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. 2024 Jun 29;25(13):7206.
doi: 10.3390/ijms25137206.

Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

Yizi Li  1 , Manlin Tian  1 , Jaceline Gislaine Pires Sanches  1 , Qingqing Zhang  1 , Li Hou  1 , Jun Zhang  1   2
Affiliations

Sorcin Inhibits Mitochondrial Apoptosis by Interacting with STAT3 via NF-κB Pathway

Yizi Li et al. Int J Mol Sci. .

Abstract

Hepatocellular carcinoma (HCC) is a common tumor. Our group has previously reported that sorcin (SRI) plays an important role in the progression and prognosis of HCC. This study aims to explore the mechanism of SRI inhibiting the mitochondrial apoptosis. Bioinformatics analysis, co-IP and immunofluorescence were used to analyze the relationship between SRI and STAT3. MMP and Hoechst staining were performed to detect the effect of SRI on cell apoptosis. The expression of apoptosis-related proteins and NF-κB signaling pathway were examined by Western blot and immunohistochemistry when SRI overexpression or underexpression in vivo and in vitro were found. Moreover, inhibitors were used to further explore the molecular mechanism. Overexpression of SRI inhibited cell apoptosis, which was attenuated by SRI knockdown in vitro and in vivo. Moreover, we identified that STAT3 is an SRI-interacting protein. Mechanistically, SRI interacts with STAT3 and then activates the NF-κB signaling pathway in vitro and in vivo. SRI interacting with STAT3 inhibits apoptosis by the NF-κB pathway and further contributes to the proliferation in HCC, which offers a novel clue and a new potential therapeutic target for HCC.

Keywords: SRI; STAT3; apoptosis; protein–protein interaction.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Figure 1
Figure 1
SRI and STAT3 are upregulated both in HCC tissues and cells. (A,B) SRI and STAT3 are highly expressed in HCC tissues (T) compared with normal liver tissues (N) in the GEO database (GDS4882). (C) Image available from Proteinatlas database showed high expression of SRI and STAT3 in tumor (T) compared with normal liver tissues (N). The expression of proteins were determined by the brown area. Scale bars = 200 μm. (D–H) Western blot showed SRI and STAT3 proteins were overexpressed in clinical HCC tissues and in Huh-7/HepG2/Hep3B cells. The red and blue spots represented the expression of different proteins in the T and N groups, respectively. (I) From the TGGA database, SRI was found to be positively correlated with STAT3 in LIHC (p < 0.001). (J,K) The expressions of SRI and STAT3 are positively correlated in HCC tissues and cells lines (p < 0.05). * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
Anti-apoptosis-related proteins are highly expressed, while pro-apoptosis-related protein is lowly expressed in HCC tissues. (A,B) Expressions of Bcl−2 and Bcl−XL were increased in clinical HCC tissues. (C) Bax expression was decreased in clinical HCC tissues. The red and blue spots represented the expression of different proteins in the T and N groups, respectively. ** p < 0.01. (D) IHC staining images of Bcl−2 and Bax in HCC (T) and normal liver tissues (N) acquired from Proteinatlas database. The expression of proteins were determined by the brown area. Scale bars = 200 μm.
Figure 3
Figure 3
SRI and STAT3 are interacting proteins. (A) SRI and STAT3 are interacting proteins in the STRING database. (B) SRI and STAT3 are interacting proteins confirmed by co-immunoprecipitation assays in Huh-7/HepG2 cells. (C,D) SRI (shown in red) co-localized with STAT3 (shown in green) was visualized by cellular immunofluorescence in Huh-7/HepG2 cells. DAPI (shown in blue) was used for nuclear staining. Scale bars = 200 μm.
Figure 4
Figure 4
SRI interacts with STAT3, inhibits apoptosis and activates the NF-κB signaling pathway in vitro and in vivo. (A–D) SRI downexpression reduced the fluorescence intensity of TMRE (shown in red) and enhanced the apoptosis sensitivities by Hoechst 33342 (shown in blue) staining assay. The results of SRI overexpression were consisted with SRI downexpression. Scale bars = 200 μm. (E–J) Western blot detected the expression of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins when SRI overexpression or downexpression were found in HCC cell and tumor xenografts. (K,L) Representative IHC images of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins when SRI overexpression or downexpression were found in tumor xenografts. The expression of proteins were determined by the brown area. Scale bars = 50 μm. * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
SRI and STAT3 interaction is crucial for anti-apoptosis. (A–D) Stattic reduced the fluorescence intensity of TMRE (shown in red) and enhanced the sensitivities to SRI induced apoptosis by Hoechst 33342 (shown in blue) staining assay. Scale bars = 200 μm. (E–H) The expression of SRI, p65, p-p65, p-IκB and apoptosis-related proteins were detected by Western blot in Stattic group. * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
SRI inhibits cells apoptosis through the NF-κB signaling pathway. (A,B) Tumor volume and weight of orthotopic xenograft models derived from upSRI cells treated with Stattic. Symbols of different colors represented the weight of tumors in different groups. (C,D) Representative IHC images of STAT3, p65, p-p65, p-IκB and apoptosis-related proteins in Stattic treatment. Scale bars = 50 μm. (C,E) Representative IHC images of p65 and apoptosis-related proteins after treatment with AL inhibitor. The expression of proteins were determined by the brown area. (F–H) AL inhibits the fluorescence intensity of TMRE (shown in red) and reduced the sensitivities to SRI-induced apoptosis by Hoechst 33342 (shown in blue) staining assay. Scale bars = 200 μm. (I–L) Apoptosis-related proteins were detected after treated with AL inhibitor by Western blot. * p < 0.05, ** p < 0.01.
Figure 7
Figure 7
A schematic illustration of SRI regulating apoptosis in HCC. SRI overexpression inhibits the apoptosis of HCC cells by interacting with STAT3 via NF-κB pathway, whereas opposing effects were observed for knockdown of SRI. Stattic, an inhibitor of STAT3, promotes apoptosis of HCC cells via NF-κB pathway. Avicularin, the inhibitor of NF-κB, which promotes apoptosis of HCC cells. ↑: represent upregulation or promotion. ↓: represent downregulation or inhibition.
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Grants and funding

This research was funded by Open Funds of State Key Laboratory of Oncology in South China (HN2023-07).