| Pancreatic ductal adenocarcinoma(PDAC)is a highly malignant cancer of digestive system.Despite the improvement of medical technology in recent years,the 5-year survival rate of pancreatic cancer is still less than 10%,which is a serious threat to human health.The early symptoms are not obvious in PDAC,and most patients have advanced cancer when diagnosed,chemotherapy is the preferred treatment.Only 15% of PDAC patients are eligible for surgery,chemotherapy is the mainly adjuvant treatment for surgery PDAC patients.Gemcitabine(2',2'-difluorodeoxycytidine)is a first-line drug for the treatment of pancreatic cancer.Although gemcitabine is effective in patients of PDAC,gemcitabine resistance ultimately leads to chemotherapy failure.Cancer stem cell(CSCs)are considered as the fundamental driving force of cancer development,occurrence,and chemoresistance.Therefore,to analyze and reveal the molecular basis for the regulation of CSCs could provide theoretical and experimental basis for the effectiveness and accuracy of pancreatic cancer treatment.It is reported that a low level of reactive oxygen species(ROS)is critical for maintaining the stemness of CSCs.Previous works in our lab have confirmed that the CSCs characteristics of the acquired gemcitabine resistance cell line Bx PC-3-Gem are significantly enhanced.This result indicates that CSCs are enriched during the evolution from gemcitabine sensitive to gemcitabine resistance cells.The level of ROS is higher after gemcitabine treatment,which leads to pancreatic cancer cell death.Therefore,this study speculated that gemcitabine resistant cells might develop a mechanism to defend ROS to avoid the chemotherapy toxicity of gemcitabine,leading to CSCs enrichment and gemcitabine resistance of pancreatic cancer.In this paper,the level of ROS in pancreatic cancer resistant cells was firstly analyzed.The results showed that low levels of intracellular ROS were observed in the gemcitabine acquired resistant cells Bx PC-3-Gem and native resistant cells PANC-1 and As PC-1.Through microarray analysis of long noncoding RNA(lnc RNA)expression profile chips,it was found that lnc RNA SLC7A11-AS1 was the most upregulated gene in Bx PC-3-Gem compared with Bx PC-3 cells.RT-q PCR confirmed that SLC7A11-AS1 was overexpressed in Bx PC-3-Gem,PANC-1,and As PC-1.Knockdown of SLC7A11-AS1 in gemcitabine resistant Bx PC-3-Gem,PANC-1,and As PC-1 cells induced significant elevation of intracellular ROS level in vivo and in vitro.Analysis of the clinical PDAC tissues and the TCGA data showed that SLC7A11-AS1 was highly expressed in the tumor tissues and correlated with the poor prognosis of pancreatic cancer patients.These results indicated SLC7A11-AS1 plays a role of oncogene as ROS inhibitor in pancreatic cancer cells.Secondly,knockdown of SLC7A11-AS1 in gemcitabine resistant cells to analyze the function of SLC7A11-AS1 in pancreatic cancer stemness and chemo-resistance.The results indicated that knockdown of SLC7A11-AS1 caused suppression of the characteristics of CSCs.The expression level of Oct4 and Nanog were decreased,and the capability of sphere-forming were decreased.In addition,compared with the adherent cells,the spheroids exhibited higher expression level of SLC7A11-AS1.Introducing the scavenger of ROS could restore the CSCs characters caused by silencing of SLC7A11-AS1.Furthermore,the involvement of SLC7A11-AS1 in chemotherapy resistance of pancreatic cancer cells was confirmed by establishing SLC7A11-AS1 knockdown stable lines and xenografts mouse model.Knockdown of SLC7A11-AS1 could increase the sensitivity of pancreatic resistant cell to gemcitabine.Compare to gemcitabine alone group,knockdown of SLC7A11-AS1 could decrease the capability of cell proliferation,colony formation,and the tumor volume and weight of xenografts.NRF2 is a core factor regulating the level of ROS in cells.In this study,NRF2 protein was found to be more highly expressed in gemcitabine resistant cells than that in gemcitabine sensitive cells.In view of the ROS was inhibited by SLC7A11-AS1 in pancreatic cancer cells,the regulation of NRF2 by SLC7A11-AS1 was investigated in this study.First,it was found that SLC7A11-AS1 could regulate the protein level of NRF2,but had no effect on its m RNA level.SLC7A11-AS1 can stabilize the expression of NRF2 protein,inhibiting the ubiquitin-proteasome degradation pathway of NRF2 protein in the nucleus,and promote the expression of downstream antioxidant genes HMOX1 and GCLM of NRF2.?-TRCP1 is responsible for NRF2 ubiquitination and proteasomal degradation in the nucleus.SLC7A11-AS1 and ?-TRCP1 were co-localized in nucleus.The results from RNA pull down showed that SLC7A11-AS1 exon3 was essential for the interaction with ?-TRCP1 protein.?-TRCP1 has main two domain,the F-box domain which is responsible for recruiting ?-TRCP1 to the SCF?-TRCP E3 ligase complex through interaction with adaptor protein SKP1,and the WD40 domain responsible for interaction with the substrates.The RIP revealed that SLC7A11-AS1 interacts with the F-box of ?-TRCP1,inhibiting the ubiquitination and degradation of NRF2 protein in the nucleus.In conclusion,this study revealed that SLC7A11-AS1 interacts with ?-TRCP1,inhibiting the ubiquitination and degradation of NRF2 protein in the nucleus.Our results demonstrate that the overexpression of SLC7A11-AS1 in gemcitabine resistant PDAC cells can scavenge ROS,leading to a low level of intracellular ROS which is required for the maintenance of cancer stemness.These findings suggest SLC7A11-AS1 as a therapeutic target to overcome gemcitabine resistance for PDAC treatment. |
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