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lncRNA SNHG9 enhances liver cancer stem cell self-renewal and tumorigenicity by negatively regulating PTEN expression via recruiting EZH2

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Abstract

Liver cancer stem cell (CSC) self-renewal and tumorigenesis are important causes of hepatocellular carcinoma (HCC) recurrence. We purposed to investigate the function of long noncoding RNA small nucleolar RNA host gene 9 (SNHG9) in liver CSC self-renewal and tumorigenesis in this study. Flow cytometry was carried out to separate CD133+ Populations and CD133− Populations from HCC cell lines. A combination of CD133+ cells and Matrigel matrix was subcutaneously injected to create the NOD-SCID mouse xenograft tumor model. Colony formation test and spheroids formation assay were carried out to clarify the impact of SNHG9 on the self-renewal of liver CSCs. RNA immunoprecipitation, RNA-pull down, and chromatin immunoprecipitation were performed on CD133+ cells to elucidate the mechanism of SNHG9 regulating PTEN expression. We found that SNHG9 was highly expressed in HCC clinical samples, HCC cells, and CD133+ cells. In vitro, interference with SNHG9 prevented the formation of colonies and spheroids in liver CSC cells and primary HCC cells. In vivo, interference with SNHG9 reduced the tumor volume and weight. SNHG9 could bind to EZH2, and SNHG9 interference suppressed EZH2 recruitment and H3K27me3 levels in the PTEN promoter region. In addition, SNHG9 inhibition promoted PTEN expression while having little impact on EZH2 levels. Interference with SNHG9 inhibited liver CSC self-renewal and tumorigenesis by up-regulating PTEN levels. In conclusion, by binding to EZH2, SNHG9 down-regulated PTEN levels, promoting liver CSC self-renewal and tumor formation, and exacerbating HCC progression.

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Data availability

The datasets used and/or analyzed during the study are available from the corresponding author upon reasonable request.

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Funding

This work was supported by Zhejiang province medical and health science and technology project (2021RC089) and Wenzhou municipal science and technology project (Y20210181).

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Authors and Affiliations

  1. Department of Hepatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Nan Bai Xiang Street, Ouhai District, Wenzhou, 325000, China

    Shouzhang Yang, Bingren Hu, Jinfu Tu & Huajie Cai

  2. Department of Gastrointestinal Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, China

    Xiaojiao Ruan

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  1. Shouzhang Yang
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  2. Xiaojiao Ruan
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Correspondence to Huajie Cai.

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Ethical approval

The study was approved by the Ethics Committee in Clinical Research (ECCR) of the First Affiliated Hospital of Wenzhou Medical University and the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University.

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Supplementary Figure 1

The expression levels of lncRNAs in CD133+ liver CSCs. (a-f) Relative levels of FENDRR, ANCR, BLACAT1, SNHG1, SNHG6, and PCAT-1 were measured using qRT-PCR. nsp > 0.05. Student’s t-test was used. (TIF 14586 KB)

Supplementary Figure 2

SNHG9 was involved in the self-renewal of primary HCC cells. Primary HCC cells were transfected with Lenti-sh-SNHG9 or Lenti-SNHG9. N = 3. (a) The levels of SNHG9 were detected by qRT-PCR. (b) The protein levels of Nanog, Sox2, and Oct4 in primary HCC cells were detected by Western blot. β-actin served as an internal control. (c-d) A colony formation assay was performed. (e-f) Onco-spheroids formation assay was performed. (g) The protein levels of E-cadherin and vimentin in primary HCC cells were detected by Western blot. β-actin served as an internal control. (h-k) A Transwell assay was performed to detect migration and invasion. Scale bar: 200 μm. One-way ANOVA followed by Tukey's multiple comparisons test was used. (TIF 24075 KB)

Supplementary Figure 3

Overexpression of SNHG9 promoted self-renewal of CD133-HCC cells. CD133- Huh7 and CD133- Hep3B cells were transfected with Lenti-SNHG9. N = 3. (a-b) The levels of SNHG9 in CD133- Huh7 and CD133- Hep3B cells were detected by qRT-PCR. (c-d) The protein levels of Nanog, Sox2, and Oct4 in CD133- Huh7 and CD133- Hep3B cells were detected by Western blot. β-actin served as an internal control. (e-g) A colony formation assay was performed. (h-j) Onco-spheroids formation assay was performed. *P < 0.05, ***P < 0.001 vs Lenti. Student’s t-test was used. (TIF 18950 KB)

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Yang, S., Ruan, X., Hu, B. et al. lncRNA SNHG9 enhances liver cancer stem cell self-renewal and tumorigenicity by negatively regulating PTEN expression via recruiting EZH2. Cell Tissue Res 394, 441–453 (2023). https://doi.org/10.1007/s00441-023-03834-x

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