LncRNA AFAP1-AS Functions as a Competing Endogenous RNA to Regulate RAP1B Expression by sponging miR-181a in the HSCR

Abstract

Background: Long noncoding RNAs (lncRNAs) have recently emerged as important regulators in a broad spectrum of cellular processes including development and disease. Despite the known engagement of the AFAP1-AS in several human diseases, its biological function in Hirschsprung disease (HSCR) remains elusive. Methods: We used qRT-PCR to detect the relative expression of AFAP1-AS in 64 HSCR bowel tissues and matched normal intestinal tissues. The effects of AFAP1-AS on cell proliferation, migration, cell cycle, apoptosis and cytoskeletal organization were evaluated using CCK-8, transwell assay, flow cytometer analysis and immunofluorescence, in 293T and SH-SY5Y cell lines, respectively. Moreover, the competing endogenous RNA (ceRNA) activity of AFAP1-AS on miR-181a was investigated via luciferase reporter assay and immunoblot analysis. Results: Aberrant inhibition of AFAP1-AS was observed in HSCR tissues. Knockdown of AFAP1-AS in 293T and SH-SY5Y cells suppressed cell proliferation, migration, and induced the loss of cell stress filament integrity, possibly due to AFAP1-AS sequestering miR-181a in HSCR cells. Furthermore, AFAP1-AS could down-regulate RAP1B via its competing endogenous RNA (ceRNA) activity on miR-181a. Conclusions: These findings suggest that aberrant expression of lncRNA AFAP1-AS, a ceRNA of miR-181a, may involve in the onset and progression of HSCR by augmenting the miR-181a target gene, RAP1B.

Keywords: AFAP1-AS; Competing endogenous RNA; Hirschsprung disease; miR-181a..

Figure 1

AFAP1-AS was down-regulated and its cytobiology change transfected with AFAP1-AS siRNA. (A) The expression of AFAP1-AS in HSCR was significantly down-regulated compared with normal tissues. Data were presented as box plot of the median and range of log-transformed relative expression level. The top and bottom of the box represent the 75th and 25th percentile. The whiskers indicate the 10th and 90th points. (B) AFAP1-AS knockdown affected abilities of cell migration and proliferation. Representative images of migrated cells were visualized as shown (left panel). Quantifications of cell migration were presented as percentage migrated cell numbers (middle panel). Absorbance at 450 nm as measured by CCK8 was expressed as Mean ± SE (right panel). (C-D) Cycle and apoptosis assays were conducted after AFAP1-AS knockdowm by flow cytometer. (E) F-actin cytoskeleton of was visualized with Rhodamine Phalloidin staining in SH-SY5Y cells.

Figure 2

AFAP1-AS acted as a ceRNA by biding miR-181a (A) Expression levels of AFAP1-AS in the nuclear and cytoplasm fractions were determined using qRT-PCR. GAPDH and U6 were used as cytoplasmic and nuclear markers, respectively. (B) The top and bottom regions are the sequences of AFAP1-AS wild-type binding to miR-181a and mutations in the 3'-UTR of AFAP1-AS, respectively. (C) The AFAP1-AS wild type or mutant vectors were co-transfected with miR-181a NC or miR-181a mimics. Firefly luciferase activities were then measured normalized to Renilla. (D) Extracts of 293T cells were applied for RNA binding protein immunoprecipitation (RIP). Relative RNA levels of AFAP1-AS and miR-181a were measured by qRT-PCR.

Figure 3

The functional assays of miR-181a were conducted in HSCR cells (A) The expression of miR-181a in HSCR was significantly over-expressed compared with normal tissues. Data were presented as box plot of the median and range of log-transformed relative expression level. The top and bottom of the box represent the 75th and 25th percentile. The whiskers indicate the 10th and 90th points. (B) Transwell analysis of 293T and SH-SY5Y cells transfected with negative control or miR-181a mimics (left panel); Migrated cells stained with crystal violet was shown (middle panel); Cell proliferation were evaluated by CCK8 assay(right panel).The data are presented as the mean ± SE. (C-D) Cycle and apoptosis assays were conducted after transfection with miR-181a mimics or negative control by flow cytometer. (E) F-actin cytoskeleton of was stained with Rhodamine Phalloidin staining after transfection with miR-181a mimics or negative control in SH-SY5Y cells.

Figure 4

Relationship between AFAP1-AS and the miR-181a target, RAP1B (A) Top: Predicted binding sites between miR-181a and 3'UTR of RAP1B were constructed as well as mutations in the 3'-UTR of RAP1B. (B) The RAP1B wild type or mutant vectors were co-transfected with negative control or miR-181a mimics. Firefly luciferase activities were then measured normalized to Renilla. (C) Expression levels of RAP1B in the previous tissues were examined by qRT-PCR analysis. Data were presented as box plot of the median and range of log-transformed relative expression level. The top and bottom of the box represent the 75th and 25th percentile. The whiskers indicate the 10th and 90th points. (D) A correlation between AFAP1-AS and RAP1B was observed according to the qRT-PCR results (r²=0.4726; P<0.0001). (E) Western blots of RAP1B were measured in HSCR and normal tissues. (F) Western blot and qRT-PCR analysis of RAP1B were conducted after transfection with si-NC, si-AFAP1-AS, and inhibition of AFAP1-AS in combination with miR-181a inhibitor. GAPDH was used as control. All results were presented as mean ± SE.