LncRNA Lnc‐APUE is Repressed by HNF4α and Promotes G1/S Phase Transition and Tumor Growth by Regulating MiR‐20b/E2F1 Axis

Abstract Many long noncoding RNAs (lncRNAs) have been annotated, but their functions remain unknown. The authors found a novel lnc‐APUE (lncRNA accelerating proliferation by upregulating E2F1) that is upregulated in different cancer types, including hepatocellular carcinoma (HCC), and high lnc‐APUE level is associated with short recurrence‐free survival (RFS) of HCC patients. Gain‐ and loss‐of‐function analyses showed that lnc‐APUE accelerated G1/S transition and tumor cell growth in vitro and allows hepatoma xenografts to grow faster in vivo. Mechanistically, lnc‐APUE binds to miR‐20b and relieves its repression on E2F1 expression, resulting in increased E2F1 level and accelerated G1/S phase transition and cell proliferation. Consistently, lnc‐APUE level is positively associated with the expression of E2F1 and its downstream target genes in HCC tissues. Further investigations disclose that hepatocyte nuclear factor 4 alpha (HNF4α) binds to the lnc‐APUE promoter, represses lnc‐APUE transcription, then diminishes E2F1 expression and cell proliferation. HNF4α expression is reduced in HCC tissues and low HNF4α level is correlated with high lnc‐APUE expression. Collectively, a HNF4α/lnc‐APUE/miR‐20b/E2F1 axis in which HNF4α represses lnc‐APUE expression and keeps E2F1 at a low level is identified. In tumor cells, HNF4α downregulation leads to lnc‐APUE upregulation, which prevents the inhibition of miR‐20b on E2F1 expression and thereby promotes cell cycle progression and tumor growth.

To create pCDH-E2F1 and pCDH-HNF4, the coding sequence of E2F1 and HNF4 were respectively inserted into the EcoRI/BamHI sites of pCDH-Ctrl.
To construct pCDH-shNC and pCDH-shAPUE vectors, the complementary oligonucleotides that contained both sense and antisense siRNA sequences, the spacer sequence (5'-CTCGAG) and the flanking EcoRI and BamHI sites were chemically synthesized, annealed and then inserted into the EcoRI/BamHI sites in the pCDH-U6 vector, which was produced based on the pCDH-CMV-MCS-EF1-copGFP vector by replacing the CMV promoter with the U6 promoter.
To verify whether miR-20b and miR-17 targeted lnc-APUE, psi-APUE-wt and psi-APUE-mut were constructed. Full-length lnc-APUE with wild-type sequence or mutant miR-20b-binding sites was inserted into the XhoI/NotI sites downstream of the To construct the firefly luciferase reporter plasmids for verifying the lnc-APUE promoter region, the genomic fragments upstream of lnc-APUE were cloned into the KpnI/HindIII sites upstream of the firefly luciferase gene in pGL3-basic vector (Promega).
All constructs were verified by sequencing. The sequences of primers are listed in Purification of S1m-tagged RNA-RNA complexes: HepG2 (2×10 7 ) sublines with stable expression of APUE, S1m-APUE or S1m-APUE-mut were washed with 5 mL 1×PBS three times, pelleted by centrifugation at 500×g for 5 minutes, resuspended in 8 mL 1×PBS containing 0.37% formaldehyde and rotated at RT for 5 minutes to crosslink cellular components, followed by incubation with 250 mM glycine at RT for another 5 minutes to abort crosslinking reaction. Cell pellets were washed 2 times with 5 mL n, the number of paired tissues. For (C), the data are presented as mean ± SEM; P values were assessed by paired Student′s t-test *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.  B) The level of cellular lnc-APUE was reduced by shRNA targeting lnc-APUE.
HepG2, Huh-7 and SK-Hep-1 cells were infected with lentiviruses carrying pCDH-APUE or pCDH-Ctrl for 96 hours before qPCR analysis. For (A-C), the data from at least three independent experiments are presented as mean ± SEM; P values were assessed by unpaired Student′s t-test. **, P < 0.01; ***, P < 0.001. Figure S4. The impact of siAPUE on the levels of key regulators of pRb phosphorylation. HepG2 cells were exposed to RNAiMAX or transfected with NC or siAPUE for 24 hours or 36 hours before immunoblotting.    Figure S8. Lnc-APUE was enriched in the precipitates from S1m-APUE-and S1m-APUE-mut-transfected cells. A) The schematic diagram of S1m-APUE and S1m-APUE-mut construct. The short vertical lines indicate the binding sites of miR-20b in S1m-APUE, while the crosses depict the mutated sites in S1m-APUE-mut.
B) HepG2-APUE, HepG2-S1m-APUE and HepG2-S1m-APUE-mut sublines were subjected to S1m-tagged RNA affinity purification. Lnc-APUE in the S1m-pulldown product and the input were analysed by qPCR. The levels of lnc-APUE in the pulldown product were adjusted by that in the input. The mean value of the adjusted RNA level in the pulldown product of the HepG2-APUE group from three experiments was set as relative RNA level 1. For (B), the data from at least three independent experiments are presented as mean ± SEM; P values were assessed by unpaired Student′s t-test. **, P < 0.01.