To screen microRNAs that are positively correlated with radiation resistance in rectal cancer, and to investigate their regulatory mechanism.Fresh rectal cancer tissues were obtained intraoperatively and divided into two equal parts, one of which was pre-stored in a refrigerator at -150℃, and the other was constructed with Patients Derived Xenograft (PDX) models for rectal cancer and irradiated to transplantation tumor screen out radiation-sensitive tumor tissues and radiation-resistant tumor tissues. According to the screening results, the pre-stored samples were screened for differential microRNAs in radiation reactivity, and the microRNAs that were positively correlated with irradiation resistance were obtained. On the base of screening results, the pre-stored samples were screened for differential microRNAs in irradiation reactivity by miRNAs microarrays, and the microRNAs that were positively correlated with irradiation resistance were obtained. Biological prediction software was used to predict the target genes regulated by microRNA, and the prediction results were verified in radiation-resistant rectal cancer cell lines. Lentivirus plasmid transfection technique was used to construct rectal cancer cell lines with down-regulated target microRNA expression, in order to further verify the effects of the changes in the expression of the microRNA on the radiation reactivity, proliferation, invasion and migration of rectal cancer cells. The target genes regulated by microRNAs were screened and verified, and the expression of the key genes in the signal transduction pathways involved in the regulation were detected.The results of microRNA screening and validation showed that there were 14 microRNAs with fold change > 1.5. Among them, miRNA-552-3p, miRNA-96-5p, miRNA-182-5p, and miRNA-183-5p were up-regulated in irradiation-resistant tumor tissues. The enrichment signaling pathway analysis showed that the Wnt signal transduction pathway was highly active in irradiation-resistant tumor tissues. The clone formation experiment under radiation interference showed that HR-8348 was the most radiation-resistant cell line among the five cell lines, and the expression level of miRNA-96-5p was positively correlated with the radiation-resistant ability of rectal cancer cell lines. The results of prediction and validation showed that the expression of GPC3 gene was directly regulated by miRNA-96-5p, and the down-regulated expression of miRNA-96-5p inhibited the activity of Wnt signal transduction pathway.The high expression of miRNA-96-5p enhanced the radiation resistance of rectal cancer cells, and its effect may be related to the down-regulation of GPC3 expression and the abnormal activation of Wnt signal transduction pathway.Copyright © 2021. Published by Elsevier Inc.