METTL3 methylated KIF15 promotes nasopharyngeal carcinoma progression and radiation resistance by blocking ATG7-mediated autophagy through the activation of STAT3 pathway

Abstract

Background: Resistance to radiotherapy is a major component in the failure of nasopharyngeal carcinoma (NPC) treatment. Enhancing autophagy in nasopharyngeal carcinoma may increase its radiation sensitivity, making it critical to find autophagy-modulating targets.

Methods: The level of KIF15 was determined in NPC patients. Then, radiation-resistant NPC cells were produced to explore the mechanism in NPC. KIF15 was suppressed, and cell function and autophagy-related variables were examined in radiation-resistant NPC cells. Then the autophagy pathway was blocked, and the link between KIF15 and autophagy was confirmed. Finally, an NPC murine model was established, with tumors implanted in aberrant sites, and the relationship discovered at the cell level was confirmed in vivo. All statistical significance was determined using the student's t-test and one-way ANOVA.

Results: Elevated amounts of KIF15 were discovered to be significantly expressed in NPC tissues and played a role in the radioresistance of NPC, a phenomenon attributed to METTL3-mediated m6A methylation. Blocking KIF15 resulted in decreased cell proliferation, increased cell death, and the activation of autophagy, ultimately making NPC more sensitive to radiation. This also resulted in decreased tumor development and increased levels of autophagy and apoptosis in vivo KIF15 interacted with STAT3, retaining it in the cytoplasm. Overexpression of STAT3 reversed the inhibitory effects of KIF15 knockdown on NPC and also reversed the influence of sh-KIF15 on autophagy activation. Inhibition of KIF15 decreased the inhibitory effect of STAT3 on ATG7, thereby upregulating autophagy activation in radio-resistant NPC cells.

Conclusion: The increased expression of KIF15 was found to be associated with the progression of NPC and play a role in the development of radioresistance in NPC. Inhibiting KIF15 was shown to impede tumor growth and improve the sensitivity of NPC to radiotherapy by triggering autophagy via the STAT3/ATG7 pathway.

Keywords: Autophagy; KIF15; NPC; Radioresistance.

Fig. 1

KIF15 was found to be significantly upregulated in NPC tissues and was associated with resistance to radiotherapy in NPC. (A) KIF15 levels were determined by qPCR in 40 pairs of NPC tissues and surrounding tissues. (B) Kaplan-Meier survival curves indicate the connections between KIF15 expression and five-year overall survival. (C and D) KIF15 levels in radioresistant and radiosensitive NPC tissues were determined using qPCR and IHC. (E and F) KIF15 levels in NP69, HNE3 and C666–1cell lines were determined using qPCR and immunoblot analysis, and the relative expression level in comparison to the NP69 cell line was calculated.

Fig. 2

KIF15 expression was raised in nasopharyngeal cancer radiation resistant cell line. C666–1-IR and HNE3-IR radiation resistant NPC cell lines were created. (A) CCK-8 were used to monitor the differences in cell viability between C666–1, HNE3, C666–1-IR and HNE3-IR under increasing doses of radiation (0, 2, 4, 6, 8, 10 Gy). (B) CCK-8 were used to monitor the differences in cell viability between C666–1, HNE3, C666–1-IR and HNE3-IR under 6 Gy doses of radiation. (C) The survival fractions of NPC cells with and without 6 Gy doses of radiation were examined using a colony formation assay. (D) A flow cytometry test was conducted to determine the rate of apoptosis with or without 6 Gy doses of radiation. (E) Immunoblot analysis was used to determine the relative expression levels of Bax and Bcl-2 in NPC cells, which were normalized to GAPDH. (F, G) KIF15 levels in normal NPC cells and radiation resistant NPC cells were determined using qPCR and immunoblot analysis.

Fig. 3

Silencing KIF15 activated autophagy and increased death in NPC radiation resistant cells. C666–1-IR and HNE3-IR cells were treated with sh-KIF15 or the negative control (sh-NC). (A and B) KIF15 levels in NPC cells were evaluated by qPCR and immunoblot analysis. Then another 3-MA was added. (C) CCK-8 assessed cell viability, and (D) colony formation assay assessed cell proliferation ability. (E) The rate of apoptosis was evaluated by flow cytometry. (F) IF examined the levels of LC3B in NPC cells. (G) Immunoblot analysis was utilized to assess the relative expression levels of Bax, Bcl-2, LC3, and p62 in NPC cells, which were normalized to GAPDH.

Fig. 4

The METTL3-mediated m6A modification increased KIF15 expression in NPC. (A and B) METTL3 levels in 40 pairs of NPC tissues and surrounding tissues were evaluated using qPCR and IHC. (C) Correlation study of KIF15 and METTL3 expression levels in NPC. (D) Me-RIP detected m6A methylation of KIF15 in NPC tissues and adjacent tissues. (E, F) METTL3 and KIF15 levels were assessed by qPCR in C666–1-IR and HNE3-IR cells after sh-METTL3 transfection. (G) KIF15 levels in m6A antibody precipitated products in C666–1-IR and HNE3-IR cells were evaluated using MeRIP following sh-METTL3 transfection. (H) SRAMP predicted the m6A modification site in KIF15. (I, J) MeRIP was used to detect the amount of KIF15 after transfection of KIF15 mutant plasmids and overexpressed METTL3. (K) The half-life of KIF15 mRNA in each group was determined following actinomycin D treatment.

Fig. 5

By activating STAT3-mediated autophagy, KIF15 silencing reduces radiation resistance in NPC. (A) The CCK-8 assay was employed to assess variations in C666–1-IR and HNE3-IR cells viability among different groups subjected to escalating doses of radiation (0, 2, 4, 6, 8, 10 Gy). (B) The survival rates of C666–1-IR and HNE3-IR cells following sh-KIF15 or oe-STAT3 treatment under 6 Gy radiation doses were investigated using a colony formation assay. (C) The apoptotic rate was determined through flow cytometry analysis. (D) Immunofluorescence was used to examine the levels of LC3B in distinct groups. (E) Immunoblot analysis was employed to evaluate the relative expression levels of Bax, Bcl-2, LC3, and p62 in C666–1-IR and HNE3-IR cells subjected to different treatments, with normalization to GAPDH.

Fig. 6

KIF15 directly interacted with STAT3, thereby inhibiting the transcription of the autophagy-related gene ATG7. (A) Immunoblot analysis was utilized to assess the relative expression levels of KIF15, STAT3, and p-STAT3 in C666–1-IR and HNE3-IR cells following sh-KIF15 treatments, with normalization to GAPDH. (B) Co-IP was employed to detect the interaction between KIF15 and p-STAT3 in C666–1-IR and HNE3-IR cells. (C) IF was utilized to observe the cellular localization of KIF15 and p-STAT3 in C666–1-IR and HNE3-IR cells. (D) The qPCR method was used to evaluate ATG7 levels in C666–1-IR and HNE3-IR cells. (E) ChIP assay was conducted to verify the relationship between STAT3 and ATG7, and (F) JASPAR was utilized to predict the binding site of STAT3 to ATG7. (G) ChIP assay confirmed the binding between STAT3 and two regions (−1866 to −1858, −506 to −498) in ATG7. (H-I) The ATG7 promoter mutation area construct was shown and was detected by dual luciferase reporter gene assay. (J) Immunoblot analysis was employed to assess the relative expression levels of KIF15, STAT3, p-STAT3, ATG7, LC3, and p62 in C666–1-IR and HNE3-IR cells, with normalization to GAPDH.

Fig. 7

Inhibiting KIF15 expression in vivo induces autophagy and improves NPC sensitivity to radiation. (A–C) The growth curves of subcutaneous tumors, and tumor weight was measured at the endpoint in each group. (D) IHC was used to detect the expression of KIF15 and Ki67 in tumor tissues. (E) TUNEL assay detected the apoptosis in tumor tissues. (F) Immunoblot analysis was employed to assess the relative expression levels of KIF15, STAT3, p-STAT3, ATG7, Bax, Bcl-2, LC3, and p62 in tumor tissues, with normalization to GAPDH.