Integrated gene expression analysis of blood and liver tissue in precancerous condition [mRNA]

Patients with diabetes mellitus (DM) have an epidemiologically higher risk for hepatocellular carcinoma (HCC). In mouse models, administration of streptozotocin (STZ) induces insulin-dependent DM by causing islet beta-cell dysfunction and also induced hepatocellular carcinoma (HCC) after 12 weeks. We attempted to elucidate the carcinogenic mechanism in the precancerous state by using hepatic miRNAs and create precancerous marker using exosomal miRNA. Serum and liver tissues were collected from STZ mice and non-treated mice (CTL mice) at 6, 10 and 12W. Total RNA in liver and exosome were extracted. Histological examination in liver in HE stain and hepatic and exosomal miRNA analysis were serially performed. miRNA and mRNA was analyzed by next-generation sequencing (NGS). The raw data of NGS was analyzed by Principal Component analysis. No inflammation or fibrosis was found in the liver in CTL mice during the observation period. In STZ-treated mice, regeneration and inflammation of hepatocytes was found at 6W and then nodules of atypical hepatocytes were found at 10 and 12 W. Expression of let-7f-5p, miR-21a-5p, 22-3p, and 26a-5p in liver of STZ mice was significantly increased during 6-10W, and these miRNAs controlled several tumor suppressor genes as target genes. We also identified six novel miRNAs associated with precancerous status. Furthermore, miR-122-5p and miR-192-5p in exosome were significantly upregulated in STZ mice in precancerous state. The expression of miRNAs that regulate tumor suppressor genes was enhanced even before carcinogenesis, and the expression of these miRNAs was not affected by liver fibrosis. In addition, the expression pattern of miRNAs in exosome is expected to be a marker for predicting carcinogenesis. Patients with diabetes mellitus (DM) have an epidemiologically higher risk for hepatocellular carcinoma (HCC). In mouse models, administration of streptozotocin (STZ) induces insulin-dependent DM by causing islet beta-cell dysfunction and also induced hepatocellular carcinoma (HCC) after 12 weeks. We attempted to elucidate the carcinogenic mechanism in the precancerous state by using hepatic miRNAs and create precancerous marker using exosomal miRNA. Serum and liver tissues were collected from STZ mice and non-treated mice (CTL mice) at 6, 10 and 12W. Total RNA in liver and exosome were extracted. Histological examination in liver in HE stain and hepatic and exosomal miRNA analysis were serially performed. miRNA and mRNA was analyzed by next-generation sequencing (NGS). The raw data of NGS was analyzed by Principal Component analysis. No inflammation or fibrosis was found in the liver in CTL mice during the observation period. In STZ-treated mice, regeneration and inflammation of hepatocytes was found at 6W and then nodules of atypical hepatocytes were found at 10 and 12 W. Expression of let-7f-5p, miR-21a-5p, 22-3p, and 26a-5p in liver of STZ mice was significantly increased during 6-10W, and these miRNAs controlled several tumor suppressor genes as target genes. We also identified six novel miRNAs associated with precancerous status. Furthermore, miR-122-5p and miR-192-5p in exosome were significantly upregulated in STZ mice in precancerous state. The expression of miRNAs that regulate tumor suppressor genes was enhanced even before carcinogenesis, and the expression of these miRNAs was not affected by liver fibrosis. In addition, the expression pattern of miRNAs in exosome is expected to be a marker for predicting carcinogenesis. Patients with diabetes mellitus (DM) have an epidemiologically higher risk for hepatocellular carcinoma (HCC). In mouse models, administration of streptozotocin (STZ) induces insulin-dependent DM by causing islet beta-cell dysfunction and also induced hepatocellular carcinoma (HCC) after 12 weeks. We attempted to elucidate the carcinogenic mechanism in the precancerous state by using hepatic miRNAs and create precancerous marker using exosomal miRNA. Serum and liver tissues were collected from STZ mice and non-treated mice (CTL mice) at 6, 10 and 12W. Total RNA in liver and exosome were extracted. Histological examination in liver in HE stain and hepatic and exosomal miRNA analysis were serially performed. miRNA and mRNA was analyzed by next-generation sequencing (NGS). The raw data of NGS was analyzed by Principal Component analysis. No inflammation or fibrosis was found in the liver in CTL mice during the observation period. In STZ-treated mice, regeneration and inflammation of hepatocytes was found at 6W and then nodules of atypical hepatocytes were found at 10 and 12 W. Expression of let-7f-5p, miR-21a-5p, 22-3p, and 26a-5p in liver of STZ mice was significantly increased during 6-10W, and these miRNAs controlled several tumor suppressor genes as target genes. We also identified six novel miRNAs associated with precancerous status. Furthermore, miR-122-5p and miR-192-5p in exosome were significantly upregulated in STZ mice in precancerous state. The expression of miRNAs that regulate tumor suppressor genes was enhanced even before carcinogenesis, and the expression of these miRNAs was not affected by liver fibrosis. In addition, the expression pattern of miRNAs in exosome is expected to be a marker for predicting carcinogenesis. Overall design: Examination of hepatic mRNA in pre-cancer state of liver流行病学研究表明，糖尿病（diabetes mellitus, DM）患者罹患肝细胞癌（hepatocellular carcinoma, HCC）的风险显著升高。在小鼠模型中，链脲佐菌素（streptozotocin, STZ）可通过损伤胰岛β细胞功能诱导胰岛素依赖型糖尿病，且造模12周后可诱发肝细胞癌（HCC）。本研究旨在通过肝脏微小RNA（microRNA, miRNA）阐明癌前状态下的致癌机制，并利用外泌体（exosome）源性miRNA构建癌前病变标志物。 本研究分别于造模第6、10、12周，采集链脲佐菌素处理小鼠（STZ组）与未处理对照小鼠（CTL组）的血清及肝脏组织。提取肝脏组织与外泌体中的总RNA，依次开展肝脏组织苏木精-伊红（HE）染色组织学检查、肝脏及外泌体miRNA表达分析。采用下一代测序技术（next-generation sequencing, NGS）对miRNA与信使RNA（mRNA）进行测序，并通过主成分分析（Principal Component Analysis, PCA）处理NGS原始测序数据。 观察期内，对照组小鼠肝脏未出现炎症或纤维化病变。链脲佐菌素处理组小鼠在造模第6周即可观察到肝细胞再生与炎症反应，第10、12周则出现异型肝细胞结节。STZ组小鼠肝脏中let-7f-5p、miR-21a-5p、miR-22-3p及miR-26a-5p的表达水平在6~10周间显著上调，上述miRNA可靶向调控多种肿瘤抑制基因。本研究同时鉴定出6种与癌前状态相关的新型miRNA。此外，处于癌前状态的STZ组小鼠外泌体中miR-122-5p与miR-192-5p的表达水平显著升高。 研究结果显示，调控肿瘤抑制基因的miRNA在致癌前即出现表达上调，且该类miRNA的表达不受肝脏纤维化影响。此外，外泌体miRNA的表达谱有望成为预测肝细胞癌发生的潜在标志物。 总体实验设计：针对肝脏癌前状态下的肝脏mRNA开展检测。