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. 2017 Feb 8;8:14422.
doi: 10.1038/ncomms14422.

LncRNA AK023948 Is a Positive Regulator of AKT

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Free PMC article

LncRNA AK023948 Is a Positive Regulator of AKT

Pratirodh Koirala et al. Nat Commun. .
Free PMC article

Abstract

Despite the overwhelming number of human long non-coding RNAs (lncRNAs) reported so far, little is known about their physiological functions for the majority of them. The present study uses a CRISPR/Cas9-based synergistic activation mediator (SAM) system to identify potential lncRNAs capable of regulating AKT activity. Among lncRNAs identified from this screen, we demonstrate that AK023948 is a positive regulator for AKT. Knockout of AK023948 suppresses, whereas rescue with AK023948 restores the AKT activity. Mechanistically, AK023948 functionally interacts with DHX9 and p85. Importantly, AK023948 is required for the interaction between DHX9 and p85 to hence the p85 stability and promote AKT activity. Finally, AK023948 is upregulated in breast cancer; interrogation of TCGA data set indicates that upregulation of DHX9 in breast cancer is associated with poor survival. Together, this study demonstrates two previously uncharacterized factors AK023948 and DHX9 as important players in the AKT pathway, and that their upregulation may contribute to breast tumour progression.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Identification of lncRNAs capable of activating AKT by SAM library screen along with an AKT reporter.
(a) Schematic description of principle for the AKT reporter used to screen the SAM library. SAM gRNAs that can activate AKT activity are enriched by puromycin selection. (b) Detection of AKT activity for cells before selection (BS) and cells after selection (AS) by western blot. (c) AK023948 SAM gRNA increases the pAKT level. A mixed pool of five SAM gRNAs against AK023948 were introduced into MCF-7 cells carrying dCas9-VP64 and pMS2-p65-HSF1 by infection and cellular extract was prepared for western blot 2 days after infection.
Figure 2
Figure 2. AK023948 regulates the pAKT level.
(a) While AK023948 overexpression increases, AK023948 knockdown decreases phosphorylation of AKT. MCF-7 cells were transfected with either AK023948 expression vector or AK023948 siRNA. Cellular extract was prepared for western blot 48 h after transfection. (b) AK023948 KO suppresses the pAKT level. AK023948 KO was performed in MCF-7 using a procedure as described in Methods. (c) Re-expression of AK023948 in the AK023948 KO cells increases the pAKT level. AK023948 expression vector or control vector was introduced into KO cells, and total cellular extract was prepared for western blot. (d) High levels of AK023948 and pAKT in breast tumour specimens with representative images for the same field of a tumour. The same TMA was first detected for AK023948 by ISH and then the signal was stripped, followed by IHC to detect pAKT. Scale bar, 100 μm. (e) A positive correlation between AK023948 and pAKT from the same TMA with P-value of Fisher's exact test for the association <0.0001. (f) The pAKT level is lower in xenograft tumours derived from AK023948 KO than in vector control tumours, as detected by IHC. Scale bar, 50 μm.
Figure 3
Figure 3. DHX9 is an AK023948-binding partner and is involved in regulation of AKT activity.
(a) RNA precipitation using the biotin-labelled AK023948 probe, followed by PAGE and silver staining. Red star indicates a unique band bound to AK023948. Mass spectrometry analysis suggested DHX9 as a candidate. (b) Confirmation of the interaction between AK023948 and DHX9 by RNA precipitation and western blot. (c) Confirmation of the interaction between AK023948 and DHX9 by RNA immunoprecipitation using DHX9 antibody. (d) While DHX9 siRNA suppresses, ectopic expression of DHX9 increases the pAKT level. DHX9 siRNAs or DHX9 expression vector was introduced into MCF-7 cells by transfection, and cellular extract was prepared for western blot 48 h after transfection. Values in c are s.e.m. (n=3). **P<0.01 by two-tailed Student's t-test.
Figure 4
Figure 4. AK023948 is required for the interaction between DHX9 and p85.
(a) AK023948 KO primarily suppresses the p85β level, as detected by western blot. (b) AK023948 interacts with p85, as detected by RIP assay using p85 antibody. (c) AK023948 is required for the interaction between DHX9 and p85, as detected by co-immunoprecipitation (co-IP) using p85 antibody. HMLE cells express little AK023948, whereas MCF-7 cells express a high level of AK023948. (d) Rescue experiments further suggest that AK023948 is critical for the interaction between AK023948 and p85. (e) AK023948 is required for the interaction between DHX9 and p85, as determined by GST pulldown assay. The DHX9 level pulled down by GST-p85 was lower in KO cells than in gRNA control. (f) AK023948 is required for the interaction between DHX9 and p85, as detected by the Duolink in situ Fluorescence Kit (Sigma). HeLa cells were transfected with Myc-p85 plus control siRNA or AK0 siRNA. After 48 h, the cells were fixed for PLA. The red signals were lower in AK0 siRNA than in control siRNA cells. Scale bar, 100 μm. (g) DHX9 siRNAs suppress, but ectopic expression of DHX9 increases both p85 and pAKT. Values in b are s.e.m. (n=3). *P<0.05 by two-tailed Student's t-test.
Figure 5
Figure 5. AK023948 has an impact on the p85 stability.
(a) Effect of AK023948 KO on the p85 stability. MCF-7 cells were treated with cycloheximide (CHX) at 20 μg ml−1 and then were harvested for western blot at indicated time points. Half-life curve is on the right. (b) AK023948 KO reduces the interaction of p85 with p110, as detected by co-IP. (c) AK023948 KO inhibits the growth factor-induced AKT activation. Cells were first cultured in a serum-free medium for 2 h and then EGF or insulin was added at 10 ng ml−1 for 30 min. (d) AK023948 KO inhibits the acidosis-induced AKT activation. Cells were cultured at pH 7.4 or pH 6.6 for 2 h before harvesting for western blot. (e) AK023948 KO has no effect on ERK activity. Cells were treated with the same way as in c. (f) Suppression of AK023948 by RNAi can still reduce the pAKT level in PTEN-deficient BT549 cells.
Figure 6
Figure 6. Upregulation of AK023948 in breast cancer and promotion of tumorigenesis.
(a) Detection of AK023948 in the OriGene breast cancer tissue cDNA array by qPCR. (b) AK023948 is upregulated in breast cancer cells (MCF-7 and MDA-MB-231) as compared with non-malignant breast cells (MCF-10A and HMLE). (c) AK023948 KO suppresses cell proliferation of MCF-7, as detected by MTT assay. (d) AK023948 KO promotes apoptosis. Cells were seeded in slide chambers, and treated with H2O2 at 0.8 mM for 4 h before TUNEL assay. Apoptotic cells were counted from three different fields, and apoptotic cell ratio was calculated against total cells. (e) Tumour growth for vector control and AK0 KO#13 in nude mice. (f) Expression of AK023948 in breast cancer TMAs, as detected by ISH. Scale bar, 100 μm. Bottom: quantitative analysis of AK023948 expression based on the results from f. (g) Upregulation of DHX9 expression is associated with poor patient survival. About 20.9% (229 of 1091 cases analysed) are positive for DHX9 using the Onco Query Language (OQL; EXP>1.5). Values in b,c are s.e.m. (n=3). **P<0.01 by two-tailed Student's t-test.

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