国外大学申请Proposal.docx

Coding-independent regulation of colon cancer cell proliferation PROJECT DESCRIPTIONColorectal Cancer (CRC), a malignant disease derived from colon epithelial cells, is one of the major threats to public health. Australia has one of the highest rates of CRC in the world. It is the third most common type of newly diagnosed cancer in the country. Moreover, it is the second most common cancer killer, claiming lives of around 3982 Australians each year. However, there is currently no curative treatment for metastatic colorectal cancers. Investigation of genes and signaling pathways that are dysregulated in CRC is a high priority for identification of novel therapeutic targets. Only 1.5% of the mammalian genome encodes proteins, and most of the genome is transcribed to tens of thousands of long (200 nt) non-coding RNAs (lncRNAs). However, the functional consequence of the expression of lncRNAs has been largely uncharacterized. To date, a very small number of lncRNAs have been investigated, and their functional roles, revealed, in colon cancer. Preliminary studies from Professor Xu Dong Zhangs laboratory have shown that the expression of a lncRNA called REG1CP is elevated in five CRC tissues in comparison with adjacent non-cancerous colon epithelial tissues. The increase in REG1CP is further confirmed in a panel of CRC cell lines compared with a normal human colon cell line FHC. Of note, inhibition of REG1CP by siRNA reduced, whereas overexpression of REG1CP enhanced, CRC cell proliferation. To turn this information into our advantage in understanding CRC biology and in the treatment of the disease, we propose in this application to clarify the consequence of REG1CP upregulation and the mechanism involved in CRC cells, and to examine whether REG1CP-mediated signal is a potential therapeutic target in CRC. Our specific aims are to test whether targeting REG1CP inhibits proliferation of CRC cells and retards CRC xenograft growth in animal models, and to define the mechanism that drives the increase in REG1CP expression in CRC cells and the mechanism by which REG1CP regulates colon cancer growth.University of Newcastle has a strong cancer research program aiming to translate scientific advances into better clinical care. This project fits perfectly to the objectives of Cancer Program in that it will potentially lead to the development of new treatment approaches for CRC. Professor Xu Dong Zhangs laboratory is highly experienced in similar studies, and it is well equipped with techniques and other materials and support needed for carrying out this project.Background: lncRNA: It has been recently shown that over 70% of the genomes of human and higher eukaryotes are transcribed, but only 1.5% of the human genome codes for proteins. lncRNAs are a large and diverse class of transcribed RNA molecules of more than 200 nucleotides that do not encode proteins, which may exert their functions either by binding to DNA or RNA in a sequence specific manner or by binding to proteins. lncRNA in cancer: Increasing evidence shows that lncRNAs play an important role in cancer biology. Dysregulated expression of lncRNAs in cancer frequently marks disease progression and often serves as an independent predictor for poor prognosis of cancer patients. (1, 2) REG1CP: REG1CP is an RNA that lacks an open reading frame. It is located to the locus enriched with regenerating (REG) gene family members, whose expression levels are closely associated with the survival rate of CRC patients (3). Interestingly, we have found REG1CP expression levels are positively related with the levels of REG gene family members including REG1A, REG1B and REG3A, suggesting that REG1CP may plays an important role in CRC patient survival as well.Supporting data:lncRNA microarray data showed that REG1CP was the most significantly upregulated lncRNA in five CRC tissues compared with adjacent non-cancerous colon epithelial tissues. While overexpression of REG1CP promotes, inhibition of REG1CP reduces, CRC cell proliferation. Moreover, REG1CP is also commonly upregulated in cultured CRC cell lines irrespective of their origins and genetic backgrounds in comparison with the cultured normal colon epithelial cell line FHC. Of note, REG1CP expression is positively associated with REG family members, which have been reported as predictors of poor CRC patient survival.Hypothesis:In this project, we will test the hypothesis that targeting REG1CP is potentially a novel therapeutic strategy in CRC.Aims:1) To elucidate the mechanism responsible for REG1CP-mediated CRC cell proliferation;2) To determine the functional role of REG1CP in CRC xenograft models;3) To define the mechanism by which REG1CP is upregulated in CRC.Research Plan:Resources and Methods:CRC specimens resource: Xu Dong Zhangs lab has full access to the resource that currently has tissue microarrays established from 500 normal colon epithelial tissues, 500 primary, and 2000 metastatic CRC with linked clinicopathological data. Inducible gene knockdown: The technique of inducible knockdown of genes by the doxycycline-induced shRNA system has been well established in our lab.In vivo delivery of siRNA by liposome-protamine-hyaluronic acid nanoparticles (LPH-NPs): The LPH-NP delivery system that is constructed by our collaborator prof. Leaf Huang significantly extends the half-life, increases bioavailability and stringently controls the tropism of delivered therapeutics including siRNA in vivo. (4)Mouse models: Human CRC xenograft models will be established using NOD-SCID mice. How does REG1CP upregulation promote proliferation of CRC cells? (Aim 1) We will use capture hybridization analysis of RNA targets (CHART) assay to identify the DNA and protein targets of REG1CP from CRC cells expressing relatively high levels of endogenous REG1CP. The identities of captured DNA targets will be clarified by DNA sequencing, and the protein targets, analysed by Mass Spectrometry. Binding between REG1CP and the identified DNAs will be confirmed by RNA pull down assays. DNA sequences will be aligned with the genomic sequence. Protein targets will be verified by RNA immunoprecipitation (RIP) assay. These associated target genes (genes encoding the proteins) will be knocked down by siRNA to confirm whether they are involved in REG1CP-mediated promotion of CRC cell proliferation.Does REG1CP promote the growth of CRC xenografts in vivo? (Aim 2.1) Lim1215 and HT29 CRC cells that express relative high levels of endogenous REG1CP carrying the REG1CP inducible knocked down system (Lim1215.shR and HT29.shR cells, respectively) and the control cells will be used. For clarity, only Lim1215.shR and the control cells (Lim1215.shC) will be exemplified below. Viable Lim1215.shR and Lim1215.shC cells (2 x 106) will be xenografted by s.c. injection into each flank of NOD-SCID mice (5 week old) in a total volume of 200 l of PBS containing 20% Matrigel matrix. Tumour growth will be measured every 2 days using a calliper. After the smallest tumour reaches 0.25 cm3, mice carrying Lim1215.shR and Lim1215.shC tumours will be respectively divided into 2 groups (8 mice/group, statistically calculated). Mice in groups 1 and 2 will be respectively administered with doxycycline and the vehicle control DMSO via oral gavage (200 mg/kg/day). At day 28 after the commencement of treatment (according to pilot results), all mice will be euthanized, tumours removed, measured, weighed, and processed for FFPE tissue blocks. Staining with antibodies against Ki-67 and activated caspase-3 will be used to show potential changes in proliferation and potential occurrence of apoptosis, respectively. Does targeting REG1CP retard CRC xenograft growth in vivo? (Aim 2.2) Lim1215 and HT29 CRC cells will be xenografted into NOD-SCID mice and tumour growth will be monitored as described above. For clarity, only Lim1215 cells will be exemplified below. After the smallest tumour reaches 0.25 cm3, mice carrying Lim1215 tumours will be randomized into 2 groups (8 mice/group, statistically calculated). Mice in groups 1 and 2 will be respectively administered via i.v. injection with the control siRNA LPH-NPs and REG1CP siRNA LPH-NPs (0.6mg/kg) every other day for 6 times.(4) At day 28 after the commencement of treatment (according to pilot results), tumors will be harvested and processed and the results analyzed as described above.What is the mechanism responsible for REG1CP upregulation in CRC cells? (Aim 3) To identify the potential transcription factor(s) responsible for transcriptional upregulation of REG1CP in CRC cells, we will determine the transcriptional region of the REG1CP gene promoter using a series of incremental deletion luciferase reporter constructs as described previously (5). The shortest region that remains active in cells will be identified. We will query for consensus binding sites of known transcription factors for the region using the TRANSFAC database. In the event where a number of potential binding sites exist within a specified region, a second series of REG1CP promoter mutants will be constructed using a site-directed mutagenesis kit (Promega). To confirm that the identified transcription factor(s) is responsible for activating the minimal promoter region in CRC cells, we will carry out Chromatin immunoprecipitation assays (ChIP) to demonstrate binding of the transcription factor(s) to the region (6). shRNA knockdown of the identified transcription factor(s) will be used to show its functional significance. These results will identify the transcription factor (s) responsible for transcriptional upregulation of REG1CP in CRC cells. We will also examine the expression levels of the transcription factor (s) in a panel of CRC cell lines by western blotting. The relationship between REG1CP and the transcription factor (s) expression levels will be analyzed by regression analysis. Expected outcome and significance The results from this project wil