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CRISPR/Cas9辅助的长链非编码RNA敲低在女性癌细胞系中可增加Mir-143 。

Long non-coding RNA Knockdown Assisted by CRISPR/Cas9 in Female Cancer Cell Lines Increases Mir-143 .

作者信息

Montazeri-Najafabadi Behshad, Doosti Abbas, Kiani Jafar

机构信息

Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.

Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.

出版信息

Iran J Public Health. 2024 Apr;53(4):934-946. doi: 10.18502/ijph.v53i4.15571.

Abstract

BACKGROUND

The lncRNAs has been linked to several malignancies, including breast cancer. Our objective was to investigate the impact of urothelial carcinoma associated 1 () on cellular growth and death by a CRISPR/Cas9 knockdown technique.

METHODS

In 2020, the CHOPCHOP program was utilized to design two sgRNAs targeting the ne. sgRNA1 and sgRNA2 were inserted into two different CRISPR plasmids to produce two recombinant plasmids. These recombinant plasmids were simultaneously transfected into MCF-7 and MDA-MB 231 carcinoma of the breast cells. Proliferation and apoptosis were compared using the MTT test, CCK-8 assay, and flow cytometry evaluation. RNA-hybrid software, quantitative reverse transcription PCR, and luciferase assays were utilized to confirm the relationship between and miR-143.

RESULTS

Proliferated cells were less active in MTT and CCK-8 tests and fellow cytometry analysis. The PX459-sgRNA1,2 group had elevated levels of the cancer biomarker gene expression (<0.001). When WT- and miR-143 were co-transfected, the luciferase activity was drastically decreased.

CONCLUSION

One very effective method of regulating cellular proliferation in vitro is the deletion of , which CRISPR/Cas9 accomplishes.

摘要

背景

长链非编码RNA(lncRNAs)已与包括乳腺癌在内的多种恶性肿瘤相关联。我们的目的是通过CRISPR/Cas9敲低技术研究尿路上皮癌相关1()对细胞生长和死亡的影响。

方法

2020年,利用CHOPCHOP程序设计了两个靶向ne的sgRNA。将sgRNA1和sgRNA2插入两个不同的CRISPR质粒中,以产生两个重组质粒。将这些重组质粒同时转染到MCF-7和MDA-MB 231乳腺癌细胞中。使用MTT试验、CCK-8测定和流式细胞术评估比较细胞增殖和凋亡情况。利用RNA杂交软件、定量逆转录PCR和荧光素酶测定来确认与miR-143之间的关系。

结果

在MTT和CCK-8试验以及流式细胞术分析中,增殖细胞的活性较低。PX459-sgRNA1,2组癌症生物标志物基因表达水平升高(<0.001)。当野生型和miR-143共转染时,荧光素酶活性急剧下降。

结论

CRISPR/Cas9实现的缺失是体外调节细胞增殖的一种非常有效的方法。

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