LncRNA UCA1 Interaction With Components Of Hippo Pathway In Pancreatic Carcer

Objective:Previous study has demonstrated that UCA1 promoted cell migration and invasion of pancreatic cancer cells.This study aims at investigating the molecular mechanisms underlying the biological behavior of UCA1 in the migration and invasion of human pancreatic cancer cells and providing a solid theoretical basis for the molecular targeted therapy of UCA1 in pancreatic cancer.Methods:1.The plasmid sh-UCA1 was designed and constructed to change the expression of UCA1 for subsequent experiments.We used Bax PC-3 and SW1990 cells with higher expression of UCA1 in sh RNA experiments,and used Pa Tu8988 and PANC-1 cells with relatively lower expression of UCA1 in UCA1 overexpression assays.Western blot was used to test the protein expression of Hippo signaling pathway,and Realtime PCR was used to verify the m RNA expression level of UCA1 and YAP.2.We purchased p SL-MS2-12 X and p MS2-GFP plasmids and constructed p SL-MS2-12X-UCA1 plasmid.And 293 T cells were transfected with these plasmids.RNA immunoprecipitation(RIP)detected the components of Hippo pathway which UCA1 binds to.Next,the p SL-MS2-12X-UCA1 5' and p SL-MS2-12X-UCA1 3' plasmids were purchased,and the part of UCA1 was involved the interaction with the above proteins of Hippo signaling pathway were detected by RIP.The UCA1 mutant plasmid was constructed,and the previous experimental results were verified again by the RIP method.3.The UCA1-wt and UCA1-mut plasmids were transfected into Pa Tu8988 and PANC-1 cells with relatively lower expression of UCA1.The protein expression of Hippo signaling pathway was detected by Western blot.Nuclear/cytoplasmic fractionation and immunofluorescence microscopy were used to detect the subcellular localization of YAP in the pancreatic cancer cells.Luciferase reporter assay detected transcriptional activity of YAP.Results:1.The sequencing results showed that sh-UCA1 plasmids were constructed successfully.MST1,MST2,p-MOB1,p-Lats1,Lats1,and p-YAP levels were increased markedly after UCA1 knockdown in Bx PC-3 cells,and MOB1 and YAP expression level downregulated,while UCA1 overexpression in Pa Tu8988 and PANC-1cells resulted in the opposite effects.q RT-PCR analysis revealed that levels of UCA1 and YAP were significant downregulated in Bx PC-3 cells transfected with sh-UCA1.In contrast,the levels of UCA1 and YAP increased in UCA1 overexpressed Pa Tu8988 and PANC-1cells.2.We successfully constructed p SL-MS2-12X-UCA1 and UCA1-mut plasmids.RNA immunoprecipitation(RIP)demonstrated that UCA1 bound to MOB1,Lats1 and YAP,and UCA1 5' was essential for MOB1,Lats1,and YAP binding.UCA1-mut did not bind to MOB1,Lats1 and YAP.3.After UCA1-wt or UCA1-mut plasmids transfected with Pa Tu8988 and PANC1 cells,Western blot showed that MOB1 and YAP expression increased in UCA1-wt group,and MST1,MST2,p-MOB1,Lats1,p-Lats1 and p-YAP expression decreased.UCA1-mut resulted in the opposite effects,and UCA1 had no significant effect in Pa Tu8988 and PANC-1 cells transfected with UCA1-mut compared with the control group.Both nuclear/cytoplasmic fractionation and immunofluorescence microscopy indicated that UCA1 promoted YAP translocation to the nucleus.The results of luciferase reporter assay demonstrated that UCA1 enhanced luciferase activity.Conclusion:To conclude,UCA1 promotes migration and invasion in pancreatic cancer cells via Hippo signaling pathway.UCA1 exerts its oncogenic function by interacting with Lats1,MOB1 and YAP to form “shielding composites”,inhibiting their phosphorylation,and translocating YAP to the nucleus,which promotes malignant phenotype of pancreatic cancer cells.