Long Non-coding RNA UCA1/KRAS Axis Promotes Pancreatic Ductal Adenocarcinoma Tumor Growth And Stem Cell Properties

Objective: In this study,the expression of long non-coding RNA UCA1 in pancreatic ductal carcinoma and the relevance of UCA1 expression with the prognosis of pancreatic ductal carcinoma patients were analyzied by bioinformatics database.Moreover,we found that the effects on the proliferation and stem cell properties of pancreatic ductal carcinoma cells.We illuminated the potential molecular mechanism of the effects of UCA1 in pancreatic ductal carcinoma cells and it may serve as a novel target for exploring new therapeutic strategies to treat PDAC.Methods: 1.We first analyzed the m RNA levels of UCA1 in pancreatic ductal carcinoma tissues and the relevance of UCA1 expression with the overall survival of pancreatic ductal carcinoma patients.UCA1 transcript level in 6 PDAC cell lines,PANC1?Pa Tu8988?SW1990?Pa Tu8902?Mpanc96 and HPAF-?,and immortalized human pancreatic ductal epithelial cell line H6C7 were detected by q RT-PCR.2.We transfected UCA1 into pancreatic ductal carcinoma cells with lower expression of UCA1,sh-UCA1 was transfected into pancreatic ductal carcinoma cells with higer expression of UCA1.CCK-8 and colony formation assays showed the effects of UCA1 on proliferative activity and colony formation in pancreatic ductal carcinoma cells.Experiments with xenograft mouse model detected the ability of tumor growth.3.We examined sphere formation by tumor sphere-forming assay in stem cell medium and the expression of stemness markers by Western blot.4.RNA immunoprecipitation(RIP)and co-immunoprecipitation(IP)were used to detect the interaction between UCA1 and the oncogene KRAS and KRAS-related protein hn RNPA2B1,and whether UCA1 affects the interaction between hn RNPA2B1 and KRAS.To identify the fragments of UCA1-hn RNPA2B1 binding,we used point mutation techniques.5.We further examined protein levels of hn RNPA2B1,phospho-KRAS and KRAS,while knocking down UCA1 in Mpanc96 and HPAF-? cells.We also validated colocalization of hn RNPA2B1 and KRAS in UCA1 downregulated KRAS-dependent PDAC cell lines(Mpanc96 and HPAF-? cells),by immunofluorescence microscopy.6.To confirm the effect of UCA1 on phospho-KRAS,we overexpressed full-length UCA1(UCA1-wt)and the section for UCA1 binding hn RNPA2B1 mutated UCA1(UCA1-mut)to measure expression of hn RNPA2B1,KRAS and phospho-KRAS.Results: 1.UCA1 was highly expressed in PDAC tumor specimens compared to normal tissue in TCGA database.Furthermore,we found that UCA1 was a negative prognostic factor for overall survival from TCGA database Kaplan-Meier survival curves.UCA1 levels were significantly increased in PDAC cell lines than H6C7 and the UCA1 m RNA levels remained highly abundant in Mpanc96 and HPAF-? cells,while UCA1 was weakly expressed in Pa Tu8988 and PANC-1 cells,by q RT-PCR.2.CCK-8 and colony formation assays exhibited that overexpression of UCA1 in Pa Tu8988 and PANC-1 cells promoted proliferation and colony formation.On the contrary,downregulated UCA1 largely suppressed proliferative activity and colony formation of Mpanc96 and HPAF-?cells.Furthermore,experiments with xenograft mouse model revealed that sh-UCA1 also significantly reduced tumor growth.3.In Pa Tu8988 and PANC-1 cells,we found that the sphere formation capability was increased in the UCA1 group as compared to vector control,as well as the expression of stemness markers,CD133,OCT4,NANOG and SOX2.In contrast,overexpression of UCA1 in Mpanc96 and HPAF-? cells resulted in the opposite effects.4.RNA immunoprecipitation(RIP)assays showed that UCA1 interacts with hn RNPA2B1 and further clarifies specific hn RNPA2B1-binding sites in UCA1.We confirmed the interaction of hn RNPA2B1 with KRAS,and UCA1 can promote the interaction between hn RNPA2B1 and KRAS,by co-immunoprecipitation assays.5.After downregulating UCA1,western blot and phos-tag system showed that the m RNA and protein expression levels of KRAS and KRAS phosphorylation were significantly decreased.Of interest,we observed a reduction in cytoplasmic hn RNPA2B1 and decreased colocalization level of hn RNPA2B1 and the cytoplasmically located KRAS in KRASdependent PDAC cell lines of UCA1 knockdown by immunofluorescence microscopy.6.Western blot and Phos-tag system results showed that the m RNA and protein expression levels of KRAS and KRAS phosphorylation increased in the UCA1-wt group compared with the control group.Immunofluorescence analysis demonstrated that UCA1-wt promoted cytoplasmic hn RNPA2B1 and increased colocalization of hn RNPA2B1 and KRAS compared with the control group,while there was no change in the UCA1-mut groupConclusion: Accordingly,we confirm that UCA1 is relatively high in PDAC tissues and cells,and its expression level is closely associated with the prognosis of patients with PDAC.UCA1 promotes pancreatic ductal adenocarcinoma stem cell properties and tumor growth through increasing the activity of KRAS by binding hn RNPA2B1.These findings suggest that UCA1-KRAS axis plays a crucial role in PDAC progression and UCA1 may serve as a target for new therapies of PDAC.