I investigated the upstream of RUNX2 at the transcription factor search site to find the regulator of RUNX2. The genes which scores greater than 95.00 were selected as candidate genes. Homeobox family A 9, HOXA 9, was selected as the primary candidate gene via semi-quantitative PCR and real-time PCR, because the expression of HOXA9 was changed over time with RUNX2. And the normal thyroid cell line Nthy-Ori 3-1 and PTC cell line TPC1 and BHP10-3 were used in vitro. I knockdowned and overexpressed HOXA9 in the two types of thyroid cell lines, and found the expression of RUNX2 was changed with the expression of HOXA9. HOXA9 also increased RUNX2 promoter activity and RUNX2 binding ability. Calcification was enhanced in HOXA9 overexpressed thyroid cell lines than control. Overexpression of HOXA9 significantly enhanced in vitro normal cell migration and invasion, while downregulation of HOXA9 inhibited tumor cell migration and invasion. IHC assay was administered in calcified thyroid cancer tissues, and RUNX2 and HOXA9 were simultaneously expressed. Then the expression of RUNX2 was downregulated in two types of thyroid cell lines with or without HOXA9 overexpression. ALP activity, mineralization behaviour, and cancer capacity were all considerably decreased upon RUNX2 downregulation, while all of these activities were increased with HOXA9 overexpression, as compared to those in the RUNX2-knockdown only groups.
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This is the first report demonstrating the function of HOXA9 on PTC. Based on the in vitro and in vivo data, my results suggest that HOXA9 can be linked to calcification and tumor invasion of papillary thyroid carcinoma dependent or independent of RUNX2.
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1 GTATTAAAAT ATGATCATTA GTTAAGAAAA TAAAAAATTA CTCAAATGGT entry score <--- M00101 CdxA 92.9
51 TACAATGAAG ACACACATAG CATTTAAACA CAAATACCTT GGTATTTTCA entry score ---> M00148 SRY 92.7 <--- M00100 CdxA 91.0 ---> M00100 CdxA 88.5 <--- M00241 Nkx-2. 85.3
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101 AAAGTTCAAC CAATTCTAAC ACATTTTATT CACTACAAAG CAATGGAGAA entry score ---> M00101 CdxA 92.9
151 GCAAGATACC AACAACTTTT TTTCTTTGAA ATTGATTTCA AGATCAAAAC entry score <--- M00148 SRY 90.0 ---> M00106 CDP CR 87.7 ---> M00076 GATA-2 87.4
201 TGTATTTCAT TTCCATGGCA TTAAAGTATG TGATTTATAC AGTATACCTA entry score ---> M00101 CdxA 100.0 ---> M00100 CdxA 96.2 <--- M00101 CdxA 92.1 <--- M00162 Oct-1 87.8 <--- M00101 CdxA 85.0
251 AACTTGTTGC TTACTTCAAA ACAGCAAATA CTAACATGTC CCCAGAGAGG entry score ---> M00101 CdxA 92.9 <--- M00083 MZF1 87.8 <--- M00131 HNF-3b 86.7 ---> M00148 SRY 86.4 --- M00261 Olf-1 85.4
301 ATAAAATTTT ATGGGTCTTT AAAAAGCAAA AATAAAAATA AAAATAAATC entry score ---> M00101 CdxA 92.1
- 66 -
351 TGTGGTTTTC AATTTCTGAT TATAACCATG GTATAAATCT ATAATCAAGA entry score ---> M00271 AML-1a 100.0
401 GCTTTATTTG CATTGACTTT TCTAAATCAG TATCTCTTCA CCACGTGTAG entry score ---> M00217 USF 98.0
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451 TAAGCTATTT CTAATGAAAC ACACTGTTAT CAAATGTGTA CCAATTATCA entry score ---> M00101 CdxA 94.3
501 AATGTGCCCC AGTTGTCATT GTTTTTTAAA TTTTTACAAC AAAGTAATAT entry score --- M00131 HNF-3b 94.2
551 TTGCTCCAAA GAGGATATAT TTTCGTTTTA GGAAAAAAAT TCATGTGGAT entry score ---> M00131 HNF-3b 94.2 --- M00137 Oct-1 90.5
- 68 -
601 AATGAATATA CTAACTTAAA GCACATGATT TCACTATCTT TCTCCTGCTA entry score <--- M00077 GATA-3 94.4
651 GAATTTGATA GGCCTCACAG AGGACAAAAA GTGCATATAT AACAATATCT entry score
- 69 -
701 ACTAAACTCT CAAATCCTGG TCCAAAAATT ACATGCAGGA GGATTACCTG entry score ---> M00162 Oct-1 93.9 <--- M00100 CdxA 87.2 ---> M00101 CdxA 86.4
751 CAAAAAGTGA ACAGGTAATT AAATGACAAA TAGATGTTAA AAATTACTTA entry score <--- M00100 CdxA 92.3
801 AATTCTTATC AGGTCAATTT GAAGAGCCAA AGTTTACATA AAGAAGATTT entry score <--- M00100 CdxA 96.2
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851 TAAGGAAGAA AATAGCAAAA TGTTGGGTTT GTAAGATGTT TCAGTGAATG entry score <--- M00148 SRY 87.3 <--- M00147 HSF2 85.3
901 CTAATGTAGA AACTATAAGC TTATTTGAAA AGAAGAAAAC CATTTGCTGA entry score --- M00059 YY1 85.7 ---> M00159 C/EBP 85.4 ---> M00241 Nkx-2. 85.3
951 TCATACTACA GCAGCTACCT ACATATTTTC AATTAAGTGA TTCTGCTATT entry score <--- M00241 Nkx-2. 100.0
1001 ATAGGTACAC ATGAAACCAT GTTTGGAAAT CCCACAAGCT ATAAGGACAA entry score <--- M00053 c-Rel 96.7
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---> M00059 YY1 88.9 <--- M00087 Ik-2 87.7 --- M00216 TATA 86.8
1051 AACCCTCCTT TTGTTTACTT TGGATACTAA TTAGAGATAC AGATAATATG entry score <--- M00148 SRY 100.0
1101 GATACGACTT ATTTATAAAA GAGAAAAAAT AATTTTTCTC TCCTTCACAT entry score ---> M00101 CdxA 100.0
- 72 -
--- M00217 USF 86.2 ---> M00101 CdxA 85.7 ---> M00203 GATA-X 85.3
1151 GAATAACTGC ATGTTAAATG AACGCTTTTA CCTTTCAATG TAAAATCTGT entry score ---> M00145 Brn-2 92.1
1201 TTACACATAC AATAAGGCAT GATAAATCCA TAGTTAAAAT AAAACACCAA entry score <--- M00100 CdxA 92.3
1251 AATGTATTTC TGGTTTTTTA TCACTAAAAG TTTATTCTGA AAAGGTTCTG entry score ---> M00101 CdxA 93.6 ---> M00100 CdxA 89.7 <--- M00203 GATA-X 86.7 ---> M00101 CdxA 85.0
1301 GACATTTGAA AAATAAAGGA CTAGAATCAT TCCCATACCC TACCAGTGTA entry score <--- M00087 Ik-2 89.5 <--- M00100 CdxA 85.9 ---> M00101 CdxA 85.7
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1351 TTTTGGGAAA ATCAAATTTT CTGTAAAGAA ACTTATGAAC ATATTTGTAC entry score ---> M00141 Lyf-1 93.5
1401 AGTTATTGTG ATCTAATATG AACCAAAAGC AGATAATGAA TAGCACTAGG entry score ---> M00128 GATA-1 91.2
1451 AAGAACACAG GGATATTTTA GTTCTAACAC CCTCCTGTCT CCCTAGCCCT entry score ---> M00076 GATA-2 85.4
1501 TACCTCCCTG CACATTCCAA ATAATCTTTT GTAATTCACT GTCTCCGCCC entry score
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1551 ACCCCATTTA CTTTATGCCA CTCCTAGTTA CTGTCACACT AGGAAGAAGT entry score ---> M00100 CdxA 96.2 <--- M00271 AML-1a 87.4 ---> M00101 CdxA 87.1
1601 CTAACATGCA GATTTAGAGT GGCATGGATA AATGGCAAAA AAATGCCTAG entry score <--- M00100 CdxA 92.3
1651 AAAATTGGTC TGTTCGCCTT TATAATTTTG GTTGAAAAAT ACTCCATCGC entry score <--- M00101 CdxA 98.6
1701 TCCCAACTGA TGAAAACAGG AAGCTCTATT CATAAATATA AAATTCACTG entry score <--- M00100 CdxA 100.0
- 75 -
1751 CCTATGATAT ATAATCATCC TAATAAGAAA ATGAGTTCTA TACATACTTG entry score ---> M00137 Oct-1 88.0 ---> M00101 CdxA 87.1 <--- M00101 CdxA 86.4
1801 TCCAAAGGGG CAAAAAAGGA GATAGTTTCC CAAAGATGTT TCCAATTTTC entry score <--- M00141 Lyf-1 93.5
1851 TTCTGAATCA GAATTAGCAA ATCGAGACGA CTAACATACT CTGTCTGTGG entry score <--- M00101 CdxA 92.1 ---> M00199 AP-1 91.1 <--- M00199 AP-1 89.6 <--- M00159 C/EBP 87.7
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1901 GCATTATTCC TTACTACACA CAGCATTTTG TAATTTATTT CAAAGCTTCC entry score <--- M00101 CdxA 97.9
1951 ATTAGAAACA AAAAAATACA TAGCTTCTGT TAACCCACTC TATTCTAAGC entry score ---> M00148 SRY 100.0
2001 TCATAGAATC AAATACTGAA CAATCTACAT TATAACATAA GCATTTTACT entry score -- M00100 CdxA 92.3
2051 TTATAGAAGA TCTGCTATCA GAAACTCTAT TAATGTCTAA ACTACTTAAA entry score <--- M00101 CdxA 100.0
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2101 GAACTATATA AACTGAATAC ACTTCAATGA AAGACAAAAA ATATTACAAT entry score <--- M00240 Nkx-2. 90.7
2151 CATAAAGAAA ACTAAGATTC ATCCAATAAA CTATATTACA ATCCCTGTCA entry score <--- M00100 CdxA 96.2
2201 TTCATTTTTT TAAGATCTTC AAACTAGGCA TGAGATAATG GTATACATGA entry score ---> M00077 GATA-3 91.9
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2251 AACATTACAT TTAATCTTTA TTGTAAAGGC CGCCATCTAA TAGATTGATA entry score ---> M00101 CdxA 89.3
2301 ATAAACTAGA CAGACGTGAT TTAAAATTTG TAAAAGAATG CCCAGACTAA entry score ---> M00100 CdxA 88.5
2351 CACTTTCATG ACAGCCAATT ATAGTCAAGC CTAGCAAGCA GTTTGCAACC entry score ---> M00101 CdxA 98.6 ---> M00099 S8 91.2 <--- M00241 Nkx-2. 85.3
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2401 AGACCTTAAG GTAAACTTTT TTTTTTTTTA CAATGAGTTA CAGATTCACA entry score <--- M00073 deltaE 85.0
2451 AGTTTAAGAA GACAAGAAAA AGGAAAACAG AAGGAATCCA GCCACCCAGC entry score ---> M00079 Evi-1 91.7 ---> M00148 SRY 90.0 <--- M00131 HNF-3b 89.0 ---> M00011 Evi-1 88.4
2501 AAATATGAAG CAGACCCCAG AATGTGATAC AGTCCAAAGA TGTGAATTAT entry score ---> M00162 Oct-1 91.8
2551 TGTATATCAT CACTGTTGTT CAGAATTTCA CACAGACTCT TGAGCCAATT entry score <--- M00101 CdxA 92.1 > M00101 CdxA 88.6 - M00101 CdxA 88.6
2601 TTGTTCATTT TTCCACAGAC ACAATAATGA ACTAAAAAGA GGAGGCAAAA entry score ---> M00101 CdxA 97.9 <--- M00159 C/EBP 91.5 ---> M00137 Oct-1 85.8 <--- M00101 CdxA 85.0
2651 AGGCAGAGGT TGAGCGGGGA GTAGAAAGGA AAGCCCTTAA CTGCAGAGCT entry score ---> M00083 MZF1 94.8 <--- M00053 c-Rel 92.6 <--- M00052 NF-kap 86.1
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---> M00241 Nkx-2. 85.3
2701 CTGCTCTACA AATGCTTAAC CTTACAGGAG TTTGGGCTCC TTCAGCATTT entry score <--- M00100 CdxA 87.2 ---- M00100 CdxA 87.2
2751 GTATTCTATC CAAATCCTCA TGAGTCACAA AAATTAAAAA GCTATATCCT entry score ---> M00199 AP-1 94.3
2801 TCTGGATGCC AGGAAAGGCC TTACCACAAG CCTTTTGTGA GAGAAAGAGA entry score <--- M00271 AML-1a 100.0 <--- M00159 C/EBP 88.5 <--- M00053 c-Rel 86.0 ---> M00141 Lyf-1 85.7
2851 GAGAGAGAAA GAGCAAGGGG GAAAAGCCAC AGTGGTAGGC AGTCCCACTT entry score <---- M00240 Nkx-2. 90.7
2901 TACTTAAGAG TACTGTGAGG TCACAAACCA CATGATTCTG CCTCTCCAGT entry score <--- M00271 AML-1a 100.0
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2951 AATAGTGCTT GCAAAAAAAA GGAGTTTTAA AGCTTTTGCT TTTTTGGATT entry score --- M00101 CdxA 93.6 ---> M00101 CdxA 88.6 <--- M00216 TATA 87.8 ---> M00116 C/EBPa 86.9 <--- M00148 SRY 86.4
3001 GTGTGAATGC TTCATTCGCC TCACAAACAA CCACAGAACC ACAAGTGCGG entry score <--- M00271 AML-1a 100.0 <--- M00271 AML-1a 100.0 ---> M00148 SRY 96.4
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--- M00271 AML-1a 92.0 ---> M00240 Nkx-2. 90.7 <--- M00141 Lyf-1 87.0
3051 TGCAAACTTT CTCCAGGAGG ACAGCAAGAA GTCTCTGGTT TTTAAATGGT entry score > M00271 AML-1a 92.0 <--- M00100 CdxA 88.5 ---> M00147 HSF2 85.3
Total 395 high-scoring sites found.
Max score: 100.0 point, Min score: 85.0 point
- 83 - - 국문요약 –
갑상선 유두상 암의 석회화와 암 전이에 관여하는 전사인자 HOXA9 에 대한 연구
아주대학교 대학원 의학과 김 예 란 (지도교수: 정 윤 석)
갑상선 유두상 암은 모든 갑상선 암의 75% ~ 85% 를 차지한다.
석회화는 갑상선 유두상 암의 특징적인 소견으로 진단에 중요한 역할을 한다.
RUNX2 는 중간엽 모세포에서 조골세포로 분화하는데 있어 주요한 유전자로, 갑상선 유두상 암 및 인간 갑상선 유두상 암 세포주인 BHP18-21 에서 RUNX2 유전자 발현이 증가되어 있다는 것이 보고되었으며, RUXN2 유전자 발현에 의해 갑상선 암의 전이가 증가한다는 보고가 있다. 그러나 갑상선 유두상 암에서 어떤 기전을 통하여 RUNX2 의 발현이 증가하는 지 또는 갑상선 유두상 암의 발암과정에서 RUNX2 가 어떤 역할을 하는지에 대한 연구는 부족하다.
본 연구에서는 갑상선 유두상 암에서의 RUNX2 발현 증가 및, 발암과정에 관여하는 새로운 유전자 발굴을 목표로 하였다. 갑상선 정상
- 84 -
- 85 -
본 연구를 통하여 RUNX2 의 양성 조절인자인 HOXA9 은 RUNX2 에 독립적 혹은 종속적으로 갑상선 유두상 암의 석회화와 발암기전에 관여 할 가능성이 있음을 증명하였다.
핵심어 : 갑상선 유두상 암, HOXA9, RUNX2, 석회화, 발암과정