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Research
Xueliang Zuo
Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-5204-2585
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Metabolic rewiring of cancer cells reshapes the tumor microenvironment, thereby restricting the response to immunotherapy. Circular RNAs (circRNAs) can influence various cellular processes and have been implicated in hepatocellular carcinoma (HCC). Here, we investigated the role of a novel circRNA circRHBDD1 in HCC metabolic transformation and immunotherapy resistance.
Methods
CircRNA sequencing was performed to determine the differentially expressed circRNA profile in HCC. RT-qPCR and in situ hybridization were used to verify the dysregulation of circRHBDD1 in two independent HCC cohorts. Univariate and multivariate survival analyses were employed to assess the prognostic significance of circRHBDD1. Loss- and gain-of-function approaches were adopted to evaluate the effects of circRHBDD1 on glycolysis and glutaminolysis. Patient-derived xenograft models were used for in vivo evaluation. RNA pull-down, mass spectrometry, RNA immunoprecipitation, fluorescence in situ hybridization, polysome profiling, and meRIP assays were utilized to explore the molecular mechanisms of circRHBDD1 in HCC.
Results
We found that circRHBDD1 was significantly upregulated in HCC and associated with unfavorable clinicopathological characteristics and poor survival outcomes. In vitro and in vivo experiments showed that circRHBDD1 facilitated HCC glycolysis and glutaminolysis. Mechanistic studies revealed that circRHBDD1 could recruit YTHDF1 to PIK3R1 mRNA and augment PIK3R1 translation in an m6A-dependent manner, leading to activation of the PI3K/AKT signaling. EIF4A3-mediated exon back-splicing contributed to the upregulation of circRHBDD1. Moreover, targeting of circRHBDD1 was able to improve anti-PD-1 therapy resulting in prolonged survival.
Conclusion
We identified that the circRHBDD1/YTHDF1/PIK3R1 axis was crucial to metabolic reprogramming of HCC. Suppression of circRHBDD1 could potentially sensitize HCC cells to anti-PD-1 therapy.
Keywords
Hepatocellular carcinoma, Circular RNA, N6-methyladenosine, Metabolic reprogramming, Immunotherapy
Figure 1
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This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1TableS1.docx
Additional file 1: Table S1. Primers used for RT-qPCR in this study.
- Additionalfile2Supplementarymethods.docx
Additional file 2: Supplementary methods.
- Additionalfile3TableS2.docx
Additional file 3: Table S2. Clinicopathological variables and circRHBDD1 expression level in HCC samples (n = 96).
- Additionalfile4TableS3.docx
Additional file 4: Table S3. The effects of clinicopathological variables on overall survival and disease-free survival in HCC patients (n = 160).
- Additionalfile5FigureS1.tif
Additional file 5: Figure S1. CircRHBDD1 overexpression promotes HCC viability in vitro. (A) RT-qPCR analysis of circRHBDD1 and RHBDD1 mRNA in HepG2 cells after the transfection of lentivirus overexpressing circRHBDD1. (B) RT-qPCR analysis of circRHBDD1 and RHBDD1 mRNA in MHCC97H cells after the transfection of lentivirus overexpressing circRHBDD1. (C) Proliferation ability was evaluated by colony formation assay (representative wells are presented) in circRHBDD1-overexpressing HepG2 and Huh7 cells. (D) The proliferation of HepG2 cells after circRHBDD1 overexpression was detected by CCK-8 assay. (E) The proliferation of circRHBDD1-overexpressing Huh7 cells was examined using CCK-8 assay. (F) EdU assays were conducted to assess the proliferative ability of HepG2 and Huh7 cells with circRHBDD1 overexpression. ** P < 0.01; *** P < 0.001; ns, no significance.
- Additionalfile6FigureS2.tif
Additional file 6: Figure S2. CircRHBDD1 knockdown suppresses glycolysis and glutaminolysis in MHCC97H cells. (A) The expression levels of GLUT1 and HK2 mRNA were determined by RT-qPCR in circRHBDD1-silenced MHCC97H cells. (B) The mRNA levels of ASCT2 and GLS1 were assessed using RT-qPCR in MHCC97H cells with circRHBDD1 knockdown. (C) Western blotting analysis was performed to detect the expression levels of GLUT1, HK2, ASCT2, and GLS1 in circRHBDD1-silenced MHCC97H cells. (D) The ECAR data showed that silencing circRHBDD1 significantly reduced the rate of glycolysis and the glycolytic capacity in MHCC97H cells. (E) The OCR results showed that circRHBDD1-silenced MHCC97H cells displayed increased basal respiration and maximum respiratory. (F) The cellular G6P level, lactate production, and cellular ATP level were detected in MHCC97H cells with circRHBDD1 knockdown. (G) The levels of glutamine, glutamate, and α-KG were examined in circRHBDD1-silenced MHCC97H cells. ** P < 0.01; *** P < 0.001.
- Additionalfile7FigureS3.tif
Additional file 7: Figure S3. CircRHBDD1 overexpression enhances glycolysis and glutaminolysis of HCC cells. (A) The expression levels of GLUT1 and HK2 mRNA were determined by RT-qPCR in circRHBDD1-overexpressing HepG2 and Huh7 cells. (B) The mRNA levels of ASCT2 and GLS1 were assessed using RT-qPCR in HepG2 and Huh7 cells with circRHBDD1 overexpression. (C) Western blotting analysis was performed to detect the expression levels of GLUT1, HK2, ASCT2, and GLS1 in circRHBDD1-overexpressing HepG2 and Huh7 cells. (D) The ECAR data showed that upregulating circRHBDD1 significantly elevated the rate of glycolysis and the glycolytic capacity in HepG2 and Huh7 cells. (E) The OCR results showed that circRHBDD1-overexpressing HepG2 and Huh7 cells exhibited decreased basal respiration and maximum respiratory. (F) The cellular G6P level, lactate production, and cellular ATP level were detected in HepG2 and Huh7 cells with circRHBDD1 overexpression. (G) The levels of glutamine, glutamate, and α-KG were examined in circRHBDD1-overexpressing HepG2 and Huh7 cells. * P < 0.05; ** P < 0.01; *** P < 0.001.
- Additionalfile8FigureS4.tif
Additional file 8: Figure S4. CircRHBDD1 augments HCC viability via PIK3R1. (A) Western blotting analysis confirmed the efficiency of PIK3R1 overexpression in circRHBDD1-silenced HCCLM3 cells. (B) CCK-8 assays were performed to examine the cell proliferation of sh-circRHBDD1#1 HCCLM3 cells after PIK3R1 overexpression. (C) EdU assays were conducted to assess the proliferative capability of sh-circRHBDD1#1 HCCLM3 cells upon PIK3R1 overexpression. * P < 0.05; ** P < 0.01; ns, no significance.
- Additionalfile9FigureS5.tif
Additional file 9: Figure S5. CircRHBDD1 promotes glycolysis and glutaminolysis of HCC cells through PIK3R1. (A) Glycolysis rate and glycolystic capacity were calculated using the ECAR data in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (B) Basal respiration and maximum respiratory were detected using the OCR data in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (C) The cellular G6P level, lactate production, and cellular ATP level were detected in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (D) The levels of glutamine, glutamate, and α-KG were examined in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (E) Western blotting analysis showing the expression levels of p-AKT, AKT, GLUT1, HK2, ASCT2, and GLS1 in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance.
- Additionalfile10FigureS6.tif
Additional file 10: Figure S6. YTHDF1 proteins predominantly associate with a specific region of circRHBDD1. (A) Schematic of biotin pull-down of YTHDF1 using three nonoverlapping biotinylated RNAs (P1: 1-100 nt; P2: 101-200 nt; P3: 201-290 nt) tiling the length of circRHBDD1. (B) Western blotting of YTHDF1 in samples pulled down by full-length (FL) or truncated circRHBDD1. (C) Schematic structures of YTHDF1 protein and two truncated mutants (YTHDF1-N: 1-359 aa; YTHDF1-C: 360-559 aa) of YTHDF1 variants used in this study. The pink boxes represent YTH domains. (D) RIP assays were performed using anti-Flag antibody and cells transfected with vectors expressing the Flag-tagged FL and the truncated mutants of YTHDF1. *** P < 0.001; ns, no significance.
- Additionalfile11FigureS7.tif
Additional file 11: Figure S7. CircRHBDD1 knockdown does not change the mRNA and protein levels of EIF4A3. (A) The levels of EIF4A3 mRNA were detected by RT-qPCR in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (B) Western blotting analysis showing the expression levels of EIF4A3 in circRHBDD1-silenced cells. ns, no significance.
- Additionalfile12FigureS8.tif
Additional file 12: Figure S8. Representative CT imaging of HCC patients treated with anti-PD-1 in the responder and non-responder group is shown.
- Additionalfile13FigureS9.tif
Additional file 13: Figure S9. The knockdown efficiency of circRHBDD1 was detected by RT-qPCR in Hepa1-6 cells after the transfection of sh-circRHBDD1#1.
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A new preprint design is coming!
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Xueliang Zuo
Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital
Corresponding Author
ORCiD: https://orcid.org/0000-0001-5204-2585
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
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CURRENT STATUS: Posted
Version 1
Posted 07 May, 2021
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License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Metabolic rewiring of cancer cells reshapes the tumor microenvironment, thereby restricting the response to immunotherapy. Circular RNAs (circRNAs) can influence various cellular processes and have been implicated in hepatocellular carcinoma (HCC). Here, we investigated the role of a novel circRNA circRHBDD1 in HCC metabolic transformation and immunotherapy resistance.
Methods
CircRNA sequencing was performed to determine the differentially expressed circRNA profile in HCC. RT-qPCR and in situ hybridization were used to verify the dysregulation of circRHBDD1 in two independent HCC cohorts. Univariate and multivariate survival analyses were employed to assess the prognostic significance of circRHBDD1. Loss- and gain-of-function approaches were adopted to evaluate the effects of circRHBDD1 on glycolysis and glutaminolysis. Patient-derived xenograft models were used for in vivo evaluation. RNA pull-down, mass spectrometry, RNA immunoprecipitation, fluorescence in situ hybridization, polysome profiling, and meRIP assays were utilized to explore the molecular mechanisms of circRHBDD1 in HCC.
Results
We found that circRHBDD1 was significantly upregulated in HCC and associated with unfavorable clinicopathological characteristics and poor survival outcomes. In vitro and in vivo experiments showed that circRHBDD1 facilitated HCC glycolysis and glutaminolysis. Mechanistic studies revealed that circRHBDD1 could recruit YTHDF1 to PIK3R1 mRNA and augment PIK3R1 translation in an m6A-dependent manner, leading to activation of the PI3K/AKT signaling. EIF4A3-mediated exon back-splicing contributed to the upregulation of circRHBDD1. Moreover, targeting of circRHBDD1 was able to improve anti-PD-1 therapy resulting in prolonged survival.
Conclusion
We identified that the circRHBDD1/YTHDF1/PIK3R1 axis was crucial to metabolic reprogramming of HCC. Suppression of circRHBDD1 could potentially sensitize HCC cells to anti-PD-1 therapy.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
This is a list of supplementary files associated with this preprint. Click to download.
- Additionalfile1TableS1.docx
Additional file 1: Table S1. Primers used for RT-qPCR in this study.
- Additionalfile2Supplementarymethods.docx
Additional file 2: Supplementary methods.
- Additionalfile3TableS2.docx
Additional file 3: Table S2. Clinicopathological variables and circRHBDD1 expression level in HCC samples (n = 96).
- Additionalfile4TableS3.docx
Additional file 4: Table S3. The effects of clinicopathological variables on overall survival and disease-free survival in HCC patients (n = 160).
- Additionalfile5FigureS1.tif
Additional file 5: Figure S1. CircRHBDD1 overexpression promotes HCC viability in vitro. (A) RT-qPCR analysis of circRHBDD1 and RHBDD1 mRNA in HepG2 cells after the transfection of lentivirus overexpressing circRHBDD1. (B) RT-qPCR analysis of circRHBDD1 and RHBDD1 mRNA in MHCC97H cells after the transfection of lentivirus overexpressing circRHBDD1. (C) Proliferation ability was evaluated by colony formation assay (representative wells are presented) in circRHBDD1-overexpressing HepG2 and Huh7 cells. (D) The proliferation of HepG2 cells after circRHBDD1 overexpression was detected by CCK-8 assay. (E) The proliferation of circRHBDD1-overexpressing Huh7 cells was examined using CCK-8 assay. (F) EdU assays were conducted to assess the proliferative ability of HepG2 and Huh7 cells with circRHBDD1 overexpression. ** P < 0.01; *** P < 0.001; ns, no significance.
- Additionalfile6FigureS2.tif
Additional file 6: Figure S2. CircRHBDD1 knockdown suppresses glycolysis and glutaminolysis in MHCC97H cells. (A) The expression levels of GLUT1 and HK2 mRNA were determined by RT-qPCR in circRHBDD1-silenced MHCC97H cells. (B) The mRNA levels of ASCT2 and GLS1 were assessed using RT-qPCR in MHCC97H cells with circRHBDD1 knockdown. (C) Western blotting analysis was performed to detect the expression levels of GLUT1, HK2, ASCT2, and GLS1 in circRHBDD1-silenced MHCC97H cells. (D) The ECAR data showed that silencing circRHBDD1 significantly reduced the rate of glycolysis and the glycolytic capacity in MHCC97H cells. (E) The OCR results showed that circRHBDD1-silenced MHCC97H cells displayed increased basal respiration and maximum respiratory. (F) The cellular G6P level, lactate production, and cellular ATP level were detected in MHCC97H cells with circRHBDD1 knockdown. (G) The levels of glutamine, glutamate, and α-KG were examined in circRHBDD1-silenced MHCC97H cells. ** P < 0.01; *** P < 0.001.
- Additionalfile7FigureS3.tif
Additional file 7: Figure S3. CircRHBDD1 overexpression enhances glycolysis and glutaminolysis of HCC cells. (A) The expression levels of GLUT1 and HK2 mRNA were determined by RT-qPCR in circRHBDD1-overexpressing HepG2 and Huh7 cells. (B) The mRNA levels of ASCT2 and GLS1 were assessed using RT-qPCR in HepG2 and Huh7 cells with circRHBDD1 overexpression. (C) Western blotting analysis was performed to detect the expression levels of GLUT1, HK2, ASCT2, and GLS1 in circRHBDD1-overexpressing HepG2 and Huh7 cells. (D) The ECAR data showed that upregulating circRHBDD1 significantly elevated the rate of glycolysis and the glycolytic capacity in HepG2 and Huh7 cells. (E) The OCR results showed that circRHBDD1-overexpressing HepG2 and Huh7 cells exhibited decreased basal respiration and maximum respiratory. (F) The cellular G6P level, lactate production, and cellular ATP level were detected in HepG2 and Huh7 cells with circRHBDD1 overexpression. (G) The levels of glutamine, glutamate, and α-KG were examined in circRHBDD1-overexpressing HepG2 and Huh7 cells. * P < 0.05; ** P < 0.01; *** P < 0.001.
- Additionalfile8FigureS4.tif
Additional file 8: Figure S4. CircRHBDD1 augments HCC viability via PIK3R1. (A) Western blotting analysis confirmed the efficiency of PIK3R1 overexpression in circRHBDD1-silenced HCCLM3 cells. (B) CCK-8 assays were performed to examine the cell proliferation of sh-circRHBDD1#1 HCCLM3 cells after PIK3R1 overexpression. (C) EdU assays were conducted to assess the proliferative capability of sh-circRHBDD1#1 HCCLM3 cells upon PIK3R1 overexpression. * P < 0.05; ** P < 0.01; ns, no significance.
- Additionalfile9FigureS5.tif
Additional file 9: Figure S5. CircRHBDD1 promotes glycolysis and glutaminolysis of HCC cells through PIK3R1. (A) Glycolysis rate and glycolystic capacity were calculated using the ECAR data in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (B) Basal respiration and maximum respiratory were detected using the OCR data in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (C) The cellular G6P level, lactate production, and cellular ATP level were detected in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (D) The levels of glutamine, glutamate, and α-KG were examined in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. (E) Western blotting analysis showing the expression levels of p-AKT, AKT, GLUT1, HK2, ASCT2, and GLS1 in sh-circRHBDD1#1 HCCLM3 cells with or without PIK3R1 overexpression. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance.
- Additionalfile10FigureS6.tif
Additional file 10: Figure S6. YTHDF1 proteins predominantly associate with a specific region of circRHBDD1. (A) Schematic of biotin pull-down of YTHDF1 using three nonoverlapping biotinylated RNAs (P1: 1-100 nt; P2: 101-200 nt; P3: 201-290 nt) tiling the length of circRHBDD1. (B) Western blotting of YTHDF1 in samples pulled down by full-length (FL) or truncated circRHBDD1. (C) Schematic structures of YTHDF1 protein and two truncated mutants (YTHDF1-N: 1-359 aa; YTHDF1-C: 360-559 aa) of YTHDF1 variants used in this study. The pink boxes represent YTH domains. (D) RIP assays were performed using anti-Flag antibody and cells transfected with vectors expressing the Flag-tagged FL and the truncated mutants of YTHDF1. *** P < 0.001; ns, no significance.
- Additionalfile11FigureS7.tif
Additional file 11: Figure S7. CircRHBDD1 knockdown does not change the mRNA and protein levels of EIF4A3. (A) The levels of EIF4A3 mRNA were detected by RT-qPCR in circRHBDD1-silenced HCCLM3 and MHCC97H cells. (B) Western blotting analysis showing the expression levels of EIF4A3 in circRHBDD1-silenced cells. ns, no significance.
- Additionalfile12FigureS8.tif
Additional file 12: Figure S8. Representative CT imaging of HCC patients treated with anti-PD-1 in the responder and non-responder group is shown.
- Additionalfile13FigureS9.tif
Additional file 13: Figure S9. The knockdown efficiency of circRHBDD1 was detected by RT-qPCR in Hepa1-6 cells after the transfection of sh-circRHBDD1#1.
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