| BACKGROUND&OBJECTIVEPSME3(proteasome activator complex subunit 3)is an important activator of the 20S proteasome.It has been found that PSME3 participated in multiple kinds of malignancies’ development and progression.However,the functional roles of PSME3 in colorectal carcinoma(CRC)and its molecular mechanisms on radioresistance have not been studied.Therefore,searching for therapeutic targets related to the enhancement of radiosensitivity in CRC is a major issue to be solved in the medical field.METHODS1.The effects of MIER3 overexpression on proliferation and migration of colorectal cancer cells in vitro were detected by CCK-8 proliferation assay and Transwell invasion assay.2.The interaction protein of MIER3 was screened out by mass spectrometry analysis.3.Immunofluorescence,quantitative real-time polymerase chain reaction(qRT-PCR),western blot and immunohistochemistry were used to detect the localization and expression of PSME3 in colorectal cancer cells and tissues.4.The effects of knockdown of PSME3 on proliferation and migration of colorectal cancer cells in vitro were detected by CCK-8 proliferation assay,transwell migration assay,colony-forming assay and wound healing assay.5.Effects of stable silencing PSME3 on radioresistance of CRC cells(1)SW480/shPSME3 and SW480/NC cells,SW620/shPSME3 and SW620/NC cells were respectively irradiated with 6Gy dose,and then the proportion of G2/M phase cells was analyzed by cell cycle assay at Oh before irradiation and 24h after irradiation.(2)SW480/shPSME3 and SW480/NC cells,SW620/shPSME3 and SW620/NC cells were respectively irradiated with different radiation doses(0Gy,2Gy,4Gy,6Gy and 8Gy),and then the size of cell colonies was detected by colony-forming assay.(3)SW480/shPSME3 and SW480/NC cells,SW620/shPSME3 and SW620/NC cells were collected at Oh before irradiation and 24h after irradiation(6Gy dose).Then expression of CyclinB1 and p-cdc2 protein in G2/M phase was detected by western blot assay.RESULTS1.CCK-8 proliferation assay(FDLD 1=173.686,P<0.001;FSW480=82.253,P<0.001)and Transwell invasion assay(tDLDl=5.54,P=0.005;tSW480=8,766,P<0.001)showed that the proliferation and invasion potential of SW480 and DLD1 CRC cells was significantly weakened after overexpressing MIER3 compared with the control group.2.Two-dimensional electrophoresis experiments and mass spectrometry technique analysis were carried out to obtain MIER3-related interaction proteins,HMGB1,SNP29 and PMSE3.3.The expression of PMSE3 in colorectal cancer(1)Immunofluorescence,quantitative real-time polymerase chain reaction(qRT-PCR)and western blot were used to detect the localization and expression of PSME3 in 7 CRC cell lines(LS-174-T,Caco-2,HCT116,HT29,SW620,SW480 and LoVo).The results showed that PSME3 was mainly localized in CRC cell nucleus and highly expressed in SW480 and SW620 CRC cells,but downregulated in HCT116 and HT29 CRC cells.(2)PMSE3 expression in paraffin-embedded tissue of 163 CRC patients was detected by immunohistochemistry.The results showed that PMSE3 expression in CRC tissues was higher than that in adjacent non-tumor tissues(94/163).4.CCK-8 proliferation assay(FSW480=325.4,P<0.0001;FSW620=218.4,P<0.0001),colony-forming assay(tSW480=14.20,P=0.0001;tSW620=43.47,P<0.0001),Transwell migration assay(tSW480=9.189,P=0.0008;tSW620=24.15,P<0.0001)and wound healing assay(tSW480=6.741,P=0.0025;tSW620=2.854,P=0.046)showed that the proliferation and migration ability of SW480/shPSME3 and SW620/shPSME3 cells was significantly inhibited after silencing PMSE3 compared with the control group.5.Effects of stable silencing PSME3 on radioresistance of CRC cell lines(1)Cell cycle assay revealed that the proportion of G2/M phase cells was increased in the SW480/shPSME3 and SW480/NC cells,SW620/shPSME3 and SW620/NC cells after irradiation;Furthermore,the proportion of G2/M phase cells in the SW480/shPSME3 and SW620/shPSME3 increased faster compared with the control group.(2)Colony-forming assay exhibited that the number of colonies in the SW480/shPSME3 and SW620/shPSME3 group was significantly lower compared with the control group under different radiation doses(OGy,2Gy,4Gy,6Gy and 8Gy).(3)Western blot assay showed that cyclinB1 and p-cdc2 protein in G2/M phase cells was down-regulated both in the SW480/shPSME3 and SW620/shPSME3 group and in the control group after irradiation;Furthermore,expression of cyclinB1 and p-cdc2 protein in G2/M phase cells dropped faster in the SW480/shPSME3 and SW620/shPSME3 group than that in the control group.CONCLUSIONKnockdown of PSME3 may induce G2/M phase cell arrest by downregulating the expression of cyclinB1 and p-cdc2,thereby enhancing the radioresistance of CRC cells.INNOVATIONSIt is original that the clinical value of PSME3 and its molecular mechanisms in colorectal cancer radioresistance were revealed though a series of in vitro experiments. |
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