Long Non-coding RNA UCA1 Promotes KRAS And YAP1 Expression By Acting As A Competing Endogenous RNA In Pancreatic Cancer

Objective: The aim of the present study is to explore whether UCA1 can function as a competitive endogenous RNA to upregulate the expression of KRAS and YAP1 in pancreatic cancer cells.Methods: Firstly,we analysed UCA1 expression level in normal pancreatic tissues and pancreatic cancer tissues in few databases.Then,we analysed the association between UCA1 expression and pancreatic cancer clinical outcomes.Next,we analysed the correlation between UCA1 and KRAS,YAP1 and predicted shared micro RNAs between them using bioinformatics analysis.We examined the expression level of UCA1 in Patu8988,PANC-1,Bx PC-3 and SW1990 cells by q RT-PCR.We constructed UCA1 interference plasmid p LKO.1-sh-UCA1(sh-UCA1)which was then used to package lentivirus to infect cells that express higher level of UCA1.Stable cells were selected by puromycin.The UCA1 overexpression plasmid was transiently tranfected into cells that express lower level of UCA1.The efficiency of these plasmids was tested by q RT-PCR.We detected the expression levels of KRAS and YAP1 in UCA1 overexpression and knockdown pancreatic cancer cells using q RT-PCR and Western blot.We examined the direct interaction between UCA1 and putative shared micro RNAs and screened the target micro RNAs by RNA immunoprecipitation assay(RIP).We detected the expression levels of the target micro RNAs after upregulating or downregulating UCA1 in pancreatic cancer cells using q RT-PCR.We examined the expression levels of KRAS and YAP1 after overexpressing the target micro RNAs in pancreatic cancer cells by q RT-PCR and Western blot.We further verified whether the target micro RNAs can directly bind to UCA1,KRAS and YAP1 by dual-luciferase reporter assay.Results: Firstly,we found that UCA1 expression was higher in pancreatic cancer tissues than that in normal pancreatic tissues in Oncomine database.Further analysis showed that higher expression of UCA1 correlated with poor survival in Cancer Browser database.The expression of UCA1,KRAS and YAP1 were found to be positively related in pancreatic cancer tissues.We predicted that UCA1 might act as a ce RNA to share miR-590-3p,miR-1271,miR-143,miR-193 b and miR-193a-3p with KRAS m RNA and share miR-18 a,miR-18 b and miR-495 with YAP1 m RNA using micro RNA.org database.UCA1 expression was highest in SW1990 cells than that of other three pancreatic cancer cell lines,the least was PANC-1 cells.The plasmid sh-UCA1 was transfected into Bx PC-3 and SW1990 cells.The expression level of UCA1 was efficiently down-regulated(P<0.05).The UCA1 overexpression plasmid was transfected into Patu8988 and PANC-1 cells.The expression level of UCA1 was strongly up-regulated(P<0.05).We found that down-regulated expression of UCA1 inhibited KRAS and YAP1 expression and up-regulated expression of UCA1 promoted KRAS and YAP1 expression at both protein and m RNA levels(P<0.05).UCA1 could target miR-18 a,miR-18 b,miR-590-3p,miR-1271,miR-143 and miR-193a-3p(P<0.05),but had no tendency to bind with miR-495 and miR-193 b.Both of miR-590-3p and miR-18 a expression were lower in Patu8988 and PANC-1 cells transfected with UCA1 than that in control cells(P<0.05).In contrast,the expression level of miR-590-3p and miR-18 a were higher in Bx PC-3 and SW1990 cells transfected with sh-UCA1 than that in control cells(P<0.05).Both of miR-590-3p and miR-18 a inhibited the expression of UCA1 in Bx PC-3 and SW1990 cells(P<0.05).Mi R-590-3p decreased KRAS and miR-18 a decreased YAP1 both in protein and m RNA levels(P<0.05).Both of miR-193a-3p and miR-18 a reduced the luciferase activities of UCA1 wild-type reporter vector(P<0.05)but not the mutant reporter vector.Mi R-590-3p remarkably inhibited the luciferase activities of KRAS 3’-UTR wild-type reporter vector(P<0.05);miR-18 a remarkably inhibited the luciferase activities of YAP1 3’-UTR wild-type reporter vector(P<0.05).However,overexpression of UCA1 could abolish the effects of miR-590-3p to KRAS 3’-UTR wild-type reporter vector and miR-18 a to YAP1 3’-UTR wild-type reporter vector.Conclusion: UCA1 was overexpressed in pancreatic cancer tissues and associated with poor prognosis of pancreatic cancer patients.Our work suggested that UCA1 promoted KRAS expression by competitively binding with miR-590-3p and promoted YAP1 expression by competitively binding with miR-18 a.