III. RESULTS
3. HOXA9 is associated with thyroid cell migration and invasion
To evaluate the effect of HOXA9 in Nthy-Ori 3-1, TPC1, and BHP10-3 cell migration and invasion, wound healing assay and invasion assay were performed.
HOXA9-overexpressing cells migrated more than control cells, whereas in the normal Nthy-Ori 3-1 cell line, HOXA9 knockdown suppressed migration; however, this difference was not significant. In PTC cells, wound healing ability was increased by a small amount marginally in HOXA9-overexpressing cells compared to that in control cells and was significantly decreased in HOXA9-knockdown cells (Figure 9 A, B).
Furthermore, cell invasion was enhanced in HOXA9-overexpressing groups and reduced in HOXA9-knockdown groups, and the results expressed the similar appearance compare to outcome of the wound-healing assay (Figure 10 A, B). These data indicate that HOXA9 can mediate migration and invasion in two types of thyroid cells.
- 35 -
Figure 9. Migration ability of thyroid cells based on HOXA9 expression. (A) Thyroid cells were seeded in 6-well plates (6 x 10⁴ cells/well) with 2 ml of complete growth media. After 24 h, wound-healing assays were performed, and the wells were photographed every 12 h to monitor wound closure. (B) The numbers of migrating cells into the wound were counted under a microscope. Experiments were repeated three times.
Error bars represent standard deviation (n = 3 biological replicates). * p < 0.05 vs.
Control.
- 36 -
Figure 10. Invasion ability of thyroid cells based on HOXA9 expression. (A) Thyroid cells were transferred to the top of Matrigel-coated chambers with 3 x 10⁴ cells/well in serum-free media. After 24 h of incubation, the invaded cells were fixed with methanol
- 37 -
and stained with 0.5% crystal violet. (B) The numbers of invading cells was were counted under a microscope. Experiments were repeated three times. Error bars represent standard deviation (n = 3 biological replicates). * p < 0.05 vs. Control.
- 38 -
4. HOXA9 and RUNX2 can be simultaneously detected in calcified PTC tissues
To confirm the results of in vitro promoter, calcification and carcinogenesis assays, next I assessed cancer tissue specimens from thyroid carcinoma patients (Table 4). First, I performed haematoxylin and eosin (H&E) staining using paraffin tissue slides to detect cancer tissues and calcification. In the set of 42 cancer tissue samples, 25 showed calcification (Figure 11 A, B). Next, RUNX2 and HOXA9 were individually evaluated by immunohistochemistry (IHC) in serial tumor tissue slides in 25 samples which with calcification. Among these, RUNX2 was detected in 12 cases and HOXA9 was apparent in 13 cases (Figure 11 B~D). Among these 25 samples, two samples expressed RUNX2 only (Figure 11 C), whereas 3 samples had HOXA9 expression alone (Figure 11 D).
Intriguingly, RUNX2 and HOXA9 were simultaneously detected with calcification in 10 cancer tissues (Figure 11 A, B). These data suggest that HOXA9 and RUNX2 are linked to calcification and carcinogenesis in thyroid tumor.
- 39 -
- 40 -
Figure 11. The expression of HOXA9 and RUNX2 in thyroid cancer tissues. (A) H&E staining was performed using the Dako coverstainer HE autostainer. RUNX2 and HOXA9 were detected by immunohistochemistry; immunostaining is in red. Image
- 41 -
analysis was performed using SPOT Software V4.0 and Image Pro plus® . (B) Numbers of cases based on staining. Among the 42 cancer tissues, 25 cases had concurrent calcification. In these 25 cases, RUNX2 was expressed in 12 cases and HOXA9 in 13 cases. Both RUNX2 and HOXA9 were simultaneously expressed in 10 cancer tissues with calcifications. (1: calcification, 2: cancer tissue.) Scale bar: (A) 200 μm, (C, D) Tissue sample: 1000 μm; calcification: 50 μm; cancer tissue: 200 μm.
- 42 -
5. HOXA9 increase PTC calcification and tumor invasion directly or indirectly via RUNX2
To determine whether the HOXA9-mediated effect on enhanced calcification and carcinogenesis is associated with RUNX2, I utilized a RUNX2-knockdown system and HOXA9-overexpression system with the normal cell line Nthy-Ori 3-1 and PTC cell lines TPC1 and BHP10-3 (Figure 12).
Next, I performed mineralization assays, as previously described, on these cells.
ALP activities were significantly decreased in all RUNX2-downregulated cells (Figure 13 A, B). Further, mineralization was also suppressed in all RUNX2-knockdown groups (Figure 13 A, C). ALP activity in Nthy-Ori 3-1 and BHP10-3 cells was marginally enhanced with RUNX2 knockdown and HOXA9 overexpression compared to that RUNX2-knockdown in control cells, and this difference was statistically significant (*p <
0.05, **p < 0.005; Figure 13 B ).
Subsequently, wound healing assay and transwell assay were performed. Similar to the results of mineralization assay, migration was considerably decreased in RUNX2-knockdown cells compared to that in controls (Figure 14 A, B). Likewise, RUNX2 knockdown cells also suppressed invasion capacity in the three thyroid cell lines.
Additionally, invasion ability was enhanced in the groups of RUNX2-knockdown cells with HOXA9 overexpression compared to that in respective RUNX2-knockdown control cells (Figure 15 A, B).
- 43 -
These data suggest that HOXA9, as a positive regulator of RUNX2, can enhance calcification and tumor migration and invasion in normal thyroid cell and PTC cells, dependent or independent of RUNX2.
- 44 -
- 45 -
Figure 12. The expression of RUNX2 and HOXA9 in thyroid cell lines. RUNX2 was knockdowned in three control and HOXA9-overexpressing cell lines. Protein expression was assessed by western blotting (#1: Control, #2: shRUNX2, #3: HOXA9-overexpressing, #4: HOXA9-overexpressing/shRUNX2).
- 46 -
Figure 13. Effect of HOXA9 overexpression and shRUNX2 on osteoblast differentiations. (A) Alkaline phosphatase (ALP) staining (upper layer) on the 7th day and Alizarin red S staining (lower layer) on the 10th day for Nthy-Ori 3-1 cells and on the 14th day for papillary thyroid carcinoma (PTC) cells (TPC1 and BHP10-3). (B, C) Quantitative data for ALP and ARS staining in thyroid cells with control, shRUNX2, HOXA9-overexpressing, and HOXA9-overexpressing/shRUNX2 conditions. Error bars represent standard deviation (n = 3 biological replicates). *p < 0.05, **p < 0.005 vs.
control.
- 47 -
- 48 -
Figure 14. Migration ability based on HOXA9 and RUNX2 expression in thyroid cells. (A) Migration ability was demonstrated by wound-healing assay. Cells were seeded in 6-well plates (6 x 104 cells/well), and after 24 h, wound-healing assays were performed and the wells were photographed every 12 h to monitor wound closure. (B) The number of migrated cells into the wound were counted under a microscope. Experiments were repeated three times. Error bars represent standard deviation (n = 3 biological replicates).
* p < 0.05 vs. control, ** p < 0.005 vs. control.
- 49 -
Figure 15. Invasion ability based on HOXA9 and RUNX2 expression in thyroid cells.
(A) Thyroid cells were transferred to the top of Matrigel-coated chambers with 3 x 10⁴ cells/well in serum-free media. After 24 h of incubation, the invaded cells were fixed with methanol and stained with 0.5% crystal violet. (B) The number of cells that invaded
- 50 -
through the filter was counted under a microscope. Experiments were repeated three times. Error bars represent standard deviation (n = 3 biological replicates). * p < 0.05 vs.
control, ** p < 0.005 vs. control.
- 51 -
IV. DISCUSSION
In this study, I screened upstream mediator genes of RUNX2, and found one candidate as a primary candidate, HOXA9, which regulates the expression of RUNX2 in two types of thyroid cell lines. Overexpression of HOXA9 was found to enhance ALP activity and mineralization, as well as in vitro tumor cell migration and invasion, while downregulation of this marker inhibited these processes. Moreover, the expression of RUNX2 and HOXA9 were confirmed by IHC in calcified malignant thyroid tumor tissues and the simultaneous expression of both markers was found in the calcified cancer tissues. Furthermore, cells exhibited enhanced migration and invasion in RUNX2-knockdown cells with HOXA9 overexpression compared to those in RUNX2-RUNX2-knockdown control cells. This suggests that HOXA9 could be linked to the calcification and tumor invasion of PTC, which is independent or dependent of RUNX2.
RUNX2 is the master regulator in osteoblast differentiation (Pratap et al., 2003;
Soltanoff et al., 2009; Sancisi et al., 2012; Gong et al., 2013). It was also reported to have a role in thyroid carcinogenesis. Specifically, levels of this marker were found to be significantly higher in larger PTC tumors (Gong et al., 2013). Moreover, RUNX2 regulates cellular invasion in thyroid tumor cells (Sancisi et al., 2012). However, there have been few reports regarding regulators of RUNX2 during thyroid calcification and carcinogenesis. Accordingly, I identified one candidate, HOXA9, and confirmed that it regulates the expression of RUNX2 in thyroid cell lines. Homeobox (HOX) family genes encode a class of transcription factors that regulate the expression of numerous genes,
- 52 -
control cell growth, and drive specific tissue differentiation (Grier et al., 2005). Further, the expression of HOX family genes is often dysregulated in tumors (Samuel and Naora, 2005). HOXA9, a transcription factor, is most commonly altered in solid tumors (Bhatlekar et al., 2014). To demonstrate the function of HOXA9 in PTC calcification, I performed ALP and ARS staining assays. ALP activity and calcification were enhanced in overexpressing Nthy-Ori 3-1 and TPC1 cells and reduced in HOXA9-knockdown TPC1 and BHP10-3 cells. These results indicated that calcification could be regulated by HOXA9 in thyroid cell lines.
Many studies have established the function of HOXA9 in cancer cells. There are some reports indicating that knockdowned the expression of HOXA9 can significantly decrease colony formation, invasion, and migration in colorectal cancer cells (Gaspar et al., 2010; Wang et al., 2017; Bhatlekar et al., 2018). However, some others reported that HOXA9 inhibits migration and that the hypermethylation of HOXA9 is especially apparent in the early stages of lung cancer (Hwang et al., 2011; Wrangle et al., 2014;
Hwang et al., 2015). Moreover, low expression of this marker was observed in cervical cancer cells and proliferation and migration were suppressed when HOXA9 expression was restored in these cells (Alvarado-Ruiz et al., 2016). This protein was also found to restrict cell growth, survival, and invasion in T4-2 breast cancer cells (Gilbert et al.,
- 53 -
microenvironment for tumor growth (Ko et al., 2012). However, there have been no reports regarding the role of HOXA9 in thyroid carcinoma. This study showed that the overexpression of HOXA9 significantly enhances in vitro normal cell migration and invasion and that tumor cell migration and invasion can be inhibited by downregulating HOXA9. The migration and invasion ability was increased in HOXA9-overexpressing cells more than control cells in the normal Nthy-Ori 3-1 cell line; whereas this difference was not significant in PTC cell lines. However HOXA9 knockdowned group considerably suppressed the cancer ability compare to the control groups in PTC cell lines, likewise the difference was not significant in normal cell line. About thses results, I infer that TPC1 and BHP10-3 cell lines are cancer cell line, and they already has the high level ability of tumor aggressiveness, therefore, HOXA9 not impressed PTC cell lines as concurrently expressed in calcified thyroid cancer tissues indicating that HOXA9, as a positive regulator of RUNX2, could be linked to the calcification and tumor invasion of thyroid cancer via RUNX2.
As stated, HOXA9 is an upstream regulator of RUNX2. Therefore, I investigated whether its effect on enhanced calcification and carcinogenesis occurs through RUNX2.
- 54 -
Thus, I downregulated RUNX2 in two types of thyroid cell lines with or without HOXA9 overexpression. My results indicated that ALP activity, mineralization behaviour, and cell migration and invasion capacity were all significantly decreased upon RUNX2 downregulation. These results were similar to a previous report showing that the of PTC not only via RUNX2, but also independently of this marker.
HOXA9, a positive regulator of RUNX2, can enhance calcification and cancer migration and invasion ability in PTC, dependent or independent of RUNX2. My data improved the understanding of the molecular mechanisms of calcification and tumorigenesis in PTC and might lead to the development of novel diagnostic or prognostic biomarkers for this disease.
- 55 -
V. CONCLUSION
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.
- 56 -
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.
- 57 -
REFERENCES
1. Alvarado-Ruiz L, Martinez-Silva MG, Torres-Reyes LA, Pina-Sanchez P, Ortiz-Lazareno P, Bravo-Cuellar A, Aguilar-Lemarroy A, Jave-Suarez LF: HOXA9 is Underexpressed in Cervical Cancer Cells and its Restoration Decreases Proliferation, Migration and Expression of Epithelial-to-Mesenchymal Transition Genes. Asian Pac J Cancer Prev 17: 1037-1047, 2016
2. Bhatlekar S, Fields JZ, Boman BM: HOX genes and their role in the development of human cancers. J Mol Med (Berl) 92: 811-823, 2014
3. Bhatlekar S, Viswanathan V, Fields JZ, Boman BM: Overexpression of HOXA4 and HOXA9 genes promotes self-renewal and contributes to colon cancer stem cell overpopulation. J Cell Physiol 233: 727-735, 2018
4. Carcangiu ML, Zampi G, Pupi A, Castagnoli A, Rosai J: Papillary carcinoma of the thyroid. A clinicopathologic study of 241 cases treated at the University of
7. Cotran RS, Kumar V, Collins T, Robbins SL: Robbins pathologic basis of disease.
- 58 - 12. Gaspar N, Marshall L, Perryman L, Bax DA, Little SE, Viana-Pereira M, Sharp
SY, Vassal G, Pearson AD, Reis RM, Hargrave D, Workman P, Jones C: MGMT-independent temozolomide resistance in pediatric glioblastoma cells associated with a PI3-kinase-mediated HOX/stem cell gene signature. Cancer Res 70:
- 59 -
14. Gong T, Wang J: The analysis of the calcification in differentiating malignant thyroid neoplasm and the molecular mechanisms for the formation of the calcification. Lin chuang er bi yan hou tou jing wai ke za zhi= Journal of clinical otorhinolaryngology, head, and neck surgery 26: 763-766, 2012
15. Gong T, Wang J, Qian M, Zhou Y: [Detection of Runx2 mRNA expression using relatively real-time RT-PCR in papillary thyroid carcinoma]. Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 27: 193-195, 2013
16. Grier DG, Thompson A, Kwasniewska A, McGonigle GJ, Halliday HL, Lappin TR: The pathophysiology of HOX genes and their role in cancer. J Pathol 205:
154-171, 2005 hypermethylation is associated with recurrence in non-small cell lung cancer.
Mol Carcinog 54 Suppl 1: E72-80, 2015
19. Hwang SH, Kim KU, Kim JE, Kim HH, Lee MK, Lee CH, Lee SY, Oh T, An S:
Detection of HOXA9 gene methylation in tumor tissues and induced sputum samples from primary lung cancer patients. Clin Chem Lab Med 49: 699-704, 2011
- 60 -
20. Johannessen JV, Sobrinho-Simoes M: The origin and significance of thyroid psammoma bodies. Lab Invest 43: 287-296, 1980
21. Ko SY, Barengo N, Ladanyi A, Lee JS, Marini F, Lengyel E, Naora H: HOXA9 promotes ovarian cancer growth by stimulating cancer-associated fibroblasts. J Clin Invest 122: 3603-3617, 2012
24. Lloyd RV: Endocrine Pathology:: Differential Diagnosis and Molecular Advances, Springer Science & Business Media, 2010
25. Morgan R, El-Tanani M: HOX Genes as Potential Markers of Circulating Tumour Cells. Curr Mol Med 16: 322-327, 2016
26. Morgan R, El-Tanani M, Hunter KD, Harrington KJ, Pandha HS: Targeting HOX/PBX dimers in cancer. Oncotarget 8: 32322-32331, 2017
27. Niu DF, Kondo T, Nakazawa T, Oishi N, Kawasaki T, Mochizuki K, Yamane T, Katoh R: Transcription factor Runx2 is a regulator of epithelial-mesenchymal transition and invasion in thyroid carcinomas. Lab Invest 92: 1181-1190, 2012 28. Pojo M, Goncalves CS, Xavier-Magalhaes A, Oliveira AI, Goncalves T, Correia
S, Rodrigues AJ, Costa S, Pinto L, Pinto AA, Lopes JM, Reis RM, Rocha M,
- 61 -
Sousa N, Costa BM: A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide.
Oncotarget 6: 7657-7674, 2015
29. Pratap J, Galindo M, Zaidi SK, Vradii D, Bhat BM, Robinson JA, Choi JY, Komori T, Stein JL, Lian JB, Stein GS, van Wijnen AJ: Cell growth regulatory role of Runx2 during proliferative expansion of preosteoblasts. Cancer Res 63:
5357-5362, 2003
30. Pratap J, Lian JB, Stein GS: Metastatic bone disease: role of transcription factors and future targets. Bone 48: 30-36, 2011
31. Samuel S, Naora H: Homeobox gene expression in cancer: insights from developmental regulation and deregulation. Eur J Cancer 41: 2428-2437, 2005 32. Sancisi V, Borettini G, Maramotti S, Ragazzi M, Tamagnini I, Nicoli D, Piana S,
Ciarrocchi A: Runx2 isoform I controls a panel of proinvasive genes driving aggressiveness of papillary thyroid carcinomas. J Clin Endocrinol Metab 97:
E2006-2015, 2012
33. Shrestha RT, Karunamurthy A, Amin K, Nikiforov YE, Caramori ML: Multiple mutations detected preoperatively may predict aggressive behavior of papillary thyroid cancer and guide management—a case report. Thyroid 25: 1375-1378, 2015
34. Sipos JA, Mazzaferri EL: Thyroid cancer epidemiology and prognostic variables.
Clin Oncol (R Coll Radiol) 22: 395-404, 2010
- 62 -
35. Soltanoff CS, Yang S, Chen W, Li YP: Signaling networks that control the lineage commitment and differentiation of bone cells. Crit Rev Eukaryot Gene Expr 19: 1-46, 2009
36. Trotter TN, Li M, Pan Q, Peker D, Rowan PD, Li J, Zhan F, Suva LJ, Javed A, Yang Y: Myeloma cell-derived Runx2 promotes myeloma progression in bone.
Blood 125: 3598-3608, 2015
37. Underwood JC, Cross SS: General and Systematic Pathology, International Edition E-Book: with STUDENT CONSULT Access, Elsevier Health Sciences, 2009
38. Wang X, Bu J, Liu X, Wang W, Mai W, Lv B, Zou J, Mo X, Li X, Wang J, Niu B, Fan Y, Hou B: miR-133b suppresses metastasis by targeting HOXA9 in human colorectal cancer. Oncotarget 8: 63935-63948, 2017
39. Wrangle J, Machida EO, Danilova L, Hulbert A, Franco N, Zhang W, Glockner SC, Tessema M, Van Neste L, Easwaran H, Schuebel KE, Licchesi J, Hooker CM, Ahuja N, Amano J, Belinsky SA, Baylin SB, Herman JG, Brock MV: Functional identification of cancer-specific methylation of CDO1, HOXA9, and TAC1 for the diagnosis of lung cancer. Clin Cancer Res 20: 1856-1864, 2014
40. Wu G, Guo J-J, Ma Z-Y, Wang J, Zhou Z-W, Wang Y: Correlation between calcification and bone sialoprotein and osteopontin in papillary thyroid carcinoma. International journal of clinical and experimental pathology 8: 2010-2017, 2015
- 63 -
41. Wysokinski D, Blasiak J, Pawlowska E: Role of RUNX2 in Breast Carcinogenesis. Int J Mol Sci 16: 20969-20993, 2015
42. Zambotti A, Makhluf H, Shen J, Ducy P: Characterization of an osteoblast-specific enhancer element in the CBFA1 gene. J Biol Chem 277: 41497-41506, 2002
- 64 -
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
- 65 -
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
- 67 -
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
- 70 -
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
- 71 -
---> 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
- 73 -
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
1451 AAGAACACAG GGATATTTTA GTTCTAACAC CCTCCTGTCT CCCTAGCCCT entry score