The Protective Effect Of LncRNA NR₀30777 By Regulating The Expression Of Zfp326 And Cpne5 In Neurodegenerative Damage Induced By Paraquat

ObjectiveParkinson's disease?PD?is a central nervous system degenerative disease that occurs frequently in the elderly.The main cause of this disease cannot be clarified at present,and it is generally believed that the occurrence of PD is due to the interaction of environmental and genetic factors.Paraquat?PQ?,as a herbicide widely used in agricultural production,may be one of the environmental factors leading to PD.Long non-coding RNA?lncRNA?usually refers to the non-coding RNA transcripts with a length greater than 200 nucleotides.PQ changes the lncRNA expression profile in the substantia nigra of mice.NR₀30777,a specific lncRNA,was determined to be the focus of our research through a series of technical means.This study explored the role of NR₀30777 in PQ induced nerve damage in mice through gene knockdown and overexpression technology in vitro and in vivo,so as to become a new prevention and treatment target of PD induced by PQ.MethodsIn vitro:?1?The expressions of NR₀30777,zfp326 and cpne5 mRNA were detected by qRT-PCR after PQ treatment of N2a cells,MN9D cells and primary neurons in the cerebral cortex of mice?n=3?.?2?N2a cells were treated with PQ,and the protein expressions of ZFP326 and CPNE5 were detected by Western blot?n=3?.?3?NR₀30777 knockdown?N2a-KD?and overexpression?N2a-OE?N2a cell lines were constructed by siRNA transfection and lentivirus infection.?4?N2a-KD and N2a-OE cells were treated with PQ,and the expressions of NR₀30777,zfp326and cpne5 Mrna were detected by qRT-PCR?n=3?.?5?N2a-KD and N2a-OE cells were treated with PQ,and the expressions of ZFP326 and CPNE5 protein were detected by Western blot?n=3?.?6?N2a-KD and N2a-OE cells were treated with ActD,and mRNA contents of NR₀30777 and zfp326 at different time points were detected by qRT-PCR,and analyzed mRNA stability?n=3?.?7?N2a-KD and N2a-OE cells were treated with PQ,and CCK-8 assay was used to detect cell proliferation activity?n=6?.?8?N2a-KD and N2a-OE cells were treated with PQ,and apoptosis and cell cycle were detected by flow cytometry?n=3?.?9?N2a cells were treated with NAC,a ROS scavenger,followed by PQ,and the expression of NR₀30777 was detected by qRT-PCR?n=3?.?10?N2a cells were treated with H₂O₂,and the expression of NR₀30777 was detected by qRT-PCR?n=3?.In vivo:?1?Neural system-specific NR₀30777 overexpressed C57BL/6 mice and their negative control mice were constructed by CRISPR/Cas9 and Cre hybridization?constructed by Shanghai nanfang mode biotechnology co.,LTD.?;?2?The mice were poisoned with the dose of PQ and MPTP successfully constructed in the early stage,namely 10 mg/kg PQ?intraperitoneal injection once every two days,a total of 10 times?and 30 mg/kg MPTP?subcutaneous injection for 5 consecutive days?.The control group was injected with the same amount of saline.The body weight was monitored in real time during the poisoning process.?3?After the poisoning,behavioral experiments include climbing experiment,suspension experiment and open field experiment were conducted to detect the motor function of mice?n=5?.?4?Mice were harvested after the behavioral experiment,and some of them was perfusion of the heart to make the whole brain paraffin sections.Other parts of the mice were sacrificed to dissect and separate the middle substantia nigra,hippocampus,cerebral cortex,heart,liver,kidney and other important organs.?5?Morphological changes of neurons in the substantia nigra were detected by HE staining?n=3?.?6?Hoechst staining was used to detect the changes in apoptosis of neurons in the substantia nigra?n=3?.?7?The expressions of NR₀30777,zfp326and cpne5 mRNA in the substantia nigra,hippocampus and cortex were detected by qRT-PCR?n=3?.?8?The protein expressions of ZFP326 and CPNE5 in the substantia nilosum,hippocampus and cortex were detected by Western blot?n=3?.?9?The expressions of NR₀30777,zfp326 and cpne5 mRNA in heart,liver and kidney were detected by qRT-PCR?n=3?.ResultsIn vitro:?1?PQ can cause regular changes the expression of NR₀30777,zfp326 and cpne5mRNA of N2a cells,MN9D cells and primary neurons in the cerebral cortex of mice.?2?PQ changed the expression of ZFP326 and CPNE5 proteins in N2a cells.?3?SiRNA transfection and lentivirus infection reduced and increased the expression of NR₀30777,and NR₀30777 knocked down and overexpressed N2a cells were successfully constructed.?4?Knockdown and overexpression of NR₀30777 regulated the expression of Zfp326 and Cpne5 mRNA and protein in PQ-induced N2a cell damage.?5?Knockdown and overexpression of NR₀30777affected the stability of zfp326 mRNA.?6?Knockdown and overexpression of NR₀30777 affected cell proliferation,apoptosis and cell cycle in PQ-induced N2a cell damage.?7?NAC can cause decreased expression of NR₀30777 in PQ-induced N2a cell damage.?8?H₂O₂ treatment resulted in increased NR₀30777expression.In vivo:?1?Overexpression of NR₀30777 in the nervous system can cause slow weight growth of mice,but does not affect the coefficients of important organs.?2?Overexpression of NR₀30777 in the nervous system can alleviate motor function damage caused by PQ or MPTP.?3?Overexpression of NR₀30777 in the nervous system can reduce the damage of PQ or MPTP to the neurons in the substantia nigra,as well as the morphological damage of nerve cells,nuclear condensation and apoptosis of nerve cells.?4?Overexpression of NR₀307777 in the nervous system regulates the expression of Zfp326 and Cpne5 mRNA and protein in the substantia nigra.Conclusion?1?LncRNA NR₀30777 can regulate the expression of its homologous gene Zfp326 and its potential downstream target gene Cpne5,change the expression level of its mRNA and protein,and affect the stability of mRNA.?2?PQ can cause changes in the expressions of lncRNA NR₀30777,Zfp326and Cpne5.During this process,NR₀30777 regulates the expressions of its homologous gene Zfp326 and its potential downstream target gene Cpne5 at the transcription level,post-transcription level and translation level.?3?LncRNA NR₀30777 plays an important role in regulating the proliferation activity,cell cycle progression and apoptosis process of nerve cells,which is manifested as promoting the proliferation activity and cell cycle progression and inhibiting apoptosis.?4?NR₀30777 expression was induced by ROS in the process of PQ damage to nerve cells.NR₀30777 with increased expression promoted cell proliferation and activity,promoted cell cycle progression and inhibited apoptosis in the process of PQ damage to nerve cells.?5?Overexpression of NR₀30777 in the nervous system of mice can alleviate the damage of PQ or MPTP to the motor function of mice,and reduce the damage of PQ or MPTP to the morphology of mesencephalic substancin neurons and inhibit the occurrence of apoptosis while regulating the expression of Zfp326 and Cpne5 in mesencephalic substancin.In summary,NR₀30777 after ROS produced by PQ stimulate higher expression,and started the homologous genes Zfp326 potential Cpne5 downstream target genes expression and regulation,and improve the nerve cell morphology,promote the activity,the process of cell cycle and cell proliferation and inhibit apoptosis,alleviate the damage of PQ on motor function,play against PQ neurotoxicity of protection.