lncRNA MIAT/HMGB1 Axis Is Involved in Cisplatin Resistance via Regulating IL6-Mediated Activation of the JAK2/STAT3 Pathway in Nasopharyngeal Carcinoma

Abstract

Cisplatin-based chemotherapy and radiotherapy are the main first-line treatment strategies for nasopharyngeal carcinoma (NPC) patients. Unfortunately, resistance is a major obstacle in the clinical management of NPC patients. We prove that the expression level of high-mobility group box 1 (HMGB1) is dramatically increased in resistant NPC cells than that in sensitive cells. HMGB1 induces the expression and secretion of IL6, which leads to constitutive autocrine activation of the JAK2/STAT3 pathway and eventually contributes to chemoresistance in NPC cells. Long non-coding RNAs (lncRNAs) have been identified as key regulators involved in drug resistance. In this study, using GO analysis of the biological process and differential expression analysis, we find 12 significantly altered IncRNAs in NPC cell lines, which may be involved in regulating gene expression. Furthermore, we determine that elevated lncRNA MIAT level upregulates HMGB1 expression, contributing to cisplatin resistance in NPC cells. We find that the deficiency of the lncRNA MIAT/HMGB1 axis, inhibition of JAK2/STAT3, or neutralization of IL6 by antibodies significantly re-sensitizes resistant NPC cells to cisplatin in resistant NPC cells. Moreover, we provide the in vivo evidence that the deficiency of HMGB1 reduces cisplatin-resistant tumor growth. Most importantly, we provide clinical evidence showing that the expression level of the lncRNA MIAT/HMGB1/IL6 axis is elevated in resistant NPC tumors, which is highly correlated with poor clinical outcome. Our findings identify a novel chemoresistance mechanism regulated by the lncRNA MIAT/HMGB1/IL6 axis, which indicates the possibilities for lncRNA MIAT, HMGB1, and IL6 as biomarkers for chemoresistance and targets for developing novel strategies to overcome resistance in NPC patients.

Keywords: HMGB1; IL6; NPC; cisplatin resistance; lncRNA MIAT.

Figure 1

The expression and secretion levels of IL6 were elevated in resistant NPC cells. (A, B) Establishment of cisplatin-resistant NPC cells. Cell proliferation was assessed by SRB assay, ***P < 0.001. (C, D) The mRNA levels of some cytokines and chemokines in parent wild-type NPC cell lines (CNE-2 WT and HONE-1 WT) and resistant cell lines (CNE-2 CR and HONE-1 CR) were detected by qPCR, *P < 0.05, **P < 0.01, ***P < 0.001. (E, F) The protein levels of IL6, p-JAK2, JAK2, p-STAT3, STAT3, and Actin were detected by western blotting. (G, H) IL6 secretion levels were detected by an ELISA kit, ***P < 0.001.

Figure 2

Activation of IL6 promotes cisplatin resistance. (A, B) Cell proliferation was assessed by SRB assay after recombinant IL6 treatment, **P < 0.01. (C, D) Cell proliferation was assessed by SRB assay after incubation with conditional medium from wild-type cells or resistant cells, and isotype control or anti-IL6, **P < 0.01, ***P < 0.001. (E, F) The protein levels of p-JAK2, JAK2, p-STAT3, STAT3, and Actin were detected by western blotting in CNE-2 WT and HONE-1 WT cells treated with conditional medium from resistant cells, and isotype control or anti-IL6. (G, H) IL6 secretion levels were detected by an ELISA kit after IL6 siRNA transfection, ***P < 0.001. (I, J) Cell proliferation was assessed by SRB assay after IL6 siRNA transfection, ***P < 0.001. (K, L) The protein levels of IL6, p-JAK2, JAK2, p-STAT3, STAT3, and Actin were detected by western blotting after IL6 siRNA transfection.

Figure 3

HMGB1 is upregulated in NPC cisplatin-resistant cells. (A, B) The protein levels of HMGB1 and Actin were detected by western blotting. (C, D) The protein levels of HMGB1, IL6, p-JAK2, JAK2, p-STAT3, STAT3, and Actin were detected by western blotting after HMGB1 siRNA transfection. (E, F) Cell proliferation was assessed by SRB assay, after HMGB1 siRNA transfection ***P < 0.001. (G, H) A cell proliferation assay was performed using an EdU assay and analyzed by flow cytometry after siRNA transfection. *P < 0.05, **P < 0.01.

Figure 4

HMGB1 upregulates expression of IL6 in resistance. (A) Cell proliferation was assessed by SRB assay after recombinant IL6 treatment in CNE-2 CR and CNE-2 CR-KO cells, **P < 0.01, ***P < 0.001. (B) The protein levels of HMGB1 and Actin were detected by western blotting in CNE-2 CR and CNE-2 CR-KO cells. (C) Cell proliferation was assessed by SRB assay after anti-IL6 treatment in CNE-2 WT and CNE-2 HMGB1 OE cells, **P < 0.01, ***P < 0.001. (D) The protein levels of HMGB1 and Actin were detected by western blotting after HMGB1 was overexpressed in CNE-2 WT cells. (E) Cell proliferation was assessed by SRB assay after HMGB1 was overexpressed in CNE-2 CR OK cells. (F) The protein levels of HMGB1 and Actin were detected by western blotting in CNE-2 CR and CNE-2 CR KO and CNE-2 CR KO with HMGB1 overexpressed cells. (G) Cell proliferation was assessed by SRB assay after treatment with JAK2 inhibitor LY2784544 or STAT3 inhibitor stattic, ***P < 0.001.

Figure 5

HMGB1 deficiency overcomes cisplatin resistance in vivo. (A) The scheme of xenograft tumors and treatment. (B) The photograph of the representative tumors from mice in each treatment group. Ruler scale is shown in cm. (C) Growth curves of CNE-2 CR xenograft tumors treated with vehicle and cisplatin (5 mg/kg, intraperitoneally) for two weeks, n = 6 mice/group, ***P < 0.001. (D) The tumor weight in each treatment group, ***P < 0.001. (E) The body weight from mice in each treatment group. (F–G) Immunohistochemistry staining of Ki67 in xenograft tumor samples. Ki67 positive cells were quantified. NS, Non-significant.

Figure 6

lnc MIAT contributes to cisplatin resistance via regulating HMGB1 in NPC cells. (A) Volcano plot showing differentially expressed lncRNAs in CNE-2 compared with normal nasopharynx cells. A change is considered significant if the change is >2-fold with a p-value of <0.05. (B) GO analysis of the biological process in CNE-2 cells. (C) Heatmap showing the selected differentially expressed lncRNAs in CNE-2 after overlap with those in HONE-1. (D, E) lncRNA levels were detected by qPCR, **P < 0.01, ***P < 0.001. (F–I) The expression levels of HMGB1 and IL6 were detected by qPCR after siRNA transfection of lncRNAs, **P < 0.01, ***P < 0.001. (J, K) Cell proliferation was assessed by SRB assay, after MIAT siRNA transfection ***P < 0.001. (L, M) Cell proliferation assay was performed using an EdU assay and analyzed by flow cytometry after siRNA transfection. *P < 0.05.

Figure 7

Clinical evidence of the lncRNA MIAT/HMGB1/IL6 axis. (A) The protein levels of HMGB1 and Actin were detected by western blotting in patient samples. (B–D) The mRNA levels of IL6, HMGB1, and lncRNA MIAT were detected by qPCR in patient samples, **P < 0.01. (E–H) Survival rate of patients was analyzed using a Kaplan-Meier plotter (https://kmplot.com/analysis/). (I) Working model of the lncRNA MIAT/HMGB1/IL6 axis in regulating cisplatin resistance in NPC cells.