MiR-103a-3p Silencing Ameliorates Calcium Oxalate Deposition nyob rau hauv nas raum Los ntawm Activating UMOD/TRPV5 Axis

Tuav qhov sib npaug ntawmcalcium (Ca{0}}) metabolismhauv lub raum yog qhov tseem ceeb hauvtiv thaiv kev tsim ntawm lub raum pob zeb. Ua haujlwm zoo, microRNA (miRNA) koom nrog hauv cov txheej txheem no yuav tsum tau nthuav tawm. Peb ntxias NRK-52E cell raug mob los ntawmKev kho mob oxalate. Lub luag haujlwm ntawm transient receptor muaj peev xwm cation channel subfamily V tus tswv cuab 5 (TRPV5) hauv oxalate-induced hlwb tau kawm los ntawm TRPV5 overexpression transfection, qRT-PCR, Western blot, MTT, thiab crystal adhesion nrhiav kom tau. Tom qab txheeb xyuas uromodulin (UMOD) qhia hauv cov hlwb raug mob, peb tau lees paub qhov kev sib cuam tshuam ntawm TRPV5 thiab UMOD los ntawm coimmunoprecipitation (CoIP) thiab cell-surface biotinylation assays. Kev siv tau ntawm UMOD-regulating TRPV5 nyob rau hauv viability, crystal adhesion, thiab Ca2+ concentration ntawm oxalate-induced hlwb tau ua. Bioinformatics tsom xam thiab kev ntsuam xyuas luciferase tau siv los txheeb xyuas cov miRNA-targeting UMOD. Lub luag haujlwm ntawm miR-103a{13}}p-regulating UMOD/TRPV5 axis tau kuaj pom los ntawm kev sim cawm. Peb tsim cov qauv nas nrog kev kho ntawm ethylene glycol (EG) los tshawb xyuas miR-103a-3p/ UMOD/TRPV5 axis hauv vivo los ntawm hematoxylin-eosin (H&E) staining, Western blot, thiab immunohistochemistry (IHC). Upregulation ntawm TRPV5 tiv thaiv NRK-52E hlwb los ntawm kev raug mob oxalate-induced los ntawm kev txhim kho cell viability thiab inhibiting CaOx adhesion. UMOD tau depleted nyob rau hauv oxalate-induced hlwb thiab zoo interacted nrog TRPV5. UMOD silencing thim rov qab cov nyhuv ntawm TRPV overexpression ntawm oxalate-induced hlwb. miR-103a-3p tsom UMOD thiab tau kho kom haum rau hauv kev tswj hwm ntawm UMOD/TRPV5 axis hauv oxalate-induced hlwb. Downregulating miR-103a-3p mitigated EG-induced CaOxdeposition nyob rau hauv lub raum cov ntaub so ntswghauv vivo los ntawm kev ua kom lub UMOD / TRPV5 axis. miR-103a-3p silencing ameliorated CaOx deposition nyob rau hauv nas lub raum los ntawm activating UMOD/TRPV5 axis.

HERBAL FORMULATION RAUDEPOSITION IN KIDNEY TISSUES

1. Taw qhia

Urinary calculi, ib qho ntawm feem ntauCov kab mob hauv Urology, yog tshwm sim los ntawm nag lossis daus ntawm oversaturated crystals los ntawm cov zis nyob rau hauv lub raum [1]. Txawm hais tias tsis yog mob hnyav li cov qog nqaij hlav, cov zis calculi tau ntev tau muaj kev cuam tshuam loj rau tib neeg kev noj qab haus huv vim lawv qhov tshwm sim siab thiab rov tshwm sim [2]. Cov kev tshawb fawb tau lees paub tias qhov pib ntawm cov zis calculi yog multifactorial thiab koom nrog cov noob caj noob ces, metabolism, ib puag ncig huab cua, thiab kev ua neej nyob [3, 4]. Calcium oxalate (CaOx) pob zeb suav rau feem ntau ntawm cov kab mob no, thiab feem ntau cov neeg mob uas muaj Ca2+-cov pob zeb muaj kev sib xyaw ntawm hypercalciuria [5]. Cov kev tshawb fawb Physi-chemical tau txheeb xyuas tias qhov saturation ntawm CaOx nyob rau hauv cov zis muaj feem xyuam rau cov concentration ntawm Ca2+ thiab cov calcium ntau ntxiv rau cov zis los yog txhawb kev tsim ntawm Ca2+-muaj nephrolithiasis [6]. Lub raum pob zeb tuaj yeem ua rau kub siab, kab mob raum ntev, thiab kab mob raum kawg. Cov kev tshawb fawb tau tshaj tawm tias kev tiv thaiv kev tiv thaiv, suav nrog cov kev cuam tshuam tshwj xeeb ntawm cov tshuaj thiab cov lus pom zoo rau kev ua neej thiab kev noj haus, tuaj yeem ua raws tom qab kev ntsuas kev ntsuas metabolic [7, 8]. Ntau yam kev tshawb fawb tib neeg tau qhia tias kev noj zaub mov kom ntau dua ntawm zaub thiab txiv hmab txiv ntoo ua lub luag haujlwm hauv kev tiv thaiv lub raum pob zeb [9-11]. Tam sim no, ib qho kev tshawb fawb tob yuav tsum tau ua nyob rau hauv molecular mechanism ntawm hypercalciuria induced los ntawm txawv txav calcium metabolism nyob rau hauv lub tsim ntawm lub raum pob zeb.

TRPV5 yog ib qho tseem ceeb ntawm cov protein uas tswj kev thauj mus los ntawm Ca2+ hla cov cell membranes [12]. Hauv lub raum cov ntaub so ntswg, TRPV5 feem ntau pom nyob rau hauv lub distal convoluted tubule (DCT) thiab sau tubule (CNT) uas yog lub luag hauj lwm rau tswj urinary Ca2+ reabsorption thiab tswj Ca2+ homeostasis hauv lub cev [13] . Muaj pov thawj qhia tias kev txo qis TRPV5 kev qhia yuav yog ib qho tseem ceeb ntawm cov kab mob pathogenic hauv kev tsim cov hypercalciuria thiab Ca2+-muaj nephrolithiasis [14].

Table 1: Cov txheej txheem primer siv rau qRT-PCR.

Uromodulin (UMOD), tseem hu ua Tamm-Horsfall protein, yog cov glycoprotein ntau tshaj plaws hauv cov zis [15], uas yog feem ntau qhia nyob rau hauv lub raum tubular epithelial hlwb nrog ntau yam physiological functions xws li ntsuas cov dej-electrolyte metabolism, tswj kev tiv thaiv. , tiv thaiv lub raum pob zeb, thiab tiv thaiv kab mob urinary [16–18]. Tam sim no, muaj ob peb tsab ntawv ceeb toom txog cov txheej txheem molecular tshwj xeeb ntawm UMOD hauv lub raum pob zeb tsim.

MicroRNAs (miRNAs) yog ib chav kawm me me, tsis yog coding, ib leeg-stranded RNA molecules nrog ntev li ntawm 20- 22 nucleotides, dav dav hauv cov tsiaj txhu [19]. Mature miRNAs tuaj yeem tswj hwm cov lus qhia tom qab ntawm lub hom phiaj cov noob los ntawm kev khi tag nrho lossis ib nrab ntxiv rau 3′-UTR ntawm lub hom phiaj mRNA, uas ua rau muaj kev puas tsuaj lossis inhibition ntawm protein translational synthesis [20]. Txij li thaum miRNAs cuam tshuam rau tib neeg lub cev kev ua ub no xws li kev loj hlob thiab kev loj hlob, cell apoptosis, thiab metabolism, lawv tau txiav txim siab tuav cov lus cog tseg zoo rau kev tiv thaiv thiab kho cov kab mob [21]. Nyob rau hauv xyoo tas los no, kev tshawb fawb txog lub luag haujlwm ntawm miRNAs nyob rau hauv qhov pib thiab kev loj hlob ntawm lub raum pob zeb tau dhau los ua lub ntsiab lus kub [22, 23].

Yog li ntawd, txoj kev tshawb no tau soj ntsuam rau hauv kev sib raug zoo ntawm UMOD thiab TRPV5 hauv kev tiv thaiv ntawm lub raum pob zeb tsim thiab elucidated lub luag hauj lwm ntawm miR-103a-3p hauv lub hom phiaj thiab tswj UMOD, ua rau kom nkag siab tob txog qhov pathogenesis ntawm lub raum pob zeb thiab muab tswv yim tshiab rau kev tiv thaiv thiab tswj tus kab mob.

2. Cov khoom siv thiab cov txheej txheem

2.1. Cell Culture thiab Oxalate Treatment.

Epitheloid nas raum cell kab NRK{{0}}E (GNR 8) tau txais los ntawm Tuam Tshoj Center for Type Culture Collection (CCTCC) (Shanghai, Suav). DMEM (12800017, Gibco, USA) tov nrog dej distilled, 7.5% NaHCO3 tov (25080094, Gibco, USA), thiab 5% fetal bovine serum (FBS) (BC-SE-FBS02, Bio channel, Tuam Tshoj) tau npaj rau kab lis kev cai cell nyob rau hauv lub incubator (37 degree C thiab 5% CO2). Tom qab subculturing, cov hlwb raug ntxias nrog diluted oxalate (0.75 mmol / L, 135623, Sigma Aldrich, USA) rau 48 teev (h), raws li tau piav ua ntej [24]. 2.2. Kev hloov ntawm tes. Rau txoj kev tshawb nrhiav molecular mechanism, TRPV5-overexpressing plasmid (Bes-mR-001007572, Ber sinBio, Guangzhou, Tuam Tshoj), UMOD-overexpressing plasmid (Bes-mR-001278605, BersinBio, Guangzhou, Tuam Tshoj), me me cuam tshuam RNA tawm tsam UMOD (siUMOD) (5′-CTCTTT TTCACACTTGATATGA-3′, GenePharma, Shanghai, Suav teb), miR-103a-3p inhibitor (GenePharma, Shanghai, Tuam Tshoj), thiab miR-103a-3p mimic (BersinBio, Guangzhou, Tuam Tshoj) tau raug kev hloov pauv txawv hauv NRK- 52E hlwb, nrog rau kev tswj hwm raug teeb tsa. Ua ntej kev hloov pauv, NRK-52E hlwb raug coj mus rau hauv ib qho 6-zoo phaj (2 × 106 hlwb/qhov zoo). Txog thaum ncav cuag li 80% ntawm tes confluence, TRPV5-overexpressing plasmid, UMOD overexpressing plasmid, siUMOD, miR-103a-3p inhibitor, los yog mimic tau kis mus rau NRK-52E hlwb siv lipoic lub sij hawm 2000 (11668019, Invitrogen, USA) raws li cov chaw tsim tshuaj paus raws tu qauv. Tom qab ntawd, cov hloov pauv NRK- 52E cov hlwb tau txais ntau npaum li cas ntawm cov tshuaj tiv thaiv kab mob polymerase (qRT-PCR) thiab Western blot rau kev soj ntsuam zoo thiab tom qab ntawd tau kho nrog oxalate.

2.3. RNA Extraction thiab qRT-PCR.

Tag Nrho RNA thiab miRNA los ntawm NRK-52E hlwb raug rho tawm siv Cell RNA Kit (19231ES50, Yeasen, Tuam Tshoj) thiab MolPure® Cell/Tissue miRNA Kit (19331ES50, Yeasen, Suav). Tom qab ntawd, HiScript II Ib-kauj ruam RT-PCR Cov Khoom Siv (P612-01, Vazyme, Tuam Tshoj) tau ua haujlwm los cuam tshuam nrog cov extract raws li cov lus qhia ua haujlwm. Qhov kev nthuav dav tau teeb tsa raws li hauv qab no: 50 degree C rau 30 min, 94 degree C rau 3 min, ua raws li 30 cycles (94 degree C rau 30 s, 60 degree C rau 30 s, thiab 72 degree C rau 40 s), thiab 72 degree C rau 5 min. QRT-PCR tau ua nyob rau hauv ABI 7500 system (Applied Biosystems, USA). Cov txheeb ze mRNA kab lus tau txheeb xyuas los ntawm 2−ΔΔCt txoj kev [25], nrog -actin lossis U6 siv rau normalization. Tag nrho cov txheej txheem primer tau qhia hauv Table 1.

2.4. Western Blot.

Tag nrho cov protein los ntawm cov qauv (NRK-52E hlwb thiab nas raum cov ntaub so ntswg) tau lysed siv RIPA Lysis Buffer (R0010, Solarbio, Tuam Tshoj) thiab ntsuas los ntawm BCA Protein Assay Kit (BI-WB005, Sbjbio, Suav). Qhov sib npaug ntawm cov protein tau sib cais los ntawm 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (P0690, Beyotime, Tuam Tshoj) thiab xa mus rau PVDF daim nyias nyias (AR0136- 02, Boster, Suav). Tom qab raug kho nrog kev thaiv tsis pub (P0252, Beyotime, Tuam Tshoj), cov membranes tau incubated nrog anti-TRPV5 (ab137028, 83 kDa, 1/10000), anti-UMOD (ab207170, 110 kDa, 1/1000), thiab anti{ {20}}actin (ab8226, 42 kDa, 1/1000) cov tshuaj tiv thaiv ntawm 4 degree C thaum hmo ntuj. Cov tshuaj tiv thaiv kab mob thib ob suav nrog cov tshuaj tiv thaiv kab mob IgG (ab97051, 1/2000) thiab cov tshuaj tiv thaiv nas IgG (ab205719, 1/2000) tau siv los ua kom cov qog ua paug rau 2 teev ntawm chav tsev kub. Cov txheeb ze cov protein blot nrhiav tau raug khiav hauv iBright Imaging System (CL750, Invitrogen, USA) los ntawm kev siv ECL Buffer (WBKlS0100, Millipore, USA) rau kev pom. - Actin tau ua haujlwm hauv kev tswj hwm sab hauv.

2.5. Cell Viability Assay.

Kev ncua ntawm tes tau npaj raws li tau piav qhia hauv txoj kev tshawb no [24]. NRK-52E cov noob tau cog rau hauv 96- cov kab lis kev cai kab lis kev cai ntawm qhov ntom ntawm 5000 cov cell/well, thiab 10 μL methyl thiazolyl diphenyl- tetrazolium bromide (MTT) tov (40201ES72, Yeasen, Tuam Tshoj) tau siv los kho cov hlwb hauv txhua qhov dej ntawm 37 degree C rau 4 h, ua raws li kev kho SDS-HCL. Rau kev soj ntsuam ntawm cell viability, tus nqi kho qhov muag (OD) tus nqi ntawm lub wavelength ntawm 570 nm yog ntsuas los ntawm tus nyeem ntawv microplate (VL0L00D0, Thermo Fisher, USA).