Research projects

細菌叢と疾患の関連において、細菌由来細胞外小胞に含まれるRNAの機能研究は未開拓である。応募者は、細菌RNAがマクロファージを変容させ肝発がんに寄与することを実証した。本研究では、細菌RNAによる宿主細胞撹乱と発がんの分子機序を包括的に解明し、細菌・宿主・がんの関係に新機軸を構築する。これにより発がん機構の理解を拡張し、革新的な予防・治療戦略の創出を目指す。

For tumors such as pancreatic cancer, where early diagnostic technology is highly anticipated, it is hoped that circulating microRNA (miRNA) diagnosis will become practical. We used CRISPR/Cas9 to perform gene editing on pancreatic progenitor cells established from the primary culture of mouse pancreatic ducts. We created a pancreatic multistage carcinogenesis cell line in which mutations in Kras, Cdkn2a, Tp53, and Smad4 were introduced. Extracellular vesicles were collected from the culture supernatant of each cell line, and miRNA-seq was used to analyze miRNA profiles comprehensively. The cell lines were transplanted into the pancreas of nude mice, and changes in miRNA expression in mouse serum were compared, identifying miRNAs that changed in the blood due to the acquisition of Kras mutations.

We established the culturing method of pancreatic progenitor cells by chemical direct reprogramming of pancreatic exocrine cells isolated from mouse pancreas. We then investigated the alteration in intracellular and extracellular miRNAs during the exposure to bile acid, which is a potential cause of pancreatic carcinogenesis. As a result, several miRNAs including miR-296-5p and miR-135a-3p were identified as miRNAs whose secretion into the cell culture supernatant was enhanced. These miRNAs are expected to be biomarkers for the early-stage pancreatic cancer.

A large number of research projects identified genetic mutations in the process of carcinogenesis, and there have been some useful findings of studies using cancer cell lines. With next generation sequencing, genetic variants in a variety of cancers have been identified and shown to have an impact on carcinogenesis; however, it is still unclear how the mutations essentially influenced the carcinogenesis and their metastatic potential. In this research project, we introduced and quantitatively analyzed the contribution of genetic mutations involved in the carcinogenic process by CRISPR-Cas9, in order to knockout the tumor suppressor genes in primary epithelial cells, and examined the effect of each gene on carcinogenesis. Also, we tried to recapitulate the multi-step carcinogenesis in vitro. This study enables us to accurately reflect and reproduce genomic aberrations in normal cells, and to achieve validation of the actual function of driver gene mutations.

Esophageal adenocarcinoma, which is rapidly increasing in Western countries, is one of the complications of gastroesophageal reflux disease. Bile reflux and central obesity are major risk factors for esophageal adenocarcinoma. In general, plasma adiponectin levels are decreased through the progression of central obesity. We had reported that bile acids contribute to carcinogenesis through the activation of nuclear bile acid receptor (FXR) and the enhancement of miR-221/222. Herein, we identified that miR-221/222 enhance the expression of COX-2 in esophageal epithelial cells. On the other hand, COX-2 expression was suppressed during exposure to adiponectin receptor agonist. These results suggest adiponectin receptor agonist might work as a chemopreventive agent for esophageal adenocarcinoma among high-risk individuals with central obesity.

Using small molecule inhibitors, we recently reported that rodent mature hepatocytes can be reprogrammed into progenitor-like phenotype with repopulative capacity. In this study, using the same strategy, we demonstrated that hepatic progenitor cells can be induced from human infant hepatocytes. These cells, named human chemically induced progenitors (hCLiPs), exhibited significant repopulative capacity in injured mouse livers following transplantation, and contributed to reconstruction of the normal liver architecture. We also found that hCLiPs can be redifferentiated into mature hepatocytes in vitro with hepatic inducible factors. These redifferentiated cells can be induced to exhibit cytochrome P450 (CYP) enzymatic activities in response to the CYP inducing molecules with the efficiency comparable with that of primary hepatocytes. Thus, this study contributes to progress in the field of liver cell transplantation therapy and pharmacological research field.