Resistance to chemotherapy is one of the major reasons for the failure of cancer treatment. Post- transcriptional and translational control plays an important role in chemoresistance. Currently there is no available high throughput approach to investigate post-transcriptional and translational control mediated by RNA binding proteins or non-coding microRNAs from a small number of cells. Our primary objective in the proposed project is to apply our newly developed Translational Immunoprecipitation-Microarray Analysis (TrIP-Chip) (1) to discover post-transcriptionally regulated mRNA targets mediated by miR-215 from a small number of colon cancer stem cells. Our recent studies revealed that miR-215 is involved in regulating some key anticancer targets, e.g. thymidylate synthase and dihydrofolate reductase. Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to form dTMP and dihydrofolate. Because the TS-catalyzed enzymatic reaction provides the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis, TS has been an important target for cancer chemotherapy for over 50 years. The enzyme dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. This reaction provides the key intermediate in one-carbon transfer reactions. DHFR plays a critical role in folate homeostasis, and is required for the de novo synthesis of purines, thymidylate and certain amino acids. Therefore, DHFR has served as a critical target in cancer chemotherapy. The mRNAs regulated post-transcriptionally by miR-215 will be validated at the protein level by a sensitive, high throughput shotgun proteomic analysis based on multi-dimensional protein identification technology (MudPIT). We will further develop a TrIP-Seq approach to increase both the coverage and sensitivity to detecting rare transcripts potentially at the single cell level.
Three specific aims are proposed:
Specific Aim 1. Identify post-transcriptionally and translationally regulated mRNA targets of miR-215 using TrIP-Chip approach.
Specific Aim 2. Validate miR-215 mediated targets by high throughput proteomic analysis.
Specific Aim 3. We will further develop the TrIP-Chip approach by integrating next generation sequencing based expression analysis (TrIP-Seq) to increase coverage and detection sensitivity from a small number of cells.

Public Health Relevance

Currently, there is a lack of high throughput approach to study post- transcriptional and translational control with a small number of cells. We have developed a novel Translational Immunoprecipitation-Array Gene expression analysis (TrIP-Chip) technology that allow us to investigating post-transcriptional and translational regulated genes from as few as 500 cells. We believe with the improvement of the dynamic range of the next generation deep sequencing platform (e.g. Illumina Instrument), we can potentially analyze translational control from a single cell. The proposed project will develop and apply this new approach to discover miR-215 mediated targets and functional significance in colon cancer and colon cancer stem like cells. The technology will have a broad application not only in cancer but other human diseases as well.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
1R33CA147966-01A1
Application #
8035099
Study Section
Special Emphasis Panel (ZCA1-SRLB-R (O1))
Program Officer
Sorg, Brian S
Project Start
2011-08-15
Project End
2014-07-31
Budget Start
2011-08-15
Budget End
2012-07-31
Support Year
1
Fiscal Year
2011
Total Cost
$225,000
Indirect Cost
Name
State University New York Stony Brook
Department
Pathology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Wu, Changping; Zheng, Xiao; Li, Xiaodong et al. (2016) Reduction of gastric cancer proliferation and invasion by miR-15a mediated suppression of Bmi-1 translation. Oncotarget 7:14522-36
Wu, Song; Fesler, Andrew; Ju, Jingfang (2016) Implications of Circadian Rhythm Regulation by microRNAs in Colorectal Cancer. Cancer Transl Med 2:1-6
Zhai, Haiyan; Fesler, Andrew; Ba, Yufeng et al. (2015) Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-miR-140-5p mediated suppression of Smad2 and autophagy. Oncotarget 6:19735-46
Fesler, Andrew; Jiang, Jingting; Zhai, Haiyan et al. (2014) Circulating microRNA testing for the early diagnosis and follow-up of colorectal cancer patients. Mol Diagn Ther 18:303-8
Fesler, Andrew; Zhai, Haiyan; Ju, Jingfang (2014) miR-129 as a novel therapeutic target and biomarker in gastrointestinal cancer. Onco Targets Ther 7:1481-5
Popescu, Nicholas C; Goodison, Steve (2014) Deleted in liver cancer-1 (DLC1): an emerging metastasis suppressor gene. Mol Diagn Ther 18:293-302
Dong, P; Karaayvaz, M; Jia, N et al. (2013) Mutant p53 gain-of-function induces epithelial-mesenchymal transition through modulation of the miR-130b-ZEB1 axis. Oncogene 32:3286-95
Zhai, Haiyan; Fesler, Andrew; Ju, Jingfang (2013) MicroRNA: a third dimension in autophagy. Cell Cycle 12:246-50
Zhai, Haiyan; Karaayvaz, Mihriban; Dong, Peixin et al. (2013) Prognostic significance of miR-194 in endometrial cancer. Biomark Res 1:
Karaayvaz, M; Zhai, H; Ju, J (2013) miR-129 promotes apoptosis and enhances chemosensitivity to 5-fluorouracil in colorectal cancer. Cell Death Dis 4:e659

Showing the most recent 10 out of 18 publications