Research Paper Downregulation Of MiR 214-3p Attenuates .

3345Int. J. Biol. Sci. 2021, Vol. 17supplemented with 10% fetal bovine serum (FBS) and14 mM HEPES (all from Gibco) at 37 C in anatmosphere of 5% CO2. HEK-293T cells were obtainedfrom the American Type Culture Collection (USA)and cultured in DMEM containing 10% FBS at 37 C in5% CO2. Monomeric human IgA1 (Abcam) washeated and aggregated at 65 C for 150 minutes toobtain polymeric IgA1 (p-IgA1), as previouslydescribed [20]. The miR-214-3p inhibitor, negativecontrol (inhibitor NC), miR-214-3p mimic, negativecontrol (mimic NC), PTEN siRNA and control siRNAwere purchased from RiboBio (Guangzhou, China).All vectors were transfected with Lipofectamine 2000(Invitrogen) according to the recommended protocol.After transfection, the cells were starved in serum-freemedium for 12 hours, and then incubated with p-IgA1(25 µg/ml) for 24 hours to construct the IgAN MCmodel [21].Cell proliferation assayWe used a Cell Counting Kit-8 (CCK-8) assay(Dojindo, Japan) to access cell proliferation. Cells wereseeded in 96-well plates at a density of 5000 cells perwell. After treatment, 10 μl CCK-8 solution was addedto each well and incubated for 1.5 hours at 37 C.Viable cell numbers were estimated by measuring theoptical density (OD) at 450 nm.Cell cycle analysisThe treated cells were fixed with 70% ice‑coldethanol overnight at 4 C, and then stained with amixture of propidium iodide and RNase A (Cell Cycleand Apoptosis Analysis Kit, Beyotime) for 30 minutesat 37 C. All samples were analyzed using flowcytometry (BD Biosciences), and the distribution ofcell cycle phases was measured using the ModfitSoftware. The proliferation index (PI) was calculatedas the sum of the percentage of cells in S phase andG2/M phase.Luciferase reporter assayThe 3’-UTR of mouse PTEN mRNA containingwild-type or mutant miR-214-3p binding sites wascloned into the psiCHECKTM-2 vector (Promega).HEK-293T cells were co-transfected with luciferasereporter plasmid and the miR-214-3p mimic ornegative control (mimic NC) using Lipofectamine2000 (Invitrogen). After forty-eight hours, luciferaseactivity was determined using a Dual-Glo LuciferaseReporter Assay System (Promega) according to themanufacturer’s instructions. The firefly luciferaseactivity of each sample was normalized to Renillaluciferase activity.Real-time PCRTotal RNA from MCs and renal cortex tissueswas extracted with the TRIzol reagent (Invitrogen).mRNA reverse transcription was completed using thePrimeScript RT Reagent Kit with gDNA Eraser(TaKaRa). Real-time PCR was performed using SYBRGreen Master mix (TaKaRa) on a LightCycler 96System (Roche). For the quantification of miR-214-3p,stem-loop real-time PCR was adopted. The relativeexpressions levels of miR-214-3p and PTEN werenormalized to those of the internal controls U6(miR-214-3p) and GAPDH (PTEN). Each sample wasshown as 2 Ct values. All primers were purchasedfrom Sangon (Shanghai, China).Immunoblot analysisRenal cortex tissues or treated cells wereharvested and lysed on ice using RIPA lysis buffer(Beyotime), which included protease and phosphataseinhibitors (Roche). The protein levels were measuredusing a BCA protein assay kit (Thermo FisherScientific). Equivalent amounts of protein wereelectrophoresed by SDS-PAGE and transferred topolyvinylidene difluoride membranes. After blockingin 5% bovine serum albumin for 1 hour at roomtemperature, membranes were incubated overnight at4 C with the following primary antibodies: PTEN(1:10000, Abcam), JNK (1:1000, Abcam), p-JNK(1:1000, Cell Signaling Technology), c-Jun (1:1000, CellSignaling Technology), p-c-Jun (1:1000, Cell SignalingTechnology), PCNA (1:2000, Proteintech), cyclinD1(1:10000, Abcam), GAPDH (1:5000, Proteintech), andβ-actin (1:2000, Servicebio). After incubation withcorresponding horseradish peroxidase (HRP)conjugated secondary antibodies at room temperaturefor 1 hour, antigens on the blots were visualized withan enhanced chemiluminescence kit (Millipore).Histology and immunohistochemical stainingRenaltissueswerefixedin4%paraformaldehyde, embedded in paraffin, and slicedinto 4 μm sections. Hematoxylin-eosin (H&E) andperiodic acid-Schiff (PAS) staining were conducted toaccess MC proliferation and matrix expansion.Immunohistochemical staining was raffinized tissue sections were sequentiallysubjected to rehydration and antigen retrieval usingheated citrate. After subsequent incubation in 3%H2O2 and blocking solution, the slides were exposedto anti-PTEN antibody (1:200, Proteintech) at 4 Covernight and stained with HRP-linked secondaryantibodies. The color was developed with a DAB kit(Vector nce analysis of IgA depositionconducted as previously described [22].http://www.ijbs.com

3346Int. J. Biol. Sci. 2021, Vol. 17Fluorescein isothiocyanate (FITC)-labeled goatanti-mouse IgA (1:50, Abcam) was used to detect IgAin renal tissues. Immunofluorescence staining incultured cells was performed as follows. Cells werewashed with phosphate-buffered saline (PBS), fixedwith 4% paraformaldehyde and permeabilized with0.1% Triton X-100. After being blocked with 5%bovine serum albumin, the cells were sequentiallystained with anti-PTEN antibody (1:100, Proteintech)overnight at 4 C, goat anti-mouse IgG conjugatedwith Alexa Fluor 488 (1:1000, Abcam) for 1 hour atroom temperature, and DAPI (Beyotime). Sampleswere imaged using laser scanning confocalmicroscopy.In situ hybridizationIn situ hybridization was performed as describedpreviously [23]. In brief, after deparaffinization andhydration, paraffin-embedded kidney tissue sectionswere incubated with 20 μg/ml proteinase K forpermeabilization followed by a prehybridizationsolution at 37 C for 1 hour. The specimens were thentreated with digoxigenin-labeled mmu–miR-214-3pLNA probe (Servicebio) overnight at 37 C. Followingincubation with 5% BSA to remove nonspecificstaining, the samples were probed with antidigoxigenin-HRP for 1 hour at 37 C. The signal wasdisplayed by adding DAB solution and recordedusing microscopy.Statistical analysisData are expressed as the mean SD of at leastthree independent experiments. Statistical differencesbetween two groups were determined by thetwo-tailed Student’s t-test, and differences in multiplegroups were analyzed by one-way analysis ofvariance. P 0.05 was considered statisticallysignificant. All statistical analyses were performedusing GraphPad Prism 7.0 and SPSS 24.0 statisticalsoftware.ResultsThe expression of miR-214-3p was upregulatedin IgAN MCsTo investigate the potential role of miRNAs inthe pathogenesis of IgAN, we initially established amouse model of IgAN. Immunofluorescence stainingindicated that the model groups had obvious IgAdeposits in the glomeruli, while there was no IgAdeposition in the control groups (Figure 1A). H&Eand PAS staining showed that IgAN mice hadpronounced mesangial hypercellularity comparedwith the control mice (Figure 1B-C). Proteinuria wasalso significantly elevated in IgAN mice (Figure 1D).All these results showed that the IgAN mouse modelwas successfully established. Then, we screened thedifferential miRNA expression profiles in the renalcortex of control and IgAN mice by miRNAsequencing. As illustrated in Figure 1E, 17 miRNAswere markedly upregulated, whereas 8 miRNAs weredownregulated in IgAN mice compared with thecontrol group (P 0.05). Among these miRNAs,miR-214-3p was obviously elevated in IgAN mice(Figure 1F). We further performed real-time PCR onthe isolated renal cortex to validate this sequencingresult (Figure 1G). In situ hybridization analysisrevealed that miR-214-3p expression was significantlyincreased in MCs from the renal cortex followingIgAN (Figure 1H). Real-time PCR also confirmed anincrease in miR-214-3p in MCs from IgAN mice(Figure 1I).Knockdown of miR-214-3p alleviated renallesions in IgAN miceTo examine the effect of miR-214-3p on IgAN invivo, miR-214-3p antagomir or negative control wereadministered to IgAN mice. We confirmed that tailvein injection of miR-214-3p antagomir markedlyreduced the level of miR-214-3p in kidney tissues(Figure 2A). Treatment with the miR-214-3pantagomir decreased IgA deposition in the glomeruli,ameliorated mesangial hypercellularity and improvedproteinuria in IgAN mice (Figure 2B-E). Consistently,immunoblot analysis detected that the miR-214-3pantagomir inhibited the protein expression of PCNAand cyclin D1, which are markers of cell proliferation(Figure 2F-G). These results supported the conclusionthat knockdown of miR-214-3p alleviated mesangialhypercellularity and renal lesions in IgAN mice.Inhibition of miR-214-3p suppressed MCsproliferationAs shown in Figure 3A, miR-214-3p expressionwas upregulated when MCs was stimulated withp-IgA1 to promote the IgAN pathological state, butwas significantly downregulated after transfectionwith the miR-214-3p inhibitor. The CCK-8 assayindicated that p-IgA1 increased cell viability, whilethe miR-214-3p inhibitor blocked this promotion(Figure 3B). To further confirm the effect of miR214-3p on the proliferation of MCs, we performed cellcycle analysis by flow cytometry. p-IgA1 increasedthe proportion of cells in S and G2/M phases after 24hours of incubation. With the application of themiR-214-3p inhibitor, more cells were arrested in G1phase (Figure 3C-D). In addition, the expression ofcell proliferation markers, such as PCNA and cyclinD1, was upregulated in IgAN. Transfection with themiR-214-3p inhibitor reduced PCNA and cyclin D1protein levels (Figure 3E-F). Taken together, the abovehttp://www.ijbs.com

Int. J. Biol. Sci. 2021, Vol. 17results demonstrated that knockdown of miR-214-3pcould inhibit the proliferation of MCs in IgAN.Identification of PTEN as a functional target ofmiR-214-3pTo understand the mechanism wherebymiR-214-3p contributed to IgAN, we used TargetScan3347to search for target genes of miR-214-3p. As illustratedin Figure S1A, the 3’-UTR of mouse PTEN mRNAcontained a putative miR-214-3p target site.miR-214-3p is conserved in human and mouse.Analysis of the human and mouse PTEN 3’-UTRsrevealed highly conserved recognition elements withsignificant homology at the miR-214-3p seedFigure 1. miR-214-3p expression was upregulated in IgAN mesangial cells (MCs). (A) Immunofluorescence staining of IgA in the glomeruli. Original magnification, 400x.(B-C) Representative images of hematoxylin and eosin (HE) staining and periodic acid-Schiff (PAS) staining in control and IgAN mice. Original magnification, 400x. (D) Proteinuria wasreported as the ratio of urinary albumin to urinary creatinine (ACR). (E-F) Clustering map of miRNA expression in the renal cortex from control and IgAN mice (P 0.05). (G) Thedifferential expression of miR-214-3p in the renal cortex of control and IgAN mice was validated by real-time PCR. The relative level of miR-214-3p was normalized to the level of U6,and the ratio of control mice was arbitrarily set as 1. (H) In situ hybridization analysis showed that miR-214-3p was significantly elevated in MCs from the mouse renal cortex followingIgAN. Original magnification, 400x. (I) The differential expression of miR-214-3p in MCs from the control and IgAN groups was validated by real-time PCR. The relative level ofmiR-214-3p was normalized to the expression of U6, and the ratio of control mice was arbitrarily set as 1. All data are expressed as the mean SD. *P 0.05, **P 0.01.http://www.ijbs.com

Int. J. Biol. Sci. 2021, Vol. 17sequence. To examine whether PTEN was indeed atarget of miR-214-3p, we first evaluated the effect ofmiR-214-3p on PTEN expression. As shown in Figure4A-C, downregulation of miR-214-3p did not changethe levels of PTEN mRNA, but elevated PTEN proteinlevels in mice. The in-vivo suppressive effect ofmiR-214-3p on PTEN was further verified ly, in vitro, although there was nodetectable change in PTEN mRNA levels, p-IgA1induced a decrease in PTEN protein levels, whiletransfection of miR-214-3p inhibitor markedlyincreased PTEN protein levels in MCs (Figure 4E-G).Immunofluorescence also confirmed that miR-214-3pinhibited PTEN expression in MCs (Figure 4H). Todetermine whether PTEN was a direct target ofmiR-214-3p, we performed a luciferase reporter assay.Wild-type and mutant 3′-UTR luciferase reporterconstructs of PTEN were generated (Figure S1B), andthese vectors were co-transfected into HEK-293T cells3348with miR-214-3p mimic or mimic NC, respectively.The results showed that the miR-214-3p mimicsignificantly decreased the luciferase activity of thewild type reporter, but had no effect on the luciferaseactivity of the mutant reporter (Figure S1C). Thesefindings demonstrated that miR-214-3p may directlytarget PTEN mRNA to repress its translation in IgAN.miR-214-3p regulated activation of JNK/c-JunpathwayPTEN is an endogenous inhibitor of JNK/c-Junpathway activation [14, 15]. Activation of JNK/c-Junpromotes cell proliferation by accelerating G1-S cellcycle progression [16-18]. Since we found thatmiR-214-3p reduced PTEN expression and promotedthe proliferation of MCs, we investigated itsinvolvement in the activation of the JNK/c-Junpathway. In vivo, p-JNK and p-c-Jun protein levelswere significantly increased in IgAN mice comparedwith controls. However, treatment with the miR-214-Figure 2. Knockdown of miR-214-3p alleviated renal lesions in IgAN mice. IgAN mice were subjected to tail intravenous injection of 20 mg/kg miR-214-3p antagomir ornegative control (antagomir NC) every two weeks for 3 consecutive days from the sixth week to the eleventh week. (A) The expression of miR-214-3p in the renal cortex. Therelative level of miR-214-3p was normalized to the expression of U6, and the ratio of control mice was arbitrarily set as 1. (B) Immunofluorescence staining of IgA in the glomeruli.Original magnification, 400x. (C-D) Representative images of hematoxylin and eosin (HE) staining and periodic acid-Schiff (PAS) staining. Original magnification, 400x. (E) Proteinuriawas reported as the ratio of urinary albumin to urinary creatinine (ACR). (F) Immunoblot analyses and (G) quantitative determination of PCNA and cyclinD1 protein levels in therenal cortex. For densitometry, the signals of the target proteins were normalized to the β-actin signal of the same samples to determine the ratios. All data are expressed as themean SD. *P 0.05, **P 0.01.http://www.ijbs.com

Int. J. Biol. Sci. 2021, Vol. 173p antagomir significantly restored the expressions ofthese proteins in IgAN (Figure 5A-B). In vitro, p-IgA1induced a marked increase in p-JNK and p-c-Jun inMCs. Similarly, inhibition of miR-214-3p suppressedp-JNK and p-c-Jun expression in IgAN (Figure 5C-D).These results verified that miR-214-3p regulated theactivation of the JNK/c-Jun pathway in IgAN.miR-214-3p targeted PTEN to promote MCproliferation though the JNK/c-Jun pathway inIgANTo test whether miR-214-3p promoted MCproliferation though the PTEN/JNK/c-Jun signalingpathway in IgAN, MCs was incubated with p-IgA1and divided into the following four groups: inhibitor3349NC control siRNA, miR-214-3p inhibitor controlsiRNA, inhibitor NC PTEN siRNA, and miR-214-3pinhibitor PTEN siRNA. Immunoblot analysisdetected that transient transfection of the miR-214-3pinhibitor increased PTEN expression and reduced theprotein levels of p-JNK, p-c-Jun, PCNA and cyclin D1(Figure 6A-D). CCK-8 assay and cell cycle analysisprovided evidence that knockdown of miR-214-3pcould inhibit the proliferation of MCs (Figure 6E-G).However, simultaneous co-transfection with PTENsiRNA attenuated all these alterations. These dataconclusively suggested that miR-214-3p stimulatedthe proliferation of MCs by regulating thePTEN/JNK/c-Jun signaling pathway in IgAN.Figure 3. Inhibition of miR-214-3p suppressed mesangial cell (MC) proliferation. MCs were transfected with miR-214-3p inhibitor or negative control (inhibitor NC)and then treated with 25 µg/mL p-IgA1 for 24 hours. (A) The expression of miR-214-3p in MCs. The relative level of miR-214-3p was normalized to the expression of U6, andthe ratio of control mice was arbitrarily set as 1. (B) Cell viability was measured by CCK8 assay. (C-D) Cell cycle analysis was performed by staining DNA with propidium iodideprior to flow cytometry. The proliferation index (PI) was calculated as the sum of the percentage of cells in S phase and G2/M phase. (E) Immunoblot analyses and (F) quantitativedetermination of PCNA and cyclinD1 protein levels in MCs. For densitometry, the signals of the target proteins were normalized to the β-actin signal of the same samples todetermine the ratios. All data are expressed as the mean SD. *P 0.05, **P 0.01.http://www.ijbs.com

Int. J. Biol. Sci. 2021, Vol. 173350Figure 4. The effect of miR-214-3p on PTEN expression in IgAN. (A) PTEN mRNA levels in the mouse renal cortex were determined by real-time PCR. PTEN levelswere normalized to GAPDH, and the ratio of control mice was arbitrarily set as 1. (B) Immunoblot analyses and (C) quantitative determination of PTEN protein expression inthe renal cortex. For densitometry, the PTEN protein signal was normalized to the GAPDH signal of the same sample to determine the ratio. (D) Immunohistochemical stainingillustrated the repressive effect of miR-214-3p on PTEN expression in glomeruli. (E) PTEN mRNA levels in mesangial cells (MCs) were determined by real-time PCR. PTEN levelswere normalized to GAPDH, and the ratio of the control group was arbitrarily set as 1. (F) Immunoblot analyses and (G) quantitative determination of PTEN protein expressionin MCs. For densitometry, the PTEN protein signal was normalized to the GAPDH signal of the same sample to determine the ratio. (H) Immunofluorescence illustrated therepressive effect of miR-214-3p on PTEN expression in MCs. All data are expressed as the mean SD. *P 0.05, **P 0.01.DiscussionThis is the first study to investigate the biologicalfunction of miRNAs expressed in the kidney on theproliferation of MCs in IgAN, and we confirmed thefollowing major findings: (1) the differentialexpression of miRNAs obtained by miRNAsequencing demonstrated that miR-214-3p wasdramatically elevated in the kidney in IgAN, and wevalidated this finding in MCs using both in vitro andin vivo models; (2) functionally, miR-214-3p promotedMC proliferation by accelerating G1-S cell cycleprogression, indicating that miR-214-3p plays aninjurious role in IgAN and that inhibition ofmiR-214-3p could prevent the progression of IgAN;and (3) mechanistically, miR-214-3p might directlytarget and repress PTEN to activate JNK/c-Junsignaling-associated MC proliferation in IgAN (Figure7). Collectively, these findings unveiled a novelmechanism of a miRNA involved in the regulation ofMC proliferation and provided potential therapeutictargets for IgAN.Many miRNAs are involved in the developmentor progression of IgAN [9, 24]. miR 148b potentiallytargets C1GALT1 [25], miR-98-5p targets CCL3 todecrease C1GALT1 activity [26], and let-7b alters theexpression of GALNT2 [27] to modulate Gd-IgA1levels. miR-223 downregulation promoted glomerularendothelial cell proliferation and monocyte adhesionby upregulating importin a4 and a5 in IgAN [28].miRNA expression analysis in IgAN kidney biopsysamples revealed that dysregulated miRNA levels arerelated to interstitial fibrosis [29, 30] and to theimmune response correlated with disease severityand progression [31, 32]. In addition to having criticalroles in the pathogenesis and progression of IgAN,specific miRNAs in serum and urine have beendiscovered as potential noninvasive biomarkers forthe diagnosis and monitoring of patients with IgAN[32-35]. However, the role of miRNAs in theproliferation of MCs in IgAN remains largelyunknown. In the present study, we providedcompelling evidence to support that miR-214-3p wasinvolved in MC proliferation in IgAN. miRNAsequencing analysis revealed that miR-214-3p washttp://www.ijbs.com

Int. J. Biol. Sci. 2021, Vol. 17obviously upregulated in IgAN, and we identifiedthis upregulation both in vitro and in vivo. Moreover,inhibition of miR-214-3p suppressed MC proliferationand arrested G1-S cell cycle progression. In IgANmice, miR-214-3p antagomir ameliorated mesangialhypercellularity and renal lesions. Accordingly,miR-214-3p downregulation had kidney protectiveeffects in IgAN.Among the 25 differentially expressed miRNAsfound in the sequencing analysis, why did we choosemiR-214-3p for further research? Previous studieshave verified that miR-214 is extensively expressed indifferent tissues and cells and exerts differentbiological effects. Under normal conditions, miR-214did not affect kidney development or homeostasis, asmiR-214 deletion in mice showed normal glomerularand tubular architecture [36]. In diabeticnephropathy, the level of miR-214 was significantlyincreased, and its downregulation attenuated high3351glucose-induced mesangial and proximal tubular cellhypertrophy [37, 38]. It is upregulated in variouscancers, inc

Committee of Xiangya Medical School, Central South University. After one week of pre-feeding, mice were randomly divided into the following four groups: control group (control), IgAN group (IgAN), IgAN group treated with miR-214-3p antagomir (IgAN miR-214-3p antagomir) and IgAN group infec