Ubiquitin protein E3 ligase ASB9 suppresses proliferation and promotes apoptosis in human spermatogonial stem cell line by inducing HIF1AN degradation

BACKGROUND: Spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered. METHODS: To explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence. RESULTS: The results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis. CONCLUSION: ASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.

Effects of shikonin on the proliferation and apoptosis of HL-60 cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the mechanism of shikonin for inducing the apoptosis of human promyelocytic leukemia cell HL-60.</p><p><b>METHODS</b>The effects of shikonin on the HL-60 cell proliferation were detected using MTT. The apoptosis rate was analyzed by Annexin-V/PI double staining. The expression level of the bcl-2 gene was detected using semi-quantitative reverse transcriptase PCR (RT-PCR), thus analyzing the correlation between the bcl-2 expression level and the apoptosis of HL-60.</p><p><b>RESULTS</b>Shikonin could inhibit the proliferation of HL-60 cells with the concentration range of 1-8 microg/mL in a time- and concentration-dependent manner. Two microg/mL shikonin could induce the apoptosis of HL-60 cells in a time-dependent manner. The expression level of bcl-2 was obviously down-regulated at 2 microg/mL shikonin.</p><p><b>CONCLUSIONS</b>Shikonin could induce the apoptosis of HL-60 cells. Its mechanism was correlated with down-regulation of the expression level of bcl-2.</p>