N6-methyladenosine (m6A) reader IGF2BP1 facilitates clear-cell renal cell carcinoma aerobic glycolysis

doi:10.21203/rs.3.rs-1959363/v1

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N6-methyladenosine (m6A) reader IGF2BP1 facilitates clear-cell renal cell carcinoma aerobic glycolysis

https://doi.org/10.21203/rs.3.rs-1959363/v1

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It has been reported that N⁶-methyladenosine (m⁶A) modification is mainly involved in clear-cell renal cell carcinoma (ccRCC) tumorigenesis. However, the regulatory mechanisms of m⁶A reader IGF2BP1 involved in ccRCC tumor energy metabolism are currently unknown. Results showed that the m⁶A reader IGF2BP1 exhibited significantly higher expression in ccRCC cells. Functionally, results by gainloss functional assay indicated that IGF2BP1 promoted the glucolytic characteristics, including glucose uptake, lactate production and extracellular acidification rate (ECAR). Mechanistically, IGF2BP1 recognizes the m⁶A modified sites on LDHA mRNA and enhances its mRNA stability, thereby accelerating tumor energy metabolism. Thus, our work reveals a novel facet of the m⁶A that promotes mRNA stability and highlights the functional importance of IGF2BP1 as m⁶A readers in post-transcriptional gene regulation.

clear-cell renal cell carcinoma

N6-methyladenosine

IGF2BP1

LDHA

Renal cell carcinoma (RCC) is the second leading cause of urological malignant neoplasm-related death and tens of thousands of newly diagnosed cases are reported by global statistics^{1, 2}. More precisely, clear cell renal cell carcinoma (ccRCC) is a higher invasive subtype of RCC, comprising 80–90% of RCC patients^{3, 4}. Unfortunately, the chemotherapy or radiotherapy is largely ineffective for all renal cancer subtypes^{5, 6}. Therefore, the immunotherapy or targeted therapy is a very valuable alternative strategy.

N⁶-methyladenosine (m⁶A) modification was firstly described in 1971 years, which has shown critical roles in mRNA metabolism and become common focus in recent years^{7, 8}. Methyltransferase installed the m⁶A modification on mRNA, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor 1-associated protein (WTAP)^{9, 10}. Besides, the m⁶A modification was wiped by demethylase, including obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5)^{11, 12}. In ccRCC, the function of m⁶A has been gradually verified. For example, METTL3 expression is significantly higher compared with adjacent normal tissues in ccRCC tissues and METTL3 depletion significantly inhibits the cell viability, migration and invasion of ccRCC cells¹³. Thus, the critical function of m⁶A modification on ccRCC is pretty obvious.

In present, metabolic alteration is commonly observed in the development of human tumors, including ccRCC. It has been reported that m⁶A modification is significantly involved in tumor processes. In this study, we identified that the novel m⁶A reader IGF2BP1 up-regulated in the ccRCC and functional analysis determined its oncogenic role in ccRCC. Notably, IGF2BP1 targeted the LDHA mRNA to enhance LDHA mRNA stability, thereby promoting ccRCC aerobic glycolysis. Our findings suggest that IGF2BP1 could be used as a marker to detect cancer energy metabolism and is a potential therapeutic target in ccRCC.

2.1. Cell culture

Human renal proximal tubular epithelial cell line (HK2) and human RCC cell lines (786-O, ACHN) were obtained from the Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences (Shanghai, China). ccRCC cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, streptomycin (100 µg/mL), benzylpenicillin (100 U/mL) following standard culture conditions (5% CO₂) in the humidified environment at 37°C.

2.2. Vector construction and transfection

To construct stable IGF2BP1 knockdown, the specific targeting to IGF2BP1 was designed by Invitrogen online tool (https://rnaidesigner.thermofsher.com/rnaiexpress/) and then cloned into the lentiviral pLVX-shRNA vectors (sh-IGF2BP1, sh-NC). The stable IGF2BP1 overexpression and negative controls were constructed by GenePharma Biotech Co.,LTD (Shanghai, China) (IGF2BP1, vector). The 786-O or ACHN cell lines were cultured in the 6-well plate and then infected with the lentivirus following the manufacturer’s instruction.

2.3. Real‑time qPCR

RNA samples from kidney tumor cells or normal HK-2 cells were extracted using TRIzol kit (Invitrogen, cat. 15596026). cDNA was generated using the Reverse Transcription kit (Takara, cat. RR047A). Real-time qPCR was performed using SYBR Premix EX Taq kit (Takara, cat. RR420A) on ABI7500 (Applied Biosystems, Foster City, CA, USA). The primer pairs’ sequences were listed in Table S1. Gene expression was normalized to actin. Relative quantification levels were determined using the 2^−ΔΔCT method.

2.4. Western blot

The total protein in RCC cells was extracted by radioimmunoprecipitation assay (RIPA) lysis buffer containing PMSF. Then, after incubation on ice, the samples were centrifuged at 25000 g for 10 min with supernatant collected. The protein concentration was measured by bicinchoninic acid (BCA) kit with deionized water. Protein separation was performed via 10% SDS-PAGE (Beyotime Biotechnology, Shanghai, China) and then transferred to polyvinylidene fluoride (PVDF) membrane. PVDF membrane was added with Tris-buffered saline tween (TBST) containing 5% skimmed milk for 2 h. Finally, the primary antibody rabbit anti-IGF2BP1 was incubated (antibody IGF2BP1, Cat No. 22803-1-AP, Proteintech, China).

2.5. Glucose uptake, lactate production and ATP analysis

The glycolytic ability of ccRCC cells was calculated using glucose uptake, lactate production and ATP analysis. Firstly, the glucose uptake level was measured using a glucose assay kit (Sigma, St-Louis, MO, USA) as previously described¹⁴. Then, the lactate production level was detected by a lactate assay kit (Sigma). Lastly, the ATP analysis was performed using Fluorometric Assay Kit (BioVision, Milpitas, CA, USA).

2.6. Measurement of extracellular acidification rate (ECAR)

ECAR was analyzed using the XF96 Bioenergetic Analyzers (Seahorse Bioscience, Mountain View, CA, USA). All details were prepared and performed according to the manufacturers’ instructions. In brief, sequential injection of glucose (10 mM), oligomycin (1 mM) and 2-deoxyglucose was conducted (50 mM). ECAR measurements was normalized to total protein content and showed as mpH/min.

2.7. RNA immunoprecipitation (RIP) PCR

The RIP experiment was carried using EZ-Magna RIP Kit (Millipore, Billerica, MA, USA) according to manufacturer’s protocol using antibody (5 mg, antibody IGF2BP1, Cat No. 22803-1-AP, Proteintech, China). 786-O and ACHN cells were lysed in completed RIP lysis buffer. The cellular extraction was incubated with protein A/G agarose beads conjugated with antibodies or control IgG at 4°C for 2h. Beads were eluted and incubated with Proteinase. Lastly, purified RNA was subjected to qRT-PCR analysis.

2.8. RNA decay assay for mRNA stability

ccRCC cells were treated with actinomycin D (0.2 mM, cat. GC16866, GlpBio, Montclair, CA, USA) for 30 min and were designated as the 0 h samples. The0, 3 and 6 h samples were collected for RNA extraction. Then, cDNA was synthesized via reverse transcription and then performed using oligo(dT) primer. The quantitated RNA level was determined by RT-qPCR. Half-life (t_1/2) of mature LDHA mRNA was calculated using ln₂/slope and actin as normalization.

2.9. RNA m⁶A methylation quantification

As previously reported, the m⁶A RNA Methylation Assay Kit (cat. ab185912, Abcam) was performed to evaluate the content of m⁶A in total RNA. In brief, RNA (300 ng) was coated in assay wells accompanied with m⁶A standard protocols. In solution, capture antibody and secondary detection antibody solution were added. Then, the colorimetric m⁶A levels were quantified by assessing absorbance at OD450 based on the standard curve.

2.10. Statistical and data analysis

All data in present study were processed using GraphPad Prism 9.0 and expressed as mean ± standard deviation (SD). Two-sided unpaired Student’s t-test and one-way analysis of variance Tukey’s post-hoc test were utilized for two-group comparisons or multiple group comparisons. p-value < 0.05 was considered statistically significant.

3.1. IGF2BP1 up-regulated in the ccRCC

In the ccRCC tissue cohort, landscape analysis by ATGC (http://gepia.cancer-pku.cn/index.html) revealed that IGF2BP1 up-regulated in the kidney renal clear cell carcinoma (KIRC) tissue compared to normal tissue (Fig. 3A). In ccRCC cells, RT-PCR analysis revealed that IGF2BP1 mRNA level increased compared to normal cells (Fig. 3B). Besides, the same landscape analysis also revealed that the prognosis of ccRCC patients with higher IGF2BP1 level was significantly lower than that of lower IGF2BP1 level (Fig. 3C). Moreover, Kaplan-Meier Plotter analysis found that the higher IGF2BP1 level indicated worse survival prognosis (Fig. 3D). Therefore, these data illustrated that IGF2BP1 up-regulated in the ccRCC.

3.2. IGF2BP1 promoted the aerobic glycolysis of ccRCC

In the ccRCC cells (786-O, ACHN), IGF2BP1 silencing and overexpression were respectively constructed using stable transfection. The transfection efficient was detected using RT-PCR and western blot (Fig. 2A, 2B). To detect the bio-functions of IGF2BP1 was investigated, and finally we found that IGF2BP1 could remarkably regulate the energy metabolism, precisely the aerobic glycolysis. Firstly, we detected the glucose uptake, lactate production and ATP generation in ccRCC cells. Results indicated that silencing of IGF2BP1 repressed the glucose uptake (Fig. 2C), lactate production (Fig. 2D) and ATP generation (Fig. 2E) in 786-O cell. Besides, the overexpression of IGF2BP1 promoted the glucose uptake (Fig. 2C), lactate production (Fig. 2D) and ATP generation (Fig. 2E) in ACHN cell. Furthermore, the extracellular acidification rate (ECAR) analysis was detected and results demonstrated that silencing of IGF2BP1 repressed the glycolysis rate in 786-O cell Fig. 2F), while overexpression of IGF2BP1 promoted the glycolytic capacity in ACHN cell (Fig. 2G). Therefore, these data illustrated that IGF2BP1 promoted the aerobic glycolysis of ccRCC.

3.3. LDHA acted as the target modified by IGF2BP1 via m⁶A binding

In the ccRCC tissue cohort, landscape analysis by ATGC (http://gepia.cancer-pku.cn/index.html) revealed that LDHA up-regulated in the kidney renal clear cell carcinoma (KIRC) tissue compared to normal tissue (Fig. 3A). Interaction analysis revealed that LDHA was positively correlated to the level of IGF2BP1 in KIRC patients (Fig. 3B). In ccRCC cells (786-O, ACHN), the level of LDHA also significantly up-regulated (Fig. 3C). Online predictive analysis revealed that the m⁶A motif was predicted and analyzed as ‘GGACA’ (Fig. 3D). Moreover, the genomic analysis using SRAMP (http://www.cuilab.cn/sramp) showed that the m⁶A modification site mainly located in the 3’-UTR of LDHA mRNA (Fig. 3E). Therefore, these data illustrated that LDHA acted as the target modified by IGF2BP1 via m⁶A binding.

3.4. IGF2BP1 enhanced the stability of LDHA mRNA

Given that we found that LDHA acted as the target of IGF2BP1 in ccRCC, next, we tried to investigate the molecular interaction within LDHA and IGF2BP1. Firstly, the RIP-PCR analysis was performed to detect the interaction within LDHA and IGF2BP1, and results illustrated that LDHA significantly combined with IGF2BP1 in 786-O cells (Fig. 4A) and ACHN cells (Fig. 4B). Because there was positive interaction within LDHA and IGF2BP1, we imagined that IGF2BP1 could positively regulate the LDHA expression. Then, we performed the RNA decay assay to show whether IGF2BP1 could positively regulate the stability of LDHA mRNA. Results illustrated that IGF2BP1 silencing repressed the LDHA mRNA remaining level in 786-O cells (Fig. 4C), while IGF2BP1 overexpression promoted the LDHA mRNA remaining level in ACHN cells (Fig. 4D). Therefore, these data illustrated that IGF2BP1 enhanced the stability of LDHA mRNA.

To date, the progression of RCC is associated with tumorigenesis and metastasis^{15, 16}. m⁶A acts as the most abundant modification in eukaryocyte mRNA, which is regulated by m⁶A methyltransferases, demethylases and readers^{17, 18}. Here, we tried to investigate the potential roles of novel m⁶A reader IGF2BP1 in ccRCC.

Emerging evidences have suggested that the m⁶A modifications are associated with human cancer proliferation, invasion and metastasis^{19, 20}. From this research point, we could explore the deep mechanism of ccRCC. Results indicated that the expression of m⁶A reader IGF2BP1 elevated in ccRCC and functionally necessary for the aerobic glycolysis by regulating glucose uptake, lactate production and ATP level. Functional assays using loss/gain-functional assays revealed the functions of IGF2BP1 on ccRCC energy metabolism.

Currently, targeted therapies are the standard treatment options for renal cell carcinoma, functioning as an oncogene or anti-oncogene in malignant tumors^{21, 22}. On this basis of epigenetics modification, this regulatory effects of IGF2BP1 suggested that m⁶A could be utilized to develop new strategies for targeted therapies. However, among several well-known key enzymes which potentially promote mRNA stability, several m⁶A regulators was predicatively identified in renal cancers. For example, m⁶A writers METTL14 expression significantly down-regulates in ccRCC tissues, and METTL14 upregulation inhibits ccRCC’s proliferation and migration via activating the PI3K/AKT signalling pathway. METTL14 enhances the Pten mRNA stability through YTHDF1/m⁶A/dependent²³. Besides, depletion of METTL3 significantly inhibits the cell viability, migration and invasion abilities of ccRCC cell lines through regulating HHLA2 expression via m⁶A modification of HHLA2 mRNA¹³. Thus, these findings reveal that m⁶A modification could regulate the renal cancer progression.

Here, we found that m⁶A reader IGF2BP1 elevated in ccRCC and promoted the aerobic glycolysis, including glucose uptake, lactate production and ATP level. The aerobic glycolysis is critical for the ccRCC tumorigenesis. Particularly, cancer cells tend to have elevated levels of glycolysis, reducing glucose to lactate. Glycolysis may be anaerobic or aerobic, aerobic glycolysis or the Warburg effect. LDHA is a critical regulator in tumor aerobic glycolysis, and critical literature reveals its critical function in tumorigenesis. For example, LDHA is overexpressed in RCC tissues and predicts worse survival, and LDHA downregulation suppresses RCC cells migration and invasion and Warburg effect²⁴.

Mechanistically, we found that enforced IGF2BP1 expression in ccRCC enhanced LDHA mRNA expression directly, as well as that of aerobic glycolysis. IGF2BP1 could exert its regulatory roles via binding targets through transcript’s stability. For example, in gastric cancer, m⁶A reader IGF2BP1 upregulated in gastric cancer tissue and acted as a predictor of poor prognosis for gastric cancer patients and IGF2BP1 directly interacts with c-MYC mRNA via m⁶A-dependent manner to stabilize its stability²⁵. In lipopolysaccharide-induced cardiomyocytes, m⁶A reader IGF2BP1 recognizes the m⁶A modified sites on HDAC4 mRNA and enhances its mRNA stability²⁶. Thus, the critical role of IGF2BP1 might promote this tumor progression.

In conclusion, our data shows that an oncogenic m⁶A reader IGF2BP1 promotes ccRCC aerobic glycolysis and malignant phenotype. Moreover, we identified that IGF2BP1 can directly bind LDHA and induce its mRNA stability increasing. In addition, we reported a regulatory mechanism that IGF2BP1 targets LDHA to promote the aerobic glycolysis of ccRCC via m⁶A-dependent manner (Fig. 5).

Conflict of interest

All authors declare no conflicts of interest

Acknowledgement

No.

Data Availability Statement

No research data shared.

Cao H, Sun Z, Wu J, Hao C, Wang W. Metastatic Clear Cell Renal Cell Carcinoma to Pancreas and Distant Organs 24 Years After Radical Nephrectomy: A Case Report and Literature Review. Frontiers in surgery 2022; 9:894272.
Kench JG, Amin MB, Berney DM, Compérat EM, Cree IA, Gill AJ, et al. WHO Classification of Tumours fifth edition: evolving issues in the classification, diagnosis, and prognostication of prostate cancer. Histopathology 2022.
Liu YF, Zhang ZC, Wang SY, Fu SQ, Cheng XF, Chen R, et al. Immune checkpoint inhibitor-based therapy for advanced clear cell renal cell carcinoma: A narrative review. International immunopharmacology 2022; 110:108900.
Pei D, Xu C, Wang D, Shi X, Zhang Y, Liu Y, et al. A Novel Prognostic Signature Associated With the Tumor Microenvironment in Kidney Renal Clear Cell Carcinoma. Frontiers in oncology 2022; 12:912155.
Vassiliou V, Katsila T, Sodergren SC, Kardamakis D. Radiotherapy in Metastatic Urothelial Carcinoma: Rationale and Clinical Applications. Anticancer research 2022; 42:3767–78.
Yin J, Zheng M. Ipsilateral synchronous papillary and clear renal cell carcinoma: A case report and review of literature. World journal of clinical cases 2022; 10:5428–34.
Chen S, Ren H, Zhang X, Chang L, Wang Z, Wu H, et al. Research advances of N6-methyladenosine in diagnosis and therapy of pancreatic cancer. Journal of clinical laboratory analysis 2022:e24611.
Li L, Xu N, Liu J, Chen Z, Liu X, Wang J. m6A Methylation in Cardiovascular Diseases: From Mechanisms to Therapeutic Potential. Frontiers in genetics 2022; 13:908976.
Lin H, Wang Y, Wang P, Long F, Wang T. Mutual regulation between N6-methyladenosine (m6A) modification and circular RNAs in cancer: impacts on therapeutic resistance. Molecular cancer 2022; 21:148.
Luo L, Zhen Y, Peng D, Wei C, Zhang X, Liu X, et al. The role of N6-methyladenosine-modified non-coding RNAs in the pathological process of human cancer. Cell death discovery 2022; 8:325.
Qin S, Zhang Q, Xu Y, Ma S, Wang T, Huang Y, et al. m(6)A-modified circRNAs: detections, mechanisms, and prospects in cancers. Molecular medicine (Cambridge, Mass) 2022; 28:79.
Zhai G, Xiao L, Jiang C, Yue S, Zhang M, Zheng J, et al. Regulatory Role of N6-Methyladenosine (m6A) Modification in Osteoarthritis. Frontiers in cell and developmental biology 2022; 10:946219.
Zhu D, Liu Y, Chen J, Wang Q, Li Y, Zhu Y, et al. The methyltransferase METTL3 promotes tumorigenesis via mediating HHLA2 mRNA m6A modification in human renal cell carcinoma. Journal of translational medicine 2022; 20:298.
Wang W, Wang B. KDM3A-mediated SP1 activates PFKFB4 transcription to promote aerobic glycolysis in osteosarcoma and augment tumor development. BMC cancer 2022; 22:562.
Chen J, Liao X, Cheng J, Su G, Yuan F, Zhang Z, et al. Targeted Methylation of the LncRNA NEAT1 Suppresses Malignancy of Renal Cell Carcinoma. Frontiers in cell and developmental biology 2021; 9:777349.
Liu T, Wang H, Fu Z, Wang Z, Wang J, Gan X, et al. Methyltransferase-like 14 suppresses growth and metastasis of renal cell carcinoma by decreasing long noncoding RNA NEAT1. Cancer science 2022; 113:446–58.
Guimarães-Teixeira C, Barros-Silva D, Lobo J, Soares-Fernandes D, Constâncio V, Leite-Silva P, et al. Deregulation of N6-Methyladenosine RNA Modification and Its Erasers FTO/ALKBH5 among the Main Renal Cell Tumor Subtypes. Journal of personalized medicine 2021; 11.
He L, Chen S, Ying Y, Xie H, Li J, Ma X, et al. MicroRNA-501-3p inhibits the proliferation of kidney cancer cells by targeting WTAP. Cancer medicine 2021; 10:7222–32.
Akhtar J, Lugoboni M, Junion G. m(6)A RNA modification in transcription regulation. Transcription 2021; 12:266–76.
Li H, Xiao W, He Y, Wen Z, Cheng S, Zhang Y, et al. Novel Insights Into the Multifaceted Functions of RNA n(6)-Methyladenosine Modification in Degenerative Musculoskeletal Diseases. Frontiers in cell and developmental biology 2021; 9:766020.
Relier S, Rivals E, David A. The multifaceted functions of the Fat mass and Obesity-associated protein (FTO) in normal and cancer cells. RNA biology 2022; 19:132–42.
Yang Z, Yu GL, Zhu X, Peng TH, Lv YC. Critical roles of FTO-mediated mRNA m6A demethylation in regulating adipogenesis and lipid metabolism: Implications in lipid metabolic disorders. Genes & diseases 2022; 9:51–61.
Zhang L, Luo X, Qiao S. METTL14-mediated N6-methyladenosine modification of Pten mRNA inhibits tumour progression in clear-cell renal cell carcinoma. British journal of cancer 2022; 127:30–42.
Zhao J, Huang X, Xu Z, Dai J, He H, Zhu Y, et al. LDHA promotes tumor metastasis by facilitating epithelial–mesenchymal transition in renal cell carcinoma. Molecular medicine reports 2017; 16:8335–44.
Luo F, Lin K. N(6)-methyladenosine (m(6)A) reader IGF2BP1 accelerates gastric cancer aerobic glycolysis in c-Myc-dependent manner. Experimental cell research 2022; 417:113176.
Shen H, Xie K, Li M, Yang Q, Wang X. N(6)-methyladenosine (m(6)A) methyltransferase METTL3 regulates sepsis-induced myocardial injury through IGF2BP1/HDAC4 dependent manner. Cell death discovery 2022; 8:322.

No competing interests reported.

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You are reading this latest preprint version

N6-methyladenosine (m6A) reader IGF2BP1 facilitates clear-cell renal cell carcinoma aerobic glycolysis

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cell culture

2.2. Vector construction and transfection

2.3. Real‑time qPCR

2.4. Western blot

2.5. Glucose uptake, lactate production and ATP analysis

2.6. Measurement of extracellular acidification rate (ECAR)

2.7. RNA immunoprecipitation (RIP) PCR

2.8. RNA decay assay for mRNA stability

2.9. RNA m⁶A methylation quantification

2.10. Statistical and data analysis

3. Results

3.1. IGF2BP1 up-regulated in the ccRCC

3.2. IGF2BP1 promoted the aerobic glycolysis of ccRCC

3.3. LDHA acted as the target modified by IGF2BP1 via m⁶A binding

3.4. IGF2BP1 enhanced the stability of LDHA mRNA

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

N6-methyladenosine (m6A) reader IGF2BP1 facilitates clear-cell renal cell carcinoma aerobic glycolysis

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Cell culture

2.2. Vector construction and transfection

2.3. Real‑time qPCR

2.4. Western blot

2.5. Glucose uptake, lactate production and ATP analysis

2.6. Measurement of extracellular acidification rate (ECAR)

2.7. RNA immunoprecipitation (RIP) PCR

2.8. RNA decay assay for mRNA stability

2.9. RNA m6A methylation quantification

2.10. Statistical and data analysis

3. Results

3.1. IGF2BP1 up-regulated in the ccRCC

3.2. IGF2BP1 promoted the aerobic glycolysis of ccRCC

3.3. LDHA acted as the target modified by IGF2BP1 via m6A binding

3.4. IGF2BP1 enhanced the stability of LDHA mRNA

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.9. RNA m⁶A methylation quantification

3.3. LDHA acted as the target modified by IGF2BP1 via m⁶A binding