| BackgroundPrimary aldosteronism (PA) is the most common and curable form of secondary hypertension, characterized by autonomous aldosterone over-production. Some patients accompanied with hypokalemia. PA is mainly caused by idiopathic hyperaldosteronism (IHA) and aldosterone-producing adenoma (APA), both of which account for 90% of all reported cases. Previous studies show that the average long-term recovery rates of hypertension after unilateral adrenalectomy for APA range from 30% to 72%. It is not clear that the pathogenesis of APA patients with postoperative persistent hypertension. With the completion of the Human Genome Project, the high-throughput microarray technology is developing rapidly. Gene chip technology, also known as cDNA microarray (cDNA microarray). A number of high-density oligonucleotide, gene fragments were immobilized on silicon, nylon slides and film media to detect the expression of specific genes by way of microarray. Its principle is complementary hybridization of hundreds or even thousands of probes interaction with the target gene. Microarray technology is accurate, rapid, high sensitivity, and can be used to study the relationship between gene expression and disease, gene mutation, gene polymorphism and gene sequencing.Currently most scholars believe that it is a multifactorial disease. Many researchers summarized the clinical characteristics of APA patients with postoperative persistent hypertension and found that women, using fewer antihypertensive drugs and shorter duration of hypertension were associated with the cure of postoperative hypertension. Few studies have been identified the genetic mechanisms associated with postoperative persistent hypertension at home and abroad to date. Most researches mainly focused on the screening of differentially expressed genes, detecting gene mutations and analyzing of gene polymorphisms detection of APA. Previous studies have found that CYP11B2, CAMK-I, and CYP11B1 were associated with the pathogenesis of APA and postoperative persistent hypertension by comparing the adenomas of APA and normal adrenal. However, there were no studies that were specially comparative differentially expressed genes between persistent hypertension and normotension of APA after unilateral adrenalectomy nowadays.Objective:1、To investigate the clinical characteristics in APA patients with persistent hypertension after unilateral adrenalectomy.2、To screen the differentially expressed genes in APA patients with postoperative persistent hypertension after unilateral adrenalectomy in genome wide range, and establish their expression profiles. It could provide a theoretical basis for investigating the genetic mechanisms of APA patients with postoperative persistent hypertension.3、On the basis of differentially expressed genes screened by microarray, differentially expressed genes were detected in APA with postoperative persistent hypertension by qRT-PCR and immunohistochemical methods, which further validate the differentially expressed genes and reliability of microarray results. Methods:1、Retrospectively analyzed clinical characteristics of 94 APA patients who had undergone unilateral laparoscopic adrenalectomy. According to whether hypertension returned to normal after surgery, all patients divided into two groups:normotension (51 cases) and postoperative persistent hypertension (43 cases). To analyze the patient’s clinical features including family history of hypertension, duration of hypertension, types of antihypertensive drug use, anthropometric indices and biochemical indicators before and after surgery.2、Through high-throughput microarray gene chip-Human Transcriptome Array 2.0 (HTA2.0), we screened the differentially expressed genes between 9 APA patients with postoperative persistent hypertension and 12 APA patients with postoperative normotension. GO enrichment, pathway enrichment analysis and mRNA-IncRNA co-expression network construction were studied.3、Nine differentially expressed genes which were selected by microarray were detected by qRT-PCR in 40 APA patients at RNA levels. Four differentially expressed genes were detected by immunohistochemistry in 12 APA patients at protein levels.Results:1、Compared to APA patients with postoperative normotension, there was no significant differences in the age at the time of surgery, the sex ratio, family history of hypertension, preoperative blood pressure levels, preoperative serum aldosterone, renin activity, ARR, serum creatinine and 24h urinary aldosterone,24h urine potassium, triglycerides, and total cholesterol level between the two groups. BM1, duration of hypertension, numbers of the use antihypertensive drugs before surgery, and LDL levels were associated with the occurrence of postoperative persistent hypertension(P<0.05). According to a logistic regression analysis adjusted for duration of hypertension and BMI, preoperative use of two or fewer antihypertensive agents (OR:2.758, P<0.05) and LDL levels (OR:2.206, P<0.05) were independent risk factor.2、Compared to the APA patients with postoperative normotension, there were 99 differentially expressed genes in postoperative persistent hypertension including 48 mRNA and 51 IncRNA. GO analysis shows that differentially expressed genes were associated with cell differentiation, lipid metabolic process, extracellular region, membrane fraction, metal ion binding and transmembrane receptor activity. Pathway analysis indicated that the enrichment existed in glycerophospholipid metabolism, steroid biosynthesis, ether lipid metabolism and linoleic acid metabolism. The mRNA-IncRNA co-expression network map includes 48 nodes and 72 connections.3、Compared to APA patients with postoperative normotension, qRT-PCR validation show that the expressions of the COLEC12, FABP4, CST2, FAT1, and MGP were of significant differences in APA postoperative persistent hypertension. Immunohistochemistry showed that the expressions of the COLEC12, FABP4, FAT1, and MGP were of significant differences, consistent with the microarray results.Conclusion:1、In this study the rate of hypertension resolution after adrenalectomy was 54.3%. BMI, duration of hypertension, use of≥2 antihypertensive drug before surgery, and the levels of LDL were associated with the occurrence of postoperative persistent hypertension. Use of≥2 antihypertensive drug before surgery and the level of LDL were independent risk factors for APA patients with the occurrence of postoperative persistent hypertension.2、Compared to APA patients with postoperative normotension, differentially expressed genes existed in APA patients tissues with postoperative persistent hypertension, which indicates that differentially expressed genes may be associated with the occurrence of postoperative hypertension. This laid the foundation for the next functional studies of differentially expressed genes.3、The expression of differentially expressed genes were verified by qRT-PCR and immunohistoehemistry, which was consistent with the microarray results. It suggested that COLEC12, FABP4, FAT1, and MGP might be associated with the occurrence of postoperative hypertension. |
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