MiR-122-5p Increases The Radiosensitivity Of Intestinal Epithelial Cells And Aggravates Radiation-induced Rectal Injury Through CCAR1

Objective: Radiation-induced rectal injury is one of the major side-effects observed in pelvic malignancies patients who receive radiotherapy.At present,the mechanism of radiation-induced rectal injury is not fully understood.Here,we studied the role of microRNA-122-5p(miR-122-5p)on radiation-induced rectal injury.Methods: Peripheral blood of rectal cancer patients before and after radiotherapy was collected,and the expression of miR-122-5p in the serum was detected by quantitative real-time PCR(qRT-PCR).The relationship between miR-122-5p and radiation-induced rectal injury was further verified in the C57BL/6 mouse model of radiation-induced rectal injury.The expression of miR-122-5p was enhanced by transfection with miR-122-5p mimic at the cell level,and its effects on the radiosensitivity and apoptosis of human intestinal epithelial cell(HIEC)were investigated by colony formation assay,flow cytometry,mitochondrial membrane potential detection,immunofluorescence staining and western blot experiments.Further,the downstream targets of miR-122-5p were predicted through bioinformatics websites and validated by dual luciferase experiments,then the expression of target was inhibited by transfection with small interfering RNA(siRNA),and its effect on radiosensitivity and apoptosis was further studied.At the animal level,miR-122-5p antagomir was used to inhibit the expression of miR-122-5p in mouse rectal tissues,and the effects of miR-122-5p inhibition on radiation-induced rectal injury were studied by H&E staining,TUNEL staining,and IHC staining experiments.Results: The expression level of miR-122-5p in the serum of rectal cancer patients was significantly increased after radiotherapy.The expression level of miR-122-5p in the mice rectal tissues was also significantly increased,and was positively correlated with the radiation-induced rectal injury.In vitro,it showed that overexpression of miR-122-5p combined with irradiation could significantly enhance the DNA damage of HIEC cells,reduce cell survival,and promote cell apoptosis.The results showed that CCAR1 was the downstream target of miR-122-5p.Inhibition of CCAR1 combined with irradiation can significantly promote DNA damage,reduce cell survival,and increase apoptosis.Mechanism studies have shown that the p-CHK2 protein levels were significantly reduced under irradiation when miR-122-5p was overexpressed or CCAR1 was inhibited.In vivo,it showed that after inhibiting the expression of miR-122-5p in mouse rectal tissues,the radiation-induced rectal injury was significantly reduced,and the expression of apoptosis-related protein cl-caspase-3 was significantly decreased,while the expressions of CCAR1 and p-CHK2 were significantly increased.Conclusion: Irradiation can significantly increase the miR-122-5p expression in vivo.MiR-122-5p may enhance the radiosensitivity of HIEC cells by regulating the CCAR1/CHK2 signaling pathway,thereby aggravating radiation-induced rectal injury.