HMGB1-induced autophagy in Schwann cells promotes neuroblastoma proliferation

Abstract

Neuroblastoma inflicts mostly on children, and the pathogenesis remains elusive. Clinical diagnosis and therapeutic approaches are still on the incipient stage, so further understanding of the molecular and cellular mechanisms of the disease is necessary. Inflammation has been commonly regarded as a hallmark in tumorigenesis and development, and we identified a new inflammatory factor, HMGB1, is considerably increased in neuroblastoma. Our study shows that HMGB1 induces autophagy in Schwann cells through activation of TLR4, and knockdown of TLR4 obviates the HMGB1-induced autophagy. The HMGB1-induced autophagy is through classical pathway, as deficiency of Beclin 1 deprived autophagy in Schwann cells. Coculture of neuroblastoma with Schwann cells pretreated with HMGB1 promoted the proliferation of neuroblastoma cells, and if Beclin 1 is knocked down in Schwann cells, no promotion effects is observed. Taken together, our study demonstrates that HMGB1-induced autophagy in Schwann cells contributes to neuroblastoma cell proliferation, thus providing a potential therapeutic approach on neuroblastoma development.

Keywords: Autophagy; HMGB1; Schwann cells; neuroblastoma.

Figure 1

Inflammatory factor profile in neuroblastoma. A. Total RNA was prepared from neuroblastoma tissues or para-tumor tissues using TRIzol reagent, reverse transcribed, and quantitative real-time PCR was carried out with specific primers on an ABI Prism 7900HT. B. Neuroblastoma or para-tumor tissues were homogenized and centrifuged, and the supernatants were collected and subjected to Luminex assay for the concentrations of inflammatory factors. The data represent three independent experiments, and shown as Mean ± SD. P < 0.05 means statistically significant.

Figure 2

Expression of TLR4 and TLR2 in neuroblastoma cells. A. JS-1 or RSC96 cells pretreated with or without IL-1 were lysed and subjected to SDS-PAGE. After transferred to membrane, the samples were blotted with indicated antibodies. Images shown here are representatives of 3 independent experiments. B. JS-1 or RSC96 cells pretreated with or without HMGB1 were transfected with an ELAM-1 promoter-controlled luciferase reporter gene were lysed, and relative luciferase activities were determined. The data represent three independent experiments, and shown as Mean ± SD. P < 0.05 means statistically significant.

Figure 3

HMGB1 induces autophagy in Schawann cells. JS-1 cells, with or without TLR4 knockdown, pretreated with or without HMGB1 were lysed and subjected to SDS-PAGE. After transfer, the membrane was analyzed by immunoblotting with antibodies against LC3 for the conversion of LC3-I to LC3-II. Images shown here are representatives of 3 independent experiments.

Figure 4

Autophagy in Schwann cell promotes neuroblastoma cell survival. A. JS-1 cells were infected with lentivirus with scramble shRNA (Control) or shRNA against specifically Becn1 (MOI = 10). After 48 h, the cells were lysed and subject to SDS-PAGE followed by transferred to nitrocellulose membrane. The blot bands were visualized with ECL chemiluminant kit. Images shown here are representatives of 3 independent experiments. B. Medium from JS-1 and JS-1^shBeclin1 cells with or without autophagy were added to cultured SH-SY5Y cells, and MTT assay was used to determine the proliferation of the cells. The data represent three independent experiments, and shown as Mean ± SD. P < 0.05 means statistically significant.