METTL3-mediated m6A methylation of C1qA regulates the Rituximab resistance of diffuse large B-cell lymphoma cells

Abstract

Rituximab has been incorporated into the standard treatment regimen for diffuse large B-cell lymphoma (DLBCL), and induces the death of tumor cells via complement-dependent cytotoxicity (CDC). Unfortunately, the resistance of DLBCL cells to Rituximab limits its clinical usefulness. It remains unclear whether the complement system is related to Rituximab resistance in DLBCL. A Rituximab-resistant DLBCL cell line (Farage/R) was generated under the stress of Rituximab. Constituent proteins of the complement system in wild-type Farage cells (Farage/S) and Farage/R cells were analyzed by qPCR, western blotting, and immunofluorescence. In vitro and in vivo knockdown and overexpression studies confirmed that the complement 1Q subcomponent A chain (C1qA) was a regulator of Rituximab resistance. Finally, the mechanism by which C1qA is regulated by m⁶A methylation was explored. The reader and writer were identified by pull-down studies and RIP-qPCR. Activity of the complement system in Farage/R cells was suppressed. C1qA expression was reduced in Farage/R cells due to post-transcriptional regulation. Furthermore, in vitro and in vivo results showed that C1qA knockdown in Farage/S cells decreased their sensitivity to Rituximab, and C1qA overexpression in Farage/R cells attenuated the Rituximab resistance of those cells. Moreover, METTL3 and YTHDF2 were proven to be the reader and writer for m⁶A methylation of C1qA, respectively. Knockdown of METTL3 or YTHDF2 in Farage/R cells up-regulated C1qA expression and reduced their resistance to Rituximab. In summary, the aberrant downregulation of C1qA was related to Rituximab resistance in DLBCL cells, and C1qA was found to be regulated by METTL3- and YTHDF2-mediated m6A methylation. Enhancing the response of the complement system via regulation of C1qA might be an effective strategy for inhibiting Rituximab resistance in DLBCL.

Fig. 1. The complement system was related to Rituximab resistance in Farage cells.

A The survival rates of Rituximab-sensitive Farage (Farage/S) and Rituximab-resistant Farage (Farage/R) cells at different concentrations of Rituximab, and the IC50 value. B, C Apoptotic cells during treatment with 20 μg/mL Rituximab were detected by flow cytometry and TUNEL staining. D The proliferation of cells treated with 20 μg/mL Rituximab was detected by the colony formation assay. E Members of the complement pathway. F C3 chains expressed during treatment with 20 μg/mL Rituximab were detected by western blotting. G, H Recognition units of the complement system (C1q, C1r, and C1s), were detected by western blotting. ns, not significant; ***p < 0.001.

Fig. 2. C1qA was the key recognition unit of the complement system related to Rituximab resistance and might be regulated by m⁶A modification.

A The mRNA levels of 3 subunits of C1q in Farage/S and Farage/R cells were detected by qPCR. B The levels of C1qA, C1qB, and C1qC proteins were detected by western blotting. C C1qA expression was localized by immunofluorescence. D The expressions of C1qA in DLBCL tissues from the first surgery of three pairs of rituximab sensitive and rituximab-resistant patients were showed by IHC assay. E A schematic diagram of the C1qA promoter inserted into the luciferase expression vector. F The relative expression levels of 2 transcriptional regulators, GATA-1 and MafB, were detected by qPCR. G The ratios of luciferase activity in the Farage/S and Farage/R groups. H The level of total m⁶A-modified RNA. I The level of m⁶A-modified C1qA mRNA as detected by Me-RIP qPCR. ns, not significant; ***p < 0.001.

Fig. 3. Knockdown of C1qA increased the Rituximab resistance of Farage/S cells, and C1qA overexpression reduced the Rituximab resistance of Farage/R cells.

Farage/S cells were transfected with shC1qA and Farage/S cells were transfected with pcDNA 3.0-C1qA. A, B The expression of C1qA in 4 groups. C The survival rates of cells treated with different concentrations of Rituximab, and the IC50 value. D The proliferation of cells treated with 20 μg/mL Rituximab was detected by the colony formation assay. E C3 chains expressed during treatment with 20 μg/mL Rituximab were detected by western blotting. F, G Apoptotic cells during treatment with 20 μg/ mL Rituximab were detected by flow cytometry and TUNEL staining. S-shCtrl, Farage/S cells transfected with shcontrol; S-shC1qA, Farage/S cells transfected with shC1qA; R-Vector, Farage/R cells transfected with an empty vector; R-C1qA, Farage/R cells transfected with the C1qA overexpression vector. ***p < 0.001, ###p < 0.001.

Fig. 4. C1qA increased the Rituximab sensitivity of Farage cells in vivo.

A Mice were subcutaneously injected with S-shCtrl, S-shC1qA, R-Vector, or R-C1qA cells and then treated with Rituximab. On day 27 after xenograft, the mice were sacrificed and the tumor tissues were collected. B The weights of the collected tumors. C Tumor growth curves were plotted with the tumor size measured every 4 days. D, E The expression of C1qA in tumor tissues. F C3 chains detected by western blotting. G Caspase-3 and Ki67 expression in tumor tissue was detected by immunohistochemistry. ***p < 0.001, ### p < 0.001.

Fig. 5. METTL3 and YTHDF2 were bound to C1qA mRNA.

A Relative expression of readers, writers, and erasers in Farage/S and Farage/R cells as detected by qPCR. B C1qA mRNA bound by METTL3 or YTHDF2 was detected by RIP-qPCR. C The levels of METTL3 and YTHDF2 in Farage/S and Farage/R cells. D, E METTL3 and YTHDF2 binding on C1qA mRNA was detected by pull-down-western blotting. ***p < 0.001.

Fig. 6. Knockdown of METTL3 increased the Rituximab sensitivity of Farage/R cells.

A–C METTL3 and C1qA expression in Farage/R cells with METTL3 knockdown (R-shMETTL3) and in control cells (R-shCtrl). D The levels of m⁶A-modified C1qA. E Cell survival rates and the IC50 value of Rituximab. F Cell proliferation under conditions of treatment with 20 μg/mL Rituximab was detected by the colony formation assay. G C3 chains expressed during treatment with 20 μg/mL Rituximab were detected by western blotting. H The proportions of apoptotic cells during treatment with 20 μg/mL Rituximab were detected by flow cytometry. ***p < 0.001, ### p < 0.001.

Fig. 7. Knockdown of METTL3 increased the Rituximab sensitivity of Farage/R cells in vivo.

A Mice were subcutaneously injected with R-shCtrl or R-shMETTL3 cells and then treated with Rituximab. On day 27 after xenograft, the mice were sacrificed and the tumor tissues were collected. B Tumor growth curves were plotted with the tumor size measured every 4 days. C The weights of the collected tumors. D, E The expression of C1qA in tumor tissues. F C3 chains were detected by western blotting. G, H Caspase-3 and Ki67 expression in tumor tissue was detected by immunohistochemistry. ***p < 0.001.

Fig. 8. Knockdown of YTHDF2 increased the Rituximab sensitivity of Farage/R cells.

A–C The expression of YTHDF2 and C1qA in Farage/R cells with YTHDF2 knockdown (R-shYTHDF2) and in control cells (R-shCtrl). D The level of m⁶A-modified C1qA. E Cell survival rates and the IC50 value of Rituximab. F The proliferation of cells being treated with 20 μg/mL Rituximab was detected by the colony formation assay. G C3 chains expressed during treatment with 20 μg/mL Rituximab were detected by western blotting. H The proportions of apoptotic cells during treatment with 20 μg/mL Rituximab were detected by flow cytometry. ***p < 0.001.