EP2358372A1 - Microrna-mediated regulation of ubc9 expression in cancer cells - Google Patents

Abstract

Novel small expressed microRNA molecules associated with physiological regulatory mechanisms and particularly in developmental control are provided herein. More particularly, the present invention relates to microRNA molecules that suppress cancerous cell growth and other cancer-related disorders by suppressing tumor promoting factors such as, for example, Ubc9. Further, it is contemplated that the use of the microRNA molecules hereof will improve the diagnoses, prevention and/or treatment and also the identification and development of pharmaceuticals that are effective in connection with cancer cell growth.

Description

MICRORNA-MEDIATED REGULATION OF UBC9 EXPRESSION IN CANCER CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Number 61/198,672, was filed on 7 November 2008, which document is hereby incorporated by reference to the extent permitted by law.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in paper and computer readable form which are hereby incorporated by reference in their entirety. The nucleic and amino acid sequences listed in the Sequence Listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant # CA 102630 awarded by the National Institutes of Health. The government has certain rights in this invention.

BACKGROUND ART Post-translational modifications play an important role in protein function through the regulation of protein activity, turnover and localization and/or interactions. One such modification involves the covalent attachment of the small ubiquitin-related polypeptide SUMO (small ubiquitin-like modifier) to different cellular protein substrates. Although SUMO conjugation or sumoylation is similar to ubiquitination in structure, conjugation process and attachment to target proteins, the biological consequences of these two pathways can be quite distinct. Unlike ubiquitination that normally targets proteins for degradation through proteasome pathways, sumoylation has been implicated in regulation of protein stability, protein-protein interactions, transcriptional activity and subcellular localization.

Ubc9 is an E2 conjugating enzyme essential for sumoylation and it transfers the activated SUMO to protein substrates. In particular, Ubc9 has been shown to play a key role in nuclear trafficking, transcriptional regulation and protein stability through regulation of sumoylation machinery. In addition, recent evidence indicates that Ubc9 is a multi-functional protein that can exert its functions independent of sumoylation. Many important proteins, including tumor suppressors and oncoproteins as well as the cell cycle and proliferation-related proteins, are targets for sumoylation or interact with Ubc9, i.e., their expression or their activity is regulated by Ubc9. Thus, alterations of Ubc9 could ultimately have an impact on cell growth and cancer development and play a role in tumorigenesis and drug responsiveness.

Ubc9 is a single copy gene and is ubiquitously expressed in all human organs and tissues. However, levels of Ubc9 vary in different organs or tissues. In tumors Ubc9 is frequently upregulated. For example, Ubc9 is upregulated in lung adenocarcinoma as detected by microarray analysis. By semi-quantitative RT-PCR analysis, overexpression of Ubc9 in ovarian carcinoma can be detected compared to the matched normal ovarian epithelium. Moreover, Ubc9 is the most highly expressed protein in protein extracts from melanoma infiltrated lymph nodes identified by antibody array technology. However, little is known about the molecular mechanism of Ubc9 upregulation in cancer.

The following references are hereby incorporated by reference in their entirety to the extent permitted by law. These references are used to illustrate certain aspects and backgrounds of the invention. However, the right to challenge the veracity of any statements made in these references is expressly reserved.

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15524-15529. DISCLOSURE OF THE INVENTION

The present invention is generally directed to novel small expressed microRNA molecules associated with physiological regulatory mechanisms and particularly in developmental control are provided herein. More particularly, the present invention relates to microRNA molecules that suppress cancerous cell growth and other cancer-related disorders by suppressing tumor promoting factors such as, for example, Ubc9. Further, it is contemplated that the use of the microRNA molecules hereof will improve the diagnoses, prevention and/or treatment and also the identification and development of pharmaceuticals that are effective in connection with cancer cell growth. Other aspects and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments and the accompanying drawing figures.

DETAILED DESCRIPTION OF THE DRAWING The accompanying drawing forms a part of the specification and is to be read in conjunction therewith, in which like reference numerals are employed to indicate like or similar parts in the various views, and wherein:

Fig. IA is a representative microphotograph of 3 cases for each type of paraffin- embedded specimens that were stained by IHC using anti-Ubc9 antibody as described in Materials and Methods wherein strong Ubc9 signals were shown in tumors as compared to the matched normal tissues;

Fig. IB is a Western blot of the expression of Ubc9 in freshly frozen samples of matched breast tumor tissue; Fig. 1C is a graphical representation showing the relative expression levels of Ubc9 between tumors and matched normal breast tissues (n = 8) wherein the Ubc9 level was first normalized with β-actin and then compared to each other and wherein the relative value of normal tissues was set at 1. N, normal; T, tumor; Fig. 2A is a bar chart representation demonstrating ectopic expression of microRNAs in

293T cells wherein cells were first transfected with microRNA expression vectors as detailed in Materials and Methods and then harvested for extraction of total RNA 2 days later; mature microRNA levels were determined using QuantiMir real-time PCR method, and miR-30e levels were determined by the TaqMan real-time PCR method and it was found that the miR-30e levels were similar, as detected by both methods (not shown);

Fig. 2B is a representative Western blot showing specific suppression of Ubc9 by miR- 30e and miR-30c;

Fig. 2C is a bar chart representation showing the effect of miR-30e and miR-30c on Ubc9 mRNA wherein the values in (A) and (C) are average of three separate experiments ± SE; Fig. 3 is a microphotograph of HeLa cells that were transfected with miR-30e or vector

(pCDH) wherein, one day later, the transfected cells were harvested and seeded on glass cover slips in a 12-well plate and grown for an additional day before immunostaining with anti-Ubc9 antibody (red) and wherein a reduction of Ubc9 expression (red signal) was observed in miR- 30e-transfected cells (green cells in upper panels); Fig. 4A is a representation of the detection of total protein sumoylation wherein 293T cells were first transfected with miR-30e or vector control, the same number of cells were then directly lyzed in 4% hot SDS followed by sonication, and signals were detected by SUMO-I antibody; Fig. 4B is a bar chart representation showing cells that were first transfected with miR- 3Oe, or vector; miR-30e plus pCMV-Ubc9 or miR-30e plus pCMV, wherein there was virtually no difference in cell growth between miR-30e and miR-30e plus pCMV (not shown) and wherein 1 and 2, 3 = 0.00002; 3, p = 0.0003; 4, p = 0.0002; Fig. 4C is a graphical representation showing that miR-30e sensitizes cells to topotecan

(TPT) wherein cells were first transfected with miR-30e or vector control and then treated with TPT at the indicated concentrations for 4 days, wherein cell growth was determined by MTT assays for both B and C, as described in Materials and Methods, and wherein values are the average of three separate experiments ± SE; Fig. 5 is a representation of 293T cells that were transfected with pLuc-Ubc9-3'-UTR or its deletion constructs and wherein, at the same time, the cells were co-transfected with pCDH (V) or miR-30e/pCDH (30e) or miR-30c/pCDH (30c) and then harvested for luciferase assays 24 h later, as detailed in Materials and Methods;

Fig. 5 A is a schematic representation of pUbc9 3'-UTR with putative binding sites for miR-30e (30e), and miR-30c (30c);

Fig. 5B is a bar chart representation of the suppression of pLuc-Ubc9-3'-UTR luciferase activity by miR-30e and miR-30c; and

Fig. 5C is a graphical representation of a deletion analysis of the pLuc-Ubc9-3'-UTR wherein luciferase activity for each deletion construct was compared between pCDH (100%) and miR-30e and values are the averages of the three separate experiments +SE..

MODE(S) FOR CARRYING OUT THE INVENTION The following detailed description of the invention references the accompanying drawings that illustrate specific embodiments in which the invention can be practiced. The embodiments are intended to describe aspects of the invention in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments can be utilized and changes can be made without departing from the scope of the present invention. The present invention is defined by the appended claims and the description is, therefore, not to be taken in a limiting sense and shall not limit the scope of equivalents to which such claims are entitled.

MicroRNAs are endogenous small non-coding RNAs that are known to post- transcriptionally regulate gene expression. Aberrant expression of microRNAs has been reported in many types of tumors because they may function as oncogenes or rumor suppressor genes. While oncogenic microRNAs are often upregulated, tumor suppressive microRNAs are often downregulated in cancer. Since ectopic expression of the miR-family causes cell growth inhibition, the present invention is premised upon the theory that the miR-family are tumor suppressor genes that function by suppression of tumor promoting factors such as Ubc9 and that the miR-family is deregulated in tumor specimens.

Certain embodiments of the present invention provide a method of treating tumor growth through suppression of Ubc9 enzyme function which plays a causal role in tumorigenesis because, while suppression of Ubc9 function by the dominant negative Ubc9 inhibits tumor growth, ectopic expression of Ubc9 enhances tumor growth in animal models due to the fact that Ubc9 is an essential enzyme for sumoylation and numerous important proteins, such as tumor suppressors or oncoproteins, are substrates for sumoylation. Thus, deregulation of Ubc9 can lead to alterations of sumoylation pathways thereby impacting cell growth and cancer development. In this regard, Ubc9-mediated sumoylation is similar to ubiquitination. It is well known that deregulation of ubiquitination pathways could play a key role in cancer development because the timely and irreversible degradation of critical regulators is essential for normal cellular function and turnover of several regulatory proteins resulting from targeted destruction via ubiquitination. Similarly, Ubc9-mediated sumoylation has been shown to play a role in diverse cellular pathways. It will be appreciated that cancer cells may therefore have evolved mechanisms to target the basic functions of these protein modification pathways and the present invention provides for one such mechanism that can involve microRNA regulation of Ubc9 at the post- transcriptional level thereby leading to its upregulation in tumors.

Four lines of evidence support the present invention's use of Ubc9 as a direct target for the miR-family. First, the miR-family specifically suppresses Ubc9 expression as demonstrated by both Western blot and immunofluorescence staining. Second, ectopic expression of the miR- family inhibits overall protein sumoylation. Third, the miR-family also causes cell growth inhibition which can be attenuated by overexpression of Ubc9. Fourth, analyses of the luciferase reporter carrying the Ubc9 3'-UTR indicate that the miR-family directly interacts with this sequence and the putative miR-family binding sites are essential for miR-family regulation.

Therefore, the present invention is generally directed to novel small expressed microRNA molecules associated with physiological regulatory mechanisms and particularly in developmental control. More particularly, the invention relates to microRNA molecules and analogs thereof, to microRNA precursor molecules and to DNA molecules encoding microRNA or microRNA precursor molecules. The microRNA molecules hereof suppress cancerous cell growth and other cancer-related disorders by suppressing tumor promoting factors such as, for example, Ubc9. Further, it is contemplated that the use of the microRNA molecules hereof will improve the diagnoses, prevention and/or treatment and also the identification and development of pharmaceuticals that are effective in connection with cancer cell growth. In certain embodiments, the present invention is directed to an isolated nucleic acid molecule selected from the group consisting of miR-30a (SEQ ED NO: 1), miR-30b (SEQ ID NO: 2), miR-30c (SEQ ID NO: 3), miR-30d (SEQ ID NO: 4), miR-30e (SEQ ID NO: 5), miR-188 (SEQ ID NO: 6), miR- 200c (SEQ ED NO: 7), miR-195 (SEQ ID NO: 8), miR-548a (SEQ ID NO:9), miR-450b (SEQ ID NO: 10), miR-361 (SEQ BD NO: 11), miR-lOb (SEQ ID NO: 12), miR-376c (SEQ ID NO: 13), miR-200b (SEQ ED NO: 14), miR-877 (SEQ ID NO: 15), miR-802 (SEQ ID NO: 16), and miR-652 (SEQ ID NO: 17) (hereinafter, collectively the "miR-family"), and/or functional variants thereof, and/or nucleic acids or variants thereof encoding the same, and/or of a cell expressing said polypeptide or a functional variant thereof or said nucleic acids or variants thereof and/or of an antibody or an antibody fragment directed against a gene usable according to the invention or a functional variant thereof and, if appropriate, combined or together with suitable additive and/or auxiliaries, for the diagnosis, prevention and/or treatment of cancerous cell growth and related disorders, or for the identification of pharmacologically active substances for the treatment thereof. hi certain other embodiments, the present invention relates to an isolated nucleic acid molecule including: (a) a nucleotide sequence as shown in Table 1 ; (b) a nucleotide sequence which is the complement of (a); (c) a nucleotide sequence which has an identity of at least 80%, preferably of at least 90% and more preferably of at least 99%, to a sequence of (a) or (b); and/or (d) a nucleotide sequence which hybridizes under stringent conditions to a sequence of (a), (b) and/or (c). Preferably the identity of sequence (c) to a sequence of (a) or (b) is at least 90%, more preferably at least 95%. The determination of identity (percent) may be carried out as follows: I=n:L wherein I is the identity in percent, n is the number of identical nucleotides between a given sequence and a comparative sequence as shown in Table 1 and L is the length of the comparative sequence. It should be noted that the nucleotides A, C, G and U as depicted in Table 1 may denote ribonucleotides, deoxyribonucleotides and/or other nucleotide analogs, e.g. synthetic non-naturally occurring nucleotide analogs. Further nucleobases may be substituted by corresponding nucleobases capable of forming analogous H-bonds to a complementary nucleic acid sequence, e.g. U may be substituted by T.

Further, the invention hereof encompasses nucleotide sequences which hybridize under stringent conditions with the nucleotide sequence as shown in Table 1, a complementary sequence thereof or a highly identical sequence. Stringent hybridization conditions comprise washing for 1 h in 1.times.SSC and 0.1 % SDS at 45⁰C, preferably at 48°C, and more preferably at 50⁰C, particularly for 1 h in 0.2.times.SSC and 0.1% SDS.

The isolated nucleic acid molecules of the present invention preferably have a length of from 18 to 100 nucleotides, and more preferably from 18 to 80 nucleotides. It should be noted that mature microRNAs usually have a length of 19 24 nucleotides, particularly 21, 22 or 23 nucleotides. The microRNAs, however, may be also provided as a precursor which usually has a length of 50-90 nucleotides and, more particularly, 60-80 nucleotides. It should be noted that the precursor may be produced by processing of a primary transcript which may have a length of >100 nucleotides.

The nucleic acid molecules may be present in single-stranded or double-stranded form. The microRNA as such is usually a single-stranded molecule, while the mi-precursor is usually an at least partially self -complementary molecule capable of forming double-stranded portions, e.g. stem- and loop-structures. DNA molecules encoding the microRNA and microRNA precursor molecules. The nucleic acids may be selected from RNA, DNA or nucleic acid analog molecules, such as sugar- or backbone-modified ribonucleotides or deoxyribonucleotides. It should be noted, however, that other nucleic analogs, such as peptide nucleic acids (PNA) or locked nucleic acids (LNA), are also suitable.

In an embodiment of the invention the nucleic acid molecule is an RNA or DNA molecule, which contains at least one modified nucleotide analog, i.e. a naturally occurring ribonucleotide or deoxyribonucleotide is substituted by a non-naturally occurring nucleotide. The modified nucleotide analog may be located for example at the 5 '-end and/or the 3 '-end of the nucleic acid molecule.

Preferred nucleotide analogs are selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6- methyl adenosine are suitable.

The nucleic acid molecules of the invention may be obtained by chemical synthesis methods or by recombinant methods, e.g. by enzymatic transcription from synthetic DNA- templates or from DNA-plasmids isolated from recombinant organisms. Typically phage RNA- polymerases are used for transcription, such as 17, T3 or SP6 RNA-polymerases. The invention also relates to a recombinant expression vector comprising a recombinant nucleic acid operatively linked to an expression control sequence, wherein expression, i.e. transcription and optionally further processing results in a miRNA-molecule or miRNA precursor molecule as described above. The vector is preferably a DNA-vector, e.g. a viral vector or a plasmid, particularly an expression vector suitable for nucleic acid expression in eukaryotic, more particularly mammalian cells. The recombinant nucleic acid contained in said vector may be a sequence which results in the transcription of the miRNA-molecule as such, a precursor or a primary transcript thereof, which may be further processed to give the miRNA-molecule. Further, the invention relates to diagnostic or therapeutic applications of the claimed nucleic acid molecules. For example, microRNAs may be detected in biological samples, e.g. in tissue sections, in order to determine and classify certain cell types or tissue types or microRNA- associated pathogenic disorders which are characterized by differential expression of microRNA- molecules or microRNA-molecule patterns. Further, the developmental stage of cells may be classified by determining temporarily expressed microRNA-molecules.

Further, the claimed nucleic acid molecules are suitable for therapeutic applications. For example, the nucleic acid molecules may be used as modulators or targets of developmental processes or disorders associated with developmental dysfunctions, such as cancer. Furthermore, existing miRNA molecules may be used as starting materials for the manufacture of sequence-modified miRNA molecules, in order to modify the target-specificity thereof, e.g. an oncogene, a multidrug-resistance gene or another therapeutic target gene. The novel engineered miRNA molecules preferably have an identity of at least 80% to the starting miRNA, e.g. as depicted in Tables 1. Further, miRNA molecules can be modified, in order that they are symmetrically processed and then generated as double-stranded siRNAs which are again directed against therapeutically relevant targets. Furthermore, miRNA molecules may be used for tissue reprogramming procedures, e.g. a differentiated cell line might be transformed by expression of miRNA molecules into a different cell type or a stem cell. For diagnostic or therapeutic applications, the claimed RNA molecules are preferably provided as a pharmaceutical composition. This pharmaceutical composition comprises as an active agent at least one nucleic acid molecule as described above and optionally a pharmaceutically acceptable carrier. The administration of the pharmaceutical composition may be carried out by known methods, wherein a nucleic acid is introduced into a desired target cell in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE- dextran, electroporation and microinjection and viral methods. A recent addition to this arsenal of techniques for the introduction of DNA into cells is the use of cationic liposomes. The composition may be in form of a solution, e.g. an injectable solution, a cream, ointment, tablet, suspension or the like. The composition may be administered in any suitable way, e.g. by injection, by oral, topical, nasal, rectal application etc. The carrier may be any suitable pharmaceutical carrier. Preferably, a carrier is used, which is capable of increasing the efficacy of the RNA molecules to enter the target-cells. Suitable examples of such carriers are liposomes, particularly cationic liposomes. In a further embodiment of the use according to the invention, the nucleic acids are comprised in a vector, preferably in a "shuttle" vector, phagemid, cosmid, expression vector or vector applicable in gene therapy. Furthermore, the above mentioned nucleic acids can be included in "knock-out" gene constructs or expression cassettes.

The expression vectors can be prokaryotic or eukaryotic expression vectors. In general, the expression vectors also contain promoters suitable for the respective host cell. For expression in mammalian cells, for example, suitable promoters are those which allow a constitutive, regulatable, tissue-specific, cell-cycle-specific or metabolically specific expression in eukaryotic cells. Regulatable elements according to the present invention are promoters, activator sequences, enhancers, silencers and/or repressor sequences.

In order to make possible the introduction of nucleic acids as described above and thus the expression of the polypeptide in a eukaryotic or prokaryotic cell by transfection, transformation or infection, the nucleic acid can be present as a plasmid, as part of a viral or non- viral vector. Suitable viral vectors here are particularly: baculoviruses, vaccinia viruses, adenoviruses, adeno-associated viruses and herpes viruses. Suitable non- viral vectors here are particularly: virosomes, liposomes, cationic lipids, or poly-lysine-conjugated DNA. Examples of vectors having gene therapy activity are virus vectors, for example adenovirus vectors or retroviral vectors.

A further form of a vector applicable in gene therapy can be prepared by the introduction of "naked" expression vectors into a biocompatible matrix, for example a collagen matrix. This matrix can be introduced into wounds in order to transfect the immigrating cells with the expression vector and to express the polypeptides according to the invention in the cells. A further embodiment of the invention relates to the use of an antibody or an antibody fragment directed against a polypeptide useable according to the invention or a functional variant thereof, preferably of a polyclonal or monoclonal antibody or antibody fragment, for the analysis, diagnosis, prevention and/or treatment of cancerous cell growth and related disorders, and its use for the identification of pharmacologically active substances, if appropriate combined or together with suitable additives and/or auxiliaries.

EXAMPLES The following examples are for purposes of illustration only and are not intended to limit the scope of the claims that are appended hereto.

EXAMPLE 1 Materials and Methods Reagents. Primary monoclonal Ubc9 antibody from BD Biosciences (San Jose, CA) or custom made polyclonal Ubc9 antibody was used in both Western blot and immunofluorescence microscopy. Anti-SUMO-1 antibody for Western blot and secondary antibodies conjugated with Alex 566 used for immunofluorescence staining were obtained from Invitrogen (Carlsbad, CA). Secondary antibodies conjugated with IRDye 800CW were purchased from LI-COR Biosciences (Lincoln, NE). PCR primers were purchased from Sigma-Genosys (Woodland, TX).

Cell culture. All cell lines were purchased from ATCC (Manassas, VA). Both HeLa and

293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Cambrex) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO). All media contained 2 mM glutamine, 100 units of penicillin/ml, and 100 μg of streptomycin/ml. Cells were incubated at 37 ⁰C and supplemented with 5% CO₂ in the humidified chamber.

Transfection. HeLa cells were transfected using DNAfectin reagent (Applied Biological Materials, British Columbia, Canada) following the manufacturer's protocol. In brief, cells were seeded at 40% confluence in a 12 or 6- well plate and then transfected with 1 or 3 μg of microRNA expression vectors in serum free medium the following day when the cells reached about 70% confluence. The serum free media was replaced by normal growth media after 15 h of transfection. 293T cells were transfected using the calcium phosphate method. The transfected cells were grown overnight before they were harvested and lyzed for luciferase assay or extraction of protein or RNA. Plasmids. To construct pre-microRNA expression vectors, we first amplified -0.5 kb DNA fragment covering a pre-microRNA, using genomic DNA from a healthy blood donor as a template. PCR reactions were performed using the high fidelity Phusion enzyme (New England Biolabs Ipswich, MA) and corresponding specific primers: (SEQ ID NO: 18) miR-30e-5.1 (sense):

5'-AAAGCTGTGCCTTGTTCTGC

(SEQ ID NO: 19) miR-30e-Not 1-3.1 (antisense):

5'-GCGGCCGCAGCCCACAGAAAACAAGGAG

(SEQ ID NO: 20) miR-30c-5.1 (sense): 5'-TTGGGGAGTTGGAGGCAATC

(SEQ ID NO: 21) miR-30c-Not 1-3.1 (antisense):

5'-GCGGCCGCAGGTTAATGGGAAACAGGGC

(SEQ ID NO: 22) miR-188-5.1 (sense):

5 ' -CTTCCCTCTCCAGTGCATAG (SEQ ID NO: 23) miR-188-Notl-3.1 (antisense):

5'-GCGGCCGCTCCTGCAGGATCCATGTAAG (SEQ ID NO: 24) miR-200c-5.1 (sense):

5'- TAAATCGGTGTGTGTCGCGG (SEQ ID NO: 25) miR-200c-Not 1-3.1 (antisense): 5 ' -GCGGCCGCAAGGTCGACTGTGGGTTCTG

The amplified fragment was first cloned into a PCR cloning vector and subsequently cloned a pCMV vector or lentiviral vector (pCDH-CMV-MCS-EFl-copGFP from System Biosciences, Mountain View, CA) at EcoRl and Notl sites. Expression of the mature microRNAs was verified by TaqMan real-time PCR kit (Applied Biosystems) or QuantiMir kit (System Biosciences).

The luciferase-UTR reporter plasmid (pLuc-Ubc9-3'-UTR) was constructed by introducing the Ubc9 3'-UTR carrying putative microRNA binding sites into pGL3 control vector (Promega, Madison, WI). Thus, we amplified the Ubc9 3'-UTR sequence from MCFlOA cDNA by PCR using the following primers:

(SEQ ID NO: 26) Ubc9-UTR-5.1 (sense):

5'-GCAGCGACCTTGTGGCATCGT (SEQ ID NO: 27) Ubc9-UTR-Not1-3.1 (antisense): 5'-GCGGCCGCGCAGCGACCTTGTGGCATCGT

For construction of deletion mutant pLuc-Ubc9-3'-UTR clones, we used primers Ubc9- UTR-5.2 (see below) and Ubc9-UTR-Notl-3.1, resulting in pLuc-Ubc9-3'-UTR-dl where the first putative miR-30e binding site was deleted. We then used primers Ubc9-UTR-5.1 and Ubc9- UTR-Notl-3.6 (see below) to generate pLuc-Ubc9-3'-UTR-d2 where the second putative miR- 30e binding site was eliminated. Finally, to delete both sites, we used primers Ubc9-UTR-5.2 and Ubc9-UTR-Notl-3.6 to generate pLuc-Ubc9-3'-UTR-dl-d2.

(SEQ ID NO: 28) Ubc9-UTR-5.2 (sense):

5 ' -ACATTTTTGCAAATCTAAAGT (SEQ TD NO: 29) Ubc9-UTR-Notl-3.6 (antisense): 5'-GCGGCCGCAGACAAAACGCCATATAAACAC

The PCR product was also first cloned into a PCR cloning vector and then subcloned into a modified pGL3 control vector where an EcoRl and Notl sites were introduced into the Xbal site so that an insert can be unidirectionally cloned downstream of the luciferase gene. All the amplified products were verified by DNA sequencing before cloning into the final destination vector.

Luciferase Assay. Luciferase assays were carried out in 293T cells to determine the effect of microRNAs on the activity of Luc-Ubc9-3'-UTR and the deletion mutant constructs. First, cells were transfected with appropriate plasmids in 12- well plates. Then, the cells were harvested and lysed for luciferase assay 24 h after transfection. Luciferase activity was determined by using a luciferase assay kit (Promega) according to the manufacturer's protocol, β-galactosidase was used for normalization.

PCR/RT-PCR and real-time RT-PCR. PCR was performed to amplify pre-microRNA sequences or the Ubc9 3'-UTR sequence according to the standard three-step procedure.

Annealing temperature varied depending on the primers used. For RT-PCR, we isolated total

RNA using Trizol reagent (Invitrogen) per the manufacturer protocol and used lμg RNA to synthesize cDNA by SuperScriptase III (Invitrogen) with random primers. Finally, the resultant cDNA was used in regular PCR or real-time PCR reactions. To detect Ubc9 mRNA levels, we used the SYBR Green method with primers Ubc9-5.10 and Ubc9-3.1O.

(SEQ ED NO: 30) Ubc9-5.10 (sense):

5 ' -CAGGAGAGGAAAGCATGGAG

(SEQ DD NO: 31) Ubc9-3.10 (antisense):

5 '-TCGGGTGAAATAATGGTGGT To detect mature microRNA expression, we also used Trizol reagent to isolate total

RNA, which was then amplified by QuantiMir method (System Biosciences) or TaqMan stem- loop RT-PCR method using specific primer sets and TaqMan probe from Applied Biosystems. Real-time PCR reactions were performed in ABI 7900 HT thermal cycler according to the manufacturer's protocol. Average levels of U6, 5s RNA and β-actin were used as an internal control. The fold-change between vector control and pre-microRNA expression vector was calculated with the 2^"ΔACt method.

Cell growth assay. Cell growth assays were carried out by MTT [3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. In brief, cells were seeded in 96-well plates and incubated for various days before adding MTT. Absorbance at 570 nm was measured in the multi well plate reader (Thermo Scientific, Waltham, MA). The relative values were calculated by expressing the first day data as 1.

Western Blot. Cells were harvested and protein was extracted 2 days after transfection. Protein concentration was determined by protein assay kit (Bio-Rad, Hercules, CA) and samples were separated in 12% SDS polyacrylamide gels. Signals were revealed by a secondary antibody labeled with IRDye 800CW and the signal intensity was determined by Odyssey Infrared Imaging System (LI-COR Biosciences).

Immunofluorescence microscopy. To detect miR-30e-mediated suppression of Ubc9 by immunofluorescence staining, HeLa cells were first transfected with vector control or miR- 3Oe expression vector and then transferred to coated coverslips in a 12-well plate. After overnight growth, the cells were fixed with 3% paraformaldehyde (Sigma-Aldrich) and permeabilized by 80% cold methanol, followed by washing with PBS (phosphate buffered saline). Coverslips were then incubated with 3%BSA in PBS for 10 min at room temperature. Primary antibodies against Ubc9 in PBST (PBS plus 0.1% Tween 20) were then added and incubated for 1 h at room temperature. After 3 washes with PBS, the cells were incubated with a fluorescence-conjugated secondary antibody in the dark for 1 h. For nuclear staining, the cells were subsequently stained in 0.5 μg/ml Hoechst dye (Sigma-Aldrich) for 5 min before examinations under a fluorescence microscope.

Immunohistochemistry (IHC). Paraffin-embedded tissue was pretreated at 65⁰C for 2 h, followed by deparaffinization using standard procedures. Antigen retrieval was carried out in antigen retrieval solution (10 mM Tris, 1 mM EDTA, pH9.0) before applying the primary Ubc9 antibody. Thereafter, slides were incubated for 2 h at room temperature followed by extensive washes with PBST and further incubated for 1 h at room temperature with the secondary antibody conjugated with horse radish peroxidase (HRP). HRP activity was detected using

Histostain Plus kit (mvitrogen) according to the manufacturer's instruction. Finally, sections were counterstained with hematoxylin and mounted.

Patient specimens. Matched breast, head and neck, and lung tumor specimens were obtained from Cooperative Human Tissue Network (CHTN) Midwestern Division (Columbus, OH) or SIU SimmonsCooper Cancer Institute Tissue Bank. The use of these specimens in this study was approved by the Institutional Review Board of Southern Illinois University School of Medicine. Where it is necessary, total protein was isolated in protein extraction buffer using a tissue homogenizer and protein concentration was determined by protein assays kit (Bio-Rad).

Statistical analysis. Statistical analysis of data was performed using the Student's t test. Differences with p values less than 0.05 are considered significant.

EXAMPLE 2 Upregulation of Ubc9 in tumor specimens

Overexpression of Ubc9 enhances tumor growth in the xenograft mouse model. To determine the clinical relevance of this finding, expression levels of Ubc9 were examined in the matched patient specimens including breast, head and neck, and lung by IHC. From 4 cases for each of three types of cancer, it was found that the Ubc9 level was higher in tumor than the matched normal tissues. Fig. IA shows representative fields for each of three cases wherein the tumor specimens revealed intensive Ubc9 staining that was concentrated in the nucleus. However, the matched normal tissues displayed very weak staining thereby suggesting that Ubc9 is overexpressed in tumors.

To better quantitate the Ubc9 expression in tumor specimens, 8 pairs of frozen samples from the matched breast tumors were examined by Western blot analysis. Ubc9 was upregulated in all 8 cases as shown in Fig. IB. On average, breast tumors expressed a 5.7-fold higher than the matched normal tissues, as shown in Fig. 1C, which is consistent with the EHC data from paraffin-embedded samples of Fig. IA.

EXAMPLE 3

Suppression of Ubc9 by miR-30

To better understand the upregulation of Ubc9 in tumors, transcriptional regulation was examined. Therefore, the putative Ubc9 promoter was cloned into a luciferase reporter plasmid and then introduced into several cell lines which expressed different levels of Ubc9. However, no significant difference in luciferase activity was seen thereby suggesting that transcriptional regulation may not be important for the observed difference of Ubc9 expression. Furthermore, epigenetic factors, such as methylation and acetylation, did not appear to play a significant role in Ubc9 expression because the de-methylation agents such as 5-Aza-deoxycytidine or histone deacetylase inhibitors such as trichostatin A (TSA) had only a marginal effect on Ubc9 expression (not shown).

Therefore, the post-transcriptional regulation of Ubc9 was investigated. Small non- coding RNAs, known as microRNAs, have been shown to silence protein-coding genes in a variety of organisms including mammals by translation repression or mRNA degradation. MicroRNAs are believed to target mRNAs by partial sequence homology to the 3 '-untranslated region (3'-UTR) of the target gene. Thus, potential microRNAs that might play a role in regulation of Ubc9 were searched for using several commonly cited microRNA target prediction programs such as TargetScan4®, miRBase Target5® (http://microrna.sanger.ac.uk/targets/v5/), PicTar®, and miRanda® (http://microrna.org). These four prediction programs all identified 7 putative microRNAs (miR-30a-e, miR-188 and miR-200c) as shown in Table 1. In addition, some other microRNAs were identified by either two or three of these programs. In general, miRBase® target5 and miRanda® tended to predict more targets than TargetScan4® or PicTar® did presumably because the first two programs did not distinguish between conservations among different species. For example, miRBase target5 and miRanda predicted as high as 37 and 42 microRNAs for Ubc9, respectively, whereas TargetScan4 and PicTar predicted fewer than 10 microRNAs.

Table 1 Putative microRNAs targeting Ubc9 Name BS^# Predicted by* miR-30e 2 T, M, P, R mϊR-30c 2 T, M, P, R miR-30a 1 T, M, P, R miR-30b 1 T, M, P, R miR-30d 1 T, M, P, R miR-188 1 T, M, P, R miR-200c 1 T, M, P, R miR-195 1 M₃ P₃ R miR-548a 1 M₃ R miR-450b 1 M₃ R miR-361 1 M₃ R miR-10b 1 M, R miR-376c 1 M₃ R miR-200b 1 M₃ R miR-877 1 M₃ R miR-802 1 M₃ R miR-652 1 M₃ R # BS₃ binding site * T, Targetscan4@; M, miRbaseTarget5®; P, PicTar®; M, miRanda®

The miR-30 family and two other microRNAs, miR-188 and miR-200c, were chosen for further investigation. Because both miR-30e and miR-30c target Ubc9 at two potential binding sites (Table 1) and the rest of the miR-family appeared to have only one site, miR-30e and miR- 30c were chosen to represent the miR-30 family. As shown in Fig. 2A, ectopic expression generated mature microRNAs which was confirmed by real-time RT-PCR and then the effect of each microRNA on Ubc9 expression was determined. Western blot analysis revealed that both miR-30e and miR-30c suppressed Ubc9 expression at the protein level as shown in Fig. 2B. In contrast, no significant effect on Ubc9 expression for miR-188 and miR-200c was detected thereby highlighting the specificity of this suppression even though both miR-188 and miR-200c are also predicted to target Ubc9. As shown in Fig. 2C, to determine whether miR-30e and miR- 30c affect the Ubc9 mRNA level, real-time RT-PCR analysis was performed for the same cells transfected with miR-30e and im^"R-30c, and these two microRNAs had no effect on the Ubc9 mRNA level thereby suggesting that they regulate Ubc9 expression mainly through translation repression.

To further confirm the suppressive effect of miR-30e on Ubc9 expression, the miR-30e expression vector was introduced into HeLa cells and then immunostained with Ubc9 specific antibody. As shown in Fig. 3, ectopic expression of miR-30e remarkably suppressed Ubc9 expression because the red signal was clearly reduced (upper panels). In contrast, the vector control (pCDH) had no effect on Ubc9 (Fig. 3, bottom panels) thereby further supporting the conclusion that Ubc9 is a target for miR-30e.

EXAMPLE 4 Effect of miR-30e on cell growth

Given that Ubc9 is an E2 enzyme for sumoylation, suppression of Ubc9 by miR-30e would inhibit sumoylation. To test this hypothesis, the effect of miR-30e on the overall levels of protein sumoylation was determined using SUMO-I antibody. As expected, miR-30e reduced total protein sumoylation as compared to vector control as shown in Fig. 4A. In particular, miR- 3Oe suppressed the level of sumoylated RanGAPl because RanGAPl is a major SUMO substrate. Further, the free SUMO-I level was higher in miR-30e-transfected cells than in vector control, presumably because reduction of overall sumoylation leads to the accumulation of the free SUMO-I. To further determine the effect of suppression of Ubc9 by miR-30e on cellular processes, the cell growth for miR-30e-transfected cells was examined. miR-30e was found to have caused growth inhibition in a time-dependent manner. For example, for the first 2 days, there was no significant difference between vector and miR-30e but, at days 3 and 4, miR-30e inhibited cell growth by almost 30% compared to the vector control as shown in Fig. 4B. Of interest, this growth inhibition was partially reversed by overexpression of Ubc9 thereby suggesting that Ubc9 is an important target for miR-30e. Li addition, miR-30e was able to sensitize cells to the anticancer agent topotecan (Fig. 4C), which is consistent with findings that suppression of Ubc9 by Gaml can increase the sensitivity to this agent. These results suggest that as a negative regulator of Ubc9, miR-30e plays a role in cell growth and drug response, in part through suppression of Ubc9 expression.

EXAMPLE 5 Ubc9 is a direct target for miR-30e Turning to Fig. 5A-B, to determine whether miR-30e directly targets Ubc9, the Ubc9-3'- UTR was cloned into pGL3 control vector and resulted in pLuc-Ubc9-3'-UTR. After transfection of 293T cells with this reporter construct along with miR-30e or miR-30c, both miR-30e and miR-30c suppressed the luciferase activity by about 50% compared to the vector control (Fig. 5B), suggesting that Ubc9 is a direct target for these two microRNAs. As shown Fig. 5A, there are two potential microRNA binding sites in the 3'-UTR of Ubc9. To determine whether any of these two binding sites is important for microRNA suppression, the first (pLuc-Ubc9-3'-UTR- dl) or second binding site (pLuc-Ubc9-3'-UTR-d2) or both (pLuc-Ubc9-3'-UTR-dl-d2) were deleted. As shown in Fig. 5C, deletion of the first binding site impaired the suppression of luciferase activity, but about 30% suppression was detected and deletion of the second binding site had a similar effect. However, when both sites were deleted, miR-30e-mediated suppression of luciferase activity was abolished. These results therefore suggest that both binding sites are critical for microRNA regulation.

EXAMPLE 6 The miR-30 family

As shown hereinabove, miR-30e and miR-30c are capable of targeting Ubc9 and this suppression is through the putative binding site in the 3'-UTR of Ubc9. Based on in silico analysis, the binding site in the Ubc9 3'-UTR for miR-30e and miR-30c is also shared by miR-30a, miR-30b and miR-30d, although the flanking regions may be very different. Since seed sequences are preferred for classifying microRNAs, it is expected that miR-30a, miR-30b and miR-30d are also able to silence Ubc9 as shown in the sequence alignment below: (SEQ ID NO: 32) Ubc9 3'UTR ' . . . GGUUUGGCAAGAACUUGUUUACA . . .

' ACAAAUGU Seed sequence miR-30a/30b/30c/30d/30e

Claims

What is claimed is:

I. A method for preventing or treating cancer cell growth comprising administering a therapeutically-effective amount of an isolated nucleic acid molecule selected from the group consisting of (a) the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1- 17; (b) a nucleotide sequence selected from the group consisting of the complement of (a); and (c) a nucleotide sequence which has a sequence identity of at least 90% to SEQ ID NOS: 1-17 or the complement of SEQ ID NOS: 1-17.

2. The method according to claim 1, wherein said molecule is a microRNA.

3. The method according to claim 1, wherein said molecule is single-stranded.

4. The method according to claim 1, wherein said molecule is at least partially double-stranded.

5. The method according to claim 1, wherein said molecule is selected from the group consisting of RNA, DNA and modified nucleotide molecules.

6. The method according to claim 5, wherein said molecule comprises at least one modified nucleotide.

7. The method according to claim 1, wherein said nucleotide sequence in part (c) has an identity of at least 95% to a sequence of (a) or (b).

9. The method according to claim 1, wherein said at least one nucleic acid molecule is in combination with a pharmaceutically acceptable carrier.

10. The method according to claim 9, wherein said pharmaceutically acceptable carrier is suitable for diagnostic applications.

I 1. The method according to claim 9, wherein said pharmaceutically acceptable carrier is suitable for therapeutic applications.

KCP-1729811-ό

12. A pharmaceutical composition for diagnosing, preventing or treating cancer cell growth comprising administering an isolated nucleic acid molecule selected from the group consisting of (a) the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1- 17; (b) a nucleotide sequence selected from the group consisting of the complement of (a); and (c) a nucleotide sequence which has a sequence identity of at least 90% to SEQ ID NOS: 1-17 or the complement of SEQ ID NOS: 1-17.

13. The composition according to claim 12, wherein said molecule is a microRNA.

14. The composition according to claim 12, wherein said molecule is single-stranded.

15. The composition according to claim 12, wherein said molecule is at least partially double-stranded.

16. The composition according to claim 12, wherein said molecule is selected from the group consisting of RNA, DNA and modified nucleotide molecules.

17. The composition according to claim 16, wherein said molecule comprises at least one modified nucleotide.

18. The composition according to claim 12, wherein said nucleotide sequence in part

(c) has an identity of at least 95% to a sequence of (a) or (b).

19. The composition according to claim 12, wherein said at least one nucleic acid molecule is in combination with a pharmaceutically acceptable carrier.

20. The composition according to claim 19, wherein said pharmaceutically acceptable carrier is suitable for diagnostic applications.

21. The composition according to claim 19, wherein said pharmaceutically acceptable carrier is suitable for therapeutic applications.

22. A diagnostic kit for determining or classifying microRNA-associated pathogenic disorders characterized by differential expression of microRNA-molecules or microRNA- molecule patterns comprising the nucleic acid molecule of claim 12.

KCP-1729811-6 31