J Korean Neurosurg Soc.  2020 Nov;63(6):707-716. 10.3340/jkns.2019.0255.

Survival Association and Cell Cycle Effects of B7H3 in Neuroblastoma

• Zhang H
• Zhang J
• Li C
• Xu H
• Dong R
• Chen CC
• Hua W

• KMID: 2508600
• DOI: http://doi.org/10.3340/jkns.2019.0255

Abstract

Objective
: The function of B7H3, a member of the B7 family of proteins, in neuroblastoma (NB) remains poorly characterized. Here we examine the expression pattern of B7H3 in clinical NB specimens and characterize the phenotype of B7H3 knock-down in NB cell line.
Methods
: Immunohistochemical (IHC) staining was carried out to assess the expression of B7H3 in clinical NB specimens. Survival association was analyzed using five Gene Expression Omnibus (GEO) datasets (GSE85047, GSE45480, GSE62564, GSE16476, GSE49710). Clonogenic survival and flow cytometry were performed after B7H3 knockdown to assess the cellular proliferation and cell survival in vitro. Impact of B7H3 silencing on NB growth was examined in vivo using the SH-SY5Y xenograft model.
Results
: On IHC staining, B7H3 was widely expressed in clinical NB specimens. Analysis of the transcriptional profiles of five GEO datasets clinically annotated NB specimens revealed that decreased B7H3 expression was associated with improved overall survival. B7H3 knockdown suppressed the proliferation of the SH-SY5Y NB model in vitro and in vivo. Cell cycle analysis revealed that B7H3 silencing induced G1/S arrest. This arrest was associated with the suppression of E2F1 expression and induction of Rb expression.
Conclusion
: Our results demonstrate that B7H3 expression correlate with clinical survival in NB patients. Preliminary studies suggest that B7H3 may mediate the G1/S transition.

Keyword

Neuroblastoma; B7H3; Proliferation; Prognosis; Cell cycle

Figure

• 

  Fig. 1. B7H3 was associated with neuroblastoma prognosis. A : IHC staining showed that B7H3 was widely expressed in human NBs. In 18 patients, 17 cases had positive B7H3 expression, including three cases in positive expression (+), nine cases in moderate positive expression (++), five cases in strong positive expression (+++). B : High expression of B7H3 had worse overall survival than those with low expression in GSE85047 (n=283, p=2.7×10-5). C : High expression of B7H3 had worse overall survival than those with low expression in GSE62564 (n=498, p=1.1×10-3). D : High expression of B7H3 had worse overall survival than those with low expression in GSE45480 (n=476, p=7.5×10-4). E : High expression of B7H3 had worse overall survival than those with low expression in GSE16476 (n=88, p=1.0×10-3). F : High expression of B7H3 had worse overall survival than those with low expression in GSE49710 (n=498, p=1.7×10-2). IHC : immumohistochemistry, NB : neuroblastoma.

• 

  Fig. 2. B7H3 knockdown could inhibit proliferation of neuroblastoma in vitro. A : B7H3 mRNA and protein level was effectively inhibited by shRNAs than control group in SH-SY5Y cell line. B : Cell proliferation was significantly inhibited in B7H3 knockdown group in SH-SY5Y cell line at the third day after plating (p<0.01). C : Colony numbers in B7H3 knockdown group in SH-SY5Y cell line was significantly reduced by 72.3±8.5% (p<0.001). *p<0.05. †p<0.01. GAPDH : glyceraldehyde 3-phosphate dehydrogenase, NS : not significant.

• 

  Fig. 3. Cell cycle was suppressed in G1/S phase with checkpoint change after B7H3 knockdown. A : B7H3 knockdown inhibit G1/S phase transition, with G1/S : 56.22%/35.16% in control group and G1/S : 76.89%/19.73% in B7H3 knockdown group in SH-SY5Y cell line. B : RT-PCR (left) and western blot (middle and right) analysis of G1 phase checkpoint (cyclinD1, Rb, E2F1) in SH-SY5Y cell line. Relative protein expression values were normalized assigning the value of the cells in control groups to 1.0. C : Bioinformatic analysis of the correlation between B7H3 and proliferation-associated molecules. *p<0.01. †p<0.001. GAPDH : glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 4. B7H3 knockdown could inhibit proliferation of neuroblastoma in vivo. A : Subcutaneous xenograft tumor model demonstrated that B7H3 knockdown could inhibit tumor growth. B : B7H3 knockdown inhibit tumor growth (22.23±11.31 vs. 50.42±10.72 mm3, p<0.05) and decrease tumor weight (0.02±0.00 vs. 0.06±0.01 g, p<0.01). C : Immunohistochemical staining showed that B7H3 knockdown group manifested lower Ki-67 index, E2F1 expression and higher Rb expression (×200). *p<0.05. †p<0.01. H&E : hematoxylin-eosin staining.

Reference

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