HNRNPA2B1 Regulates Hypoxia-Related Tumor Angiogenesis In Hilar Cholangiocarcinoma Via Exosomal MiR-155-5p

Micro RNAs(miRs)are playing an increasingly important role in biological processes such as carcinogenesis,cancer inhibition,and drug resistance.In recent years,many reports have confirmed the association between miRs and the development of hilar cholangiocarcinoma,and miRs-based therapy is likely to become an effective treatment for hilar cholangiocarcinoma.It is well known that hypoxia is one of the important biological features of malignant solid tumors.As a malignant solid tumor,tumor cells will secrete a large number of miRs-rich exosomes under hypoxic conditions to adapt to this change to complete their biological behaviors such as angiogenesis,proliferation and metastasis.Our group found that the exosomes secreted by hilar cholangiocarcinoma cells under hypoxic conditions not only increased in particle size and quantity,but also changed the expression profile of miRs within the exosomes.To date,although the mechanisms regulating the differential expression of miRs within tumor exosomes are not yet understood,RNA-binding protein-mediated exosome sorting of miRs has been demonstrated.It has been found that heterotrimeric nuclear ribonucleoprotein A2B1(HNRNPA2B1),as an RNA-binding protein can mediate the loading of exosomal miRs through ubiquitination and quantitative changes.We sought to clarify whether HNRNPA2B1 is involved in the sorting of specific miRs in the exosomes of hilar cholangiocarcinoma cells under hypoxic conditions and exerts a pro-tumor angiogenic effect by bioinformatics analysis methods and corresponding experiments.Objective: To investigate the mechanisms associated with the entry of miRs into the exosomes of hilar cholangiocarcinoma angiogenesis under hypoxia.To elucidate whether HNRNPA2B1 is involved in the exosomal miR-155-5p sorting process in hilar cholangiocarcinoma cells under hypoxia and whether this process is involved in tumor angiogenesis.Methods:In this study,we screened the miRs differentially expressed in the exosomes of hilar cholangiocarcinoma cells under hypoxic conditions by bioinformatics,and the most effective miR associated with HNRNPA2B1 binding to tumor angiogenesis was miR-155-5p.WB and NTA were used to verify the extraction of hypoxic and normoxic QBC939 cell exosomes.WB and q RT-PCR were used to verify the expression of HNRNPA2B1 in hypoxic and normoxic conditions.The binding status of HNRNPA2B1 and miR-155-5p under hypoxia and normoxia was observed by RNA immunoprecipitation(RIP).The expression of cellular and exosomal miR-155-5p was detected by q RT-PCR after siRNA interference with HNRNPA2B1 in QBC939 cells.The effect of HNRNPA2B1 and exosomal miR-155-5p on the angiogenic capacity of HUVECs in QBC939 cells under hypoxia was investigated using a tube formation assay.Results: Based on the bioinformatics analysis,it was found that miR-155-5p in exosomes of hilar cholangiocarcinomacells under hypoxic conditions was selected as the miRs with strong binding ability to HNRNPA2B1 and closely related to tumor angiogenesis,based on the binding of miRs with more than 2-fold increase in the expression of miRs in hilar cholangiocarcinoma exosomes under hypoxic conditions,and the miRs screened by the database related to angiogenesis,which were intersected.WB assay showed that the exosomes of QBC939 cells from hilar cholangiocarcinoma expressed CD9 and TSG101 specific marker proteins under both normoxia and hypoxia induction;NTA analysis showed that the diameter of exosome particles of QBC939 cells was about 90-150 nm under normoxia and hypoxia.This indicates that exosomes were successfully isolated from the culture medium of QBC939 cells under normoxic or hypoxic conditions.Under hypoxic conditions,WB quantitative analysis showed that the expression of HNRNPA2B1 in QBC939 cells increased to 2.43 times that under normoxia(P<0.0001);while the expression of HNRNPA2B1 in QBC939 cells exosomes reached 2.96 times that under normoxia(P<0.0001).q RT-PCR showed that under hypoxic conditions,the expression of HNRNPA2B1 in QBC939 cells increased to 1.88-fold under normoxia(P<0.0001).RIP technique reflected that miR-155-5p interacted with HNRNPA2B1 under both hypoxic and normoxic conditions,with 1.43-fold miR-155-5p detected under hypoxia than under normoxia.qRT-PCR was performed to verify the cellular and exosomal miR-155-5p expression after siRNA interference with HNRNPA2B1 in QBC939 cells,and the results showed that the exosomal miR-155-5p expression was reduced by 66.4% in the knockdown group(KD)compared with the negative control group(NC),however,the cellular miR-155-5p expression was not statistically different in the KD and NC groups.The tube formation assays showed that under hypoxia,the tube numbers of HUVECs treated by the exosomes of QBC939 cells with HNRNPA2B1 knockdown(KD-EXO)decreased by 55.5% compared to the exosomes of QBC939 cells from the negetive control group(NC-EXO)(P<0.0001).When NC-EXO and miR-155-5p inhibitors simultaneously acted on HUVECs,the tube formation was reduced by 32.3% compared with NC-EXO alone(P<0.001).However,tube formation of HUVECs treated by KD-EXO and miR-155-5p mimics increased by 1.14 folds compared to KD-EXO alone(P<0.0001).Western blotting showed that the expression of VEGF-A in HUVECs induced by KD-EXO was significantly lower than that induced by NC-EXO.The expression of VEGF-A in HUVECs induced by NC-EXO combined with miR-155-5p inhibitors was down-regulated compared with NC-EXO alone.The expression of VEGF-A in KD-EXO was increased by transfected miR-155-5p mimics.The change of the expression of VEGF-A in each group was consistent with the fluctuation of tube numbers in the tube formation assays.Conclusion: HNRNPA2B1 is involved in exosomal miR-155-5p sorting in hilar cholangiocarcinoma cells under hypoxia and can promote tumor vascular growth via miR-155-5p in exosomes.