Biological Effects of CRISPR/Cas9-mediated Knockout of RAB27A in SCLC

Doğu Karadeniz Sağlık Bilimleri Dergisi Pub Date : 2023-10-31 DOI:10.59312/ebshealth.1367257

Kubilay INCI, Büşra ÇELİKKAYA, Nesrin İREP, Aziz GÜLTEKİN, Onur TOKGÜN

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Abstract

Small cell lung cancer (SCLC) is characterized by rapid growth and early metastasis. Identifying new molecular targets are important in the pathogenesis of SCLC in order to develop new treatment strategies. RAB27A is the critical protein for intracellular exosome trafficking and is a driver of tumour progression. However, demonstrating the potential impact of suppressing RAB27A in SCLC as therapeutic approach is an important deficiency. RAB27A gene knockout SCLC cell lines were generated using a CRISPR/cas9 system. qRT-PCR, Western blotting and Sanger sequencing were performed to confirm RAB27A knockout in SCLC cells. TEM and EXOCET assays were used to detect the alteration of exosomes. Proliferation and colony formation were detected by MTT and microscopy. Subsequently, we intrapulmonally injected N417 and H524 SCLC cells(control and RAB27A knockout for each cell) into SCID mice. The effects of RAB27A knockout on mouse tumor model were analysed using 18F-FDG PET/CT scans.Knocking out RAB27A significantly decreased the expression of CD9, CD63, Tsg101, exosome secretion and exosomal protein in SCLC(p

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CRISPR/ cas9介导的RAB27A敲除在SCLC中的生物学效应

小细胞肺癌(SCLC)具有快速生长和早期转移的特点。在SCLC的发病机制中发现新的分子靶点对于制定新的治疗策略具有重要意义。RAB27A是细胞内外泌体运输的关键蛋白，是肿瘤进展的驱动因素。然而，证明抑制RAB27A作为治疗方法在SCLC中的潜在影响是一个重要的缺陷。使用CRISPR/cas9系统生成RAB27A基因敲除的SCLC细胞系。采用qRT-PCR、Western blotting和Sanger测序证实SCLC细胞中RAB27A基因敲除。使用TEM和EXOCET检测外泌体的改变。MTT和显微镜检测细胞增殖和菌落形成。随后，我们向SCID小鼠肺内注射N417和H524 SCLC细胞(每个细胞分别为对照和RAB27A敲除)。采用18F-FDG PET/CT扫描分析RAB27A基因敲除对小鼠肿瘤模型的影响。敲除RAB27A可显著降低SCLC中CD9、CD63、Tsg101、外泌体分泌和外泌体蛋白的表达(p

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Doğu Karadeniz Sağlık Bilimleri Dergisi

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