The Expression And Clone Of Genes Related With Oncogenesis And Development Of Thyroid Carcinoma

Objective: It has been known that combined biological effects of multiple factors are involved in the carcinogenesis of the tumors. The genes, from the effects of carcinogen factors to the mutation of inherit genes, including the mutation of oncogenes and tumor suppressor genes, cell cycle genes regulating proliferation and differentiation, apoptosis related genes, vascular growth factors and receptors, matrix metalloproteinase and tissue inhibitor of metalloproteinase genes, are all related closely with oncogenesis and development of tumor. Thyroid carcinoma is a familiar malignant tumor, the incidence rate is 1.3% of all malignant tumor, it threaten the human health seriously. The study on pathogeny, oncogenesis, development and prognosis of thyroid carcinoma, as well as the possible mechanism of thyroid cancerization and metastasis, have crucial clinical significance for reducing the occurance rate, improving forepart diagnosis rate, guiding treatment and improving prognosis. At present, the study on molecular mechanism of thyroid carcinoma in this field is not enough and limited to single oncogene, furthermore, there is little report on cloning genes related with thyroid carcinoma by DD-RTPCR. Therefore, in order to explore the function of cell cycle regulatory genes in the carcinogenesis of thyroid tumors, we detect the DNA content, cellular proliferation activity and expression of CyclinD1, CDK4, Rb, p27, bcl-2 and PCNA in normal thyroid tissue (NT), nodular goiter (NG), thyroid adenoma (TA) and papillary carcinoma of the thyroid (PTC); in order to explore the mechanism of invasiveness and metastasis, we detect the expression of MMP-9, TIMP-1mRNA and MMP-9, TIMP-1, VEGF, Bcl-2, PCNA protein in different thyroid tissues and different clinopathological factors (age, gender, tumor size, clinical stage, lymph node, prognostic index) of PTC; in order to explore the pathogeny, we try to separate new genes related with thyroid carcinoma by DD-RTPCR. Methods: 1 Studies on cell cycle regulatory genes in thyroid tumor tissues The paraffin-embedded tissue specimens were made into single-cell suspension and the nuclear DNA was fluorescence-stained with propidium iodide. DNA content, ploidy and cell cycle of thyroid cells were detected by flow cytometry (FCM). At the same time, we analysised quantitatively the expressions of CyclinD1, CDK4, Rb, p27, bcl-2, PCNA labeled by indirect immunofluorescence by FCM, respectively. 2 Studies on genes related with invasiveness and metastasis in thyroid carcinoma The specimens of thyroidectomy were freezed by liquid nitrogen for in situ hybridization (ISH) experiment. Immunohistochemistry (IHC) were carried out in pathological paraffin specimens. All of the specimens were stained with hematoxylin-eosin (HE), and then diagnosed by pathologists again, respectively. All of the clinical pathology datum of PTC cases were collected. The expressions of MMP-9, TIMP-1mRNA were detected by ISH, and expressions of MMP-9, TIMP-1, VEGF, Bcl-2, PCNA protein were detected by IHC. The results of morphologic were measuread by Motic color pathological image analysis system, and the average optical density values were calculated by statistics. 3 Clone genes related with thyroid carcinoma Total RNA was extracted from four kinds of specimens of thyroidectomy by the use of Trizol reagent. The quality of RNA was confirmed on a 1.2% formaldehyde agarose gel to check the integrality and purity. RT-PCR reaction was done using binding primer APA and random primer A1. After amplification,PCR reaction product was analyzed by electrophoresis on a 15% agarose gel and radioselfdeveloping. The amplification fragment was cloned into plasmid vector pGEM-T,The recombinant plasmid was amplifiedin E. coli and then extracted for sequence analyzing. The sequenced results were searched in NCBI Gene Bank. 4 Statistic method ANOVA and χ² test were used for the determination of statistic significance as appropriate by statistical package for social science (SPSS) 10.0. Data are presented as mean±SEM. Statistical significance was assigned at a P value <0.05. Results: 1 Analysis of DNA content, ploidy and cell cycle and expression of cell cycle regulatory genes in thyroid tumor tissues DNA content and heteroploid rate increased while the percentage of diploid decreased among NT, NG, TA and PTC group. For example, DNA index (DI, 1.225±0.177) of PTC increased obviously comparing to other groups such as NT group(1.000±0.006), NG(1.017±0.062)and TA group(1.090±0.090), respectively (P<0.01). There was no heteroploid cell in normal thyroid group, and the heteroploid rate was 9.52% in NG group, 18.75% in TA group, 80% in PTC group. The percentage of cells in G₀/G₁ phase decreased significantly while the percentage of cells in S phase and G₂/M phase increased, and the proliferation index (PI) enhanced in PTC cells. The expressions of CyclinD1, CDK4, PCNA, Bcl-2 enhanced gradually in normal thyroid, thyroid benign node and PTC. The expression in PTC was up-regulated comparing to other groups such as NT group, NG or TA group, respectively (P<0.01), while the difference among the last three groups was not significant (P>0.05). The expression of p27 was down-regulated in PTC, TA, NG groups comparing to NT group, respectively (P<0.05), there was not significant difference among PTC, TA and NG groups (P>0.05). However, the difference of Rb expression was not significant among the four groups (P>0.05). 2 Expressions of MMP-9, TIMP-1, VEGF, genes related with proliferation and apoptosis in thyroid tumor tissues and relations with different clinopathological factors of PTCMMP-9, TIMP-1 mRNA and MMP-9, TIMP-1 protein all showed up-regulation in PTC, TA and NG groupS. Compared with normal thyroid, the expression of MMP-9 and TIMP-1 protein and mRNA increased remarkably (P<0.05 or P<0.01). Among PTC, NG and TA, MMP-9 protein and mRNA in PTC group expressed the strongest positive (P<0.05), but TIMP-1 protein and mRNA have not distinct changes (P>0.05). On the other hand, MMP-9 protein and mRNA have positive correlation with tumor size, clinical stage, lymph node, prognostic index of PTC (P<0.01 or P<0.05), that is to say, MMP-9 protein and mRNA level are higher in tumor size≥20mm, with lymph node, PI≥65 cents and clinical stage â…¢,â…£than those in tumor size<20mm, without lymph node, PI<65 cents and clinical stage â… ,â…¡. On the contrary, TIMP-1 protein and mRNA have negative correlation with tumor size, clinical stage, lymph node and prognostic index of PTC (P<0.01 or P<0.05). Compared with normal thyroid, VEGF has different up-regulation degree in PTC, NG and TA tissues (P<0.05), however, there are not obvious difference in the three types of thyroid tumor (P>0.05). The level of VEGF has positive correlation with tumor size, clinical stage, lymph node, prognostic index of PTC (P<0.01or P<0.05). Compared with normal thyroid, Bcl-2 protein level only increases in PTC tissue (P<0.01), and PCNA protein level shows up-regulation in PTC and TA tissue (P<0.05). Compared with PTC, both of Bcl-2 and PCNA protein level are decreased in 2 kinds of benign tumor (TA and NG, P<0.01). The level of Bcl-2 and PCNA protein level has positive correlation with tumor size, clinical stage, lymph node, and prognostic index of PTC. That is to say, the malignant of tumors as well as the worse of prognostic are corresponding to the high level of Bcl-2 and PCNA protein (P<0.01 or P<0.05). 3 Clone thyroid carcinoma genes by DD-RTPCR After sequence analyzed, there are eight fragments and primers related to the data of prophase. When we searched in GenBank for homologous analysis,A61 fragment (287bp) has 76 percent homology with Homo sapiens cDNA, UDP-GLUCOSE 6-DEHYDROGENASE, A62 fragment (284bp) has99 percent homology with Homo sapiens lectin mannose-binding 1 (LMAN1), C31 (278bp) has 99 percent homology with Homo sapiens ribosomal protein L23 (RPL23), B61 (179bp) has 95 percent homology with Homo sapiens chromosome 3 clone RP11-79P21, B62(274bp) has 100 percent homology with Homo sapiens lectin, mannose-binding, 1 (LMAN1), B32 (231bp) has 100 percent homology with Homo sapiens complete mitochondrial genome, haplotype B4a2, B51(201bp) has 99 percent homology with Homo sapiens ribosomal protein L35 (RPL35) pseudogene, B81(133bp) has 100 percent homology with Homo sapiens hypothetical protein LOC51234. Conclusions: The DNA content and heteroploid rate increased gradually in NT, NG, TA, PTC. The proliferation index increased while the cells of G0/G1 phase decreased in the cells of S phase, and the cells in G2/M phase increased significantly in PTC. The DNA quantitative detection by FCM may be one of the biomarkers of earlycarcinogenesis of thyroid and the distinction of benign and malignant. p27-CyclinD1/CDK4-pRb formed an access to regulate cell cycle. Positive regulatory genes, CyclinD1/CDK4, play important roles in oncogenesis of PTC and act as one of the biomarkers to distinguish benign and malignant in thyroid tumors. On the other hand, negetive regulatory genes, p27 and Rb, may not play important roles in oncogenesis of PTC. PCNA and Bcl-2 protein level are greatly higher in PTC than that in NT, NG and TA, and they have positive correlation with clinical pathology character. The result indicates that PCNA and Bcl-2 have close relation with oncogenesis, invasion and metastasis of PTC. PCNA and Bcl-2 protein level may be one of the biomarkers of early carcinogenesis of thyroid and distinction of benign and malignant.The way of apoptosis induced selectively perhaps become a good method for malignancy therapeutics. MMP-9mRNA and protein level have up-regulated trend in NT, NG, TA and PTC tissues, and have positive correlation with clinical pathology character (tumor size, clinical stage, lymph node and prognostic index). TIMP-1mRNA...