LncRNA HOTTIP Promotes Diabetic Retinopathy By Regulating P38-MAPK Signaling Pathways

Background and significance:Diabetes is a metabolic disease caused by insufficient insulin secretion and islet dysfunction.It is characterized by chronic progressive elevated blood sugar.Diabetes is a chronic disease with a high incidence.At present,depending on the cause,diabetes is mainly divided into type 1,type 2,gestational diabetes and special types of diabetes.Among them,type 2 diabetes is the most common.Diabetes has seriously endangered the health of people all over the world.According to statistics,the incidence of type 2 diabetes in China has been increasing year by year.This may be related to the improvement of our people's living standards and changes in diets.At the same time,diabetes is the serious threat to children,adolescents and adults around the world.The majority of diabetics live in developing countries and low-and middle-income countries,and the studies show that the current incidence of diabetes in the country is 11.6%.Among them,the incidence rate is 12.1%for men and 11%for women.Only 25.8%of these patients received adequate treatment and blood sugar was controlled.Diabetes can lead to a series of pathological changes,such as diabetes,which can lead to a series of vascular lesions by increasing ROS,inflammatory reactions,and damage to blood vessel walls.The vascular complications of diabetes are very serious and diverse.They mainly include microvessels in multiple organs and tissues and lesions of large blood vessels such as muscles,eyes,skin,heart,brain,and kidneys.Diabetic retinopathy is the most common vascular complication in most diabetic patients and the most common cause of blindness in diabetic patients.Therefore,the research has participated in the occurrence and advancement of diabetic retinopathy,and it is the focus of DR research to find targets for molecular level diagnosis and treatment.In the human genome,less than 2%of gene sequences are involved in encoding proteins,and more than 98%of gene sequences are classified as non-coding sequences.Among them,most of these sequence transcriptions are non-coding RNA.In these non-coding RNAs,long-stranded non-coding RNAs(lncRNAs)have more than 200 nucleotide lengths.LncRNAs are structurally similar to mRNA,but it does not participate in the protein coding process.They can regulate the expression level of genes that encode proteins at the transcription level and post-transcription levels.The transcription of long-chain non-encoded RNA also undergoes RNA polymerase transcription and is modified by capping,cutting,and tail modification.LncRNA is a product of RNA polymerase and was once considered to have no specific biological function.However,recent studies have shown that lncRNA participates in a series of biological processes and plays a corresponding regulatory role.The researchers found that lncRNA is widely involved in many important biological functions in the body,such as the regulation of epigenetics,regulation of cell cycles,and apoptosis and induction of recoding of pluripotent stem cells.In addition,IncRNA can also act as a precursor to miRNA,thus regulating the function of the latter.In general,lncRNA can mainly regulate gene expression,regulate the position of certain proteins in cells,and combine with cell substructures to play its unique biological functions.The abnormal expression of LncRNA is also closely related to the progress of many human diseases,such as cardiovascular diseases and tumors.In recent studies,studies of human beta-cell transcription groups have found that lncRNA can be dynamically regulated and expressed in humans with type 2 diabetes.Genomic association studies have shown that lncRNA-ANRIL is closely related to type 2 diabetes.The HOXA terminal transcription antisense RNA(HOXA transcriptat the distal tip,HOTTIP)is a long-chain non-encoded RNA.It was transcribed from the HOXA family and was found to be involved in the biological control process in many diseases.LncRNA HOTTIP was first found in the body's distal distal fibroblasts,whose genes are adjacent to HOXA 13 and are located at 330bp upstream of the latter.Its expression is mainly located at the far end of the body and its expression is relatively conservative.Because the 5 'end structure of the HOXA gene is relatively close,this leads to the close proximity of the nearby HOTTIP gene to multiple sequences of the HOXA gene.This close spatial structure may be related to the regulation of the HOTTIP gene.HOTTIP RNA can be combined with the WDR5/MLL complex to act on the HOXA 5 'end.HOTTIP RNA can also activate the transcription process of H3K3me3 and is necessary during the activation of the HOXA gene.The study found that the silent HOTTIP gene inhibits the expression of the HOXA5 'terminal gene.And its inhibition effect is inversely proportional to the HOTTIP gene distance.That is,the HOXA13 and HOXA11 genes closest to the HOTTIP gene are most inhibited.A number of studies have shown that HOTTIP RNA is related to the development of malignant tumors.For example,in cancers such as pancreatic cancer,gastric cancer,and colorectal cancer,the expression of HOTTIP was abnormal.LncRNA has the above-mentioned regulatory target gene pathway and it is also considered to have many regulatory effects in ophthalmopathy.For example,in cataracts,IncRNA is considered to be involved in the pathogenesis.Cataracts are a serious eye disease that can cause the transparency of the lens to be reduced until blindness,and their incidence is high,mainly in elderly patients.Studies have found an increase in the expression of IncRNA MIAT in age-related cataracts.The activity of the lens cells that expressed the decline of MIAT was also weakened by oxidative stimulation.In the eyes of another common eye disease,green light,lncRNA has also been confirmed to be involved in its development.Glaucoma is characterized by abnormal increases in Intraocular pressure,optic nerve atrophy and visual field defects,which eventually affect vision and even blindness.Evidence of the involvement of lncRNAs was also found in PVR,another intractable eye disease hyperplastic vitreous retinal ophthalmopathy.PVR is a kind of disease that causes the retina to pull away or fix in the presence of a cellular membrane structure before and after the retina and in the glass.The study found that during the occurrence of PVR,the key process of EMT appeared in retinal pigment epithelium(RPE)cells.Some scholars have found that the expression of lncRNA MALAT1 in PVR patients has been increased,and its expression level is proportional to that of Yanzhongchengdu.In diabetic retinopathy(DR),lncRNA has also been confirmed to be involved in its occurrence and progression.The occurrence mechanism of DR is very complex and may involve the interaction and influence of multiple pathways.Some scholars have found that the expression of lncRNA MIAT in retinal endothelial cells induced by high sugar was significantly increased.In recent years,some studies have found that LncRNA is involved in the occurrence and development of diabetic retinopathy.This can not help but suggest to us whether HOTIP RNA is involved in the progression of DR because the specific biological mechanism of HOTTIP in retinopathy is still unclear.This study found that HOTTIP is involved in the regulation of retinopathy,which also provides a theoretical basis for the prevention and treatment of this disease in the future.Objective:To investigate the expression of LncRNA HOTTIP in DR tissues and cells and its relationship with the biological behavior of retinal endothelial cells.Methods:1.In order to study the role of HOTTIP in diabetic ophthalmopathy,we firstly constructed a diabetic animal model.Streptozotocin STZ was intraperitoneally injected into SD rats to induce diabetes.After the animal model was successfully constructed,we used QRT-PCR to study the expression of HOTTIP in diabetic animal models.2.1n order to further study the role of HOTTIP in the regulation of retinal function,we performed an intraocular injection of shRNA.QRT-PCR test was used to verify whether HOTTIP expression was decreased.3.After the conversion of HOTTIP siRNA,the HOTTIP expression in RF/6A cells was significantly reduced.MTT results show that high sugar and oxidative stress environments can weaken cell viability.The reduction of HOTTIP can significantly reduce the viability of cells in high sugar and H202 environments.4.We used the Western blue experiment to detect whether the MAPK signal path was activated.In order to further clarify the relationship between the p38-MAPK signaling pathway and HOTTIP regulating cells,we added p38 specific inhibitors SB203580,ERK specific inhibitors U0126,and JNK specific inhibitors SP600125 in RF/6A cells.And observe changes in cell viability.Results:1.QRT-PCR results showed that the expression of HOTTIP was up regulated in the retina of STZ induced diabetic rats,and was statistically significant compared with that of non diabetic rats.The expression of HOTTIP in the retina of type 2 diabetic model dB/db mice was significantly higher than that of control mice at the same age.QRT-PCR experiments showed that HOTTIP shRNA significantly decreased the expression of HOTTIP,while shRNA NC did not alter HOTTIP expression.2.We used the Western blot method to detect the expression of inflammatory protein ICAM-1 and VEGF in the retina of diabetic rats.The results showed that the expression of ICAM-1 and VEGF in retina was significantly up-regulated by diabetes.Next,we injected HOTTIP shRNA into the eyes of rats.The results showed that down-regulation of HOTTIP significantly reduced the expression of ICAM-1 and VEGF in the retina.3.After transfection of HOTTIP siRNA,the expression of HOTTIP in RF/6A cells was significantly downregulated.MTT results showed that high glucose and oxidative stress could attenuate cell viability respectively.Down regulation of HOTTIP significantly attenuated cell viability in high glucose and H2O2 environments.4.Western Blot showed that the down-regulation of HOTTIP resulted in a significant down-regulation of phosphorylated p38,but the expression of phosphorylated ERK1/2 and JNK1/2 remained unchanged.Down regulation of HOTTIP leads to activation of p38-MAPK signaling pathway.5.We added p38 specific inhibitor SB203580 to RF/6A cells.The results showed that the overexpression of HOTTIP was also inhibited after the suppression of p38 expression,and also resulted in the improvement of the viability of RF/6A cells.We also added ERK specific inhibitor U0126 and JNK specific inhibitor,SP600125,in RF/6A cells,however,the viability of HOTTIP mediated cells was not altered.Conclusion:1.The expression of HOTTIP in diabetic rats was increased,and HOTTIP promoted the inflammatory response of the diabetic retina and participated in the regulation of diabetic microvascular lesions.2.in vitro cell culture,HOTTIP can reduce the viability of RF/6A cells.Under high sugar.HOTTIP increases cell apoptosis.3.HOTTIP regulates retinal endothelial cells through the P38-MAPK signaling pathway and plays an important role in the pathogenesis of diabetic retinopathy.