The Study Of The Relationship Between HEPN,RASSF3and Pituitary Tumorigenesis

ObjectsThe pituitary gland regulates many functions of other endocrine glands and theirtarget tissues throughout the body. Pituitary adenomas can cause mood disorders, sexualdysfunction, infertility, acromegaly, obesity, visual disturbances, hypertension, diabetesmellitus, and accelerated heart disease. Although pituitary adenomas are benign, somepituitary adenomas are considered to be aggressive or invasive, showing atypical behavior,such as invading adjacent tissues and proliferating rapidly. Invasion of surroundingstructures by pituitary adenomas increases the difficulty of complete resection and is animportant reason for post-operative recurrence. The pathogenic mechanisms underlyingpituitary adenoma formation, progression, and invasion are poorly understood. Mutationsin classic oncogenes and tumor suppressor genes (TSGs), which might be prognosticpredictors or gene therapy targets, are rarely found in pituitary tumors. Thus furtherinvestigation of new oncogenes and TSGs is needed.To understand the candidate oncogenes and TSGs involved in the pathogenesis ofpituitary adenomas, we studied the machanisms related to pituitary adenomastumorigenesis and progress in the following three aspects:(1) Expression and function of HEPN1in pituitary adenomas.(2) Methylated DNA immunoprecipitation with comparative high-densitywhole-genome microarray analysis to identify silenced TSGs in pituitary adenomas.Methylation, expression level and function of RASSF3in pituitary adenomas.MethodsBased on the three aspects above of research,the experimental methods are as follows:(1) Four normal human adenohypophyses were obtained at the time of autopsy frompatients with no evidence of endocrinopathies. Fifteen invasive somatotroph adenomas and12non-invasive adenomas were obtained at the time of surgery at Changzheng Hospitaland frozen in liquid nitrogen and stored at-80oC.(2) We analysed the expression of HEPN1in pituitary adenomas andadenohypophyses. HEPN1reduction was more frequent in invasive adenomas. Tounderstand the function of HEPN1, the pituitary adenoma cell lines, GH3and GT1.1, werestably transfected with short hairpin RNA (shRNA) targeting HEPN1or ectogenic HEPN1 by lentivirus-mediated transfection. MTT colorimetric assay, Annexin V/propidium iodide(PI) apoptosis assay, and transwell assay were performed to analyse the effect of HEPN1on cell proliferation, apoptosis, and migration. We also studied the influence of HEPN1expression on the level of MMP-2, MMP-9, BAX, p53, caspase-3and Bcl-2.(3) To identify candidate tumor suppressor genes involved in pituitary somatotrophadenoma tumorigenesis, we used HG18CpG plus Promoter Microarray in27humansomatotroph adenomas and4normal human adenohypophyses. After bioinformaticsanalysis, RASSF3was selected as a candidate TSG.(4) Pyrosequencing analysis was used to confirm the result of HG18CpG plusPromoter Microarray. QRT-PCR was performed to study expression of RASSF3.5-Aza-2deoxycytidine and trichostatin-A treatment was used in GH3and GT1.1cell lines.Tounderstand the function of RASSF3, the pituitary adenoma cell lines, GH3and GT1.1,were stably transfected with short hairpin RNA (shRNA) targeting RASSF3or ectogenicRASSF3by lentivirus-mediated transfection.Results(1) In qRT-PCR, HEPN1reduction was more frequent in the invasive group. HEPN1overexpression in GH3and GT1.1cells inhibited cell proliferation, induced apoptosis,and attenuated invasive capacity, whereas HEPN1silencing enhanced cell proliferationand invasion accompanied by decreased apoptosis. Western blot analysis revealed thatHEPN1overexpression decreased MMP-2, MMP-9, and Bcl-2expression, but increasedBAX, p53, and caspase-3expression. In contrast, HEPN1silencing increased MMP-2,MMP-9, and Bcl-2expression, but decreased BAX, p53, and caspase-3expression.(2) RASSF3was found with frequent methylation of CpG island in its promoter regionin somatotroph adenomas but rarely in adenohypophyses. This result was confirmed bypyrosequencing analysis. We also found that RASSF3mRNA level correlated negativelyto its gene promoter methylation level. RASSF3hypermethylation and downregulationwas also observed in rat GH3and mouse GT1.1somatotroph adenoma cell lines.5-Aza-2deoxycytidine and trichostatin-A treatment induced RASSF3promoter demethylation, andrestored its expression in GH3and GT1.1cell lines. RASSF3overexpression in GH3andGT1.1cells inhibited proliferation, induced apoptosis accompanied by increased Bax, p53,and caspase-3protein and decreased Bcl-2protein expression. We also found that theantitumor effect of RASSF3was p53dependent, and p53knockdown blocked RASSF3-induced apoptosis and growth inhibition.Conclusions(1) HEPN1performs multiple functions as a TSG through suppression of proliferationand invasion, and induction to apoptosis. In conclusion, silencing of HEPN1maycontribute to the progress and invasion of human pituitary somatotroph adenomas. Inpituitary adenoma cell lines, HEPN1silencing promotes proliferation, inhibits apoptosis bydecreased p53, BAX, and caspase-3expression, and promotes invasiveness by increasingthe expression of MMP-2and-9. Our study indicates that HEPN1might be a potentialprognostic predictor or gene therapy target for patients with invasive somatotrophadenomas.(2) RASSF3gene silencing by promoter methylation is an important early event insomatotroph adenoma tumorigenesis. Hypermethylation-induced silencing of RASSF3may contribute to tumor cell growth by apoptosis inhibition through the p53pathway. Inthe context of the data presented in this study,5-Aza induced re-expression of RASSF3might offer new avenues for treatment of somatotroph adenomas.