Expression Of DLC-1 In Pituitary Adenomas And Its Relationship With Invasive Behavior Of Pituitary Adenoma And Its Promoter Methylation Status

Pituitary adenoma account for about 10%to 15%of intracranial tumors[1], although it is benign, but still can lead to clinical symptoms. Mass effect can lead to headaches, decreased vision; Functional pituitary adenoma can cause hormone imbalance which can lead to emotional disorders, sexual dysfunction, infertility, obesity, hypertension, diabetes, heart disease, accelerated deterioration; Residual normal pituitary tissue pressure will appear hypopituitarism [2]. The occurrence of pituitary adenoma development involves a number of factors:gene mutation and inactivation, hypothalamus hormones, blood circulation of hormones, growth factors, cytokines interaction [3]. Mutation, deletion, reorganization have been found in pituitary adenomas. And abnormal methylation of tumor suppressor genes is one important mechanism in pituitary adenomas. More and more evidence showed that pituitary adenoma is monoclonal, and accompanied by genetic changes. However, we still know very little about the genetic changes accompanied the development of pituitary tumors [4].In order to study the occurrence and development mechanism of pituitary tumors, the research group used Affymetrix's pre-U133 Plus 2.0 gene chips to detect invasive, non-invasive pituitary tumors and normal human pituitary gene expression, and found a series of down-regulated genes, including the DLC-1. In 1998, Yuan studied a serials of liver cell lines used the reproduction of difference analysis (RDA), and found an important tumor suppressor genes DLC-1.It has been found that DLC-1 were expressed at low level in many primary tumors and cell lines, but at normal level in normal tissue of the human body; DLC-1 gene was also found methylated in a variety of primary tumors and cell lines. DLC-1 contains three domains, RhoGAP, SAM (sterile alpha motif) and START (a steroidogenic acute regulatory (StAR)-related lipid transfer). Studies have shown that DLC-1 can regulate the formation and morphological changes of the cytoskeleton, affect cellular transcription, proliferation, migration and apoptosis.To explore the role of DLC-1 gene in the occurrence and development of pituitary adenoma, we studied the expression and methylation status of DLC-1 in pituitary adenomas and its effect on biological behavior of pituitary adenoma cells; In this paper, the following three aspects were studied:1. Expression of different transcripts of DLC-1 mRNA in human pituitary adenoma and its relationship with the invasiveness of pituitary adenoma; 2. Protein expression of DLC-1 in human pituitary adenoma and its relationship with the clinicopathologic of pituitary adenoma; 3. Promoter methylation status of DLC-1 in Human pituitary adenoma research.Part one Expression of different transcripts of DLC-1 mRNA in human pituitary adenoma and its relationship with the invasiveness of pituitary adenomaObjective:To identify the expression of three different mRNA transcripts of DLC-1 in normal pituitary and various types of pituitary adenomas and its relationship with the invasiveness of pituitary adenoma.Methods:After the transcript-specific primers designed, using fluorescence quantitative polymerase chain reaction technique to detect different transcripts of DLC-1 in normal pituitary and various types of pituitary adenoma tissues. Pituitary adenoma specimens according to clinical manifestations and magnetic resonance features are divided into invasive group and non-invasive group; Adenomas were divided into functional groups and non-functional adenomas according to the clinical and immunohistochemical. Analysis of different transcripts in the performance of each group. Results:1. DLC-1 transcripts 1,2 were expressed in normal pituitary and pituitary adenomas; no transcript expression of a third.2. In normal pituitary, expression level of transcripts 1,2 was significantly higher than pituitary adenoma group (P<0.01); In invasion group, expression level of transcripts 1,2 was significantly lower than non-invasive group (P<0.01), functional adenoma group and non-functional adenomas were no significant difference (P>0.05);Conclusion:DLC-1 transcripts 1,2 were expressed in the normal pituitary and pituitary adenomas, and had a close relationship with the invasiveness of pituitary adenomas; No transcript expression of a third.Part two Protein expression of DLC-1 in human pituitary adenoma and its relationship with the clinicopathologic of pituitary adenoma. Objective:To identify DLC-1, Ki-67 protein in various types of pituitary adenomas and normal pituitary and its relationship with the clinical pathology of the pituitary adenoma, and investigate the relevance of the DLC-1 and Ki-67.Methods:The specimens of pituitary adenomas are divided into invasive group and non-invasive group according to clinical manifestations and magnetic resonance features. Adenomas were divided into functional groups and non-functional adenomas according to the clinical and immunohistochemical features. Immunohistochemistry was used to observe DLC-1 and Ki-67 protein in cells positioning, as well as different expression in the different groups to discuss the relevance of the expression of the two proteins; Using western-blot method to test DLC-1 protein expression in specimens then observe its relationship with the invasiveness of pituitary adenomas.Results:1. DLC-1 protein expressed in plasma in pituitary adenomas and normal pituitary cells; Ki-67 protein mainly expressed in the nucleus.2. DLC-1 protein expression level in normal pituitary was significantly higher than pituitary adenoma group (P<0.01); In invasion group, DLC-1 protein expression level was significantly lower than non-invasive group (P<0.01), functional adenoma group and the non-functioning adenomas had no significant difference (P> 0.05); 3. In pituitary adenoma, Ki-67 protein expression was significantly higher than the normal pituitary group (P<0.01); In invasion group, Ki-67 protein expression was significantly higher than the non-invasive group (P <0.01), functional adenoma group and the non-functional adenomas had no significant difference (P> 0.05); 4. DLC-1 and Ki-67 protein expression has a negative correlation in pituitary adenomas (r=-0.764, P<0.01).Conclusion:DLC-1 protein expression may be related with the occurrence of pituitary adenoma, invasive and proliferative ability;part three Promoter methylation status of DLC-1 in Human pituitary adenoma research.Objective:To identify whether the low expression of DLC-1 gene due to the promoter methylation. Methods:Design the methylation and non-methylated primers, using MSP (methylation-specific PCR) to detect the DLC-1 promoter methylation status.Results:Unmethylated primers can be amplified in all specimens of pituitary adenomas, while methylation primers can be amplified in only 2 cases (2/of 15) samples; methylation, unmethylated primer sequencing cloned products MSP further verify the results of tests are specific. Non-methylated and methylated primer amplified products were direct sequenced. The test results further verified that the MSP is specific.Conclusion:DLC1 gene promoter methylation may not be main reasons of the low expression of DLC-1 in pituitary adenoma, there may be other reasons.