Construction of Miro1 overexpression lentiviral vectors and establishment and identification of Miro1Hi-BMSCs stable transfection cell line
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摘要:
背景 线粒体转移是干细胞发挥免疫修复功能的重要机制之一，Miro1是线粒体转移过程的关键蛋白，但其动力学研究欠缺研究模型。 目的 构建过表达Miro1的间充质干细胞稳转细胞株，为线粒体动力学研究间充质干细胞的抗炎修复机制提供细胞模型。 方法 通过PCR扩增目的基因后进行Gibson反应、转化及Gateway等方法构建载体，并对载体进行酶切鉴定。慢病毒感染间充质干细胞后，使用嘌呤霉素进行药物筛选获得稳转细胞株。后续分为三组进行相关鉴定。(1) BMSC组：正常间充质干细胞；(2) Con-BMSC组：慢病毒空载体感染的间充质干细胞；(3) MiroHi-BMSC组：过表达Miro1的慢病毒载体感染的间充质干细胞。通过RT-qPCR、Western blot检测上述三组细胞Miro1的表达水平，并进行划痕试验、水平迁移实验、成骨及成脂诱导分化以鉴定干细胞特性。 结果 所构建的载体完成酶切鉴定，并成功获得过表达Miro1重组慢病毒载体pLV-EGFP:T2A:Puro-EF1A＞mRhot1/HA。经mRNA和蛋白水平检测，MiroHi-BMSC组的Miro1表达高于BMSC组和Con-BMSC组(P＜0.05)，干细胞水平、垂直迁移能力三组间差异无统计学意义(P＞0.05)，三组干细胞均可成功进行成脂及成骨诱导分化。 结论 通过慢病毒载体成功构建Miro1Hi-BMSCs稳转细胞株，且该稳转株仍保留干细胞特性，可用于间充质干细胞调控线粒体转移的相关研究。 Abstract:Background Mitochondrial transfer is one of the important mechanisms for stem cells to exert immune repair function. Miro1 is the key protein in the process of mitochondrial transfer, but its dynamics research lacks research models. Objective To establish a stable Miro1 protein overexpressed mesenchymal stem cell line, so as to provide a cell model for further study on the anti-inflammatory and repair mechanism of mesenchymal stem cells from the perspective of mitochondrial dynamics. Methods The amplification of the target gene by PCR was firstly performed, followed by Gibson reaction, transformation and high-throughput Gateway to construct the vector, which was further identified by the enzyme digestion. The stable transfected mesenchymal stem cell line was then acquired by lentivirus transfection and puromycin drug screening and subsequently divided into three groups for identification, which were BMSC group with normal mesenchymal stem cells, Con-BMSC group with empty lentiviral vector stably transformed mesenchymal stem cells, and Miro1Hi-BMSC group with Miro1 overexpression lentiviral vector stably transformed mesenchymal stem cells. Expression level of Miro1 of above groups was detected by RT-qPCR and Western blot. Original natures of stem cells were identified by scratch test, vertical migration test, osteogenic and adipogenic differentiation induction. Results Recombinant Miro1 overexpressed lentivirus vector pLV-EGFP: T2A: Puro-EF1A>mRhot1/HA was successfully confirmed by digestion identification. Compared with the groups of BMSC and Con-BMSC, the expression level of Miro1 in Miro1Hi-BMSC group was significantly higher by mRNA and protein level detection (P<0.05), while there were no significant differences in the horizontal and vertical migration ability among the three groups (P>0.05). The stem cells in the three groups could successfully differentiate into adipogenic and osteogenic cells. Conclusion Miro1Hi-BMSCs stable transfection cell line is established by lentivirus vector, additionally, it retains original nature, which can be utilized for the further research on mitochondrial donation regulated by mesenchymal stem cells. -
Key words:
- Miro1 /
- lentiviral vectors /
- mesenchymal stem cells /
- mitochondria /
- contact-dependent transfer
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图 1 过表达载体与对照空载体设计图谱
A：pLV-EGFP:T2A:Puro-EF1A>mRhot1[NM_021536.8]/HA；B：pLV-EGFP:T2A:Puro-EF1A>mCherry
Figure 1. Gene mapping of the overexpression and control vectors
A: pLV-EGFP:T2A:Puro-EF1A>mRhot1[NM_021536.8]/HA; B: pLV-EGFP:T2A:Puro-EF1A>mCherry
图 2 终载体的酶切鉴定结果
A：SnapGene软件查找酶切位点；B：限制性内切酶酶切产物琼脂糖凝胶电泳
Figure 2. Identification of the final vector by enzyme digestion
A: Finding the digestion sites by the SnapGene software; B: Agarose gel electrophoresis map of the products from the enzyme digestion
图 3 病毒载体感染HEK293T细胞后48 h荧光图(100×)
A：质粒DNA转染HEK293T细胞包装病毒明场图；B：质粒DNA转染HEK293T细胞包装病毒EGFP荧光图；C：病毒感染HEK293T细胞明场图；D：病毒感染HEK293T细胞EGFP荧光图(MOI=5)
Figure 3. Photos of HEK293T cells taken by fluorescence microscopy after 48 h of transduction (100×)
A: Plasmid DNA transfected virus packaged by HEK293T cells which observed through bright-field microscope; B: Plasmid DNA transfected virus packaged by HEK293T cells which observed through fluorescence microscopy; C: The virus transduced HEK293T cells which observed through bright - field microscope; D: The virus transduced HEK293T cells which observed through fluorescence microscopy (MOI=5)
图 4 慢病毒载体转染BMSCs荧光图(MOI=40，转染11 d，药筛6 d，200×)
A：对照慢病毒载体转染BMSCs明场图 ；B：对照慢病毒载体转染BMSCs mCherry荧光图；C：对照慢病毒载体转染BMSCs EGFP荧光图；D：Miro1 过表达载体转染BMSCs明场图；E：Miro1过表达载体转染BMSCs EGFP荧光图
Figure 4. Photos of BMSCs taken by fluorescence microscopy after transduction of lentiviral vectors (MOI=40, 11 days after the transduction, 6 days after screening test by puromycin, 200×)
A: Control lentiviral vectors transduced BMSCs which observed through bright-field microscope; B: Control lentiviral vectors transduced BMSCs which observed through mCherry fluorescence microscopy; C: Control lentiviral vectors transduced BMSCs which observed through EGFP fluorescence microscopy; D: Miro1 protein overexpressed lentiviral vectors transduced BMSCs which observed through bright-field microscope; E: Miro1 protein overexpressed lentiviral vectors transduced BMSCs which observed through EGFP fluorescence microscopy
图 5 BMSC、Con-BMSC及Miro1Hi-BMSC细胞株的Miro1表达水平鉴定
A：蛋白水平Western blot结果；B：mRNA水平RT-qPCR结果
Figure 5. Identification of Miro1 expression levels among groups of BMSC, Con-BMSC and Miro1Hi-BMSC
A: Western blot results of protein; B: RT-qPCR results of mRNA
图 6 BMSC、Con-BMSC及Miro1Hi-BMSC细胞株的迁移能力鉴定
A：水平迁移实验结果及统计分析(40×)，P＞0.05；B：垂直迁移实验结果及统计分析(100×)，P＞0.05
Figure 6. Migration capacity identification among groups of BMSC, Con-BMSC and Miro1Hi-BMSC
A: Results of scratch test and statistical analysis (40×), P＞0.05; B: Results of vertical migration test and statistical analysis (100×), P＞0.05
图 7 BMSC、Con-BMSC及Miro1Hi-BMSC细胞株的分化能力鉴定
A：成骨诱导分化26 d (200×)；B：成脂诱导分化20 d (400×)
Figure 7. Differentiative capacity identification among groups of BMSC, Con-BMSC and Miro1Hi- BMSC
A: 26 days after osteogenesis induces differentiation (200×); B: 20 days after adipogenesis induces differentiation (400×)
表 1 mRhot1引物序列
Table 1. Primer sequences of mRhot1
mRNA 引物序列 mRhot1-F CAACTTTGTACAAAAAAGCAGGCTGCCACCATGCGTGCGGGCCGAGTG mRhot1-R TCAAGCGTAATCTGGAACATCGTATGGGTATCGCTGTTTCAGTAGTGCTCTGTAC 
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表 2 反应体系构成
Table 2. Composition of the reaction system
组分 体积/μL 2⊆SYBR Green PCR MasterMix 10.0 引物 0.3 cDNA 3.0 去离子水 6.7
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[1] López-Doménech G,Covill-Cooke C,Ivankovic D,et al. Miro proteins coordinate microtubule- and actin-dependent mitochondrial transport and distribution[J]. EMBO J,2018,37(3): 321-336. doi: 10.15252/embj.201696380 [2] König T,Nolte H,Aaltonen MJ,et al. MIROs and DRP1 drive mitochondrial-derived vesicle biogenesis and promote quality control[J]. Nat Cell Biol,2021,23(12): 1271-1286. doi: 10.1038/s41556-021-00798-4 [3] Golan K,Singh AK,Kollet O,et al. Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma[J]. Blood,2020,136(23): 2607-2619. doi: 10.1182/blood.2020005399 [4] Han JB,Liu YX,Liu H,et al. Genetically modified mesenchymal stem cell therapy for acute respiratory distress syndrome[J]. Stem Cell Res Ther,2019,10(1): 386. doi: 10.1186/s13287-019-1518-0 [5] Wang JC,Liu X,Qiu Y,et al. Cell adhesion-mediated mitochondria transfer contributes to mesenchymal stem cell-induced chemoresistance on T cell acute lymphoblastic leukemia cells[J]. J Hematol Oncol,2018,11(1): 11. doi: 10.1186/s13045-018-0554-z [6] Ahmad T,Mukherjee S,Pattnaik B,et al. Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy[J]. EMBO J,2014,33(9): 994-1010. [7] Jackson MV,Morrison TJ,Doherty DF,et al. Mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS[J]. Stem Cells,2016,34(8): 2210-2223. doi: 10.1002/stem.2372 [8] Tang H,Kuhen KL,Wong-Staal F. Lentivirus replication and regulation[J]. Annu Rev Genet,1999,33: 133-170. doi: 10.1146/annurev.genet.33.1.133 [9] Hotta A,Ellis J. Retroviral vector silencing during iPS cell induction:an epigenetic beacon that signals distinct pluripotent states[J]. J Cell Biochem,2008,105(4): 940-948. doi: 10.1002/jcb.21912 [10] Fisher CL,Fisher AG. Chromatin states in pluripotent,differentiated,and reprogrammed cells[J]. Curr Opin Genet Dev,2011,21(2): 140-146. doi: 10.1016/j.gde.2011.01.015 [11] Ng HH,Surani MA. The transcriptional and signalling networks of pluripotency[J]. Nat Cell Biol,2011,13(5): 490-496. doi: 10.1038/ncb0511-490 [12] Wen S,Zhang HM,Li YS,et al. Characterization of constitutive promoters for piggyBac transposon-mediated stable transgene expression in mesenchymal stem cells (MSCs)[J]. PLoS One,2014,9(4): e94397. doi: 10.1371/journal.pone.0094397 [13] Liew CG,Draper JS,Walsh J,et al. Transient and stable transgene expression in human embryonic stem cells[J]. Stem Cells,2007,25(6): 1521-1528. doi: 10.1634/stemcells.2006-0634 [14] Li M,Zhong A,Wu YJ,et al. Transient inhibition of p53 enhances prime editing and cytosine base-editing efficiencies in human pluripotent stem cells[J]. Nat Commun,2022,13(1): 6354. doi: 10.1038/s41467-022-34045-7 [15] Tseng N,Lambie SC,Huynh CQ,et al. Mitochondrial transfer from mesenchymal stem cells improves neuronal metabolism after oxidant injury in vitro:the role of Miro1[J]. J Cereb Blood Flow Metab,2021,41(4): 761-770. doi: 10.1177/0271678X20928147 [16] Lahiri V,Klionsky DJ. Functional impairment in RHOT1/Miro1 degradation and mitophagy is a shared feature in familial and sporadic Parkinson disease[J]. Autophagy,2017,13(8): 1259-1261. doi: 10.1080/15548627.2017.1327512 [17] Safiulina D,Kuum M,Choubey V,et al. Miro proteins prime mitochondria for Parkin translocation and mitophagy[J]. EMBO J,2019,38(2): e99384. [18] Birsa N,Norkett R,Wauer T,et al. Lysine 27 ubiquitination of the mitochondrial transport protein Miro is dependent on serine 65 of the Parkin ubiquitin ligase[J]. J Biol Chem,2014,289(21): 14569-14582. doi: 10.1074/jbc.M114.563031 [19] Hsieh CH,Li L,Vanhauwaert R,et al. Miro1 marks parkinson’s disease subset and Miro1 reducer rescues neuron loss in parkinson’s models[J]. Cell Metab,2019,30(6): 1131-1140. doi: 10.1016/j.cmet.2019.08.023 [20] Shaltouki A,Hsieh CH,Kim MJ,et al. Alpha-synuclein delays mitophagy and targeting Miro rescues neuron loss in Parkinson’s models[J]. Acta Neuropathol,2018,136(4): 607-620. doi: 10.1007/s00401-018-1873-4 [21] Conejeros C,Parra V,Sanchez G,et al. Miro1 as a novel regulator of hypertrophy in neonatal rat cardiomyocytes[J]. J Mol Cell Cardiol,2020,141: 65-69. doi: 10.1016/j.yjmcc.2020.03.014 -
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