Mirnas as therapeutic targets in cancer

Abstract

MicroRNAs (miRNAs) are a class of non-coding small RNA molecules that regulate gene expression at the post-transcriptional level by interacting with 3′ untranslated regions (UTRs) of their target mRNAs. The invention relates to the application of miR-192 and miR-215. Both of these miRNAs impact cellular proliferation through the p53-miRNA circuit, and interact with dihydrofolate reductase (DHFR) and thymidylate synthase (TS). Particularly, upregulation of these miRNAs reduces cellular proliferation. The invention relates to this discovery. For example, inhibiting miR-192 and/or miR-215 sensitizes a neoplasm or a subject with a neoplasm to chemotherapeutic agents. Furthermore, measuring the levels of miR-192 and/or miR-215 provides one with information regarding whether the neoplasm or subject will respond to chemotherapeutic agents. Accordingly, the invention relates to composition and methods relating to the identification, characterization and modulation of the expression of miR-192 and miR-215.

What is claimed is: 
     
         1 . A method of modulating expression of a component of a cell, comprising contacting the cell with a nucleic acid comprising an inhibitory nucleic acid having a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 9, 10, 25, a complementary sequence thereof, a variant thereof on a fragment thereof in an amount sufficient to modulate the cellular component. 
     
     
         2 . The method of  claim 1 , wherein the nucleic acid is an antisense nucleic acid. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid is an siRNA or an shRNA. 
     
     
         4 . The method of  claim 1 , wherein the cellular component is selected from the group consisting of miR-192 and miR-215. 
     
     
         5 . The method of  claim 1 , wherein the cellular component is p21 or p53. 
     
     
         6 . The method of  claim 1 , wherein the cellular Component is regulated by p53. 
     
     
         7 . The method of  claim 1 , wherein the cellular component is DHFR or TS. 
     
     
         8 . A method of increasing proliferation of a cell, comprising contacting the cell with a nucleic acid complementary to at least a portion of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 9, 10, 25 and an antisense sequence of SEQ ID NO:25, in an amount effective to increase proliferation of the cell. 
     
     
         9 . A method of increasing the sensitivity of a cell to a chemotherapeutic agent, comprising contacting the cell with a nucleic acid complementary to a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 9, 10, 25 and an antisense sequence of SEQ ID NO:25, in an amount effective to sensitize the cell to the chemotherapeutic agent. 
     
     
         10 . The method of  claim 9 , wherein the chemotherapeutic agent is selected from methotrexate, fluorouracil (5-FU), and ralitrexed. 
     
     
         11 . A method of increasing the sensitivity of a cell to radiation, comprising contacting the cell with a nucleic acid complementary to at least a portion of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 9, 10, 25 and an antisense sequence of SEQ ID NO:25, in an amount effective to sensitize the cell to radiation. 
     
     
         12 . The method of any one of  claims 8  to  11 , wherein the nucleic acid is an antisense nucleic acid. 
     
     
         13 . The method of any one of  claims 8  to  11 , wherein the nucleic acid is an siRNA or an shRNA. 
     
     
         14 . The method of any one of  claims 8  to  11 , wherein the cell is a cancer stem cell. 
     
     
         15 . The method of any one of  claims 8  to  11 , wherein the cell is a neoplastic cell. 
     
     
         16 . A method of treating a neoplasm in a subject, comprising administering to the subject an effective amount of an inhibitory molecule that inhibits expression of miR-192 or miR-215, and a second therapy, wherein inhibition of expression of miR-192 or miR-215 sensitizes the neoplasm to the second therapy. 
     
     
         17 . The method of  claim 16 , wherein the second therapy comprises administering a chemotherapeutic agent. 
     
     
         18 . The method of  claim 17 , wherein the chemotherapeutic agent is selected from methotrexate, fluorouracil (5-FU), and ralitrexed. 
     
     
         19 . The method of  claim 16 , wherein the second therapy comprises administering radiation to the subject. 
     
     
         20 . The method of any one of  claims 16  to  19 , wherein the neoplasm is cancer. 
     
     
         21 . The method of  claim 20 , wherein the cancer is selected from the group consisting of colon cancer, pancreatic cancer, lung cancer, breast cancer, cervical cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer. 
     
     
         22 . A method of diagnosing a neoplasm in a subject comprising determining the level of expression of miR-192 or miR-215. 
     
     
         23 . A method of identifying a neoplasm resistant to chemotherapy comprising determining the level of expression in the neoplasm of a microRNA (miRNA) selected from the group consisting of miR-192 and miR-215, and identifying the neoplasm as resistant to therapy if the level of the miRNA is elevated. 
     
     
         24 . A method of determining whether a neoplasm is a candidate for treatment with a chemotherapeutic agent comprising evaluating the level of expression of a microRNA (miRNA) selected from the group consisting of miR-192 and miR-215, and rejecting the candidate if expression of the miRNA is elevated. 
     
     
         25 . A kit for analysis of a pathological sample, the kit comprising in a suitable container a hybridization reagent for, determining the level of a miRNA selected from the group consisting of miR-192 or miR-215. 
     
     
         26 . The kit of  claim 25 , wherein the hybridization reagent comprises a hybridization probe. 
     
     
         27 . The kit of  claim 25 , wherein the hybridization reagent comprises amplification primers. 
     
     
         28 . A method of identifying an agent that inhibits expression of a microRNA (miRNA) selected-from the group consisting of miR-192 and miR-215 comprising
 contacting a cell that expresses the miRNA with an agent; and   comparing an expression level of the miRNA in the cell contacted by the agent with an expression level of the miRNA in the absence of the agent, wherein the agent is an inhibitor of the miRNA if expression of the miRNA is reduced.   
     
     
         29 . The method of  claim 28 , wherein the test cell overexpresses the miRNA. 
     
     
         30 . An isolated inhibitory molecule comprising a nucleic acid, wherein the nucleic acid consists essentially of a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:25, a complementary sequence of SEQ ID NO:9, a complementary sequence of SEQ ID NO:25, fragments thereof and variants thereof. 
     
     
         31 . The isolated inhibitory molecule, whenever the sequence is selected from the group consisting of the complementary sequence of SEQ ID NO:9, the complementary sequence of SEQ ID NO:25, fragments thereof and variants thereof. 
     
     
         32 . The isolated inhibitory molecule, wherein the sequence is SEQ ID NO:25 or a complementary sequence of SEQ ID:25.