Kasumi-1 cells (cat: TCHu202), HL-60 (cat: TCHu 23), THP-1 (cat: SCSP-567), HEL (cat: TCHu 71), TF-1 (cat: TCHu139), and U-937 (cat: TCHu159) were obtained from the Chinese Academy (Shanghai, China). SKM-1 cells were obtained from the Pricella Biotechnology Co., Ltd. (Wuhan, China). Fetal bovine serum (Siji Qing) was purchased from Tianhang Biotechnology Co., Ltd (cat: 13011-8611, Zhejiang, China). RPMI 1640 medium was acquired from Gibco (cat: 11875093, GrandIsland, NE, USA). Penicillin and streptomycin were sourced from Suolaibao Technology Co., Ltd (cat: P1400, Beijing, China). Reverse transcription (RT) and RT-quantitative real-time polymerase chain reaction (qPCR) reagents were purchased from Takara (cat: RR037A, Osaka, Japan). The Cell Counting Kit-8 (CCK-8) reagent was purchased from MedChemExpress (cat: HY-K0301, Newark, NJ, USA). Annexin V-APC/7-amino-actinomycin D reagent was purchased from Procell (cat: P-CA-107, Wuhan, China). Primary antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000), phosphatidylinositide 3-kinases (PI3K, 1:1500), protein kinase B(AKT, 1:1500), phosphorylated-Akt (p-AKT, 1:1500), Bcl-2-associated protein x (BAX, 1:1000), B-cell leukemia/lymphoma 2 protein (BCL-2, 1:1000), proliferating cell nuclear antigen (PCNA, 1:1500), G Protein-Coupled Receptor Kinase 2 (GRK2, 1:1500), and horseradish peroxidase (HRP, 1:5000)-labeled ancillary antibodies were procured from Sanying Biotechnology Co., Ltd (Wuhan, China). The cleaved caspase-3 antibody was purchased from Cell Signaling Technology (cat: 9664S, Danvers, MA, USA). The electrochemiluminescence (ECL) reagent was purchased from Millipore (cat: WBKLS0100-2, Bedford, MA, USA). The ANRIL gene lentivirus was purchased from Genesis Co., Ltd (Shanghai, China). Both GRK2 overexpression plasmid (pCDH-CMV-MCS-EF1-copGFP-T2A-Puro) and siRNA sequences were acquired from Aiji Biotechnology Co., Ltd (Guangzhou, China).

Peripheral blood samples were collected from 31 patients with AML. Their basic information and medical characteristics were recorded. AML diagnosis was confirmed according to the French American British classification standard. The control group consisted of peripheral blood from 13 healthy individuals. This project was approved by the Affiliated Hospital of Guizhou Medical University, and informed consent was obtained from all patients. The ethical approval number is 2016 (65).

The cells of the human AML line (Kasumi-1) were provided by the Chinese Academy. Kasumi-1 cells were cultured in RPMI 1640 complete medium with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO₂ at 37 °C, and were mycoplasma-free (Pulilai Gene Technology Co., Ltd., Mycoplasma Detection Kit, cat: E2036, Beijing, China). The medium was changed and the cells sub-cultured every 2 days. The cell line was authenticated shortly before use by the Short Tandem Repeat (STR) validation, carried out by Fuheng Biotechnology Co., Ltd (Shanghai, China).

Total RNA extraction was extracted from the human AML cells following the manufacturer’s instructions. The RNA was reverse-transcribed into complementary DNA (cDNA) using Prime Script RT kits (cat: RR036A, Takara, Osaka, Japan). ACTB (F: 5^′-GCGTGACATTAAGGAGAAGC-3^′, R: 5^′-CCACGTCACACTTCATGATGG-3^′) was used as the internal control. The expression patterns of ANRIL (F: 5^′-ATAAGCCTCATTCTGATTCAACAGC-3^′ and R: 5^′-AGCAGTACTGACTCGGGAAAG-3^′) and GRK2 (F: 5^′-GCGCTCACTCCTTTCAA-3^′, R: 5^′-GAGATCTGGAGGCACCTGCTT-3^′) were detected by RT-qPCR at the following cycling condition: 95 °C for 30 s, 1 cycle; 95 °C for 5 s, 60 °C for 30 s, 40 cycles. Gene expression was calculated using the 2^{-Δ⁢Δ⁢Ct} method.

Kasumi-1 cells were seeded at a concentration of 5 $\times$ 10⁵ cells/mL in six-well plates, with 500 µL per well. The cells were divided as follows: upregulated ANRIL (LV-ANRIL) and its control cell (LV-ANRIL NC) groups, upregulated GRK2 (oe-GRK2) and its control cell (oe-GRK2 NC) groups, downregulated ANRIL co-transfection with upregulated GRK2 (sh-ANRIL+oe-GRK2) group, upregulated ANRIL co-transfection with GRK2 siRNA (LV-ANRIL+siGRK2) group. The target sequences of GRK2 siRNA were shown in Supplementary Table 1. And the sequence of downregulated ANRIL (sh-ANRIL) were shown in Supplementary Table 2. Kasumi-1 cells were transfected at the logarithmic growth phase, following the manufacturer’s instructions. The culture medium was completely replaced 24 h after transfection. Puromycin was added 48 h after transfection, and the cells were selected with a complete medium consisting of 1 µg/mL puromycin. The medium was replaced every 2 to 3 days. After 1 week of uninterrupted screening, Green fluorescent protein (GFP) expression pattern was measured using a fluorescence microscope (Nikon Corporation, Tokyo, Japan) to assess the transfection effects, which was quantified by RT-qPCR and western blotting.

According to the instructions of the RIP Kit (cat: FI8709, FITGENE, Guangzhou, China), Kasumi-1 cells lysates were collected. And extracted 50 µL protein A/G agarose beads into a 1.5 mL centrifuge tube, resuspended protein A/G agarose beads with 100 µL NT-2, added 5 µg of target antibody or IgG antibody, and incubated at room temperature for 1 hour. The magnetic bead antibody complex was subsequently washed and used for spare. Extracted 100 µL of cell lysate supernatant into 900 µL of protein A/G and antibody complex, and incubate overnight at 4 °C. The bead protein complexes were subsequently washed. RNA was extracted from the complex, and ANRIL expression was quantitatively analyzed by RT-qPCR.

Cells were adjusted to a density of 1 $\times$ 10⁵ cells/mL. Next, cells in the LV-ANRIL NC and LV-ANRIL groups were treated with DNR (cat: HY-13062, MedChemExpress, Newark, NJ, USA) and LY294002 (cat: HY-10108, MedChemExpress, Newark, NJ, USA) , respectively. Subsequently, 50 µL of the cell suspension was seeded into 96-well plates (at a density of 50 µL/well), and 50 µL complete medium was added when there were no drugs. Thereafter, 50 µL final concentration was 0.4 µmol/L DNR medium when DNR was acting, added 50 µL final concentration was 40 µmol/L LY294002 medium when LY294002 was acting. Meanwhile, 100 µL complete medium was added to the blank group, and 100 µL of PBS was introduced into the well plate to prevent the evaporation of the medium from affecting the experimental results. Subsequent to introducing 10 µL of CCK-8 reagent at intervals of 0 h, 24 h, 48 h, and 72 h, the cells were placed back in the incubator for a duration of 2 h to 4 h. Each well’s absorbance level was gauged with a microplate reader (Thermo Fisher Scientific, GrandIsland, NE, USA) operating at 450 nm.

Cells were adjusted to a density of 5 $\times$ 10⁵ cells/mL. Next, cells in the LV-ANRIL NC and LV-ANRIL groups were treated with DNR at a final concentration of 0.4 µmol/L and LY294002 (40 µmol/L), and allowed to culture for 24 h. Subsequently, cells were collected from each group and centrifuged at a speed of 200 rpm for 7 min. After rinsing with precooled PBS according to the reagent instructions, and followed by the addition of 500 µL of 1$\times$binding buffer for cell resuspension. Following double staining with the Annexin V-APC/7AAD reagent (cat: P-CA-107, Procell, Wuhan, China), the parameters were adjusted on the machine to detect the apoptosis rates.

Total cell lysates were prepared using RIPA lysis buffer (cat: R0010, Solarbio, Beijing, China) consisting of a protease inhibitor cocktail. After mixing with 5$\times$loading buffer, the samples were boiled at 100 °C for 10 min. A total of 30 µg of protein underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel, was blocked for 2 hours, and then left to incubate with the fundamental antibody at 4 °C throughout the night. Subsequently, the protein underwent incubation with a secondary antibody at ambient temperature for 2 h. Ultimately, the protein signals were detected using ECL agents (WBKLS0100-2, Millipore, Bedford, MA, USA). The intensities of the western blot bands were analysed using Image J software (version 1.51j8, LOCI, University of Wisconsin, Madison, WI, USA), with GAPDH protein acting as a loading control.

Kasumi-1 cells lysates were collected. According to the instructions of the Nuclear and Cytoplasmic Extraction Kit (cat: P0028, Beyotime, Shanghai, China). Added 200 µL of cytoplasmic extraction reagent A and 10 µL cytoplasmic extraction reagent B every 20 µL of cell precipitation. Subsequently, centrifuged the mixture at 12,000 g for 10 minutes and transfered supernatant into a tube to obtain the extracted cytoplasmic. For precipitation, added 50 µL of nuclear extraction reagent. Then centrifuged the mixture at 12,000 g for 10 minutes. Sucked the supernatant into the tube, which was the nuclear. RNA in nuclear and cytoplasmic was extracted following the manufacturer’s instructions. The RNA was reverse-transcribed into complementary DNA (cDNA) using Prime Script RT kits (cat: RR036A, Takara, Osaka, Japan). GAPDH (F: 5^′-GGAGCGAGATCCCTCCAAAAT-3^′, R: 5^′-GGCTGTTGTCATACTTCTCATGG-3^′) was designated as a positive control of cytoplasmic, and U6 (F: 5^′-GTGCAGGGTCCGAGGT-3^′, R: 5^′-CTCGCTTCGGCAGCACA-3^′) was used as positive control of nucleus.

The GEPIA2 database (http://gepia2.cancer-pku.cn/#index) was utilized to assess ANRIL expression in AML specimens. The Multi Experiment Matrix database (https://neic.no/mem-multiexperimentmatrix/) was utilized to predict potential downstream interacting proteins of ANRIL. The RPISeq database (http://pridb.gdcb.iastate.edu/RPISeq/#) was performed to predict the interaction probability between ANRIL and GRK2. The RNAfold software (SnapGene 6.1, GSL Biotech, San Diego, CA, USA) was adopted to predict the secondary structure of ANRIL. Moreover, the catRAPID database (http://service.tartaglialab.com/) was carried out to predict the interaction site between ANRIL and GRK2.

Statistical analysis were conducted using SPSS 2.0 (IBM Corp., Chicago, IL, USA). Each experiment was conducted in triplicate, with the data recorded as the mean $\pm$ standard deviation (SD). GraphPad prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) was adopted to draw figures. T-test was utilized for contrasting data among different groups. A p-value 0.05 indicated statistical significance.

First, the GGEPIA2 database was utilized to compare ANRIL expression between the AML patients and individuals without leukemia. High ANRIL expression was observed in AML patients (Fig. 1A). ANRIL expression was further validated using peripheral blood specimens from the patients and human AML cell lines, including THP-1, HEL, Kasumi-1, HL-60, U937, SKM-1, and TF-1. ANRIL was substantially overexpressed in AML patients and Kasumi-1 cells, compared with healthy controls (Fig. 1B,C). Kasumi-1 cells were selected for the subsequent experiments. The cells have been validated for mycoplasma and Short Tandem Repeat (STR), no cross contamination of human derived cells or mycoplasma contamination (Supplementary Fig. 1A,B).

Fig. 1.

ANRIL overexpressed in AML patients and Kasumi-1 cells. (A) GGEPIA2 database was utilized to compare the differential expression of ANRIL between the AML patients and individuals without leukemia. (B) RT-qPCR was performed to detect the expression of ANRIL in AML patients. (C) RT-qPCR was adopted to detect the expression of ANRIL in AML cell lines (n = 3) (*p 0.05, **p 0.01, ***p 0.001). ANRIL, antisense noncoding RNA in the INK4 locus; LAML, acute myeloid leukemia-like; AML, acute myeloid leukemia; RT-qPCR, real-time quantitative polymerase chain reaction; THP-1, human monocytic leukemia cells; HEL, human erythroleukemia cells; HL-60, human acute promyelocytic leukemia cells; U937, human histiocytic lymphoma cells; SKM-1, human myelodysplastic syndrome cells; TF-1, human blood leukemia cells.

Recombinant lentivirus was adopted to establish the Kasumi-1 cells with upregulated ANRIL. The expression of green fluorescent protein (GFP) in both groups were observed via fluorescence microscopy (Fig. 2A), RT-qPCR analysis indicated a 7.5-fold increase in ANRIL expression in the LV-ANRIL group, compared with the LV-ANRIL NC group (Fig. 2B). The CCK-8 assay demonstrated that upregulated ANRIL significantly promoted Kasumi-1 cell proliferation (p 0.001) (Fig. 2C). After treatment with 0.4 µmol/L DNR for 24 h, 48 h and 72 h, the proliferation suppression rates were significantly lower in the LV-ANRIL group (31.38 $\pm$ 1.99%), (63.01 $\pm$ 1.46%), and (80.42 $\pm$ 1.39%) respectively, than in the LV-ANRIL NC (44.96 $\pm$ 1.25%) (p 0.01), (76.93 $\pm$ 0.34%) (p 0.001), and (86.64 $\pm$ 2.43%) (p 0.05) respectively (Fig. 2D). Flow cytometry findings suggested that the apoptosis rate was (2.13 $\pm$ 0.10%) in the LV-ANRIL group and (6.47 $\pm$ 0.53%) in the LV-ANRIL NC (p 0.001) (Fig. 2E,F). After DNR treatment for 24 h, the apoptosis rate in the LV-ANRIL group was significantly lower than in the LV-ANRIL NC group (p 0.01) (Fig. 2G,H). Taken together, upregulated ANRIL expression may promote Kasumi-1 cell proliferation and inhibit apoptosis while antagonizing the cell-killing effect of DNR.

Fig. 2.

ANRIL upregulation promoted Kasumi-1 cell proliferation and reduced the cytotoxic effect of DNR. (A) A fluorescence microscope was utilized to observe green fluorescent protein (GFP) expression and DAPI fluorescent staining (scale bar: 20 µm). (B) RT-qPCR was performed to detect the overexpression efficiency of ANRIL in Kasumi-1 cells (n = 3). (C) CCK-8 assay was carried out to detect the effect of upregulation of ANRIL on the proliferation of Kasumi-1 cells (n = 3). (D) CCK-8 assay was applied to detect the effect of upregulation of ANRIL on the cytotoxic effect of DNR on Kasumi-1 cells (n = 3). (E,F) Flow cytometry was adopted to detect the effect of upregulation of ANRIL on Kasumi-1 cell apoptosis. (G,H) Flow cytometry was used to detect the apoptosis rate of Kasumi-1 cells under DNR after ANRIL upregulation (n = 3). (*p 0.05, **p 0.01, ***p 0.001). NC, negative control, with or without DNR treatment; LV-ANRIL, upregulation of ANRIL, with or without DNR treatment; DNR, daunorubicin; GFP, green fluorescent protein; DAPI, 4^′,6-diamidino-2^′-phenylindole; CCK-8, Cell Counting Kit-8; 7AAD, 7-amino-actinomycin D; APC, allophycocyanin.

The PI3K/AKT pathway regulates cell proliferation, apoptosis, migration, and invasion [17]. Therefore, a western blot assay was conducted to identify the expression patterns of the PI3K/AKT pathway and associated proteins after increased ANRIL express in Kasumi-1 cells. ANRIL upregulation significantly increased PI3K/AKT pathway activity in Kasumi-1 cells, evidenced by enhanced protein expressions of PI3K, PCNA, and BCL-2 as well as AKTphosphorylation. Moreover, it reduced BAX expression, suggesting that ANRIL upregulation may promote activation of the PI3K/AKT signaling pathway, thereby altering the expression or activity of related molecules. Subsequently, Kasumi-1 cells with upregulated ANRIL expression were treated with LY294002, a PI3K/AKT inhibitor. Compared with the untreated group, protein expressions of PI3K, PCNA, and BCL-2 as well as AKT phosphorylation were diminished in the LY294002 treatment group. Additionally, BAX expression was enhanced in the LY294002 treatment group, suggesting that it could reverse activation of the PI3K/AKT/BCL-2 pathway induced by the upregulated ANRIL expression, restore BAX protein expression, and inhibit PCNA protein expression (Fig. 3A,B). Flow cytometry analysis suggested that the apoptosis rate in LY294002 treated group was (11.04 $\pm$ 0.44%) and the untreated group was (4.71 $\pm$ 0.09%) (p 0.001) (Fig. 3C,D). The CCK8 results showed that the cell proliferation in the LY294002 treatment group was significantly lower than that in the untreated group (Fig. 3E). Thus, LY294002 can reverse the function of upregulated ANRIL in the proliferation and apoptosis of Kasumi-1 cells. Taken together, these results further validate our previous conclusion [16], ANRIL can enhance Kasumi-1 cells proliferation and inhibit apoptosis by triggering the PI3K/AKT/BCL-2 pathway.

Fig. 3.

ANRIL promoted proliferation and inhibited apoptosis of Kasumi-1 cells via PI3K/AKT pathway. (A,B) Western Blot assay was performed to detect the expression of related proteins after upregulation of ANRIL and LY294002 (n = 3). (C,D) Flow cytometry was utilized to detect the apoptosis of cell treated with LY294002 after ANRIL upregulation. (n = 3). (E) CCK-8 assay was carried out to detect cell proliferation after ANRIL upregulation (n = 3) (*p 0.05, **p 0.01, ***p 0.001, ns, no significance). LV-ANRIL NC, negative control group, with or without LY294002 treatment; LV-ANRIL, upregulation of ANRIL group, with or without LY294002 treatment. PI3K, phosphatidylinositide 3-kinases; AKT, protein kinase B; p-AKT, phosphorylation-AKT; BCL-2, B-cell lymphoma-2; BAX, Bcl-2 assaciated X protein; PCNA, proliferating cell nuclear antigen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

ANRIL activates the PI3K/AKT/BCL-2 pathway, enhancing Kasumi-1 cell growth and inhibiting apoptosis, however, its underlying remains unclear.

Karyoplasm isolation experiments suggested that ANRIL is distributed in both the cytoplasm and nucleus of Kasumi-1 cells (Fig. 4A). We used bioinformatics to predict potential downstream proteins interacting with ANRIL, and observed an interaction with GRK2 (Fig. 4B,C). The RIP assay demonstrated significantly enriched ANRIL by the anti-GRK2 antibody, compared with non-specific IgG control (p 0.001) (Fig. 4D,E), confirming the physical binding between ANRIL and GRK2 protein. RT-qPCR and western blot results elucidated the regulatory association between ANRIL and GRK2, the GRK2 mRNA levels were not affected by ANRIL upregulation or downregulation. However, ANRIL positively regulated the GRK2 protein. The GRK2 protein level increased with ANRIL expression and vice versa (Fig. 4F,G). Thus, ANRIL may regulate GRK2 protein expression at the post-transcriptional level by regulating GRK2 activity and structure, thereby affecting its function.

Fig. 4.

ANRIL interacted with GRK2 in Kasumi-1 cells. (A) Nucleocytoplasmic separation assay was carried out to detect the expression of ANRIL in cytosolic and nuclear in Kasumi-1 cells (n = 3). (B) Bioinformatics was adopted to predict potential downstream proteins interacting with ANRIL. (C) RPISeq database was used to predict the possibility of interacting between ANRIL and GRK2. (D,E) RIP assay was applied to detect the interaction between ANRIL and GRK2 (n = 3). (F) RT-qPCR was performed to detect the mRNA level of GRK2 after upregulation or downregulation of ANRIL (n = 3). (G) Western Blot assay was adopted to detect the expression of GRK2 protein after upregulation or downregulation of ANRIL (*p 0.05, ***p 0.001). RIP, RNA-binding protein immunoprecipitation; Anti-GRK2, Anti-G Protein Coupled Receptor Kinase; Anti-IgG, Anti-Immunoglobulin G; sh-ANRIL, downregulation of ANRIL; NC, negative control; RF classifier, random forest classifier; SVM classifier, support vector machine classifier; GRK2, G Protein-Coupled Receptor Kinase 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Researchers have established the role of GRK2 in regulating tumor cell proliferation and invasion [18]. Nonetheless, its role in the AML-M2 Kasumi-1 cells remains elusive. Consequently, we investigated the impact of GRK2 on Kasumi-1 cell proliferation and apoptosis by upregulating or downregulating GRK2 expression.

The GRK2 overexpression plasmid was transfected into Kasumi-1 cells, and GFP expression was observed via fluorescence microscopy (Fig. 5A), and western blot assay was performed to detect the effect of GRK2 overexpression transfection (Fig. 5B). Three GRK2 siRNAs (siGRK2#1/2/3) were transfected into Kasumi-1 cells, with the interference efficiency determined through the western blot assay. siGRK2#1 and siGRK2#2 displayed noticeable interference effects (Fig. 5C). To guarantee the precision and objectivity of the outcomes, two sequences, namely siGRK2#1 and siGRK2#2, were used in subsequent experiments. The flow cytometry assay indicated that the apoptosis rate of the oe-GRK2 group was significantly lower in the oe-GRK2 group (4.73 $\pm$ 0.35%) than in the oe-GRK2 NC group (8.19 $\pm$ 0.42%) (p 0.001) (Fig. 5D,E). Furthermore, CCK-8 assay results demonstrated that upregulation of GRK2 expression promoted Kasumi-1 cell proliferation (p 0.001), whereas, downregulated expression suppressed cell proliferation (p 0.01) (Fig. 5F). Moreover, GRK2 upregulation increased the protein expressions of PI3K, AKT, PCNA, and BCL-2 as well as AKT phosphorylation. And it decreased the expression of BAX and cleaved caspase-3 in Kasumi-1 cells. Conversely, the GRK2 downregulation reversed the upregulation-induced changes in PI3K/AKT expression, restored BAX and cleaved caspase-3 levels, and inhibited BCL-2 and PCNA expressions (Fig. 5G,H). Thus, GRK2 and ANRIL have similar biological functions, which could promote Kasumi-1 cell proliferation and inhibit the apoptosis by activating the PI3K/AKT/BCL-2 pathway.

Fig. 5.

GRK2 promoted Kasumi-1 cell proliferation and inhibited apoptosis via activation of the PI3K/AKT pathway. (A) A fluorescence microscope was utilized to observe green fluorescent protein (GFP) expression (scale bar: 100 µm). (B) Western blot assay was performed to detect the effect of GRK2 overexpression transfection. (C) Western blot assay was performed to detect the interference effect of GRK2 siRNA#1/2/3. (D,E) Flow cytometry was adopted to detect the effect of upregulation of GRK2 on Kasumi-1 cell apoptosis (n = 3). (F) CCK-8 assay was carried out to detect cell proliferation after knockdown and upregulation of GRK2 (n = 3). (G,H) Western blot assay was applied to detect the expression of related proteins after knockdown and upregulation of GRK2 (n = 3) (*p 0.05, **p 0.01, ***p 0.001, ns, no significance). GRK2, G Protein-Coupled Receptor Kinase 2; GFP, green fluorescent protein; oe-GRK2, upregulation of GRK2; NC, negative control; siGRK2, downregulation of GRK2.

We elucidated the role of GRK2 in regulating the PI3K/AKT/BCL-2 pathway and affecting Kasumi-1 cell proliferation and apoptosis. Next, we constructed downregulated ANRIL co-transfection with upregulated GRK2 (sh-ANRIL+oe-GRK2) and upregulated ANRIL co-transfection with GRK2 siRNA (LV-ANRIL+siGRK2) in Kasumi-1 cells for rescue experiments. Downregulated GRK2 expression reversed the proliferation-promoting effects of ANRIL overexpression on Kasumi-1 cells (Fig. 6A). Conversely, upregulated GRK2 expression reversed the inhibitory effects of ANRIL downregulation on cell proliferation (Fig. 6B). Flow cytometry results suggested that the apoptosis rate was significantly lower in the sh-ANRIL+oe-GRK2 group (1.20 $\pm$ 0.08%) than in the sh-ANRIL (7.37 $\pm$ 0.72%) and sh-ANRIL+oe-GRK2 NC (6.91 $\pm$ 0.38%) groups (p 0.001) (Fig. 6C,D). Thus, GRK2 overexpression partially inhibited cell apoptosis induced by downregulated ANRIL expression. Hence, ANRIL may modulate the proliferation and apoptosis of Kasumi-1 cells via the GRK2 protein mediated. Additionally, western blot analysis suggested that GRK2 downregulation inhibited activation of the PI3K/AKT/BCL-2 pathway caused by ANRIL overexpression and restored BAX and cleaved caspase-3 expressions. Conversely, GRK2 overexpression reversed the inhibitory effect of ANRIL downregulation on the PI3K/AKT/BCL-2 signaling pathway (Fig. 6E,F). Taken together, ANRIL may activate the PI3K/AKT/BCL-2 pathway, promote Kasumi-1 cell proliferation, and inhibit apoptosis via the GRK2 protein mediated.

Fig. 6.

ANRIL regulated Kasumi-1 cell proliferation and apoptosis by interacting with GRK2 protein. (A) CCK-8 assay was carried out to detect the effect of co-transfection of LV-ANRIL and siGRK2 on Kasumi-1 cell proliferation (n = 3). Compare the data of group siNC and siGRK2#1/2. (B) CCK-8 assay was performed to detect the effect of co-transfection of sh-ANRIL and oe-GRK2 on Kasumi-1 cell proliferation (n = 3). (C,D) Flow cytometry was utilized to detect the effect of co-transfection of sh-ANRIL and oe-GRK2 on Kasumi-1 cell apoptosis (n = 3). Compare the data of group (sh-ANRIL + oe-GRK2 NC) and (sh-ANRIL + oe-GRK2). (E,F) Western Blot assay was applied to detect the effect of co-transfection of (sh-ANRIL and oe-GRK2) and (LV-ANRIL and siGRK2) on the PI3K/AKT signaling pathway (n = 3) (*p 0.05, **p 0.01, ***p 0.001, ns, no significance).

LncRNAs can interact with DNA, RNA, and proteins through secondary space structures to regulate gene expression [19]. The RNAfold software predicted the secondary structure of ANRIL. ANRIL regions at positions 1550 to 1880 nt (Fig. 7A,a), 2140 to 2620 nt (Fig. 7A,c) and 200 to 500 nt (Fig. 7A,b) were folded to form a complex stem-loop structure, which may serve as a protein interaction site. The catRAPID database was utilized to predict the interaction points between ANRIL and GRK2. The heatmap illustrates that the 279 to 418 nt regions of ANRIL (Fig. 7A,b) have possible interaction site with the 439 to 640, 251 to 351 and 39 to 101 regions of the GRK2 amino acid sequence. Additionally, the interaction score was the highest with the 439 to 640 region (Fig. 7B), corresponding to the carboxyl-terminal Pleckstrin Homolgy (PH) domain of the GRK2 protein structure. Thus, the 279 to 418 nt regions of ANRIL may interact with the PH domain of GRK2 protein (Fig. 7C).

Fig. 7.

Predicted interaction sites between ANRIL and GRK2. (A) Secondary structure of ANRIL predicted by RNAfold software (SnapGene 6.1, GSL Biotech, San Diego, CA, USA). (A,a) Secondary structure of ANRIL regions at positions 1550 to 1880 nt. (A,b) Secondary structure of ANRIL regions at positions 200 to 500 nt. (A,c) Secondary structure of ANRIL regions at positions 2140 to 2620 nt. (B) Interaction sites between ANRIL and GRK2 predicted by the catRAPID database (http://service.tartaglialab.com/). (C) Interaction sites between ANRIL and GRK2. N, Amino-terminal domain; C, Carboxyl-terminal domain; PH, Pleckstrin Homolgy domain; RH, RGS Homology domain.