LncRNA Uca1 Regulates SRPK1 Expression Through MiR-99b-3p In Ovarian Cancer

Ovarian cancer(OC)is one of the most common types of malignancy in women with high mortality.Therefore,studies on the early diagnosis,treatment and prognosis of OC are urgently required.Accumulating evidence has indicated that long noncoding RNAs(lnc RNAs)play an important role in OC.However,the dysregulated lnc RNAs and their function mechanisms in OC have not been extensively studied.In the present study,differences in lnc RNA and m RNA expression profiles between OC samples and normal ovarian epithelium samples were investigated using microarray analysis.DAVID 6.8 database was used for GO enrichment and KEGG analysis of differentially expressed m RNAs.The results showed that differentially expressed m RNAs were closely related to the functions of "cell division" "plasma membrane" "protein binding" and were mainly concentrated in the cancer-related pathways including "Wnt signaling pathway" "Pathways in cancer" "TGF-beta signaling pathway" "Rap1 signaling pathway" and so on.The results of lnc RNAs difference analysis showed that compared to normal ovarian epithelial tissue,the expression of lnc RNA UCA1 was significantly up-regulated in ovarian cancer samples.The variation of UCA1 in ovarian cancer samples in TCGA database was 16%,which indicated that UCA1 was strongly associated with ovarian cancer.Therefore,UCA1 was determined as the target lnc RNA.Based on previous reports,we will focus on the ce RNA mechanism of UCA1.The UCA-associated ce RNA regulatory network which provides a range for the selection of subsequent target genes for OC were predicted based on bioinformatics analysis generated from lnc RNA-mi RNA online prediction tool(Reg RNA 2.0)and mi RNA target gene prediction database(mi RDB,mi RTar Base,Target Scan Human 7.2).The regulation route of UCA1/mi R-99b-3p/SRPK1 was found to have high research value in ovarian cancer by bioinformatics analysis and literature retrieval.To verify the prediction results of bioinformation methods,four recombinant vectors,p RL-UCA1-WT/p RL-SRPK1-WT,p RL-UCA1-MUT/p RL-SRPK1-MUT,were successfully constructed.The results of double luciferase activity assay indicated that there were direct binding sites of mi R-99b-3p on both UCA1 and SRPK1 3’UTR.Therefore,mi R-99b-3p may be involved in the regulation of SRPK1 expression by UCA1.To further verify the above results and understand the regulation modes of UCA1/mi R-99b-3p on SRPK1 better,we evaluated the SRPK1 m RNA and protein expression level in A2780 cells after treatment with mi R-99b-3p mimic/inhibitor、siUCA1 or si-UCA1 plus mi R-99b-3p inhibitor for 48 h by q RT-PCR and western blot.The expression of SRPK1 was reduced in both m RNA(0.7)and protein(0.56)when A2780 cells were transfected with mi R-99b-3p mimic.Whereas its expression levels of both m RNA(1.3)and protein(1.37)were increased when endogenous mi R-99b-3p was inhibited by transfection with mi R-99b-3p inhibitor.The results showed that mi R-99b-3p had negative regulation on SRPK1 m RNA and protein expression.The expression of mi R-99b-3p was increased significantly when UCA1 was down-regulated;the overexpression of mi R-99b-3p inhibited the expression of UCA1,while the knockdown of mi R-99b-3p had the opposite effect.Which showed that the expression of UCA1 was negatively correlated with the expression of mi R-99b-3p.Si-UCA1 inhibited the expression of SRPK1 m RNA and protein,while mi R-99b-3p inhibitor could partially reverse the inhibition of si-UCA1 on SRPK1 expression.Thus,mi R-99b-3p is involved in regulation of SRPK1 expression by UCA1.In order to investigate the effects of UCA1/mi R-99b-3p on the proliferation or apoptosis of A2780 cells.Mi R-99b-3p mimic/inhibitor,si-UCA1 or si-UCA1 plus mi R-99b-3p inhibitor was transferred into A2780 cells.The viability or apoptosis of cells in each treatment group were measured by MTT or apoptosis detection kit.The results showed that si-UCA1 impaired cell viability,while mi R-99b-3p inhibitor could partially reverse the inhibitory effect of si-UCA1 on cell viability.However,there was no significant difference in apoptosis among different treatment groups.In conclusion,this study reveals a novel ce RNA regulatory pathway in which UCA1 maybe regulate the expression of SRPK1 by competitively binding to mi R-99b-3p,which may provide a new potential target for the treatment of ovarian cancer.