Efè Naringenin sou pwofil MiRNA-mRNA nan selil HepaRG
Mar 12, 2022
Pou plis detay, kontakte:tina.xiang@wecistanche.com
Résumé: Naringenin, yon natirèlflavonoidlajman jwenn nan fwi Citrus, yo te rapòte yo posede pwopriyete antioksidan, anti-enflamatwa, ak epatoprotective kòm yon sipleman natirèl dyetetik. Sepandan, mekanis regilasyon nan naringenin nan fwa imen an rete klè. Nan etid la prezan, yo te itilize sekans RNA mesaje (mRNA-seq), sekans microRNA (miRNA-seq), ak qPCR an tan reyèl pou distenge diferans ki genyen ant espresyon kontwòl ak selil HepaRG trete ak naringenin. Nou te jwenn 1037 diferan eksprime mRNA ak 234 miRNAs.Selon prediksyon sib la ak analiz entegrasyon nan silico, nou te jwenn 20 potansyèl pè miRNA-mRNA ki enplike nanfwametabolis. Etid sa a se premye moun ki bay yon pèspektiv nan entèraksyon miRNA-mRNA nan règleman an nan naringenin atravè analiz entegre nan mRNA-seq ak miRNA-seq nan selil HepaRG, ki plis karakterize valè a nutraceutical nan naringenin kòm yon aditif manje.
Mo kle: naringenin; mRNA-seq; miRNA-seq; Selil HepaRG
klike pou jwenn plis enfòmasyon sou pwodwi
1. Entwodiksyon
Naringenin, yon flavonoid natirèl, yo jwenn anpil nan fwi Citrus ak lòt fwi manjab, tankou chadèk, zoranj, bergamot, tomat, ak fig frans [1]. Apre administrasyon oral, naringenin distribye lajman nan aparèy gastwoentestinal la ak fwa [2,3] epi li montre yon pwopriyete antioksidan dirèk pa aktivite radikal gratis sou kont estrikti molekilè li yo, epi li pwovoke sistèm antioksidan andojèn nan fwa a [4]. Prèv k ap grandi nan tou de etid in vitro ak in vivo te idantifye divès kalite pwoteksyon kapasite nan naringenin, tankouanti-enflamatwa[5], antioksidan [6], anti-fibwoz [Z], ak epatoprotective 4,8| aktivite yo. Kapasite sa yo sijere ke naringenin dyetetik ta ka aplike pou anpeche sendwòm metabolik ak maladi malfezan, ki gen ladan epatit fulminant, gra.maladi fwa, fibwoz, elatriye 19,10]. Sepandan, gen kèk etid detaye sou efè regilasyon naringenin sou jèn yo an jeneral nan fwa a [4].
MikroRNAs (miRNAs) se yon klas molekil RNA ki pa kode, ki gen yon longè 18-26 nukleotid ki kode pa jèn andojèn, ki montre yon pakèt fonksyon regilasyon byolojik nan filojeni, diferansyasyon, pwopagasyon, ak apoptoz. [11]. miRNA, RNAs mesaje obligatwa (mRNAs) pa rekonesans sekans-espesifik, negatif kontwole ekspresyon jèn nan nivo pòs-transcriptional nan degradasyon nan mRNA sib [12]. Anba eksitasyon ekzojèn, ekspresyon miRNA chanje, ak Lè sa a, ekspresyon mRNA sib reglemante. Evantyèlman, anpil fonksyon fizyolojik yo chanje pou fè fas ak defi ki te koze pa eksitasyon ekzojèn [13]. Yon metòd ki pi serye pou predi relasyon sib miRNA-mRNA se ansanm analiz mRNA-seq ak analiz miRNA-seq lè l sèvi avèk yon kontèks pwosesis patikilye nan silico [14].
Se poutèt sa, objektif nou an se te etidye efè naringenin sou jèn mondyal epi mete aksan sou posib mekanis regilasyon pè miRNA-mRNA nan fwa a. Nan etid aktyèl la, chanjman ekspresyon mRNA ak pwofil ekspresyon miRNA yo te envestige nan HepaRGcells lè yo fè mRNA-seq, miRNA-seq, analiz bioenfòmatik, ak an tan reyèl qPCR bay prèv pou potansyèl la nan naringenin kòm yon sipleman natirèl dyetetik.
2. Rezilta yo
2.1.Analiz sekans transcriptom nan repons a Naringenin
Pou idantifye chanjman ekspresyon mRNA nanSelil HepaRGan repons a naringenin, uit bibliyotèk konplemantè ADN(cDNA) nan gwoup kontwòl la (CK-1, CK-2, CK-3, ak CK-4) ak gwoup eksperimantal la. (T-1, T-2, T-3, ak T{-4) yo te konstwi ak RNA total epi yo te sibi sekans Illumina HiSeq2500 (Genedenovo Biotechnology Co., Ltd, Guangzhou , Lachin). Apèsi sou rezilta yo sekans ak asanble pou gwoup kontwòl la ak gwoup eksperimantal yo montre nan Tablo 1. Chanjman ekspresyon jen yo te analize lè yo konpare gwoup trete ak kontwòl yo. Jan yo montre nan Figi la a, gwoup la naringenin-ekspoze eksprime 1037 diferan eksprime jèn (DEGs) konpare ak gwoup kontwòl la. Yon kat jeyografik chalè 1037 DEG te montre analiz gwoup kontwòl gwoup la ak gwoup naringenin (Figi 1b; 381 monte-ak 656 jèn ki reglemante; Tablo S1, Materyèl siplemantè).
Dapre sistèm klasifikasyon Gene Ontology (GO), 1037 DEG yo te klase nan twa gwo kategori fonksyonèl (pwosesis byolojik, eleman selilè, ak fonksyon molekilè) ak 61 sou-kategori (Figi 2). Pami pwosesis byolojik la, pwosesis selilè a (731) te pi souvan reprezante a, ki te swiv pa pwosesis la yon sèl-òganis (672) ak règleman byolojik (595). Nan kategori a nan eleman selilè, yon pwopòsyon enpòtan nan grap yo te asiyen nan selil (727), pati selil (721), ak òganèl (580). Jèn ki enplike nan gwoup obligatwa (666) ak aktivite katalitik (238) yo te miyò reprezante nan kategori fonksyon molekilè.
Yo te jwenn klasifikasyon Kyoto Encyclopedia of Genes and Genomes (KEGG) pou 1037 DEGs ki te plis klase nan ven pi gwo chemen byochimik dapre pi piti q-valè ak pi gwo GeneNumber nan anotasyon chemen (Figi 3). GeneNumber ak rapò jèn anote nan senk pi gwo chemen yo se lupus sistemik eritematos (33,8.4 pousan), alkolis (38,9.67 pousan), move regleman transcription nan kansè (22, 5.6 pousan), chemen siyal PI3K-Akt (34, 8.65 pousan), ak konpleman ak kaskad koagulasyon (12, 3.05 pousan o). Te gen plis pase 10 jèn anrichi pami senk chemen yo. An jeneral, sibi tretman naringenin te gen yon enpak siyifikatif sou pwofil ekspresyon jèn mondyal selil HepaRG yo. Rezilta sa yo implique ke jèn ki enplike nan chemen sa yo ka jwe wòl enpòtan nan règleman naringenin.
2.2. Analiz de nivo transkripsyon miRNA an repons a Naringenin
Nan etid sa a, nou te vize pou detèmine sinaringeninekspoze chanje nivo ekspresyon miRNAs nan selil HepaRG yo. Apre ekspoze, nou kolekte ti RNA ak mezire abondans relatif yo lè l sèvi avèk Illumina HiSeq2500 (Genedenovo Biotechnology Co., Ltd, Guangzhou, Lachin). Jan yo montre nan Tablo 2, li pwòp nan uit echantiyon yo te pwodwi, respektivman, apre yo fin retire lekti kontaminan yo. Yon apèsi sou lekti pou ti RNA sekans soti nan done anvan tout koreksyon nan bon jan kalite ak bon jan kalite filtraj yo bay nan Tablo 2. Distribisyon longè ti RNA yo te menm jan an pami bibliyotèk nan ke 21-23 nt RNA yo te pi abondan (Figi 4). ).
Tout ti RNA yo te aliyen nan baz done GeneBank (Release 209.0) ak baz done Rfam (11.0) pou idantifye ak retire RNA ribozomal (rRNA), ti RNA kondisyonèl (scRNA), ti nukleolè (snoRNA), ti nikleyè (snRNA), ak transfere RNA (tRNA). An akò ak jenom referans la, yo te retire ti RNA sa yo ki te trase nan ekson, entron, ak sekans repete yo tou. Yo te fouye ti RNA filtraj yo kont baz done miRBase (Release 21) pou idantifye miRNA yo. Kat jeyografik chalè 3373 miRNA yo montre analiz gwoup kontwòl gwoup la ak gwoup naringenin nan Figi 5a. Profile Illumina HiSeq2500 nan 3373 miRNA yo analize nan ekspoze naringenin kont echantiyon kontwòl te montre ke yon total de 234 miRNA eksprime diferans (DEMs, 174up-ak 60 desann-reglemante; Tablo S2, Materyèl siplemantè) te detekte nan selil HepaRG (Figi 5b) .
2.3.Prediksyon sib ak analiz entegrasyon pwofil ekspresyon mRNA ak miRNA an repons a Naringenin
Aji nan nivo pòs-transkripsyon, miRNAs silans ak/oswa desann-regleman ekspresyon jèn selilè mRNA pa klivaj RNA sib. .01), Miranda (v3.3a), ak TargetScan (Vèsyon:7.0). Profilage similtane 234 DEM ak nivo 1037 DEG nan silico ka idantifye mRNA yo sipoze sib nan miRNAs. Nou te chwazi entèseksyon DEG yo ak jèn sib DEM yo epi nou te fè analiz bioenfòmatik sou jèn entèseksyon sa yo. Yo te jwenn yon total de 5607 pè miRNA-mRNA negatif pou tretman naringenin, ak patisipasyon 216 DEM ak 681 DEG (Tablo S3, Materyèl siplemantè). Nan liy ak sistèm nan klasifikasyon GO, 681 DEG yo te klase nan 58 sou-kategori (Figi 6). Twa premye sous-kategori yo pa t chanje nan twa gwo kategori fonksyonèl konpare ak analiz sekans transcriptom (Figi 2 ak 6).
Analiz anrichisman chemen pou 681 DEG nan 5607 pè miRNA-mRNA negatif idantifye ven pi gwo chemen yo dapre pi piti q-valè a ak pi gwo GeneNumber nan anotasyon chemen an apre ekspoze naringenin (Figi 7): ak chemen siyal PI3K-Akt (25,8.96). pousan) yo te wityèm chemen an ak dezyèm ki pi abondan.
2.4.Validasyon qPCR an tan reyèl nan Règleman Naringenin nan metabolis fwa ak potansyèl regilasyon pè miRNA-mRNA
Dapre notasyon fonksyon jèn mondyal, revizyon literati, ak relasyon potansyèl yo ak naringenin-responsive miRNAs, 19 DEGs (ALOX15, CA9, TH, HKDC1, NDUFA4L2, RRM2, ACSL5, PLA2G4C, LIPT2, UGDH, FTCD, ABAT, AZTIN2, HS6STIN2 , B4GALT6, GUSB, DCT, ALAS2, ak MAT1A) yo te chwazi manyèlman kòm reprezantan pou wòl potansyèl yo nan metabolis fwa. Anplis de sa, chemen siyal PI3K-Akt la te siyifikativman anrichi (katriyèm nan analiz KEGG nan RNA-seq, Figi 3; wityèm nan KEGG nan analiz miRNA-RNA-seq, Figi 7); Se poutèt sa.11 DEG (PDGFRB. CSF1R. FGFR2, IL2RG, IL7R, ITGB4, GNG4, PCK1, CREB3L3, CREB3L1, ak NFkB1) nan mitan chemen siyal PI3K-Akt yo te depistaj.
Isit la nou dekri entèraksyon ki genyen ant 30 DEG yo ak 11 miRNA imen (hsa-miR-1306-5p, hsa-miR-627-3p, hsa-miR-194-3p, hsa-miR{{9 }}p, hsa-miR-6837-5p, hsa-miR-429, hsa-miR-100-3p, hsa-miR-194-5p, hsa-miR{{19} }a-3p, hsa-miR-7-5p, ak hsa-miR-200a-5p), ki gen ladan 20 entèraksyon negatif miRNA-mRNA (Figi 9). Jan yo montre nan Figi 9, yon sèl miRNA ka kontwole plizyè mRNA sib ak vis vèrsa (egzanp, ABAT HS6ST3, B4GALT6, ak DCT ta ka pètèt an menm tan reglemante pa hsa-miR-429; HS6ST3 ka an menm tan reglemante pa hsa-miR. -429, hsa-miR-100-3p, hsa-miR-519a-3p, hsa-miR-676-3p, hsa-miR-7-5 p, ak hsa-miR-194-5p; kèk DEG pa te gen okenn DEM pè). Pwofil ekspresyon 19 DEG ki gen rapò ak metabolis fwa ak 11 DEG nan mitan chemen siyal PI3K-Akt yo te plis valide lè l sèvi avèk qPCR an tan reyèl (Figi 9a, b). Nivo ekspresyon 11 miRNAs imen (de moute-ak nèf desann-reglemante) te evalye plis pou chanjman naringenin pwovoke pa qPCR an tan reyèl ki konsistan avèk rezilta sekans (Figi 9c). Malgre ke te gen kèk diferans quantitative ant de platfòm analyse yo, resanblans ki genyen ant done RNA-seq ak PCR an tan reyèl sijere ke done RNA-seq yo te repwodiktif ak serye.
![ure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR. (a) The 19 genes involved in liver metabolism; (b) 11 genes involved in the PI3K–Akt signaling pathway; (c) 11 putative regulatory miRNAs. Y-axis represents log2 (FC); FC (fold change) = the naringenin group/the control group. The dashed line indicated fold change data of 2.0. Values are the mean ± SD (n = 4). 3. Discussion and Conclusions The findings discussed here reveal the first detailed information regarding parallel mRNA and miRNA expression changes in HepaRG cells in response to naringenin. We performed an integrative analysis of these data including 234 DEMs and 1037 DEGs induced by naringenin, which provide global insight into the miRNA–mRNA interactions of naringenin in the regulatory mechanism. According to the gene function annotations and literature review, 19 DEGs related to metabolism were screened out. In particular, the PI3K–Akt signaling pathway was significantly enriched both in analysis of transcriptome sequencing (the third-most abundant, and ranked fourth) and integration analysis of miRNA-mRNA expression profiles (the second-most abundant, and ranked eighth) in responses to naringenin. In addition, 11 DEGs in the PI3K–Akt signaling pathway were further validated using real-time qPCR analysis. In this work, we constructed a miRNAmRNA regulatory network according to the DEMs and DEGs datasets and miRNA-targeting information. Some studies have demonstrated that the miRNA–mRNA regulatory network responds to liver damage, including hepatocellular carcinoma and oxidative stress [15]. Although several miRNA-induced RNA activation phenomena were identified [16], under most circumstances, the negative correlation between miRNAs and their target mRNAs is often considered support for miRNA targeting [17]. Ultimately, 20 miRNAmRNA negative correlation pairs were identified with the involvement of liver metabolism and the PI3K–Akt signaling pathway. With regard to global genes, we addressed our particular research question using pathway analysis to highlight 19 DEGs related to the functional clusters: metabolism, including Lipid metabolism (ALOX15, ACSL5, PLA2G4C, and B4GALT6), Energy metabolism (CA9 and NDUFA4L2), Metabolism of cofactors and vitamins (TH, LIPT2, and Figure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR](/Content/uploads/2022842169/20220310100538dcd6c59958f541a08c523a32322fd47d.png "ure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR. (a) The 19 genes involved in liver metabolism; (b) 11 genes involved in the PI3K–Akt signaling pathway; (c) 11 putative regulatory miRNAs. Y-axis represents log2 (FC); FC (fold change) = the naringenin group/the control group. The dashed line indicated fold change data of 2.0. Values are the mean ± SD (n = 4). 3. Discussion and Conclusions The findings discussed here reveal the first detailed information regarding parallel mRNA and miRNA expression changes in HepaRG cells in response to naringenin. We performed an integrative analysis of these data including 234 DEMs and 1037 DEGs induced by naringenin, which provide global insight into the miRNA–mRNA interactions of naringenin in the regulatory mechanism. According to the gene function annotations and literature review, 19 DEGs related to metabolism were screened out. In particular, the PI3K–Akt signaling pathway was significantly enriched both in analysis of transcriptome sequencing (the third-most abundant, and ranked fourth) and integration analysis of miRNA-mRNA expression profiles (the second-most abundant, and ranked eighth) in responses to naringenin. In addition, 11 DEGs in the PI3K–Akt signaling pathway were further validated using real-time qPCR analysis. In this work, we constructed a miRNAmRNA regulatory network according to the DEMs and DEGs datasets and miRNA-targeting information. Some studies have demonstrated that the miRNA–mRNA regulatory network responds to liver damage, including hepatocellular carcinoma and oxidative stress [15]. Although several miRNA-induced RNA activation phenomena were identified [16], under most circumstances, the negative correlation between miRNAs and their target mRNAs is often considered support for miRNA targeting [17]. Ultimately, 20 miRNAmRNA negative correlation pairs were identified with the involvement of liver metabolism and the PI3K–Akt signaling pathway. With regard to global genes, we addressed our particular research question using pathway analysis to highlight 19 DEGs related to the functional clusters: metabolism, including Lipid metabolism (ALOX15, ACSL5, PLA2G4C, and B4GALT6), Energy metabolism (CA9 and NDUFA4L2), Metabolism of cofactors and vitamins (TH, LIPT2, and Figure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR")
![ure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR. (a) The 19 genes involved in liver metabolism; (b) 11 genes involved in the PI3K–Akt signaling pathway; (c) 11 putative regulatory miRNAs. Y-axis represents log2 (FC); FC (fold change) = the naringenin group/the control group. The dashed line indicated fold change data of 2.0. Values are the mean ± SD (n = 4). 3. Discussion and Conclusions The findings discussed here reveal the first detailed information regarding parallel mRNA and miRNA expression changes in HepaRG cells in response to naringenin. We performed an integrative analysis of these data including 234 DEMs and 1037 DEGs induced by naringenin, which provide global insight into the miRNA–mRNA interactions of naringenin in the regulatory mechanism. According to the gene function annotations and literature review, 19 DEGs related to metabolism were screened out. In particular, the PI3K–Akt signaling pathway was significantly enriched both in analysis of transcriptome sequencing (the third-most abundant, and ranked fourth) and integration analysis of miRNA-mRNA expression profiles (the second-most abundant, and ranked eighth) in responses to naringenin. In addition, 11 DEGs in the PI3K–Akt signaling pathway were further validated using real-time qPCR analysis. In this work, we constructed a miRNAmRNA regulatory network according to the DEMs and DEGs datasets and miRNA-targeting information. Some studies have demonstrated that the miRNA–mRNA regulatory network responds to liver damage, including hepatocellular carcinoma and oxidative stress [15]. Although several miRNA-induced RNA activation phenomena were identified [16], under most circumstances, the negative correlation between miRNAs and their target mRNAs is often considered support for miRNA targeting [17]. Ultimately, 20 miRNAmRNA negative correlation pairs were identified with the involvement of liver metabolism and the PI3K–Akt signaling pathway. With regard to global genes, we addressed our particular research question using pathway analysis to highlight 19 DEGs related to the functional clusters: metabolism, including Lipid metabolism (ALOX15, ACSL5, PLA2G4C, and B4GALT6), Energy metabolism (CA9 and NDUFA4L2), Metabolism of cofactors and vitamins (TH, LIPT2, and Figure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR](/Content/uploads/2022842169/20220310100554574bd67b0bf24e35b08ba4e023cc742c.png "ure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR. (a) The 19 genes involved in liver metabolism; (b) 11 genes involved in the PI3K–Akt signaling pathway; (c) 11 putative regulatory miRNAs. Y-axis represents log2 (FC); FC (fold change) = the naringenin group/the control group. The dashed line indicated fold change data of 2.0. Values are the mean ± SD (n = 4). 3. Discussion and Conclusions The findings discussed here reveal the first detailed information regarding parallel mRNA and miRNA expression changes in HepaRG cells in response to naringenin. We performed an integrative analysis of these data including 234 DEMs and 1037 DEGs induced by naringenin, which provide global insight into the miRNA–mRNA interactions of naringenin in the regulatory mechanism. According to the gene function annotations and literature review, 19 DEGs related to metabolism were screened out. In particular, the PI3K–Akt signaling pathway was significantly enriched both in analysis of transcriptome sequencing (the third-most abundant, and ranked fourth) and integration analysis of miRNA-mRNA expression profiles (the second-most abundant, and ranked eighth) in responses to naringenin. In addition, 11 DEGs in the PI3K–Akt signaling pathway were further validated using real-time qPCR analysis. In this work, we constructed a miRNAmRNA regulatory network according to the DEMs and DEGs datasets and miRNA-targeting information. Some studies have demonstrated that the miRNA–mRNA regulatory network responds to liver damage, including hepatocellular carcinoma and oxidative stress [15]. Although several miRNA-induced RNA activation phenomena were identified [16], under most circumstances, the negative correlation between miRNAs and their target mRNAs is often considered support for miRNA targeting [17]. Ultimately, 20 miRNAmRNA negative correlation pairs were identified with the involvement of liver metabolism and the PI3K–Akt signaling pathway. With regard to global genes, we addressed our particular research question using pathway analysis to highlight 19 DEGs related to the functional clusters: metabolism, including Lipid metabolism (ALOX15, ACSL5, PLA2G4C, and B4GALT6), Energy metabolism (CA9 and NDUFA4L2), Metabolism of cofactors and vitamins (TH, LIPT2, and Figure 9. Relative mRNA and miRNA expression of the control group and the naringenin group, in respect to RNA-seq and real-time qPCR")
3. Diskisyon ak konklizyon
Konklizyon yo diskite isit la revele premye enfòmasyon detaye konsènan chanjman paralèl mRNA ak ekspresyon miRNA nan selil HepaRG an repons anaringenin. Nou te fè yon analiz entegre done sa yo ki gen ladan 234 DEM ak 1037 DEG ki te pwovoke pa naringenin, ki bay insight mondyal sou entèraksyon miRNA-mRNA nan naringenin nan mekanis regilasyon an. Dapre notasyon fonksyon jèn yo ak revizyon literati, 19 DEG ki gen rapò ak metabolis yo te depistaj. An patikilye, chemen siyal PI3K-Akt te siyifikativman anrichi tou de nan analiz de sekans transcriptome (twazyèm ki pi abondan, epi ki klase katriyèm) ak analiz entegrasyon nan pwofil ekspresyon miRNA-mRNA (dezyèm ki pi abondan, epi ki klase wityèm) an repons a naringenin. Anplis de sa, 11 DEG nan chemen siyal PI3K-Akt yo te plis valide lè l sèvi avèk analiz qPCR an tan reyèl. Nan travay sa a, nou konstwi yon rezo regilasyon miRNA-mRNA dapre ansanm done DEM ak DEG yo ak enfòmasyon miRNA-sib. Gen kèk etid ki demontre ke rezo regilasyon miRNA-mRNA reponn adomaj nan fwa, ki gen ladan karsinom epatoselilè ak estrès oksidatif [15]. Malgre ke plizyè miRNA-induit fenomèn aktivasyon RNA yo te idantifye [16], nan pifò sikonstans, korelasyon negatif ki genyen ant miRNAs ak mRNA sib yo souvan konsidere kòm sipò pou vize miRNA [17]. Alafen, yo te idantifye 20 pè korelasyon negatif miRNA-mRNA ak patisipasyon metabolis fwa ak chemen siyal PI3K-Akt.
Konsènan jèn mondyal, nou te adrese kesyon rechèch patikilye nou an lè l sèvi avèk analiz chemen pou mete aksan sou 19 DEG ki gen rapò ak grap fonksyonèl yo: metabolis, ki gen ladan metabolis lipid (ALOX15, ACSL5, PLA2G4C, ak B4GALT6), metabolis enèji (CA9 ak NDUFA4L2) , Metabolis kofaktè ak vitamin (TH, LIPT2, ak FTCD), metabolis asid amine (RRM2, AZIN2, DCT, ALAS2, ak MAT1A), biosentèz Glycan ak metabolis (HS6ST3 ak GUSB), ak metabolis idrat kabòn"(HKDC1, PCK1, UGDH, ak ABAT).Sa yo 19 DEG anrichi metabolis yo prensipalman enplike nan sansiblite ensilin, akimilasyon lipid, depo glikojèn, ak depans enèji.Etid anvan yo te rapòte ke desann-règleman nan ALOX15 nan domaj alkòl-pwovoke fwa sourit [18], CA9 nan sourit BALB/C [19], maladi fwa gra ki pa alkòl THin (NAFLD) [20], HKDC1 [21], ak regilasyon UGDH [22] ka soulaje siyifikativman estrès oksidatif, akimilasyon lipid, ak domaj nan fwa. Knockdown nan NDUFA4L2 siprime ROS akimile syon ak apoptoz nan selil karsinom epatoselilè (HCC) [23]. Silans RRM2 anpeche pwopagasyon selil NCI-H929 [24], ak ekspresyon FTCD siprime pwopagasyon selil pa ankouraje domaj ADN ak pwovoke apoptoz selil nan selil HCC [25]. Ablation nan ACSL5 amelyore sansiblite ensilin ogmante depans enèji, ak reta absòpsyon trigliserid nan sourit [26]. Sipleman DCT amelyore steatosis fwa ki asosye ak laj ak enflamasyon [27]. Fòmasyon ti gout lipid sou asid gra ak eksitasyon viris epatit C nan selil knockdown PLA2G4C te andikape [28]. Règleman anba LIPT2 anpeche sentèz asid gra [29]. Règleman ABAT [30], AZIN2 [31], B4GALT6 [32], HS6ST3 [33], ak GUSB [34] te kapab ankouraje dekonpozisyon asid gra ak glikojèn. ALAS2 twòp ekspresyon te kapab amelyore kapasite fwa ematopoyetik [35l, ak MAT1A eksprime nan epatosit kenbe eta a diferansye nan selil sa yo [36]. Konfòm ak rezilta ki dekri pi wo a, règleman nan jèn metabolik sa yo nan rezilta nou yo ka favorab pou amelyore metabolis an repons a naringenin.
Chemen siyal PI3K-Akt ka ofri endikasyon pou mekanis molekilè ki enplike nan metabolis, enflamasyon, ak estrès oksidatif [37], ki jwe yon wòl esansyèl nan repons a naringenin. Chemen siyal PI3K-Akt a gen divès efè en sou metabolis selilè atravè swa règleman dirèk transpòtè eleman nitritif ak anzim metabolik oswa kontwòl faktè transkripsyon ki kontwole ekspresyon eleman kle nan chemen metabolik yo [38,39], ki gen ladan metabolis glikoz, byosentèz. nan makromolekil, ak antretyen nan balans redox. Yo te rapòte ke PDGFRB silans inibit aktivasyon an ak pwopagasyon nan selil epatik stellate ak amelyore fibwoz fwa [40]. Anpèchman an kolaborasyon nan CSF1R ak FGFR2 espere amelyore efè antitumoral pa vize evazyon iminitè ak anjojenèz nan mikroanvironman timè a [41]. Frape ITGB4 siprime glikoliz nan fibroblast ki asosye ak kansè [42]. Knockdown jenetik nan PCK1 anpeche yon ogmantasyon asid gra-pwovoke nan flux oksidatif, estrès oksidatif, ak enflamasyon [43], ki te tou korelasyon ak chemen siyal ki gouvène pa ensilin [44]. Yo te montre ke twòp ekspresyon nan nikleyè CREB3L3 pwovoke lipoliz sistemik, ketogenesis epatik, ak sansiblite ensilin ak depans enèji ogmante [45]. Anpèchman nan CREB3L1 rapòte bloke envazyon kansè ak metastaz [46]. NFkB se yon regilatè kle nan devlopman iminitè, repons iminitè, enflamasyon, ak kansè [47]. Li te byen etabli ke siprime NFkB trans-duce siyal anti-enflamatwa ak diminye enflamasyon [48]. Nan chemen siyal PI3K-Akt, rezilta nou yo te montre ke naringenin siyifikativman desann-reglemante ekspresyon mRNA PDGFRB, PCK1, CREB3L1, ak NFkB1 ak miRNA ki gen rapò (has-miR-1306-5p ak hsa-miR{{40} }p) ke yo te siyifikativman desann-reglemante. Se poutèt sa, done nou yo sijere ke naringenin ka jwe yon wòl saluter nan anti-enflamatwa, estrès anti-oksidatif, ak metabolis amelyore atravè anpèchman nan chemen an siyal PI3K-Akt.
Etid sa a se te premye moun ki entegre analiz mRNA-seq ak miRNA-seq nan fwa a an repons a naringenin epi bay yon pèspektiv nan metabolis nan règleman naringenin. An tèm de metabolis ak chemen siyal PI3K-Akt, 11 DEM, 30 DEG ak 20 pè miRNA-mRNA bezwen plis rechèch pou aktivite naringenin yo. Gen kèk limit nan rechèch sa a; pou egzanp, efè yo depann de dòz ak tan-depandan nan naringenin nan entèraksyon miRNA-mRNA yo te toujou klè. Malgre ke yo bay miRNAs analize nan silico sijere kapasite regilasyon, fonksyon yo bezwen plis sètifye nan yon kontèks espesifik nan yon sistèm k ap viv. An rezime, nou te bay rechèch preliminè analize mRNA ak miRNA ekspresyon ak pwofil metabolis. Mekanis regilasyon pè miRNA-mRNA ta ka plis prèv posib pou anote valè nutraceutical naringenin.
4. Materyèl ak Metòd
4.1. Pwodwi chimik ak reyaktif
Yo te achte liy selil HepaRG nan men Biopredic International (Rennes, Frans). Yo te jwenn solisyon RPMI-1640 mwayen ak penisilin-streptomycin-glutamine nan Gibco (Gaithersburg, MD, USA). Serum fetal bovine (FBS) te achte nan men Corning (Auckland, New Zeland). Naringenin ak dimethyl sulfoxide (DMSO) te soti nan Sigma-Aldrich (St.Louis, MO, USA). TRIzolTM reyaktif te apwovizyone pa Thermo Fisher (Carlsbad, CA, USA). Dlo ultrapur te pirifye pa yon sistèm pirifikasyon dlo akademik Milli-O (Millipore, Bedford, MA, USA). Tout lòt reyaktif yo te komèsyalize pwodwi nan pi wo klas analyse ki disponib.
4.2. Kilti Selil HepaRG
Selil HepaRG yo te simen nan 5 × 10 * selil/cm- nan plak sis-pou epi yo te grandi nan mwayen RPMI-1640, kiltive avèk oswa san 100 uM naringenin pou 48 èdtan, epi yo te konplete ak 10 pousan FBS ak 1 pousan antibyotik. (100 U/mL penisilin ak 100 ug/mLstrepto-mycin) at37 degre nan yon imidite 5 pousan CO, enkibatè. CK-1, CK-2, CK-3, ak CK-4 refere a gwoup chèk kontwòl la; T-1, T-2, T-3, ak T-4 refere a gwoup tretman 100 μM naringenin. Nimewo 1, 2, 3, ak 4 reprezante echantiyon nan kat eksperyans endepandan repete.
4.3.Total RNA Ekstraksyon
Selil HepaRG yo te lave de fwa ak saline ki gen tanpon fosfat glas (PBS) epi yo te rekòlte ak reyaktif TRIzolTM jan manifakti a rekòmande. Thermo Scientific NanoDropTM2000 c Spectrophotometers (Wilmington, DE, USA) yo te itilize pou mezire kalite RNA ak kantite echantiyon chak dapre pwotokòl manifakti a.
4.4.RNA Sekans
Te mRNA an rich pa pèl Oligo (dT), Lè sa a, mRNA anrichi yo te fragmenté ak ranvèse-transkripsyon nan cDNA ak primè o aza pa OiaOuick PCR ekstraksyon twous (Qiagen, Venlo, Netherlands). Molekil RNA nan yon seri gwosè 18-30 nt yo te anrichi pa elektwoforèz jèl poliakrilamid. Yo te ajoute adaptè 3'ak 5' yo, Lè sa a, RNAs anrichi yo te ranvèse transkripsyon pa twous ekstraksyon QiaQuick PCR la, dapre enstriksyon manifakti a (Qiagen, Venlo, Netherlands). Pwodwi ligasyon yo te chwazi gwosè pa elektwoforèz jèl agarose. Te gen kat echantiyon nan gwoup la naringenin ak kat echantiyon nan gwoup la kontwòl. Chak echantiyon te pwodwi de bibliyotèk cDNA: youn pou mRNA-seq ak lòt la pou miRNA-seg. PCR anplifye pwodwi yo te anrichi respektivman jenere 16 bibliyotèk cDNA ak sekans lè l sèvi avèk Ⅲlumina HiSeq2500 pa Genedenovo Biotechnology Co (Guangzhou, Lachin). Done sekans RNA ak ti RNA yo te depoze nan Achiv Lekti Sekans NCBI (nimewo admisyon soti nan SRR13675952 rive nan SRR13675963).
4.5. An tan reyèl qPCR
Transkripsyon mRNA nan cDNA te fèt ak GoScriptTM Reverse Transcription System (Promega, Madison, WI, USA) soti nan 3 ug RNA total, dapre enstriksyon manifakti a. Pou analiz miRNA, cDNA-sentèz te fèt ak miRNA First Strand cDNA sentèz la (Reaksyon Tailing, Sangon Biotech, Shanghai, Lachin) soti nan 2 ugs nan RNA total.
ak GoTaq® qPCR Master Mix（Promega, Madison, WI, USA) sou yon LightCycler 480 (Roche, Mannheim, Almay), jan manifakti a rekòmande. Pwosedi sikilasyon tèmik la te kòmanse ak yon premye denaturasyon nan 95C pou 10 min. Sa a te swiv pa 45cycles nan denaturasyon pou 10s nan 95 degre, primè obligatwa pou 20s nan 60 degre, ak elongasyon pou 20 sat 72 degre. Pwosedi a te fini ak yon anplifikasyon final nan 95 degre pou 5s, 65 degre C pou 1 min, adisyon a nan yon etap koub disosyasyon, ak yon etap refwadisman. Premye yo te achte nan men Sangon Biotech (Shanghai, Lachin). Yo dekri sekans pè primè yo itilize pou validation siyati a nan Tablo 3 ak 4. Ct-valè yo te kalkile an referans a -aktin oswa U6.
4.6.Bioinformatik analiz ak estatistik
Yo te idantifye DEG ak DEM yo lè l sèvi avèk yon pake lojisyèl ki baze sou R. Valè papòt pou seleksyon DEG ak DEM se te valè q (valè p ajiste) Pi piti oswa egal a 0.05 ak chanjman pliye (FC) Pi gwo oswa egal a 2 oswa mwens. pase oswa egal a 0.5. Klasifikasyon KEGG ak GO ki gen ladan fonksyon molekilè, pwosesis byolojik, ak eleman selilè yo te itilize pou analize DEG ak DEM.
Yo prezante rezilta tès byolojik la sou fòm mwayen ± SD ki baze sou kat eksperyans endepandan nan GraphPad Prism 8.
Materyèl Siplemantè: Sa ki annapre yo disponib sou Entènèt nan https://www.mdpi.com/1422-0 067/22/5/2292/s1: Tablo S1, Idantifikasyon 1037 mRNA ki eksprime diferan an repons a naringenin; Tablo S2, Idantifikasyon 234 miRNA ki eksprime diferansye an repons a naringenin; Tablo S3, Idantifikasyon 5607 pè miRNA-mRNA negatif an repons a naringenin, ak patisipasyon 216 DEM ak 681 DEG nan total
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