SIRT5 promote malignant advancement of chordoma by regulating the desuccinylation of c-myc

Abstract

Chordoma is a relatively rare and locally aggressive malignant tumor. Sirtuin (SIRT)5 plays pivotal roles in various tumors, but the role of SIRT5 in chordoma has not been found. This study was performed to investigate the regulatory effects of SIRT5 on cell proliferation, migration, and invasion and the underlying mechanism in chordoma. A xenograft tumor mouse model was established to assess tumor growth. Reverse transcription-quantitative polymerase chain reaction was used to analyze the mRNA levels of SIRT5 and c-myc. The effects of SIRT5 and c-myc on cell proliferation, migration, and invasion of chordoma cells were detected by cell counting kit-8, colony formation, and Transwell assays. The interaction between SIRT5 and c-myc was evaluated by co-immunoprecipitation (IP) assay. The succinylation of c-myc was analyzed by IP and Western blot. The results showed that SIRT5 expression was upregulated in chordoma tissues and cells. SIRT5 interacted with c-myc to inhibit the succinylation of c-myc at K369 site in human embryonic kidney (HEK)-293T cells. Silencing of SIRT5 suppressed the cell proliferation, migration, and invasion of chordoma cells, while the results were reversed after c-myc overexpression. Moreover, silencing SIRT5 suppressed tumor growth in mice. These findings suggested that SIRT5 promoted the malignant advancement of chordoma by regulating the desuccinylation of c-myc.

Keywords: Chordoma; Desuccinylation; Migration; Proliferation; SIRT5; c-myc.

Fig. 1

High expression of SIRT5 in chordoma tissues and cells. A, RT-qPCR and B, Western blot were performed to analyze the mRNA and protein levels of SIRT5 in normal and tumor tissues; The expression of SIRT5 in HUVEC, U-CH1, and U-CH2 cells was analyzed by C, RT-qPCR and D, Western blot. SIRT, sirtuin; HUVEC, human umbilical vein endothelial cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction

Fig. 2

Knockdown of SIRT5 inhibited cell proliferation, migration, and invasion of U-CH1 and U-CH2 cells. The expression of SIRT5 in sh-NC and sh-SIRT5 groups in U-CH1 and U-CH2 cells was detected by A, RT-qPCR and B, Western blot; C, CCK-8 assay was performed to assess the cell viability of U-CH1 and U-CH2 cells; D, Cell colonies were evaluated by colony formation analysis (scale bars = 0.5 cm); E, The colony number in sh-NC and sh-SIRT5 groups in U-CH1 and U-CH2 cells; F, Transwell assay was performed to detect cell migration (magnification, ×200); G, The migration cell number in sh-NC and sh-SIRT5 groups in U-CH1 and U-CH2 cells; H, Cell invasion was detected by Transwell assay (magnification, ×200); I, The invasion cell number in sh-NC and sh-SIRT5 groups in U-CH1 and U-CH2 cells. SIRT, sirtuin; RT-qPCR, reverse transcription-polymerase chain reaction; CCK-8, cell counting kit-8; sh-RNA, short hairpin RNA.

Fig. 3

SIRT5 interacted with c-myc to inhibit the succinylation of c-myc at K369 site. A, The succinylation level in sh-NC and sh-SIRT5 groups in HEK-293T cells was assessed by Western blot; B, STRING database was used to screen SIRT5-related proteins; C, Co-IP assay was performed to detect the interaction between SIRT5 and c-myc; D, The protein levels of c-myc and c-myc-succ in HEK-293T cells were detected by IP and Western blot after SIRT5 knockdown; E, The succinylation sites for c-myc were predicted using GPSuc database; F, IP and Western blot assays were used to analyze the succinylation sites of c-myc; G, The protein expression of c-myc was assessed by Western blot at the different time points (0, 8, 16, and 24 h) in HEK-293T cells; H, Quantification of the existing c-myc protein level at different time points (0, 8, 16, and 24 h) in HEK-293T cells. SIRT, sirtuin; STRING, Search Tool for Recurring Instances of Neighbouring Genes; Co-IP, co-immunoprecipitation; succ, succinylation

Fig. 4

Overexpressing c-myc reversed the decreased cell viability, colony number, migration and invasion cell numbers caused by silencing SIRT5 in U-CH1 and U-CH2 cells. The expression of c-myc after c-myc overexpression in U-CH1 and U-CH2 cells was detected by A, RT-qPCR and B, Western blot; C, CCK-8 assay was performed to assess the viability of U-CH1 and U-CH2 cells in each group; D, Cell colonies were evaluated by colony formation analysis(scale bars = 0.5 cm); E, The colony number in each group in U-CH1 and U-CH2 cells; F, Transwell assay was performed to detect cell migration (magnification, ×200); G, The migration cell number in each group in U-CH1 and U-CH2 cells; H, Cell invasion was detected by Transwell assay (magnification, ×200); I, The invasion cell number in each group in U-CH1 and U-CH2 cells. SIRT, sirtuin; RT-qPCR, reverse transcription-polymerase chain reaction; CCK-8, cell counting kit-8; sh-RNA, short hairpin RNA.

Fig. 5

Silencing SIRT5 inhibited tumor growth of mice. A, The tumor size, B, weight, and C, volume in sh-SIRT5 and sh-NC groups; D, IHC assay was performed to assess the SIRT5 and c-myc protein levels in sh-SIRT5 and sh-NC groups (scale bar = 100 μm). SIRT, sirtuin; sh-RNA, short hairpin RNA; IHC, immunohistochemistry