看看人家都是怎樣做lncRNA功能缺失性研究
LncRNA起初被認(rèn)為是不具有生物學(xué)功能的轉(zhuǎn)錄“垃圾”。但近年來的研究發(fā)現(xiàn)lncRNAs參與了許多重要生物學(xué)過程的調(diào)控，如基因組印記、染色質(zhì)修飾、轉(zhuǎn)錄激活或抑制、轉(zhuǎn)錄后調(diào)控等。lncRNA基因的過表達(dá)、缺失或突變與許多人類疾病有關(guān)；目前，大多數(shù)lncRNAs的功能仍然是未知的。因此，對于lncRNAs的功能研究已成為科研工作者關(guān)注的重點。
lncRNA功能研究一般采用gain/loss of function策略，即通過過表達(dá)或沉默lncRNA后觀察其對細(xì)胞增殖、凋亡、侵襲、遷移、克隆形成以及病毒轉(zhuǎn)錄復(fù)制等的影響。其中l(wèi)ncRNA功能缺失性研究的方法多種多樣，常見的是通過siRNA或shRNA轉(zhuǎn)染細(xì)胞，但很多時候發(fā)現(xiàn)細(xì)胞轉(zhuǎn)染效率很高，但干擾效率卻很低，這可能是方法選擇的問題。今天小編就帶大家一起看看人家都是怎樣做lncRNA功能缺失性研究~~~
lncRNA功能缺失性研究的方法
大家都清楚lncRNA在發(fā)揮調(diào)控功能時，會與DNA、RNA或蛋白分子形成緊密復(fù)合物，且lncRNA定位多樣性，選擇性分布在核內(nèi)和胞質(zhì)中，所謂位置決定功能，這些特性也因此決定了lncRNA功能研究的復(fù)雜性。
LncRNA定位更加多樣化
所以，選擇何種lncRNA功能缺失性研究方法還得看lncRNA定位在哪里。針對這個問題，Kim A等人給出的答案是：當(dāng)lncRNA位于核內(nèi)時，使用ASO效果更佳；當(dāng)lncRNA位于胞質(zhì)中時，使用RNAi效果更佳；當(dāng)lncRNA在細(xì)胞質(zhì)和細(xì)胞核中共定位時，兩種方法都可以（偷偷告訴你們，這里有一個叫做Smart Silencer的神器，它是RNAi與ASO的結(jié)合體，包含3條siRNA和3條ASO）。
lncRNA功能缺失性研究之Smart Silencer
小編整理了部分使用了lncRNA?Smart Silencer的文章，發(fā)現(xiàn)lncRNA不管是定位在細(xì)胞質(zhì)或細(xì)胞核，還是細(xì)胞質(zhì)與細(xì)胞核共定位，抑或是定位不清楚，均可使用lncRNA Smart Silencer，并且具有很好的沉默效果。
lncRNA smart silencer研究模型與細(xì)胞類型（部分統(tǒng)計結(jié)果）
1、An interferon-independent lncRNA promotes viral replication by modulating cellular metabolism. Science, 2017.
模型：病毒復(fù)制
細(xì)胞類型：A549
定位：細(xì)胞質(zhì)（FISH）
濃度：10nM
(A).?Schematic diagram showed human lncRNA-ACOD1?LOC105370268 locus. Labeled with half arrows are Q-PCR primers and short line indicated the target site of siRNA and antisense oligonucleotides in?Ribo??lncRNA Smart Silencer. (B).?Numbers of PR8?PFU in culture medium supernatant of A549 cells in left determined by plaque assays?on MDCK cell monolayers. N=3. *P < 0.05 (two-tailed Student’s t test).
2、A novel lnc-PCF promotes the proliferation of TGF-β1-activated epithelial cells by targeting miR-344a-5p to regulate map3k11 in pulmonary fibrosis. Cell death & disease, 2017.
模型：肺纖維化
細(xì)胞類型：RLE-6TN
定位：主要位于細(xì)胞質(zhì)（FISH）
濃度：50nM
(A). RNA FISH detecting the location?of endogenous lnc-PCF (red) in cells. The result showed that lnc-PCF mainly localized in the cytoplasm.?(B).?Cells were transfected with lnc-PCF smart silencer?/NC. And The smart silencer could significantly inhibit the expression of lnc-PCF.
3、Long non‐coding RNA NEAT1 promoted ovarian cancer cells’ metastasis through regulation of miR‐382‐3p/ROCK1 axial. Cancer science, 2018.
模型：卵巢癌
細(xì)胞類型：SKOV3, HO8910
定位：細(xì)胞核（FISH）
濃度：N/A
(A). NEAT1 and miR-382-3p were localized to a similar position in nucleus of SKOV3?and HO8910 cells as presented by an RNA FISH assay.?(B). Depression of NEAT1 by transfection of NEAT1 silencer?significantly suppressed SKOV3 and HO8910 cells’ migration and invasion abilities as determined by a transwell assay.
4、A New Long Noncoding RNA ALB Regulates Autophagy by Enhancing the Transformation of LC3BI to LC3BII during Human Lens Development. Molecular Therapy-Nucleic Acids, 2017.
模型：人類晶狀體發(fā)育/自噬
細(xì)胞類型：HLECs
定位：細(xì)胞質(zhì)，小部分位于細(xì)胞核
濃度：N/A
(A and B). Quantitative real-time PCR analysis of lncRNA?ALB and LC3B in HLECs with lncRNA ALB?smart silencer?or NC. (C). Quantitative real-time PCR analysis of lncRNA ALB?expression in the nucleus and cytoplasm of HLECs.
5、Long non-coding RNA CEBPA-AS1 correlates with poor prognosis and promotes tumorigenesis via CEBPA/Bcl2 in oral squamous cell carcinoma. Cancer biology & therapy, 2018.
模型：口腔鱗狀細(xì)胞癌
細(xì)胞類型：Tca8113、Cal-27
定位：細(xì)胞質(zhì)和細(xì)胞核周圍的細(xì)胞區(qū)室（FISH）
濃度：N/A
(A and B). Localization of CEBPA-AS1 by RNA-FISH in OSCC cells. Nuclei are stained blue?(DAPI), 18S (Cytoplasm positive), U6 (Nuclei positive) and CEBPA-AS1 labeled with CY3 are?stained red. Magnification is 60 × with 4.5 × zoom.?(C). qRT-PCR detects the CEBPA-AS1 smart silencers?transfection efficiency.
6、Identification of cardiac long non-coding RNA profile in human dilated cardiomyopathy. Cardiovascular research, 2018.
模型：擴(kuò)張型心肌病
細(xì)胞類型：HCMs, HCFs，HCMECs
定位：N/A
濃度：50nM
Level of lncRNAs in HCMs transfected with the indicated siRNAs (smart silencer). n=5, *p<0.05 vs. si-control.
更多lncRNA Smart Silencer應(yīng)用案例見下表：
lncRNA功能缺失性研究之ASO
ASO（反義寡核酸）是經(jīng)化學(xué)修飾的單鏈RNA&DNA雜合體，主要（但并非完全）存在于細(xì)胞核中，能干擾核內(nèi)的lncRNA，依賴RNase H發(fā)揮功能，可直接用于體內(nèi)實驗(in vivo純化)。通過部分已發(fā)表文章發(fā)現(xiàn)，當(dāng)lncRNA定位于細(xì)胞核時，多數(shù)客戶會選擇ASO；細(xì)胞核與細(xì)胞質(zhì)共定位時，也會選擇ASO，另外再結(jié)合siRNA一起使用，沉默效果顯著。
1、Insertion of an Alu element in a lncRNA leads to primate-specific modulation of alternative splicing. Nature Structural and Molecular Biology, 2016.
模型：可變剪切
細(xì)胞類型：human (HeLa，293T)、mouse (N2a，3T3)
定位：細(xì)胞核（FISH）
濃度：N/A
(A). Double FISH of human and mouse 5S-OT RNA (red) and 5S rDNA genomic loci (green) in human (HeLa,?n = 42) and mouse (N2a, n = 36) cells. 5S-OT is localized in the nucleus (blue; DAPI nuclear stain); mouse 5S-OT is confined at the genomic loci?of 5S rDNA, whereas human 5S-OT is not.?(B). Knocking down 5S-OT in human (Hela and 293T) cells with ASO?did not affect the total levels of 5S rRNA, but decreased the?transcription of 5S but not the 5S-OT.
2、LncRNA Dum interacts with Dnmts to regulate Dppa2 expression during myogenic differentiation and muscle regeneration. Cell Research, 2015.
模型：肌肉再生/肌發(fā)生
細(xì)胞類型：C2C12
定位：細(xì)胞核與細(xì)胞質(zhì)共定位（FISH）
濃度：100 nM
(A). Visualization?of Dum or U1 in C2C12 myoblasts by RNA-FISH. Scale bar, 50 μm. (B). Knockdown of?Dum by an ASO oligo?in C2C12 cells decreased the expression of Dum and the indicated myogenic genes, myogenin, MyHC?and troponin.
更多ASO應(yīng)用案例見下表：
lncRNA功能缺失性研究之siRNA
siRNA是常見的lncRNA基因沉默方法，需要通過RISC系統(tǒng)發(fā)揮沉默作用，而RISC主要存在于細(xì)胞質(zhì)中，所以理論上siRNA對核內(nèi)lncRNA進(jìn)行有效沉默的效果不如在細(xì)胞質(zhì)中的沉默效果好。從已發(fā)表的研究可發(fā)現(xiàn)，當(dāng)lncRNA是細(xì)胞質(zhì)定位或是細(xì)胞質(zhì)與細(xì)胞核共定位時，siRNA有很好的沉默效果；除此之外，部分定位于細(xì)胞核的lncRNA，使用siRNA也可獲得較好的沉默效果。
1、LncRNA KCNQ1OT1 regulates proliferation and cisplatin resistance in tongue cancer via miR-211-5p mediated Ezrin/Fak/Src signaling. Cell death & disease, 2018.
模型：舌癌
細(xì)胞類型：CAL27, SCC9
定位：細(xì)胞質(zhì)（ISH）
濃度：50 nM
(A). Representative?images of in situ hybridization for KCNQ1OT1?in paraffin-embedded TSCC tissues and adjacent normal tissues (scale bar: 100 μM).?(B). Efficiency of KCNQ1OT1 knockdown in CAL27-res/CAL27 and SCC9-res/SCC9 cells by two siRNAs?was verified by RT-qPCR.
2、LncRNA ZFAS1 as a SERCA2a Inhibitor to Cause Intracellular Ca2+ Overload and Contractile Dysfunction in a Mouse Model of Myocardial Infarction. Circulation research, 2018.
模型：心肌梗死
細(xì)胞類型：neonatal mouse?cardiomyocytes (NMCMs)
定位：細(xì)胞質(zhì)與細(xì)胞核共定位（ISH）
濃度：100 nM
(A). Representative images of In Situ Hybridization(ISH) in NMCMs showing an increase in ZFAS1 expression after hypoxia treatment for 12 h. Note that?ZFAS1 was distributed evenly in both cytosol and nucleus.?(B). Mitigation of increased intracellular Ca2+?concentration ([Ca2+]i) by ZFAS1 siRNA?(siZFAS1; n=13~19; left panel) and restoration of the decreased?rate of Ca2+?reuptake into sarcoplasmic reticulum (SR) by siZFAS1?(n=9~13; right panel) in NMCMs?exposed to hypoxic environment.
3、Quantitative proteomics reveals that long non-coding RNA MALAT1 interacts with DBC1 to regulate p53 acetylation. Nucleic acids research, 2017.
模型：癌癥
細(xì)胞類型：HepG2
定位：細(xì)胞核（已發(fā)表文章數(shù)據(jù)）
濃度：N/A
The efficiencies of MALAT1 knockdown using two?siRNAs?were examined by qRT-PCR.
4、Long noncoding RNA derived from CD244 signaling epigenetically controls CD8+ T-cell immune responses in tuberculosis infection. PNAS, 2015.
模型：結(jié)核病感染
細(xì)胞類型：CD8+ T細(xì)胞
定位：細(xì)胞核（FISH）
濃度：50nM
(A). Confocal microscopic images of RNA FISH assay of lncRNACD244?and immunofluorescence analysis of EZH2 show that EZH2 colocalizes with?lncRNA-CD244 in nucleus of CD8+ T cells from patients with active TB.?(B). Representative CBA assays of PBMCs from a patient with active TB showing that, compared with siRNA-Ctrl or transfection medium, siRNA-lncRNA-CD244?induced significant increases in concentrations of IFN-γ/TNF-α. (C and D).?Pooled data showing the concentrations of IFN-γ/TNF-α upon transfection of indicated siRNAs?or medium (n=7).
更多siRNA應(yīng)用案例見下表：
大概總結(jié)一下就是：
1、當(dāng)lncRNA定位于細(xì)胞質(zhì)時，可選擇Smart Silencer、siRNA
2、當(dāng)lncRNA定位于細(xì)胞核時，可選擇Smart Silencer、ASO，不推薦siRNA
3、當(dāng)lncRNA定位于細(xì)胞核與細(xì)胞質(zhì)時，可選擇Smart Silencer、siRNA、ASO
4、當(dāng)lncRNA定位不清楚時，可選擇Smart Silencer
哪種lncRNA功能缺失性研究產(chǎn)品適合您？
銳博生物可提供LncRNA siRNA、LncRNA Smart Silencer、LncRNA ASO等多種高效的lncRNA抑制工具，請根據(jù)下表選擇適合您需求的lncRNA沉默產(chǎn)品。
lncRNA功能研究的急先鋒——lncRNA FISH Probe
由銳博生物自主研發(fā)的lncRNA FISH檢測探針，創(chuàng)新性地改進(jìn)了lncRNA分子的特異性核酸探針數(shù)量及標(biāo)記方式，極大地提高了探針靈敏度，能滿足細(xì)胞中l(wèi)ncRNA準(zhǔn)確定位要求，結(jié)合激光共聚焦顯微鏡可清晰地展示lncRNA的分布情況，為lncRNA功能及作用機(jī)制研究提供強有力的工具。
● 定位準(zhǔn)確：核內(nèi)核外清晰明了，功能機(jī)制更易確定；
● 靈敏度高：信號放大倍數(shù)平均達(dá)到160倍，輕松定位低拷貝數(shù)lncRNA；
● 特異性強：集群式的FISH探針最大程度地保證FISH信號的高特異性。
采用激光共聚焦顯微鏡觀察兩個lncRNA FISH實驗結(jié)果，18S與U6為內(nèi)參。紅色：Cy3標(biāo)簽，顯示lncRNA FISH Probe標(biāo)記的lncRNA及內(nèi)參；藍(lán)色：Hoechst染色，顯示細(xì)胞核。圖中清晰顯示，18S幾乎均位于細(xì)胞質(zhì)，而U6幾乎均位于細(xì)胞核。lncRNA-1主要分布在細(xì)胞質(zhì)，而lncRNA-2分布于細(xì)胞質(zhì)和細(xì)胞核中。
