RAB22A Promotes Chemoresistance Of Colorectal Cancer By Regulating Exosome Release

Objective: Chemotherapy is one of the main treatments of colorectal cancer(CRC),but chemoresistance often occurs and limits the drug efficacy.However,the mechanism is still poorly understood.Therefore,investigating the underlined chemoresistance mechanism is of theoretical and clinical value.We found that RAB22A is a novel candidate oncogene in our previous study,however,the detailed role of RAB22A in chemoresistance remains unclear.Here,we intend to clarify the potential mechanism of RAB22A which promotes CRC chemoresistance in vitro.This study will provide novel ideas and strategies to overcome CRC chemoresistance.Methods: The diagnosed advanced CRC patients who had received 5-FU/LOHP-based palliative chemotherapy were selected retrospectively.Then,the blood and histopathological sections samples were collected.Plasma exosomes were extracted by using commercial kits,and the size and concentration of exosomes were detected using Nanosight NS 300 system.The expression of RAB22A was detected by immunohistochemistry(IHC),and the correlations among the number of plasma exosomes,RAB22A expression of tumor tissues and the effect of chemotherapy were analysed.RAB22A levels of different CRC cells were detected by Western blot,and exosomes were extracted from cell culture supernatant and detected by Nanosight NS 300 system.Correlation analyses were performed among the IC50,RAB22A levels and the number of cell culture supernatant exosomes from different CRC cells.The p WPXL-NC and p WPXL-RAB22A plasmids were cotransfected into SW480 cells along with the packaging plasmid to establish RAB22A stable-overexpression cell lines.The p LKD-CMV-NC or p LKD-CMV-sh RAB22A plasmids were cotransfected into SW620 cells along with the packaging plasmid to establish RAB22A stable-knockdown cell lines.Next,the correlations among the IC50,RAB22A expression levels and the number of exosomes from CRC cell culture supernatant were analysed.In addition,the expression of celluar surface CD63 was detected by flow cytometry in these stable-expression CRC cells.The secretion of exosomes on the surface of these stable-expression CRC cells were observed by immunofluorescence.And co-localization of RAB22A and exosomes in CRC cells was also detected using immunofluorescence.The poteintial effector molecules of RAB22A were screened using Gene function prediction tools.Results:(1)In clinical CRC samples,we found that the concentration of plasma exosomes was higher compare with normal controls and CRC cells with higher RAB22A expression were more resistant to chemotherapeutic drugs.(2)There was a positive correlation among RAB22A levels,cell chemoresistance and the secretion of exosomes in different CRC cell lines.(3)Alteration of RAB22A levels in CRC cells could affect secretion of exosomes and chemoresistance of cells.(4)The co-localization of RAB22A and exosomes was found by immunofluorescence assays,and the increase or decrease of RAB22A levels could promote or inhibit exosomes secretion.(5)A total of 21 effector molecules which have potential interactions with RAB22A were predicted by gene function prediction tools.Then,4 possible effector genes(CDC14B,EEA1,RAB21 and RAB5A)were further screened based on the expression levels in different CRC cell lines and CRC patients from the TCGA database.(6)PRK7-Flag-RAB22A plasmid was successfully constructed.And 6 potential effector proteins(RAB2A,EIF5A2,RAB31,RAB6 C,TBC1D15 and EEA1)were screened according to the result of IP experiment.Conclusion: The increase of RAB22A expression in CRC could promote the secretion of exosomes,thereby making CRC cells resistant to chemotherapy.