| Hath1 is homologous to the Drosophila atonal and mouse Math1, its location is at 4q22 and its CDS has 1065bp. Hath1 encodes Ath1 which is a basic helix-loop-helix (bHLH) transcription factor. Previous studies in the developing brain and inner ear have shown that Math1 induced cerebellar granule neurons and inner ear hair cells of mice to differentiate, respect- tively. More importantly, Math1 also results mice intestinal secretory cells, including goblet cells, to differentiate and induces Muc2 expression, so Math1 is a critical positive regulator of terminal cell differentiation . How ever, a potential role for Hath1 in colon cancer is yet unclear and remains to be determined. In the course of human colon carcinogenesis, goblet cells have gradually decreased and goblet cells are few or disappearance in colon cancer. The majority of colon carcinomas produce a little mucin except for mucinous adenocarcinoma and signet-ring cell carcinoma. Therefore, we presume that Hath1 plays an important role in colon carcinogenesis. To confirm the hypothesis,we performed following experiments.We collected samples from 12 cases of colon adenocarcinomas not inclu- ding mucinous adenocarcinomas and signet-ring cell carcinomas. Each sample included normal mucosa, mucosa adjacent to cancer and cancer from the same patient. We used Real Time RT-PCR and S-P immunohistochemistry to detect Hath1 mRNA, Muc2 mRNA and Ath1, Muc2 respectively. Simultaneously, we also detected these indexes expressing in HT29 cells which belong to colon carcinoma cell line. To determine whether introduction of Hath1 would alter the proliferative ability of colon cancer cells, HT29 colon cancer cells were transfected with pcDNA3.1(+)-Hath and pcDNA3.1(+) respectively. Following G418 selec- tion, we obtained an clone each transfection. Then, Real Time RT-PCR and immunocytochemistry were used to determine whether the clone trans- fected pcDNA3.1(+)-Hath has higher expression of Hath1 mRNA and Ath1 comparing with the expression in untransfected HT29. When we confirmed the clone transfected pcDNA3.1(+)-Hath1 can express Hath1 stably, we used colony formation assay in soft agar and xenografts in athymic nude mice to observe whether introduction of Hath1 would alter the proliferative ability of HT29 cells. By Real Time RT-PCR and immunocytochemistry, we compared the level of Muc2 mRNA, Cyclin D1 and P27 between trans- fected HT29 and untransfected HT29.Results:⑴In normal mucosa, mucosa adjacent to cancer and cancer, the relative levels of Hath1 mRNA were 6.15±1.83, 3.37±1.27, 0.35±0.12 respectively (P<0.01), the relative levels of Muc2 mRNA were 2.24×105±7.39×104, 5.12×104±6.19×103, 8.03±0.97 respectively (P<0.01), the area density of Ath1 positive region was 0.1716±0.0761, 0.1585±0.0961 and 0.0598±0.0162 respectively,the differences of Ath1 expression were signi- ficant between cancer group and other two groups(P<0.05), and the area density of Muc2 positive region was respectively 0.1634±0.0515, 0.1611±0.0753 and 0.0652±0.0259, the differences of Muc2 were significant between cancer group and other two groups (P<0.05).⑵In HT29 cells, the relative levels of Hath1 mRNA and Muc2 mRNA were respectively 2.0×10-2±7.63×10-3, 2.46×10-4±1.0×10-4, they were very low.⑶In group untransfected , group transfected pcDNA3.1(+) and group transfected pcDNA3.1(+)-Hath1, the relative levels of Hath1 mRNA were 2.0×10-2±7.63×10-3, 1.96×10-2±6.62×10-3 , 507.83±115.93, respectively, the level in group transfected pcDNA3.1(+)-Hath1 was significantly higher (P<0.05) than in other two groups, and relative levels of Muc2 mRNA were res- pectively 2.46×10-4±1.0×10-4, 2.43×10-4±9.57×10-3, 3.41×10-2±9.93×10-3,the expression in group transfected pcDNA3.1(+)-Hath1 was significantly up- regulated (P<0.01).⑷In group untransfected, group transfected pcDNA3.1(+) and group transfected pcDNA3.1(+)-Hath1, the relative area density of Ath1 positive region were respectively 0.2094±0.0667, 0.2153±0.0694 and 0.4736±0.0895, the expression in group transfected pcDNA3.1(+)-Hath1 was significantly higher (P <0.005), the relative area density of Cyclin D1 positive region was 0.0422±0.0122, 0.0389±000108 and 0.0197±0.0075, the level in transfected pcDNA3.1(+)-Hath1 was significantly down- regulated (P <0.005), the relative area density of P27 positive region was 0.0143±0.0064, 0.0136±0.0071 and 0.0377±0.0161, the P27 expression in group transfected pcDNA3.1(+) was significant up-regulation (P<0.001).⑸By xenografts in athymic nude mice, we found the mean tumor volume of group transfected pcDNA3.1(+)-Hath1 was smallest (P <0.005), its mean tumor volume was (225.4±168.4)mm3, otherwise, (3077.8±1717.7) mm3 of group untransfected, (2740.8±1038.5) mm3 of group transfected pcDNA3.1(+).⑹Colony formation assay in soft agar shown that cloning efficiency was (3.83±0.75)% of group transfected pcDNA3.1(+)-Hath1, (9.33±1.21)% of untransfected group and (8.50±1.05)% of group transfected pcDNA3.1(+), the proliferation of HT29 cells transfected pcDNA3.1(+)-Hath1 was significantly inhibited (P<0.001).Accordingly, we can conclude:⑴The expression of Muc2 and Hath1 is down-regulated in human colon cancer and HT29 cells.⑵Hath1 can inhibit the proliferation of HT29 cells.⑶Up-regulating expression of Hath1 can result down-regulation of Cyclin D1 and up-regulation of P27.⑷Muc2 is a downstream gene of Hath1. In a word, down-regulation of Hath1 may concern with colon cancer. Hath1 can increase the expression of P27 and decrease the expression of Cyclin D1, this may be one of the reasons that Hath1 inhibit the proliferation of colon carcinoma cell line. |
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