LncRNA DHRS4-AS1 Regulate Gastric Cancer Apoptosis And Cell Proliferation By Destabilizing DHX9 And Inhibited The Association Between DHX9 And ILF3

Objective: To investigate the expression of LncRNA DHRS4-AS1 in gastric cancer and its clinical significance,and investigate the effect of DHRS4-AS1 on the proliferation and apoptosis of gastric cancer cells and its molecular mechanism.Methods:1)Quantitative reverse transcriptase PCR(q RT-PCR)assay was used to detect expression levels of LncRNA DHRS4-AS1 in gastric cancer tissues.Analyzed the correlation between DHRS4-AS1 expression in GC tissues and patient clinicopathological characteristics and prognosis.2)Constructing stably over-expressing or knock-down DHRS4-AS1 gastric cancer cell lines by lentiviral transfection.Detected the effect on proliferation after over-expressed of DHRS4-AS1 in gastric cancer cell by Ed U assay and Clone formation assay.Flow cytometry and TUNEL assay were also performed to detect the effect of DHRS4-AS1 on GC apoptosis.Western blot and immunohistochemistry(IHC)were also used to detect the expression level of apoptosis related proteins.We injected the stable overexpressing knock-down DHRS4-AS1 GC cell into nude mice to investigate the effects of DHRS4-AS1 on GC cell tumorigenesis in vivo.3)We identified and analyzed the RNA binding protein of DHRS4-AS1 by pull-down assay and found DHX9 was regulated by DHRS4-AS1.Western-blot and RIP assay verified the interaction of DHRS4-AS1 and DHX9.Western-blot assay also performed to detect the expression level of DHX9 after over-expressing or knock-down DHRS4-AS1.Then,we detect the ubiquitination levels of DHX9 by manipulating the expression of DHRS4-AS1 in gastric cancer.Finally,the specific molecular mechanism of DHRS4-AS1 regulating DHX9 ubiquitination was explored by protein immunoprecipitation and in vitro ubiquitination experiment.4)Further CO-IP assay was performed to verified the interaction of DHX9 with ILF3.Mapping the regions of DHX9 and ILF3 responsible for their interaction by Constructed DHX9-Myc and ILF3-Flag full-length plasmid as well as their ILF3-Flag and Myc-tagged truncated.Explored the effect of DHX9 on NF-κB signaling pathway and further investigated function of ILF3 on activation of NF-κB signaling pathway by DHX9.Finally,CO-IP assay was performed to explore the effect of DHRS4-AS1 on association between DHX9 and ILF3.Results:1)The expression of LncRNA DHRS4-AS1 was significant down-regulated in gastric cancer tissue.And its expression in gastric cancer was significant correlated with tumor size,invasion depth,TNM stage and vascular invasion.In addition,the overall survival of the high DHRS4-AS1 group was significantly better than patients in the low DHRS4-AS1 group.2)Ed U assays and Colony formation assay demonstrated that DHRS4-AS1 significantly inhibited cell proliferation after over-expressed the expression of LncRNA DHRS4-AS1 in HGC-27 and MGC-803 cells.Furthermore,flow cytometry analysis was performed and observed that DHRS4-AS1 over-expression significantly increased apoptosis in AGS cells.On the contrary,Ed U and colony formation assays revealed cell proliferation was significantly increased with LncRNA DHRS4-AS1knock-down.Also,flow cytometry showed that GC cell apoptosis was decreased by DHRS4-AS1 knockdown in vitro.In addition,tumors derived from DHRS4-AS1 over-expressed HCG-27 cells were smaller than Vector group.Conversely,the tumors derived from DHRS4-AS1knock-down AGS cells were larger than in the negative controls.Besides,TUNEL assays and IHC assay found that DHRS4-AS1 significantly promoted tumor apoptosis in vitro.3)Western blotting using DHRS4-AS1 pull-down extracts and RIP assay confirmed that LncRNA DHRS4-AS1 and DHX9 interacted with each other.Furthermore,DHX9 protein abundance was significantly decreased after DHRS4-AS1 over-expression.However,q RT-PCR revealed that DHRS4-AS1 did not alter DHX9 m RNA levels.The proteasome inhibitor MG132 could reverse down-regulation of DHX9 by DHRS4-AS1 induced.Furthermore,endogenous DHX9 was immunoprecipitated from GC cells,which showed that ubiquitin signals were increased by DHRS4-AS1.Bioinformatics Analysis and Co-IP assay demonstrated that LncRNA DHRS4-AS1 acts as a scaffold to facilitate interactions between DHX9 and the E3 ligase MDM2,thus accelerating DHX9 degradation.4)CO-IP assay was demonstrated that DHX9 and ILF3 interacted with each other.To further map the regions of DHX9 and ILF3 responsible for their interaction.Co-IP assay with ILF3-Flag and Myc-tagged truncated as well as full-length DHX9.CO-IP results showed that ILF3-Flag was only present when the Myc3(636-1200aa)domains was present as well as DHX9-Myc full-length.Besides,we also constructed Flag-tagged domains to test in co IPs with DHX9-Myc.ILF3-Flag1(1-378aa)and ILF3-Flag3(592-894aa)pulled down DHX9-Myc at a similar efficiency as full-length.Those resu Lts demonstrated that ILF3-Flag1 and ILF3-Flag3 with DHX9-Myc3 were critical elements for complex formation between DHX9 and ILF3.Western Blot analysis showed that DHX9 over-expression up-regulated the expression of p-IκBα and p-p65.However,co-transfection of DHX9 overexpression and si-ILF3 indicated that DHX9 increased the p-IκB and p-p65 would be reversed by ILF3 knock-down.It means the interaction between ILF3 plays a crucial role for DHX9 to activate the NF-κB signaling pathway.Finally,CO-IP results showed that DHRS4-AS1over-expression decreased the associateion between ILF3 and DHX9.Conclusions:1)The expression of LncRNA DHRS4-AS1 was significant down-regulated in gastric cancer tissue.And its lower expression in gastric cancer was significant correlated with poor prognosis.LncRNA DHRS4-AS1 promotes gastric cancer cell apoptosis and inhibits proliferation in vitro and in vivo.2)LncRNA DHRS4-AS1 acts as a scaffold to facilitate interactions between DHX9 and the E3 ligase MDM2,thus accelerating DHX9 degradation via ubiquitin-proteasome system.3)LncRNA DHRS4-AS1 inhibited association between DHX9 and ILF3 and inhibiting the activation of NF-κB signaling pathway by DHX9.