GENE RELEVANT TO PAPILLARY THYROID TUMORS

Abstract

The invention relates to a gene relevant to papillary thyroid tumors and an application thereof. According to the base sequence of the gene, real-time and quantitative PCR (Polymerase Chain Reaction) primers are designed and synthesized; the expression level of long-chain non-coding RNA (Ribonucleic Acid) transcribed by the gene is detected in a papillary thyroid carcinoma clinical case specimen; the result shows remarkable reducing of the expression level of the long-chain non-coding RNA in papillary thyroid tumor tissues and the long-chain non-coding RNA of the gene silencing can remarkably promote the growth of thyroid cancer cells. The gene relevant to the papillary thyroid tumors is expected to prepare preparations used in papillary thyroid carcinoma auxiliary diagnosis, gene therapy, curative effect prediction or prognosis.

Claims

1. Use of a nucleic acid molecule hybridizing with the gene represented by SEQ ID NO:1 under stringent conditions, for preparing reagents for screening, detection or auxiliary diagnosis of papillary thyroid carcinoma.

2. The use according to claim 1, wherein the nucleic acid molecule has a sequence represented by SEQ ID NO:2 or SEQ ID NO:3.

3. A detection kit comprising the nucleic acid molecule hybridizing with the gene represented by SEQ ID NO:1 under stringent conditions, wherein the kit is used for screening, detection or auxiliary diagnosis of papillary thyroid carcinoma.

4. The kit according to claim 3, wherein, the kit is a real-time and quantitative PCR detection kit.

5. The kit according to claim 3, wherein, the nucleic acid molecule has a sequence represented by SEQ ID NO:2 or SEQ ID NO:3.

6. Use of the gene represented by SEQ ID NO:1 as an auxiliary diagnosis marker for papillary thyroid carcinoma.

7. Use of the gene represented by SEQ ID NO:1 as a therapeutic target in papillary thyroid carcinoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Further objects, functions and advantages of the present invention will be elucidated with reference to the embodiments described hereinafter and the accompanying drawings in which:

[0019] FIG. 1 is a schematic diagram illustrating mutations of lncRNA GAS8-AS1 gene and genes related thereof;

[0020] FIG. 2 is an RNA secondary structural diagram of lncRNA GAS8-AS1 predicted by the RNAfold software;

[0021] FIG. 3 is a schematic diagram illustrating expression levels of lncRNA GAS8-AS1 in papillary thyroid carcinoma tissues and para-carcinoma normal tissues detected by Zhejiang Queue and Huai'an Queue;

[0022] FIG. 4 is a schematic diagram illustrating proliferation of papillary thyroid carcinoma cell lines GLAG66, NPA and TPC-1 after transfecting plasmids carrying lncRNA GAS8-AS1 gene;

[0023] FIG. 5 is a schematic diagram illustrating expression levels of lncRNA GAS8-AS1 in papillary thyroid carcinoma cell lines GLAG66, NPA and TPC-1 after transfecting plasmids carrying lncRNA GAS8-AS1 gene by a real-time and quantitative PCR detection;

[0024] FIG. 6 is a schematic diagram illustrating proliferation of papillary thyroid carcinoma cell lines GLAG66 and TPC-1 after transfecting siRNAs directed against lncRNA GAS8-AS1; and

[0025] FIG. 7 is a schematic diagram illustrating expression levels of lncRNA GAS8-AS1 in papillary thyroid carcinoma cell lines GLAG66 and TPC-1 after transfecting siRNAs directed against lncRNA GAS8-AS1 by a real-time and quantitative PCR detection.

DETAILED DESCRIPTION

[0026] The above contents of the present invention will be explained and described in further details through specific embodiments hereinafter, so that persons skilled in the art will readily understand the present invention, however, the scope of the subject matter described herein should not be constructed as being limited to the following examples, or a limit to any or all claims of the present invention, or to depart from the spirit of the present invention.

[0027] One object of the present invention is to provide a gene, named GAS8-AS1, having a sequence represented by a sequence listing SEQ ID NO:1.

[0028] The GAS8-AS1 gene (NCBI-GeneID: 750), also known as C16orf3 gene (chromosome 16 open reading frame 3), is located in the intron 2 of human chromosome 16 GAS8 gene. The GAS8-AS1 gene doesn't contain any intron sequence that is transcribed in the opposite direction to the GAS8 gene to generate a long non-coding RNA (lncRNA) that cannot be translated into a protein. Currently, biological functions of the gene and its transcription product are still unclear.

[0029] The sequence of GAS8-AS1 gene (NCBI Reference Sequence: NR_122031.1) is shown as follows:

TABLE-US-00001 (SEQIDNo.1) 1 acctgcagtcccagctactgggcagcctgaagcagcaggatggtgtgaacccaggaggtg 61 gagcttgcagtgagccgaggtcgcgccaccgcactccagcctgggccacacagcgagatt 121 ccgtcagaatcagttacttttcgggcacagccccaggccacttactgtgagcctttttct 181 ttctcaacaccacattccccacagggaaaacacatttctcacctcaaaagaagacaagac 241 aacgagcaaacaagaaggagcagcaggaggggttctgagccgaggatgccgggcagacat 301 gagggagacacgcacccccgaatccaaccagtgcctcggcacaacgacaaatgtcttcac 361 gtcacagacctttagaggctcctgggcagagcctgaaccagggctcctgactggtctgtt 421 tggctcacatggtgttgagattttgccatcactcaatattcagatttcttataaatatcc 481 agatttccagcttctcttggaaaatcagaaaaaaacagcactgaactcctaggcccacaa 541 ggcactccccagtgaacagatgaaactgtcctctgctgcggggcaggagtctccaggtca 601 cccccatccctccccacctgcctggaccctgaagaagccttctgagtctgtggctcaacg 661 tgcgatgtgcagtgcaagggcctgccccgtagcctgccccgtaggctgccccatagcctg 721 ccccgtaagctgccccgtagcctgccccgtaggctgccccgtaggctccatggccactgc 781 cccacaaggcctgtctccacaggaatgggaagcggacagggagacgggcagcagctcaca 841 tgctgggacaacgcagtgttcaatccattctccatccagcagctccagacatctttccag 901 aacacaaacctgaccccatcacctctctgcttagccactggcttaaactgccaatggttt 961 gcctgcatgtaaaataaagccattctttaccattaaaaaa

[0030] Long non-coding RNA (long non-coding RNA, lncRNA) is a kind of non-coding RNA with a transcript of more than 200 nucleotides in length, it is found in recent researches that, lncRNA is a kind of RNA with important biological functions, participating in various important regulating processes, such as genome imprinting, chromosome silencing, chromatin modification, transcriptional activation, transcriptional interference, and intra-nuclear transport, and playing an important role in regulation and control of life activities, such as cell differentiation and development, gene transcription and translation, genetics and epigenetics.

[0031] Another object of the present invention is to provide use of the GAS8-AS1 gene for preparing reagents used in screening, detection or auxiliary diagnosis of papillary thyroid carcinoma.

[0032] Another object of the present invention is to provide a nucleic acid molecule hybridizing with the GAS8-AS1 gene under stringent conditions, to prepare reagents for detecting the GAS8-AS1 gene. The nucleic acid molecule has a sequence represented by SEQ ID NO:2 or SEQ ID NO:3.

[0033] Another object of the present invention is to provide a detection kit including the nucleic acid molecule hybridizing with the GAS8-AS1 gene under stringent conditions, and the kit can be used to detect the GAS8-AS1 gene. Obviously, after learning a gene sequence represented by SEQ ID NO:1 and a primer sequence represented by SEQ ID NO:2 or SEQ ID NO:3 disclosed in the present invention, persons skilled in the art can prepare other primers and kits for detecting the GAS8-AS1 gene without creative efforts, on the basis of the present invention, and the detection method of the kit includes, but is not limited to the real-time and quantitative PCR method. The kit can include the nucleic acid molecule represented by SEQ ID NO:2 or SEQ ID NO:3 or other molecules hybridizing with the GAS8-AS1 gene under stringent conditions. Optionally, the kit also can include auxiliary reagents required for performing conventional gene detection.

[0034] Another object of the present invention is to provide use of the GAS8-AS1 gene as an auxiliary diagnosis marker for papillary thyroid carcinoma. As discussed in the present disclosure, the GAS8-AS1 gene in a patient with papillary thyroid carcinoma has a significant level of mutation. Therefore, detecting the level of mutation can be used as an effective method for early screening and auxiliary diagnosis of papillary thyroid carcinoma.

[0035] Another object of the present invention is to provide use of the GAS8-AS1 gene as a therapeutic target in papillary thyroid carcinoma. As discussed in the present disclosure, inducing the GAS8-AS1 gene overexpression in a patient with papillary thyroid carcinoma can significantly inhibit the growth of papillary thyroid carcinoma cells. Therefore, GAS8-AS1 can be used as an effective therapeutic target in papillary thyroid carcinoma.

Embodiment 1: Mutation Sequencing Method

[0036] 1.1 Acquiring a Tumor Tissue Sample of a Subject

[0037] 1.2 Genomic DNA Extraction

[0038] Get ready an autoclaved mortar, pour into liquid nitrogen for pre-cooling after drying; an appropriate amount of tissue is ground with the mortar under addition of liquid nitrogen, grind into powder then thaw; collect the tissue in the mortar with 800 l of PBS solution then put the tissue in a centrifuge tube (1.5 ml); centrifuge at 12000 rpm for 1 min, and then remove the supernatant. Then add 200 l of buffer GA and shake to thoroughly suspend.

[0039] Add 20 l of protease K (20 mg/ml), mix thoroughly, incubate at 56 C. for 2 h, and shake once every 20 min till the tissue is dissolved.

[0040] Add 200 l of GB buffer and mix uniformly upside down and incubate at 70 C. for 10 min until the solution becomes clear.

[0041] Add 200 l of absolute alcohol, fully oscillate for 15 sec, then flocculent precipitate should appear.

[0042] Transfer the above solution and flocculent precipitate to a CB3 adsorption column, then centrifuge at 12,000 rpm for 30 sec and remove the liquid in the collection tube.

[0043] Add 500 l of GD to the CB3 adsorption column, centrifuge at 12,000 rpm for 30 sec and remove the liquid in the collection tube.

[0044] Add 600 l of PW (check whether alcohol has been added before use) to the CB3 adsorption column, centrifuge at 12,000 rpm for 30 sec, and remove the liquid in the collection tube. Repeat this step 2 times.

[0045] Centrifuge at 12,000 rpm for 2 min, remove waste liquid, and then stand for a few minutes to dry the residual rinse liquid.

[0046] Replace the collection tube, add 50 l to 200 l of TE to the CB3 adsorption column for dissolving DNA, stand for 5 min at room temperature and centrifuge for 2 min at 12,000 rpm, and store the collected DNA at 20 C. for later use.

[0047] 1.3 Template Preparation

[0048] The solid-phase PCR (Illumina's Hiseq) method is used, that is, the amplification process is carried out on glass slides. High-density forward and reverse primers are covalently linked on these glass slides, and the ratio of templates to primers determines the density of the amplified clusters. The solid-phase PCR can produce one to two hundred million spatially isolated template clusters and provide free ends for universal sequencing primers to initiate sequencing reactions.

[0049] 1.4 Exome Trapping

[0050] High-coverage exon region trapping is performed using the Agilent 50 Mb SureSelectXT Human All Exon V5 kit on human exon liquid-phase targeted sequence enrichment system. The All Exon 50 Mb kit is a human all exon trapping kit jointly developed by the Agilent and the Wellcome Trust Sanger Institute and Gencode consortium. Exon trapped can reach up to 50 Mb. Objects to be trapped: 1) exons found in the GENCODE project (about 12M); 2) exons in the NCBI Consensus CDS database (CCDS, March 2009); 3) miRNAs in the Sanger V13 database; 4) over 300 human non-coding RNAs (e.g., snoRNAs and scaRNAs).

[0051] 1.5 Targeted Sequencing and Bioinformatics Analysis

[0052] The basic principle of Illumina's Hiseq 2000 sequencing is sequencing by synthesis, also referred to as cyclic reversible termination. DNA polymerases, linker primers and four dNTPs with base-specific fluorescent labels are added simultaneously to the reaction system. Since the 3hydroxyl groups of these dNTPs are chemically protected, only one dNTP can be added to each round of synthesis reaction. After dNTP is added to the synthetic chain, all unused free dNTPs and DNA polymerases will be eluted. Add the buffer required to stimulate the fluorescence, excite the fluorescence signal with a laser, record the fluorescence signal with an optical device, and then convert into sequencing results via computer analysis. After the recording of the fluorescent signal is completed, add chemical reagents to quench the fluorescent signal, remove the 3 hydroxy protecting group of dNTP, restore the 3 end viscosity, and continue to polymerize the second nucleotide. This continues until all the template sequences are completely polymerized into double-strand. In this way, make statistics to the results of the fluorescence signals collected in each round, to learn the sequence of DNA fragment on each template. One advantage of this sequencing method is to reduce the time for sample separation and preparation, the read length of the paired end can reach up to 250 bp, more than 20 GB of high quality filter data can be obtained after each running, and the running cost is relatively low, and therefore, it is a new generation of sequencing technology with a higher cost performance.

[0053] Targeted sequencing of papillary thyroid carcinoma is carried out with the method of constructing Gaussian mixture model, to align the obtained short fragment sequence and the reference sequence (mapping), find mutation (variant calling) and filter and screen the mutation.

Embodiment 2: Taking Genes Such as GAS8-AS1 as a Target for Screening or Detecting Papillary Thyroid Carcinoma

[0054] 2.1 Experimental Method

[0055] According to the present invention, targeted sequencing of 91 pairs of paired tissues (thyroid carcinoma tissues and peripheral blood samples) of papillary thyroid carcinoma patients is carried out with the whole genome exon technique described in Embodiment 1 to obtain gene mutation. Finally, genes, such as GASB-AS1 are determined to be papillary thyroid carcinoma susceptibility genes for the Chinese Han populations. The paired tissues of papillary thyroid carcinoma patients refer to thyroid carcinoma tissues and peripheral blood samples of the patient, and the Cancer Institute and Hospital of the Chinese Academy of Medical Sciences and Zhejiang Cancer Hospital are entrusted to collect the 91 pairs of tissue samples of papillary thyroid carcinoma patients

[0056] 2.2 Experimental Result

[0057] 2.21 Gene Mutation Frequency Statistics

[0058] Next generation sequencing is carried out on 91 pairs of paired tissues of papillary thyroid carcinoma, and the high-frequency mutant genes are shown in Table 1.

[0059] 2.22 Papillary Thyroid Carcinoma Susceptibility Gene

[0060] The high-frequency mutant genes are analyzed with MutsigCV software, and GAS8-AS1 is finally determined to be papillary thyroid carcinoma susceptibility gene for the Chinese Han populations. See Table 1.

TABLE-US-00002 TABLE 1 Papillary thyroid carcinoma susceptibility gene for the Chinese Han populations Number of non- Number of Number of coding False non-silent silent region discovery Gene mutations.sup.1 mutations.sup.2 mutations.sup.3 P value.sup.4 rate.sup.5 BRAF 53 1 0 <1.0E16 <1.0E16 GAS8-AS1 15 0 0 <1.0E16 <1.0E16 Notes: .sup.1the number of non-silent mutations in the gene; .sup.2the number of silent mutations in the gene; .sup.3the number of non-coding region mutations in the gene; .sup.4calculated by MutSigCV software; .sup.5multiple testing correction results.

Embodiment 3: Taking GAS8-AS1 Gene Detection as a Target for Screening or Detecting Papillary Thyroid Carcinoma

[0061] The GAS8-AS1 gene (NCBI-GeneID: 750), also known as C16orf3 gene (chromosome 16 open reading frame 3), is located in the intron 2 of human chromosome 16 GAS8 gene. The GAS8-AS1 gene doesn't contain any intron sequence that is transcribed in the opposite direction to the GAS8 gene to generate a long non-coding RNA (lncRNA) that cannot be translated into a protein. Currently, biological functions of the gene and its transcription product are still unclear.

[0062] The sequence of GAS8-AS1 gene (NCBI Reference Sequence: NR_122031.1) is shown as follows:

TABLE-US-00003 (SEQIDNo.1) 1 acctgcagtcccagctactgggcagcctgaagcagcaggatggtgtgaacccaggaggtg 61 gagcttgcagtgagccgaggtcgcgccaccgcactccagcctgggccacacagcgagatt 121 ccgtcagaatcagttacttttcgggcacagccccaggccacttactgtgagcctttttct 181 ttctcaacaccacattccccacagggaaaacacatttctcacctcaaaagaagacaagac 241 aacgagcaaacaagaaggagcagcaggaggggttctgagccgaggatgccgggcagacat 301 gagggagacacgcacccccgaatccaaccagtgcctcggcacaacgacaaatgtcttcac 361 gtcacagacctttagaggctcctgggcagagcctgaaccagggctcctgactggtctgtt 421 tggctcacatggtgttgagattttgccatcactcaatattcagatttcttataaatatcc 481 agatttccagcttctcttggaaaatcagaaaaaaacagcactgaactcctaggcccacaa 541 ggcactccccagtgaacagatgaaactgtcctctgctgcggggcaggagtctccaggtca 601 cccccatccctccccacctgcctggaccctgaagaagccttctgagtctgtggctcaacg 661 tgcgatgtgcagtgcaagggcctgccccgtagcctgccccgtaggctgccccatagcctg 721 ccccgtaagctgccccgtagcctgccccgtaggctgccccgtaggctccatggccactgc 781 cccacaaggcctgtctccacaggaatgggaagcggacagggagacgggcagcagctcaca 841 tgctgggacaacgcagtgttcaatccattctccatccagcagctccagacatctttccag 901 aacacaaacctgaccccatcacctctctgcttagccactggcttaaactgccaatggttt 961 gcctgcatgtaaaataaagccattctttaccattaaaaaa

[0063] Long non-coding RNA (long non-coding RNA, lncRNA) is a kind of non-coding RNA with a transcript of more than 200 nucleotides in length, it is found in recent researches that, lncRNA is a kind of RNA with important biological functions, participating in various important regulating processes, such as genome imprinting, chromosome silencing, chromatin modification, transcriptional activation, transcriptional interference, and intra-nuclear transport, and playing an important role in regulation and control of life activities, such as cell differentiation and development, gene transcription and translation, genetics and epigenetics. In recent years, more and more authoritative studies confirm that lncRNA plays a role of inhibiting or promoting tumors in the formation and development of tumors, and also plays a very important role in the regulation of tumor cell proliferation, apoptosis, cell cycle, invasion and metastasis, etc.

[0064] 3.1 Experimental Method

[0065] 3.1.1 According to the present invention, targeted sequencing of 91 pairs of paired tissues of papillary thyroid carcinoma is carried out with the whole genome exon technique described in Embodiment 1.

[0066] 3.1.2 A real-time quantitative PCR method is used to detect the gene expression of 87 pairs of RNA samples from thyroid carcinoma tissues and para-carcinoma normal tissues of patients with papillary thyroid carcinoma collected by Zhejiang Provincial Tumor Hospital and the Second People's Hospital of Huai'an (referred to as Zhejiang Queue and Huai'an Queue, respectively in the present invention). The real-time quantitative PCR detection kit includes primers designed for real-time quantitative PCR according to the sequence of the GAS8-AS1 gene: GAS8-AS1-F and GAS8-AS1-R, having sequences of SEQ ID No. 2 and SEQ ID No. 3 shown in the table below:

TABLE-US-00004 Prime Sequence(5 to3) GAS8-AS1-F CAACGAGCAAACAAGAAGGAG(SEQIDNo.2) GAS8-AS1-R TGAGCCAAACAGACCAGTCA(SEQIDNo.3)

[0067] 3.1.3 The papillary thyroid carcinoma cell lines GLAG66, NPA and TPC-1 are cultured in vitro, plasmids carrying the lncRNA GAS8-AS1 gene or siRNAs directed against lncRNA GAS8-AS1 are transfected into the above cells, cells are counted at 24 hours and 48 hours after transfection, to observe the influence of the lncRNA GAS8-AS1 gene on the proliferation of papillary thyroid carcinoma cells.

[0068] 3.2 Experimental Result

[0069] 3.2.1 As shown in Table 1, targeted sequencing of 91 pairs of paired tissues of papillary thyroid carcinoma is carried out with the whole genome exon technique, and it is determined that 8 patients carry GAS8-AS1 gene mutation with a mutation rate of 8.8%, and thus determined that GAS8-AS1 gene is a papillary thyroid carcinoma susceptibility gene for the Chinese Han populations. FIG. 1 is a schematic diagram illustrating mutations of lncRNA GAS8-AS1 gene and genes related thereof. The RNA secondary structure of lncRNA GAS8-AS1 predicted by the RNAfold software is as shown in FIG. 2.

[0070] 3.2.2 FIG. 3 is a schematic diagram illustrating expression levels of lncRNA GAS8-AS1 in papillary thyroid carcinoma tissues and para-carcinoma normal tissues of papillary thyroid carcinoma patients detected by Zhejiang Queue and Huai'an Queue. As shown in FIG. 3, after detecting the expression of the GAS8-AS1 gene in 87 pairs of papillary thyroid carcinoma tissues and normal tissues from Zhejiang Queue and Huai'an Queue with the real-time quantitative PCR method, it is found that the expression thereof is significantly down-regulated in tumor tissues, notifying the GAS8-AS1 gene is a brand new anti-oncogene of thyroid carcinoma.

[0071] 3.2.3 FIG. 4 is a schematic diagram illustrating proliferation of papillary thyroid carcinoma cell lines GLAG66, NPA and TPC-1 after transfecting plasmids carrying the lncRNA GAS8-AS1 gene. As shown in FIG. 4, after transfecting plasmids carrying the lncRNA GAS8-AS1 gene, proliferation of GLAG66, NPA and TPC-1 is significantly lower than the carrier of not transfecting plasmids carrying the lncRNA GAS8-AS1 gene. Therefore, lncRNA GAS8-AS1 can significantly inhibit the proliferation of GLAG66, NPA and TPC-1.

[0072] FIG. 5 illustrates expression levels of lncRNA GAS8-AS1 in papillary thyroid carcinoma cell lines GLAG66, NPA and TPC-1 after transfecting plasmids carrying lncRNA GAS8-AS1 gene detected by a real-time and quantitative PCR method. As shown in FIG. 5, the expression levels of lncRNA GAS8-AS1 in GLAG66, NPA and TPC-1 in the carrier of not transfecting plasmids carrying the lncRNA GAS8-AS1 gene are very low, substantially 1.0 or so; the expression levels of lncRNA GAS8-AS1 in GLAG66, NPA and TPC-1 in the carrier of transfecting plasmids carrying the lncRNA GAS8-AS1 gene are about 49, 41, 55, respectively, which is significantly higher than the carrier of not transfecting plasmids carrying the lncRNA GAS8-AS1 gene.

[0073] After transfecting GAS8-AS1-siR-1 and GAS8-AS1-siR-2 directed against lncRNA GAS8-AS1, proliferation of papillary thyroid carcinoma cell lines GLAG66 and TPC-1 is increased significantly, as shown in FIG. 6, which indicates that gene silencing of the lncRNA GAS8-AS1 can significantly promote the proliferation of the above cells. After transfecting GAS8-AS1-siR-1 and GAS8-AS1-siR-2 directed against lncRNA GAS8-AS1, expression levels of lncRNA GAS8-AS1 in papillary thyroid carcinoma cell lines GLAG66 and TPC-1 learned by the real-time and quantitative PCR detection are all lower than 0.6, while expression levels if not transfecting GAS8-AS1-siR-1 and GAS8-AS1-siR-2 are 1.0, as shown in FIG. 7.

[0074] In summary, overexpression of GAS8-AS1 gene can significantly inhibit the growth of thyroid carcinoma cells in papillary thyroid carcinoma cells cultured in vitro. In contrast, gene silencing of the GAS8-AS1 gene can significantly promote growth of thyroid carcinoma cells.

[0075] With the progress of the later research results, the function and action mechanism of GAS8-AS1 in PTC will be clarified gradually that this novel long non-coding RNA not only becomes a diagnosis-related biomarker, but also is expected to become a new PTC therapeutic target so as to improve the clinical PTC treatment effect, having a very important practical significance.

[0076] Those described above are just preferred embodiments of the present invention, it should be noted that, various improvements and additions can be made by persons skilled in the art without departing from the method of the present invention, and such improvements and additions shall be deemed to fall within the protection scope of the present invention.