METTL3 Promotes PGC-1α Transcrition And Alleviates Mitochrondrial Dysfunction Of Diabetic Nephropathy By Targeting LncRNA TUG1 M~6A Modification

Objective:According to the International Diabetes Federation(IDF),the number of people with diabetes worldwide in 2021 is about 537 million,but this number will rapidly increase to 783 million in 2045.Diabetic kidney disease(DKD)is one of the most common and serious chronic microvascular complications of diabetes.DKD is a complex polygenic disease,and the interaction and expression regulation of multiple genes often affect the occurrence and development of the disease.Among them,the epigenetic system has become one of the important mechanisms involved in the occurrence and development of diabetic nephropathy by regulating gene transcription and translation.It is well known that RNA is subject to various modifications,among which internal modifications play important roles in RNA metabolism.The most abundant internal RNA modification is N6-methyladenosine(m~6A).The m~6A methylation modification is an emerging epigenetic regulatory mechanism and first discovered in the 1970s,and recent advances in RNA modification have identified the key roles of N6-methyladenosine(m~6A)in nc RNA metabolism and biological processes.m~6A modification occurs best in the consensus motif RRACH(R=G or A;H=A,C or U).The m~6A modification machinery consists of"writers","erasers"and"readers"that are precisely balanced to support normal cellular function.m~6A modification plays an important role in a variety of biological processes,such as tumorigenesis,fat metabolism,glucose metabolism,biological rhythms,reproductive development,immune regulation,viral replication,myocardial remodeling,and stress response.However,the role of m~6A in the pathogenesis of DKD is not fully understood.Numerous studies have found that m~6A modification not only functions in m RNA,but also regulates the production and function of non-coding RNAs(such as mi RNAs,lnc RNAs,and circ RNAs).Studies have shown that m~6A modifications may modulate the function of lnc RNAs by providing binding sites for m~6A reader proteins or by modulating the structure of local RNAs to induce entry of RNA-binding proteins.Long non-coding RNAs(lnc RNAs)greater than 200 nucleotides in length covers a large class of transcribed RNA molecules that are specific,generally poorly conserved,and expressed at low levels.lnc RNAs have multiple modes of action,are also important transcriptional regulators.lnc RNAs are crucial in epigenetic regulation,gene transcription,gene translation,and m RNA processing,regulating a variety of biological processes,including homeostasis,cell metabolism,proliferation,apoptosis,and differentiation.Studies have shown that TUG1 plays an important role in many human kidney diseases,lnc RNA TUG1 interacts with the lnc RNA TUG1 binding site upstream of the Ppargc1a promoter region,increasing transcription of Ppargc1a m RNA,which in turn regulates mitochondrial function in podocytes by targeting Ppargc1a-encoded transcription factor PPARγcoactivator 1α(PGC-1α).Decreased levels of PGC-1αlead to impaired mitochondrial function leading to energy depletion,increased production of reactive oxygen species(ROS),and ultimately the development of diabetic nephropathy.Our research group used the Arraystar m~6A single-base resolution chip method to find that the m~6A level of lnc RNA TUG1 was reduced in HK-2 cells cultured with high glucose.According to the full-length sequence of the transcript,it was found that there were four m~6A sites in lnc RNA TUG1 in human HK-2 cells,and two identical m~6A sites in lnc RNA TUG1 on chromosome 11 corresponding to mouse.The purpose of this study was to analyze the biological pattern of RNA m~6A modification in HK-2 cells cultured with high glucose,and to explore whether the dynamic regulation of RNA m~6A methylation/demethylation imbalance is involved in the occurrence and development of diabetic nephropathy;to explore the m~6A of lnc RNA TUG1 modification mechanism,and to clarify whether the changes in the m~6A modification level of lnc RNA TUG1 cause the expression changes of lnc RNA TUG1 in high glucose-induced HK-2 cells,and whether the targeted regulation of methylation regulatory enzymes can promote PGC-1αtranscrition,alleviate mitochrondrial dysfunction and diabetic renal injury of diabetic nephropathy by targeting lnc RNA TUG1m~6A modification.Methods:The first part:Colorimetric method was used to detect the m~6A modification level of total RNA in human proximal tubular epithelial cells(HK-2).Western Blot and RT-q PCR was used to detect the levels of m~6A modification related regulatory enzymes in HK-2 cells.The second part:Maz F-RT-q PCR was used to detect the m~6A modification level at the four m~6A sites of lnc RNA TUG1 in HK-2 cells,respectively.The level of lnc RNA TUG1 in HK-2 cells was determined by RT-q PCR,and RIP(RNA immunoprecipitation)-PCR was used to detect whether lnc RNA TUG1 can bind to METTL3,IGF2BP2,RT-q PCR detects the RNA degradation of TUG1.The third part:Colorimetric method was used to detect the m~6A modification level of total RNA in HK-2 cells and mouse kidney tissue.Western Blot and RT-q PCR were used to detect the level of m~6A modification related regulatory enzymes in HK-2 cells and mouse kidney tissue.Western Blot,RT-q PCR and immunohistochemistry were used to detect the expressions of PGC-1αand downstream targets including Nrf1,Nrf2 and TFAM in HK-2cells and mouse kidney tissue.The expressions of mitochondrial DNA content in HK-2cells and mouse kidney tissue were detected by RT-q PCR,the content of mitochondrial ATP and the activities of mitochondrial respiratory chain complexes I,II,III,and IV in HK-2 cells and mouse kidney tissue were determined by UV spectrophotometry,Mito SOX Red was used to detect the level of ROS produced by HK-2 cells.Flow cytometry was used to detect apoptosis in HK-2 cells under different stimuli.The morphology of mitochondria in HK-2 cells and mouse kidney tissue and the changes of foot process in kidney tissue were observed by transmission electron microscope.Results:The first part:In HK-2 cells cultured with high glucose,the m~6A modification level of total RNA was significantly decreased,the expression of methyltransferase METTL3 was down-regulated,and the expression of modification recognition protein YTHDF1 was significantly up-regulated,while the expression of modification recognition protein IGF2BP2 was significantly down-regulated,and the changes of demethylase FTO,methyltransferase METTL14 and modification recognition protein YTHDF2 were not statistically different.The second part:In HK-2 cells cultured with high glucose,the m~6A modification levels at sites of 2047 and 4944 of lnc RNA TUG1were significantly down-regulated.The expression level of lnc RNA TUG1 decreased in high glucose-cultured HK-2 cells.RIP-PCR analysis showed that lnc RNA TUG1 could bind to METTL3,and indicated that METTL3 could enrich the expression of lnc RNA TUG1,and overexpression of methyltransferase METTL3 could significantly up-regulate the m~6A modification level of total RNA in HK-2 cells,and the m~6A modification level at sites of 2047 and 4944 of lnc RNA TUG1 were significantly up-regulated.RIP-PCR analysis showed that IGF2BP2 can recognize m~6A-modified lnc RNA TUG1 and enrich the expression of lnc RNA TUG1,high glucose stimulation can shorten the half-life of lnc RNA TUG1 and reduce its stability,while IGF2BP2 can prolong the half-life of lnc RNA TUG1 and improve its stability.The third part:Overexpression of METTL3 in HK-2 cells cultured with normal or high glucose induced their protein and m RNA levels of PGC-1α,Nrf1,Nrf2 and TFAM significantly up-regulated,mitochondrial DNA content,ATP content significantly increased,mitochondrial respiratory chain complex I activity and mitochondrial respiratory chain complex III activity were significantly up-regulated,ROS production and apoptosis significantly reduced,mitochondrial morphological damage alleviated.After transfection of lnc RNA TUG1 si RNA,the protein and m RNA levels of PGC-1α,Nrf1,Nrf2 and TFAM were significantly down-regulated,and the mitochondrial DNA content,ATP content,mitochondrial respiratory chain complex I activity and mitochondrial respiratory chain complex III activity were significantly down-regulated,ROS production and apoptosis were significantly increased,and mitochondrial morphological damage was aggravated.Compared with db/m mice,the m~6A modification level in kidney tissue of db/db mice was significantly down-regulated.Western Blot,RT-q PCR and immunohistochemical analysis showed that methyltransferase METTL3,the expression of modified recognition protein IGF2BP2 was significantly up-regulated,blood glucose,body weight,serum creatinine,urine albumin/creatinine ratio were significantly increased,plasma albumin levels were significantly decreased,and Blood urea nitrogen had no significant difference;Compared with db/db mice,after injection of AAV-METTL3 overexpression,the m~6A modification level in kidney tissue of db/db mice was significantly increased,methyltransferase METTL3 and modification recognition protein IGF2BP2 was significantly up-regulated,the levels of blood glucose,body weight,serum creatinine and urine albumin/creatinine ratio were significantly decreased,and the level of plasma albumin was significantly increased There were no significant changes in blood urea nitrogen.HE staining showed that compared with db/m mice,the kidneys of db/db mice had enlarged,dilated glomeruli,glomerular hyaline degeneration,and tubular dilatation,and PAS staining showed that compared with db/m mice,The glomerular mesangial matrix increased,the mesangial matrix expanded,and the ratio of matrix-positive area increased in the kidneys of db/db mice.The degree of tubulointerstitial fibrosis and the interstitial injury score were elevated,and METTL3 overexpression attenuated high glucose-induced renal damages.Compared with db/m mice,the expression levels of lnc RNA TUG1 in kidney tissue and serum of db/db mice were significantly down-regulated,Western Blot,RT-q PCR and immunohistochemical analysis showed that the levels of PGC-1α,Nrf1,Nrf2 and TFAM were significantly down-regulated,the content of mitochondrial DNA and ATP,the activities of mitochondrial respiratory chain complex I and mitochondrial respiratory chain complex III were significantly down-regulated.Mitochondrial morphological damage was aggravated,foot process fusion,and compared with db/db mice,after AAV-METTL3 overexpression injection,the level of lnc RNA TUG1 in kidney tissue and serum of db/db mice were significantly up-regulated,the levels of PGC-1α,Nrf1,Nrf2 and TFAM were significantly up-regulated,the mitochondrial DNA content was significantly up-regulated,ATP content was significantly increased,mitochondrial respiratory chain complex I activity and mitochondrial respiratory chain complex III activity were significantly up-regulated,mitochondrial morphological damage and foot process lesions were alleviated.Conclusions:The first part:The level of m~6A modification in human proximal tubular epithelial cells cultured with high glucose was significantly down-regulated,the level of RNA m~6A modification may be mainly regulated by methyltransferase METTL3,and the dynamic regulation of RNA m~6A methylation/demethylation imbalance may be involved in diabetic nephropathy occurrence and development.The second part:The m~6A modification level of lnc RNA TUG1 under high glucose is downregulated,resulting in a decrease of lnc RNA TUG1 level in HK-2 cells,and methylated transferase METTL3 can promote the expression of TUG1 through regulation of m~6A modification in a IGF2BP2-dependent manner in the HK-2 cells.The third part:METTL3 upregulates the expression of lnc RNA TUG1 through lnc RNA TUG1 m~6A modification,and regulates the PGC-1αpathway to improve the activity of mitochondrial respiratory chain complex I and III in HK-2 cells induced by high glucose,increase mitochondrial DNA content and ATP content,reduce reactive oxygen species,reduce mitochondrial morphological changes and apoptosis,also improve mitochondrial respiratory chain complex I and III activity,mitochondrial morphology and foot process changes in db/db mice,and increase mitochondrial DNA content and ATP content,reduce blood sugar,body weight,serum creatinine and urine microalbumin/urine creatinine ratio levels,up-regulate serum albumin levels,reduce glomerular hypertrophy,increases in mesangial matrix,collagen deposition and tubulointerstitial fibrosis.