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. 2021 Oct 19:11:670292.
doi: 10.3389/fonc.2021.670292. eCollection 2021.

Quantitative Expression of Key Cancer Markers in the AS-30D Hepatocarcinoma Model

Marco A Briones-Orta  1 , Blanca Delgado-Coello  2 , Roxana Gutiérrez-Vidal  2 , Marcela Sosa-Garrocho  3 , Marina Macías-Silva  3 , Jaime Mas-Oliva  2
Affiliations

Quantitative Expression of Key Cancer Markers in the AS-30D Hepatocarcinoma Model

Marco A Briones-Orta et al. Front Oncol. .

Abstract

Hepatocellular carcinoma is one of the cancers with the highest mortality rate worldwide. HCC is often diagnosed when the disease is already in an advanced stage, making the discovery and implementation of biomarkers for the disease a critical aim in cancer research. In this study, we aim to quantify the transcript levels of key signaling molecules relevant to different pathways known to participate in tumorigenesis, with special emphasis on those related to cancer hallmarks and epithelial-mesenchymal transition, using as a model the murine transplantable hepatocarcinoma AS-30D. Using qPCR to quantify the mRNA levels of genes involved in tumorigenesis, we found elevated levels for Tgfb1 and Spp1, two master regulators of EMT. A mesenchymal signature profile for AS-30D cells is also supported by the overexpression of genes encoding for molecules known to be associated to aggressiveness and metastatic phenotypes such as Foxm1, C-met, and Inppl1. This study supports the use of the AS-30D cells as an efficient and cost-effective model to study gene expression changes in HCC, especially those associated with the EMT process.

Keywords: AS-30D cells; AS30D model; Spp1; cancer markers; epithelial-mesenchymal transition; hepatocellular carcinoma; osteopontin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AZH declared a shared affiliation, with several of the authors, BDC, RGV, MSG, MMS, JMO, to the handling editor at the time of the review.

Figures

Figure 1
Figure 1
Comparative expression of SPP1 in human HCC samples compared with normal liver. SPP1 expression is 7.5-fold higher in HCC than in normal liver. Box plot obtained from Oncomine database (30).
Figure 2
Figure 2
Expression of genes involved in angiogenesis in rat hepatocytes and AS-30D cells. Rat hepatocytes were freshly isolated, and AS-30D samples were obtained from cells from the ascitic fluid of experimental rats. Data represented as mean ± SD. ***p ≤ 0.001; and ****p ≤ 0.0001.
Figure 3
Figure 3
Expression of several genes related to metabolic processes or regulation of cell cycle in rat hepatocytes and AS-30D cells. Rat hepatocytes were freshly isolated, and AS-30D samples were obtained from cells isolated the ascitic fluid of experimental rats. Data represented as mean ± SD. *p-values ≤0.05; **p ≤0.01; ***p ≤ 0.001.
Figure 4
Figure 4
Expression of genes encoding EMT markers in rat hepatocytes and AS-30D cells. Rat hepatocytes were freshly isolated, and AS-30D samples were obtained from cells from the ascitic fluid of experimental rats. Data represented as mean ± SD. *p-values ≤0.05; **p ≤0.01; ***p ≤ 0.001; and ****p ≤ 0.0001.
Figure 5
Figure 5
Gene expression of canonical receptors recognizing OPN in rat hepatocytes and AS-30D cells. Rat hepatocytes were freshly isolated, and AS-30D samples were obtained from cells from the ascitic fluid of experimental rats. Data represented as mean ± SD.
Figure 6
Figure 6
Expression of genes encoding for the plasma membrane Ca2+-ATPases (Atp2b1-Atp2b4) in rat hepatocytes and AS-30D cells. Rat hepatocytes were freshly isolated and AS-30D samples were obtained from cells from the ascitic fluid of experimental rats. Data represented as mean ± SD. *p-values ≤0.05; **p ≤0.01.
Figure 7
Figure 7
AS-30D cells express high protein levels of PMCA and OPN and increase the expression of mesenchymal markers after TGF-β stimulation. (A) Protein levels for PMCA and OPN in hepatocytes and AS-30D cells were detected by Western blot. One hundred micrograms of protein (first lane) and 200 μg (second lane) were run for each cell type. (B) AS-30D cells were treated with 2 ng/ml of TGF-β for 24 and 48 h OPN, vimentin, and phospho-Smad2 protein levels were detected by Western blot. One hundred micrograms of protein were run in each lane. β-Actin was used as a loading control.
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