous cell carcinoma, and rectal adenocarcinoma.
  • RNA extraction and cDNA synthesis Use Trizol kit to extract RNA according to conventional methods. Then a reverse transcriptase kit was used to synthesize cDNA, all performed according to conventional methods.
  • Primer design Design primers from the NCBI gene sequence.
  • MOGAT2 upstream primer 5′-TCTCTTCGATCTTCCCCGGTA-3′
  • downstream primer 5′-GATGCCCAGCAAGTTTCCG-3′.
  • the length of the amplified target fragment is 167bp; the internal reference ⁇ -actin gene is upstream.
  • Primer 5′-CAGCAAGGACTGGTCTTTCTAT-3′
  • downstream primer 5′-GCGGCATCTTCAAACCTC-3′
  • the length of the amplified target fragment is 162 bp.
  • the melting curve of the PCR amplification product was measured and the product was analyzed by 2% agarose gel electrophoresis.
  • test results were calculated using SPSS 22.0 statistical analysis software as the mean ⁇ standard deviation, coefficient of variation (CV) and P value, and the test level ⁇ was set to 0.05.
  • the mRNA expression levels of MOGAT2 in 40 pairs of hepatocellular carcinoma and adjacent tissues were detected by qRT-PCR. As shown in Figure 5, it was found that the expression of MOGAT2 mRNA in hepatocellular carcinoma tissues was significantly lower than that in the corresponding para-cancerous tissues.
  • the ROC curve results showed that MOGAT2 mRNA expression can distinguish hepatocellular carcinoma and para-cancerous normal liver tissues.
  • the area under the curve (AUC ) is 0.9247 (95% CI, 0.8644-0.9849), the cutoff is 0.75, and the sensitivity and specificity are 92.5% and 82.5% respectively.
  • Distilled water 2.0ml; 30% Acr-Bis (29:1): 4.0ml; 1mol/l Tris-HCl pH 8.8: 3.8ml; 10% SDS: 0.1ml; 10% AP: 0.1ml; TEMED: 0.004ml.
  • Distilled water 2.7ml; 30% Acr-Bis (29:1): 0.67ml; 1mol/l Tris-HCl pH 6.8: 0.5ml; 10% SDS: 0.04ml; 10% AP: 0.04ml; TEMED: 0.004ml.
  • the protein expression level of MOGAT2 in 40 groups of fresh hepatocellular carcinoma and adjacent tissues was detected by Western blot method. As shown in Figure 6, the results showed that the expression of MOGAT2 protein in hepatocellular carcinoma tissues was significantly lower than that in corresponding adjacent normal tissues.
  • hepatitis specimens 100 hepatic cavernous hemangioma specimens, 70 liver cirrhosis specimens and 42 focal nodular hyperplasia specimens were selected during the same period. All selected cases were pathologically determined by two experienced pathologists, and the patients had not received anti-tumor treatments such as immunotherapy, chemotherapy, or radiotherapy before surgery.
  • the tissue array block is heated and fused in a constant-temperature oven at 55°C for 10 minutes, and allowed to cool to room temperature before melting quickly, so that the recipient wax block and the donor tissue can be integrated; the tissue chip is frozen at 4°C for about 4 hours. , and then use a fully automatic tissue slicer to correct the tissue array block at a speed of 20mm/revolution until all tissue cores are completely exposed;
  • Antigen retrieval After dewaxing, rinse with water for a period of time, microwave antigen retrieval, and rinse with running water. Incubate in 3% H 2 O 2 at room temperature for 10 minutes to remove endogenous peroxidase, rinse with 0.01mol/LPBS for 3 minutes ⁇ 3 times;
  • Color development and counterstaining Take the slices out of the incubator and rinse them with 0.01mol/l PBS for 3 minutes ⁇ 3 times. Immerse the slices in DAB working solution for 5 minutes. At the same time, observe and control the degree of color development under a microscope until a positive reaction occurs. Rinse under running water to terminate the reaction in time; counterstaining: hematoxylin counterstaining for 30 seconds to 2 minutes, rinse with running water, differentiate with 0.1% hydrochloric acid, rinse with running water, and blue;
  • the final score is the product of the proportion of positive cells and the staining rate.
  • the ROC curve was drawn based on the scores, and the cutoff point was set to 120 using X-tile software (Rimm Lab, Yale University; http://www.tissuearray.org/rimmlab). Among them, 0 to 119 are low expression or no expression (-), and 120-300 are high expression (+). SPSS22.0 statistical software was used, and ⁇ 2 test or Fisher's exact probability method was used for statistical analysis to determine the relationship between target gene expression and various pathological indicators. Kaplan-Meier method was used to draw survival curves, log-rank test was used to compare between groups, and Cox proportional hazard regression was used for prognostic analysis. Finally, asymptotic Sig. bilateral P ⁇ 0.05 was used as the standard for statistically significant differences.
  • MOGAT2 protein is mainly located in the cytoplasm of normal liver tissue next to cancer and appears brown. However, there is low or no expression in hepatocellular carcinoma tissues. The positive rate of MOGAT2 protein expression in hepatocellular carcinoma tissue (36.9%, 134/363) was significantly lower than that in adjacent normal liver tissue (63.1%, 229/363). Moreover, MOGAT2 protein expression can differentiate between hepatocellular carcinoma and adjacent normal liver tissue. The area under the curve is 0.9150 (95% CI, 0.8955-0.9345), the cutoff is 0.7217, and the sensitivity and specificity are 93.11% and 79.06 respectively. %.
  • MOGAT2 can be used as a molecular marker to assist in the diagnosis and prognosis of hepatocellular carcinoma.
  • MOGAT2 can be used to assist in the diagnosis of hepatocellular carcinoma.
  • the expression level of the molecular marker MOGAT2 will make the operation simpler and easier.

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Abstract

MOGAT2在制备肝细胞癌诊断和预后判断产品中的应用，以及用于检测MOGAT2表达水平的试剂在制备预测肝细胞癌患者的预后情况的试剂盒中的应用。通过实验验证MOGAT2在肝细胞癌组织中低表达，在癌旁正常肝组织中高表达，通过检测分子标志物MOGAT2的表达水平进行肝细胞癌诊断和/或预后判断，能够补充和改善肝癌诊断和预后当前筛选策略的不足。

Description

MOGAT2在制备肝细胞癌诊断和预后判断产品中的应用 技术领域
本发明涉及分子标志物领域，特别是涉及MOGAT2(单酰甘油酰基转移酶2)在制备肝细胞癌诊断和预后判断产品中的应用。
背景技术
根据相关报道，肝癌是全球第六大常见人类恶性肿瘤(841，80例/年)，也是癌症相关死亡的第四大主要原因(781，631例/年)。肝细胞癌(HCC)起源于肝细胞，占绝大多数(约75-85％)。尽管最近在早期检测和管理方面取得了进展，但由于早期诊断率较低，肝细胞癌的死亡率仍然很高。脂代谢异常与肝细胞癌密切相关，其在脂代谢方面不同于正常肝组织，也不同于其他肿瘤，目前很少有基于代谢相关基因的模型对肝细胞癌的诊断和肝癌患者的预后进行预测。因此，迫切需要建立一种能够可靠预测肝细胞癌预后的新的代谢相关生物标志物和治疗靶点，以补充和改善肝细胞癌诊断和预后的当前筛查策略。
MOGAT2是MOGAT基因家族的一员，属于二酰基甘油酰基转移酶(DGAT)基因家族的膜结合酰基转移酶，在合成三酰基甘油的单酰基甘油途径中起重要作用，在人的小肠和肝脏中高度表达。最近的研究表明，MOGAT2是治疗肥胖、糖尿病和非酒精性脂肪性肝炎的有吸引力的靶点。但MOGAT2在肝细胞癌中的具体表达情况和对诊断及预后的判断价值至今尚未有研究。
发明内容
本发明的目的是提供MOGAT2在制备肝细胞癌诊断和预后判断产品中的应用，以解决上述现有技术存在的问题，可以快速诊断肝细胞癌并判断肝细胞癌患者预后情况，补充和改善肝细胞癌诊断和预后的当前筛选策略的不足。
为实现上述目的，本发明提供了如下方案：
本发明提供MOGAT2脂质代谢相关生物标志物在制备用于肝细胞癌诊断和/或预后判断的产品中的应用。
优选的是，所述产品包括用于检测MOGAT2的表达水平的试剂和/或试剂盒。
本发明还提供一种用于检测MOGAT2表达水平的试剂在制备预测肝细胞癌患者的预后情况的试剂盒中应用。
优选的是，所述试剂盒包括扩增MOGAT2的引物和扩增内参β-actin基因的引物。
优选的是，扩增所述MOGAT2的引物为：
上游引物：5′-TCTCTTCGATCTTCCCCGGTA-3′，
下游引物：5′-GATGCCCAGCAAGTTTCCG-3′；
扩增所述内参β-actin基因的引物为：
上游引物：5′-CAGCAAGGACTGGTCTTTCTAT-3′，
下游引物：5′-GCGGCATCTTCAAACCTC-3′。
本发明公开了以下技术效果：
本发明通过筛选得到一种新的辅助预测肝细胞癌预后的分子标志物MOGAT2，实验验证结果显示MOGAT2在肝细胞癌组织中低表达，在癌旁正常肝组织中高表达，通过检测患者组织中的MOGAT2的表达水平就能快速的对其诊断并预测肝细胞癌患者的预后情况。因此，本发明提供的分子标志物MOGAT2能够补充和改善肝细胞癌诊断和预后当前筛选策略的不足。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为GEPIA及UALCAN数据库分析MOGAT2在肝细胞癌组织mRNA的表达水平图；图中LIHC代表肝细胞癌，Normal代表正常肝组织，Primary tumor代表原发性肿瘤；
图2为UALCAN及GEPIA数据库分析MOGAT2的表达与肝细胞癌患者及其他肿瘤患者术后生存的相关性；图中LIHC代表肝细胞癌，BLCA代表膀胱尿路上皮癌，HNSC代表头颈鳞状细胞癌，READ代表直肠腺癌；
图3为GEPIA数据库分析MOGAT2在肝细胞癌组织中的表达与部分肝细胞癌相关基因(AFP，GPC3，HSPA4及TP53)的相关性；
图4为GEPIA数据库分析MOGAT2在肝细胞癌组织中的表达增殖相关因子(MKI67，MYC)的相关性；
图5为比较40对新鲜肝细胞癌及癌旁组织中MOGAT2 mRNA的表达水平结果图；A为利用qRT-PCR法检测肝细胞癌及其癌旁组织中MOGAT2的mRNA水平，β-actin 为内参，B为A的ROC曲线图；图中Adjacent normal tissue代表癌旁组织，HCC代表肝细胞癌组织；
图6为比较40对新鲜肝细胞癌及癌旁组织中MOGAT2的蛋白表达水平结果图；A为利用Western blot法检测肝细胞癌及其癌旁组织中MOGAT2的蛋白水平，GAPDH为内参，B是A的量化柱形图；图中Adjacent normal tissue及AN代表癌旁组织，HCC及H代表肝细胞癌组织；
图7为组织芯片结合IHC观察MOGAT2在363对肝细胞癌及癌旁石蜡组织中的表达结果图；A为免疫组化结果图，棕褐色表示强阳性(+++)、浅棕色表示阳性(++)、黄色表示弱阳性(+)、蓝色表示阴性(-)，放大倍数：左侧×40(bar＝500μm)，右侧×400(bar＝50μm)；B是A的ROC曲线图；图中Adjacent normal代表癌旁组织，HCC代表肝细胞癌组织；
图8为MOGAT2表达对肝细胞癌患者五年生存率的Kaplan-Meier plotter图，(P＜0.05，差异具有统计学意义)。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
下面结合具体实施例对本发明进一步进行说明。
以下实施例中使用的主要试剂为：
(1)qRT-PCR试剂；Trizol：Invitrogen公司；qRT-PCR引物：广州锐博生物科技有限公司，GAPDH引物：上海生工生物工程股份有限公司；逆转录试剂盒：美国Thermo公司，SYBR GreenⅠmix：北京百泰克生物技术有限公司；基因组DNA(gDNA)提取试剂盒：北京天根生化科技有限公司；DNA Marker：上海生工生物工程股份有限公司。
(2)Western blot试剂：1×TBST：取Tris2.42g、NaCl8.0g、Tween-200.5mL，混合溶解，定容至1L，常温保存。1×转膜Buffer：甘氨酸14.4g、Tris3.03g，加适量双蒸水搅拌溶解，再加200mL无水甲醇，定容至1L，混合均匀(用时配制)。封闭液100mL：取脱脂奶粉5g，加入100mL1×TBST，混合溶解即可(需用时配制)。BCA蛋白测定试剂盒：Biosharp；MOGAT2多克隆抗体(Westemblot试验用)：Invitrogen公司；辣根过氧化物酶标记山羊抗兔二抗(Westemblot试验用)：美国CST公司；ECL发光试剂盒：美国Thermofisher公司。
(3)二步法免疫组化检测试剂盒：兔抗人MOGAT2多克隆抗体(免疫组化试验用)：Invitrogen公司；辣根过氧化物酶标记山羊抗兔二抗(免疫组化试验用)：美国CST公司；0.01mol/L柠檬酸缓冲液(pH6.0)：北京中山生物技术有限公司；DAB染色液试剂盒：中国福州迈新生物技术；二甲苯、中性树胶等由病理科提供。
以下实施例中使用的主要仪器如下：
分子杂交炉：美国UVP公司；超微量分光光度计：美国T hermo公司；普通PCR扩增仪：美国BIO-RAD公司；Light Cycler cobasZ480PCR仪：美国Roche公司；组织芯片制作仪：美国Beecher Instruments公司；自动免疫组化染色仪(2D)：美国LABVISION公司；凝胶成像系统：美国BIO-RAD公司；多功能酶标仪：美国Thermo公司；全自动病理成像系统：Perkin ElmerVectra，美国；电泳仪：Bio-Rad公司，型号miniprotean 3cell；电转仪：大连竞迈科技有限公司，型号PS-9；一体式化学发光成像仪：ChemiScope 5300Pro；倒置相差显微镜：德国leica公司；光学显微镜：XDS-1A。
实施例1
1、通过对GEPIA及UALCAN数据库的数据分析，得出MOGAT2在肝细胞癌中mRNA的表达水平图，如图1所示，MOGAT2在肝细胞癌组织中低表达。
2、通过GEPIA及UALCAN数据库的数据分析，得出MOGAT2的表达与肝细胞癌患者及其他肿瘤患者术后生存的相关性，如图2所示，MOGAT2的表达与肝细胞癌患者及其他肿瘤如膀胱尿路上皮癌，头颈鳞状细胞癌及直肠腺癌等的患者术后生存正相关。
3、通过GEPIA数据库的基因相关性数据分析，得出MOGAT2在肝细胞癌组织中的表达与部分肝细胞癌相关基因AFP(R＝-0.16；P＝0.0017)，GPC3(R＝-0.28；P＝5.8e-08)，TP53(R＝-0.24；P＝4.5e-06)，HSPA4(R＝-0.3；P＝5.3e-09)的表达负相关。
4、通过GEPIA数据库的基因相关性数据分析，得出MOGAT2在肝细胞癌组织中的表达与增殖相关基因MKI67(R＝-0.22；P＝1.4e-05)及MYC(R＝-0.19；P＝0.00018)的表达负相关。
5、qRT-PCR法检测样本
40组新鲜肝细胞癌手术患者的癌组织和癌旁正常组织的新鲜标本由南通大学附属医院提供。上述组织手术离体后去除坏死组织，生理盐水冲洗血污，于30min内液氮冷冻处理，之后置于-80℃冰箱保存备用。具体操作如下：
(1)RNA提取及cDNA合成：应用Trizol试剂盒按常规方法提取RNA。然后应用反转录酶试剂盒合成cDNA，均按常规方法进行。
(2)引物设计：从NCBI基因序列中设计引物，MOGAT2上游引物：5′-TCTCTTCGATCTTCCCCGGTA-3′，下游引物：5′-GATGCCCAGCAAGTTTCCG-3′，扩增目的片段长度167bp；内参β-actin基因上游引物：5′-CAGCAAGGACTGGTCTTTCTAT-3′，下游引物：5′-GCGGCATCTTCAAACCTC-3′，扩增目的片段长度162bp。
(3)荧光定量PCR检测
1)荧光定量PCR反应体系
表1 荧光定量PCR反应体系(20.0μl)
Figure PCTCN2022104290-appb-000001
Figure PCTCN2022104290-appb-000002
2)反应条件
93℃预变性2min，然后94℃5s、61℃30s、82℃1s，40个循环。反应结束后计算机根据循环阈值(crossing point，Cp)与其对应的不同定量模板拷贝数对数值自动拟合作图，绘制出靶基因和GAPDH的标准曲线。
3)方法的特异性
为了验证SYBR GreenⅠ染料方法的特异性，测定PCR扩增产物的熔解曲线并将产物进行2％琼脂糖凝胶电泳分析。
4)重复性试验
将5个不同稀释度的标准品(5.0×10 9～5.0×10 5copies/ml)分5批次在不同时间进行检测，计算批间变异系数；将上述标准品同批次各重复5管检测计算批内变异系数。
5)标本中人靶基因及内参GAPDH基因表达水平的检测
将不同标本的cDNA模板与不同稀释度的标准品分别加入17μlPCR反应液中，根据标准曲线得到不同样本中靶基因和GAPDH拷贝数及相应的Cp值。为了消除样本、逆转录和PCR反应的差异，以靶基因mRNA和GAPDH浓度对数比值作为评价靶基因表达水平的指标。
6)数据处理
检测结果应用SPSS 22.0统计分析软件计算均数±标准差、变异系数(CV)和P值,检验水准α设为0.05。
通过qRT-PCR法检测40对肝细胞癌和其癌旁组织中MOGAT2的mRNA表达水平。如图5所示，结果发现MOGAT2 mRNA在肝细胞癌组织中表达明显低于对应的癌旁组 织，ROC曲线结果显示MOGAT2 mRNA表达能够区分肝细胞癌及癌旁正常肝组织，曲线下方面积(AUC)为0.9247(95％CI，0.8644-0.9849)，临界值(cut off)为0.75，灵敏度和特异度分别为92.5％和82.5％。
6、蛋白免疫印迹法标本
(1)提组织蛋白
1)将备用的40对新鲜组织从-80℃冰箱取出放碎冰上，每份称取组织100mg，用剪刀剪碎放入10ml的EP管中(插入碎冰中)；
2)取裂解液8ml,加80μl PMSF混匀(每100mg组织加1ml混匀裂解液)；
3)每管加1ml含PMSF的裂解液在电动匀浆器上匀浆；
4)匀浆后倒入2ml EP管内，低温(4℃)12000rpm离心15min；
5)取上清分装(1ml EP管)，每管40μl(或者根据需要量进行分装)；
6)放入-20℃冰箱(用Parafilm膜封口)，可先加入上样buffer煮15min,也可在电泳前再煮。
(2)蛋白浓度的测定(BCA试剂盒)
1)根据样品数量，按50体积BCA试剂A加1体积BCA试剂B(50:1)配制适量BCA工作液，充分混匀；
2)完全溶解蛋白标准品，取10μl稀释至100μl，使终浓度为0.5mg/ml，用PBS稀释标准品；
3)将标准品按0、1、2、4、8、12、16、20μl加到96孔板的标准品孔中，加用于稀释标准品的溶液补足至20μl；
4)加适当体积样品到96孔板的样品孔中，加用于稀释标准品的溶液至20μl；
5)各孔加入200μl BCA工作液，37℃放置30分钟；
6)测定A 562，根据标准曲线计算出蛋白浓度。
(3)配胶
1)选择所需胶的浓度及所需体积(配2块胶需10ml分离胶和4ml浓缩胶)；
2)12％分离胶的配制
蒸馏水：2.0ml；30％Acr-Bis(29:1)：4.0ml；1mol/l Tris-HCl pH 8.8：3.8ml；10％SDS：0.1ml；10％AP：0.1ml；TEMED：0.004ml。
3)5％浓缩胶的配制
蒸馏水：2.7ml；30％Acr-Bis(29:1)：0.67ml；1mol/l Tris-HCl pH 6.8：0.5ml；10％SDS：0.04ml；10％AP：0.04ml；TEMED：0.004ml。
4)将配胶的玻璃片洗净干燥后组装在一起(用力夹紧，防止漏胶)，然后先加分离胶，加至距上端1.5cm，静置20min后加浓缩胶至顶端，插入梳子(要不断的补胶防止浓缩时出现空隙)室温放2h或4℃冰箱过夜。
(4)上样电泳
1)将50μg总蛋白与5×loading buffer混合(蛋白：5×Loading buffer＝4:1)在1ml EP管中用封口膜封口，沸水煮3～5min；
2)将预先配好的胶从冰箱内拿出，放入电泳槽内支架上(如两块胶同时电泳，放的位置是：两块薄的玻璃板都向内，若只跑一块胶，另一块用塑料板代替，有字的一面向里)，夹玻璃片的时候要用力向下按紧同时转动夹子；
3)将夹好后的架子放入电泳槽内，内部用电泳液加满，外部加1/3至1/2体积；
4)上样，枪头要紧贴玻片，另外两边不加样的孔可加1×loading buffer进行平衡；
5)接通电源，先80V约20min再换成100V约90min；
6)电泳结束(溴酚兰跑到胶底，Marker分开且条带清晰)，将电泳后的胶与玻片同时拿下，切下多余的部分(根据Marker的位置)；
(5)转膜
1)将棉垫、滤纸提前放入1×转膜缓冲液平衡20min，剪合适大小的PVDF膜，甲醛极化3s，再放入ddH 2O中2min；
2)组装转膜夹层：阴极(黑色)-棉垫-滤纸-胶-PVDF膜-滤纸-棉垫-阳极(红色)，膜不能干，亦不能产生气泡(注意膜的正反面，转膜板黑色在下，有孔的白色面在上)；
3)将夹层放入转移槽中，注意正负极(黑色-黑色，红色-红色)；放入冰盒，加入1×转膜缓冲液至满；
4)将电转移仪放在碎冰中，上面盖上冰袋，接通电源(300mA，2h，分子量小的转膜时间短)；
5)关闭电源，取出转印膜。
(6)丽春红染膜(检查蛋白是否已经转移到膜上)
1)将膜取出直接放入丽春红染液(现配)中染1min；
2)取出放入ddH 2O中清洗2min；
3)1×TBS-T(不含脱脂奶粉)清洗5min×3次，若不明显可复染。
(7)封闭，加一抗、二抗孵育
1)将PVDF膜放入封闭液(含5％脱脂奶粉的1×TBS-T)中，4℃过夜或室温摇2h，洗膜5min×3次；
2)将洗好的膜放在平板上，配含5％脱脂奶粉1×TBS-T稀释的一抗(MOGAT2一抗稀释1000倍，内参GAPDH稀释1000倍，各稀释1ml放EP管中)，膜的正面向上，均匀滴加在膜上；
3)室温约1h，再放入4℃过夜，也可直接放4℃(时间可以更长)；
4)将膜取出后用1×TBS-T液洗10min×3次；
5)配含5％脱脂奶粉的1×TBS-T稀释的二抗(二抗稀释2000倍，稀释1ml放EP管中)；
(6)将稀释好的二抗均匀滴加在膜上室温或4℃放置2h(与二抗混合的比例可根据说明书)不超过多3h；
(7)1×TBS-T(不含脱脂奶粉)清洗5min×3次；
(8)使用吸水纸将膜上的多余液体吸干，显影；
(9)使用Image J图像处理软件对图像进行灰度统计分析。
通过Westrn blot法，检测40组新鲜肝细胞癌和其癌旁组织中MOGAT2的蛋白质表达水平。如图6所示，结果显示MOGAT2蛋白在肝细胞癌组织中表达明显低于对应的癌旁正常组织。
7、组织芯片结合免疫组织化学标本：
(1)组织标本
选取2004年1月至2012年12月在南通大学附属医院病理科的363例肝细胞癌患者的癌组织及癌旁组织的石蜡标本，所有患者的临床及病理资料收集均完整，可经电话等追踪随访，无失访(随访截止日期为2017年12月31日)。患者本人或其家属均知情并均签署知情同意书，组织标本的获得和使用均获得南通大学附属医院伦理委员会批准同意。TNM分期采用国际癌症控制联盟(Union for International Cancer Control UICC)以及美国癌症联合委员会(AJCC)推荐的第八版TNM分类。另外选取同期128例肝炎标本、100例肝海绵状血管瘤标本、70例肝硬化标本及42例局灶性结节状增生标本。所有选取的病例均是由两名经验丰富的病理学专家进行病理组织学确定， 患者术前没有接受过免疫治疗、化疗或放疗等抗肿瘤治疗。
(2)组织芯片的制作
所有标本均用10％甲醛固定，石蜡包埋，蜡块经过筛选无明显缺陷。制作成组织芯片。主要流程为：
1)根据HE染色切片的镜检结果，在蜡块上有代表性的病变区域作标记；
2)按照体积比1∶1混合石蜡与蜂蜡，制作空白受体蜡块：在蜡块上设计10×7孔的组织阵列，然后用组织芯片仪制成TMA空白蜡块；
3)将供体蜡块在标记的点上选取最有代表性的病变区域，取直径2mm的组织块，每例各取1个芯；将取好的组织芯转移到受体蜡块的孔中；
4)组织阵列块在55℃的恒温烤箱中加热融合10min，在快融化之前放至室温冷却，使受体蜡块与供体组织融为一体；将组织芯片置于4℃条件下冷冻4h左右，随后用全自动组织切片机对组织阵列块进行修正，速度为20mm/转，等修到所有组织芯完全曝露；
5)切片机对组织阵列块进行切片，将连续切片分别漂在凉水中，使其自然展开，再将切片转移至45℃温水中展片2min左右，待展开后将其贴在经过防脱片处理的载玻片上晾干；
6)将切片置于60℃的环境下烤片3min，58℃继续烤片16h；
7)将做好的组织芯片保存于切片盒，置于冰箱4℃冷藏室备用。
(3)免疫组化染色(EnVision两步法)
1)常规脱蜡水化：脱蜡前，将组织芯片放在60℃的恒温箱中，烘烤约20min。二甲苯15min×3次(60℃)→无水乙醇10min×2次→95％乙醇5min×2次→80％乙醇5min→70％乙醇5min；二甲苯起脱蜡作用，依据相似相容原理；一般在每个试剂中放10min，天气热可以少放几分钟，相反，天气较冷可延长脱蜡时间至12～15min；
2)抗原修复：脱蜡后用清水冲洗一段时间，微波抗原修复，流水冲洗。3％H 2O 2室温孵育10min去除内源性过氧化物酶，0.01mol/LPBS冲洗3min×3次；
3)加一抗、二抗：滴加一抗，用抗体稀释液分别作1:150稀释，4℃过夜，滴加生物素标记的第二抗体室温孵育30min和SP溶液室温孵育30min，每步骤间PBS洗涤；用PBS缓冲液代替一抗作阴性对照；
4)显色、复染：片子从温箱中取出，0.01mol/l PBS冲洗3min×3次，将切片浸入 DAB工作液中5min，同时在显微镜下观察控制显色程度，待出现阳性反应，置流水下冲洗及时终止反应；复染：苏木素复染30s～2min，流水冲洗，0.1％盐酸分化，流水冲洗，蓝化；
5)脱水：70％乙醇3min→80％乙醇3min→95％乙醇5min→无水乙醇5min→二甲苯8min×2次，60℃烘干；
6)封片：用中性树胶滴在组织旁边，再用盖玻片盖上，要先放平一侧，然后轻轻放下另一侧，以免产生气泡，封好片子后置于通风柜中晾干。
(4)结果判读：
所有的免疫组化染色切片由两位独立的有经验的病理学家(双盲)根据阳性染色肿瘤细胞的比例和染色的强度进行评估和打分。在400倍视野下，观测每张切片中具有代表性的区域，随机选取5个视野，计数500～1000个细胞，计算细胞的阳性率根据染色阳性的肿瘤细胞数目所占百分比计为0～100％，染色细胞的强度分为以下四个等级：0(无染色，-)，1(染色弱；黄色，+)，2(染色中等；浅棕色，++)，3(染色强烈；棕褐色，+++)。染色比例得分是0到100分。最终得分是阳性细胞的比例与染色率的乘积。根据得分绘制ROC曲线，另外利用X-tile软件(Rimm Lab,Yale University；http://www.tissuearray.org/rimmlab)设定的截止点为120。其中0至119为低表达或无(-)，120-300为高表达(+)。应用SPSS22.0统计学软件，采用χ 2检验或Fisher确切概率法进行统计分析，判断靶基因表达与各病理指标的相互关系。Kaplan-Meier法绘制生存曲线、组间比较采用对数秩检验，Cox比例风险回归进行预后分析。最终以渐近Sig.双侧P<0.05为差异有统计学意义的标准。
免疫组化染色结果如图7所示，MOGAT2蛋白主要定位于癌旁正常肝组织的细胞质，呈棕褐色。而肝细胞癌组织中低或无表达。肝细胞癌组织MOGAT2蛋白表达阳性率(36.9％，134/363)显著低于癌旁正常肝组织(63.1％，229/363)。并且MOGAT2蛋白表达能够区分肝细胞癌及癌旁正常肝组织，曲线下方面积为0.9150(95％CI，0.8955-0.9345)，临界值(cut off)为0.7217，灵敏度和特异度分别为93.11％和79.06％。
非癌组织中，肝炎组织中MOGAT2阳性率为82.8％(106/128)，海绵状血管瘤为82.0％(82/100)，肝硬化为75.7％(53/50)，局灶性结节状增生为70.7％(29/41)，与肝细胞癌组织相比，差异具有统计学意义(χ2＝,199.460，P＜0.0001)(表2)。
应用Personχ2分析和Studem t检验，MOGAT2的表达水平与血AFP值，T分期， 淋巴结转移(N)，TNM分期，肝硬化和血管侵犯密切相关，差异有统计学意义(表3)。Cox回归结果显示MOGAT2表达和T分期均为肝细胞癌患者的独立预后因素(表4)，Kaplan-Meier plotter生存曲线显示MOGAT2高表达组比低或无表达组总体生存率高，见图8。
表2 肝良性组织、肝细胞癌和癌旁正常肝组织中MOGAT2蛋白表达情况
Figure PCTCN2022104290-appb-000003
表3 MOGAT2蛋白表达对肝细胞癌患者临床病理特征的影响
Figure PCTCN2022104290-appb-000004
Figure PCTCN2022104290-appb-000005
Figure PCTCN2022104290-appb-000006
表4 MOGAT2与肝细胞癌患者预后的COX回归分析结果
Figure PCTCN2022104290-appb-000007
Figure PCTCN2022104290-appb-000008
上述结果表明MOGAT2可作为一种辅助诊断肝细胞癌预后的分子标志物，通过将该分子标志物的引物用于制备肝细胞癌诊断和/或预后的试剂盒中，检测肝细胞癌中辅助诊断的分子标志物MOGAT2的表达水平，会使得操作更加的简便易行。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (5)

  1. MOGAT2作为脂质代谢相关生物标志物在制备用于肝细胞癌诊断和/或预后判断的产品中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述产品包括用于检测MOGAT2的表达水平的试剂和/或试剂盒。
  3. 一种用于检测MOGAT2表达水平的试剂在制备预测肝细胞癌患者的预后情况的试剂盒中的应用。
  4. 根据权利要求3所述的应用，其特征在于，所述试剂盒包括扩增MOGAT2的引物和扩增内参β-actin基因的引物。
  5. 根据权利要求4所述的应用，其特征在于，所述扩增MOGAT2的引物为：
    上游引物：5′-TCTCTTCGATCTTCCCCGGTA-3′，
    下游引物：5′-GATGCCCAGCAAGTTTCCG-3′；
    所述扩增内参β-actin基因的引物为：
    上游引物：5′-CAGCAAGGACTGGTCTTTCTAT-3′，
    下游引物：5′-GCGGCATCTTCAAACCTC-3′。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180113135A1 (en) * 2015-04-13 2018-04-26 Johann Wolfgang Goethe-Universität Frankfurt am Main Serum Biomarker For Hepatocellular Carcinoma (HCC)
CN109609650A (zh) * 2019-01-31 2019-04-12 泰山医学院 用于诊断和治疗肝细胞癌的生物标志物
CN112280857A (zh) * 2020-10-19 2021-01-29 郑州大学 一种用于肝细胞癌诊断的生物标志物及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180113135A1 (en) * 2015-04-13 2018-04-26 Johann Wolfgang Goethe-Universität Frankfurt am Main Serum Biomarker For Hepatocellular Carcinoma (HCC)
CN109609650A (zh) * 2019-01-31 2019-04-12 泰山医学院 用于诊断和治疗肝细胞癌的生物标志物
CN112280857A (zh) * 2020-10-19 2021-01-29 郑州大学 一种用于肝细胞癌诊断的生物标志物及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BLANC VALERIE, RIORDAN JESSE D., SOLEYMANJAHI SAEED, NADEAU JOSEPH H., NALBANTOGLU ILKE, XIE YAN, MOLITOR ELIZABETH A., MADISON BL: "Apobec1 complementation factor overexpression promotes hepatic steatosis, fibrosis, and hepatocellular cancer", JOURNAL OF CLINICAL INVESTIGATION, vol. 131, no. 1, 4 January 2021 (2021-01-04), XP093127541, ISSN: 0021-9738, DOI: 10.1172/JCI138699 *
PIÑERO FEDERICO, DIRCHWOLF MELISA, PESSÔA MÁRIO G.: "Biomarkers in Hepatocellular Carcinoma: Diagnosis, Prognosis and Treatment Response Assessment", CELLS, vol. 9, no. 6, pages 1370, XP093127867, ISSN: 2073-4409, DOI: 10.3390/cells9061370 *
WANG ZHIHAO, EMBAYE KIDANE SIELE, YANG QING, QIN LINGZHI, ZHANG CHAO, LIU LIWEI, ZHAN XIAOQIAN, ZHANG FENGDI, WANG XI, QIN SHENGHU: "A Novel Metabolism-Related Signature as a Candidate Prognostic Biomarker for Hepatocellular Carcinoma", JOURNAL OF HEPATOCELLULAR CARCINOMA 2014, vol. Volume 8, pages 119 - 132, XP093127860, ISSN: 2253-5969, DOI: 10.2147/JHC.S294108 *

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