Abstract: Abstract: Objective To predict and validate the targeting regulation of HOX antisense RNA (HOTAIR) on human leukocyte antigen-G (HLA-G) by upregulating miR-152-3p. Methods Bioinformatics websites and software were used to predict the binding sites of miR-152-3p targeting to HOTAIR and HLA-G 3′untranslated region (UTR). The 3′-UTR and mutant sequences of HOTAIR and HLA-G genes containing the binding sites with miR-152-3p were designed and synthesized, and the wild-type and mutant double luciferase reporter gene vectors of HOTAIR and HLA-G were constructed. After two-enzyme digestion electrophoresis and sequencing, HOTAIR and HLA-G wild-type and mutant double luciferase reporter gene vectors were co-transfected HTR-8/SVneo cells with miR-152-3p mimics and microRNA negative sequence control (mimics NC) respectively. Luciferase activity was detected, and the effects of miR-152-3p on the expression of HOTAIR and HLA-G were observed. Results The results of double enzyme digestion electrophoresis and sequencing showed that the size and sequence of wild type and mutant type HOTAIR and HLA-G gene vectors were consistent with the experimental expectations, and the vectors were successfully constructed. After transfection of miR-152- 3p mimics, the activity of HOTAIR and HLA-G wild-type luciferase decreased significantly (P＜0.05), but the expression of luciferase in mutant HOTAIR and HLA-G vectors was not significantly inhibited (P＞0.05). Conclusion HOTAIR regulates HLA-G targeting by upregulating the expression of miR-152-3p.

Key words: HLA-G antigens, genes, reporter, HOTAIR, miR-152-3p, HTR-8/SVneo, dual fluorescein reporter gene