Alteration Of Message RNA And Long Non-coding RNA Expression In Aldosterone-Producing Adenomas

Background and objectPrimary aldosteronism (PA) is the most frequent form of endocrine hypertension, accounting for up to5%to15%of all hypertensive patients, and is characterized by the chronic, excessive, and autonomous secretion of aldosterone by the adrenal gland. The diagnosis of this form of hypertension is fundamental because, compared with essential hypertensives with similar risk profiles, patients with PA are more prone to stroke and myocardial infarction and display an increase in cardiovascular damage and metabolic complications. Aldosterone-producing adenomas (APA) are a common underlying cause of PA and are found in40%to50%of PA patients, whereas bilateral adrenal hyperplasia is present in50%to60%of patients. Unilateral adrenalectomy normalizes, or at least markedly improves, the blood pressure in patients with APA, and therefore, APA is the most common, specifically treatable, and potentially curable form of hypertension.Long non-coding RNA(lncRNA) are defined as transcripts longer than200nt without coding capacities, but play an important role in the regulation of gene expression. Multiple lines of evidence indicate that lncRNA have a variety of regulation ways by interacting with its partner DNA, RNA, and/or protein. Although the pathogenic mechanism of lncRNA remain unclear, a large number of clinical observations and experimental evidence indicate that lncRNA can regulate gene expression at transcription and post-transcription levels and widely take part in species evolution, embryonic development, stem cell maintenance, cell proliferation and differentiation and tumorigenesis.However, the molecular mechanisms underlying the alterations in cell growth in the adrenal cortex and the hypersecretion of aldosterone have not yet been defined. Previous genomic analyses have provided evidence for differential gene expression in APA compared with normal adrenals (NA). The present study was undertaken to better define upregulated gene transcripts in highly selected APA and to investigate the functional role of such genes specifically with regard to autonomous aldosterone secretion and deregulated cell growth. The APA used in this study were removed from patients studied in our hypertension unit who had been homogeneously selected following a rigorous diagnostic flowchart that includes adrenal venous sampling and postadrenalectomy evaluation. Genetic regulation is crucial for the occurrence of many diseases, including tumor osteogenic oncogenesis. The human genome comprises not only sequences encoding proteins but also a myriad of non-protein coding RNAs that may be involved in important biological processes and the regulation of molecular mechanisms. Many long noncoding RNAs (lncRNAs) have been found in human and mouse using large-scale analyses of full-length cDNA sequences and other methods. An increasing amount of evidence suggests that lncRNAs are involved in a variety of regulatory processes, including transcriptional regulation and epigenetic gene regulation, as well as disease. lncRNAs are over200nucleotides in length and are separate from the other known ncRNA subsets. Previous research has focused primarily on short ncRNAs, such as microRNAs, transfer RNAs and short interfering RNAs. MicroRNAs, which are the most-studied short RNAs, play an important role in certain processes and pathways involved in development, differentiation and proliferation as well as in apoptosis and tumor. Over the last decade, much research has led to considerable progress in understanding lncRNAs; however, the exact function of most lncRNAs is currently unknown. Some preliminary studies have reported that alterations in the levels of several lncRNAs have been detected in PCGEM1, MEG3, p15AS和HOTAIR. However, the biological functions of lncRNAs in APA as well as the correlation between APA and the expression levels of these lncRNAs remain unclear. Therefore, study the molecular mechanism of APA and detection specific molecular biomarkers for APA, which will have important significance for the early diagnosis of APA. We primarily studied the altered expression of lncRNA in the development process of APA. And then, The whole LncRNA genomic expression was investigated with microarray to explore the possible mechanism of hyperaldosterone. The article was divided into four parts.Part1Pathology in aldosterone-producing adenomaObjectivesTo studay the pathologic type and character of6cases of aldosterone-producing adenomas and6cases of normal adrenal that were collected, in order to determine whether put these into the studies or not. We try to work out the contructive charactors of aldosterone-producing adenomas using electron microscope.MethodsThe treatment group received6cases of APA validated by history, clinical manifestation, chemical examination, imaging examination and pathological examination.The control group received6normal adrenal cases validated by pathological examination from radical nephrectomy.Every case was separated by three specimens,and the first performed HE stain, the second performed electron microscopy examination and the third performed Mircoarray. results1.HE stain result of adrenal tissuesThe aldosterone-producing adenomas, in the optical microscope, most of are hybrid cells. Hyaline cell often is the most and another cell also exist.2. The aldosterone-producing adenomas in the electron microscopeThe aldosterone-producing adenomas, in the electron microscope, these cells have the special charaters, the plastosome of globular or long and of long tracheid crista, obvious Golgi complex, a heap of parallel disposed rough endoplasmic, reticulum, many lipid droplets, obvious lipofuscin granulation, sometimes intranuclear pseudo-inclusion baly also can be see.Conclusions1. All samples include6APA specimens and6normal adrenal specimens pathological analysis showed that all were classifiable as adenomas and normal adrenal.2. The aldosterone-producing adenomas in the electron microscope showed that the most obvious feature of the cells forming the APA was the presence of large lipid droplets occupying a large part of the cell volume.They mostly appeared grey in colour and showed clear spaces in the centre and/or at the periphery.Part2Alteration of message RNA in Aldosterone-Producing Adenomas objectiveThe pathogenesis of aldosterone over-secretion in aldosterone-producing adenomas is still not clear. The whole genomic expression including aldosterone synthesis related enzyme and associated regulatory factor genes in adrenal glands with aldosterone-producing adenomas was investigated with microarray to explore the possible mechanism of hyperaldosterone.Methods1.6aldosterone-producing adenoma and6normal adrenal tissues were studied, which were confirmed by clinical and pathologic diagnosis. Chips Illumina HumanRef-6v2were applied to analyze these gene expressions of the three groups.2. Matlab and Graphviz softwarewas used to do data analysis including TwoClassDif, GO-Analysis, Path-Finder and GeneFunNet.ResultsUsing the Screening criteria of2FC up-or-down-regulated in their expression level and probability values (P<0.01) to select different express mRNA, we identified82mRNA that statistically, differently expressed in aldosterone-producing adenoma. These included529mRNA that were overexpressed and323mRNA that were underexpressed. According to significant GO category, APA main activate positive regulation of protein amino acid dephosphorylation, focal adhesion formation, regulation of exocytosis and inhibit regulation of NF-kappaB import into nucleus, defense response to protozoan, negative regulation of myeloid cell differentiation. APA downregulation in pathways of MAPK, PPAR, Apoptosis, ErbB and Bladder cancer may have partly same pathogenesis. In addition, APA may also correlate with the abnormal of JAK-STAT pathway.conclusionsCYP11B2was over-expressed in aldosterone synthesis related enzymes. Most of the regulators in aldosterone synthesis down regulated but RENBP and NR1H2suggested that a special aldosterone synthesis stimulating pathway may exist. According to the changes of main gene expression pathways, APA has the characteristics of tumor.Part3Alteration of LncRNA in Aldosterone-Producing AdenomasobjectiveStudy the molecular mechanism of Aldosterone-Producing Adenomas and detection specific molecular biomarkers for Aldosterone-Producing Adenomas, which will have important significance for the early diagnosis of Aldosterone-Producing Adenomas. We study the altered expression of lncRNA in the development process of Aldosterone-Producing Adenomas. And then, select the specific expression lncRNA in the development process of Aldosterone-Producing Adenomas as potential molecular biomarkers of Aldosterone-Producing Adenomas, and provide some experimental evidence.Methods1. The treatment group received6cases of APA validated by history, clinical manifestation, chemical examination, imaging examination and pathological examination.The control group received6normal adrenal cases validated by pathological examination from radical nephrectomy.Every case was separated by three specimens,and the first performed HE stain, the second performed electron microscopy examination and the third performed Mircoarray. Real-time RT-PCR was used to confirm the LncRNA level of the changed expressions among APA and normal adrenal and associated regulatory factor genes.2.Total RNA was extracted from6primary APA samples and their paired normal adrenal tissues using TRIzol reagent (Invitrogen, CA, USA). Total RNA from each sample was quantified using a NucleoSpin(?) RNA clean-up, and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. The RNA samples were sent for microarray hybridization,hybridization and cleaning.The GeneChips were washed, stained, and then scanned with a Agilent G2565CA Microarray Scanner. The hybridization data were analyzed using Agilent Feature Extraction Software.Chip imaging collection and data analysis.Finally,RT-PCR verification.3. Statistial analysesAll statistical analysis was done with SPSS13.0software. Values are expressed as mean±SD. All experiments were done at least three times. Differences between groups were analyzed by double-sided Student’s t-test or Mann-Whitney tests for the compare of two groups, according to data feature. The differences were considered to be significant when P<0.05.Results1.Analysis the different express of lncRNA in expression profilesUsing the Screening criteria of2-fold up-or-down-regulated in their expression level and probability values (P<0.01) to select different express lncRNA, we identified883lncRNA that statistically, differently expressed in APA group compare to normal adrenal tissue of control group. These included649lncRNA that were overexpressed and234lncRNA that were underexpressed.2. RT-PCR to verify the microarray resultsThe result show that the expression level of lncRNA was consistent with the microarray results.Conclusions1. The expression of lncRNA significant change in aldosterone-producing adnomas, suggest that lncRNA involved in the occurrence and development of the tumor.2. The lncRNA of LINC00290,RP11-460H9.1,LSAMP-AS2,RP11-457M11.5, RP11-525J21.1,RP11-998D10.4,AC128709.4,RP11-79P5.2and RP11-738B7.1were different expression between APA and normal adrenal, they may be potential molecular markers for the diagnosis of APA.Part4The relationship of LncRNA and mRNA in Aldosterone-Producing AdenomasObjectivesTo speculate the relationship between lncRNA and mRNA based on the research data, so that reveal the mechanism for lncRNA involved in the occurrence and development of the aldosterone-producing adenomas and provide the experimental evidence of mechanism for aldosterone-producing adenomas.MethodsThrough the Pearson product moment correlation coefficient of bioinformatics analysis method, to construct LncRNA and mRNA co-expression regulation relation network, the interaction between LncRNA and mRNA display.1. Calculation of LncRNA and the relationship between the expression of mRNA, according to the set thresholdselection of lncRNA and mRNA on the relationship between LncRNA and mRNA, construction of co-expression network.2. Expression of lncRNA and mRNA and the correlation between the lncRNA and mRNA genome location based on the close relationship, the potential target genes of lncRNA, functional annotation and functional enrichment analysis of lncRNA target gene differential expression3. Co-expression of lncRNA and mRNA network topology characteristics, degree of screening important network topology based on the lncRNA, the lncRNA is most likely associated with the background of lncRNA.Results1. The mutual regulation among ENST00000512487, ENST00000506917and ENST00000496217, these three LncRNA are not involved in the regulation of topic selected importantmRNA; negative regulation among ENST00000496217, ENST00000512487and ENST00000506917, however between ENST00000512487and ENST00000506917was positively regulated.2. ENST00000547523and ENST00000514661were negative, and ENST00000547523was not directly involved in the regulation of mRNA. 3. The regulation among ENST00000514661, ENST00000445908and ENST00000497854is the most complex, they can mutual regulation, but also regulates the expression of ATR, CYB5A and AKR1C3.4. Regulation of HTR4is not affected by LncRNA, positive regulation of it only by KCNJ5.5. ENST00000547523can express the positive regulation of CYB5A and AKR1C3and can negatively regulate the expression of PCP4.6. ENST00000320322can positively regulate ENST00000445908and ENST00000497854, and it can also express the positive regulation of AKR1C3.7. The regulation of CYP11B2express by ENST00000497854, and the ENST00000497854control of CYPl1B2is positive regulation.Conclusions1. ENST00000512487,ENST00000506917and ENST00000496217, these three LncRNAs do not participate in the regulation of research selected important mRNA, so it is not the experimentresearch key content.2. ENST00000547523is not directly involved in the regulation of topic selected important mRNA, its action is through the negative regulation of ENST00000514661expression and indirectly regulation of ATR,CYB5Aand AKR1C3, the control relationship and control processure are complex, so it is not the experimental research key content.3. The regulation among ENST00000514661,ENST00000445908and ENST00000497854is the most complex in our experimental, they can mutual regulation, but also regulates the expression of ATR,CYB5A and AKR1C3, they are not the experimental research key content.4. Regulation of HTR4is not affected by LncRNA, positive regulation of it only by KCNJ5, not the experimental research key content.5. The expression of CYP11B2was only regulated by ENST00000497854and the regulation is positive. This relation of LncRNA and mRNA was the simplest form in our research, so the experiment and research focus on the contents of CYP11B2and ENST00000497854.