| Backgroud: Tumours of the thyroid are the most common diseasees in our head and neck disease. The papillary thyroid carcinoma derives from the epithelium of the thyroid , which is the most common carcinoma in the thyroid tumors. But our fundamental understanding of the molecular events in thyroid carcinoma is still limited. The development and prognosis of the carcinoma are very complicated. In order to better understand and therapy the papillary thyroid carcinoma, we need to educidate the molecular mechanisms.Recently, a Ras homologue member I(ARHI)has been identified in normal breast and ovarian epithelial cells by differential display PCR, which is a novel imprinted tumour suppressor gene. ARHI can inhibit the cells pass GI phase by inhibiting cyclin-CDK activities. A large number of studies have shown that ARHI is loss or low-expression in breast and ovarian cancers, when contrasted to the normal breast and ovarian epithelial cells. Then these show ARHI is a tumour suppressor gene, which inhibits proliferation of cancer cells. It havs been found that E2F1can bind to the ARHI promoter regien (-385to-409) in ovarian cancer cell lines (SKOV3) by EMSA. And they also show that E2F1 negtively regulates ARHI promoter activity in ovarian cancer cell lines. It has been confirmed that ARHI can form a complex with STAT3 in the cytoplasm by yeast two-hybrid and coimmunoprecipitation, while this can inhibit interleukin-6–induced STAT3 accumulation in the nucleus to inhibit STAT3 binding to DNA and STAT3-dependent promoter activity.Objective: The expression of ARHI, E2F1 and STAT3 protein and ARHI mRNA in the papillary thyroid carcinoma were detected with immunohistochemical staining and RT-PCR respectively. Then, the role and relevance of ARHI, E2F1 and STAT3 in the papillary thyroid carcinoma were analyzed. Method:1 Research objectThe cases used to investigate with complete clinical information were collected from patients diagnosed from 2000 to 2010 at the Second Hospital of HeBei Medical University. 88 cases of papillary thyroid carcinoma, 22 cases of thyroid adnoma, 15 cases of follicular thyroid carcinoma, 12 cases of medullary thyroid carcinoma and 8 cases of undifferentiated thyroid carcinoma were studied. At the same time, 20 cases of normal thyroid tissues were selected as control. Formalin-fixed and paraffin-embedded tissue blocks from all cases were used in this study with immunohistochemical staining. 11 cases of fresh papillary thyroid carcinama, 2 cases of fresh medullary thyroid carcinoma, 1 cases of fresh nodular goiter and 1 cases of fresh Hashimotos thyroiditis were obtained from patients at the Fourth Hospital of HeBei Medical University. Because the cases of medullary thyroid carcinoma, nodular goiter and Hashimotos thyroiditis were limited, so only 11 cases of fresh papillary thyroid carcinama were used to study the ARHI mRNA. At the same time, 10 cases of fresh normal thyroid tissues were used as control. All cases were fresh-frozen and stored at -80℃until processed for RNA extraction.2 Immunohistochemical stainingThe protein expression of ARHI, E2F1, STAT3 in all cases were detected with immunohistochemisty S-P two-step. The procession was carried out acccording to manufacturer's instructions.Hematoxylin stsining, grad alcohol dehydration, dimethyl benzene transparence,neutral gum mounting,observel under light microscope.3 RNA extraction, cDNA synthesis and PCRTotal RNA from fresh tissues was isolated with guanidinium isothiocyanate. The integrity of total RNA was identfied at 90V on 1% agarose gels containing EB(0.5ug/ml). The UV Spectrophotometer was used for the quatitation of the total RNA.Total RNA was revers transcribed using reverse trascriptase with oligoDT at 43℃for 90min to synthsize cDNA.The PCR was carried out in a duplex mixture such as cDNA, TaqDNA polymerase, primers and so on, denaturation for 5 mins, 94℃50s, 62℃30s, 72℃3 0s, 36 circulations. After amplification, each samples was analyzed by 1.5% agarose gel electrophoresis for 40mins. At the same time, we used GAPDH as an internal control.4 Statistical analysisThe experimental data were anlyzed with Chi-square test and correlation with stastics software of SPSS17.0 edition.Results:1 The protein expression of ARHI in the papillary thyroid carcinomaThe positive expression rate of ARHI was 65.91%(58/88) in papillary thyroid carcinoma. The positive expression of ARHI was significantly higher in the cases of papillary thyroid carcinoma than that in the normal thyroid tissues(P<0.05). Then at the same time, The positive expression of ARHI was significantly higher in the cases of papillary thyroid carcinoma than that in thyroid adenoma, medullary thyroid carcinoma and undifferentiated thyroid carcinoma (P<0.05). No signifficant difference was found about the expression of ARHI in papillary thyroid caecinoma and follicular thyroid carcinomas. There were also no signifficant differences among thyroid adenoma, medullary thyroid carcinoma, undifferentiated thyroid carcinoma and normal thyroid tissues on the expression of ARHI.2 The protein expression of E2F1 in the papillary thyroid carcinomaThe positive expression rate of E2F1 was 70.45%(62/88) in papillary thyroid carcinoma, which was signifficantiy higher than that in the normal thyroid tissues(P<0.05). The positive expression rates of E2F1 in normal thyroid tissues, thyroid adnoma, follicular thyroid carcinoma, medullary thyroid carcinoma and undifferentiated thyroid carcinoma were 0%(0/20), 31.82%(7/22), 80%(12/15), 83.33%(10/12) and 50%(4/8) respectively. We could see that the positive expression rates of E2F1 in thyroid adnoma, follicular thyroid carcinoma, medullary thyroid carcinoma and undifferentiated thyroid carcinoma were higher than that in normal thyroid tissues, but the statistical differences were not found among papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma or undifferentiated thyroid carcinoma(P>0.05).3 The protein expression of STAT3 in the papillary thyroid carcinomaThe positive expression rate of STAT3 was 79.55%(62/88) in papillary thyroid carcinoma, which was signifficantiy higher than that in normal thyroid tissues(P<0.05). The positive expression rates of STAT3 in normal thyroid tissues, thyroid adnoma, follicular thyroid carcinoma, medullary thyroid carcinoma and undifferentiated thyroid carcinoma were 35%(7/20), 72.73%(16/22), 86.67%(13/15), 100%(12/12) and 87.5%(7/8) respectively. The positive expression rate of STAT3 in follicular thyroid carcinoma, medullary thyroid carcinoma and undifferentiated thyroid carcinoma were higher than that in normal thyroid tissues (P<0.05), but the statistical differences were not found among papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma or undifferentiated thyroid carcinoma(P>0.05).4 Relationship among ARHI, E2F1 and STAT3 expression in papillary thyroid carcinomaIn papillary thyroid carcinoma, positive correlation could be found between the protein expression of ARHI and E2F1 (r=0.238,P=0.026). No correlation could be found between the protein expression of ARHI and STAT3.5 Relationship among the protein expression of ARHI, E2F1, STAT3 and clinical pathological features in papillary thyroid carcinomaOur studies found that the protein expression of ARHI, E2F1 and STAT3 were not correlated to age, sex and lymphoid node metastasis in papillary thyroid carcinoma.6 ARHI mRNA expression in papillary thyroid carcinomaThe result of RT-PCR showed that ARHI mRNA expression was detected in 8 of 11 papillary thyroid carcinoma, 7 of 10 normal thyroid tissues. The positive expression rates of ARHI mRNA in papillary thyroid carcinoma and normal thyroid tissues were 72.73% and 70% respectively. There was no signifficant difference between papillary thyroid carcinoma and normal thyroid tissues (P>0.05).Conclusion:1 The expression of ARHI, an anti-oncogene in papillary and follicular thyroid carcinoma is significant higher than that in normal thyroid tissues, medullary and undifferentiated thyroid carcinoma, but no significant difference of mRNA, which suggesting that ARHI may play different roles in different tissues, and the up-regulation may be contribute to the oncogenesis of papillary and follicular thyroid carcinoma.2 Compared with normal thyroid tissues, the expression of E2F1 and STAT3 protein were significant higher, which showed that the high expression may play a important role in the oncogenesis of papillary, follicular, medullary and undifferentiated thyroid carcinoma.3 The positive correlation that the up-regulation of ARHI by E2F1 was found in papillary thyroid carcinoma.4 The protein expression of ARHI, E2F1 and STAT3 were not correlated to age, sex and lymphoid node metastasis in papillary thyroid carcinoma.
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