| PartⅠ MicroRNA-21-5p Regulating p38Mitogen-ActivatedProtein Kinases Signaling PathwayObjectiveTo verify the targeting regulatory relationship between microRNA（miR）-21-5p andmitogen-activated protein kinase kinase3(MKK3)Methods1. miR-21-5p and MKK3targeted relationship analysed by Bioinformatics databasepredict. Constructing MKK33’UTR reporter vector (MKK3-3U) and mutationreporter vector (MKK3-3U-M); synthesizing miR-21-5p mimics (mimic),miR-21-5p inhibitor (inhibitor), nonsense miR (NC).2. NC (NC1group), mimics (mimic1group), inhibitor (inhibitor1group) togetherwith MKK3-3U co-transfected human embryonic kidney (HEK293) cellsrespectively, The dual luciferase assay detected fluorescence ratio.3. Empty vector-pYr-MirTarget (Control group, C group), MKK3-3U (Wt group),MKK3-3U-M (Mut group) together with mimic co-transfected HEK293respectively. The dual luciferase assay detected fluorescence ratio.4. NC (NC2group), mimics (mimic2group), inhibitor (inhibitor2group) intervenedhuman renal tubular epithelial (HK-2) cells, respectively.5. RT-PCR and Western blot tested cell MKK3express level in HK-2. Results1. Bioinformatics database predicted miR-21-5p blinding with MKK33’UTR.2. The fluorescence ratio of NC1group, mimics1group, inhibitor1group were100%,67.99%,133.17%.3. The fluorescence ratios of C group, Wt group, Mut group were100%,65.3%,98.48%. There was no significant difference between C and Mut group (P>0.05).4. NC (NC2group), mimics (mimic2group), inhibitor (inhibitor2group) intervenedHK-2, MKK3mRNA relative expression levels were1.36±0.02、1.01±0.04、1.43±0.06; relative protein expression levels were0.97±0.05,0.62±0.06、1.36±0.32. Mimic2group MKK3mRNA and protein levels were significantly lowercompared with NC2group (P<0.05).ConclusionMKK3is the targed gene of miR-21-5p. MiR-21-5p regulates expression of MKK3both in mRNA and protein levels. It is suggested may be an important regulatorymolecule in p38MAPK signaling pathway. PartⅡ Expression and significance of miR-21/mitogen-activated protein kinase kinase3/interleukin-6andtumor necrosis factor-α in mice kidney withischemia-reperfusion injuryObjectiveTo explore the expression and significance of miR-21, MKK3and interleukin-6(IL-6)/tumor necrosis factor-α (TNF-α) in mice kidney with ischemia-reperfusion injuryMethods1. Grouping：Male C57BL/6J mice were randomly divided into three groups: controlgroup (C group), ischemia-reperfusion group (IR group), sham surgery group (Sgroup). The later two groups were divided into nine sub-groups according toreperfusion time respectively:0h,3hs,6hs,12hs,24hs,48hs,72hs,5ds,7ds.2. Animal Models: AKI mouse model: bilateral renal pedicle were clampped for30mins following by reperfusion. Sham group: Bilateral renal pedicle were exposedonly without clipping.3. Experimental Methods: Detecting serum creatinine (Scr), blood urea nitrogen(BUN); HE staining tested renal pathological damage. Real-time quantitativeRT-PCR detected t miR-21expressoin. RT-PCR and immunohistochemistry (IHC)tested MKK3, IL-6, TNF-α expression.Results1. Changes of kidney function and renal pathology score: IR group Scr, BUN, renalpathological lesions gradually increased, reaching a peak at24hours, thengradually declined. 2. Changes of miR-21expression: MiR-21expression after reperfusion in IR groupgradually increased most from12h to24h, and reached the peak of286.0times thebaseline value at the24h, ther remained stable at this high level.3. Changes of MKK3expression: IR group MKK3mRNA and protein expressionincreased gradually after reperfusion,24h reached the highest value, then decreasedgradually.4. Changes of IL-6and TNF-α: IL-6mRNA and protein expression levels of IR micesharply rise after ischemia-reperfusion in kidney,6h peaked, and then graduallydeclined. TNF-α mRNA expression increased gradually after reperfusion,24hpeaked; different with mRNA expression trendency. IR group ischemic TNF-αprotein expression gradually increased after reperfusion,48h reperfusion peaked,48h to72h to maintain a high value, then gradually declined.ConclusionMiR-21and mitogen-activated protein pathway and downstream inflammatorycytokines---interleukin-6/tumor necrosis factor-α is closely related to renalischemia-reperfusion injury, miR-21and MKK3and downstream factor-IL-6/TNF-αplays an important role in the process of renal ischemia-reperfusion injury. PartⅢ Renal Ischemic Preconditioning protected Kidneys ofAcute Kidney Injury by Upregulating Mir-21Down-regulatingMitogen-Activated Protein Kinase Kinase3And DownstreamInterleukin-6/Tumor Necrosis Factor-αObjectiveExplore the protective effect of miR-21-mediated mitogen-activated protein kinasekinase3understated interleukin6/tumor necrosis factor-α for the kidneys.Methods1. Ggrouping：Male C57BL/6J mice were randomly divided into three groups:ischemia-reperfusion group (IR group), Reperfusion of ischemic preconditioning(IPC+IR group), sham ischemic preconditioning reperfusion group (S+IR group.Every group was divided into nine sub-groups according to reperfusion timerespectively:0h,3hs,6hs,12hs,24hs,48hs,72hs,5ds,7ds.2. Animal Models:①R enal ischemia-reperfusion injury model: Clipping bilateralrenal pedicle for30mins following by reperfusion;②Ischemic preconditioningmodel: First, clipping bilateral renal pedicle for15mins,4days later, clippingbilateral renal pedicler for30mins;③sham ischemic preconditioning model: freebilateral renal pedicle wihtout clipping, then clipping bilateral renal pedicler for30mins after4ds.3. Methods: Detecting serum creatinine (Scr), blood urea nitrogen (BUN); testingrenal pathological damage with HE staining, detecting miR-21expressoin byReal-time quantitative RT-PCR, testing MKK3, IL-6and TNF-α expression byRT-PCR and immunohistochemistry (IHC) test. Results1. Changes of kidney function and renal pathology score:(1) Kidney function: IR group’s Scr, BUN, renal pathological lesions graduallyincreased, reaching peak at24hours, then gradually declined. IPC+IR group’s Scr,BUN, renal pathological injury trendency were consistent with the IR group, butthe extent of damage wer lower than IR group.(2) Renal pathology score: tubulointerstitial pathological score (points) between IRgroup and IPC+IR group at0h,3h,6h,12h,24h,48h,72h,5d and7d point were:1.00±0.12v0.30±0.11,1.50±0.15v1.50±0.15,2.00±0.15v1.30±0.16,2.60±0.23v1.5±0.16,3.80±0.22v1.70±0.12,3.10±0.24v1.30±0.13,2.50±0.12v1.30±0.11,2.30±0.24v1.00±0.11,1.60±0.11v0.50±0.07. There were ignificantly pathologicaldifferences at each time point between IR group and IPC+IR group (P<0.01).2.Changes of miR-21in kidney: MiR-21expression in IR group gradually increased,rose most significantly from12h to24h, peaked at24hours, then maintained a highvalue; IPC+IR group reached a peak value which was286times comparing to thebase line level and maintained for a long time.3. Chang of MKK express: IR group MKK3mRNA and protein expression increasedgradually after reperfusion, reached the highest value24hs later, and then decreasedgradually; IR group MKK3mRNA trendency was consistent with the IR group, butthe value of each point of time was lower than the IR group. MKK3mRNA levelsbetween IR group and IPC+IR group at0h,3h,6h,12h,24h,48h,72h,5d,7d timepoint were:0.89±0.10v0.51±0.06,1.45±0.13v0.52±0.07,1.65±0.18v0.54±0.09,2.15±0.22v0.61±0.11,3.19±0.31v0.81±0.09,2.66±0.28v0.76±0.10,1.87±0.22v0.68±0.08,1.68±0.15v0.66±0.07,1.62±0.17v0.63±0.08, MKK3protein expresslevels (OD/10000) were:15.51±1.56v14.36±1.51,34.24±2.82v21.14±1.67,71.54±5.43v31.52±2.01,145.16±8.29v45.39±2.03,159.50±7.32v60.34±3.67,209.74±8.13v95.12±4.02,95.58±5.47v62.13±2.31,87.32±5.12v42.51±2.57,45.13±3.25v33.18±1.99. Between IR group and IPC+IR group, MKK3and protein expression’s differences from the reperfusion3thh~7thd same among eachpoint subgroup were statistically significant (P<0.05).4. Changes of IL-6and TNF-α express:(1)IL-6express: IR group IL-6mRNA and protein expression rise sharply afterischemia-reperfusion, peaked6hours later and then gradually decreased, whileIPC+IR group’s IL-6mRNA and protein expression levels remained low level.IL-6mRNA levels between IR group and IPC+IR group at0h,3h,6h,12h,24h,48h,72h,5d,7d time point were:0.65±0.11v0.30±0.07,1.53±0.14v0.39±0.05,2.82±0.24v0.43±0.07,2.21±0.26v0.41±0.09,1.73±0.21v0.44±0.09,1.55±0.18v0.46±0.07,1.38±0.14v0.43±0.06,1.16±0.08v0.41±0.06,0.89±0.06v0.40±0.05,expression level of IL-6protein (OD/10000) were45.22±1.58v35.16±2.53,68.13±2.72v42.73±1.99,135.28±8.42v58.33±2.23,108.99±5.49v50.11±2.41,91.18±6.32v47.29±4.12,72.63±5.15v46.63±3.87,66.57±4.47v43.18±2.67,58.74±5.82v40.12±2.85,46.33±2.27v36.47±1.82. IL-6mRNA expression hadsignificantly difference at3h~5d time point between IR group and IPC+IR group(P<0.05); IL-6protein expression had significantly difference at0h~7d time pointbetween IR group and IPC+IR group (P <0.01).(2) TNF-α express: IR group’s TNF-α mRNA expression increased gradually afterreperfusion, reached the highest value after24hours, and then gradually declined,IPC+IR group TNF-α mRNA is consistent with the IR group. However, theexpression level of each of time was lower than IR group. IR group’s TNF-αprotein expression gradually increased after reperfusion, achieved high value48hours later, maintained a high value form48h to72h, finally declined, IPC+IRgroup MKK3protein trends were consistent with IR group, but the expressionlevels at various time points were lower than the IR group. TNF-α mRNA levelsbetween IR group and IPC+IR group at0h,3h,6h,12h,24h,48h,72h,5d,7d timepoint were0.800.20v0.50±0.07,1.66±0.21v0.51±0.11,2.02±0.26v0.50±0.09,2.63±0.31v0.54±0.09,3.57±0.42v0.54±0.12,2.82±0.47v0.55±0.15,2.19±0.32v0.52±0.16,1.73±0.22v0.52±0.14,1.12±0.17v0.50±0.09. TNF-α protein express(OD/10000) were63.67±3.56v62.31±4.22,82.36±5.82v66.47±4.41,97.23±6.4316 v70.39±4.57,106.67±6.29v77.12±4.63,124.37±7.32v81.36±4.74,167.53±7.23v91.16±5.01,158.36±5.43v81.82±4.45,98.42±5.22v70.11±4.87,82.31±3.17v65.35±4.39. TNF-α mRNA express has ignificantly difference at3h~5d time pointbetween IR group and IPC+IR group (P<0.05); TNF-α protein expression hassignificantly difference at3h~7d time point between IR group and IPC+IR group(P<0.01). |
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