| [Background]Colorectal cancer (CRC) is one of the most common cancers in the western countries but the incidence of CRC has increased in recent years in China. Large-scale population screening in China has revealed CRC is the fourth common tumor type. Metastases at distant sites are the main cause of death, while liver is the main metastatic organ. It is not possible to accurately predict the risk of liver metastasis development in individual patients using the current prognostic markers. There is a needed to screen the specific metastasis-related genes and biomarkers in CRC liver metastasis.CRC liver metastasis is a complex multi-step process and only a sub-populations tumor cells with high metastatic potential could form distant metastases. It is the best way to investigate the detailed molecular mechanisms using orthotopic metastasis animal model. By injecting SW620cells into the cecum of nude mouse, we have established an orthotopic CRC liver metastasis animal model. We performed gene expression analysis of SW620and SW620M, which was isolated from the liver metastasis of animal model. We found HNF6was abundantly expressed in SW620M and colorectal liver metastases.Hepatocyte nuclear factor6(HNF6) belongs to a liver-enriched transcription factors which contains six members-HNF1, HNF3, HNF4, HNF6, C/EBP,DBP. HNF6locate on chromatin15q21.1-q21.2, express a465aa protein which control a wide spectrum of biological processes such as:gene expression of liver, development of early liver、biliary system, pancreas、metabolism. It has been founed that HNF6regulate expression of NEUROG3、FOXM1、PDX1、HNF1、FOXA1.FOXA2, most of which have been found involved in cancer tumorigenicity and metastases. Recent researches indicated that HNF6itself also involved in those process. Prevot PP et al found HNF6was required for pancreatic acinar-to-ductal metaplasia (ADM) and a new biomarkers of acinar-to-ductal metaplasia which lead to cancer.Lehner F et al found HNF6and its target gene FOXA2,CXCL1. CXCL2was up-regulated in liver metastases by immunostaining in CRC liver metastases and primary tumor. But there is little report about the effect of HNF6on cancer cells.In the present research we constructed a recombinant lentiviral vector that stably express HNF6and successfully established a colorectal cancer cell line SW620-HNF6by infecting lentivirus.Then we further examined its effects on the invasive ability of SW620cells.Our investigation provides us preliminary understanding of the role of HNF6in colorectal liver metastasis process and is helpful to further study the detailed mechanism.[Objective]The purpose of the study is to construct a recombinant lentiviral vector-pLeno-DCE-HA-HNF6,establish a stable expressing HNF6colorectal cancer cell line and examin its effects on the behaviors of SW620cells.[Methods]1. HNF6gene was amplified by PCR and then cloned into recombinant lentiviral vector-pLeno-DCE-HA and verified by sequencing.2. pLeno-DCE-HA-HNF6, pRsv-REV, pMD1g-pRRE and pMD2G were cotransfected into293T cells and the supematant containing lentivirus particles was harvested and determined the virus titer.3. The stable cell line was established by infecting the letivirus particles and the transfection efficiency was examined by flow cytometry, western blot, RT-PCR.4. The morphology changes was observed by microscope and invasion ability of SW620-HNF6cens was detected by wound bealing and transwell assays.5. The changes of proliferation and colony formation of SW620-HNF6were tested by counting cells and colony formation assay.6. The effect of HNF6on tumorigenicity was investigated using in vivo animal model.[Results]1. The recombinant lentiviral vector pLeno-DCE-HA-HNF6was constructed correctly and verified by PCR and sequencing.2. Lentivirus particles were produced by packeting cell293T cells and virus titer was up to5.1x108TU/ml 3. The stable HNF6expression cell line was established successfully and transfection efficiency of SW620-HNF6, SW620GFP were up to82.3%and93.8%. Western blot and RT-PCR demonstrated that HNF6expression was significantly upregulated in SW620-HNF6cells.4. SW620-HNF6cells showed a square-like epithelial shape compared with the elongated, fibroblast-like shape of the control cells. Wound healing showed invasion ability of SW620cells was inhibited. Transwell assays showed invased cell of SW620, SW620-EGF6and SW620-HNF6was65.7±5.5,72.3±12.9and25.0±2.0.5. Growth curve revealed SW620-HNF6cells were proliferated at a significantly higher rate than that of SW620-EGFP. Colony formation assay showed the colony formation numbers and rate of SW620-EGF6and SW620-HNF6were53.00±4.00,26.5%and82.33±4.73,41.17%.6. In vivo reseach showed2x105SW620-HNF6cells could form tumor while SW620-HNF6cells could not. The tumor volume of SW620-EGF6and SW620-HNF6were789.9±266.3mm3and1808±475.5mm3.[Conclusion]pLeno-DCE-HA-HNF6was constructed correctly and high quality lentivirus particles were produced. Recombinant lentiviral vector carrying HNF6can efficiently infect SW620cells. HNF6changed morphological appearance of SW620cells and inhibited its invasion ability. HNF6enhanced cells growth and colony formation ability significantly. Further investigation revealed that overexpression of HNF6significantly increased tumorigenicity and enhanced tumor growth in vivo. |
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