However, the biological functions of ITGA9 in HCC and the underlying molecular mechanisms have not been studied yet, therefore placing the restrictions on developing novel anticancer-targeted therapies.
To quantify the level of ITGA9 protein expression, each tumor was assigned with a score according to the intensity of cell membrane staining and the proportion of stained tumor cells (score 0 = 0-5%, score 1 = 6-30%, score 2 = 31-70%, and score 3 = 71-100%).
The human ITGA9 ORF (NM_002207.2) was subcloned into the pEZlv105 vector (GeneCopoeia, China) to generate pEZ-lv105ITGA9 plasmid.
For ITGA9 mRNA level in HCC cell lines, [DELTA]Ct value of THLE-2 cell line was used as the reference.
The primary antibodies against ITGA9 (ab140599; Abcam), FAK (ab81293; Abcam), p-FAK Tyr397 (ab81298; Abcam), Src (2108; Cell Signaling Technology), p-Src Tyr527 (2105; Cell Signaling Technology), and a-tubulin (T6199; Sigma-Aldrich) were incubated for overnight at 4[degrees]C, followed by incubating with species-specific antibodies (926-32213; 926-68051; LI-COR, Lincoln, NE) for 1 h.
ITGA9 Is Significantly Downregulated in HCC and Correlates with Vascular Invasion and Prognosis.
Furthermore, ITGA9 protein level associated well with alpha-fetoprotein, vascular invasion, tumor thrombosis, tumor size, and TNM stage (Table 1).
ITGA9 Affects HCC Cell Growth Both In Vitro and In Vivo.
Interestingly, novel Wnt/[beta]-catenin targets genes ranked between known targets and included the serine/threonine kinase 32A Stk32a (ID: 364858; nominal p value = 1.3 x 10-3; FC = 1.8), the integrin subunit alpha 9 Itga9 (ID: 586004; nominal p value = 1.7 x 10-3; FC = 2.0), and the transmembrane protein 72 TMEM72 (ID: 362424; nominal p value = 2.0 x 10-3; FC = 1.7), among others (Table 1).
First, Itga9 (integrin subunit alpha 9) is among the 10 top upregulated genes reported in this study.
Fibrinogen gamma chain (Fgg), fibrinogen beta chain (Fgb), fibrinogen alpha chain isoform 2 precursor (Fga), integrin alpha 9 (Itga9
), protein-glutamine gamma-glutamyl transferase 2 (Tgm2), T-kininogen 2 (Kng2), and inter-alpha-inhibitor H4 heavy chain precursor (Itih4) were implicated in coagulation and hemostasis.