LncRNA RP11-753H16.3调控ITGA5对AIDS患者肠粘膜损伤作用机制研究

HIV infection is an important factor in accelerating disease process of AIDS patients by causing the intestinal mucosal injury. Previous works have revealed that lncRNAs played crucial role on cell differentiation and apoptosis. However, the mechanism for intestinal mucosal injury of AIDS patients is still unclear. Our previous work illustrated that the expression of lncRNA RP11-753H16.3 was significantly decreased in the intestinal mucosal tissue for AIDS patients, this was in line with the expression of protein ITGA5. Further bioinformatics statistic analysis and bioinformatics prediction indicated that ITGA5 was the core target protein for lncRNA RP11-753H16.3, which suggested that lncRNA RP11-753H16.3 may regulate the mucosal injury for AIDS patients via ITGA5. The decreased expression tendency of lncRNA RP11-753H16.3 and ITGA5 were confirmed in the intestinal mucosal tissue of AIDS patients and health control, as well as the Caco-2-HIV-1 cell model. Thus, we hypothesized that the intestinal mucosal damage of AIDS patients would be regulated by lncRNA RP11-753H16.3 via ITGA5, as well as its downstream pathways PI3K/Akt and focal adhesion. In this project, to verity our hypothesis, we intend to confirm the role of ITGA5 on mucosal injury for AIDS patients by constructing the intestinal cell model with Caco-2 cell line; and prove the role of lncRNA RP11-753H16.3 on regulating ITGA5, as well as its downstream signal pathway PI3K/Akt and focal adhesion during the process of intestinal mucosal injury. This project will find target protein, such as ITGA5, which may affect the intestinal mucosal injury of AIDS patients, and prove the role of lncRNA RP11-753H16.3 on regulating the intestinal mucosal injury via ITGA5. Our work will provide an important target for treating intestinal mucosal injury of AIDS patients, and benefiting for intestinal mucosal immune reconstitution of AIDS patients.

HIV感染致AIDS肠黏膜损伤是加快该病死亡的重要因素。国内外研究表明lncRNAs在细胞分化和凋亡中发挥主要作用，但对AIDS肠黏膜损伤机制不清。我们前期发现lncRNA RP11-753H16.3和ITGA5在AIDS肠黏膜上皮细胞中表达均显著下降，生物信息学预测ITGA5可能是该lncRNA作用的核心靶标，在AIDS、正常肠黏膜组织及细胞模型中证实两者存在显著差异。提出假说，lncRNA RP11-753H16.3可能通过调控ITGA5参与AIDS肠黏膜损伤机制。为阐明该假说，我们基于AIDS肠黏膜细胞损伤模型，拟验证lncRNA和ITGA5过表达和干扰对细胞损伤模型的影响，确定该lncRNA与ITGA5的调控关系，明确该lncRNA调控ITGA5及其下游PI3K/Akt等通路对AIDS肠黏膜损伤机制。该课题从非编码RNA水平揭示调控AIDS肠黏膜损伤机制，为该病治疗提供新视角。

为了确定lncRNA RP11-753H16.3、hsa-miR-32-5p（miRNA-32）及ITGA5在HIV/AIDS肠上皮细胞生长、迁移及渗透性的功能，探讨HIV/AIDS肠粘膜损伤的细胞分子生物学机制，我们利用Caco-2细胞和Tat蛋白，构建HIV/AIDS肠黏膜上皮细胞模型。利用细胞活性实验、侵袭实验及渗透性实验分别检测干扰lncRNA RP11-753H16.3、干扰ITGA5及过表达miRNA-32对HIV/AIDS Caco-2细胞生长、迁移及渗透性的影响。利用Western Blot检测干扰lncRNA RP11-753H16.3、干扰ITGA5及过表达miRNA-32对HIV/AIDS Caco-2细胞中ITGA5、p-p38、p-ERK、p-JNK、p-cMET、p-beta-catenin、p53、γH2AX及AcH4表达的影响。结果表明：干扰lncRNA RP11-753H16.3使Caco-2细胞活力降低，增加了Caco-2细胞渗透性，抑制了ITGA5、p-p38、p-JNK、p-β-catenin及p-53的蛋白表达，促进了p-cMET、γH2AX及AcH4的蛋白表达。干扰ITGA5和过表达miRNA-32使Caco-2细胞活力降低，增加了Caco-2细胞渗透性。干扰ITGA5抑制了p-p38、p-JNK及γH2AX的蛋白表达，促进了p-ERK、p-cMET和p-β-catenin的蛋白表达。过表达miRNA-32抑制了p-p38、p-β-catenin、p53、γH2AX及AcH4的蛋白表达，促进了p-cMET的表达。.这些结果说明干扰lncRNA RP11-753H16.3对HIV/AIDS肠黏膜上皮细胞生长和迁移有一定的抑制作用，对HIV/AIDS肠黏膜上皮细胞渗透性有一定的促进作用。干扰lncRNA RP11-753H16.3抑制肠黏膜上皮细胞生长和迁移，促进其渗透性，可能通过抑制ITGA5、p-p38和/或促进p-cMET发挥作用。干扰ITGA5和过表达miRNA-32抑制HIV/AIDS肠黏膜上皮细胞生长和迁移，促进其渗透性；可能相互调节，并且可能通过抑制p-p38和/或促进p-cMET发挥。