| Background: Head and neck cancer(HNC)has become a serious public health issue due to its high morbidity and mortality.Apparently,the treatment of OCC(oral cancer cells)remains a formidable challenge for clinicians.Radiotherapy is one of the most common and effective treatments for this cancer besides surgery and chemotherapy.Objective: The aim of the present study was to reveal the changes in the expression of ARPC3,and illuminate the mi RNA functions in effect on the function of actin dynamics of OCC cells.As well,whether the ARP2/3 could act as a key regulator of radiation sensitivity in oral cancer development is one of proposal of this study,and detailed investigate the structures of the F-actin cytoskeleton after X-ray.Methods: The current report is organized into four main parts.First,to constructed the radiomic models,all of three different kinds of cells(Cal27,DOK,TSCC)were divided into four groups: control(non-irradiated),irradiated after30 min,1h,and 6h.The expression level of mi R-26a-5p and ARPC3 m RNA were detected by quantitative realtime(PCR).Levels of ARPC3 protein were assessed via Western blotting(WB)and immunofluroscence(IF).Second,F-actin fibers both pre-and post-irradiation were visualized using STORM microscope.Third,to inhibit the activity of the Arp2/3complex,we used 100 μM of CK666.Transwell assay(Corning)was used to detect the capacity of oral cancer cell invasion and migration.WB was performed to detect MMP-9 protein expression.Fourth,constructed the recombinant plasmid,then the reporter plasmid mi R-26a-5p mimic,NC and ARPC3 plasmids were co-transfected into Cal27 cells.Dual luciferase report confirmed whether the mi R-26a-5p targeted to regulate ARPC3.Statistical significance analysis was performed by SPSS software,Graphpad Prism7 and Image J.Results: First,the WB results showed an obviously suppressed expression of ARPC3 protein in 30 min,but upgraded in 1h,recovered in 6h which was consistent with the q RT-PCR and IF results.Meanwhile,the level of mi R-26a-5p reduced in 30 min,increased in 1h and 6h.Second,the STORM microscope observed the result of F-actin depolymerization after X-ray of 30 min.However,the recovery process of F-actin was present after 1h,the arrangement and structure of cytoskeleton was similar between 6h and control group.Third,we found that cells migration and invasion ability was inhibited in ARPC3 inhibitor group(CK666)comparing to the control groups,which indicated that ARPC3 protein induced the radiosensitivity in oral cancer cells.The recombinant plasmid prim GLO was successfully constructed and confirmed by sequencing.Luciferase activity in Cal27 cells co-transfected with mi RNA-26a-5p mimic/NC and a significant decrease of luciferase activity about 63.33% was indicated in mi R mimic group.Conclusion: During the six hours after radiation,the expression of ARPC3 decreased first and then increased,but the level of mi R-26a-5p expression first increases and then decreases that indicated ARPC3 was a bona fide negatively target for mi R-26a-5p.The regulation of ARPC3 attribute to dynamic change of F-actin during radiation and induces re-arrangement of actin cytoskeleton,as well as the increase radiation sensitivity in OCC.At the same time,our study also provided an experimental basis for further study of the mechanism of cytoskeleton rearrangements happened after radiation.Provide direction for further application research in radiation biology and radiation biophysics. |
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