| [Objective]Low back pain has become one of the most common musculoskeletal diseases in the world. Studies have shown that intervertebral disc degeneration (IDD) is an important factor leading to low back pain. The process of disc degeneration is an aberrant, cell-mediated response which include: 1.The degradation of matrix components in nucleus pulposus (NP) region; 2.Increased levels of matrix metalloproteinases; 3.NP cell apoptosis or death. It has been demonstrated that non-coding RNA (ncRNA) is widely present in living organisms and can play an important role in many biological processes by regulating gene expression. As a competitive endogenous RNA(ceRNA), circRNA can act as microRNA(miRNA)sponge to regulate the expression of target gene by binding to miRNA and eliminating the inhibition of its target gene. The aim of this study is to use bioinformatics to find circRNA that play a key role in IDD process and to explore its regulatory mechanisms in IDD process.[Method]1. Morphological changes of degenerated NP tissues were observed by HE staining. 2. The apoptosis of NP cells in NP tissues was observed by TUNEL fluorescence staining. 3. Differential expression of circRNA and microRNA microarray data from normal NP tissues and degenerated NP tissues were downloaded from the GEO database. Looking for key circRNAs and microRNAs that are negatively correlated with the key circRNAs. 4. Detection of key circRNAs and microRNAs that are significantly negative correlated with key circRNAs in NP cells by fluorescence in situ hybridization. 5. The binding sites of key circRNA and microRNA were detected by luciferase reporter assay. 6. Followed by miRPathDB v1.0, DIANA-miRPath v3.0, TargetScan database to predict the microRNA regulatory pathway. Using the DIANA, miRTarBase, TargetScan, miRDB database to predict the microRNA target gene. 7. Overexpressing key circRNA and key microRNAs in NP cells and then detected the expression of target genes by Western blotting and RT-PCR. 8.The effect of key circRNA on NP cell apoptosis was detected by flow cytometry.[Results]In this study, 104 differentially expressed circRNA were screened by bioinformatic method and predicted that 343 microRNA response elements. Then,the interaction relationship between circ-GRB10 and miR-328-5p was predicted by combining the microRNA chip data from the same sample source and correlation analysis. The presence of circ-GRB10 and miR-328-5p in the cytoplasm of the nucleus pulposus was verified by situ hybridization. And the changes in the expression levels of circ-GRB10 and miR-328-5p in nucleus pulposus cells were observed, which confirmed the results of the chip data analysis. It is speculated that circ-GRB10 may play a potential regulatory role in IDD process by competitive binding of miR-328-5p through the mechanism of ceRNA.The interation of circ-GRB10 as miR-328-5p sponge was confirmed by luciferase reporter assay, and the interaction sites of circ-GRB10 and miR-328-5p were confirmed. Incombination with the predicted pathway information, this study concluded that ERBB2 was the target gene of miR-328-5p. In the functional verification section, this study confirmed: 1. overexpression of miR-328-5p in NP cells could down-regulate the expression of ERBB2, and promote the apoptosis of NP cells. And circ-GRB10 can inhibit the function of miR-328-5p, up regulate the content of ERBB2 in NP cells, and inhibit the apoptosis of NP cells. 2. Similarly,inhibit the expression of circ-GRB10, can reduce the level of ERBB2 expression,and promote apoptosis of NP cells.[Conclusion]This study demonstrated that a large number of circRNAs are involved in the development and progression of IDD, and circ-GRB10 plays an important role in inhibiting NP cell apoptosis as a key circRNA in NP cell. And revealed important role of the balance of circ-GRB10-miR-328-5p-ERBB2 network in regulates NP cell apoptosis. |
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