Abstract:

Objective To analyze the effect of expression of microRNA-17-5p（miR-17-5p） in the colorectal cancer cell-derived exosomes （Exo） on the chemotherapy sensitivity of colorectal cancer cells，and to clarify its possible mechanism. Methods Real-time fluorescence quantitative PCR （qRT-PCR） method was used to detect the expression levels of miR-17-5p in the human colorectal cancer HCT116 cells， CT26 cells， LoVo cells， HT29 cells， SW620 cells， SW480 cells， and human normal colorectal mucosal epithelial HIEC cells.The CT26 cells were divided into control group，miR-NC inhibitor group， and miR-17-5p inhibitor group， and the exosomes were extracted；the morphology of the Exo was observed under transmission electron microscope； the distribution of particle size was detected by nanoparticle tracking analysis； the expressions levels of marker proteins CD9， CD63， apoptosis-inducing factor 6 interacting protein （Alix），and tumor susceptibility gene 101 （TSG101） in the Exo were detected by Western blotting method； the expression level of miR-17-5p in the Exo was detected by RT-qPCR method. The CT26 cells were divided into control group， Exo group， Exo-miR-NC inhibitor group， and Exo-miR-17-5p inhibitor group. The CT26 cells were treated with CT26 cell-derived Exo in different transfection groups， and the uptake of Exo by CT26 cells were observed by Exo green fluorescence marker PKH67 dye trace method； MTT assay was used to detect the inhibitiory rates of proliferation of the CT26 cells after treated with 1.25， 2.50， 5.00， 10.00， 20.00， and 40.00 mg·L^－1 5-fluorouracil（5-FU）； flow cytometry was used to detect the apoptotic rates of the CT26 cells in various groups； cell immunofluorescence staining was used to observe the expression intensities of microtubule-associated protein 1B light chain 3 （LC3B） in the CT26 cells in various groups； Western blotting method was used to detect the expression levels of autophagy-related proteins microtubule-associated protein light chain 3Ⅱ （LC3-Ⅱ）， microtubule-associated protein light chain 3Ⅰ （LC3-Ⅰ）， autophagy effector protein Beclin-1， and P62 proteins in CT26 cells in various groups，and the ratio of LC3-Ⅱ/LC3-Ⅰ was calculated. Results The expression levels of miR-17-5p in the human colorectal cancer HCT116， CT26， LoVo， HT29， SW620， and SW480 cells were significantly higher than that in the HIEC cells （P<0.05）； the isolated particles showed typical spherical vesicles with a peak particle size at about 140 nm， and the CD9， CD63， Alix and TSG101 proteins were all significantly expressed， indicating that Exo was successfully isolated.Compared with control group，the expression level of miR-17-5p in the Exo in miR-17-5p inhibitor group was significantly decreased （P<0.05）； compared with control group， obvious PKH67 staining could be observed around the CT26 cells in Exo group， Exo-miR-NC inhibitor group， and Exo-miR-17-5p inhibitor group， indicating that the CT26 cells could take in the Exo. Compared with control group， the inhibitory rate of proliferation and the apoptotic rate of the CT26 cells in Exo group were decreased after treated with 1.25， 2.50， 5.00， 10.00， 20.00，and 40.00 mg·L^－1 5-FU （P<0.05）， the intracellular LC3B fluorescence intensity was increased （P<0.05）， the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression level of Beclin-1 protein were increased （P<0.05）， while the expression level of P62 protein was decreased（P<0.05）； compared with Exo group， the inhibitory rate of proliferation and apoptotic rate of the CT26 cells in Exo-miR-17-5p inhibitor group were increased after treated with 1.25， 2.50， 5.00， 10.00， 20.00，and 40.00 mg·L^－1 5-FU（P<0.05）， the intracellular LC3B fluorescence intensity was decreased （P<0.05）， the ratio of LC3-Ⅱ/LC3-Ⅰand the expression level of Beclin-1 protein were decreased （P<0.05），and the expression level of P62 protein was increased （P<0.05）. Conclusion The inhibition of expression of miR-17-5p in Exo derived from colorectal cancer cells can improve the chemosensitivity of the colorectal cancer cells， and the mechanism may be related to the inhibition of cell autophagy levels.

Key words: Colorectal neoplasm, Exosomes, MicroRNA-17-5p, Chemosensitivity, Cell autophagy