Panoply™ Human CCR9 Knockdown Stable Cell Line

Here, researchers utilized the GEO database (GSE87630) and clinical samples to demonstrate that C-C motif chemokine receptor 9 (CCR9) is highly expressed in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and predicts poor overall survival. Its overexpression was associated with HBsAg-positive HCC. Both univariate and multivariate Cox regression analyses demonstrated that CCR9 was an independent risk factor for poor overall survival in HCC patients. In vitro results further demonstrated that HBV structural proteins, small HBV surface antigens (SHBs), can trigger CCR9 upregulation. Functional analyses revealed that SHBs enhanced HCC cell proliferation, migration, and invasion, increased ABCB1 and ABCC1 expression, and promoted regorafenib resistance through CCR9. Interestingly, both HBV overexpression plasmid and AAV-HBV mouse models exhibited significantly elevated global N6-methyladenosine (m6A) levels. Further studies showed that SHBs elevated these m6A levels, upregulated CCR9 and stabilized CCR9 mRNA through KIAA1429-mediated m6A modification, with sites 1373 and 1496 on CCR9 mRNA being critical for modification. In summary, SHBs promoted HCC progression and regorafenib resistance via KIAA1429-mediated m6A modification of CCR9.

The researchers used the CCK-8 assay to determine the IC50 level of regorafenib treatment in Huh7 cells after overexpressing CCR9. The results showed that overexpression of CCR9 significantly increased the IC50 and enhanced the viability of Huh7 cells (Figure 1A). Conversely, CCR9 knockdown in HCCLM3 cells significantly reduced their resistance to regorafenib (Figure 1B). Flow cytometry and a nude mouse xenograft model were used to further assess whether CCR9 induces regorafenib resistance in HCC cells. Flow cytometry results showed that CCR9 overexpression effectively counteracted regorafenib-induced apoptosis (Figure 1C). In the xenograft model, CCR9 knockdown in regorafenib-treated HCCLM3 cells resulted in reduced xenograft tumor weight and size compared with the control group (Figures 1D, E). This further confirms that CCR9 knockdown significantly reduces regorafenib resistance in HCC cells.

Figure 1. The role of CCR9 in the resistance of HCC to regorafenib. (Lv Z, et al., 2024)