The Role And Mechanism Of LncRNA In Autophagy Induced By Oxidative Stress In Human TMCs

Primary open-angle glaucoma（POAG）is one of the most common types of glaucoma.The main risk factor of optic nerve damage in POAG patients is the dysfunction of aqueous humor drainage caused by the changes of function and tissue structure of trabecular meshwork cells（TMCs）.The longer the intraocular pressure（IOP）increases,the more serious the damage of optic function.At present,the most effective way to treat POAG is to dredge the angle of atrium and relieve the block of aqueous humor to reduce intraocular pressure.It has been confirmed that reactive oxygen species（ROS）are abundantly expressed in the aqueous humor and trabecular meshwork of POAG patients,indicating that POAG patients are under oxidative stress.As an important pathogenic factor,oxidative stress can cause abnormal deposition of extracellular matrix（ECM）in the trabecular meshwork schlem canal（TM/SC）,change the normal physiological structure of trabecular meshwork（TM）,increase the resistance of aqueous outflow,and increase the pathological intraocular pressure.Autophagy is a highly conserved catabolic process in cells.It can maintain homeostasis through lysosomal degradation of excess proteins and damaged organelles.The results show that autophagy under oxidative stress can accelerate the removal of ROS,and autophagy in fibrotic diseases can also degrade ECM related proteins such as collagen.In the study of apparent regulation,long-chain noncoding RNAs（lnc RNAs）and interacting proteins play an important role.lnc RNAs usually do not encode proteins and were once considered as by-products in the transcription process and have no biological function.However,in recent years,many studies have shown that lnc RNAs participates in the regulation of gene expression,including transcription and post transcriptional regulation,in the form of transcriptional enhancer and other regulatory elements,and is involved in many important regulatory processes such as chromosome silencing,chromatin modification,proto oncogene activation,and so on.Previous studies have shown that lnc RNAs are differentially expressed in different types of ophthalmopathy,which may be involved in the occurrence and development of ophthalmopathy,but the specific mechanism has not been revealed.Although autophagy plays a key role in the removal of ROS and ECM related proteins,the regulatory role and mechanism of lnc RNAs in oxidative stress-induced autophagy of TMCs are unclear.[Research purpose]To explore the effect of lnc RNAs on autophagy in TMCs induced by oxidative stress,and to reveal its specific regulatory mechanism.[Research contents and methods]1.Different concentrations of H₂O₂ stimulated TMCs to induce oxidative stress in vitro.The expression level of ROS was detected by fluorescence probe.The activity and apoptosis of TMCs were detected by CCK-8 and flow cytometry.The expression level of ECM related genes and proteins was detected by real-time quantitative PCR（RT-q PCR）and western blot.Finally,the appropriate stimulation concentration was determined;2.Autophagy under oxidative stress was observed by double labeled adenovirus tracing GFP-m RFP-LC3,and fluorescent probe technique was used to detect ROS expression level.Autophagy related markers and ECM related proteins expression level were detected by western blot.Using 3-methyladenine（3-m A）,an autophagy inhibitor,to explore the role of autophagy in TMCs under oxidative stress;3.High throughput sequencing of transcriptome.Bioinformatics was used to analyze the differentially expressed lnc RNAs and m RNAs,to screen the RNAs that may be related to autophagy,to predict the expression correlation and to carry out RT-q PCR verification;4.We designed specific si RNA to down regulate the expression of the selected genes,detected the expression of ROS and autophagy,detected the expression level of autophagy related markers and ECM related proteins,and confirmed that the selected genes participate in autophagy regulation;5.The lnc RNA interacting protein was found by RNA pull down and mass spectrometry（MS）,and the binding site of lnc RNA was predicted by RNA binding protein immunoprecipitation（RIP）;6.Specific si RNA was used to knockdown the interaction protein expression level,then detect the target m RNA and its coding protein expression level to confirm the role of the interaction protein on target m RNA.[Results]1.After hydrogen peroxide（H₂O₂）stimulated h TMCs,the average fluorescence intensity of ROS increased,the expression of ECM associated proteins and their coding genes increased,the number of autophagosomes increased,and the expression level of autophagy membrane protein increased,which had statistical significance compared with the control group（P < 0.05）.2.The results of transcriptome sequencing showed that the expression of lncrna-ENST000523905 and TP53INP1 was significantly increased under oxidative stress of TMCs,the difference multiples were larger than 2,P < 0.05.3.RT-q PCR and Western blot showed that inhibition of ENST00000523905 could down regulate the expression of TP53INP1.4.In accordance with the results of 3-m A treatment,knocking down the expression of TP53INP1 or ENST00000523905 can reduce the number of autophagic bodies of TMCs induced by H₂O₂,down regulate the expression of autophagic membrane protein,promote the increase of the average fluorescence intensity of ROS and the expression of ECM related protein.Compared with the control group,the difference is statistically significant（P < 0.05）.5.The results of RNA pull down,MS and rip suggested that ENST00000523905 could bind directly to C/EBPβ.6.The results of RT-q PCR and Western blotting showed that down regulating the expression of C/EBPβ could inhibit the expression of TP53INP1,the difference was statistically significant（P < 0.05）.[Conclusion]1.Autophagy can promote the degradation and clearance of ECM in TM under oxidative stress,which may be involved in the pathological process of glaucoma.2.ENST00000523905 played a positive role in the regulation of TP53INP1 transcription,both of which were involved in the regulation of autophagy level of TMCs under oxidative stress.3.ENST000523905 may enhance autophagy by promoting TP53INP1 transcription expression by binding with C/EBPβ,thereby reducing ROS and ECM related protein expression induced by oxidative stress in TMCs.