m6A-methylated KCTD21-AS1 regulates macrophage phagocytosis through CD47 and cell autophagy through TIPR

Blocking immune checkpoint CD47/SIRPα is a useful strategy to engineer macrophages for cancer immunotherapy. However, the roles of CD47-related noncoding RNA in regulating macrophage phagocytosis for lung cancer therapy remain unclear. This study aims to investigate the effects of long noncoding RNA (lncRNA) on the phagocytosis of macrophage via CD47 and the proliferation of non-small cell lung cancer (NSCLC) via TIPRL. Our results demonstrate that lncRNA KCTD21-AS1 increases in NSCLC tissues and is associated with poor survival of patients. KCTD21-AS1 and its m6A modification by Mettl14 promote NSCLC cell proliferation. miR-519d-5p gain suppresses the proliferation and metastasis of NSCLC cells by regulating CD47 and TIPRL. Through ceRNA with miR-519d-5p, KCTD21-AS1 regulates the expression of CD47 and TIPRL, which further regulates macrophage phagocytosis and cancer cell autophagy. Low miR-519d-5p in patients with NSCLC corresponds with poor survival. High TIPRL or CD47 levels in patients with NSCLC corresponds with poor survival. In conclusion, we demonstrate that KCTD21-AS1 and its m6A modification promote NSCLC cell proliferation, whereas miR-519d-5p inhibits this process by regulating CD47 and TIPRL expression, which further affects macrophage phagocytosis and cell autophagy. This study provides a strategy through miR-519-5p gain or KCTD21-AS1 depletion for NSCLC therapy by regulating CD47 and TIPRL.

of the TME 10 .miR-200a can affect nasopharyngeal carcinoma cell proliferation, migration, and invasion by regulating CD47, thereby providing a potential form of cancer therapy 11 .miR-128 can also inhibit cancer cell proliferation, clonogenicity, and invasion, as well as promote phagocytosis in macrophages via CD47 12 .However, little is known about the role of CD47-related noncoding RNA in regulating the phagocytosis of macrophage for lung cancer therapy.In this study, we investigated the role of CD47-related noncoding RNA in the tumorigenesis of lung cancer.Results demonstrated that KCTD21-AS1 inhibited the phagocytic function of macrophages through CD47 and promoted the tumorigenesis of NSCLC.
The main histological type of NSCLC is lung adenocarcinoma.Therefore, RNA was isolated from lung adenocarcinoma tissues and controls and analyzed by lncRNA array and qRT-PCR.The results further supported that KCTD21-AS1 levels were higher in lung adenocarcinoma tissues than in the control (Fig. 1c-e).Then, we analyzed the effect of KCTD21-AS1 expression on the overall survival of patients with lung adenocarcinoma through the Kaplan-Meier plotter (data from GDC TCGA Lung Adenocarcinoma, https://xenabrowser.net/).The results indicated that high KCTD21-AS1 levels in patients with lung adenocarcinoma were related to poor survival compared with low levels (p < 0.05; Fig. 1f).Results of in situ hybridization showed that KCTD21-AS1 expression increased in NSCLC tissues compared with controls (Fig. 1g).These results indicated that KCTD21-AS1 may play an oncogenic role in the tumorigenesis of NSCLC.KCTD21-AS1, m6A modification by Mettl14, and promoted NSCLC cell proliferation LncRNAs participate in the tumorigenesis and progression of cancers 14 .Next, we detected its expression in cells and constructed lentiviral KCTD21-AS1-overexpressed with siRNA-KCTD-AS vectors as in our previous reports 15,16 to investigate the roles of KCTD21-AS1 in NSCLC (Supplementary Fig. 1e).The results showed that KCTD21-AS1 expression was higher in NSCLC cell lines than in control BEAS-2B cells (Supplementary Fig. 1f, g).The results of in situ hybridization demonstrated that KCTD21-AS1 was overexpressed in lv-KCTD21-AS1-treated A549 and H1975 cells compared with the control (Fig. 1h; Supplementary Fig. 1h).KCTD21-AS1 overexpression was promoted, but its downregulation inhibited NSCLC cell proliferation compared with the controls (Fig. 2a-d; Supplementary Fig. 2a-d).KCTD21-AS1 overexpression also increased whereas its downregulation decreased the colony number of NSCLC cells (Fig. 2e, f; Supplementary Fig. 2e, f).
Considering that miR-519-5p can suppress NSCLC growth, we then investigated the effect of TIPRL-3ʹ-UTR recovery on the role of miR-519d-5p in regulating cell growth and migration.The results demonstrated that TIPRL-3ʹ-UTR treatment attenuated miR-519d-5psuppressing cell proliferation compared with the control (Fig. 6e, f).Similar results were found in the migration study (Fig. 6g, h).These results supported that miR-519d-5p can regulate cell growth and migration by negatively regulating TIPRL expression.
TIPRL, an essential PP2A inhibitory protein, plays an important role in cell apoptosis and cell proliferation.The protein is upregulated in various carcinomas, including NSCLC 21 .TIPRL affects cell growth and autophagy through light chain 3 (LC3)-II/I 22 .Then, the results demonstrated that LC3-II/I was found to be increased in miR-519d-5p-treated cells compared with the control.KCTD21-AS1 overexpression downregulated the LC3-II/I ratio, whereas siRNA-KCTD21-AS1 increased the LC3-II/I ratio (Fig. 6i).These results supported that miR-519d-5p may promote cell autophagy through regulating TIPRL, whereas KCTD21-AS1 may inhibit such a process.
Innate immune checkpoints, such as the CD47/SIRPα axis, PD-1/PD-L1 axis, and MHC-I/LILRB1 axis, play important roles in tumor-mediated immune escape from the clearance by phagocytosis 23 .Then, human peripheral blood mononuclear cells (PBMC) were sorted, and macrophages were induced (Supplementary Fig. 10b) and co-cultured with A549/GFP+ cells.The results showed that lv-miR-519d-5p treatment enhanced the phagocytic function compared with the control (Fig. 7f).KCTD21-AS1 overexpression reduced the phagocytic function of macrophages, whereas its downregulation promoted such a function (Fig. 7g, h).

miR-519d-5p, TIPRL, and CD47 levels in NSCLC tissues
The above-mentioned results indicated that miR-519d-5p can effectively suppressed NSCLC cell proliferation and metastasis.However, the role of miR-519d-5p in the pathogenesis of NSCLC requires clarification.First, the miR-519d-5p levels in carcinoma tissue lysates prepared from 20 patients with NSCLC were further analyzed.Compared with lung adjacent control samples, miR-519d-5p levels were reduced in lung carcinoma samples (Fig. 8a).The Kaplan-Meier plot showed that low miR-519d-5p levels in patients with squamous carcinoma corresponded with poorer survival than high levels (http://www.kmplot.com/,Fig. 8b).As the targets of miR-519d-5p, the levels of TIPRL and CD47 of NSCLC tissues were relatively higher in NSCLC tissues than in adjacent control samples through qRT-PCR and IHC analyses, respectively (Fig. 8c-e).Second, the Kaplan-Meier plot further showed that high TIPRL (http://www.kmplot.com/) or CD47 (gene expression RNAseq from GDC TCGA LUSA, https://xenabrowser.net/) levels in patients with NSCLC corresponded with poorer survival than low levels (Fig. 8f).These results supported that miR-519d-5p played tumor suppressive roles, but CD47 and TIPRL promoted tumorigenesis in NSCLC.

Discussion
Cancer cells can evade immune surveillance via the immune checkpoint CD47/SIRPα, and blocking this immune checkpoint is a useful strategy to engineer macrophages for cancer immunotherapy 24 .Given the limited understanding of the regulation of CD47 and the function of TAMs, the immunosuppressive status of CD47-related factors remains poorly understood.In this study, we revealed that KCTD21-AS1 inhibited the phagocytic function of macrophages through CD47, whereas miR-519d-5p promoted such a function.In addition, KCTD21-AS1 promoted lung cancer proliferation by regulating TIPRL, whereas miR-519d-5p suppressed this proliferation (Fig. 8g).
The immune cells within TME play crucial roles in the tumorigenesis of different kinds of tumors.TME contains a series of immune cells, cancerassociated fibroblasts, and endothelial cells, and a crosstalk was observed between cancer cells and immune cells 25 .In contrast to traditional radiotherapy or chemotherapy, immunotherapy primarily exert its effect through the culture and modification of the immune cells to further recognize and attack tumor cells with higher specificity and lower side effect 26 .Based on tumor immune evasion, a series of immunotherapy approaches such as blocking CD47/SIRPα has been developed and clinically applied to tumor therapy 27 .
Tumor cells express "self" signals (e.g., CD47 molecules) and prevent the phagocytosis of macrophage.The siRNA specific to CD47 increases the phagocytosis of tumor cells by macrophages 28 .Noncoding RNAs have also been used to regulate CD47 expression to further promote the phagocytotic function of macrophages.Tan et al. demonstrated that miR-708 directly binds to CD47 and plays an important tumor suppressive role in the selfrenewal of breast cancer stem cells and TAM-mediated phagocytosis 29 .Similarly, by directly targeting and regulating CD47, miR-708 may serve as an effective and attractive candidate for the immunotherapy of T-ALL 30 .miR-340 overexpression can downregulate CD47 and inhibit tumor growth via regulating macrophage phagocytosis.MiR-340 also promotes the function of macrophages in tumor immune microenvironments and increases CD8 + T cells 31 .In the present study, we demonstrated that miR-519d-5p potentially bound the 3ʹ-UTR sequence of CD47 to further downregulate CD47 expression.Through ceRNA with miR-519d-5p, KCTD21-AS1 treatment suppressed the numbers of phagocytosis of macrophages compared with the control, indicating that KCTD21-AS1 promoted the tumorigenesis of lung cancer via miR-519d-5p-blocking CD47.
LncRNAs play an important role in human cancer types via ceRNAs to regulate targeted gene expression 32 .lncRNA MIAT can reportedly suppress EZH2 expression and promote papillary thyroid carcinoma cell invasion via miR-150 33 .The downregulation of lncRNA NEAT1 increases miR-381 and reduces IGF1 levels, thereby suppressing the apoptosis of ovarian granulosa cells 34 .Here, in situ hybridization results showed that cytosolic presence of KCTD21-AS1 was higher in NSCLC tissues compared with adjacent control, indicating that KCTD21-AS1 might regulate gene expression by ceRNA.In vitro results also demonstrated that the cytosolic and nuclear KCTD21-AS1 levels were higher in KCTD21-AS1-overexpressed cells compared with control treatment.Higher levels of KCTD21-AS1 in cytoplasm in vitro may be related to regulate gene expression, and the higher levels of KCTD21-AS1 in nucleus in vitro may be attributed to high transcription levels of KCTD21-AS1 lentiviral vector.Our results further demonstrated that KCTD21-AS1 regulated TIPRL expression and promoted lung cancer proliferation via ceRNA with miR-519d-5p.
TIPRL, an essential PP2A inhibitory protein, increases in various cancers, including NSCLC and hepatocellular carcinomas 21 .TIPRL plays important roles in cell apoptosis and proliferation of cancer through the TIPRL/PP2A axis.The depletion of circ_0010235 or gain of miR-433-3p would repress NSCLC cell proliferation and autophagy by regulating TIPRL 22 .TIPRL upregulation potentially brings metabolic benefits to lung cancer cells and promotes cancer cells survival 35 .TIPRL downregulation significantly reduces LC3 and CD133 expression, indicating that the TIPRL/ LC3/CD133 complex may serve as a valuable biomarker for liver cancer diagnosis 36 .Our results demonstrated that TIPRL was high in NSCLC tissues, which correlated with the poor overall survival of patients with lung cancer.The downregulation of TIPRL evidently increased the LC3-II levels in miR-519d-5p-treated NSCLC cells, indicating that miR-519d-5p suppressing NSCLC proliferation may be related to cell autophagy.
This study demonstrated that KCTD21-AS1 promoted NSCLC cell proliferation and metastasis by regulating CD47 levels, whereas miR-519d-5p inhibited such a processes.Moreover, the oncogenic role of KCTD21-AS1 was related to TIPRL expression regulation via miR-519d-5p, thereby affecting cell autophagy.Our results provided a strategy through miR-519-5p gain or KCTD21-AS1 depletion for NSCLC therapy by regulating CD47 and TIPRL.

Methods
Clinical specimens A total of 20 specimens from patients with NSCLC after operation were collected from Yantai Mountain Hospital (Supplementary Table 1).The collected clinical tumors and their adjacent tissues were stored at −80 °C.This study was approved by the Binzhou Medical College Ethics Committee.Prior to inclusion in the study, patients were fully informed of the study procedure, and they signed a written informed consent.All ethical regulations relevant to human research participants were followed.

RNA extraction and qRT-PCR
Total RNA (or small RNA) from tissues and cells was extracted by RNAiso Plus (Takara, Dalian, China), and <1 μg of the extracted RNA was reverse transcribed into cDNA by using the SPARKscript II RT Plus Kit (AG0304-B, SpakeJade, Jinan, China).Quantitative real-time PCR was performed using the Quantitect SYBRGreen Kit (Takara) and a StepOnePlus Real-time system (Thermo Fisher, Shanghai, China).GAPDH and 5S gene served as an endogenous control gene.The primers used to amplify miR-519d-5p, TIPRL, KCTD21-AS1, etc. are described in Supplementary Table 2.

Cell culture and lentiviral vectors
The human cell lines, including NSCLC cells (A549 and NCI-H1975), normal lung epithelial cells (BEAS-2B as NSCLC cell control) and 293T, were obtained from Shanghai Institute of Cell Biology, China.Cells were cultured in RPMI-1640 medium (Gibco, USA) or DMEM medium, (high glucose; Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco).All cells were cultured in an atmosphere containing 5% CO 2 at 37 °C.
Lentiviral-mediated KCTD21-AS1-overexpressed and siRNA-KCTD-AS vectors were constructed and produced according to the previous report 15 .BamH I-EcoR I element containing KCTD21-AS1 was inserted into FUGW vector to construct the overexpression vector.U6 promoter was used to drive siRNA expression, which was cloned into FUGW vector by Xba I and Xho I. 293T cells were cultured to package lentivirus.In brief, viral vector (1 mg), the gag/pol expression vector (△8.9, 0.9 mg), and VSVG vector (0.1 mg) were transfected 293 T cells.The virus supernatant was then harvested.

CCK-8 assay
After transfection, about 1 × 10 3 A549 and H1975 cells were seeded in each well of 96-well plates.A CCK-8 assay reagent (10 μL) was added into each well after the cells were attached to the well.After 2 h, the culture plate was removed, and the OD value was measured at 450 nm.Each experiment was repeated at least three times.
Cell transwell assay A549 and H1975 cells were collected and re-suspended in a serum-free culture medium.The suspension was adjusted to 1 × 10 5 cells/mL, and 100 μL of cell suspension was directly added into the upper chamber of the transwell culture plate (Corning, NY, USA).The lower chamber contains 600 μL of 30% FBS medium.The subsequent processing is according to the previous reports 37,38 .
Colony formation A549 and H1975 cells were collected after transfection.About 1 × 10 3 cells were added to a 10 cm dish and cultured continuously for 12-14 days.The colonies were washed with PBS and stained with 1% crystal violet, and the colony formation of cells was observed and counted as previously described in refs. 37,39.

In situ hybridization
The 5′-Cy3 labeled probe of KCTD21-AS1 was designed and synthesized by GenePharma (Shanghai, China).In accordance with the instructions of the FISH kit (no.F11201, GenePharma, Shanghai, China), cells were fixed with 4% paraformaldehyde for 20 min.After membrane-disruption treatment, cells were incubated with 1 μM probe and hybridized at 37 °C overnight.Nuclei were stained with DAPI for 2-3 min.Cells were observed under a confocal microscope (Leica TCS SPE, Leica, Dresden, Germany).The tissue sections were dewaxed before incubation, treated with proteinase K, denatured, and incubated with 1 μM probe before hybridizing at 37 °C overnight.Nuclei were stained with DAPI.

Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) analysis
Xenografts or tissues were fixed with 4% paraformaldehyde and embedded with paraffin.Then, the sections were dewaxed in xylene and rehydrated in alcohol.Antigen retrieval was performed using sodium citrate, and then endogenous peroxidase was blocked.The sections were incubated with specific rabbit anti-human CD44 (1:100, Boster Biological Technology, China), and TIPRL (1:200, Abcam, USA) at 4 °C overnight.After incubation with secondary antibodies at 37 °C for 1 h, DAB was used for detection.The sections were observed under an EVOSTM M7000 Imaging System (Thermo Fisher Scientific, USA).

Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100, and incubated with rabbit anti-human CD47 (1:200 dilution; Boster Biological Technology, China) at 4 °C overnight.Then, cells were incubated with Alexa Fluor 488 donkey anti-rabbit IgG (H + L) at 37 °C for 1 h.Immunofluorescence images were captured under a microscope (DM6000B, Leica) in accordance with a previous study 16 .

Luciferase assay
Luciferase levels were analyzed using a luciferase assay system (E1500, Promega, WI, USA).The wild-type, mutant fragments of KCTD21-AS1 or mRNA 3′-UTR fragments were cloned into pcDNA3.1-lucivector.Cells were transfected with the pcDNA3.1-lucivector or control and lysed at room temperature for 15 min, and the supernatants were collected.Next, 70 μL of lysate were added onto a 96-well plate, and then 20 μL of luciferase assay reagent II was added.The luciferase activity was determined using a chemiluminescence analyzer (Infinite 200 PRE, Tecan Austria GmbH).
Migratory cancer cell detection in vivo A549-GFP cells were infected with lentivirus, and 1.5 × 10 6 cells were collected.Then, the cells were injected into the tail vein of nude mice.After 6-7 weeks, the migration of GFP positive cells was observed.

Macrophage extraction
This study was approved by the Binzhou Medical College Ethics Committee.Human peripheral blood was collected from a central blood station in Yantai.Prior to inclusion in the study, volunteers were fully informed of the procedure, and they signed a written informed consent.Human PBMCs were obtained after being treated with lymphocyte (P8900, Solarbio China).In accordance with the instructions, CD14 + CD16 -monocytes were obtained by sorting using the EasySep Human Monocyte Isolation Kit (19359, STEMCELL, USA).Afterward, the sorting efficiency of monocytes was measured by flow cytometry.

Phagocytic function of macrophages
Macrophages were collected at the 5th day after stimulation of differentiation, and A549-EGFP cells were collected.Then, 5 × 10 4 macrophages and 10×10 4 A549-EGFP cells were inoculated onto a 48-well plate.After coculture for 1 h, the suspension cells were washed, the adherent cells were collected, and APC anti-Human CD45 antibody was added.The cells were washed with PBS and resuspended.For the detection of phagocytosis, 10,000 cells per sample were analyzed using a FACS flow cytometer (BD, USA), and unstained control and single stained cells were prepared for gating.After CD45 + macrophages were selected using the APC fluorescent channel gate, and the phagocytosis was detected by selecting CD45 + GFP + cells using the FITC channel.Therefore, phagocytosis was calculated as the percentage of APC + GFP + cells among APC + macrophages 31 .
Xenograft mouse model A549 cells were treated with lentivirus and stably expressed KCTD21-AS1 or miR-519d-5p.Then, 1 × 10 7 cells were injected subcutaneously into the shoulder and back of BALB/c nude mice (female, 6-8 weeks old; HFK Bio-Technology, Beijing, China).The tumor width and height were measured using a vernier scale, and tumor volume was calculated using the following formula: tumor volume = (length × width 2 )/2.About a month later, the mice were euthanized with intraperitoneal injection of barbiturate.All animal experiments were approved by the Ethics Committee of Binzhou Medical University.We have complied with all relevant ethical regulations for animal use.
Statistics and reproducibility SPSS 22.0 (IBM Corp., Armonk, NY, USA) was used to analyze experimental data.Normally distributed data are shown as the mean ± SD.Two averages and multiple groups were analyzed by Student's t test and ANOVA, respectively.For homogeneous variance assumption, the LSD test or Games-Howell test was used to compare the means.Abnormally distributed data are shown as median (interquartile range), and analyzed by Mann-Whitney U test or Kruskal-Wallis H test. Kaplan-Meier survival analysis was performed to analyze the survival of patients and gene expression.Luciferase assay and imaging analysis were performed at least twice to make sure that similar results could be reproduced.Experiments for CCK8, cell transwell assay, colony formation, RT-PCR, and immunoblotting experiments were repeated in A549 and H1975 cells.p < 0.05 was considered as statistically significant difference.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Fig. 1 |
Fig. 1 | Expression of KCTD21-AS1 increased in lung cancer.a Number of different lncRNA expression levels.b Different expression levels of some lncRNAs including KCTD21-AS1 between lung cancer and adjacent normal tissues from GSE70880.**p < 0.01, Mann-Whitney U test.Data were shown as median (interquartile range).c Results of lncRNA microarray showed the up-regulated or downregulated genes between lung adenocarcinoma and adjacent controls.d Different expression levels of some lncRNAs from lncRNA microarray, including KCTD21-AS1.e Levels of lncRNA were detected by qRT-PCR in lung adenocarcinoma tissues and adjacent normal tissues (N = 20).Data were shown as median (interquartile range), **p < 0.01, Mann-Whitney U test.f Overall survival analysis of patients with lung adenocarcinoma.g In situ hybridization showed that KCTD21-AS1 expression in lung adenocarcinoma tissues.Red indicates the expression of KCTD21-AS1.Bar = 20 μm.h In situ hybridization detection of KCTD21-AS1 in A549 cells.Red indicates the expression of KCTD21-AS1.Bar = 20 μm.

Fig. 6 |
Fig. 6 | KCTD21-AS1 and miR-519d-5p regulated TIPRL expression.a The sites of TIPRL-3'-UTR or Mu-TIPRL-3'-UTR targeted by miR-519d-5p.b, c Luciferase levels were analyzed in both pc3.1-luci-TIPRL-3'-UTR and miR-519d-5p-treated H1975 or A549 cells, respectively.Data were expressed as mean ± SD for triplicate experiments.**p < 0.01, Student's t-test.d immunoblotting detection from different membrane, and the sample loading amount according to the reference control, which was run on a different gel than the corresponding sample of interest.e, f Effect of TIPRL recovery on the role of miR-519d-5p in H1975 or A549 cell growth using CCK8 assay, respectively.Data were expressed as mean ± SD for triplicate experiments.*p < 0.05, **p < 0.01; ANOVA.g, h Effect of TIPRL-3′UTR recovery on miR-519d-5p-regulating H1975 or A549 cell metastasis, respectively.Data were expressed as mean ± SD for triplicate experiments.Bar = 100 μm.*p < 0.05, **p < 0.01; ANOVA.i immunoblotting detection from different membrane, and the sample loading amount according to the reference control, which was run on a different gel than the corresponding sample of interest.