RETRACTED ARTICLE: LNCAROD enhances hepatocellular carcinoma malignancy by activating glycolysis through induction of pyruvate kinase isoform PKM2

Lipofectamine 2000 transfection reagents and total RNA extraction agent (TRIzol) were purchased from Invitrogen (Grand Island, NY, USA). Antibodies against PKM1, PKM2, fibronectin, and β-actin were obtained from Abcam (Cambridge, MA, USA), whereas those against E-cadherin, N-cadherin, and vimentin were from Cell Signaling Technology (Danvers, MA, USA). α-Catenin antibodies were purchased from Becton Dickinson. Unless otherwise stated, all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

All studies involving human samples were reviewed and approved by the ethics committee of The First Affiliated Hospital of Guangxi Medical University, and written informed consent was obtained from all patients based on the Declaration of Helsinki. Tumor and adjacent non-tumor tissues were collected from patients with HCC (n = 92) at the First Affiliated Hospital of Guangxi Medical University. The clinicopathological characteristics examined included age, sex, tumor nodule number, etiology, serum AFP level, tumor stage, tumor size, tumor differentiation, and vascular invasion. Tumor classification and grade were determined based on the pTNM classification advocated by the International Union against Cancer.

HCC cell lines including SNU-449, SNU-182, Huh7, HepG2, and HCC-LM3 were obtained from Guangzhou Cellcook Biotech Co., Ltd. SMMC-7721 was from Shanghai Saily Biotechnologies Co., Ltd. Cells were cultured in an appropriate medium containing 10 % fetal bovine serum (FBS; Gibco) supplemented with penicillin (100 U/mL) and streptomycin (100 µg/mL) to minimize the chance of germ contamination in a humidified atmosphere containing 5 % CO₂/95 % air at 37 °C. Normal human hepatocyte line THLE-2 was from Zhejiang Meisen Cell Technology Co., Ltd. and cultured according to ATCC guidelines for culturing this cell line, which are available at: https://www.atcc.org/products/crl-2706↗. All cell lines were confirmed to be mycoplasma negative during the experiments. The hypoxia model was established using 1 % O₂/5 % CO₂/94 % N₂ or via the introduction of CoCl₂, a hypoxia mimetic agent [14].

Short hairpin RNAs (shRNAs) specifically targeting LNCAROD, serine-and arginine-rich splicing factor 3 (SRSF3), and PKM2, and the small interference RNA (siRNA) targeting Mettl3, IGF2BP1, and HIF1α were all obtained from GenePharma (Shanghai, China). These shRNAs or siRNAs were transfected into HCC cells using Lipofectamine 2000. For in vivo experiments, sh-LNCAROD was delivered into HCC cells via lentiviral transfection.

Total RNA was extracted using an RNA simple Total RNA Kit (TIANGEN, DP419), whereas cytoplasmic and nuclear RNA was isolated using a Nuclear/Cytoplasmic Isolation Kit (BioVision, San Francisco, CA, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, #K1622) and poly (A) polymerase reaction buffer (NEB, M0276s) according to the manufacturer’s instructions. RT-qPCR was performed using the iTaqTM Universal SYBR Green Supermix. The fold-change in RNA expression was quantified using the 2^−ΔΔCt method. The primer sequences used in this study are listed in Table S1.

LncRNA fluorescence in situ hybridization (FISH) was performed to determine the subcellular localization of LNCAROD. Cells were fixed with 4 % paraformaldehyde for 10 min at room temperature. After permeabilization in 0.5 % Triton X-100 for 5 min, cells were treated with pre-hybridization buffer at 37ºC for 30 min and then were subjected to hybridization with the LNCAROD probe (RiboBio, Guangzhou, China) overnight at 37 ºC in the dark. Subsequently, the cells were washed in turn with 4×SSC containing 0.1 % Tween 20 thrice, 2×SSC once, 1×SSC once at 37 ºC, and 1×PBS once at room temperature in the dark. Finally the nuclei were counterstained with DAPI for observation using a fluorescence microscope.

IHC was performed on paraformaldehyde-fixed, paraffin-embedded tissue specimens using heat-mediated antigen retrieval citrate (0.01 M, pH 6.0). Endogenous peroxidase activity was blocked using 3 % H₂O₂ for 15 min at room temperature (RT). Thereafter, the sections were incubated with goat serum for 1 h to block the non-specific binding sites and then with primary antibodies overnight at 4 °C. After rinsing three times for 5 min each with PBS, the sections were further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. Each section was then rinsed with PBS thrice for 5 min each, and the reactions were developed using diaminobenzidine tetrahydrochloride (DAB) as a substrate. Cellular nuclei were counterstained using hematoxylin, and the sections were sealed with neutral gum. Images were obtained using an Olympus X71 inverted microscope (Olympus Corp., Tokyo, Japan).

The glycolytic capacity of living cells was evaluated through ECAR measurement using Seahorse Bioscience XF96 Extracellular Flux Analyzer and software (Seahorse Bioscience, North Billerica, MA, USA). Briefly, 4 × 10⁴ cells were seeded in a cell culture microplate XF96 in triplicate. ECAR readings were initially measured under basal conditions and further determined after the successive addition of glucose, oligomycin (an oxidative phosphorylation inhibitor), and 2-deoxy-D-glucose (2-DG, a glycolytic inhibitor) at the indicated time points using a Seahorse XF Glycolysis Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions.

The MeRIP-qPCR assay was performed to determine the level of LNCAROD m⁶A. Total intracellular RNA was extracted using TRIzol reagent. Anti-m⁶A antibodies or anti-immunoglobulin G (IgG; Cell Signaling Technology) (3 µg) was first conjugated to protein A/G magnetic beads and mixed with 100 µg aliquot of total RNA in IP buffer containing RNase/protease inhibitors. m⁶A-modified RNA was eluted twice with 6.7 mM N⁶-methyladenosine 5’-monophosphate sodium salt at 4 ºC for 1 h. Subsequently, RT-qPCR analysis was performed to determine the m⁶A enrichment on LNCAROD using the following primer sequences: F, 5’-AGGGCTAGAGTAAGAGCCCC-3’; and R, 5’-CCGTTGTGGCCAGAAGTTGA-3’.

Four-week-old BALB/c nude mice were purchased from the Animal Center of Guangxi Medical University (Nanning, China). All animal experimental protocols were performed in accordance with the National Institutes of Health Animal Use Guidelines on the Use of Experimental Animals. HCC-LM3 cells with silenced LNCAROD or the negative control were subcutaneously injected into the flank region of the mice at 2 × 10⁶ cells. Tumor volume was measured weekly and calculated as follows: V = (length × width²)/2. After 4 weeks, the tumors were excised and weighed. To evaluate the extent of lung metastasis, 1 × 10⁶ control or LNCAROD knockdown HCC-LM3 cells were injected into nude mice via the tail vein of nude mice. On the 6 weeks, the mice were sacrificed, and the lung tissues were harvested and fixed in 4 % formaldehyde solution. The tumors were counted on the hematoxylin-eosin (H&E) stained lung tissues.

A Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was used according to the manufacturer’s instructions. Briefly, cell extracts were immunoprecipitated with sepharose beads conjugated antibodies against AGO2, IGF2BP1, SRSF3, or IgG at 4 °C for 6 h. To remove proteins from the complex, 0.1 % SDS/Proteinase K (0.5 mg/mL, 30 min at 55 °C) was used. Immunoprecipitated proteins and RNAs were detected using western blot and RT-qPCR, respectively.

A Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, 20164) was used according to the manufacturer’s instructions. In brief, cell lysates were treated with RNAase-free DNAase I and incubated with biotinylated LNCAROD in the presence of streptavidin magnetic beads, which can capture the proteins/miRNAs potentially interacting with LNCAROD. A Pierce™ RNA 3’ End Desthiobiotinylation Kit (Thermo, 20,163) was used for LNCAROD biotinylation labeling. Proteins and RNAs in the captured protein–RNA complex were analyzed using western blot and RT-qPCR, respectively.

Numerical data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Student’s t-test or one-way analysis of variance (ANOVA) was used for comparison between groups. Qualitative data were analyzed using the chi-square test. Kaplan-Meier analysis was used for survival analysis with inter-group comparison performed using the log-rank test. Linear regression analysis was performed to evaluate the correlation between gene expression levels. Statistical analysis was conducted in the GraphPad Prism v8.0 (GraphPad, Inc., USA) and the Statistical Software Package for Social Sciences (v 22.0; SPSS, Inc., Chicago, IL, USA). Differences were considered statistically significant at P < 0.05.

Previously we performed a lncRNA microarray assay of 18 HCC and adjacent para-tumorous tissues and identified 26 significantly upregulated lncRNAs (all fold change > 6 and P < 0.01) [1], suggesting that these lncRNAs minght play a critical role in HCC tumorigenesis. Among these upregulated lncRNAs, LNCAROD (ENST00000443523.1) was the fourth most upregulated gene. However, its role in HCC tumorigenesis is completely unclear and thus interested us. Here, we found a significant upregulation of LNCAROD in HCC tissues upon comparison with adjacent normal liver tissues across TCGA datasets, and this was verified by RT-qPCR assay results of matched HCC tumor and adjacent normal tissues (Fig. 1 A-B). The enhanced expression of LNCAROD was also observed in HCC cell lines (SNU-182, HepG2, Huh7, SNU-449, SMMC-7721, and HCC-LM3) compared to the normal THLE-2 liver cell line (Fig. 1 C). Further, as shown in Fig. 1D, Kaplan-Meier analysis showed that HCC patients with high level of LNCAROD had poorer prognosis (P = 0.0039). Correlation analysis with clinicopathological parameters demonstrated that high LNCAROD levels are associated with unfavorable tumor-node-metastasis (TNM) stage, tumor size, differentiation grade, and microvascular invasion (Table S2). Collectively, these findings strongly suggest that LNCAROD is highly expressed in HCC tissues, is positively associated with HCC malignancies, and might function as a chemoresistant molecule in HCC.

To identify the role of LNCAROD in HCC, HCC cell lines (SNU-449, HCC-LM3, SMMC-7721, and Huh7) were transfected with pcDNA3.1(+) vectors encoding human LNCAROD inserts or shRNAs, and transfection efficiency was verified by RT-qPCR (Figure S1A, B). To identify whether LNCAROD facilitates HCC cell growth, CCK8 and colony formation assays were performed. As shown in Fig. 2 A-B, LNCAROD silencing in SNU-449 and HCC-LM3 cell lines decreased cell proliferation compared with their control counterparts, whereas LNCAROD overexpression significantly enhanced their proliferation (Figure S2A-B), demonstrating that LNCAROD has a potent HCC-promoting effect.

Subsequently, we examined the migration and invasion properties of HCC cells with LNCAROD knockdown or overexpression. LNCAROD knockdown significantly decreased the migration and invasion of SNU-449 and HCC-LM3 cells (Fig. 2 C), whereas its overexpression significantly promoted the migration and invasiveness of SMMC-7721 and Huh7 cells (Figure S2C). Epithelial-to-mesenchymal transition (EMT) is closely related to cancer cell migration and invasion. In line with this, we found that the epithelial markers E-cadherin and α-catenin were increased, whereas the mesenchymal markers N-cadherin, fibronectin, and vimentin were decreased in HCC cell lines upon LNCAROD knockdown (Fig. 2D); LNCAROD overexpression produced the opposite results (Figure S2D), indicating that EMT in HCC cells is positively regulated by LNCAROD. In summary, these findings suggest that LNCAROD accelerates HCC cell migration and invasiveness.

We further investigated the effect of LNCAROD on HCC chemoresistance to 5-fluorouracil (5-FU). Although this agent has greatly improved the prognosis of HCC patients, acquired or inherent chemoresistance compromises its overall efficacy in HCC treatment [15, 16]. As seen in Fig. 2E-F, LNCAROD knockdown sensitized HCC cells to 5-FU treatment, whereas its overexpression promoted 5-FU resistance in SMMC-7721 and Huh7 cells (Figure S2E-F). Besides, flow cytometry assay showed that LNCAROD knockdown notably facilitated 5-FU-induced HCC cell apoptosis (Fig. 2G).

Consistent with the in vitro findings above, LNCAROD deficiency evidently decreased tumor growth rate and ki67-positive cell number in both the normal saline (NS) and 5-FU-treated nude mice xenografts (Fig. 2 H-J). In addition, in the lung metastasis model, we observed that LNCAROD knockdown significantly inhibited the lung metastasis capacity of HCC cells as evidenced by the dramatic decrease in both the number of mice with lung tumors and that of tumors in mouse lungs treated with NS or 5-FU (Fig. 2 K-L). Collectively, these results indicate that LNCAROD facilitates the malignancy of HCC cells, thereby rendering chemoresistance to 5-FU.

Subsequently, we explored the mechanism underlying the upregulation of LNCAROD in HCC cells. The m⁶A modification is emerging as a critical mechanism for the modulation of RNA life cycle, expression, and function [17]. Methyltransferase-like 3 (Mettl3) plays an important role in mediating m⁶A modification in mammalian cells, and its silencing led to the decreased expression of LNCAROD in HNSCC [10].

By searching the TCGA database, we found that Mettl3 levels were significantly higher in HCC tissues than in corresponding para-cancerous tissues (Fig. 3 A). Many m⁶A sites were found with LNCAROD using the RMBase (http://rna.sysu.edu.cn/rmbase/index.php↗) prediction. Thus, we hypothesized that LNCAROD upregulation might be due to its m⁶A modification. m⁶A RNA IP (RIP) and RT-qPCR results showed that m⁶A was significantly more abundant in HCC cell lines (SNU-449 and HCC-LM3) than in normal THLE-2 liver cells (Fig. 3B). Furthermore, Mettl3 silencing significantly decreased LNCAROD level (Fig. 3 C), whereas its overexpression increased it (Fig. 3D). RIP assays showed that Mettl3 depletion reduced the m⁶A modification of LNCAROD in HCC cells (Fig. 3E). In the presence of actinomycin D, an agent that blocks the de novo synthesis of RNA, Mettl3 depletion decreased the stability of LNCAROD, whereas Mettl3 overexpression produced the opposite result (Fig. 3 F). Collectively, these data suggest that Mettl3 acts as an m⁶A writer and plays a critical role in maintaining the high level of LNCROAD in HCC.

The insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is a “reader” of m⁶A and is essential in many cancers as it maintains the stability of m⁶A-modified noncoding RNAs [18]. Here, we confirmed the direct binding between IGF2BP1 and LNCAROD using an RIP assay (Fig. 3G). IGF2BP1 silencing in HCC cells significantly decreased the LNCAROD level, whereas its overexpression increased it (Fig. 3 H-I). In the presence of actinomycin D, LNCAROD stability was significantly impaired following IGF2BP1 silencing but was enhanced by IGF2BP1 overexpression (Fig. 3 J). Furthermore, the interaction between IGF2BP1 and LNCAROD was remarkably interrupted upon Mettl3 depletion (Fig. 3 K). Taken together, our data indicate that the m⁶A modification maintained by both the “writer” Mettl3 and the “reader” IGF2BP1 is pivotal in maintaining the stability and upregulation of LNCAROD in HCC cells.

To explore the molecular mechanism by which LNCAROD triggers HCC malignancy and chemoresistance, we performed RNA-sequencing analysis (Fig. 4 A). Using KEGG analysis, we found that the terms including vitamin B6 metabolism, type II diabetes, pyruvate metabolism, proteasome, and glycolysis/gluconeogenesis were significantly enriched in HCC cells with LNCAROD knockdown, of which glycolysis was the most significantly enriched (Fig. 4B). As shown in Fig. 4 C, LNCAROD knockdown remarkably inhibited glucose consumption, lactate production, and ATP production. In contrast, dramatically increased glucose consumption, lactate production, and ATP production were observed in LNCAROD-overexpressing HCC cells (Figure S3A-B). In addition, LNCAROD knockdown effectively decreased the extracellular acidification rate (ECAR). In contrast, ECAR was significantly increased upon LNCAROD overexpression (Fig. 4D and Figure S3C-D). These results indicate that LNCAROD increases glycolysis metabolism in HCC cells.

We next determined whether glycolysis is implicated in LNCAROD-mediated promotion of HCC cell growth, migration, invasion and found that these processes were significantly inhibited by 2-deoxy-D-Glucose (2-DG), a glycolysis inhibitor (Fig. 4E-G). Moreover, 2-DG effectively reversed the LNCAROD-induced upregulation of N-cadherin, fibronectin, and vimentin, as well as the downregulation of E-cadherin and α-catenin (Fig. 4 H). Taken together, these data suggest that LNCAROD induces HCC malignancies via increased cell glycolysis.

In addition, we examined the influence of LNCAROD on the expression of key glycolysis regulatory genes in HCC cells using RT-qPCR. As shown in Fig. 4I, LNCAROD knockdown significantly suppressed the expression of PKM2 and PFKL but did not affect that of PKM1. Other glycolysis regulatory genes, including GLUT3, HK-2, PDK1, and ENO1, were not significantly affected by LNCAROD (data not shown).

Thus far, our data showed that LNCAROD acts as an important oncogene in HCC progression. We next explored the mechanism underlying its HCC-promoting effect. First, we evaluated the subcellular localization of LNCAROD in SNU-449 and HCC-LM3 cells using RT-qPCR. As shown in Fig. 5 A-B, LNCAROD was well-distributed in both the cytoplasm and the nucleus. As such, we hypothesized that LNCAROD exerts its HCC-promoting effect via both nuclear and cytoplasmic pathways.

LncRNAs can regulate the biological functions of specific nuclear proteins that are critically involved in tumor progression via interacting with proteins. We predicted the potential LNCAROD–protein interactions using catRAPID (http://service.tartaglialab.com/page/catrapid_group↗). Bioinformatics analysis revealed that the serine-and arginine-rich splicing factor 3 (SRSF3), a splicer of PKM that induces PKM switching from PKM1 to PKM2, is a potential LNCAROD-interacting protein (Table S3). Furthermore, we observed that SRSF3 deficiency decreased PKM2 levels but increased those of PKM1 in HCC cells (Figure S4A-D).

Given these findings, we speculate that LNCAROD exert its oncogenic role by directly binding to SRSF3 to promote PKM isoform switching from PKM1 to PKM2. This hypothesis is supported by previous studies reporting that PKM switching towards PKM2 plays an oncogenic role in various cancers [19–21]. Consistent with this, we found that higher PKM2 levels in HCC patients were significantly correlated with decreased OS patient survival (Figure S5). We performed a RIP assay and observed LNCAROD enrichment with SRSF3 in the interactome (Fig. 5 C) that was further validated by the RNA pull-down assay results (Fig. 5D).

In addition, we identified the implication of SRSF3 in LNCAROD-mediated regulation of PKM alternative splicing and found that SRSF3 knockdown greatly alleviated LNCAROD-induced PKM switching (Fig. 5E-F). Furthermore, knockdown of SRSF3 in LNCAROD-overexpressed cells greatly blunted the increase of HCC cell proliferation, migration, invasion, and glycolysis (Fig. 5G-M).

We also explored the critical effect of LNCAROD/SRSF3/PKM2 in HCC tumorigenesis. As shown in FigureA-B, LNCAROD or SRSF3 knockdown significantly inhibited HCC cell proliferation. The combination of shLNCAROD and shSRSF3 yielded a stronger inhibitory effect on HCC proliferation than their individual knockdown, whereas a stronger proliferation-promoting effect was observed for the combined overexpression of LNCAROD and SRSF3 (FigureA-B). Notably, the growth-suppressing effect of the combined LNCAROD/SRSF3 knockdown was significantly reversed by PKM2 overexpression (FigureA-B). In contrast, the growth-promoting effect of their combined overexpression was significantly alleviated by PKM2 knockdown (FigureA-B). The synergistic effects on HCC cell migration, invasion, and glycolysis were also observed between LNCAROD and SRSF3, which were also PKM2 dependent (FigureC-G and FigureC-G). S6 S7 S6 S7 S6 S7

Considering the nuclear and cytoplasmic localization of LNCAROD, we hypothesized that the ceRNA mechanism might also contribute to the oncogenic effect of LNCAROD in HCC. To verify this assumption, we first predicted potential LNCAROD target miRNAs using the bioinformatics database The Encyclopedia of RNA Interactomes (ENCORI, previously known as starBase v2.0) [22] and randomly selected five miRNAs for subsequent experimental verification.

According to the results of the RNA pull-down assay, miR-145-5p was the most enriched among all other miRNA candidates in the precipitate of LNCAROD (Figure S8A-B). Furthermore, miR145-5p significantly decreased the luciferase activity of LNCAROD WT but did not affect that of LNCAROD MUT (Fig. 6 A). We also observed that miR-145-5p levels were downregulated in HCC cells upon the overexpression of LNCAROD (Fig. 6B). These findings indicate that LNCAROD targets and sequesters miR-145-5p to prevent RISC-mediated downstream mRNA silencing. Thus, we tested this hypothesis using RIP and RNA pull-down assays. LNCAROD and miR-145-5p were highly enriched in the Ago2 precipitate (Fig. 6 C). In agreement with this, higher enrichments of miR-145-5p and Ago2 were observed in the biotin-labeled LNCAROD group than the control (Fig. 6D). These data suggest that LNCAROD acts as a molecular sponge for miR-145-5p in HCC via the ceRNA mechanism.

Then, we examined the role of miR-145-5p in HCC progression by screening for its potential targets and predicted a potential binding between PKM and miR-145-5p, indicating that PKM might be a direct target of miR-145-5p. To confirm this, dual-luciferase reporter assays were performed. We found that miR-145-5p significantly decreased the luciferase activity of PKM WT, but no significant inhibition of PKM MUT was detected (Fig. 6E). Furthermore, miR-145-5p significantly inhibited the expression of PKM1/2 (Fig. 6F-G). Subsequently, we analyzed the involvement of miR-145-5p in LNCAROD-mediated PKM2 alterations and HCC tumorigenesis. As seen in Fig. 6 H-I, the LNCAROD-induced PKM2 upregulation was significantly suppressed by miR-145-5p mimics. Collectively, these findings suggest that LNCAROD increases PKM2 level through targeting miR-145-5p.

Furthermore, the oncogenic effects of LNCAROD in terms of cell proliferation, migration, invasion, and glycolysis were suppressed by the miR-145-5p mimic (FigureA-G), thereby suggesting an indispensable role of miR-145-5p in LNCAROD-mediated modulation of PKM2 and HCC tumor phenotypes. SRSF3 silencing did not affect miR-145-5p expression (data not shown), demonstrating that LNCAROD/SRSF3/PKM2 and LNCAROD/miR-145-5p/PKM2 exerted their oncogenic function in HCC in a relatively independent manner. S9

The hypoxia-inducible factor-1α (HIF-1α), a master cancer driver under hypoxic conditions, regulates aerobic glycolysis in many cancer types, including HCC in a PKM2-dependent manner [23]. Therefore, we compared the oncogenic efficiency of LNCAROD under hypoxic and normoxic conditions and found that its overexpression resulted in significantly higher proliferation of HCC cells under hypoxia than normoxia (Fig. 7 A-B). The combination of LNCAROD and hypoxic treatment synergistically enhanced tumor cell migration, invasion, and glycolysis (Fig. 7 C-G). A more noticeable increase in PKM2 expression was also observed following LNCAROD overexpression under hypoxic conditions (Fig. 7 H-I).

Subsequently, we determined the role of HIF1α in LNCAROD-mediated biological effects under hypoxia and observed that the induction of PKM2 upregulation by LNCAROD was greatly attenuated by HIF1α knockdown (FigureA-B). Similarly, LNCAROD-promoted HCC cell proliferation, migration, invasion, and glycolysis under hypoxia were also remarkably inhibited by HIF1α knockdown (FigureC-I). Collectively, these results indicate that LNCAROD has a more potent oncogenic role in HCC under hypoxic conditions, probably via a HIF1α-dependent mechanism. S10 S10

All participants provided written informed consent, and the study was approved by the ethics committee of The First Affiliated Hospital of Guangxi Medical University. The animal experimental protocols were in accordance with institutional guidelines approved by the Animal Care and Use Committee.

All authors agree with the content of the manuscript.

The authors declare that they have no competing interests.

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.