Ovarian Cancer

HOST2[LncRNA]

let-7b[miRNA]

c-Myc[mRNA]

Ovcar3 Cell Line

Homo sapiens (human)

Hum Mol Genet 2015 Feb 1 24, 841-52 doi:10.1093/hmg/ddu502 PMID:25292198

LncRNA-HOST2 regulates cell biological behaviors in epithelial ovarian cancer through a mechanism involving microRNA let-7b.

qRT-PCR

A luciferase reporter vector encoding 1500–2000 nt of the HOST2 sequence (harboring all of complementary positions of seven miRNAs, renamed pGL3-HOST2-wt) and a Renilla luciferase (Rluc) gene in the 3' UTR and a downstream, constitutively expressed firefly luciferase gene, which served as an internal control for normalization, were constructed. These vectors were then sequentially co-transfected with mimics of the above-mentioned, predicted miRNAs and a scramble mimic (as the control, renamed miCon) into OVCAR-3 cells (Aiferase activity of pGL3-HOST2-wt (WT), whereas little to no significant reduction in luciferase activity was observed when the cells were co-transfected with the other five predicted miRNAs. TThe data indicated that mutating the seed regions for let-7b and miR-1266 barely abrogated its luciferase activity. We also found that the inhibition effect of let-7b was more stable than that of miR-1266. Given that let-7 regulates target-gene expression by binding to imperfect complementary sequences in messenger RNAs (mRNAs), leading to translational repression and/or mRNA destabilization {(32)}, and because we knew nothing about miR-1266, we only focused on the regulatory role of let-7b for the HOST2 transcript in the further work. Meanwhile, the expression of let-7b was detected in the 30 OBT and 50 EOC tissue samples and was shown to be lower in EOC than in OBT (P < 0.01). After transfecting the let-7b mimic into OVCAR-3 cells, HOST2 expression was observed to be markedly down-regulated at the mRNA level when compared with that in cells transfected with miCon. These results indicated that let-7b repressed HOST2 expression in EOC, but the reason for let-7b expression being lower in EOC than in OBT was uncertain.When HOST2 was down-regulated, the expression of HMGA2, c-Myc, Dicer and Imp3 were also significantly reduced at the RNA and protein level, which was consistent with an increase in let-7b expression. Concomitantly, cell migration, invasion and proliferation were all inhibited, similar to what we have shown. Thus, the expression of the four targets were analyzed using reciprocal, HOST2 over-expressed experiments, where pcDNA3.0-HOST2 and a control empty vector (pcDNA3.0) were, respectively, transfected into HEK293 cells to increase the endogenous HOST2 level. Cells transfected with pcDNA3.0-HOST2 expressed HOST2 5-fold higher than cells transfection with pcDNA3.0. Significant increases of HMGA2, c-Myc, Dicer and Imp3 at the mRNA and protein levels in response to HOST2 over-expression were all observed. Together, these HOST2 loss- and gain-of-function studies suggest that ectopically expressed HOST2 sequesters endogenous let-7b and inhibits its functions, leading to derepression of the target genes of let-7b. To confirm that these effects were indeed due to down-regulated let-7b, we performed transfection experiments using ilet-7. As expected, increased mRNA as well as protein levels of HMGA2, c-Myc, Dicer and Imp3 were all observed in ilet-7-treated HEK293 cells when compared with iCon-treated cells.