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. 2024 Mar 12;24(1):334.
doi: 10.1186/s12885-024-12065-4.

Knockdown of ribosome RNA processing protein 15 suppresses migration of hepatocellular carcinoma through inhibiting PATZ1-associated LAMC2/FAK pathway

Tongtong Pan #  1 , Jinhai Li #  2 , Ouyang Zhang  3 , Yuqin Zhu  3 , Hongfei Zhou  1 , Mengchen Ma  3 , Yanwen Yu  3 , Jiaojian Lyu  4 , Yongping Chen  5 , Liang Xu  6
Affiliations

Knockdown of ribosome RNA processing protein 15 suppresses migration of hepatocellular carcinoma through inhibiting PATZ1-associated LAMC2/FAK pathway

Tongtong Pan et al. BMC Cancer. .

Abstract

Background: Ribosomal RNA processing protein 15 (RRP15) has been found to regulate the progression of hepatocellular carcinoma (HCC). Nevertheless, the extent to which it contributes to the spread of HCC cells remains uncertain. Thus, the objective of this research was to assess the biological function of RRP15 in the migration of HCC.

Methods: The expression of RRP15 in HCC tissue microarray (TMA), tumor tissues and cell lines were determined. In vitro, the effects of RRP15 knockdown on the migration, invasion and adhesion ability of HCC cells were assessed by wound healing assay, transwell and adhesion assay, respectively. The effect of RRP15 knockdown on HCC migration was also evaluated in vivo in a mouse model.

Results: Bioinformatics analysis showed that high expression of RRP15 was significantly associated with low survival rate of HCC. The expression level of RRP15 was strikingly upregulated in HCC tissues and cell lines compared with the corresponding controls, and TMA data also indicated that RRP15 was a pivotal prognostic factor for HCC. RRP15 knockdown in HCC cells reduced epithelial-to-mesenchymal transition (EMT) and inhibited migration in vitro and in vivo, independent of P53 expression. Mechanistically, blockade of RRP15 reduced the protein level of the transcription factor POZ/BTB and AT hook containing zinc finger 1 (PATZ1), resulting in decreased expression of the downstream genes encoding laminin 5 subunits, LAMC2 and LAMB3, eventually suppressing the integrin β4 (ITGB4)/focal adhesion kinase (FAK)/nuclear factor κB kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway.

Conclusions: RRP15 promotes HCC migration by activating the LAMC2/ITGB4/FAK pathway, providing a new target for future HCC treatment.

Keywords: Focal adhesion kinase; Hepatocellular carcinoma migration; LAMC2; Ribosome RNA processing protein 15.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Upregulation of RRP15 in HCC tissues and cells. (A) The expression level of RRP15 mRNA in HCC tissues (T) and normal liver tissues (N) in the TCGA database. (B) Overall survival of HCC patients according to Kaplan-Meier database in the TCGA database. (C, D) The expression of RRP15 mRNA and protein in HCC tissues and adjacent non-tumor tissues. (E) Representative images and quantitative analysis of TMA stained with IHC for RRP15. Scale bar = 50 μm. (F, G) RRP15 protein and mRNA levels in five HCC cell lines and a normal liver cell line. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. RRP15: ribosomal RNA processing protein 15; HCC: hepatocellular carcinoma; TMA: tissue microarray
Fig. 2
Fig. 2
RRP15 knockdown attenuated the migration and invasion of HCC. (A) The knockdown efficiency of RRP15 in HCC cells. (B) Downregulation of RRP15 inhibited migration of HCC cell in the wound healing assay. Scale bars = 100 μm. (C-D) Migration and invasion of MHCC-97 H or LM3 cells transfected with shNT or shRRP15 in the transwell assay. Scale bars = 100 μm. (E) Downregulation of RRP15 inhibited adhesion of HCC cells. Scale bars = 100 μm. (F) Expression levels of EMT-related proteins in MHCC-97 H or LM3 cells transfected with shNT or shRRP15. (G) Representative images of lung metastases observed under a stereoscope. Scale bars = 1 cm. (H) Number of metastatic nodules in the lung tissues in the indicated groups (n = 8 per group). (I) Representative images of H&E-stained lung tissue sections in shNT/shRRP15 mice. Scale bars = 100 μm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. RRP15: ribosomal RNA processing protein 15; HCC: hepatocellular carcinoma. H&E: hematoxylin and eosin
Fig. 3
Fig. 3
RRP15 knockdown inactivated the LAMC2/ITGB4/FAK pathway in HCC cells. (A) The volcano plot of differentially expressed genes (DEGs). The blue dots represent downregulated genes, the grey dots are non-differentially expressed genes and the red dots are upregulated genes. (B) KEGG pathway analysis showing the significantly associated signaling pathways in the RRP15-knockdown MHCC-97 H cells. (C) Heatmap of DEGs. Up- and down-regulated genes are shown in red and blue respectively. (D-E) Effect of RRP15 knockdown on the expression of LAMC2, LAMB3 and LAMA3 mRNAs. (F) Effect of RRP15 knockdown on the expression of LAMC2 and LAMB3 proteins. (G) Immunoblots showing ITGB4, pFAK, pERK, p-p65, FAK, ERK and p65 levels in RRP15-knockdown and control cells. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01. RRP15: ribosomal RNA processing protein 15; LAMC2: laminin subunit gamma 2; ITGB4: integrin subunit beta 4; FAK: focal adhesion kinase; HCC: hepatocellular carcinoma; LAMB3: laminin subunit beta 3; ERK: mitogen-activated protein kinase 1
Fig. 4
Fig. 4
RRP15 enhanced migration of HCC cells through the LAMC2/ITGB4/FAK pathway. (A) The knockdown efficiency of LAMC2 in HCC cells. (B) The overexpression of RRP15 in HCC cells. (C) Expression levels of the indicated proteins in RRP15-overexpressing MHCC-97 H and LM3 cells with or without LAMC2 knockdown. (D-E) Migration and invasion of RRP15-overexpressing MHCC-97 H and LM3 cells with or without LAMC2 knockdown in the transwell assay. Scale bars = 100 μm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. RRP15: ribosomal RNA processing protein 15; LAMC2: laminin subunit gamma 2; ITGB4: integrin subunit beta 4; FAK: focal adhesion kinase; HCC: hepatocellular carcinoma
Fig. 5
Fig. 5
Knockdown PATZ1 suppressed LAMC2 and LAMB3 transcription in HCC cells. (A) Transcriptional binding site of PATZ1. (B) Correlation of PATZ1 mRNA level with RRP15, LAMC2 and LAMB3 mRNA levels. (C) Protein and mRNA levels of PATZ1 in RRP15 knockdown cells. (D) The knockdown efficiency of PATZ1 in HCC cells. (E-F) Expression levels of the indicated mRNAs in RRP15-overexpressing MHCC-97 H and LM3 cells with or without PATZ1 knockdown. (G) Migration and invasion of RRP15-overexpressing MHCC-97 H and LM3 cells with or without PATZ1 knockdown in the transwell assay. Scale bars = 100 μm. (H) Schematic diagram showing that knockdown of RRP15 inhibits the growth and metastasis of HCC cells via inactivation of the LAMC2 /FAK signaling. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. PATZ1: POZ/BTB and AT hook containing zinc finger 1; LAMC2: laminin subunit gamma 2; LAMB3: laminin subunit beta 3; HCC: hepatocellular carcinoma
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