HHLA2 promotes tumor progression by long non‑coding RNA H19 in human gallbladder cancer

Abstract

Advanced gallbladder cancer (GBC) is one of the most malignant of all types of biliary tract cancers that is associated with poor prognosis and high mortality. Accumulating evidence suggest that the B7 family of proteins serve an essential role in various types of cancers, including GBC. However, the potential function and regulatory mechanism of human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2; also known as B7-H7 or B7H5) in GBC remain poorly understood. In the present study, immunohistochemistry was used to examine the expression pattern of HHLA2 in samples from 89 patients with GBC. The possible association between HHLA2 expression and the clinicopathological parameters, including prognosis, were then assessed. Using lentiviruses, overexpression of HHLA2 plasmid or short-hairpin RNA (shRNA) of HHLA2 were transfected into GBC-SD cells to overexpress or knock down HHLA2 expression, respectively. The effects of HHLA2 overexpression and knockdown on the epithelial-mesenchymal transition (EMT) process on GBC-SD cells were measured by the western blotting and immunofluorescence staining of collagen I, N-cadherin, E-cadherin, vimentin and α-SMA. By contrast, changes in cell proliferation were measured using EdU assay. Cell invasion and migration were assessed using Transwell and wound-healing assays, respectively. In addition, a xenograft mouse model was established to evaluate the tumorigenic ability of the GBC cell line in vivo after stable transfection with lentivirus for HHLA2 overexpression or shRNA for HHLA2 knockdown. The regulatory relationships among TGF-β1, long non-coding RNA (lncRNA) H19 (H19) and HHLA2 were then investigated. The mRNA expression of lncRNA H19 were assessed using reverse transcription-quantitative PCR, whereas the expression levels of HHLA2 were detected by western blotting and immunofluorescence staining. HHLA2 expression was found to gradually increase as the stages of the GBC samples become more advanced. In addition, the expression level of HHLA2 was calculated to be positively associated with the Nevin stage, American Joint Committee on Cancer stage, tumor invasion and regional lymph node metastasis but was negatively associated with the overall patient survival (OS). In vitro experiments demonstrated that overexpression of HHLA2 promoted GBC migration, invasion, proliferation and EMT, whereas in vivo experiments found a promoting role of HHLA2 overexpression on GBC tumor growth. After transfection with lentiviruses encoding the overexpression plasmid of lncRNA H19, GBC migration, invasion, proliferation and EMT were increased. By contrast, knocking down HHLA2 expression suppressed TGF-β1- or lncRNA H19 overexpression-induced GBC migration, invasion, proliferation and EMT. In addition, HHLA2 knockdown significantly reduced the sizes of the GBC tumors in vivo . These results suggest that HHLA2 overexpression can promote GBC progression. Conversely, ablation...

Overexpression and Role of HHLA2, a Novel Immune Checkpoint, in Colorectal Cancer

“…Expression of EMT markers, such as E-cadherin, N-cadherin, and Vimentin, were significantly changed after the knockdown of HHLA2 expression in human ccRCC [ 27 ]. Zhang et al found out that HHLA2 via an impact on EMT in advanced gallbladder cancer can promote tumor progression [ 28 ]. They presented the hypothesis that HHLA2 may be involved in the Wnt/βcatenin axis.…”

The Importance of HHLA2 in Solid Tumors—A Review of the Literature

“…Studies show that HHLA2 could play a significant role in gallbladder cancer (GBC) as well. Zhang et al found that among 89 examined patients, all cases exhibited HHLA2 staining [36]. In another study, high HHLA2 expression was observed in 53.68% of samples in the training group and 60.19% of cases in the testing group.…”

“…Notably, according to other authors, higher Nevin and TNM stages were associated with the high expression of HHLA2, B7-H3, and B7-H4 in both the training and testing groups. Elevated HHLA2 expression was also correlated with a lower density of CD8+ TILs in the training group (p = 0.001), but there was no relationship between HHLA2 expression and Foxp3 + TILs [36,37].…”

“…B7-H3 and HHLA2 were expressed together in 43.16% and 48.54% of samples, while B7-H4 was co-expressed with HHLA2 in 37.89% and 43.69% of cases in the training and the testing groups, respectively. These co-expression patterns could influence the effects of HHLA2 targeting in immunotherapies, therefore it is important to be mindful of this data [36,37]. Moreover, HHLA2 expression in GBC was more prominent than PD-L1 expression, suggesting that it could be a better therapeutic target in gallbladder cancer than PD-L1 [79].…”

Immune checkpoints and cancer immunotherapies: insights into newly potential receptors and ligands

“…[173][174][175][176] In in vivo experiments of human gallbladder cancer, the HHLA2 overexpression promoted tumor progression while its knockdown reduced the sizes of the GBC tumors. 177 Conversely, the HHLA2 ablation inhibited both TGF-β1-and long noncoding RNA H19-induced GBC progression in vitro. However, in a recent study on human ccRCC that evaluated the HHLA2 prognostic value, a positive correlation between increased HHLA2 and survival rates is reported.…”