Identification of HIV1-specific cascaded microRNA-mRNA regulatory relationships by parallel mRNA and microRNA expression profiling with AIDS patients after antiviral treatment

The molecular pathogenesis of infection by HIV (human immunodeficiency virus) is extremely complex and is not clearly defined despite intensive research efforts. MiRNA as a class of regulators in cancer development can affect multiple gene pathways by targeting various mRNAs. Thus, studies aimed at transcription and regulation after antiviral treatment combined with beneficial researches related to miRNAs are required. With the purpose of identifying the cascaded miRNA-mRNA regulatory relationships related to infection of HIV in gene level, the parallel miRNA and mRNA expression profiles were analyzed in our study. The differential expressed miRNAs and mRNAs were selected primarily. The differentially regulated miRNA-mRNA interactions were further identified according to the Pearson correlation analysis of gene expression level. In our study, the calculation of the regulatory impact factors (RIF) was also conducted, and it revealed several essential regulators related to antiviral treatment with AIDS. To give a further insight into these TF (transcription factors) regulators, the co-expression gene modules were identified using a threshold-based hierarchical clustering method. Meanwhile, the regulatory network was generated during the computing process. Understanding these regulatory elements may bear significant implications on understanding the pathogenesis of AIDS occurrence. The microarray profiles utilized in our study is derived from Chengdu University of TCM in Sichuan, China. All donors as experimental group are confirmed as AIDS clinical patients according to 1994 revised classification system for HIV infection in America using protein western blotting by local epidemic station. The diagnosis standards to patients were referred to the Guidelines for Diagnosis and Treatment of HIV/AIDS in China (2005). The patients were accepted if they conformed to the following criteria: (a) corresponding to the criteria for diagnosis and treatment of HIV/AIDS; (b) ranging in age from 4 to 78 years old; (c) agreeing to sign an informed consent form. In our study, there are fifty AIDS patients and seven healthy persons as control group donated blood samples. Controls were all healthy, HIV-1-negative donors. Whereas, two more representative groups named LVL and HVL were further defined according to the viral load of the patients in order to explore the relationship between virus load and gene expression level after antiviral therapy. The LVL group includes 14 samples from patients while HVL group includes 16 AIDS samples. In addition, a further 36 independent blood samples from 33 AIDS patients and 3 healthy donors were applied to test and verify the accuracy of the differential expression results. The clinical characteristics of the three groups (CON, LVL and HVL) were assumed in Table 6. The majority of HIV-positive carriers being treated with ART (antiretroviral therapy) as in-patients got improvements of the AIDS disease in our research. mRNA and miRNA profiling of three groups: CON (n=7), LVL (n=14), HVL (n=16).

HIV（人类免疫缺陷病毒，human immunodeficiency virus）感染的分子发病机制极为复杂，尽管已有大量深入的研究投入，目前仍未得到明确阐明。微小RNA（microRNA, miRNA）作为一类参与肿瘤发生发展的调控分子，可通过靶向多种mRNA影响多条基因通路。因此，亟需开展抗病毒治疗后的转录与调控机制研究，并结合miRNA相关的前沿探索。
本研究旨在从基因层面鉴定与HIV感染相关的级联miRNA-mRNA调控关系，对平行获取的miRNA和mRNA表达谱进行了系统分析。首先初步筛选出差异表达的miRNA与mRNA，随后通过基因表达水平的Pearson相关分析，进一步鉴定出差异调控的miRNA-mRNA互作关系。
本研究同时开展了调控影响因子（regulatory impact factors, RIF）的计算分析，筛选出多个与艾滋病抗病毒治疗相关的关键调控因子。为深入解析这些转录因子（transcription factors, TF）的调控机制，我们采用基于阈值的层级聚类方法鉴定了共表达基因模块，并在计算过程中构建了调控网络。阐明上述调控元件对于理解艾滋病发生的发病机制具有重要意义。
本研究使用的微阵列表达谱数据来源于中国四川成都中医药大学。本研究实验组的所有供者均经当地疾控中心采用蛋白质印迹法检测，符合美国1994年修订的HIV感染分类标准，确诊为艾滋病临床患者。患者诊断标准参照《中国艾滋病诊疗指南（2005版）》。入组患者需满足以下标准：（a）符合艾滋病诊断与治疗标准；（b）年龄介于4至78岁之间；（c）同意签署知情同意书。
本研究共纳入50名艾滋病患者及7名健康个体作为对照组，采集血液样本。对照组受试者均为HIV-1阴性的健康供者。此外，为进一步探索抗病毒治疗后病毒载量与基因表达水平的关联，我们根据患者的病毒载量额外划分了LVL和HVL两个亚组：LVL组包含14份患者样本，HVL组包含16份艾滋病患者样本。另有36份独立血液样本（来自33名艾滋病患者及3名健康供者）用于验证差异表达分析结果的准确性。
三组（CON、LVL及HVL）的临床特征详见表6。本研究中接受抗逆转录病毒治疗（antiretroviral therapy, ART）的多数HIV阳性住院患者的艾滋病病情得到改善。本研究的三组样本表达谱信息如下：CON组（n=7）、LVL组（n=14）、HVL组（n=16）。