| Background and purposeObstructive nephropathy(ON)refers to urinary calculi,ureteral stenosis,bladder dysfunction and other post-renal factors that cause urinary excretion disorders,resulting in renal pelvis hydronephrosis,resulting in renal parenchymal damage,ultimately impaired renal function and renal fibrosis.ON is common in clinical practice,renal fibrosis is the pathological outcome of ON,When urine is not excreted smoothly,the pressure in the renal pelvis and calyx increases,the renal tubules expand,and urine leaks into the interstitial space,activating a series of fibrosis mechanisms,and a large number of collagen fibers are deposited,replacing the original functional structure.For the clinical treatment of early ON,surgical removal of urinary tract obstruction is the first choice to save renal function,but many patients’renal function still cannot recover after the removal of urinary tract obstruction,or even progressively worsen.The reason may be related to the progressive development of obstructive renal fibrosis.However,there is still no effective measure to prevent and reverse the progress of obstructive renal fibrosis,and the research on its molecular mechanism is still unclear.So in-depth study about the molecular mechanisms of obstructive renal fibrosis is still necessary.The most abundant RNA modification of eukaryotes is N6-methyladenosine(m6A),which is an important part of epigenetics and highly conserved in mRNA and non-coding RNAs of many species.RNA m6A is the methylation of the 6th N atom of the RNA base adenine,which is catalyzed by methyltransferases and demethylases and recognized by reading proteins.Methyltransferase is responsible for catalyzing the addition of methyl groups.The RNA m6A methylases that have been found in mammalian cells mainly include WTAP,METTL14,KIAA1429,METTL3,among which WTAP,METTL3 and METTL14 form the core methyltransferase complex.ALKBH5 and FTO are major demethylases,which are responsible for removing added methyl groups.Thus,RNA m6A level in tissues of different organisms and at various stages are constantly changing and reversible.Numerous literatures have reported that RNA m6A plays an important role in the process of organ fibrosis.Current studies mostly believe that the level of RNA m6A is elevated in the development of fibrosis and plays a promoting role,and is involved in apoptosis,inflammation,oxidative stress and non-coding RNA regulation and other mechanisms,but its research in renal fibrosis is still very limited,worthy of further study.In recent years,the mechanism research on the occurrence and development of renal fibrosis has gradually shifted from proteins and mRNAs to non-coding RNAs,including microRNAs,lncRNAs,and circRNAs.The role of microRNAs in animal and plant genomes has been extensively explored and studied.The vast majority of microRNAs consist of 21-23 nucleotides,and by binding to specific sites on target genes,they affect the mRNA degradation and protein synthesis of many genes,thereby participating in various biological processes.Numerous research reports have confirmed that microRNAs play an important role in the occurrence and development of renal fibrosis,among which miR-21 is one of the most frequently mentioned microRNAs in the field of renal fibrosis.The dysregulated expression of miR-21 can be found in many renal fibrosis models and clinical kidney samples,and most studies agree that miR-21 has the function of promoting the development of renal fibrosis,but its maturation process and downstream signaling pathway is unclear and warrants further exploration.SPRY1 is a target gene of miR-21.Studies have reported that SPRY1 inhibits the ERK/NF-κB pathway in angiotensin Ⅱ-induced rat liver fibrosis and bleomycin-induced pulmonary fibrosis,while ERK/NF-κB pathway also plays an important role in the occurrence and development of organ fibrosis inflammation;then whether miR-21 activates the ERK/NF-kB signaling pathway by targeting SPRY1,promotes the development of inflammation in obstructive kidneys,and then promotes obstructive renal fibrosis has not yet been determined.Mature microRNAs originate from long primary transcripts called pri-microRNAs,which are recognized in the nucleus by the microprocessor complex formed by DGCR8 and nuclear RNase III DROSHA,where DGCR8 mainly recognizes pri-miRNAs hairpin stems and flanking single-stranded RNAs,then recruit DROSHA to cleave the RNA duplex to produce a precursor miRNA product(pre-miRNA),which is transported into cytoplasm for further splicing processing.Studies have revealed that m6A modification in mammals can mark pri-microRNAs,which are then recognized by DGCR8 to participate in the maturation of microRNAs.Altering the level of m6A may affect the processing of pri-microRNAs,thereby changing the expression level of mature microRNAs,and ultimately affecting the biological process.A previous study reported that m6A is involved in the maturation of miR-126 and promotes the development of pulmonary fibrosis,so whether m6A modification affects the maturation of miR-21 and regulates downstream pathways in obstructive renal fibrosis remains to be studied.In this study,we collected human normal and obstructive kidney tissue samples,and unilateral ureteral obstruction(UUO)in mice,a commonly used model of obstructive renal fibrosis,were constructed,and mice with conditional knockout of METTL3 gene in kidney were used,and the expression of METTL3 and miR-21-5p in HK-2 cells was enhanced or inhibited by plasmid transfection,to explore the molecular mechanism of METTL3-mediated m6A modification promoting obstructive renal fibrosis,that is,whether it plays a role by affecting the maturation of miR-21-5p and the activation of downstream SPRY1/ERK/NF-κB pathway and inflammatory response.Therefore,our research is divided into the following four parts.Part 1.The m6A modification and miR-21-5p expression in adult obstructed kidneyObjectiveTo detect the changes of m6A modification,the expression of m6A-related methylase and demethyl ase,the level of miR-21-5p,the downstream SPRY1/ERK/NF-κB signaling pathway and inflammatory indexes in adult obstructive kidney tissue,and explore the role of m6A modification and miR-21-5p in obstructive renal fibrosis.Materials and Methods1.Obstructed kidney group:a total of 28 kidney specimens were collected,which were from patients with severe hydronephrosis who underwent nephrectomy for loss of renal function.Control group:After radical nephrectomy in patients with renal cancer,in the area at least 5 cm away from the tumor site and without obvious tumor invasion,kidney specimens were taken as normal kidney tissue,and a total of 16 cases were collected2.The pathological changes and collagen deposition of kidney tissue were detected by HE and Masson’s trichrome staining.3.The protein expression levels of fibrosis indicators a-SMA,Collagen I,FN,TNF-α and IL-6 were detected by western blot or immunohistochemical techniques.4.RNA dot blot was utilized to test m6A levels in obstructed kidney and normal kidney tissue.5.qRT-PCR was utilized to test the expression levels of m6A-related methylases and demethylases:WTAP,KIAA1429,METTL3,METTL14,ALKBH5 and FTO.6.qRT-PCR was utilized to test the level of miR-21-5p.The expression levels of SPRY1,p-ERK/ERK,p-NF-κB/NF-κB were detected by western blot.7.Statistical analysis:Statistical analysis was performed using SPSS 21.0,and mean±standard deviation(mean ± SD)described measurement data,comparative analysis between two groups applied independent samples t-test.One-way analysis of variance was utilized for three groups or more than three groups.α=0.05 served as the test level,and P<0.05 was considered statistically significant.Results1.The results of HE staining revealed that the structure of the obstructive kidney changed significantly comparing with the control group,the cortex was significantly thinned,the cortical structure was destroyed,glomerular sclerosis atrophy,renal tubules were dilated accompanied with casts,and numerous inflammatory cells infiltrated the interstitium.The distal medullary canal also sclerosed and atrophied.2.The results of Masson’s trichrome staining demonstrated that numerous glue fibers deposited in the obstructive renal tissue,which were interconnected to form a grid,the normal structure was destroyed,the glomerular sclerosed and atrophied.And the area of collagen deposition was increased significantly contrasting with the control group(P<0.05).3.Western blot revealed that the levels of α-SMA,Collagen I and FN in the obstructed kidney were higher than those in the control group(P<0.05).The results of immunohistochemistry also showed that the expressions of fibrosis indexes α-SMA,Collagen I and FN were increased(P<0.05).4.RNA dot blot detection showed that the m6A level in obstructive kidney tissue was increased,and qRT-PCR detected the expression levels of related methylases and demethylases and found that change of METTL3 was the most and its expression increased obviously(P<0.05),western blot and immunohistochemical results also confirmed the enhanced expression of METTL3 in obstructive kidney tissue.5.qRT-PCR detection revealed that the level of miR-21-5p was increased significantly in obstructive kidney tissue(P<0.05),Western blot demonstrated that the expression level of SPRY1 decreased in obstructive kidney tissue,while the expression levels of p-ERK and p-NF-κB increased,and the expression levels of inflammatory factors TNF-α and IL-6 increased(P<0.05).SummaryThe results confirm that adult obstructed kidneys have obvious fibrosis,and the level of m6A is increased.Among the methylases and demethylases,the up-regulation of METTL3 is the most.It is the main enzyme that mediates the increase of m6A.We observed up-regulation of miR-21-5p expression and activation of SPRY1/ERK/NF-κB signaling pathway in obstructed kidneys,and these findings provide clues to further study the molecular mechanism of ORF.Part 2.The role of METTL3/m6A and miR-21-5p in the development of obstructive renal fibrosisObjectiveTo construct a model of hydronephrosis in mice with different obstructive time,to observe the changes of METTL3/m6A,miR-21-5p and downstream SPRY1/ERK/NF-κB pathway under different obstructive time,and to explore the role of METTL3/m6A and miR-21-5p in the development of obstructive renal fibrosisMaterials and Methods1.Sixty C57BL/6 mice were used to construct unilateral ureteral obstruction models(UUO),and they were randomly divided into UUO group and Sham group(sham operation group),30 mice in each group.On the 3rd,7th,and 14th days after the operation,small animal ultrasound was used to observe the hydronephrosis,and the width of the renal pelvis was measured.The blood of the inferior vena cava was collected to test the renal function,then the mice were sacrificed to obtain left kidneyspecimens,10 mice at each time point.2.The pathological changes and collagen deposition of renal tissue at different time of obstruction were detected by HE and Masson’s trichrome staining.3.The mRNA and protein expression levels of fibrosis indicators α-SMA,Collagen I,and FN were detected by qRT-PCR,western blot or immunohistochemistry.4.RNA dot blot was used to detect the changes of m6A levels in different obstructed kidney tissues,and western blot or immunohistochemical techniques were used to detect the changes of METTL3 expression levels with the time of obstruction.5.qRT-PCR was utilized to test the level of miR-21-5p,the expression of SPRY 1/ERK/NF-κB pathway protein was detected by western blot,and the expressions of inflammatory factors TNF-α and IL-6 were detected by western blot and immunohistochemistry6.Statistical analysis:Statistical analysis was performed using SPSS 21.0,and mean±standard deviation(mean±SD)described measurement data,comparative analysis between two groups applied independent samples t-test.One-way analysis of variance was utilized for three groups or more than three groups.α=0.05 served as the test level,and P<0.05 was considered statistically significant.Results1.After ligation of the left ureter in mice,ultrasound showed that the width of the left renal pelvis gradually increased as the obstruction time prolongation,and the thickness of the renal cortex gradually became thinner(P<0.05),which revealed that the construction of UUO model in mice was successful.Compensation of renal function,the levels of BUN and SCr were generally within the normal range,but with the accumulation of urine in the left kidney,the upward trend of BUN and SCr within the normal range was still evident(P<0.05).2.HE staining showed that proximal tubules in renal cortex were dilated and aggravated with the prolongation of obstruction time,the structure of renal tubules was damaged,many glomeruli collapsed,and a large number of inflammatory cells infiltrated,especially in the kidneys after 7 and 14 days of obstruction,and even formed inflammation foci,the score of tubulointerstitial injury gradually increased(P<0.05).Masson staining showed that the area of collagen deposition increased with the prolongation of obstruction time.It was also observed that the collagen deposition in the inflammatory foci was more obvious(P<0.05).3.qRT-PCR results showed that the mRNA expression levels of α-SMA,Collagen I and FN were significantly increased in the UUO-7d and UUO-14d groups(P<0.05);consistent with this,Western blot and immunohistochemical staining showed that the expressions of α-SMA,Collagen I and FN were up-regulated in a time-dependent manner,which further confirmed the occurrence of fibrosis in the obstructed kidneys in mice(P<0.05).4.RNA dot blot demonstrated that the m6A level in mice kidney tissue increased as the obstruction time prolongation,the results of Western blot showed that the protein level of METTL3 was elevated significantly after ureter ligation,especially in the UUO-7d group and UUO-14d group group(P<0.05),which was consistent with immunohistochemical staining of METTL3(P<0.05),so the trend of METTL3/m6A modification level was consistent with the development trend of fibrosis.5.The expression of miR-21-5p in renal tissue with different obstruction time after UUO operation was detected by qRT-PCR,which gradually increased with the prolongation of obstruction time(P<0.05),and accompanied by p-ERK,p-NF-kB protein expression was up-regulated and SPRY1 protein expression was down-regulated(P<0.05).At the same time,western blot and immunohistochemical staining results showed that the expressions of inflammatory factors TNF-α and IL-6 also increased with the prolongation of obstruction time(P<0.05).SummaryThe UUO models were constructed in this part,and it was observed that with the prolongation of renal obstruction time,fibrosis gradually aggravated,the change trend of METTL3/m6A modification level was consistent with the fibrosis development,and with the development of obstructive renal fibrosis,miR-21-5p was gradually up-regulated and the SPRY 1/ERK/NF-κB pathway was activated and the inflammatory response was aggravated.So METTL3/m6A and miR-21-5p were closely related to the development of obstructive renal fibrosis.Part 3.Effects of METTL3 knockout in kidney on miR-21-5p and obstructive renal fibrosisObjectiveThe m6A level was changed by conditional knockout of mouse kidney METTL3 gene,and the therapeutic effect of METTL3/m6A on miR-21-5p expression and obstructive renal fibrosis was investigated.Materials and Methods1.There were 20 kidney METTL3 gene conditional knockout mice and 20 wild-type mice.Grouped:wild-type mice Sham group(WT-Sham group)10 mice,wild-type mice UUO group(WT-UUO group)10 mice,METTL3 knockout mouse Sham group(KO-Sham group)10 mice,METTL3 knockout mouse UUO group(KOUUO group)10 mice.2.Masson’s trichrome staining was used to detect the collagen deposition in the kidney tissue of each group.3.Histoimmunofluorescence was applied to detect the levels of METTL3 and Collagen I in the kidney of each group.4.Immunohistochemistry be utilized to test METTL3,α-SMA and FN in the kidney of each group.5.RNA dot blot be utilized to test the m6A levels in the kidney of each group.6.qRT-PCR be utilized to detect the level of miR-21-5p in the kidney of each group,and western blot was applied to test the levels of TNF-α and IL-6 in the kidney of each group.7.Statistical analysis:Statistical analysis was performed using SPSS 21.0,and mean±standard deviation(mean±SD)described measurement data,comparative analysis between two groups applied independent samples t-test.One-way analysis of variance was utilized for three groups or more than three groups.α=0.05 served as the test level,and P<0.05 was considered statistically significant.Results1.The results of Masson’s trichrome staining showed that there was no collagen fibers deposition in the renal interstitium of the WT-Sham group and the KO-Sham group,and no statistical difference,P>0.05.The renal interstitial collagen deposition in the KO-UUO group was significantly improved comparing with the WT-UUO group,with a statistical difference,P<0.05.2.Tissue immunofluorescence showed that the fluorescence intensity of Collagen I protein was significantly enhanced in the WT-UUO group.Although the fluorescence intensity of Collagen I protein in the KO-UUO group was enhanced compared with WT-Sham group and KO-Sham group,it was significantly lower than WT-UUO group(P<0.05).3.The level of α-SMA detected by immunohistochemistry was up-regulated significantly in the WT-UUO group,while the level of α-SMA in the KO-UUO group was obvious lower than the WT-UUO group(P<0.05).The trend of immunohistochemical detection of FN was consistent with that of α-SMA,that is,it was significantly up-regulated in the WT-UUO group,but significantly decreased in the KO-UUO group compared with the WT-UUO group(P<0.05).4.RNA dot blot showed that the level of m6A in the kidney tissue of the WT-UUO group was higher than WT-Sham group,and METTL3 gene knockout significantly decreased the level of m6A in the kidney tissue of the KO-UUO group.5.qRT-PCR showed that the WT-UUO group miR-21-5p level was significantly up-regulated,while the expression of miR-21-5p in the kidney tissue of KO-UUO group with METTL3 knockout was significantly inhibited(P<0.05).The expressions of TNF-α and IL-6 in the WT-UUO group were significantly increased,while the expressions of TNF-α and IL-6 was reduced in the KO-UUO group with METTL3 gene knockout(P<0.05).SummaryThis part confirms that METTL3 knockout reduces m6A level in obstructive kidney tissue,which can alleviate obstructive renal fibrosis significantly,and METTL3 knockout inhibits the expression of miR-21-5p and inflammatory response,further indicating that the profibrotic effect of METTL3/m6A is closely related to miR-21-5p.Part 4.The mechanism of METTL3/m6A-mediated miR-21-5p maturation in regulating renal fibrosisObjectiveEnhancing or inhibiting METTL3 and miR-21-5p in HK-2 cells to explore the mechanism of METTL3/m6A-mediated miR-21-5p maturation and activation of SPRY1/ERK/NF-κB signaling pathway in regulating renal fibrosis.Materials and Methods1.The human renal proximal tubule epithelial cell line(HK-2)was cultured in a humidified environment of 37℃ and 5%CO2.HK-2 cells were seeded on 6-well plates,Lipofectamine 3000 kit was used for transfection,and 48h after transfection we collected the cells for subsequent experiments.2.After the HK-2 cells were transfected with PGV657-METTL3 plasmid to overexpress METTL3 gene,the expressions of METTL3,a-SMA,Collagen I and FN were detected by western blot.And then m6A modification level in HK-2 cells were detected by RNA dot blot.The expression changes of pri-miR-21 and miR-21-5p were detected by qRT-PCR,and RIP experiment was performed with m6A antibody to detect the level of pri-miR-21-5p modified by m6A,and RIP experiment was performed with DGCR8 antibody to detect the level of pri-miR-21 combined with DGCR8.3.After transfecting miR-21-5p-mimics into HK-2 cells to overexpress miR-21-5p,qRT-PCR was applied to test the level of miR-21-5p,and the expression of Collagen I、FN、SPRY1、p-ERK/ERK、p-NF-κB/NF-κB、TNF-α and IL-6 was detect by western blot.And the expression changes of α-SMA were detected by cellular immunofluorescence.4.The PGV657-METTL3 plasmid and miR-21-5p-inhibitor were co-transfected into HK-2 cells.The expressions of METTL3 and α-SMA were detected by cell immunofluorescence,and the levels of α-SMA,Collagen I and FN were tested by western blot.5.Statistical analysis:Statistical analysis was performed using SPSS 21.0,and mean ± standard deviation(mean±SD)described measurement data,comparative analysis between two groups applied independent samples t-test.One-way analysis of variance was utilized for three groups or more than three groups.α=0.05 served as the test level,and P<0.05 was considered statistically significant.Results1.After transfecting the pGV657-METTL3 plasmid into HK-2 cells to overexpress the METTL3 gene,RNA dot blot revealed that the m6A level of the transfection group was higher than the control group,and qRT-PCR demonstrated that the level of miR-21-5p increased in the transfection group,while the level of pri-miR-21 decreased(P<0.05),these results indicated that overexpression of METTL3 gene promoted the maturation of miR-21-5p,and western blot showed that α-SMA,Collagen I,and FN were increased in the transfection group comparing with the control group(P<0.05),indicating that overexpression of METTL3 gene promoted the fibrosis of HK-2 cells.2.After transfection of pGV657-METTL3 plasmid into HK-2 cells,the cellular immunofluorescence demonstrated that the fluorescence of α-SMA was enhanced,while co-transfection of PGV657-METTL3 plasmid and miR-21-5p-inhibitor suppressed the effect significantly.Compared with the PGV657-METTL3 plasmid transfection group,the levels of α-SMA,Collagen I and FN was decreased in the co-transfection group(P<0.05).These results confirmed that the pro-fibrotic effect of METTL3 in HK-2 cells might be achieved by the miR-21-5p maturation.3.In HK-2 cells overexpressed METTL3 gene,the results of RIP experiment with DGCR8 antibody showed that the level of pri-miR-21 binding to DGCR8 was significantly increased(P<0.05),the results of RIP experiment with m6A antibody showed that the level of m6A-modified pri-miR-21 was significantly increased(P<0.05),indicating that overexpression of METTL3 gene increased m6A modification of pri-miR-21 and enhanced the binding of pri-miR-21 to DGCR8 protein,and then promoted the mature processing of pri-miR-21,and the m6A modification site of pri-miR-21 was predicted by SRAMP(http://www.cuilab.cn/sramp/),a total of 7 m6A modification sites were found,of which 2 were high-confidence sites.4.Cellular immunofluorescence showed that the fluorescence of α-SMA protein was enhanced after transfection of the pGV514-miR-21-5p-mimic plasmid into HK-2 cells,and the western blot results showed that,the protein expressions of Collagen I,FN,TNF-α and IL-6 were increased comparing with the control group(P<0.05),indicating that overexpression of miR-21-5p in HK-2 cells significantly promotes the expression of fibrosis and inflammatory factors.Western blot also showed that p-ERK1/2,p-NF-κB expression levels were increased,while SPRY1 expression was inhibited(P<0.05),indicating that overexpression of miR-21-5p in HK-2 cells activated SPRY1/ERK1/2/NF-κB pathway.SummaryThis part confirms that METTL3/m6A mediates the processing and editing of pri-miR-21,thereby promoting the maturation of miR-21-5p.The results indicate that METTL3/m6A promotes the maturation of miR-21-5p,and then activation of SPRY1/ERK/NF-κB signaling pathway and inflammatory response promotes renal fibrosis.Conclusion1.Elevated m6A modification is mediated by METTL3 in obstructive kidneys.2.METTL3/m6A in obstructive kidney exerts a profibrotic effect via promoting the expression of miR-21-5p and inflammatory response.3.METTL3 knockout in kidney can inhibit the expression of miR-21-5p and inflammatory response in obstructive renal tissue,thereby improving obstructive renal fibrosis.4.METTL3 promotes miR-21-5p maturation by mediating m6A modification of pri-miR-21,and then activates the SPRY1/ERK/NF-κB pathway to regulate renal fibrosis. |
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