LncRNA LINRIS stabilizes IGF2BP2 and promotes the aerobic glycolysis in colorectal cancer
Long noncoding RNAs (lncRNAs) play nonnegligible roles in the epigenetic regulation of cancer cells. This study aimed to identify a specific lncRNA that promotes the colorectal cancer (CRC) progression and could be a potential therapeutic target.
We screened highly expressed lncRNAs in human CRC samples compared with their matched adjacent normal tissues. The proteins that interact with LINRIS (Long Intergenic Noncoding RNA for IGF2BP2 Stability) were confirmed by RNA pull-down and RNA immunoprecipitation (RIP) assays. The proliferation and metabolic alteration of CRC cells with LINRIS inhibited were tested in vitro and in vivo.
LINRIS was upregulated in CRC tissues from patients with poor overall survival (OS), and LINRIS inhibition led to the impaired CRC cell line growth. Moreover, knockdown of LINRIS resulted in a decreased level of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), a newly found N6-methyladenosine (m6A) ‘reader’. LINRIS blocked K139 ubiquitination of IGF2BP2, maintaining its stability. This process prevented the degradation of IGF2BP2 through the autophagy-lysosome pathway (ALP). Therefore, knockdown of LINRIS attenuated the downstream effects of IGF2BP2, especially MYC-mediated glycolysis in CRC cells. In addition, the transcription of LINRIS could be inhibited by GATA3 in CRC cells. In vivo experiments showed that the inhibition of LINRIS suppressed the proliferation of tumors in orthotopic models and in patient-derived xenograft (PDX) models.
LINRIS is an independent prognostic biomarker for CRC. The LINRIS-IGF2BP2-MYC axis promotes the progression of CRC and is a promising therapeutic target.
KeywordsAutophagy CRC IGF2BP2 LINRIS MYC
- Baf A1
Earle’s balanced salt solution
Extracellular acidification rate
Exponentially modified protein abundance index
Esophageal squamous cell carcinoma
Fluorescence in situ hybridization
Hematoxylin and eosin
Insulin-like growth factor 2 mRNA-binding protein
Long Intergenic Non-coding RNA for IGF2BP2 Stability
Long noncoding RNAs
MS2 coat protein
Pancreas ductal adenocarcinoma
Quantitative real-time PCR
RNA recognition motifs
Short hairpin RNAs
Small interfering RNAs
The Cancer Genome Atlas
TdT-mediated dUTP nick end labeling
YT521-B homology domain-containing proteins
Colorectal cancer (CRC) is an aggressive primary intestinal malignancy with the third leading incidence and second highest mortality of all types of cancers worldwide . In China, over 380,000 new cancer cases are projected to be discovered in the colon and rectum annually . Therefore, finding new therapeutic strategies for CRC is of great significance.
Long noncoding RNAs (lncRNAs) are special RNA molecules that are longer than 200 nucleotides long and have no protein-coding potential . As epigenetic regulators in various diseases, including cancers, lncRNAs are involved in biological processes with diverse mechanisms, such as mediating interactions between DNA and proteins, adsorbing microRNAs, and binding to proteins as decoys [4, 5, 6]. Accumulating evidence has shown that, just as oncogenes affect the prognosis of patients, some lncRNAs influence the progression and death of cancer cells [7, 8, 9], indicating that targeting lncRNAs could be a new approach for CRC treatment.
As another critical epigenetic regulator, the N6-methyladenosine (m6A) modification has attracted the attention of researchers worldwide . During the biological processes of m6A modifications, there are three types of proteins (‘readers’, ‘writers’ and ‘erasers’) that play irreplaceable roles [11, 12, 13]. YT521-B homology domain-containing proteins (YTHDFs) are the well-known m6A ‘readers’ that participate in the recognition of m6A-modified mRNAs [13, 14]. The insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family consists of three members, IGF2BP1–3, which are newly reported m6A ‘readers’ . Unlike YTHDFs, which regulate pre-mRNA splicing and facilitate translation, these proteins are responsible for targeted mRNA stability and are associated with thousands of targets, such as MYC, KRAS and MDR1 [15, 16, 17]. In brief, IGF2BPs recognize m6A-modified mRNAs and maintain their stability by recruiting RNA stabilizers to promote the progression of cancers [14, 18]. However, the biological mechanism of IGF2BP2 in CRC remains largely unclear.
In this study, we found a highly expressed lncRNA called LINRIS (Long Intergenic Noncoding RNA for IGF2BP2 Stability) in CRC. LINRIS blocked the degradation of IGF2BP2 through the ubiquitination-autophagy pathway. As a consequence, MYC-mediated glycolysis was downregulated, inhibiting the proliferation of CRC cells in vitro and in vivo.
Cell lines and cell culture
All human CRC cell lines described in this article were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in basic RPMI-1640 medium (1×) or DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. All cells tested negative for mycoplasma contamination, and this result was verified by short tandem repeat fingerprinting before use.
Reagents and antibodies
The reagents and antibodies are listed in Additional file 1: Table S1.
RNA-sequencing (RNA-seq) analysis
With the raw reads from sequencing, we first retained the qualified reads (also known as clean reads) that passed the quality control step by FastQC software. Reads with low base quality, contamination or containing more than 10% N were removed from further analysis. By using STAR , clean reads of each sample were then aligned to the GRCh38 human reference genome from GENCODE. Gene and transcript expression were sequentially estimated with RSEM . To compare the lncRNAs, the genes were grouped in terms of “lncRNA”, “non_coding” and “antisense” according to the annotation from GENCODE. Differential expression analysis was performed using DESeq2 , and those RNAs with an adjusted P value < 0.05 and a fold change > 1.5 were considered differentially expressed genes. In addition, the genes with < 1 fragments per kilobase of transcript per million fragments mapped were removed.
Lentivirus and plasmid transfection
The expression of LINRIS was knocked down by short hairpin RNAs (shRNAs) targeting human LINRIS or by a nonspecific oligonucleotide that was ligated into the LV-3 (pGLVH1/GFP + Puro) vector. The lentiviruses were synthesized by OBiO Technology Co., Ltd. (Shanghai, China) and the sequences are listed in Additional file 2: Table S2. HCT116 and DLD-1 cells were transfected with the lentivirus according to the manufacturer’s instructions. To obtain stably transfected cell lines, these cells were treated with puromycin (2–3 μg/mL) for 2 weeks. After the knockdown efficiency was confirmed by quantitative PCR (qPCR) and Western blotting analyses, the cells were used for subsequent experiments.
The expression vectors for 3FLAG-tagged MS2 coat protein (MCP) and MS2-tagged LINRIS were provided by OBiO Technology Co., Ltd. (Shanghai, China), and FLAG-tagged expression vectors for full-length IGF2BP2 and site-directed mutants (K77R and K139R) were provided by Kidan BioTechnology Co., Ltd. (Guangzhou, China). The plasmids were transfected into the cells with Lipofectamine 3000 as recommended by the manufacturer.
Human tissue specimens
Clinical samples were collected from Sun Yat-sen University Cancer Center (Guangzhou, China). All patients were histologically diagnosed with CRC before the operation. Written informed consent was obtained from all patients. The study was approved by the Medical Ethics Committee of Sun Yat-sen University.
Immunoprecipitation (IP) assay
An anti-IGF2BP2 antibody (1–2 mg per test, Abcam, ab124930) and an anti-FLAG/DYKDDDDK Tag (1–2 mg per test, Cell Signaling Technology, 8146 s) were used in the IP assays, and the proteins were detected by Western blotting with an anti-ubiquitin antibody (1:1000, Cell Signaling Technology, #3933) according to the manufacturer’s instructions.
RNA pull-down and RNA immunoprecipitation (RIP) assays
Expression vectors for full-length LINRIS and its N-terminal (1–570 nt) and C-terminal (571–913 nt) regions used for the in vitro synthesis of RNA were provided by OBiO Technology (Shanghai, China). The lncRNAs were transcribed in vitro using a MEGAscript™ T7 Transcription Kit (Invitrogen, Carlsbad, CA, USA) and were biotinylated with a Pierce RNA 3′ End Desthiobiotinylation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The proteins were extracted from HCT116 and DLD-1 cell lines using Pierce IP Lysis Buffer. Then, RNA pull-down assays were performed with a Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the biotinylated lncRNAs were captured with streptavidin magnetic beads and incubated with the cell lysates at 4 °C for 6 h. Then, the mixture was washed and eluted. The eluate was subjected to mass spectrometry or Western blotting analysis. RIP assays were performed with a Magna RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. The mixture was digested with proteinase K before the immunoprecipitated RNAs were extracted, purified and subjected to qPCR. The RNA levels were normalized to the input RNA levels (10%).
Extracellular acidification rate (ECAR) and the measurement of intracellular metabolites
ECAR was measured according to the XF Glycolysis Stress Test protocol on a Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA), and 13C-labeled intracellular metabolites were identified as previously described . CRC cells (approximately 1 × 107 cells) were incubated with 2 g/L 13C-labeled glucose for 2 h. Metabolites were extracted and detected with a liquid chromatography system equipped with a TripleTOF 5600 mass spectrometer (SCIEX, Framingham, MA, USA). The concentration of the 13C-labeled metabolites was normalized to the cell number.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed with a ChIP kit from Merck Millipore (Billerica, MA, USA) according to the manufacturer’s instructions. qPCR analysis was performed to detect the DNA fragments that coimmunoprecipitated with GATA3.
RNA interference (RNAi)
The small interfering RNAs (siRNAs) used for in vivo treatment were provided by RiboBio (Guangzhou, China) according to the same sequences as the sh-LINRIS (sh-1 and sh-2). The resulting constructs were verified by sequencing. siRNAs were injected into the tumors at two or more spots each time.
In vivo therapeutic study
All female BALB/c nude mice (3–4 weeks old) used in our study were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. and then housed in specific pathogen-free units.
For the orthotopic models, 2 × 106 cells with negative control (NC, sh-NC), sh-1 or sh-2 in 0.5 mL of PBS were subcutaneously injected into the dorsal flank of 2 mice respectively. After the tumors grew up to 1 cm3, they were resected and equally divided into small pieces. Then 15 mice were separated into 3 groups (sh-NC, sh-1 and sh-2), of which the tumor pieces were tied to the base of the ceca. The growth of the tumors was monitored every 2 weeks after intraperitoneal injection of D-luciferin with a Xenogen IVIS 100 Bioluminescent Imaging System. All mice were sacrificed 4 weeks after the surgery.
For the PDX models (PDX#1–3), the tumor tissues were obtained from patients receiving surgeries at our cancer center . After taking a biopsy of a small part of the tumor, we conserved the tissue in ice cold culture medium with 5% penicillin and streptomycin. The tissue was separated into several pieces, which were implanted into the dorsal flanks of mice. To investigate the antitumor effects of RNAi in each PDX model (#1 and #2), 15 female BABL/c nude mice were randomly assigned into 3 groups (NC, si-LINRIS#1 and #2) 2 weeks later. The RNAi solution (20 nmol) was injected directly into the tumor bodies of si-LINRIS#1 and #2 groups twice per week. PDX#3 was used to investigate the combination effect of RNAi and oxaliplatin. Twenty female BALB/c nude mice were randomly assigned into the following 4 groups: control, oxaliplatin (injected into the abdominal cavity, 5 mg/kg twice per week), RNAi (injected directly into the tumor bodies, 20 nmol twice per week), or combined treatment. The tumor volumes were recorded twice weekly. After treatment for approximately 4 weeks, the mice were sacrificed and all the tumors were extracted and weighed. Furthermore, the tumor tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) or immunohistochemically (IHC) stained with antibodies against Ki-67, IGF2BP2 and MYC according to previously reported protocols . Apoptotic cells in situ were also identified by using a Cell Death Detection Kit (Biotool, Houston, TX, USA) for TdT-mediated dUTP nick end labeling (TUNEL) staining according to the manufacturer’s instructions. The animal study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.
All data are presented as the mean ± SD. Student’s paired or unpaired t-tests and chi-square tests were used for the comparison of significant differences between groups with GraphPad Prism software. Correlations between the LINRIS levels and MYC, GLUT-1, PKM2 and LDHA expression were analyzed with Pearson’s correlation analysis. Survival analyses were performed using the Kaplan-Meier method and assessed using the log-rank test with SPSS and MedCalc statistical software. The levels of significance were set at *, representing P < 0.05 and **, representing P < 0.01.
Additional methods are described in Additional file 3.
LINRIS is highly expressed in CRC with poor prognosis
According to The Cancer Genome Atlas (TCGA) database, LINRIS expression was upregulated in most kinds of tumors and CRC cell lines compared with the expression in normal cells (Fig. 1d and Fig. 1e); these results indicate that LINRIS generally acts as an oncogene. Our samples from patients with esophageal squamous cell carcinoma (ESCC), gastric cancer (GC) and pancreas ductal adenocarcinoma (PDAC) also showed the oncogenic status of LINRIS in digestive cancers (Fig. 1f). Furthermore, by measuring the expression of LINRIS in CRC cell lines and 8 human CRC samples, we found that the increased copy number was account for the upregulation of LINRIS in CRC (Fig. 1e and Additional file 4: Figure S1D-S1F). Subsequently, we decided to use two cell lines (HCT116 and DLD-1) with a relatively high LINRIS copy number for further research.
Next, we knocked down LINRIS in CRC cells with shRNAs (Additional file 4: Figure S1G). BrdU and the 3D-culture assays were used to compare CRC cells transfected with LINRIS-specific shRNAs (sh-1 and sh-2) with the nagative control (sh-NC), and the results identified the oncogenic function of LINRIS in assisting the growth of cancer cells (Fig. 1g, h, i and Additional file 4: Figure S1H). With RNA FISH assays, we found that LINRIS was mainly located in the cytoplasm (Fig. 1j and Additional file 4: Figure S1I), which was further confirmed by the RNAScope® ISH assays and the qPCR analysis of the nuclear and cytosolic extractions (Fig. 1k and l).
LINRIS interacted with IGF2BP2 and maintained its expression
By measuring the expression of LINRIS and IGF2BP2 in human CRC cell lines (Fig. 2f and Additional file 7: Figure S2G), we found a positive correlation between them (Fig. 2g). Intriguingly, IGF2BP2 was obviously downregulated with LINRIS knockdown in both HCT116 and DLD-1 cells (Fig. 2h). Moreover, knocking down LINRIS significantly increased the ubiquitination of IGF2BP2 but repressed IGF2BP2-regulated mRNAs (Fig. 2i and Additional file 7: Figure S2H). Therefore, we assumed that IGF2BP2 might be the key to the molecular mechanism of LINRIS and that the degradation of IGF2BP2 was probably prevented by this lncRNA.
LINRIS protected IGF2BP2 from autophagic degradation
To elucidate the degradation pattern of IGF2BP, the CRC cells with downregulated LINRIS expression were treated with the protein synthesis inhibitor cycloheximide (CHX) and exhibited a shorter IGF2BP2 half-life than the untreated control cells (Fig. 3e). However, in sh-LINRIS-transfected cells, endogenous IGF2BP2 expression could not increase when cells were treated with the proteasome inhibitor MG132 (Fig. 3f), indicating that the degradation of IGF2BP2 may be linked to a more complex mechanism than the ubiquitin-proteasome pathway.
Apart from the ubiquitin-proteasome system (UBS), cellular proteins could be degraded from the autophagy-lysosome pathway (ALP) after ubiquitination (Fig. 3g) . As shown in Fig. 3h and Additional file 8: Figure S3A, the reduction in the endogenous IGF2BP2 protein was successfully reversed by the autophagy inhibitors bafilomycin A1 (Baf A1), NH4Cl and 3-methyladenine (3-MA). In contrast, the autophagy activators Earle’s balanced salt solution (EBSS) and Rapamycin (Rap) decreased the levels of IGF2BP2 (Fig. 3i). EBSS-treated cells also exhibited an increased colocalization of IGF2BP2 and LC3B (Fig. 3j and Additional file 8: Figure S3B). Furthermore, we used small guide RNAs (sgRNAs) against autophagy-related gene 5 (ATG5) in DLD-1 cells to block the autophagy system (Additional file 8: Figure S3C). As shown in Additional file 8: Figure S3D, the IGF2BP2 protein levels remained stable when LINRIS was inhibited in ATG5-depleted cells. Overall, the above observation suggests that the LINRIS-mediated degradation of IGF2BP2 was realized through the ubiquitination-autophagy pathway.
MYC-mediated glycolysis was influenced by the interaction between LINRIS and IGF2BP2
Moreover, we transfected CRC cells with plasmids overexpressing IGF2BP2. As shown in Fig. 4g-i and Additional file 9: Figure S4B-S4E, the impaired proliferation and glycolysis induced by LINRIS knockdown was rescued to some extent in HCT116 and DLD-1 cells. In addition, transfecting CRC cells with K139R-mutated IGF2BP2 could fully reverse or even enhance the proliferation impaired by LINRIS (Additional file 9: Figure S4F), further confirming the mechanism of the LINRIS-IGF2BP2-MYC axis.
Inhibition of LINRIS suppressed CRC growth in vivo
In addition, we tested the in vivo effect of ‘anti-LINRIS therapy’ with 2 patient-derived xenograft (PDX) models. As shown in Fig. 5c, d and Additional file 10: Figure S5A, inhibition of LINRIS via in vivo-optimized RNA interference (RNAi) significantly suppressed the growth of tumors. Besides, no obvious side effects, such as toxicity or weight loss, were observed (Additional file 10: Figure S5B). IHC staining of the excised tumor sections showed that the expression of Ki-67, which was consistent with that of IGF2BP2 and MYC, decreased with the depletion of LINRIS (Fig. 5e and f). Moreover, we explored the clinical perspective of downregulating LINRIS in combination with chemotherapy as previous reports suggested [32, 33]. In particular, targeting glycolysis has been reported to be a strategy to overcome chemoresistance . As shown in Additional file 10: Figure S5C and S5D, LINRIS inhibition could be performed simultaneously with oxaliplatin treatment. While the proliferation index was decreased, the percentage of apoptotic cells after RNAi and oxaliplatin treatment were higher than the percentage in the control cells (Additional file 10: Figure S5E and S5F). Overall, blocking the LINRIS-IGF2BP2-MYC axis is a promising approach for CRC treatment.
GATA3 inhibited the transcriptional activity of LINRIS
The LINRIS-IGF2BP2-MYC axis was deeply correlated with the development of CRC
Moreover, IGF2BP2 expression was significantly higher in the tumor tissues from these patients compared with the matched normal tissues (Fig. 7c and d). Higher IGF2BP2 expression was also associated with a poor prognosis for patients with CRC (Fig. 7e). Then we established a combination scoring system separating the tissues into three groups, namely LINRIS/IGF2BP2-high, LINRIS/IGF2BP2-low and intermediate. As expected, the LINRIS/IGF2BP2-high group showed a poorer prognosis than the other two groups (Fig. 7f, clinicopathological features are listed in Additional file 13: Table S6). In summary, the LINRIS-IGF2BP2-MYC axis deeply influenced the development and prognosis of CRC and acts as a potential therapeutic target.
Epigenetic regulation is deeply involved in the genesis and development of cancer cells [35, 36, 37]. Among the complex regulatory networks, lncRNAs play a crucial role in affecting the fate of tumors [38, 39, 40, 41]. In this study, we used RNA-seq to compare advanced tumors with their paired adjacent normal tissues, and we discovered that LINRIS is a highly expressed oncogenic lncRNA that was related to the poor prognosis of patients with CRC. LINRIS is located at chromosome 16q21, and few studies have investigated its function or molecular mechanism. We found that downregulating the expression of LINRIS resulted in the inhibition of CRC cell proliferation. Moreover, we observed that LINRIS interacted with IGF2BP2, whose protein levels were positively correlated with LINRIS expression. IGF2BPs recognize m6A-modified mRNAs via KH domains and maintain their stability by recruiting RNA stabilizers, such as ELAV-like RNA-binding protein 1 (ELAVL1; also known as HuR), matrin 3 (MATR3) and poly (A)-binding protein cytoplasmic 1 (PABPC1) [14, 42]. Therefore, our findings built a bridge between the epigenetic networks of lncRNAs and m6A.
Autophagy is a double-edged sword that determines the survival and death of cells under different circumstances, including interacting with lncRNAs [43, 44, 45]. By degrading cellular materials, autophagic degradation is able to change the environmental or nutritional conditions and eliminate damaged organelles [46, 47]. In addition, it is associated with ubiquitination, forming a large degradation system instead of an isolated pathway [29, 48]. In our study, we found that LINRIS bound to a ubiquitination site of IGF2BP2, and this binding blocked IGF2BP2 degradation through the ubiquitination-autophagic pathway. Therefore, its downstream mRNAs including MYC mRNA were stabilized. To the best of our knowledge, our study is the first to elucidate the degradation pathway of a member of the IGF2BP family.
Because of the Warburg effect, glycolysis is the major method of glucose utilization and determines the progression of cancer cells [49, 50, 51]. As MYC mRNA is a typical target of IGF2BP2 and one of the core regulators of glycolysis [14, 52, 53], we detected the expression of MYC and its downstream enzymes. The downregulation of MYC-related metabolic enzymes resulted in a reduction in glycolysis, which could also account for the proliferation arrest following LINRIS knockdown. In contrast, the suppression of cancer cell progression and glycolysis could be reversed by overexpressing IGF2BP2, especially by the K139R mutant without the LINRIS-binding site.
GATA3 has been discovered to be linked with the development and invasion of cancer cells [54, 55, 56]. In this study, we identified GATA3 as a tumor suppressor gene that interacts with LINRIS. A decreased GATA3 level was accompanied by upregulation of LINRIS and a better prognosis for CRC patients. In contrast, overexpression of GATA3 downregulated the transcriptional activity of LINRIS in CRC cells.
Furthermore, the in vivo experiments further identified the antitumor effects of inhibiting LINRIS in CRC, and the analysis of LINRIS/IGF2BP2 expression in the tissues from patients indicated their unique role in the development of CRC; all of these experimental results confirmed the therapeutic potential of targeting the LINRIS-IGF2BP2-MYC axis.
In conclusion, without inhibition factors such as GATA3, LINRIS binds to the K139 ubiquitination site of IGF2BP2 and prevents it from degradation via the ALP, maintaining the MYC-mediated glycolysis and the proliferation of CRC cells (Fig. 7g).
We thank doctor Zhi-Ling Li from Sun Yat-sen University Cancer Center for their advice and assistance in the investigation of IGF2BP2 degradation.
Conception and design: YW, Q-NW, H-QJ and R-HX; Development of methodology: YW, J-HL, Q-NW, H-QJ, YJ and R-HX; Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): YW, J-HL, Q-NW, YJ, D-SW, JL, X-JL, QM, Y-NW; Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): YW, J-HL, Y-XC, QZ and Z-XL; Writing, review, and/or revision of the manuscript: YW, R-HX and H-QJ; Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): YW, J-HL, H-YP, P-SH and Z-LZ; Study supervision: R-HX and H-QJ. Suggestions: RD and X-FZ. All authors read and approved the final manuscript.
This research was supported by National Natural Science Foundation of China (81930065, 81871951, 81802438); Natural Science Foundation of Guangdong Province (2014A030312015); Science and Technology Program of Guangdong (2019B020227002); Science and Technology Program of Guangzhou (201904020046, 201803040019, 201704020228); CAMS Innovation Fund for Medical Sciences (2019-I2M-5-036) and Pearl River S&T Nova Program of Guangzhou (201806010002).
Ethics approval and consent to participate
The clinical CRC specimens were conducted with the permission by the Institutional Research Ethics Committee of Sun Yat-sen University Cancer Center, China. All animal experiments were performed in accordance with a protocol approved by the Ethics Committee of the Institutional Animal Care of Sun Yat-sen University Cancer Center, China.
Consent for publication
The content of this manuscript has not been previously published and is not under consideration for publication elsewhere.
The authors declare that they have no competing interests.
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