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本文所用到的细胞系Calu-1和H1299均来自于ATCC细胞库。细胞培养液由DMEM高糖培养基(Gibco,澳洲)、10%胎牛血清(Gibco,澳洲)、另外添加1% 青霉素和链霉素混合液(其中青霉素工作浓度为100 U/mL,硫酸链霉素的工作浓度为0.1 mg/mL)配置而成。细胞在37 ºC培养箱培养,CO2的体积分数为5%。
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本文中所有的射线处理均为X射线。主要采用苏州大学放射医学与辐射防护国家重点实验室仪器平台的PXi X-RAD 225Cx/SmART小动物放疗模拟定位机进行(Precision X-Ray Inc., 美国)。管电压225 kV,照射功率3 kW,开放野剂量率为4.48 Gy/min。细胞照射剂量为4 Gy,照射后2 h收集细胞样品。
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细胞密度达到80%后,加入100 nmol/L Swinholide A处理6 h,然后用RNA提取试剂盒(碧云天,中国)收集总RNA;样品总RNA利用NanoDrop ND-2000 (Thermo Scientific,美国)定量后逆转录为cDNA;进而用Cyanine-3-CTP(Cy3)标记的cDNA;标记好的cDNA和芯片杂交,洗脱后利用Agilent Scanner G2505C(Agilent Technologies)扫描得到原始图像;采用Feature Extraction 软件 (version10.7.1.1, Agilent Technologies, 美国)进行数据分析和处理;统计变化差异并采用T检验进行差异lncRNA筛选,筛选的标准为上调或者下调倍数变化值
$\geqslant$ 2.0倍且P$ \leqslant $ 0.05。 -
细胞在4 Gy照射后2 h,将TRIzol试剂(Invitrogen,美国)按照每1×106个细胞加入1 mL的量加入培养皿中。将加入TRIzol裂解的细胞样品在冰上静置2 min后,用移液器反复吹打,直至所有的细胞都被裂解完毕。用RNA提取试剂盒提取TRIzol中的总RNA并用Nanodrop测定总RNA浓度。抽取1 μg RNA,采用All-in One TM RNA qRT-PCR检测试剂盒(Genecopoeia, 美国)将RNA逆转录为cDNA备用。采用SYBR Green PCR master Mix荧光定量试剂盒(GeneCopoeia,美国)进行测定。所有目的基因或RNA的引物由上海生工生物科技有限公司(上海,中国)合成,PCR引物序列见表1。PCR程序采用Chromo4系统(Bio-Rad, 美国),然后分别预变性95 ºC 5 min、变性95 ºC 5 s、退火60 ºC 20 s、延伸72 ºC 20 s,共设计42个循环。用Ct数值比较法检测基因的表达差异。设置独立的3次生物学重复。
lncRNA或
基因名称引物序列5’-3’ 扩增产物
长度(bp)GAPDH 正向:ATTCCACCCATGGCAAATTCC
反向:GACTCCACGACGTACTCAGC145 XR_923281 正向:GCCAAGGTTTGTAGCTGTGC
反向:TGGTTTCAGGACAACGCAGT276 XR_930632 正向:AAGGGAAAGTCCTGAACGCC
反向:GCAACTCTCCTCCTACTCGC152 XR_942150 正向:AGCACAGACCTCAGGGTGAA
反向:TATGTGGAGGCTGACTCCCT732 XR_001755972 正向:CAAAGGGAAGCCGTAGCAGA
反向:GCTCAGACCAGACAGGGTTC266 XR_002956697 正向:AGGCTGCCTTAAAACCCCAG
反向:TGAGGTCAACACGATTTCCGT114 XR_923639 正向:GGTTCCATCCCATCCGCCT
反向:AAAAACCGCAGAGTCCACCT202 XR_01739734 正向:AGCGACAAAGACGGTTTCCT
反向:ACCTTTTCCGCCAGTGCATA264 XR_923426 正向:GAAGTGGGACAGGGAGCATT
反向:CAGTAGGCTTGTTCCTTGCC129 -
将细胞种植在预置15 mm直径圆形玻璃片的培养皿中,细胞在4 Gy X射线照射后2 h或100 nmol/L Swinholide A处理后6 h后,在培养皿中加入用预热的4%多聚甲醛室温固定15 min后,使用PBS润洗培养皿并覆盖细胞,向培养皿中加入工作浓度5 μg/mL 的Fluo488标记的鬼笔环肽(Phalloidin, CST,美国)染色20 min。最后,用PBS洗去鬼笔环肽染色液,用DAPI (Sigma,美国)染核后,将15 mm玻璃片取出并在共聚焦显微镜下镜检、拍照、获取实验数据。
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对获取的数据进行双尾Student’s t检验比较。目标基因或RNA的表达统计为平均值±SEM。在所有实验中,P值小于0.05被认为有显著差异,其中P值小于0.05标记为*,P值小于0.01标记为**,P值小于0.001标记为***。使用GraphPad Prism 7软件(GraphPad Software Inc., 美国)作图并统计。
Long Non-coding RNA XR_923426 Participates in the Regulation of Microfilament Dynamics Induced by Ionizing Radiation
doi: 10.11804/NuclPhysRev.39.2022061
- Received Date: 2022-05-18
- Rev Recd Date: 2022-06-05
- Publish Date: 2022-12-20
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Key words:
- ionizing radiation /
- long non-coding RNA /
- microfilament /
- cytoskeleton
Abstract: Microfilament is a multi-functional sub-cell structure, and it is also a sensor for ionizing radiation. In order to clarify the regulatory effect of long non-coding RNAs(lncRNA) in the alteration of microfilament dynamics caused by ionizing radiation, Swinholide A was used to depolymerize the microfilament and then, the lncRNA chip was used to detect the differentially expressed lncRNAs. Microfilament staining and network/structure analysis were used to evaluate the changes in microfilament skeleton. It was found that the expression of lncRNA XR_923426 was decreased after the microfilament depolymerization. Meanwhile, the overexpression of lncRNA XR_923426 could significantly alleviate the microfilament depolymerization caused by ionizing radiation. This provides a new clue for the research on the regulatory relationship between lncRNA targeted microfilament dynamics and radiation induced tumor death or metastasis, which is expected to develop into a new target for tumor treatment or normal tissue protection.
Citation: | Yu ZHANG, Luheng SHEN, Guangming ZHOU, Hailong PEI. Long Non-coding RNA XR_923426 Participates in the Regulation of Microfilament Dynamics Induced by Ionizing Radiation[J]. Nuclear Physics Review, 2022, 39(4): 505-511. doi: 10.11804/NuclPhysRev.39.2022061 |