牛LncRNA-133a對(duì)骨骼肌衛(wèi)星細(xì)胞增殖分化的影響

李燕，陳明明，張俊星，張林林，李新，郭宏，丁向彬，劉新峰

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牛LncRNA-133a對(duì)骨骼肌衛(wèi)星細(xì)胞增殖分化的影響

李燕，陳明明，張俊星，張林林，李新，郭宏，丁向彬，劉新峰

（天津農(nóng)學(xué)院動(dòng)物科學(xué)與動(dòng)物醫(yī)學(xué)學(xué)院，天津 300384)

【目的】探討長(zhǎng)鏈非編碼RNA LncRNA-133a對(duì)牛骨骼肌衛(wèi)星細(xì)胞增殖分化過程的影響。【方法】利用測(cè)序樣品3、6、9月齡胎牛及24月齡成年和牛骨骼肌肌肉組織，qRT-PCR法檢測(cè)LncRNA-133a的組織時(shí)序表達(dá)譜。構(gòu)建牛骨骼肌衛(wèi)星細(xì)胞的體外成肌誘導(dǎo)分化模型，模擬牛骨骼肌的生長(zhǎng)發(fā)育過程，qRT-PCR法檢測(cè)LncRNA-133a和肌細(xì)胞分化標(biāo)記因子MyoG、MHC的細(xì)胞時(shí)序表達(dá)譜。利用過表達(dá)LncRNA-133載體（pCDNA3.1-EGFP-LncRNA-133a） 或LncRNA-133a抑制物（si-LncRNA 133a） 轉(zhuǎn)染牛骨骼肌衛(wèi)星細(xì)胞，qRT-PCR法檢測(cè)轉(zhuǎn)染效率以及各轉(zhuǎn)染處理組LncRNA-133a、MyoD、MyoG及MHC基因mRNA的表達(dá)水平，Western blotting檢測(cè)MHC 基因的蛋白表達(dá)水平；同時(shí)，通過EdU細(xì)胞增殖檢測(cè)、免疫熒光蛋白染色技術(shù)檢測(cè)牛骨骼肌衛(wèi)星細(xì)胞增殖階段的細(xì)胞增殖量和分化階段的肌管融合程度?！窘Y(jié)果】組織表達(dá)譜分析發(fā)現(xiàn)LncRNA-133a在3月齡胎牛肌肉組織中表達(dá)量最高，6月齡胎牛肌肉組織中次之，9月齡胎牛及成年牛肌肉組織中表達(dá)量最低，時(shí)序表達(dá)呈下降趨勢(shì)；利用成功構(gòu)建的牛骨骼肌衛(wèi)星細(xì)胞體外誘導(dǎo)分化模型，進(jìn)行LncRNA-133a、MyoG、MHC的細(xì)胞時(shí)序表達(dá)譜分析，結(jié)果發(fā)現(xiàn)在牛骨骼肌衛(wèi)星細(xì)胞分化過程中（D0-D3），肌分化標(biāo)記因子MyoG、MHC的表達(dá)水平逐漸升高，LncRNA-133a的表達(dá)在分化階段呈上升趨勢(shì)，且分化48 h時(shí)（D2）表達(dá)量最高；成功構(gòu)建的過表達(dá)LncRNA-133a或抑制LncRNA-133a的牛骨骼肌衛(wèi)星細(xì)胞模型，在增殖期（D0）：與對(duì)照組相比，過表達(dá)LncRNA-133a處理組EdU增殖染色檢測(cè)得到EdU陽(yáng)性細(xì)胞數(shù)顯著增加（＜0.01），而LncRNA-133a抑制處理組EdU陽(yáng)性細(xì)胞數(shù)顯著減少（＜0.01）；在分化48 h時(shí)（D2）：與對(duì)照組相比，LncRNA-133a過表達(dá)處理組肌細(xì)胞分化標(biāo)記因子MyoD、MyoG及MHC的mRNA表達(dá)水平顯著升高（＜0.05），Western blotting檢測(cè)MHC蛋白表達(dá)量顯著增加（＜0.01），且MHC蛋白的免疫熒光蛋白染色檢測(cè)觀察到融合肌管的體積占比更大；而LncRNA-133a抑制處理組MyoD、MyoG及MHC的mRNA表達(dá)水平均降低，其中MyoG顯著降低（＜0.05）, MHC蛋白表達(dá)量顯著減少（＜0.01），同時(shí)MHC蛋白融合肌管的體積占比也降低。【結(jié)論】研究證實(shí)LncRNA-133a具有促進(jìn)牛骨骼肌衛(wèi)星細(xì)胞增殖及分化的作用，為進(jìn)一步挖掘LncRNA-133a調(diào)節(jié)牛骨骼肌衛(wèi)星細(xì)胞增殖分化調(diào)控網(wǎng)絡(luò)機(jī)制奠定了基礎(chǔ)。

LncRNA-133a；牛;骨骼肌衛(wèi)星細(xì)胞；增殖；分化

0 引言

【研究意義】骨骼肌作為動(dòng)物軀體最重要的組織之一，占重比可高達(dá)40%[1]，因此骨骼肌的發(fā)育對(duì)經(jīng)濟(jì)動(dòng)物肉牛來(lái)說(shuō)意義重大。肌細(xì)胞作為骨骼肌的基本形成單位，它的分化發(fā)育直接影響到骨骼肌的發(fā)育形成[2]。隨著人們對(duì)LncRNAs研究的不斷深入，研究者已經(jīng)發(fā)現(xiàn)許多LncRNAs可以參與肌細(xì)胞的增殖和分化過程[3-5]。【前人研究進(jìn)展】linc-MD1及l(fā)nc-mg作為內(nèi)源性競(jìng)爭(zhēng)性RNA（ceRNA）促進(jìn)肌細(xì)胞的分化發(fā)育[6-7]；lncRNA MAR1發(fā)揮其“sponge”吸附作用促進(jìn)肌分化和再生[8]；Linc-YY1則通過與靶基因的互作作用促進(jìn)肌分化和再生[9]；此外，還有Lnc-SEMT，H19等，也都在肌形成過程中發(fā)揮其重要的生物學(xué)功能[10-12]]。【本研究切入點(diǎn)】有關(guān)LncRNAs參與肌肉發(fā)育的研究已有很多，但多集中于模式動(dòng)物小鼠的研究上。近年來(lái)，隨著生物檢測(cè)技術(shù)及生物信息學(xué)分析技術(shù)的發(fā)展，通過對(duì)牛骨骼肌肌肉組織進(jìn)行高通量測(cè)序分析，已經(jīng)獲得了大量與肌肉發(fā)育潛在相關(guān)的LncRNAs[13-15]。研究者針對(duì)這些 LncRNAs開展了一系列的功能研究，并證實(shí)了其中一些LncRNA如lncMD[14]、LncRNA-AK143003[15]、LncRNA HZ-5[16]、LncRNA H19[17]等，確實(shí)參與調(diào)節(jié)牛骨骼肌細(xì)胞的分化發(fā)育。但相較小鼠C2C12成肌細(xì)胞生長(zhǎng)發(fā)育相關(guān)LncRNA的報(bào)道，參與調(diào)節(jié)牛骨骼肌細(xì)胞生長(zhǎng)發(fā)育的LncRNAs的相關(guān)報(bào)道仍然較少，測(cè)序獲得的與牛肌肉發(fā)育相關(guān)的海量LncRNAs，還需要研究者不斷挖掘和證實(shí)它們的功能?！緮M解決的關(guān)鍵問題】本研究利用前期在牛肌肉組織中鑒定到的長(zhǎng)鏈非編碼RNA LncRNA-133a，通過表達(dá)譜分析，并進(jìn)一步以體外分化牛骨骼肌衛(wèi)星細(xì)胞為模型，通過過表達(dá)/抑制LncRNA-133a處理，探究其對(duì)牛骨骼肌衛(wèi)星細(xì)胞增殖和分化過程的影響，旨在為牛骨骼肌生長(zhǎng)發(fā)育相關(guān)LncRNAs的功能研究積累更多的有益資料。

1 材料與方法

試驗(yàn)于2017—2018年在天津市農(nóng)業(yè)動(dòng)物繁育及健康養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室完成。

1.1 材料

1.1.1 肌肉組織與細(xì)胞來(lái)源 肌肉組織為天津市農(nóng)業(yè)動(dòng)物繁育及健康養(yǎng)殖重點(diǎn)實(shí)驗(yàn)室凍存的3、6、9月齡胎牛及24月齡成年和牛骨骼肌肌肉；細(xì)胞為該實(shí)驗(yàn)室分離凍存的原代牛骨骼肌衛(wèi)星細(xì)胞。

1.1.2 主要儀器與試劑 CO2恒溫培養(yǎng)箱（日本三洋）；LightCycle 96實(shí)時(shí)熒光定量PCR儀（Roche）；Nano-Drop ND 2000c Spectrophotometer（Thermo- scientific）；熒光顯微鏡（Leica）。

pCDNA3.1-EGFP（武漢淼靈生物科技有限公司）；siRNA oligos（上海吉瑪基因）；Lipofectamine? 3000（Invitrogen）；Trizol reagent（Invitrogen）；PrimeScript II 1st Strand cDNA Synthesis Kit（Takara）；All-in- OneTMqPCR Mix（GeneCopoeia）；Hoechst 33342染色液（碧云天）；一抗，Anti-Myosin VⅡα antibody ab3481（abcom）；二抗，CY3-羊抗兔IgG（BA1032，博士德生物）；Cell-Light EdU Apollo 567 In Vitro Imaging Kit（RN:R11053.2，廣州市銳博生物科技有限公司）；qRT-PCR檢測(cè)相關(guān)引物（蘇州金唯智生物科技有限公司）。

1.2 方法

1.2.1 牛肌肉組織中LncRNA-133a時(shí)序表達(dá)譜鑒定 qRT-PCR檢測(cè)：3、6、9月齡胎牛及成年牛肌肉組織全基因組檢測(cè)LncRNA-133a的表達(dá)情況，其中3月齡胎牛肌肉組織作為對(duì)照。

1.2.2 細(xì)胞培養(yǎng) 常規(guī)復(fù)蘇實(shí)驗(yàn)室前期凍存的原代牛骨骼肌衛(wèi)星細(xì)胞[18]，利用原代牛骨骼肌衛(wèi)星細(xì)胞體外培養(yǎng)模擬牛肌肉的發(fā)育進(jìn)程[19]，使用增殖培養(yǎng)基：含體積分?jǐn)?shù)20%的胎牛血清、100 IU·mL-1青霉素和鏈霉素的DMEM，置于37℃、5%CO2、飽和濕度培養(yǎng)箱中培養(yǎng)。待增殖細(xì)胞融合度達(dá)80%—90%時(shí)將培養(yǎng)基更換為含體積分?jǐn)?shù)2%馬血清的DMEM的分化培養(yǎng)基。

1.2.3 牛骨骼肌衛(wèi)星細(xì)胞轉(zhuǎn)染及收集 24孔培養(yǎng)板內(nèi)增殖期細(xì)胞融合度達(dá)50%時(shí)，按生產(chǎn)商家說(shuō)明書利用Lipfectamine3000轉(zhuǎn)染已構(gòu)建的過表達(dá)載體pCDNA3.1-EGFP-LncRNA-133a（簡(jiǎn)寫為pcDE-LNC）及抑制物 si-lncRNA133a，以空質(zhì)粒pCDNA3.1-EGFP（簡(jiǎn)寫為pcDE-NC）及si-NC轉(zhuǎn)染細(xì)胞作為對(duì)照，轉(zhuǎn)染后24 h時(shí)培養(yǎng)基更換為分化培養(yǎng)基。

細(xì)胞總RNA及總蛋白收集。增殖期細(xì)胞（D0）：更換分化培養(yǎng)基前收集；分化期細(xì)胞（D1—D3）：于更換培養(yǎng)基后24 h（D1）、48 h（D2）、72 h（D3）時(shí)收集。按生產(chǎn)商家說(shuō)明書利用Trizol及蛋白裂解液裂解并提取細(xì)胞總RNA及蛋白。

1.2.4 LncRNA-133a、分化標(biāo)記基因表達(dá)水平的檢測(cè) qRT-PCR檢測(cè)：收集24孔板中增殖期（D0）、分化期（D1—D3）未處理細(xì)胞、各轉(zhuǎn)染處理細(xì)胞以及相應(yīng)對(duì)照組細(xì)胞。取4 μg總RNA利用 All-in- OneTM First-Strand cDNA Synthesis Kit 試劑盒反轉(zhuǎn)錄為第一鏈cDNA，再采用qRT-PCR法檢測(cè)各基因的表達(dá)。

qRT-PCR反應(yīng)體系：10 μmol·L-1上游引物、10 μmol·L-1下游引物、2.0 μL 5x稀釋 cDNA、10 μL 2×All-in-OneTMqPCR Mix，Nuclease-free water 將體系補(bǔ)至20 μL。反應(yīng)條件：95℃ 10 min；95℃ 10 s、60℃ 20 s、72℃ 15 s，重復(fù)35個(gè)循環(huán)。qRT-PCR反應(yīng)引物信息如表1所示。

1.2.5牛骨骼肌衛(wèi)星細(xì)胞增殖、分化能力的檢測(cè) qRT-PCR檢測(cè)：24孔板中細(xì)胞在轉(zhuǎn)染后DM2期時(shí)，檢測(cè)肌細(xì)胞分化標(biāo)記因子MYOD（muscle regulatory factors, MRFs成員）、MYOG（myogenin）及MHC（Myosin heavy chain）的轉(zhuǎn)錄組表達(dá)情況。

EdU細(xì)胞增殖檢測(cè)：96孔板中細(xì)胞在轉(zhuǎn)染后D0期時(shí)，按Cell-Light EdU Apollo 567 In Vitro Imaging Kit試劑盒說(shuō)明書檢測(cè)各處理細(xì)胞的增殖期細(xì)胞數(shù)。

MHC細(xì)胞免疫熒光檢測(cè)：48孔板中各處理細(xì)胞在DM2期時(shí)，進(jìn)行肌衛(wèi)星細(xì)胞分化標(biāo)記因子MHC的免疫熒光蛋白染色檢測(cè)。

1.2.6 牛骨骼肌衛(wèi)星細(xì)胞分化標(biāo)記基因蛋白表達(dá)水平的檢測(cè) Western blotting檢測(cè)：6孔板中細(xì)胞在DM2期時(shí)被蛋白裂解液裂解，收集蛋白，通過Western blotting檢測(cè)分化標(biāo)志基因MHC的蛋白表達(dá)。

表1 qRT-PCR引物信息

2 結(jié)果

2.1 LncRNA-133a的組織表達(dá)譜

LncRNA-133a是天津農(nóng)學(xué)院動(dòng)物分子育種與轉(zhuǎn)基因創(chuàng)新中心實(shí)驗(yàn)室前期從3、6、9月齡胎牛肌肉組織的高通量測(cè)序數(shù)據(jù)中篩選鑒定出的組織時(shí)序表達(dá)呈下降趨勢(shì)的一條LncRNA。本研究利用qRT-PCR法，驗(yàn)證不同月齡肌肉中LncRNA-133a的時(shí)序表達(dá)情況。如圖1：除在6月齡肋間肌及背腰肌中有相對(duì)上調(diào)外，LncRNA-133a在3、6、9月齡胎牛及成年牛各肌肉組織中均呈下降的表達(dá)趨勢(shì)。表明測(cè)序結(jié)果可靠，該LncRNA可進(jìn)行下一步的功能研究。

圖1 3、6、9月齡胎牛及成年牛不同肌肉組織中LncRNA-133a的表達(dá)

2.2 牛骨骼肌衛(wèi)星細(xì)胞LncRNA-133a及肌分化標(biāo)記因子的表達(dá)水平

光鏡下觀察D0-D3時(shí)期牛骨骼肌衛(wèi)星細(xì)胞的分化狀態(tài)并于對(duì)應(yīng)時(shí)期收集細(xì)胞總RNA，qRT-PCR法檢測(cè)LncRNA-133a及肌分化標(biāo)記因子的正常表達(dá)水平。如圖2、圖3：隨著骨骼肌衛(wèi)星細(xì)胞進(jìn)入誘導(dǎo)分化階段，牛骨骼肌衛(wèi)星細(xì)胞分化形成的肌管清晰可見（圖2），且肌分化標(biāo)記因子MHC及MyoG均呈上升表達(dá)趨勢(shì)，表明牛骨骼肌衛(wèi)星細(xì)胞的體外誘導(dǎo)分化模型構(gòu)建成功；LncRNA-133a在牛骨骼肌衛(wèi)星細(xì)胞分化期間高表達(dá)，且分化48 h表達(dá)量最高，這提示其可能參與牛骨骼肌衛(wèi)星細(xì)胞分化發(fā)育的調(diào)節(jié)過程。

圖2 牛骨骼肌衛(wèi)星細(xì)胞分化進(jìn)程（100×）

圖3 牛骨骼肌衛(wèi)星細(xì)胞分化階段 LncRNA-133a及分化標(biāo)記因子的表達(dá)

2.3 LncRNA-133a的過表達(dá)/抑制效率

牛骨骼肌衛(wèi)星細(xì)胞轉(zhuǎn)染過表達(dá)LncRNA-133a載體pcDE-LncRNA-133A或LncRNA-133a的抑制物si-lncRNA133a 及相應(yīng)對(duì)照載體pcDE-NC或?qū)φ找种莆飐i-NC。利用qRT- PCR法檢測(cè)轉(zhuǎn)染處理后D0及D2期過表達(dá)/抑制效率。相比pcDE-NC組，pcDE- LNC組LncRNA-133a在D0和D2期均有顯著過表達(dá)效果（圖4-A）；si-lncRNA133a 組相比si-NC組，si-lncRNA133a組LncRNA-133a在D0和D2期均有顯著抑制效果（圖4-B）。結(jié)果表明構(gòu)建過表達(dá)/抑制LncRNA-133a的牛骨骼肌衛(wèi)星細(xì)胞模型成功，可進(jìn)行下一步試驗(yàn)驗(yàn)證。

2.4 LncRNA-133a對(duì)牛骨骼肌衛(wèi)星細(xì)胞增殖能力的影響

過表達(dá)/抑制LncRNA-133a 后D0期對(duì)牛骨骼肌衛(wèi)星細(xì)胞進(jìn)行EdU染色檢測(cè)牛骨骼肌衛(wèi)星細(xì)胞增殖數(shù)。結(jié)果顯示：過表達(dá)LncRNA-133a組EdU陽(yáng)性細(xì)胞數(shù)顯著增多；而抑制LncRNA-133a后，EdU陽(yáng)性細(xì)胞數(shù)則顯著減少。這表明LncRNA-133a可以促進(jìn)牛骨骼肌衛(wèi)星細(xì)胞的增殖，對(duì)牛骨骼肌衛(wèi)星細(xì)胞的增殖有著正向調(diào)控作用（圖5）。

圖4 qRT-PCR檢測(cè)LncRNA-133a的過表達(dá)（A）和抑制（B）效率

圖5 EdU檢測(cè)過表達(dá)/抑制LncRNA-133a后牛骨骼肌衛(wèi)星細(xì)胞的增殖細(xì)胞

2.5 LncRNA-133a對(duì)牛骨骼肌衛(wèi)星細(xì)胞分化能力的影響

qRT-PCR及Western blotting檢測(cè)過表達(dá)/抑制LncRNA-133a后D2期牛骨骼肌衛(wèi)星細(xì)胞分化標(biāo)記因子MHC、MyoD及MyoG 的mRNA水平及蛋白水平表達(dá)。過表達(dá)LncRNA-133a后，MHC、MyoD及MyoG的mRNA水平表達(dá)均呈顯著上調(diào)趨勢(shì)（圖6-A）；同時(shí)MHC在蛋白水平均也顯著上調(diào)（圖6-B）。而抑制LncRNA-133a后，MHC、MyoD及MyoG的mRNA水平表達(dá)均呈下調(diào)趨勢(shì)（圖7-A）；MHC在mRNA水平雖然下調(diào)不顯著，但蛋白水平下調(diào)顯著（圖7-B）。此外，通過免疫熒光染色試驗(yàn)檢測(cè)MHC融合細(xì)胞也得到一致結(jié)果，如圖8：過表達(dá)LncRNA-133a后，MHC融合肌管數(shù)增多（圖8-A）；而抑制LncRNA-133a表達(dá)后，MHC融合肌管數(shù)減少（圖8-B）。

圖6 過表達(dá)LncRNA-133a促進(jìn)牛骨骼肌衛(wèi)星細(xì)胞分化

圖7 抑制LncRNA-133a表達(dá)阻滯牛骨骼肌衛(wèi)星細(xì)胞分化

3 討論

肌肉發(fā)育分化是發(fā)育生物學(xué)研究的重要主題。肌細(xì)胞作為骨骼肌的基本形成單位，它的分化發(fā)育直接影響到骨骼肌的發(fā)育形成。肌細(xì)胞的分化和生長(zhǎng)發(fā)育過程是內(nèi)在遺傳、表觀遺傳和外在各種信號(hào)互作的結(jié)果，受到機(jī)體肌肉發(fā)育調(diào)節(jié)因子和調(diào)控通路的多層次精細(xì)調(diào)節(jié)。lncRNA作為一類調(diào)控因子參與肌肉的發(fā)育已是不爭(zhēng)的事實(shí)，相關(guān)報(bào)道也不斷涌現(xiàn)。

有研究表明通過EdU、CCK8或流式細(xì)胞儀檢測(cè)技術(shù)，甚至是細(xì)胞劃痕試驗(yàn)得到如Sirt1[20]、lncRNA AK017368[21]、長(zhǎng)非編碼RNA-GTL2[22]等LncRNAs具有促進(jìn)肌細(xì)胞增殖的作用。本研究中，在過表達(dá)/抑制LncRNA-133a處理后24h（D0），通過EdU細(xì)胞增殖檢測(cè)初步驗(yàn)證LncRNA-133a具有促進(jìn)牛骨骼肌衛(wèi)星細(xì)胞增殖的作用。

圖8 免疫熒光檢測(cè)過表達(dá)（A）/抑制（B）LncRNA-133a牛骨骼肌衛(wèi)星細(xì)胞的分化標(biāo)記因子MHC

在肌細(xì)胞分化過程中，骨骼肌特異性標(biāo)記基因如MyoD 、MyoG 及MHC 開始表達(dá)[23]。其中，MyoD誘導(dǎo)細(xì)胞周期的退出同時(shí)開啟細(xì)胞分化[24-25]，骨骼肌分化決定因子MyoG受MyoD的啟動(dòng)開始表達(dá)，并調(diào)控成肌細(xì)胞融合和肌纖維形成[26- 27]，而作為骨骼肌纖維內(nèi)粗肌絲主要成分的MHC[28]，在肌細(xì)胞分化后期表達(dá)量逐漸升高。在各肌分化相關(guān)的研究中，通常把MyoD、MyoG、MHC作為肌分化標(biāo)記因子[29-30]，利用qRT-PCR、Western blotting及免疫熒光蛋白染色分析等方法，檢測(cè)它們mRNA水平、蛋白水平及基因蛋白融合表達(dá)的變化來(lái)驗(yàn)證肌細(xì)胞的分化進(jìn)程。

通過這樣的驗(yàn)證手法，現(xiàn)已證實(shí)如Linc-YY1、LncRNA Dum、LncMyoD、linc-MD1[9, 31-33]等LncRNAs參與調(diào)節(jié)肌細(xì)胞分化，且這類LncRNAs在成肌細(xì)胞分化階段呈時(shí)序性上升表達(dá)，所以后續(xù)的功能研究基本都集中在這些LncRNAs的高表達(dá)時(shí)期。此外，Albrecht等[34]研究發(fā)現(xiàn)，在3月齡牛胎兒的初級(jí)纖維中便可檢測(cè)到作為肌纖維成熟標(biāo)記物的肌球蛋白，說(shuō)明肌纖維的發(fā)育主要在妊娠早期。本研究中，通過對(duì)LncRNA-133a的時(shí)序表達(dá)譜分析發(fā)現(xiàn)：組織上，LncRNA-133a主要在3月齡胎牛肌肉組織中高表達(dá)；細(xì)胞上，LncRNA-133a在牛骨骼肌衛(wèi)星細(xì)胞分化早期（D2）表達(dá)量最高。據(jù)此我們推測(cè)LncRNA-133可能參與調(diào)節(jié)牛骨骼肌衛(wèi)星細(xì)胞的早期分化發(fā)育。在牛骨骼肌衛(wèi)星細(xì)胞分化早期（D2）進(jìn)行LncRNA-133a的功能驗(yàn)證發(fā)現(xiàn)，有效過表達(dá)LncRNA-133a后，肌分化標(biāo)記因子如MHC、MyoG、MyoD的mRNA水平均顯著上升，同時(shí)MHC在蛋白水平也顯著上調(diào)。同樣，在有效抑制LncRNA-133a后，對(duì)MyoG mRNA表達(dá)水平和MHC蛋白表達(dá)水平的下調(diào)影響是顯著的，對(duì)MHC、MyoD mRNA表達(dá)水平有不顯著的下調(diào)影響，這有可能是該檢測(cè)時(shí)期這些分化標(biāo)記因子自身表達(dá)特性的影響。此外，MHC融合細(xì)胞的免疫熒光試驗(yàn)分析也得到了一致結(jié)果。這些結(jié)果均表明LncRNA-133a可以促進(jìn)牛骨骼肌衛(wèi)星細(xì)胞分化。

現(xiàn)階段研究對(duì)LncRNAs在肌細(xì)胞增殖分化過程中扮演的經(jīng)典調(diào)節(jié)角色已很明確：在肌細(xì)胞的增殖分化過程中，肌生長(zhǎng)發(fā)育相關(guān)LncRNAs對(duì)成肌細(xì)胞的增殖與分化兩個(gè)生物學(xué)過程的促進(jìn)或抑制調(diào)節(jié)作用是相反的，或只參與兩者其中一個(gè)，但這并不排除LncRNAs 對(duì)肌細(xì)胞增殖及分化有一致的促進(jìn)或抑制作用，進(jìn)而促進(jìn)或抑制成肌纖維的形成，影響肌肉的生長(zhǎng)發(fā)育。YUE[35]等研究發(fā)現(xiàn)，過表達(dá)lncYYW后通過mRNA microarray分析得到，GH1及其下游基因AKT1和PIK3CD上調(diào)，上調(diào)的GH1激活JAK促進(jìn)成肌細(xì)胞增殖，同時(shí)也發(fā)現(xiàn)過表達(dá)lncYYW促進(jìn)了成肌細(xì)胞的分化。相對(duì)于LncRNAs在其他生物學(xué)過程中（如腫瘤）調(diào)節(jié)作用機(jī)制的研究，LncRNAs對(duì)肌細(xì)胞增殖分化的研究仍有短缺，如2017年研究發(fā)現(xiàn)與乳腺癌轉(zhuǎn)移相關(guān)的LncRNA H19，在腫瘤轉(zhuǎn)移的不同階段（EMT和MET），通過吸附不同的miRNA均發(fā)揮其促腫瘤轉(zhuǎn)移作用[36]。綜上，本研究中LncRNA-133a促進(jìn)肌衛(wèi)星細(xì)胞的增殖分化，可能是LncRNA-133a通過某種互作機(jī)制對(duì)肌細(xì)胞增殖分化相關(guān)靶標(biāo)的調(diào)控所致。

4 結(jié)論

利用前期鑒定的牛肌肉發(fā)育相關(guān)的長(zhǎng)鏈非編碼RNA LncRNA-133a為研究靶標(biāo)，經(jīng)組織表達(dá)譜分析發(fā)現(xiàn)，LncRNA-133a在3、6、9月齡胎牛及成年牛骨骼肌中時(shí)序表達(dá)呈下降趨勢(shì)；進(jìn)一步利用牛骨骼肌衛(wèi)星細(xì)胞體外分化模型，經(jīng)過表達(dá)和抑制LncRNA-133a后發(fā)現(xiàn)，LncRNA-133a對(duì)牛骨骼肌衛(wèi)星細(xì)胞的增殖及分化均有促進(jìn)作用。

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Effects of Bovine LncRNA-133a on the Proliferation and Differentiation of Skeletal Muscle Satellite Cells

LI Yan, CHEN MingMing, ZHANG JunXing, ZHANG LinLin, LI Xin, GUO Hong, DING XiangBin, Liu XinFeng

(College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384)

【Objective】The objective of this paper was to investigate the effects of long non-coding RNA LncRNA-133a on the proliferation and differentiation of bovine skeletal muscle satellite cells. 【Method】This study used qRT-PCR to detect the expression level of LncRNA-133a in the skeletal muscle tissues of 3, 6 and 9 months old fetal cattle and 24 months old adult bovine skeletal muscle, and obtained the tissue temporal expression profile of LncRNA-133a. The in vitro induced myoblast differentiation model of bovine skeletal muscle satellite cells was constructed to simulate the growth and development of bovine skeletal muscle. The qRT-PCR was used to detect the cells temporal expression profiles of LncRNA-133a and myocyte differentiation markers MyoG and MHC. The bovine skeletal muscle satellite cells were transfected with LncRNA-133a overexpression vector (pCDNA3.1-EGFP- LncRNA-133a) or LncRNA-133a inhibitor (si-LncRNA 133a), and the transfection efficiency and the mRNA expression levels of LncRNA-133a, MyoD, MyoG and MHC were detected by qRT-PCR in each transfection treatment group, then the protein expression level of MHC gene was detected by western blotting. In addition, the cell proliferation of the bovine skeletal muscle satellite cells and the extent of myotube fusion at the differentiation stage were detected by EdU cell proliferation assay and immunofluorescence protein staining, respectively. 【Result】Tissue expression profiling revealed that LncRNA-133a had the highest expression in the muscle tissue of 3 months old fetal bovine, followed by the 6-month-old fetal bovine muscle tissue, and the lowest expression in the 9-month-old fetus and adult bovine muscle tissue, which demonstrated that the time expression showed a downward trend. Cell-time expression profiles of LncRNA-133a, MyoG, and MHC were analyzed by a successfully constructed bovine skeletal muscle satellite cell differentiation model in vitro, and the results showed that the expression levels of myogenic differentiation markers MyoG and MHC gradually increased during the differentiation of bovine skeletal muscle satellite cells (D0-D3). The expression of LncRNA- 133a increased in the differentiation stage, and the expression level reached the highest at 48 h of differentiation (D2). The bovine skeletal muscle satellite cell model of overexpressing LncRNA-133a or inhibiting LncRNA-133a was constructed successfully, and in the proliferative phase (D0): the number of EdU positive cells in the overexpressed LncRNA-133a-treated group was significantly increased (＜0.01), and the number of EdU positive cells in the LncRNA-133a inhibition treatment group was significantly decreased (＜0.01), compared with the control group. At 48 h of differentiation (D2): compared with the control group, the results of LncRNA-133a overexpression treatment showed that mRNA expression levels of myocyte differentiation markers MyoD, MyoG and MHC were significantly increased (＜0.05). Western blotting showed that the expression of MHC protein was also significantly increased (＜0.01), and the immunofluorescence protein staining of MHC protein showed that the volume of fusion myotubes was larger. On the contrary, in the LncRNA-133a inhibition treatment group, the mRNA expression levels of MyoD, MyoG and MHC were decreased, and MyoG was significantly decreased (＜0.05). Meanwhile, the expression of MHC protein was significantly decreased (＜0.01), and the volume fraction of MHC protein fusion myotubes was also decreased. 【Conclusion】Thus, this study confirmed that LncRNA-133a promoted the proliferation and differentiation of bovine skeletal muscle satellite cells, which laid a foundation for further research on the regulatory network mechanism of LncRNA-133a regulating the proliferation and differentiation of bovine skeletal muscle satellite cells.

LncRNA-133a; bovine; skeletal muscle satellite cells; proliferation; differentiation

10.3864/j.issn.0578-1752.2019.01.013

2018-05-18；

2018-09-28

國(guó)家自然科學(xué)基金青年項(xiàng)目（31501938）、天津市“131人才工程第二層人選”項(xiàng)目（J01009030725）

李燕，E-mail：lihongjiliyan@163.com。通信作者丁向彬，E-mail：xiangbinding@aliyun.com。通信作者劉新峰，E-mail：lxf20001924036@126.com

（責(zé)任編輯 林鑒非）