Circular RNA circ_0076631 promotes coxsackievirus B3 infection through modulating viral translation by sponging miR-214-3p

FIGURE 1. The source of circ_0076631 and association with CVB3 infection. (A) The genomic location of circ_0076631 in the transcript of SLC29A1 gene. (B) Ring formation by circ_0076631 was performed on the basis of the amplification curve and dissolution curve, which are shown. (C) HeLa cells were infected with CVB3 (MOI = 10) and incubated for 6 h. Circ_0076631 levels were analyzed by RT-qPCR. n = 3, ***P < 0.01.

FIGURE 2. Circ_0076631 knockdown suppresses CVB3 replication. (A) HeLa cells were transfected with circ_0076631 siRNA (si-1, si-2, si-3) and cultured for 24 h. The abundance level of circ_0076631 was assayed by RT-qPCR. n = 3. *P < 0.05, ***P < 0.001. (B–E) HeLa cells were transfected separately with si-NC and si-circ-0076631 for 24 h. After infection with CVB3 (MOI = 10) for 6 h. (B) The relative abundance level of circ_0076631 and CVB3 3D-coding RNA detected by RT-qPCR. (C,D) Immunofluorescence staining was performed to detect the level of CVB3 3D protein. (E) The relative level of CVB3 3D protein was detected by western blotting. n = 3. **P < 0.01, ***P < 0.001 compared with the si-NC. #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the si-NC + CVB3 group.

FIGURE 3. Prediction of miRNAs that may bind to targeting circ_0076631. (A) Screening the potential miRNAs that may bind to circ_0076631. (B,C) Screening of the miRNAs related to the inflammatory pathway and viral infection. (D) Comprehensive analysis of the three aforementioned screening results. (E) HeLa cells were infected with CVB3 (MOI = 10) for 6 h, and the abundance of miRNAs was measured by RT-qPCR. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 4. Verification of miR-214-3p binding to circ_0076631. (A) FISH assay was performed to observe the cellular localization of circ_0076631 in HeLa cells with CVB3 infection. Circ_0076631, 18S rRNA, U6 are showed in red. Nuclei are stained with DAPI (blue). (B) The miR-214-3p-binding sequence in wild-type circ_0076631 and the mutated circ_0076631. Plasmids expressing Renilla luciferase (Rluc) fused with wide-type circ_0076631 (containing miR-214-3p-targeting site, WT-circ_0076631) or with mutated circ_0076631 (without miR-214-3p-targeting site, Mut-circ_0076631) sequence were co-transfected with miR-214-3p mimic (miR-214-3p), miR-NC, AMO-miR-214-3p (AMO-214-3p), or AMO-miR-NC (AMO-NC) into HEK293T cells, respectively. Luciferase activity was detected with dual luciferase assay. The activity of Rluc was normalized with luciferase (Luc). n = 3. ***P < 0.001.

FIGURE 5. Prediction and verification of miR-214-3p tergetig CVB3 genomic RNA. (A) Identification of the potential miR-214-3p-binding sites in CVB3 genomic RNA by RNAhybrid. The binding ability of miR-214-3p with CVB1- CVB6 3D and EV-A71 3D sequence. (B) The miR-214-3p-binding sequence in the CVB3 3D-coding sequence and the mutated CVB3 3D sequence. Wide-type CVB3 3D sequence (containing miR-214-3p-binding sequence, WT-CVB3-3D) or mutated CVB3 3D sequence (without miR-214-3p-binding sequence, Mut-CVB3-3D) was cloned into a luciferase-expressing plasmid. The plasmid was co-transfected separately with miR-214-3p, miR-NC, AMO-214-3p, or AMO-NC into HEK293T cells, respectively. The luciferase activity was detected with dual luciferase assay. The acitivity of Rluc was normalized with Luc. n = 3. ***P < 0.001. (C) HEK293T cells were co-transfected with pEGFP-CVB3-3D (EGFP-3D) or pEGFP-CVB3-3Dmut (EGFP-3Dmut) and miR-214-3p for 24 h. Immunofluorescence staining was performed to detect the expression level of the CVB3 3D protein.

FIGURE 6. Circ_0076631 knockdown suppresses CVB3 replication through miR-214-3p targeting 3D-coding sequence. (A) HeLa cells were transfected separately with si-NC + AMO-NC, si-2 + AMO-NC, and si-2 + AMO-214-3p for 24 h, and infected with CVB3 (MOI = 10) for 6 h. (A–C) The relative abundance level of circ_0076631, miR-214-3p, and CVB3 3D was detected by RT-qPCR. (D,E) Immunofluorescence staining was performed to detect the level of the CVB3 3D protein. (F) The relative level of CVB3 3D protein was detected by western blotting. n = 3. ***P < 0.001 compared with the si-NC + AMO-NC group. ##P < 0.01, ###P < 0.001 compared with the CVB3 + si-NC + AMO-NC group. $P < 0.05, $$$P < 0.001 compared with the CVB3 + si-2 + AMO-NC group.

FIGURE 7. The putative mechanism of circ_0076631 regulating CVB3 infection by sponging miR-214-3p. At the early stage of CVB3 infection, miR-214-3p is abundant and can suppress viral biosynthesis by targeting the 3D-coding sequence (black lines). Later, CVB3 infection can somehow trigger circ_0076631 generation. Circ-0076631 facilitates CVB3 replication through sponging miR-214-3p and relieving its suppression on CVB3 biosynthesis (red lines).