Analysis Of MRNA And MicroRNA Expression Profiles Of Pituitary ACTH Adenoma Based On Complete Transcriptome Sequencing

Background:pituitary adenoma is one of the most common intracranial benign tumor, the incidence of PA occupies the second position in intracranial tumors, accounting for about 10%-15% of intracranial tumors, and increased year by year. As the tumor grows, around compression of sellar region, such as optic nerves, cavernous sinus, cerebral artery, hypothalamus, frontal lobe, even involving the brainstem, and cause severe dysfunction. At the same time, tumor growth can also lead to pituitary hormone secretion disorder. According to the level of hormone in peripheral blood, pituitary adenoma is divided into functional pituitary adenoma (NFPA) and functional pituitary adenoma, including prolactinomas (PRL),ACTH-secreting pituitary adenoma (Cushing’s disease), growth hormone (GH)-secreting pituitary adenoma and thyroid stimulating hormone-secreting pituitary adenoma (TSH), gonadotrophin hormone-secreting pituitary adenomas (PGA) and mixed hormone secreting adenomas and. The,ACTH-secreting pituitary adenoma(ACTH-PAs) is also called Cushing’s disease, which causes excess production of adrenocorticotropic hormone (ACTH).,ACTH-secreting pituitary adenoma accounted for about 14% of all pituitary adenomas, accounted for Cushing’s syndrome (70% of Cushing’s syndrome. The incidence of the disease in Europe and the United States is 39/million, China is still a lack of large-scale epidemiological data. Due to the diagnosis of the disease mainly based on laboratory examination in hormone levels, clinical symptoms of patients, imaging diagnosis,pathological examination and immunohistochemical detection,therefore in the early diagnosis of Cushing’s disease is very difficult and easy to be misdiagnosed. Patients often have a variety of serious complications, such as hypertension, diabetes, hyperlipidemia, osteoporosis and mental depression. In the treatment, for the vast majority of Cushing’s patients, pituitary adenoma resection surgery by endonasal transsphenoidal approach remains the first choice for clinical treatment, radiotherapy and chemotherapy,such as pasireotide, ketoconazole, is often used as a adjuvant therapy for refractory Cushing’s disease after. Although the literature reports the success rate of surgical treatment in 65%～90%, but due to differences in the biological behavior of the tumor and in operation level, tumor recurrence rate was 3%～47% and average recurrence time was 16-49 months,as well as recurrence of patients with a poor prognosis, mortality rate high. In recent years for Cushing’s disease at the molecular level research mainly concentrated in the tumorigenesis, tumor invasiveness and hormone secretion, but so far, the pathogenesis of Cushing’s disease is not fully understood, so clinical urgently looking for help a potential target for Cushing’s disease diagnosis, treatment and estimate prognosis.The primary aim of this study was to screen the differentially expressed genes and microRNAs in ACTH-secreting pituitary adenoma by whole transcriptome next generation sequencing(NGS) technology; In addition, considering the free serum miRNA has the potential as a tumor marker, we investigated whether miRNAs present in the tumor tissue and serum of ACTH-secreting pituitary adenoma patients can be used as diagnostic or prognostic biomarkers and whether they are associated with biological behavior of the tumor and clinical treatment. In order to realize personalized treatment of patients with pituitary adenoma.Section One:Differential mRNA expression profiles in ACTH-secreting pituitary adenoma by Next generation sequencing technologiesContext:Adrenocorticotrophin(ACTH)-secreting pituitary adenoma is also called Cushing’s disease(CD),which is rare and causes metabolic syndrome, such as cardiovascular disease, hypertension and diabetes, due to hypercortisolism. However the molecular pathogenesis of CD is still unclear because of lack of human cell lines and animal models.Objective:The primary aim of the study was to elucidate altered miRNA expression profiles between human ACTH-secreting pituitary adenomas and adjacent nontumors pituitary tissues(ANPT) by the next generation sequencing technology. In order to uncover the mechanism of tumorgenisis and to provide a complete basis of molecular biology for microRNA subsequent researchMethods:We obtained 68 pathologically proven fresh ACTH-secreting pituitary adenoma specimens and adjacent nontumors pituitary tissues(ANPT),20 cases of non functioning pituitary adenomas,18 cases of growth hormone pituitary adenomas and 8 cases of prolactinomas for this study. At the same time collected clinical information of patients. We used new generation sequencing technologies to discovery abnormally expressed mRNA in 9 pairs of tumors and adjacent nontumors pituitary tissues. Then the differentially expressed genes were obtained by bioinformatics analysis. Further, according to Go term and the pathway analysis, to uncover the molecular regulation mechanism of human ACTH-secreting pituitary adenomas.Results:According to bioinformatics analysis, we screened 166 up-regulated differentially expressed genes and 288 down-regulated differentially expressed gene Go term and PATHWAY analysis indicated that tumorigenesis, tumor invasiveness and hormone secretion were correlated with Cushing’s disease. TIMP3 gene was selected for further verification, our findings showed that TIMP3 expression was significantly correlated with Ki-67 expression, and invasiveness of pituitary adenomas. TIMP3 mRNA and protein expression was reduced in ACTH-secreting pituitary adenomas and the other three types of pituitary adenomas compared to adjacent non-tumorous pituitary tissuesConclusions:Our findings provide new insight into the potential mechanisms of tumorgenisis and development of ACTH-secreting pituitary adenomas,and may open new potential avenues for intervention and treatment in Cushing’s diseaseSection Two:Differential miRNA expression profiles in ACTH-secreting pituitary adenoma by Next generation sequencing technologiesContext:MicroRNA is a class of small RNA molecules involved in the regulation of gene expression. It has been confirmed that there are abnormal expression in tumor tissues and serum of patients. Therefore, the abnormal expression of microRNA may play an important role in the tumorgenisis and development of human tumors, serum microRNA may serve as a molecular marker for tumor diagnosis and prognosis.Objective:The primary aim of the study was to. analyze of ACTH-secreting pituitary adenoma related microRNAs by next generation sequencing technologies.Methods:ACTH-secreting pituitary tumor samples andadjacent non-tumorous pituitary tissues (ANPT) were obtained during transphenoidal surgery frompatients with Cushing disease. The relative expression of miRNAs was measured by new generation sequencing technologies, further verification in 55 cases of tumor tissues and 15 cases of adjacent nontumors pituitary tissues,and Then the detected serum miRNAs were validated in 197 patients and 120 healthy people using fluorescence quantitative PCR, correlation analysis between prediction of target genes and the results of mRNA-seq in the first section showed that using this microRNA panel may have potential as a molecular markers for Cushing’s disease diagnosis and prognosis.Results:Our next generation sequencing obtained 10 million reads per sample. We found 67 microRNAs differentially expressed between paired tumour samples and ANPT. Then mirnas were selected and further quantified using real-time qPCR in more than 300 samples, including tumor tissues and serum of patients with Cushing’s disease. We identified 6 up-regulated (miR-9-5p, miR-9-3p, miR-190b, miR-137, miR-885-5p, miR-592) and 1 down-regulated miR-551b-3p in tumor tissue. However, we discovered not all selected miRNAs were consistently detected in serum,because of high Ct values (Ct values> 35).Only miR-551b-3p was significantly down-regulated in both tissue and serum samples. The expression levels of miR-551b-3p in tissue and plasma showed significant positive correlation. The receiver operating characteristic curve analysis showed that mir-551b-3p with high sensitivity and specificity for discriminating CD patients from healthy controls.The expression level of mir-551b-3p in serum was significantly correlated with the tumor size, serum cortisol and ACTH levels.Conclusion:Our results support the possibility that altered miRNA expression profile might be involved in corticotrophic tumorigenesis. Serum microRNA could serve as a minimally invasive biomarker for Cushing’s disease detection.