Piezo1 promoted hepatocellular carcinoma progression and EMT through activating TGF-β signaling by recruiting Rab5c

Background Piezo1 has been revealed to play a regulatory role in vascular development and progression of variety tumors. However, whether and how the progression of hepatocellular carcinoma (HCC) regulated by Piezo1 remains elusive. This study aimed to elucidate the effect and mechanisms of Piezo1 in HCC. Methods The mRNA and protein expression level of Piezo1 in HCC samples and cell lines was determined by qRT-PCR, western blot and immunohistochemistry analyses. Two independent study cohorts containing 280 patients were analyzed to reveal the association between Piezo1 expression and clinicopathological characteristics. Series of in vitro and in vivo experiments were used to validate the function of Piezo1 in HCC. Gene set enrichment analysis (GSEA) was performed to explore the signaling pathway of Piezo1. Immunoprecipitation, immunofluorescence and in vitro and in vivo experiments were used to explore the molecular mechanism of Piezo1 in HCC progression. Results Our results demonstrated the Piezo1 expression was significantly upregulated in HCC tissues and cell lines, and upregulation of Piezo1 closely correlated with aggressive clinicopathological features and poor prognosis. Knockdown of Piezo1 in HCCLM3 and Hep3B cells significantly restrained proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of HCC cells in vitro, and tumor growth, metastasis, EMT in vivo. TGF-β signaling pathway was most significant enriched pathway in GSEA. Finally, tumor promotion effect of Piezo1 was found to exerted through recruiting and combining Rab5c to activating TGF-β signaling pathway. Conclusions Piezo1 significantly related to poor prognosis and promotes progression of hepatocellular carcinoma via activating TGF-β signaling, which suggesting that Piezo1 may serve as a novel prognostic predictor and the potential therapeutic target for HCC patients. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-022-02574-2.


MTT assay and colony formation assay
For MTT assay, 5 × 103 cells were seeded in 96-well plates, incubated for 0-7 days, stained with MTT, and absorbance values were determined at 570 nm. The relative cell number was normalized by the absorbance from the control cells. For colony formation assays, 500 cells were seeded per well in 6-well plates and cultured for 14 days. The colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Only colonies containing more than 50 cells were counted.

Wound healing and transwell invasion assay
For wound healing assay, 5 × 105 cells were seeded into 6-well plates and grown to confluence. Mitomycin C (10 μg/mL) was used to suppress cell proliferation before scratching [27]. Wounds were created by scraping the confluent cell monolayers with a 10 μL pipette tip. After extensively rinsed to remove cellular debris, cells were cultured in serum-free medium. The wound closure rate was monitored every 12 h and images were taken using an inverted microscope TE-2000S (Nikon, Tokyo, Japan). Transwell invasion assay was performed in a 24-well transwell plate with 8-μm polyethylene terephthalate membrane filters (Corning Costar Corp, Corning, NY). 1×105 cells in 200 μL of serum-free medium were added to the upper chambers, which containede Matrigel coated membranes (BD Biosciences). Each lower chamber was filed with 500 μL medium with 10% FBS. After 18 h or 24 h of incubation, cells that invaded to the bottom chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Invasive cells were counted in five randomly chosen fields (magnification, × 200) per well.
Cancer 10-pathway reporter arrays A Cignal Finder 10-Pathway Reporter Array (SABiosciences, Valencia, CA) was performed to explore the signaling pathways that were regulated by Piezo1 in HCC cells. The assay was conducted according to the manufacturer's protocol. Relative firefly luciferase activity was calculated and normalized to the constitutively expressed Renilla luciferase. Experiments were done in triplicates.

Gene set enrichment analysis (GSEA)
The GSEA (http://www.broadinstitute.org/gsea) tool was used to determine the differential enrichment of gene sets in the HCC patient samples belonging to high and low Piezo1 expression groups. Gene expression data from TCGA were divided into high and low expression groups according to the median level of Piezo1 mRNA expression, and GSEA was used to explore the influence of Piezo1 expression level on each gene and to analyze the mechanism underlying the involvement of Piezo1 in the invasion and metastasis of HCC. The genome was sequenced 1000 times per analysis. In addition, the level of Piezo1 was used as a phenotypic marker. The nominal p-value (NOM p) and the normalized enrichment score (NES) were used to classify enrichment pathways in each phenotype.

Co-immunoprecipitation (co-IP) assay
For Co-IP, pre-cleared protein from whole cell lysates were incubated with antibody against Piezo1 or Rab5c at 4 °C overnight, which was conjugated to AminoLink Plus Resin (Pierce, Rockford, IL), The IP targets were disassociated from the immobilized antibodies on the AminoLink Plus Resin by the gentle elution buffer. Eluted proteins were resolved using 10% SDS-PAGE, followed by western blot with appropriate antibodies.

Cell immunofluorescence staining
Indicated HCC cells (2 × 10 4 cells) were seeded into 12-well plate with glass coverslips for 24 h. Then cells were successively fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 1% BSA and incubated with primary antibody at 4 °C overnight. After washed with PBS, cells were incubated with appropriate DyLight-conjugated secondary antibody and DAPI (Vector laboratories, Burlingame, CA). Finally, the slides were mounted and images were captured using an inverted fluorescence microscope DMI4000-B (Leica, Wetzlar, Germany).