Characterization Of SOD1Post-transcriptional Regulation And Mechanisms On Increasing Radiation-induced Effect In Cancer

Purpose:Known strategies of reversing resistance to radiotherapy in cancer werecommonly unsatisfied. It has been reported that a high expression of SOD1is related tochemo-radiotherapy resistance in cancer, the mechanism underlying an increasedexpression of SOD1are unknown. It is well known that the precise regulation of geneexpression is relative to post-transcriptional regulation through3’UTR. But little wasknown about SOD1post-transcriptional regulation in cancer, which was investigated inthis project, as well as the impact on radiation induced effect in cancer. These studieswill possibly provide new insights for strategies targeted SOD1in increasing cancerradiosensitivity.Methods：The ovarian cancer cell line A280and the pancreatic cancer cell linePANC1were the main cell models in this project.(1) The fragment of SOD-3’UTR wasinserted into pGL3-Promoter vector.The influences of3’UTR on the expression ofSOD1/2in cancer cell were explored,as well as after treatment by the hydrogenperoxide and X-ray radiaiton.Meanwhile,the SOD1mRNA and protein level weremeasured before and after treatment by hydrogen peroxide.(2)SOD1-3’UTR mediatedrelative luciferase activity was conpared between the two cancer cell line,and therelative firely luciferase mRNA level was measured by RT-PCR.The influence of foredexpression of SOD-3’UTR on endogenous SOD protein expression was explored bywesturnblot assay.Using pGL3-Promoter-SOD1-3’UTR sense vector as a template,thecloning of serially deleted SOD-13’UTR reporter vectors and that including differentdeletions of AREs or miRNA binding site was finished.By using these vectors andmiRNA mimics or inhibitors,the impact of different RNA elements(AREs and miRNAbinding site)on SOD1-3’UTR mediated relative luciferase acitivty or SOD1proteinexpression were examined.The SOD1-3’UTR fragment was also inserted intopGL3-SOD1-Promoter reporter vector,and the vector including different deletions of AREs were cloned again,then the impact of AREs on SOD1-Promoter-SOD1-3’UTRsense mediated relative luciferase activity with or without X-ray radiation wereinvestigated.(3)The SOD1protein expression and SOD1-3’UTR mediated luciferaseactivity was measured by westurnblot assay after forced expression or knockdown ofAUF1and HuR in cancer cell. The influence of AUF1knockdown on radiation inducedROS was measured by confocal microscope.In further,we immunoprecipitated AUF1from cell lysates prepared from PANC1cells and analyzed SOD1-3’UTR expression inthe precipitates.The expression of AUF1and SOD1in pancreatic cancer tissue sampleswere detected by immunohistochemistry.In the preliminary studies, we first found an increased stability of SOD1mRNA bySOD1-3’UTR in cancer cells, six AREs in the SOD13’UTR which interacted with theRNA-binding protein, and several miRNAs binding sites. The expression level of SOD1was significantly correlated to the RNA-binding protein AUF1expression in cancertissues examined. Furthermore, we also found knock-down of AUF1significantlyincreased radiation-induced effect in cancer cells.Results：（1）To investigate whether SOD1/23’UTR influences SOD1/2expression,A2780cell line was transfected with the pGL3-promoter-SOD1/23’UTR sense orpGL3-promoter-SOD1/23’UTR antisense vector, respectively. Inclusion of the SOD1/23’UTR dramatically enhanced the reporter gene activity by20fold in A2780cells,respectively. Hydrogen peroxide at100μM significantly increased the SOD1/2mediatedluciferase activtiy in A2780,the similar results was got by treatment with10Gy X-rayradiation.Both were consistent with the increase of SOD1mRNA and protein level withtreatment of hydrogen peroxide.(2) A2780and PANC1cell lines were transfected withthe pGL3-Promoter vector, pGL3-promoter-SOD13’UTR sense orpGL3-promoter-SOD13’UTR antisense vector, respectively. Inclusion of the SOD13’UTR dramatically enhanced the reporter gene activity by109-,219-fold in PANC1,A2780cells, respectively. Inclusion of the SOD13’UTR remarkably increased firelyluciferase mRNA levels, indicating that the high luciferase activity is attributed toincreased mRNA stability.Transfection of SOD13’UTR sense vector, which sharesidentical sequence with endogenous SOD13’UTR, decreased endogenous SOD1expression in a concentration-dependent manner, suggesting that the exogenousintroduced SOD13’UTR competes with endogenous SOD13’UTR for the trans-acting factors that are required for maintaining SOD1gene expression. Interestingly, when theSOD13’UTR construct was introduced into A2780cells, endogenous SOD2proteinexpression was also significantly down-regulated, indicating the two SODs likely sharea common post-transcriptional mechanism. Truncated deletion of SOD13’UTRgradually decreased luciferase activity and deletion of any part of SOD13’UTRdramatically affects the reporter activity. Deletion of the binding sites for miR-224andmiR-377, but not miR-621in SOD13’UTR, significantly down-regulated luciferasereporter activity, which was not in accordance with the general belief that miRNAsnegatively regulate gene expression. In addition, application of miR-377or miR-621mimics/inhibitors did not affect endogenous SOD1expression or the SOD13’UTR-driven reporter gene activity, indicating that the SOD13’UTR’s function insustaining SOD1expression is less likely attributed to these miRNA species.Deletion ofany of the four AREs remarkably reduces luciferase activity with the ARE6being themost important one, indicating that SOD1expression is significantly sustained by theAREs in its3’UTR. However, when the vectors, containing only two or four of theputative AREs, were transfected to A2780or PANC1cells, luciferase activity wasalmost undetectable, further suggesting that the importance of the AREs is related totheir contribution to the integrity of the3’UTR, which is essential for sustaining SOD1expression. Deletion of ARE6reversed the increased SOD1-Promoter-SOD1-3’ UTR-driven luciferase activity induced by X-ray radiation in A2780.(3)The knockdown ofAUF1and HuR was confirmed by Western blot analysis. We found that knockdown ofAUF1but not HuR significantly reduces SOD1protein expression levels.Co-transfection of the SOD13’UTR reporter constructs with the AUF1siRNAsignificantly down-regulated the SOD13’UTR-driven reporter gene activity. In contrast,the reporter gene activity in cells transfected with control vector or the SOD13’UTRantisense vector was unaffected by AUF1knockdown. Forced expression of AUF1increases the SOD13’UTR-driven reporter gene activity in a concentration-dependentmanner in both cell lines. We then immunoprecipitated AUF1using cell lysatesprepared from PANC1cells and analyzed SOD13’UTR expression in the precipitates.In contrast to IgG-precipitated sample, expression of SOD13’UTR was only detectedby RT-PCR in AUF1-precipitated sample, indicating a direct interaction of AUF1withSOD13’UTR in these cells. Transfection of shRNA-AUF1to PANC1cells was foundto promote basal ROS generation compared with that of shRNA-NC transfection. Furthermore, transfection of shRNA-AUF1significantly enhanced radiation-inducedROS levels. Similar results were obtained in A2780cells with or without irradiation.Immunohistochemistry analysis showed that, in all93pancreatic cancer tissuesexamined, expression of SOD1is significantly correlated to AUF1expression levels(rho=0.558, P <0.001).Conclusion:(1)SOD1-3’UTR maintains the SOD1expression of cancer cell inbasic and oxidative conditions.（2）SOD1-3’UTR increased SOD1mRNA stability incancer cells,which was attributed to AREs(ARE6) more than miRNA binding site in3’UTR.(3)Post transcriptional regulation of SOD1by3’UTR was attributed to theinteract between AUF1and SOD1-3’UTR,which in further modulated the cellular andradiation induced ROS in cancer cells.