Mechanism Of MicroRNA-27A Regulated Apoptosis Of Nucleus Pulposus Cells In Human Intervertebral Disc

IntroductionIntervertebral disc degeneration diseases(IDDD) is considered to be the pathologicalbasis of spinal degenerative diseases, which bring a global burden with severe healthcareand socioeconomic costs. The mechanisms underlying IDDD remain largely unknown,although a variety of factors have been suggested to influence its etiology. Recent studiesboth in vitro and in vivo suggest that an important contributor to the development of IDDis the cellular loss due to excessive apoptosis of disc cells. There is a growing body ofevidence in support of the hypothesis that the newly defned small noncoding RNAs,miRNAs, regulate the apoptotic machinery. This research is to aim at providing therapeuticeffect and experimental base of IDD, and finding the pathway for early repairment of IDD.MethodsThirty-two Human NP specimens were collected from idiopathic scoliosis patients[n=20; average age21.4(range18–38) years] and not disc herniation. NP cells werereleased from the NP tissues. Primary cultures of NP cells were divided into two groups,the experimental group was given artificial compressed air (0.5%CO2+99.5%compressed air) in a pressurized cabin, and the control group was cultured in a1.0MPaincubator. Apoptotic NP cells were identified by staining with FITC Annexin V/PI,andthen analyzed by FCM. The upregulation of miR-27a in cultured NP cells was achieved bytransfection with lentiviral Has-miR-27a, while the knockdown of miR-27a was achievedby transfection with lentiviral antigomiR-27a. The expression levels of PIK3CD, p-AKT,Bcl-2and NF-κB in NP cells were detected using Western Blot. Flow cytometry withFITC Annexin V/PI staining was used to detect the apoptosis in NP cells with modulatedmiR-27a in control and damaged samples.ResultsWe analyzed degenerative nucleus pulposus (NP) cells and found that the expressionof miR-27a was increased. The overexpression of miR-27a was further verified usingreal-time RT-PCR. Bioinformatics target prediction identified phosphoinositide-3kinases(PI3K) as putative targets of miR-27a. Furthermore, miR-27a inhibited PI3K expression bytargeting their3’-UTRs,and that was abolished by mutation of the miR-27a binding sites.Various cellular processes including cell growth, proliferation, migration and adhesion areregulated by activation of the PI3K/AKT signaling pathway, and nucleus pulposus cells areknown to strongly express the phosphorylated survival protein AKT. We investigated the role of PI3K/AKT pathway in miR-27a induced apoptosis by targeting PIK3CD and foundthat specific transient miR-27a expression reduced expression of PIK3CD, p-AKT, Bcl-2and NF-κB in accordance with the results in damaged cells. Whereas knockdown ofmiR-27a with con-miR-27a led to overexpression of PIK3CD, p-AKT, Bcl-2and NF-κB inNP cells. Caspase3is activated in the apoptotic cell both by intrinsic and extrinsicpathways. We examined the levels of cleaved caspase3and found that expression ofmiR-27a increased cleaved caspase3levels.ConclusionIn conclusion,we have identified PI3K as a novel target of miR-27a. In human IDD,upregulated of miR-27a promotes apoptosis by targeting PI3K, which implicated a role ofmiR-27a in the pathology of IDD. Moreover, miR-27a could provide a potentialtherapeutic target for IDD using the lentiviral Has-miR-27a.