Küçük Hücreli Akciğer Kanseri’nde RAB27A’nın CRISPR/Cas9 Sistemi Aracılığıyla Susturulmasının Biyolojik Etkileri

Özet

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<0.0001). We found that RAB27A knockout dramatically reduced proliferation and colony formation in SCLC cells(p< 0.001, p<0.0001). Furthermore, RAB27A knockout decreased proliferation and especially metastasis in mouse model (p<0,0001). These studies clearly demonstrated that RAB27A plays an important role in the pathogenesis of SCLC, and targeting the RAB27A gene in SCLC cell lines significantly reduces the activity of the exosomal pathway. RAB27A, therefore, can be a promising cancer therapeutic strategy.

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<0.0001). We found that RAB27A knockout dramatically reduced proliferation and colony formation in SCLC cells(p< 0.001, p<0.0001). Furthermore, RAB27A knockout decreased proliferation and especially metastasis in mouse model (p<0,0001). These studies clearly demonstrated that RAB27A plays an important role in the pathogenesis of SCLC, and targeting the RAB27A gene in SCLC cell lines significantly reduces the activity of the exosomal pathway. RAB27A, therefore, can be a promising cancer therapeutic strategy.