郭云鵬,譚皓云,郭宏,符夢云,李新,胡德寶,張林林,丁向彬,郭益文
Lnc721靶向調(diào)控MMP9對牛骨骼肌衛(wèi)星細(xì)胞增殖分化的影響
郭云鵬,譚皓云,郭宏,符夢云,李新,胡德寶,張林林,丁向彬,郭益文
天津農(nóng)學(xué)院動物科學(xué)與動物醫(yī)學(xué)學(xué)院/天津市農(nóng)業(yè)動物繁育與健康養(yǎng)殖重點實驗室,天津 300384
【背景】肌肉系統(tǒng)是維持動物體生存生長的重要基礎(chǔ),在哺乳動物肌肉中,將近一半的肌肉為骨骼肌,骨骼肌通過細(xì)胞繁育增殖到遷移融合,逐步形成了具有收縮能力的附著在骨骼上的成熟肌束。在機(jī)體內(nèi),骨骼肌不僅參與動物生長,也參與呼吸、代謝等生理活動。目前許多研究表明,lncRNA具有調(diào)控肌肉生長發(fā)育的作用,是影響骨骼肌功能、疾病的關(guān)鍵因子。但由于lncRNA作用機(jī)制復(fù)雜,方式多樣,在物種間保守型很低,故不同種屬動物 lncRNA之間作用關(guān)系不大,且大多數(shù)研究存在于人和小鼠等生物體內(nèi),而對牛肌肉生長影響的研究內(nèi)容極少。近年來,人們發(fā)現(xiàn)lncRNA會與某些靶基因相互作用,進(jìn)而調(diào)控肌肉細(xì)胞的發(fā)生過程?!灸康摹吭谇捌诓杉煌慢g不同組織部位的牛肌肉,進(jìn)行高通量測序,獲得高表達(dá)差異的lncRNA,在對其調(diào)控成肌過程進(jìn)行機(jī)制研究的基礎(chǔ)上,探究長鏈非編碼RNA lnc721與其靶基因MMP9的互作關(guān)系,以及MMP9對牛骨骼肌細(xì)胞生長發(fā)育的影響,以期為牛骨骼肌發(fā)育調(diào)控機(jī)制的研究提供參考?!痉椒ā壳捌谠囼灠l(fā)現(xiàn)干擾lnc721對牛骨骼肌衛(wèi)星細(xì)胞增殖具有正調(diào)控作用,且對其分化具有負(fù)調(diào)控作用。為了進(jìn)一步探究lnc721 對牛肌肉發(fā)育的調(diào)控通路。分別在牛骨骼肌衛(wèi)星細(xì)胞的分化期設(shè)置了3 個干擾lnc721牛骨骼肌衛(wèi)星細(xì)胞組,與3個對照組采用 NGS 技術(shù)進(jìn)行轉(zhuǎn)錄組測序,以期獲得lnc721 差異靶基因,進(jìn)一步研究lnc721 對牛骨骼肌發(fā)育的調(diào)控通路。根據(jù)篩選結(jié)果以及qRT-PCR的驗證結(jié)果,試驗選取MMP9作為lnc721的靶基因,并通過CatRAPID網(wǎng)站對lnc721與MMP9結(jié)合能力進(jìn)行預(yù)測。設(shè)計并合成lnc721及MMP9干擾序列,轉(zhuǎn)染至牛骨骼肌衛(wèi)星細(xì)胞中,通過qRT-PCR、Western blot技術(shù)在mRNA水平與蛋白質(zhì)水平分析下調(diào)lnc721對MMP9表達(dá)情況的影響。并且通過下調(diào)MMP9,采用qRT-PCR、Western blot與EdU技術(shù)檢測增殖標(biāo)志因子Ki67、Pax7以及分化標(biāo)志因子MyHC與MyOG的表達(dá)情況,以反映下調(diào)MMP9對牛骨骼肌衛(wèi)星細(xì)胞生長發(fā)育的影響?!窘Y(jié)果】CatRAPID結(jié)果顯示,lnc721與MMP9結(jié)合傾向為0.75,共有9個結(jié)合位點,二者存在互作結(jié)合。干擾lnc721之后,采用qRT-PCR分析,結(jié)果表明在細(xì)胞增殖期下調(diào)lnc721可以極顯著抑制MMP9表達(dá)(<0.01),而在分化期則極顯著促進(jìn)MMP9的表達(dá)(<0.01),證實了MMP9為lnc721調(diào)控牛骨骼肌衛(wèi)星細(xì)胞互作的靶基因。進(jìn)而下調(diào)MMP9,檢測對增殖期細(xì)胞的影響,發(fā)現(xiàn)Ki67的mRNA水平表達(dá)極顯著上調(diào)(<0.01),Pax7蛋白表達(dá)量顯著上調(diào)(<0.05),EdU觀察下調(diào)MMP9后的陽性細(xì)胞率也明顯升高。同樣檢測下調(diào)MMP9之后對細(xì)胞分化期的影響,發(fā)現(xiàn)下調(diào)MMP9后鏡下觀察發(fā)現(xiàn)下調(diào)MMP9會抑制肌管形成,同時MyHC的mRNA和蛋白表達(dá)量極顯著下降(<0.01), MyoG蛋白表達(dá)量顯著下調(diào)(<0.05)。【結(jié)論】lnc721與MMP9相互結(jié)合。在細(xì)胞增殖期干擾lnc721極顯著抑制MMP9表達(dá),而在分化期促進(jìn)MMP9的表達(dá),且干擾MMP9可促進(jìn)細(xì)胞增殖并抑制分化。證明lnc721靶向MMP9調(diào)控牛骨骼肌衛(wèi)星細(xì)胞的發(fā)育。
lnc721;MMP9;牛骨骼肌衛(wèi)星細(xì)胞;增殖;分化
【研究意義】牛骨骼肌作為人類優(yōu)質(zhì)蛋白質(zhì)來源之一,其發(fā)生發(fā)育是一個極其精準(zhǔn)且十分繁復(fù)的調(diào)控過程[1]。近年來有報道lncRNA會參與調(diào)控肌肉生長發(fā)育的過程,本文主要研究lncRNA對牛骨骼肌發(fā)生發(fā)育的調(diào)控機(jī)制,以期改善牛肉質(zhì)量,為市場提供優(yōu)質(zhì)牛肉?!厩叭搜芯窟M(jìn)展】Wang等[2]發(fā)現(xiàn)lncRNA Sirt1AS通過保護(hù)Sirt1 mRNA(細(xì)胞周期抑制劑的抑制因子)免受miR-34a介導(dǎo)的降解,促進(jìn)成肌細(xì)胞增殖。同樣的lncRNA Mir22hg在肌母細(xì)胞分化過程中顯著上調(diào),并在骨骼肌中高度表達(dá)。Li等[3]驗證了Mir22hg在體外可以促進(jìn)肌母細(xì)胞分化。并且在機(jī)制上發(fā)現(xiàn)Mir22hg可以抑制其靶基因組蛋白脫乙酰酶4(HDAC4),從而增加下游肌細(xì)胞增強(qiáng)因子2C(MEF2C),最終促進(jìn)肌母細(xì)胞分化?!颈狙芯壳腥朦c】本研究團(tuán)隊先前的研究已知,lnc721 的長度為 311 bp,在 NCBI 對比發(fā)現(xiàn)lnc721位于18號染色體,編號為OX344707.1,是一個未研究的 lncRNA。通過建立體外的lnc721干擾模型,并分析相關(guān)標(biāo)志因子,發(fā)現(xiàn)干擾lnc721之后,肌肉細(xì)胞增殖期標(biāo)志因子Pax7與Ki67表達(dá)水平均顯著上調(diào),而分化期標(biāo)志因子MyHC與MyoG表達(dá)水平均極顯著降低。證明lnc721參與調(diào)控了肌肉細(xì)胞的生長發(fā)育,但對其作用的分子機(jī)制并未探明。鑒于此,本研究選取前期研究的lnc721,通過抑制其表達(dá)后RNA測序獲得差異基因,篩選出與肌肉發(fā)育相關(guān)的差異基因,隨后根據(jù)剩余候選基因在測序結(jié)果中的差異表達(dá)倍數(shù)的高低,最終確定MMP9為最適基因。MMP9是MMPs家族明膠酶的成員中以被糖化的形式存在的酶[4-5]。基質(zhì)金屬蛋白酶(MMPs)是一個龐大的家族[6-7]。自1962年MMPs被發(fā)現(xiàn)開始[8],MMPs家族的28個成員[9]調(diào)控骨骼肌的功能被不斷發(fā)掘出來。有研究表明,MMPs通過促進(jìn)細(xì)胞遷移和融合,在成肌細(xì)胞融合中發(fā)揮著重要的作用[10]。MMP13[11]、MMP14[12]等MMPs家族的成員也被證實會影響成肌細(xì)胞的增殖分化?!緮M解決的關(guān)鍵問題】本試驗選取前期篩選的MMP9進(jìn)行試驗,在體外建立MMP9的干擾模型,觀察MMP9對于骨骼肌衛(wèi)星細(xì)胞增殖分化的影響,并探究MMP9與lnc721的互作結(jié)合關(guān)系,以期為研究MMP9在肌肉中發(fā)揮的作用提供參考。
1.1.1 細(xì)胞系 本研究所用的細(xì)胞樣品為原代牛骨骼肌衛(wèi)星細(xì)胞,細(xì)胞的分離培養(yǎng)凍存過程均由天津市農(nóng)業(yè)動物繁育與健康養(yǎng)殖重點實驗室完成。
1.1.2 試驗時間及地點 試驗地點為天津市農(nóng)業(yè)動物繁育與健康養(yǎng)殖重點實驗室,試驗時間為2021年至2022年12月。
1.1.3 主要試劑及儀器 Magna RIP? RNA-Binding Protein Immunoprecipitation Kit購自Sigma-Aldrich公司;PAGE凝膠快速制備試劑盒購自上海雅酶生物公司;MMP9抗體購自上海Abmart公司;Ribo? ASO-lncRNA與Cell-Light? EdU Apollo 567 In Vitro Kit均購自廣州銳博生物科技有限公司;0.25%胰蛋白酶、DAPI溶液、4%多聚甲醛與PMSF均購自北京Solarbio公司;ECL超敏發(fā)光液購自上海生物工程有限公司;提RNA試劑盒購自北京艾德萊公司;Lipofectamine? 3000轉(zhuǎn)染試劑購自Invitrogen公司;DMEM 高糖培養(yǎng)基購自Hyclone公司;Gel Extraction Kit、BCA 蛋白定量試劑盒與PBS 緩沖液均購自北京康為世紀(jì)公司;PrimeScript II 1st Strand cDNA Synthesis Kit購自大連Takara公司;正常山羊血清、鼠抗 MHC與鼠抗Pax7均購自DSHB公司;胎牛血清(FBS)、馬血清(HS)與opti-MEM培養(yǎng)基均購自Gibco公司;鼠抗GAPDH購自北京中杉金橋公司;羊抗鼠二抗和羊抗兔二抗均購自北京中杉金橋公司;NE-PER Nuclear and CytoplasmicExtraction Reagents Kit與蛋白Marker均購自Thermo-Scientific公司;All-in-One? qPCR Mix購自GeneCopoeia公司。
脫色搖床購自IKA公司;離心機(jī)購自Eppendorf公司;電泳儀購自PowerPac Basic公司;普通PCR儀與LightCycle 96 實時熒光定量PCR儀均購自Roche公司;二氧化碳培養(yǎng)箱購自SANYO公司;熒光顯微鏡購自Leica公司;高壓滅菌鍋購自TOMY公司;Nano-Drop ND 2000cSupectrophotometer購自Thermo公司;垂直電泳槽與ChemiDoc? Imaging System均購自Bio-Rad公司;酶標(biāo)儀購自Thermo- scientific公司。
1.2.1 MMP9的生物信息學(xué)篩選 構(gòu)建lnc721干擾模型文庫,將原始數(shù)據(jù)進(jìn)行分類統(tǒng)計,然后利用Cutadapt去除接頭序列及低于Q20的Reads,并對堿基質(zhì)量及含量進(jìn)行評估。通過HTSeq將過濾后的序列與牛全基因組進(jìn)行比對,統(tǒng)計比對到每一個基因上Read Count值,并最終還原出轉(zhuǎn)錄本序列,接下來與已知的mRNA和LncRNA轉(zhuǎn)錄本進(jìn)行比較,通過FPKM將表達(dá)量進(jìn)行標(biāo)準(zhǔn)化處理,進(jìn)一步分析基因間表達(dá)水平相關(guān)性。使用DESeq軟件包分析PCA主成分,通過繪制火山圖進(jìn)一步分析表達(dá)差異結(jié)果。根據(jù)測序結(jié)果以及qRT-PCR結(jié)果最終選取MMP9為候選差異基因,并采用catRAPID在線軟件預(yù)測lnc721與MMP9的結(jié)合能力及結(jié)合位點。
1.2.2 培養(yǎng)基的制備 增殖培養(yǎng)基為10%胎牛血清(FBS)+10%DMEM,分化培養(yǎng)基為5%孕馬血清+95%DMEM。
1.2.3 細(xì)胞培養(yǎng)及誘導(dǎo)分化 復(fù)蘇原代細(xì)胞后使用增殖培養(yǎng)基進(jìn)行原代、傳代培養(yǎng),待細(xì)胞密度適宜時進(jìn)行轉(zhuǎn)染處理,24 h后將培養(yǎng)基更換為分化培養(yǎng)基,每12 h觀察一次,直至肌管形成。
1.2.4 細(xì)胞轉(zhuǎn)染 待增殖期與分化期細(xì)胞密度約為60%時即可開始轉(zhuǎn)染,分別將3個合成的干擾序列與對照轉(zhuǎn)染至細(xì)胞當(dāng)中,每組設(shè)置3個生物學(xué)重復(fù),3條si-MMP9與一條lnc721干擾序列均由廣州瑞博生物技術(shù)有限公司設(shè)計并合成:si-bta-MMP9_001(si-1):5′-AAGAAAUGCAAGCGGUUCC-3′;si-bta-MMP9_002(si-2):5′-AUGUCGUGCGUGCUAAUGG-3′;si-bta- MMP9_003(si-3):5′-UUGUCUUUGUCGAAGUUGG -3′;ASO-bta-lnc721_001:5′-TTTCAAGCAGCCAGAC AAAG-3′。
1.2.5 細(xì)胞總RNA的提取與cDNA的合成 分別收取增殖期(GM)與分化第一天(DM1)、第二天(DM2)、第三天(DM3)4個時期的細(xì)胞總RNA,利用HiFiScript cDNA Synthesis Kit進(jìn)行cDNA第一鏈的合成,反應(yīng)條件為 42 ℃ 15 min,84 ℃ 5 min。反應(yīng)結(jié)束后,加無酶水稀釋5倍,裝入袋中標(biāo)記批次及樣品,置于-20 ℃冰箱保存?zhèn)溆?,為后續(xù)使用qRT-PCR技術(shù)提供原材料。
1.2.6 qRT-PCR 采用qRT-PCR技術(shù)檢測lnc721表達(dá)水平、MMP9表達(dá)水平得到干擾效率并檢測增殖期和分化期標(biāo)志因子(Pax7、Ki67與MyHC、MyoG)的mRNA表達(dá)水平。引物序列(表1)均在NCBI中查詢得到,PCR反應(yīng)體系共20 μL:2×All-in-One? qPCR Mix 10.0 μL;Forward primer (F) 0.5 μL;Reverse primer (R) 0.5 μL;cDNA template 2.0 μL;RNase Free dH2O 7.0 μL,PCR程序:預(yù)變性10 min,95 ℃變性10 s,60 ℃退火20 s,72 ℃延伸15 s,共40個循環(huán)。結(jié)果采用2-△△Ct方法計算。
1.2.7 RIP 采用Magna RIP? RNA-Binding Protein Immunoprecipitation Kit 進(jìn)行RIP 試驗。將細(xì)胞培養(yǎng)至分化第3天,加入預(yù)混的RIP裂解液冰上孵育5 min后-80 ℃保存。磁珠處理及免疫沉淀按照說明書操作后用Trizol法提RNA。
表1 qRT-PCR 引物序列
1.2.8 EdU檢測 試驗操作按照Cell-Light? EdU Apollo 567 In Vitro Kit說明書進(jìn)行,接種至96孔板的細(xì)胞分為試驗組與空白組并設(shè)置3個生物學(xué)重復(fù)。在熒光顯微鏡下觀察拍照并計數(shù)。
1.2.9 Western blotting檢測 前期使用RIPA蛋白裂解液處理細(xì)胞,提取細(xì)胞蛋白后采用BCA法通過酶標(biāo)儀測量蛋白濃度。采用Western blotting分別檢測內(nèi)參與增殖期標(biāo)志因子(Pax7、Ki-67)和分化期標(biāo)志因子(MyHC、MyoG)。SDS-PAGE參數(shù)設(shè)定為80V 30 min,120V 45 min;轉(zhuǎn)膜參數(shù)設(shè)置為300mA 2 h。轉(zhuǎn)膜后在適宜位置裁剪條帶,于4℃冰箱中孵育一抗,12 h后使用TBST清洗3次后孵育相應(yīng)二抗,1 h后使用TBST清洗3次,上機(jī)滴加ELC發(fā)光液曝光。
1.2.10 統(tǒng)計分析 采用SPSS 17.0軟件進(jìn)行統(tǒng)計分析。EdU陽性細(xì)胞率用卡方檢驗分析,qRT-PCR采用GAPDH 作為內(nèi)參,2-ΔΔCt方法計算基因相對表達(dá)水平,t檢驗做差異顯著性分析。<0.01表示差異極顯著(**),<0.05表示差異顯著(*),>0.05表示差異不顯著(N.S.),Western blotting條帶的量化采用Image J 軟件分析。
CPC網(wǎng)站預(yù)測lnc721編碼潛能為-1.33129(圖1-A)。核質(zhì)分離試驗表明lnc721主要定位于細(xì)胞質(zhì)內(nèi)(圖1-B)。并且通過qRT-PCR技術(shù)驗證lnc721組織表達(dá),發(fā)現(xiàn)其在3月齡胎牛、6月齡胎牛、9月齡胎牛及成年牛體內(nèi)的各種組織中均有表達(dá)(圖2),在6月齡胎牛組織中表達(dá)量相對較低(圖2-B),而在9月齡初生牛犢肌肉組織內(nèi)表達(dá)量較高(圖2-C)。表明lnc721主要在9月齡胎牛肌肉組織中高表達(dá)。
A:CPC 網(wǎng)站蛋白編碼能力預(yù)測;B:lnc721 亞細(xì)胞定位檢測 A: Prediction of CPC website protein coding ability; B: Subcellular localization detection of lnc721
A:3月齡胎牛;B:6月齡胎牛;C:9月齡胎牛;D:成年牛 A: 3-month-old fetus; B: 6-month-old fetus; C: 9-month-old fetus; D: Adult cattle
實驗室前期已研究發(fā)現(xiàn)干擾lnc721對牛骨骼肌衛(wèi)星細(xì)胞增殖具有正調(diào)控作用且對其分化具有負(fù)調(diào)控作用。為了進(jìn)一步探究lnc721對牛肌肉發(fā)育的調(diào)控機(jī)制,通過對干擾lnc721后轉(zhuǎn)錄組測序結(jié)果進(jìn)行過濾、質(zhì)量評估、表達(dá)量以及表達(dá)差異分析,以|log2(Flodchange)| ≥1以及<0.05為篩選條件篩選出差異基因并作出火山圖。采用NGS轉(zhuǎn)錄組測序,尋找lnc721調(diào)控的下游靶基因。結(jié)果發(fā)現(xiàn),共有499個上調(diào)表達(dá)的差異基因,93個下調(diào)表達(dá)的差異基因(圖3)。
為驗證測序結(jié)果的準(zhǔn)確性,在表達(dá)倍數(shù)較高的基因中選取了5個上調(diào)與4個下調(diào)差異基因,采用qRT-PCR技術(shù)對其mRNA水平進(jìn)行驗證,結(jié)果如圖4所示,檢測9個差異基因,其中7個基因差異顯著且與測序結(jié)果一致,有2個基因差異不顯著,表明測序數(shù)據(jù)的可靠性。進(jìn)一步對驗證的差異靶基因加以分析,并根據(jù)文獻(xiàn)及基因功能分析篩選出與肌肉發(fā)育相關(guān)的差異基因,根據(jù)基因差異表達(dá)倍數(shù)的高低,最終篩選MMP9為lnc721候選靶基因。
圖3 轉(zhuǎn)錄組測序基因表達(dá)差異分析火山圖
A:上調(diào)表達(dá)基因的qRT-PCR檢測;B:下調(diào)表達(dá)基因的qRT-PCR檢測
網(wǎng)絡(luò)數(shù)據(jù)計算預(yù)測lnc721與MMP9的結(jié)合能力,結(jié)果指出lnc721與MMP9共有9個結(jié)合位點,結(jié)合傾向(RBP propensity)為0.75,(圖5),這一結(jié)果證實了差異高表達(dá)基因MMP9可作為lnc721互作蛋白進(jìn)行后續(xù)功能性研究分析。
圖5 catRAPID預(yù)測lnc721與MMP9的結(jié)合位點
通過RIP測定MMP9免疫沉淀物上lnc721的富集程度來證明二者的結(jié)合關(guān)系。以10% input為陽性對照,以與MMP9一抗同種屬的IgG為陰性對照進(jìn)行實驗。如圖6所示,lnc721富集于MMP9免疫沉淀物中,證明了lnc721與MMP9存在互作結(jié)合關(guān)系。
圖6 RIP檢測MMP9與lnc721相互關(guān)系
經(jīng)過上述試驗已經(jīng)確定lnc721與MMP9存在互作關(guān)系,為了進(jìn)一步研究二者的關(guān)系,干擾lnc721檢測MMP9的表達(dá)情況。將lnc721特異的siRNA序列轉(zhuǎn)染至細(xì)胞中,收取增殖期和分化期的mRNA與蛋白質(zhì),分別采用qRT-PCR與Western blotting技術(shù)檢測MMP9的表達(dá)情況。結(jié)果發(fā)現(xiàn)在下調(diào)lnc721之后,qRT-PCR結(jié)果顯示出在增殖期MMP9的表達(dá)被極顯著抑制(<0.01),而分化期截然相反,MMP9的表達(dá)被極顯著促進(jìn)(<0.01)(圖7);Western blotting結(jié)果表明下調(diào)lnc721之后,對于MMP9在增殖期與分化期表達(dá)情況的影響均不顯著,這可能與不同時期蛋白翻譯水平變化相關(guān)(圖7-B、C)。
為了探究MMP9對牛骨骼肌生長發(fā)育的影響,設(shè)計合成3條干擾序列轉(zhuǎn)染到細(xì)胞中,培養(yǎng)至增殖期收取RNA,篩選出最佳干擾序列(圖8-A),qRT-PCR結(jié)果表明3條干擾序列均極顯著降低了MMP9的表達(dá)量(<0.01),證明3條干擾序列均構(gòu)建成功。由于si-1干擾效果最佳,因此后續(xù)將si-1轉(zhuǎn)染進(jìn)細(xì)胞中檢測增殖期標(biāo)志因子Pax7和Ki67的表達(dá)情況。結(jié)果如圖8-B、D所示,干擾MMP9后Ki67的mRNA表達(dá)極顯著上調(diào)(<0.01),而Pax7蛋白表達(dá)量沒有顯著變化。下調(diào)MMP9后采用EdU技術(shù)觀察陽性細(xì)胞,發(fā)現(xiàn)干擾組陽性細(xì)胞率也顯著高于對照組(圖8-C)。
A:下調(diào)lnc721后MMP9增殖期與分化期mRNA水平變化;B:增殖期下調(diào)lnc721,MMP9蛋白表達(dá)水平檢測;C:分化期下調(diào)lnc721, MMP9蛋白表達(dá)水平檢測
A:定量PCR篩選牛骨骼肌衛(wèi)星細(xì)胞中MMP9干擾siRNA;B:qRT-PCR檢測增殖標(biāo)志因子在mRNA表達(dá)水平;C:EdU染色增殖陽性細(xì)胞數(shù) D:Western blotting檢測增殖標(biāo)志因子 Pax7蛋白表達(dá)量
將MMP9的干擾序列轉(zhuǎn)染進(jìn)細(xì)胞中,收取細(xì)胞分化3d(DM3)的RNA和蛋白質(zhì),收取前鏡下觀察發(fā)現(xiàn)下調(diào)MMP9會抑制肌管形成(圖9-A)。檢測分化期標(biāo)志因子MyHC和MyoG的mRNA與蛋白質(zhì)表達(dá)情況。下調(diào)MMP9后,MyHC的mRNA水平表達(dá)量顯著下降(<0.01)(圖9-B),MyHC的蛋白表達(dá)量極顯著下降(<0.01),MyoG蛋白表達(dá)量顯著下調(diào)(<0.05)(圖9-C)。
A:轉(zhuǎn)染ASO-MMP9后,倒置顯微鏡觀察分化期細(xì)胞生長狀態(tài)(200×);B:細(xì)胞分化標(biāo)志因子mRNA表達(dá)水平;C:Western blotting檢測分化標(biāo)志因子
一般mRNA和蛋白質(zhì)的表達(dá)水平應(yīng)該有一定的比例關(guān)系,但是二者不一定完全一致,這可能與mRNA翻譯過程的調(diào)控與蛋白質(zhì)的降解有關(guān)。真核生物的翻譯包括起始、延伸和終止[13-14],翻譯的起始主要通過m7GpppN以帽蓋依賴的形式發(fā)生[15],但是某些胞內(nèi)核糖體會繞過該結(jié)構(gòu)直接結(jié)合在起始密碼子上[16],即KOZAK序列[17]。影響翻譯起始的因素有幾個,例如,核糖體可以與位于mRNA 5'UTR中的上游開放閱讀框(uorf)結(jié)合,并以競爭的方式改變主要開放閱讀框的翻譯水平;二級結(jié)構(gòu)也通過減慢核糖體的傳代來影響翻譯,而次優(yōu)的Kozak序列會對起始產(chǎn)生負(fù)面影響;翻譯延伸過程中三種主要的伸長因子(eEF1A, eEF1B和eEF2)在響應(yīng)多種刺激時通過磷酸化/去磷酸化進(jìn)行調(diào)節(jié)[18]。延伸率還受到起始速率的變化、密碼子的選擇以及相應(yīng)的tRNA豐度的影響。再有蛋白質(zhì)降解也有可能導(dǎo)致二者表達(dá)水平不一致,蛋白質(zhì)降解是高度特異性和嚴(yán)格調(diào)控的,它包括兩個主要系統(tǒng)。溶酶體降解和泛素介導(dǎo)的內(nèi)吞作用、胞飲作用、吞噬作用和自噬作用[19]。本試驗中干擾lnc721之后,在增殖期與分化期檢測了MMP9的表達(dá)水平,qRT-PCR結(jié)果顯示增殖期MMP9的mRNA表達(dá)水平極顯著降低(<0.01),分化期MMP9的mRNA表達(dá)水平極顯著升高(<0.01),然而Western blotting結(jié)果卻顯示干擾lnc721之后MMP9的蛋白質(zhì)水平卻沒有顯著的變化。同樣的干擾MMP9之后。分化期標(biāo)志因子MyOG的mRNA表達(dá)量沒有顯著變化而蛋白質(zhì)水平顯著下調(diào)(<0.05)。前者有可能是mRNA發(fā)生了轉(zhuǎn)錄后修飾,而后者發(fā)生了蛋白質(zhì)的降解。
隨著學(xué)者對骨骼肌生長發(fā)育調(diào)控方式的深入研究,越來越多的lncRNA與靶蛋白的協(xié)同調(diào)控機(jī)理被發(fā)掘出來。Wang等[20]發(fā)現(xiàn)lnccDLEU2的過表達(dá)會促進(jìn)SEPP1mRNA與蛋白水平,來抑制肌肉分化和再生。同樣的,Cai等[21]發(fā)現(xiàn)過表達(dá)lncORA可以抑制肌肉分化,并且lncORA可以與IGF2BP2相互作用,抑制肌肉生長發(fā)育。CHEN等[22]發(fā)現(xiàn)被敲低干擾和過表達(dá)lncKBTBD10,均會抑制牛骨骼肌衛(wèi)星細(xì)胞的增殖和分化。同時檢測lncKBTBD10的鄰近基因KBTBD10是否參與肌生成,發(fā)現(xiàn),無論 lncKBTBD10 被敲低還是過表達(dá),KBTBD10 的蛋白水平均降低。研究認(rèn)為lncKBTBD10可以抑制KBTBD10,進(jìn)而影響牛骨骼肌的生成[23]。本研究前期發(fā)現(xiàn)lnc721正向調(diào)控骨骼肌的增殖,負(fù)調(diào)控牛骨骼肌的分化。在此基礎(chǔ)上,通過lnc721轉(zhuǎn)錄組測序鎖定了候選下游基因MMP9,并通過RIP實驗驗證了lnc721與MMP9相互結(jié)合。通過qRT-PCR檢測干擾lnc721對MMP9的影響,結(jié)果發(fā)現(xiàn)在細(xì)胞分化期干擾lnc721后顯著促進(jìn)了MMP9的表達(dá),推測lnc721可能通過MMP9影響肌衛(wèi)星細(xì)胞生長發(fā)育。
MMP9是最復(fù)雜的基質(zhì)金屬蛋白酶形式之一。MMP9具有降解細(xì)胞外基質(zhì)(ECM)成分的能力,在病理生理功能中具有重要作用,MMP9的過表達(dá)和失調(diào)與各種疾病有關(guān)[24]。比如心力衰竭[25]、胃癌[26]、肝癌[27]等。研究發(fā)現(xiàn)過表達(dá)腫瘤壞死因子相關(guān)的弱凋亡誘導(dǎo)劑(TWEAK)會增加基質(zhì)金屬蛋白酶(MMP)在骨骼肌細(xì)胞肌管中的表達(dá)。TWEAK轉(zhuǎn)基因小鼠的肌纖維中MMP9的水平也較高。TWEAK增加了核因子-kappaB(NF-kappaB)信號通路的激活。干擾NF-kappaB 活性阻止了TWEAK誘導(dǎo)的肌管中 MMP9 的產(chǎn)生。此外,據(jù)大量研究報道,MMP9對骨骼肌發(fā)育也具有調(diào)控作用[28]。LI等[29]已在mdx小鼠中研究了MMP9在肌肉功能中的合作相互作用,其中MMP9活性的抑制導(dǎo)致骨骼肌結(jié)構(gòu)惡化,壞死和纖維化減少,并對骨骼肌再生具有補(bǔ)救作用。同樣的,KHERIF等[30]在肌營養(yǎng)不良蛋白缺陷MDX小鼠的骨骼肌中觀察到MMP9的上調(diào)。此外,據(jù)報道,MMP9能夠處理β-肌營養(yǎng)不良聚糖,并通過DMD和肌糖病患者骨骼肌中的肌營養(yǎng)不良聚糖復(fù)合物破壞ECM與細(xì)胞膜之間的連接,從而干擾肌肉細(xì)胞的生長發(fā)育[31-32]。CHENETTE等[33]研究敲除小鼠auf1基因后,骨骼肌會隨小鼠年齡的增長而萎縮,在auf1敲除小鼠中阻斷MMP9活性可恢復(fù)骨骼肌修復(fù)。ZIMOWSKA等[34]在擠壓損傷大鼠SOL模型中證實了MMP9抑制對肌肉再生正向調(diào)節(jié)的作用,特別是對減少纖維化的正向調(diào)節(jié)作用。本研究重點關(guān)注MMP9與骨骼肌生長發(fā)育之間的關(guān)系,進(jìn)一步驗證MMP9是否對牛骨骼肌衛(wèi)星細(xì)胞具有調(diào)控作用。使用MMP9的siRNA干擾MMP9后,qRT-PCR結(jié)果與WB結(jié)果均顯示顯著促進(jìn)了細(xì)胞的增殖,并抑制細(xì)胞分化,此結(jié)果與lnc721下調(diào)會使細(xì)胞分化受阻結(jié)果相一致。故而lnc721與MMP9對牛肌衛(wèi)星細(xì)胞調(diào)控趨勢一致,lnc721可以結(jié)合MMP9蛋白,共同作用對細(xì)胞增殖有正向調(diào)控的作用,對細(xì)胞分化過程具有負(fù)向調(diào)控的作用。綜合前期研究結(jié)果,本試驗證明了lnc721與MMP9結(jié)合參與了肌肉細(xì)胞發(fā)育初期的調(diào)控,揭示了lnc721調(diào)控牛肌衛(wèi)星細(xì)胞增殖分化的機(jī)制。
干擾lnc721在細(xì)胞增殖期極顯著抑制MMP9表達(dá),而在分化期促進(jìn)MMP9的表達(dá),且干擾MMP9可升高增殖期標(biāo)志因子Pax7與Ki67D的表達(dá)水平并下調(diào)分化標(biāo)志因子MyHC與MyoG的表達(dá)水平。證明了MMP9具有可負(fù)向調(diào)控細(xì)胞增殖并正向調(diào)控細(xì)胞分化的作用。結(jié)合lnc721與MMP9的調(diào)控關(guān)系,可以得出結(jié)論lnc721促進(jìn)MMP9的表達(dá)進(jìn)而對細(xì)胞的增殖起到抑制作用,對細(xì)胞分化起到促進(jìn)作用,lnc721與MMP9互作結(jié)合共同調(diào)控牛骨骼肌衛(wèi)星細(xì)胞的發(fā)育。
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LNC721 Targeted Regulation MMP9 Affects Bovineskeletal Muscle Satellite Cell Proliferation and Differentiation
GUO YunPeng, TAN HaoYun, GUO Hong, FU MengYun, LI Xin, HU DeBao, ZHANG LinLin, DING XiangBin, GUO YiWen
School of Animal Science and Animal Medicine, Tianjin Agricultural College/Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, Tianjin 300384
【Background】The muscular system is an important basis for maintaining the survival and growth of the animal body. In the mammalian muscle, nearly half of the muscle is skeletal muscle, skeletal muscle through cell multiplication to migration fusion, and gradually formed a mature muscle bundle attached to the bone with contractile ability. In the animal body, skeletal muscle is not only involved in animal growth, but also in physiological activities, such as respiration and metabolism. At present, many studies have shown that lncRNA had the effect of regulating muscle growth and development, and was a key factor affecting skeletal muscle function and diseases. However, due to the complex mechanism of action of lncRNA, the variety of methods, and the very low conservation type between species, the relationship between lncRNAs of different species is not large, and most of the studies exist in organisms such as humans and mice, and there are few studies on the effect on bovine muscle growth. In recent years, it has been discovered that lncRNAs interact with certain target genes to regulate the process of muscle cell genesis. In the early stage of this experiment, the high-throughput sequencing was performed by collecting bovine muscles of different tissues of different months, the lncRNA with high expression difference was obtained, and the mechanism of its regulation of the myogenic process was studied. 【Objective】This study aimed to explore the interaction between long non-coding RNA lnc721 and its target gene MMP9, and the effects of MMP9 on the growth and development of bovine skeletal muscle cells, in order to provide a reference for the study of the regulatory mechanism of bovine skeletal muscle development.【Method】 The previous experiments showed that interference with lnc721 had a positive regulatory effect on the proliferation of bovine skeletal muscle satellite cells and negatively regulated its differentiation. Three groups of interfering lnc721 bovine skeletal muscle satellite cells and three control groups were set up to sequence transcriptome using NGS technology in the differentiation stage of bovine skeletal muscle satellite cells, in order to obtain the lnc721 differential target gene and to further study the regulatory pathway of lnc721 on bovine skeletal muscle development. According to the screening results and the verification results of qRT-PCR, MMP9 was selected as the target gene of lnc721, and the binding ability of lnc721 and MMP9 was predicted through the CatRAPID website. The interference sequences of lnc721 and MMP9 were designed and synthesized, transfected into bovine skeletal muscle satellite cells, and the effect of lnc721 on MMP9 expression was down-regulated by qRT-PCR and Western blot technology at the mRNA level and protein level. After down-regulation of MMP9, qRT-PCR, Western blot and EdU were used to detect the expression of proliferation marker factors Ki67 and Pax7 and differentiation marker factors MyHC and MyOG, so as to reflect the effect of down-regulation of MMP9 on the growth and development of bovine skeletal muscle satellite cells. 【Result】 MMP9 was identified as a target gene for lnc721 to regulate the interaction of bovine skeletal muscle satellite cells. They were found to interact and bind to each other by RIP. After interfering with lnc721, qRT-PCR analysis showed that down-regulation of lnc721 significantly inhibited MMP9 expression (<0.01) during the proliferative phase, while it significantly promoted MMP9 expression (<0.01) during the differentiation phase. Downregulation of MMP9 resulted in a highly significant upregulation of Ki67 mRNA level expression in proliferating cells (<0.01) and the Pax7 protein expression (?0.05). As also, it could significant increase the positive cell rate of EdU labled cells. At the stage of cell differentiation, the downregulation of MMP9 could inhibit muscle myotube formation. On the other hand, the mRNA and protein expressions of MyHC were significantly decreased (<0.01); MyoG protein expression was significantly down-regulated (<0.05). 【Conclusion】 lnc721 could bind to MMP9. Interfering with lnc721 was significantly inhibited MMP9 expression during the proliferative phase of cells, while promoting MMP9 expression during the differentiation phase. MMP9 inhibition could promoted cell proliferation and inhibited differentiation. This study demonstrated that lnc721 targeting MMP9 regulated the development of bovine skeletal muscle satellite cells.
lnc721; MMP9; bovine skeletal muscle satellite cells; proliferation; differentiation
10.3864/j.issn.0578-1752.2023.24.012
2023-05-06;
2023-07-04
天津市農(nóng)業(yè)動物繁育與健康養(yǎng)殖重點實驗室開放基金課題、天津市自然基金(20JCQNJC00640)、天津市教委科研計劃項目(2020KJ089)
郭云鵬,E-mail:guoyunpeng1225@163.com。通信作者郭益文,E-mail:yiwenguo33@163.com
(責(zé)任編輯 林鑒非)