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Cancer-associated fibroblast exosome LINC00355 promotes epithelial-mesenchymal transition and chemoresistance in colorectal cancer through the miR-34b-5p/CRKL axis

Cancer Gene Therapy volume 31, pages 259–272 (2024)Cite this article

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Abstract

This study was designed to investigate the role and mechanism of cancer-associated fibroblasts (CAFs)-derived exosomes (CAFs-exo) in metastatic and chemoresistant colorectal cancer (CRC). First, CAFs and normal fibroblasts (NFs) were isolated from CRC tissues and histologically normal adjacent tissues. Then, CAFs-exo and NFs-exo were separated with the help of ultracentrifugation. Next, the morphology, diameter and marker expression of exos were evaluated by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot, respectively. Besides, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of LINC00355, miR-34b-5p, and CRKL in clinical tissue samples, CRC cells, fibroblasts and exos; MTT assay and cell colony formation assay to assess the chemoresistance and colony formation ability of CRC cells, respectively. Subsequently, the targeting relationship among LINC00355, miR-34b-5p, and CRKL (a target gene of miR-34b-5p) was verified by Luciferase reporter assay; and the binding relationship between LINC00355 and miR-34b-5p was assessed by a pull-down assay. Finally, the expression of epithelial-mesenchymal transition (EMT)-related proteins, and CRKL in cells or exos were detected using western blot. After a series of treatments, CAFs and NFs, CAFs-exo and NFs-exo were successfully isolated and identified. It could be observed that CAFs-exo promoted EMT, colony formation and multidrug resistance in CRC cells by secreting LINC00355. Further studies demonstrated that CAFs-exo-secreted LINC00355 increased the expression of CRKL via inhibiting the expression of miR-34b-5p, thereby enhancing chemoresistance and promoting EMT progression in CRC cells. Collectively, CAFs-exo-derived LINC00355 promotes EMT and chemoresistance in CRC by regulating the miR-34b-5p/CRKL axis.

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Fig. 1: The schematic of study design.
Fig. 2: Identification of fibroblasts and fibroblast exos.
Fig. 3: CAFs-exo promotes chemoresistance and EMT progression in CRC cells.
Fig. 4: LINC00355 secreted by cancer-associated fibroblasts exosome transferred to colorectal cancer cells.
Fig. 5: LINC00355 secreted by CAFs-exo promotes chemoresistance and EMT progression in CRC cells.
Fig. 6: LINC00355 regulates CRKL expression via sponging miR-34b-5p.
Fig. 7: LINC00355 secreted by CAFs-exo promotes chemoresistance and EMT process in CRC cells via the miR-34b-5p/CRKL axis.
Fig. 8: Schematic representation of the potential molecular mechanisms by which exosomal LINC00355 regulates CRC progression and chemical resistance.

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Data availability

The data used to support the findings of this study are available from the corresponding author upon request.

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Funding

This research was supported by the Henan Medical Science and Technology Public Relations Project (SBGJ202102121).

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Authors and Affiliations

  1. Department of Colorectal Anal Surgery, The First Affiliated Hospital of Zhengzhou University, 450052, Zhengzhou, Henan Province, China

    Jun-Hong Hu & Yu-Hang Wang

  2. Department of Colorectal Anal Surgery, Huaihe Hospital of Henan University, Kaifeng, Henan Province, China

    Hong-Na Tang

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J-HH conceptualized and designed the study and drafted the initial manuscript. H-NT and Y-HW collected the data and carried out the initial analyses. J-HH critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.

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Correspondence to Jun-Hong Hu.

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Hu, JH., Tang, HN. & Wang, YH. Cancer-associated fibroblast exosome LINC00355 promotes epithelial-mesenchymal transition and chemoresistance in colorectal cancer through the miR-34b-5p/CRKL axis. Cancer Gene Ther 31, 259–272 (2024). https://doi.org/10.1038/s41417-023-00700-4

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