Gene and Protein Relating to Hepatocellular Carcinoma and Methods of Use Thereof

Abstract

The present invention provides a novel human gene ZNFN3A1 whose expression is markedly elevated in a great majority of HCCs compared to corresponding non-cancerous liver tissues. The gene encodes a protein having a zinc finger domain as well as a SET domain and has been found to form a regulatory complex with RNA helicase and RNA polymerase.

1 . An isolated DNA selected from the group consisting of:
 (a) a DNA encoding a protein consisting of the amino acid sequence of SEQ. ID. NO. 2;   (b) a DNA consisting of the coding region of the nucleotide sequence of SEQ. ID. NO. 1;   (c) a DNA encoding a protein comprising one or more substitutions in the amino acid sequence of SEQ ID NO: 2 wherein the encoded protein is a functional equivalent of the protein consisting of the amino acid sequence of SEQ. ID. NO. 2; and   (d) a DNA hybridizing under stringent conditions with a DNA consisting of the nucleotide sequence of SEQ. ID. NO.1, such that the encoded protein is a functional equivalent to the protein consisting of the amino acid sequence SEQ. ID. NO.2;   (e) a DNA encoding a partial peptide of the protein consisting of the amino acid sequence of SEQ. ID. NO.2.   
     
     
         2 . A vector comprising the DNA of  claim 1 . 
     
     
         3 . A transformed cell comprising the DNA of  claim 1 . 
     
     
         4 . A transformed cell comprising the vector of  claim 2 . 
     
     
         5 . An isolated protein encoded by the DNA according to  claim 1 , wherein said protein promotes cell proliferation or activates transcription of a target gene. 
     
     
         6 . An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or fragment thereof. 
     
     
         7 . A method for producing a protein, comprising the steps of culturing the transformed cell of  claim 4 , and collecting the protein expressed from the cells or the culture supernatant thereof. 
     
     
         8 . A transcription activation complex comprising the protein of  claim 5  and at least one-co-activator. 
     
     
         9 . The complex of  claim 8 , the co-activator is selected from the group consisting of an RNA helicase, and an RNA polymerase II. 
     
     
         10 . An antibody that bind immunospecifically to the protein of  claim 5  or the complex of  claim 8 . 
     
     
         11 . A polynucleotide comprising at least 15 nucleotides, wherein said polynucleotide hybridizes under stringent conditions to the nucleotide sequence of SEQ. ID. NO.1 or the complement of said nucleotide sequence. 
     
     
         12 . A composition comprising the antibody of  claim 10  or the polynucleotide of  claim 11 . 
     
     
         13 . A method of screening for a compound that binds to the protein of  claim 5 , comprising the steps of:
 (a) contacting a subject sample, containing at least one test compound, with the protein of  claim 5  or a fragment thereof;   (b) detecting the binding activity of the subject sample with the protein or fragment thereof; and   (c) selecting the test compound that binds to the protein or fragment thereof.   
     
     
         14 . A compound identified by the method of  claim 13 . 
     
     
         15 . A method of screening for a compound that inhibits the activity of the protein of  claim 5 , comprising the steps of:
 (a) culturing cells which express the protein of  claim 5  or fragment thereof in the presence of a subject sample which contains at least one test compound;   (b) detecting the proliferation of the cell; and   (c) selecting the test compound that inhibits the proliferation as compared to the proliferation detected in the absence of the subject sample.   
     
     
         16 . A compound identified by the method of  claim 15 . 
     
     
         17 . A method of screening a compound for anti-cancer activity, comprising the steps of:
 (a) contacting a subject sample, containing at least one test compound, with the protein of  claim 5 , a co-activator thereof and a DNA containing the target sequence of said protein under suitable conditions to allow formation of the complex of said protein with the DNA; and   (b) selecting the test compound that inhibits the formation of the complex.   
     
     
         18 . The method of  claim 17 , wherein said target sequence comprises a CBS sequence flanking the 5′ region of EGFR. 
     
     
         19 . A method of screening a compound for anti-cancer activity, comprising the steps of:
 (a) contacting a subject sample, containing at least one test compound, with the complex of  claim 7  and a reporter gene with a transcriptional regulatory region recognized by said complex; and   (b) selecting the test compound that inhibits the expression of the reporter gene.   
     
     
         20 . The compound identified by the method of  claim 17 . 
     
     
         21 . The compound identified by the method of  claim 18 . 
     
     
         22 . The compound identified by the method of  claim 19 . 
     
     
         23 . An anti-cancer composition comprising the compound of  claim 20 . 
     
     
         24 . An anti-cancer composition comprising the compound of  claim 21 . 
     
     
         25 . An anti-cancer composition comprising the compound of  claim 22 . 
     
     
         26 . An anti-cancer composition comprising an antisense oligonucleotide, ribozyme, or small interference RNA that binds to the DNA of  claim 1 . 
     
     
         27 . A method diagnosing cancer, wherein said method comprises the steps of:
 (a) determining a expression level of the ZNFN3A1 gene in biological sample of specimen;   (b) comparing the expression level of ZNFN3A1 gene with that in normal sample, and   (c) defining a high expression level of the ZNFN3A1 gene in the sample as having a cancer.   
     
     
         28 . The method of  claim 27 , wherein the cancer is hepatocellular carcinoma. 
     
     
         29 . A diagnostic agent for diagnosing hepatocellular carcinoma comprising a compound that binds to the DNA of  claim 1 . 
     
     
         30 . A diagnostic agent for diagnosing hepatocellular carcinoma comprising a compound that binds to protein of  claim 5 . 
     
     
         31 . A method of inhibiting tumor cell growth in a subject, comprising administering to said subject a composition comprising a ZNFN3A1 small interfering RNA (siRNA). 
     
     
         32 . The method of  claim 31 , wherein said siRNA comprises a sense ZNFN3A1 nucleic acid and a anti-sense ZNFN3A1 nucleic acid. 
     
     
         33 . The method of  claim 32 , wherein said tumor cell is colorectal cancer cell or liver cancer cell. 
     
     
         34 . The method of  claim 33 , wherein said colorectal cancer cell is an adenocarcinoma cell. 
     
     
         35 . The method of  claim 33 , wherein said liver cancer cell is a hepatocellular carcinoma cell. 
     
     
         36 . The method of  claim 32 , wherein the siRNA is specific for a ZNFN3A1 target selected from the group consisting of nucleotides 451-471, 532-552, 623-643, 625-645, 636-656, 726-746, 923-943, 1065-1085, and 1258-1278 of SEQ ID NO:1. 
     
     
         37 . The method of  claim 36 , wherein the siRNA has the general formula 5′-[A]-[B]-[A′]-3′, wherein [A] is a ribonucleotide sequence corresponding to a sequence selected from the group consisting of nucleotides 451-471, 532-552, 623-643, 625-645, 636-656, 726-746, 923-943, 1065-1085, and 1258-1278 of SEQ ID NO:1,
 [B] is a ribonucleotide sequence consisting of 3 to 23 nucleotides, and 
 [A′] is a ribonucleotide sequence consisting of the complementary sequence of [A]. 
 
     
     
         38 . The method of  claim 31 , wherein said composition comprises a transfection-enhancing agent. 
     
     
         39 . An isolated polynucleotide comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence selected from the group consisting of nucleotides 451-471, 532-552, 623-643, 625-645, 636-656, 726-746, 923-943, 1065-1085, and 1258-1278 of SEQ ID NO:1, and said antisense strand nucleic acid consists of complementary sequence thereof, respectively. 
     
     
         40 . The isolated polynucleotide of  claim 39 , wherein said sense strand nucleic acid and antisense strand nucleic acid are on the same strand. 
     
     
         41 . The isolated nucleic acid molecule of  claim 39 , wherein said sense strand nucleic acid consists of a nucleotide sequence shorter than about 100 nucleotides. 
     
     
         42 . The isolated nucleic acid molecule of  claim 41 , wherein said sense strand nucleic acid is shorter than about 75 nucleotides. 
     
     
         43 . The isolated nucleic acid molecule of  claim 42 , wherein said sense strand nucleic acid is shorter than about 50 nucleotides. 
     
     
         44 . The isolated nucleic acid molecule of  claim 43 , wherein said sense strand nucleic acid is shorter than about 25 nucleotides. 
     
     
         45 . The isolated nucleic acid molecule of  claim 44 , wherein said sense strand nucleic acid is between about 19 and about 25 nucleotides in length. 
     
     
         46 . A vector comprising a nucleic acid molecule comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence selected from the group consisting of nucleotides 451-471, 532-552, 623-643, 625-645, 636-656, 726-746, 923-943, 1065-1085, and 1258-1278 of SEQ ID NO:1, and said antisense strand nucleic acid consists of complementary sequence thereof, respectively. 
     
     
         47 . The vector of  claim 46 , wherein said nucleic acid molecule has the general formula 5′-[A]-[B]-[A′]-3′, wherein [A] is a nucleotide sequence selected from the group consisting of nucleotides 451-471, 532-552, 623-643, 625-645, 636-656, 726-746, 923-943, 1065-1085, and 1258-1278 of SEQ ID NO:1,
 [B] is a nucleotide sequence consisting of 3 to 23 nucleotides, and 
 [A′] is a nucleotide sequence consisting of the complementary sequence of [A]. 
 
     
     
         48 . A composition comprising at least one siRNA comprising a combination of a sense strand nucleic acid and an antisense strand nucleic acid, and pharmaceutically acceptable carrier, wherein said sense strand nucleic acid comprises ribonucleotide sequence corresponding to a sequence selected from the group consisting of nucleotides 451-471, 532-552, 623-643, 625-645, 636-656, 726-746, 923-943, 1065-1085, and 1258-1278 of SEQ ID NO:1, and said antisense strand sequence consists of complementary sequence thereof, respectively. 
     
     
         49 . A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a ribonucleotide sequence corresponding to a ZNFN3A1 target sequence, and wherein the antisense strand comprises a ribonucleotide sequence which is complementary to said sense strand, wherein said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said double-stranded molecule, when introduced into a cell expressing the ZNFN3A1 gene, inhibits expression of said gene. 
     
     
         50 . The double-stranded molecule of  claim 49 , wherein said ZNFN3A1 target sequence comprises at least about 10 contiguous nucleotides from SEQ ID No:1. 
     
     
         51 . The double-stranded molecule of  claim 50 , wherein said ZNFN3A1 target sequence comprises from about 19 to about 25 contiguous nucleotides from SEQ ID No:1. 
     
     
         52 . The double-stranded molecule of  claim 51 , wherein said ZNFN3A1 target sequence is selected from the group consisting of nucleotides 451-471, 532-552, 623-643, 625-645, 636-656, 726-746, 923-943, 1065-1085, and 1258-1278 of SEQ ID NO:1. 
     
     
         53 . The double-stranded molecule of  claim 49 , wherein a single ribonucleotide transcript comprises the sense strand and the antisense strand, said double-stranded molecule further comprising a single-stranded ribonucleotide sequence linking said sense strand and said antisense strand. 
     
     
         54 . The double-stranded molecule of  claim 49 , wherein the double stranded molecule is an oligonucleotide of less than about 100 nucleotides in length. 
     
     
         55 . The double-stranded molecule of  claim 54 , wherein the double stranded molecule is an oligonucleotide of less than about 75 nucleotides in length. 
     
     
         56 . The double-stranded molecule of  claim 55 , wherein the double stranded molecule is an oligonucleotide of less than about 50 nucleotides in length. 
     
     
         57 . The double-stranded molecule of  claim 56 , wherein the double stranded molecule is an oligonucleotide of less than about 25 nucleotides in length. 
     
     
         58 . The double-stranded nucleic acid molecule of  claim 57 , wherein the double stranded molecule is an oligonucleotide of between about 19 and about 25 nucleotides in length. 
     
     
         59 . A vector encoding the double-stranded molecule of  claim 49 . 
     
     
         60 . The vector of  claim 59 , wherein the vector encodes a transcript having a secondary structure, wherein the transcript comprises the sense strand and the antisense strand. 
     
     
         61 . The vector of  claim 59 , wherein the transcript further comprises a single-stranded ribonucleotide sequence linking said sense strand and said antisense strand.