非酒精性脂肪性肝病恶性转化中CD44异常激活及其分子调控机制

Prevalence of non-alcoholic fatty liver diseases (NAFLD)-related hepatocellular carcinoma (HCC) is still increasing. Previously, we found that CD44 (cluster of differentiation 44, as a marker of cancer stem cells) closely associated with fatty liver malignant transformation with mitochondrial carnitine palmitoyl transferase-II (CPT-II) inactivity. However, the regulatory mechanism CD44 activation remains to be clarified. Based on these previous observations, we plan to carry out the following researches. First, with the use of NAFLD malignant transformation models, the dynamic changes of CD44 activation, CPT-II inactivation and Wnt3a expression will be investigated in hepatocarcinogenesis. Second, the expressions of hepatic CD44 and CPT-II gene transcription, specific concentration (μg/mg wet tissue) and their clinico-pathological features will be revealed using surgically removed human NAFLD-related HCC tissues, and exploring the relationship between exon 4 promoter mutation of CPT-II gene by sequencing or CD44 expression and lipid metabolism. Third, the genes of CD44 and Wnt3a will be knockouted by a CRISPR-Cas9-sgRNA technology to observe them on effects of hepatoma cell biological behaviors through RhoA to regulate YAP signaling or Wnt pathway. And finally, circulating CD44, CPT-II, and Wnt3a levels will detected in population-based case-control study and retrospective cohort study of the high-risk NAFLD chronic hepatitis, liver cirrhosis, liver cancer and healthy controls to confirm CD44 activation with NAFLD progression and analyze the interaction of CD44 and Wnt3a with NAFLD malignant transformation. This study will elucidate a novel mechanism of CD44-regulating NAFLD malignant progression and establish a novel monitoring biomarker for possible preventive-therapeutic strategies of NAFLD-related HCC occurrence.

非酒精性脂肪性肝病（NAFLD）恶性转化致癌呈增长趋势。前期研究新发现NAFLD恶性转化中肝细胞CD44异常激活伴线粒体CPT-II失活，其分子调控机制待阐明。本课题拟通过NAFLD恶性转化模型观察CD44激活、CPT-II失活及其动态表达规律；分析人NAFLD相关肝癌组织CD44和CPT-II转录、比浓度及病理学特征；测序分析CPT-II第4号外显子变异、CD44表达与脂代谢的关系；以CRISPR-Cas9-sgRNA技术敲除CD44和Wnt3a基因，探讨经RhoA调节YAP或Wnt通路对肝癌细胞生物学行为影响；比较NAFLD相关慢性肝炎、肝硬化、肝癌和健康对照组血CD44、CPT-II和癌胚型Wnt3a表达，验证CD44激活与NAFLD恶性转化的关系。分析CD44-NAFLD恶性转化与Wnt3a的交互作用，阐明CD44调控NAFLD恶性转化的机制，为早期监测提供靶标依据。

非酒精性脂肪性肝病（NAFLD）恶性转化致癌呈增长趋势。介于前期NAFLD恶性转化中肝细胞CD44异常激活伴线粒体CPT-II失活的研究发现，其分子调控机制待阐明，本课题从基础和临床方面进行了研究。通过NAFLD恶性转化模型，首次报道CD44从NAFLD到肝癌（HCC）发生的过程呈动态激活及进行性升高表达，与对照组CD44积分光密度值相比，NAFLD组、变性组、癌前病变组、 HCC鼠肝CD44均显著升高（P<0.001）。肝细胞癌及癌前病变组模型鼠血清CD44水平，也明显高于其他各组(P<0.001)。肝CD44、CD44 mRNA与血清CD44呈正相关，肝CD44 mRNA与血清CD44呈正相关。此外，肝CD44与肝AFP、血清CD44与血清AFP、CD44 mRNA与AFP mRNA均有显著相关。NAFLD患者伴有肝细胞损伤和血脂异常，NAFLD和慢性肝病患者血清 CD44水平，均显著高于正常对照组，NAFLD患者CD44阳性淋巴细胞比率为78.19±16.33% ，慢乙肝组为68.47±20.91%，均显著高于对照组46.51±20.52%。慢乙肝伴NAFLD组CD44为181.42±49.36 ng/mL显著高于单纯慢乙肝组(142.52±53.87) ng/mL和正常对照组99.47±15.23 ng/mL。肝癌组CD44/GAPDH比率为1.306±0.614明显高于癌旁组0.477±0.291(t=3.451，P=0.004)。NAFLD相关肝癌组CD44积分光密度为25.721±5.881显著高于癌旁组14.155±4.001(t=14.544，P<0.001)；CD44高表达与HBV感染、肿瘤大小、单中心/多中心、淋巴结转移、分化程度、TNM分级、 Child-Pugh评分、门静脉癌栓和肝外转移有关。NAFLD 组血清CD44为234.62 ± 69.40 ng/ml 明显高于非 NAFLD 组186.49 ± 58.89 ng/ml (t = 3.191，P= 0.002)。上述结果提示CD44可能是调控 NAFLD恶性转化的新机制，CD44异常可作为NAFLD相关性肝癌发生新的监测标志物。