Sleeping Beauty transposon system harboring HRAS, c-Myc and shp53 induces sarcomatoid carcinomas in mouse skin

The Sleeping Beauty transposon system is used as a tool for insertional mutagenesis and oncogenesis. However, little is known about the exact histological phenotype of the tumors induced. Thus, we used immunohistochemical markers to enable histological identification of the type of tumor induced by subcutaneous injection of the HRAS, c-Myc and shp53 oncogenes in female C57BL/6 mice. The tumor was removed when it reached 100 mm3 in volume. Subsequently, we used 13 immunohistochemical markers to histologically identify the tumor type. The results suggested that the morphology of the tumor was similar to that of sarcomatoid carcinoma.ope

Transgenic mouse model expressing P53(R172H), luciferase, EGFP, and KRAS(G12D) in a single open reading frame for live imaging of tumor

Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).ope

Investigation of Oncogenic Cooperation in Simple Liver-Specific Transgenic Mouse Models Using Noninvasive In Vivo Imaging

iver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. Here, we report a simple methodology by which oncogenic cooperation between cancer-related genes can be efficiently investigated in the liver. We developed various non-germline transgenic mouse models using hydrodynamics-based transfection which express HrasG12V, SmoM2, and a short-hairpin RNA down-regulating p53 (shp53) individually or in combination in the liver. In this transgenic system, firefly luciferase was co-expressed with the oncogenes as a reporter, allowing tumor growth in the liver to be monitored over time without an invasive procedure. Very strong bioluminescence imaging (BLI) signals were observed at 4 weeks post-hydrodynamic injection (PHI) in mice co-expressing HrasG12V and shp53, while only background signals were detected in other double or single transgenic groups until 30 weeks PHI. Consistent with the BLI data, tumors were observed in the HrasG12V plus shp53 group at 4 weeks PHI, while other transgenic groups failed to exhibit a hyperplastic nodule at 30 weeks PHI. In the HrasG12V plus shp53 transgenic group, BLI signals were well-correlated with actual tumor growth in the liver, confirming the versatility of BLI-based monitoring of tumor growth in this organ. The methodology described here is expected to accelerate and facilitate in vivo studies of the hepatocarcinogenic potential of cancer-related genes by means of oncogenic cooperation.ope

DNA 혈관 주입 방법을 통한 다양한 이중 형질전환 간암모델의 개발

Dept. of Medical Science/박사Liver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. This study aims to develop transgenic liver cancer models using a hydrodynamics-based transfection method with the Sleeping Beauty transposon system. Using this time-saving and cost-effective methodology, transgenic models have been developed that express various types of oncogenes, individually or in combination to elucidate the genetic mechanisms underlying liver cancer. Transposon vectors each encoding an oncogene or down-regulating a tumor suppressor gene (HrasG12V, β-cateninS33Y, cMyc, SmoM2, and shP53) were constructed. Tumors were observed when livers were hydrodynamically transfected with HrasG12V plus shP53, cMyc plus shP53 and cMyc plus HrasG12V. In tumor of HrasG12V plus shP53 model, expression levels of genes related to cell cycle and epithelial-mesenchymal transition were significantly elevated when determined by a quantitative RT-PCR method. Along with the simple transgenic approach, Cre-mediated conditional transgenic mice were developed using the traditional transgenic methodology that express two oncogenes (KrasG12D and P53R172H) and two reporter genes (green fluorescent protein and luciferase) upon DNA recombination by Cre. Liver tumors were induced using adenovirus encoding Cre and tumor development was subsequently monitored in vivo using bioluminescence imaging. Also, transposons expressing various isoforms of oncogenic Ras (KrasG12V, NrasG12V, KrasG12D, Hras61L, and Nras61K) were generated and used for hydrodynamic transfection. Although different tumorigenic potentials among Ras isoforms have been suggested in liver, we found no difference in intrinsic hepato-carcinogenic potentials among the isoforms.Lastly, using the hydrodynamic transfection model which successfully expressed HrasG12V and shP53, we have observed repression of liver cancer growth in low-carbohydrate diet groups.The methodology described here is expected to accelerate and facilitate in vivo studies of tumorigenic potentials of cancer-related genes by means of oncogenic cooperation. Due to the versatility of our transposon-based liver cancer models in testing the efficacy of anti-cancer therapeutics, they can be applied to various anti-cancer research leading to the development of new drugs and novel therapeutic strategies.prohibitio

Analysis of miRNA expression patterns in human and mouse hepatocellular carcinoma cells

AIM: Hepatocellular carcinoma (HCC), one of the most common malignancies in adults displays aberrant miRNA expression during its pathogenesis. We assessed expression of miRNA in surgically resected human HCC of an early stage and murine HCC with a high malignancy in order to find miRNA overexpressed in HCC regardless of tumor stage and underlying etiology. Further, the role of the deregulated miRNA in HCC pathogenesis was investigated. METHODS: miRNA were isolated from HCC tissues and surrounding non-tumorous tissues from HCC patients and a murine transgenic model of HCC. A quantitative reverse transcription polymerase chain reaction was performed to determine expression levels of miRNA. Human HCC cell lines stably expressing individual miRNA were generated to investigate the biological function of overexpressed miRNA. RESULTS: We found that levels of miR-221, -181b-1, -155-5p, -25 and -17-5p were significantly upregulated in both human and murine HCC regardless of tumor stage, underlying etiology or the presence of fibrosis. Using HCC cell lines stably expressing respective miRNA, we found that miR-221 increased the proliferation of hepatoma cells, while miR-17-5p induced cell migration. CONCLUSION: We identified miRNA that are consistently upregulated in HCC. The overexpressed miRNA could potentially be used as a bona fide biomarker for HCC.restrictio

Combination of preS deletions and A1762T/G1764A mutations in HBV subgenotype C2 increases the risk of developing HCC.

BACKGROUND: The interactions among hepatitis B virus (HBV) mutations in developing hepatocellular carcinoma (HCC) remain unclear and thus we investigated the risk of HCC related with single or multiple HBV mutations in Korean patients infected with HBV subgenotype C2. METHODS: From January 2003 to December 2008, HBV isolates from 135 patients with HCC were compared with those from 135 patients without HCC, matching for age, gender, and HBeAg status. The prevalence of preS deletions and G1896A and A1762T/G1764A mutations was evaluated. RESULTS: The frequency of preS deletions significantly differed between the non-HCC and HCC groups, with 6 (4.4%) versus 25 (18.5%) patients, respectively (p < 0.001). Additionally, the frequency of A1762T/G1764A mutations was higher in the HCC than the non-HCC group [82 (60.7%) versus 30 (22.2%), p < 0.001]. For combined mutations, the odds ratio (OR) was highest in patients with both preS deletions and the A1762T/G1764A mutation, with 1 (0.7%) versus 11 (8.1%) patients (p = 0.005; OR 11.887). CONCLUSIONS: HCC was associated with preS deletions and A1762T/G1764A mutations, and the combination of both mutations had a stronger association with HCC in Korean patients infected with HBV subgenotype C2.ope