Expression Of MiR-15a In Human Knee Articular Chondrocytes And Effects Of MiR-15a On Proliferation And Apoptosis Of Articular Chondrocytes

BackgroundOsteoarthritis, also known as degenerative arthritis, is one of the most prevalentconnective tissue disorders, which has a tremendous influence on health of older peoplearound the world. Osteoarthritis is a kind of age-related degenerative joint condition, andis becoming an increasingly crucial risk factor of disability of the elderly. With theacceleration of aging of the population in our country, osteoarthritis has become a growingburden of the whole country. The etiology of osteoarthritis, however, is still unknown yet,involving various risk factors, such as aging, BMI (Body Mass Index), and joint injury.In-depth research of the mechanisms participating in the progression of osteoarthritis nowis desperately needed to better elucidate pathogenesis of osteoarthritis. Osteoarthritis,characterized by progressive loss of articular chondrocytes, osteophyte formation, is morelikely to occur among weight-bearing joints, such as hip and knee, and ultimately leads tothe destruction of articular cartilage structure, joint deformity and loss of function of joint.MicroRNAs, a family of endogenous noncoding RNA molecules with17-25 nucleotides, is highly conserved and prevalent in eukaryotic cells. MicroRNAs play animportant role in the negative post-transcriptional regulation of their target genes bybase-pairing with them. They usually directly cleave target mRNAs or repress thetranslation of them. Previous researches have shown that microRNAs participate invarious biological processes, including cell proliferation, cell death, organ developmentand many diseases. It is of great importance to investigate the role microRNAs play in thepathogenesis of osteoarthritis. Thus we focus on miR-15a and strive to figure out itseffects on the pathogenesis of osteoarthritis in vitro.Objective1. To detect the expression of miR-15a in normal human articular chondrocytes and inosteoarthritic articular chondrocytes.2. To make a preliminary research on the effects of miR-15a on the proliferation andapoptosis of articular chondrocytes.3. To investigate the effects of miR-15a on the expression of Col2a1in human articularchondrocytes in order to supply experimental evidence for the application of miR-15a as atarget for molecular therapy.Methods1. Sequential enzymatic digestion was used to harvest human knee articular chondrocytes.Chondrocytes were identified through toluidine blue staining and immunocytochemicalstaining of collagen type Ⅱ.2. Real-time quantitative PCR was used to detect the expression of miR-15a in normalhuman articular chondrocytes and in osteoarthritic articular chondrocytes.3. Lipofectamine2000was adopted to facilitate the transfection of miR-15a mimics orNegative Control into articular chondrocytes.4. MTT assay was used to verify the effects of miR-15a on proliferation of chondrocytesafter transfection.5. The influence of miR-15a on the apoptosis of chondrocytes was detected by flowcytometry.6. Western blot was used to detect the effects of miR-15a on the expression of Col2a1in chondrocytes.Results1. The chondrocytes were successfully harvested after digestion with trypsin andcollagenase Ⅱ. Immunocytochemical staining of collagen type Ⅱ and Toluidine bluestaining showed positive results and the purity of chondrocytes met the standard of thisexperiment.2. Real-time quantitative PCR showed that the expression level of miR-15a inosteoarthritic articular chondrocytes was higher compared with the counterpart of normalhuman articular chondrocytes (P<0.05).3. MiR-15a mimics and miR-15a Negative Control were successfully transfected intoarticular chondrocytes with a high efficiency.4. The results of MTT assay suggested that the proliferation of chondrocytes wassignificantly inhibited in the experimental group which was transfected with miR-15amimics, compared with the blank group (P<0.05).5. Flow cytometry showed the apoptosis rate of chondrocytes in the experiment group wassignificantly higher compared with the blank group (P<0.05).6. The results of Western blot showed the expression of Col2a1in chondrocytes of theexperiment group was significantly inhibited compared with the blank group (P<0.05)Conclusion1. The chondrocytes could be successfully harvested after digestion with trypsin andcollagenase Ⅱ with a high purity and vitality.2. The expression level of miR-15a in osteoarthritic articular chondrocytes was highercompared with the counterpart of normal human articular chondrocytes.3. Transfection of miR-15a mimics could increase the expression of miR-15a inchondrocytes.4. miR-15a could inhibit the proliferation and promote the apoptosis of human articularchondrocytes in vitro, which suggested that miR-15a might play crucial role in thepathogenesis of osteoarthritis.5. miR-15a could inhibit the expression of Col2a1in human articular chondrocytes in vitro, which could make miR-15a a potential target of molecular therapy of osteoarthritis.