| Thyroid cancer is the most commom endocrinal and craniocervical malignancy, among which papillary thyroid carcinoma (PTC) is the most comman type. Along with the advance of diagnostic techniques, the incidence rate of thyroid cancer is significantly increasing. As a developing country, China is facing a huge challenge of preventing and treating thyroid cancer.With far-reaching molecular and immunological stydies on carcinogenesis, the known mechanisms underly the onset and progression of thyroid cancer have been switched from simple morphological changes to genic alterations. The mechanisms underlying the abnormal gene expression is complicated, including genetic, epigenetic, environmental and susceptible factors. It has been well accepted that genetic alterations play significant roles in the carcinogenesis. However, recent findings suggested epigenetic modifications, such as abnormal gene promotor DNA methylation which may partly replace the role of gene mutation, is identified to be an essential, common mechanism in many kinds of tumors. Hence, epigenetic modifications could work together with genetic alterations during the genesis and progression of tumorigenesis.Recent studies have focused on the genes related to the initiation, progression and prognosis of thyroid cancer. Down-regulation or inactivation of Deleted in liver cancer-1(DLC1) is crucial for initiation, progression and metastasis of many types of tumors. Loss of heterozygosity and DNA methylation are the main two inactivation pathway of DLC1. However, up to now, few reports about the relationships among DLC1gene expression, promotor DNA methylation and the metastasis of PTC can be found in pulished papers. Hence, an in-depth study of DLC1will be useful for understanding the mechanisms of initiation, progression and metastasis of PTC, as well as provide novel insights into the therapeutic strategies for PTC. This research planed to deeply study the biological features of DLC1gene in PTC, including the mRNA and protein level of DLC1and also addressed the significance of DLC1in clinical samples from human patients and explore the potential epigenetic mechanisms beneath the DLC1aberrant expression. In addition, on the basis of above-mentioned studies, in order to study the interrelationship between DLC1gene expression and lymphangiogenesis, this research further explored the mechanism underlying DLC1-induced regulation of lymphangiogenesis. This study will provide the theoretical clues for understanding its tumor suppressor function, and for applying DLC1as a new target for anti-thyroid cancer therapies.This study mainly included the following two parts:Part1:Expression and promoter methylation of the Deleted in liver cancer-1gene in papillary thyroid carcinoma and pericarcino-matous thyreoid tissue and its clinicopathological signifycanceObjective:To investigate the expression and promoter methylation of DLC1gene in PTC and pericarcinomatous thyreoid tissue (PCTT) and its clinicopathological significants.Methods:Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mRNA expression differences of DLC1gene in PTC and PCTT. Western-blotting, immunohistochemistry (IHC), Quantum dot-based immunofuorescence histochemistry (QDs-IHC) and Methylation-specific PCR (MSP) were used to analyze the protein expression differences and promoter methylation status of DLC1gene in tissues of PTC and PCTT. The DLC1mRNA expression rate, DLC1protein expression rate and methylation rate and clinicopathological parameters including patient s age, tumor maximum size, lymph nodes metastasis and TNM stage between PTC and PCTT were analysed with chi square test, one way ANOVA and Spearman rank correlation analysis.Results:(1) The mean expression of DLC1mRNA of PCTT was0.47±0.25, while the mean expression of DLC1mRNA of PTC was0.20±0.23, the DLC1mRNA expression of PTC was lower than that of PCTT, with statistical significance (F25.403, p<0.05). In40PTC cases,19cases expressed DLC1mRNA, the positive expression rate was47.5%(19/40), while in corresponding PCTT, there was32cases detected positive expression, the positive rate was80%(32/40). Compared to PCTT, the difference of DLC1mRNA positive expression rate was statistically significant in PTC (χ2=9.141, p<0.05).(2) The mRNA expression of DLC1in PTC was correlated with tumor size, lymph nodes metastasis and TNM stage, the difference was statistically significant (χ2=10.948,12.031,3.879, p<0.05). There were no significant correlations between the expression of DLC1mRNA and patient’s gender and age (p>0.05).(3) Compared with the DLC1protein expression in PCTT (0.33±0.14), the DLC1protein was significantly reduced in PTC (0.18±0.12), the difference has statistical significance (F=27.014, p<0.05).(4) The DLC1protein positive signal was located in the cytoplasm of follicular epithelial cell. In40cases of PTC,7cases show (++),21cases display (+),21cases have no expression, the positive rate was47.5%(19/40); in corresponding PCTT,7cases show (+++),21cases display (++),8cases display (+),4cases negatively expressed, the positive rate was90.0%(36/40). Compared with PCTT, the difference of DLC1positive expression rate was statistically significant in PTC (χ2=26.36, p<0.05). The expression of DLC1protein was negatively correlated with tumor size, lymph nodes metastasis and TNM stage (χ2=18.05,15.132,7.02, p<0.05). There were no significant correlations between the positive expression of DLC1and patient s gender and age (p>0.05). Spearman rank correlation analysis also reveal that DLC1protein expression was associated with tumor size, lymph nodes metastasis and TNM stage of PTC, respectively (rs=-0.552,-0.607,-0.381, p<0.05), there was no correlation between DLC1protein expression and gender and age (p>0.05).(5) The DLC1protein positive signal detected by QDs-IHC was also located in the cytoplasm of follicular epithelial cell. In PTC,7cases show (++),14cases display (+),19cases have no expression, the positive rate was52.5%(21/40), the positive rate was slightly higher than that detected by IHC. While in corresponding PCTT,9cases show (+++),19cases display (++),8cases display (+),4cases negatively expressed, the positive rate was90.0%(36/40). The positive rate detected by QDs-IHC are same as that detected by IHC. Compared with PCTT, the difference of DLC1positive expression rate was statistically significant in PTC (χ2=25.96, p<0.05). The expression of DLC1protein detected by QDs-IHC was negatively correlated with tumor size and lymph nodes metastasis (χ2=23.594,20.338, p<0.05). There were no significant correlations between the positive expression of DLC1and patient s gender, age and TNM stage (p>0.05). Spearman rank correlation analysis reveal that DLC1protein expression was associated with tumor size, lymph nodes metastasis and TNM stage of PTC, respectively (rs=-0.651,-0.713,-0.342,p<0.05), there was no correlation between DLC1protein expression and gender and age (p>0.05).(6) Consistency test reveal that the DLC1protein in total thyroid tissue, PTC and PCTT detected by IHC and QDs-IHC has a good consistant (K=0.924,0.919,0.546;p<0.05).(7) The expression of DLC1mRNA and DLC1protein in PTC and PCTT has a positive correlation (rs=0.953,0.695, p<0.05).(8) The methylation rate of DLC1in PTC was40%, while in PCTT, the rate was12.5%. Compared with the PCTT, the promoter methylation of DLC1was more common in PTC, the methylation rates of DLC1gene between PCTT and PTC has statistical significance (χ2=7.813, p<0.05). The methylation of DLC1gene in PTC and PCTT was negatively correlated with expression of DLC1mRNA (χ2=13.099,12.857, rs=-0.572,-0.567, p<0.05). And the methylation of DLC1gene in PTC and PCTT was negatively correlated with DLC1protein expression (χ2=5.657,13.333, rs=-0.376,-0.577, p<0.05). The methylation of DLC1gene in PTC had no significant correlation between clinicopathological parameters, such as gender, age, lymph node metastasis and TNM stage (p>0.05), but is associated with tumor size (χ2=12.857, p<0.05).Conclusions:(1) The decreased or loss expression of DLC1gene may play important biological roles in carcinogenesis and metastasis of PTC.(2) Promoter methylation in the promoter region of DLC1in PTC may cause decreased or loss expression of DLC1gene. Part2:The expression of vascular endothelial growth factor C and microlymphatic vessel density in papillary thyroid carcinoma and pericarcinomatous thyreoid tissue and its clinicopathological signific-ance and the relationship between the expression of DLC1, vascular endothelial growth factor C, and microlymphatic vessel densityObjective:To investigate the expression and clinicopathological significance of vascular endothelial growth factor C (VEGF-C) and microlymphatic vessel density (MLVD) in PTC and PCTT. And investigate the relationship between DLC1, VEGF-C expression and MLVD and their associations with PTC lymph node metastases.Methods:The expression of VEGF-C was detected by IHC in40cases of PTC and corresponding PCTT. MLVD was counted with D2-40antibody as specific immunomarker of lymphatic vessel. The relationship between VEGF-C, MLVD and clinicopathologic parameters including patient’s ages, tumor maximum size, lymph nodes metastasis and TNM stages between PTC and PCTT were analysed. And the correlation between DLC1, VEGF-C expression and MLVD were also analysed. Results:(1) The VEGF-C positive signal was observed in the cytoplasm staining of follicular epithelial cell. In40cases of PTC,9cases show (+++),14cases display (++),3cases present (+),14cases show negatively expression, the positive rate was65%(26/40); in40cases of corresponding PCTT, there was no (+++) cases,2cases present (++),11cases display (+),27cases show natively expression, the positive rate was32.5%(13/40). Compared to PCTT, the difference of VEGF-C positive expression rate was statistically significant in PTC (χ2=20.693, p<0.05).(2) The increased protein expression of VEGF-C of PTC was positively correlated with tumor size and lymph nodes metastasis, the difference was statistically significant (χ2=21.626,30.380, p<0.05). But there were no significant correlations between the positive expression of VEGF-C and patient’s gender, age and TNM stage (p>0.05). Spearman rank correlation analysis reveal that VEGF-C protein expression was associated with tumor size, lymph nodes metastasis and TNM stage of PTC, respectively (rs=0.405,0.838,0.396,p<0.05).(3) The VEGF-C protein expression was negatively correlated with the DLC1protein expression, with statistical significance (rs=-0.739, p<0.05).(4) The mean MLVD of peritumor area of PTC was11.63±4.19, while the mean MLVD of PCTT was4.40±2.50, the MLVD of peritumor area of PTC was higher than that of PCTT, with statistical significance (t=9.363, p<0.05).(5) The MLVD of PTC was positively correlated with tumor size and lymph nodes metastasis, the difference was statistically significant (χ2=21.897,32.169, p<0.05). There were no significant correlations between the MLVD and patient’s gender, age and TNM stage (p>0.05).(6) Spearman rank correlation analysis reveal the MLVD has significant positive correlation with VEGF-C protein expression of PTC, the correlation was statistically significant (rs=0.873, p<0.05). And the MLVD has significant negative correlation with DLC1protein expression of PTC, with statistical significance (rs=-0.701, p<0.05).Conclusions:(1) Increased protein expression of VEGF-C of PTC indicates that it may have an important role for lymph node metastasis of PTC and positive correlated with late TNM stage.(2) MLVD of PTC is closely related with lymph node metastasis, the relatively high MLVD of the peritumor area may be act an important role in lymph node metastasis of PTC.(3) Decreased expression or absence of DLC1in PTC may regulate VEGF-C upregulation in promoting Lymphangiogenesis during tumorigenesis.(4) Combined detection of DLC1, VEGF-C expression and MLVD in PTC, will be conducive to evaluate the biological behavior and predict lymph node metastasis of PTC. |
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