Junctional adhesion molecule-like protein promotes tumor progression via the Wnt/β-catenin signaling pathway in lung adenocarcinoma

Abstract

Background: Lung adenocarcinoma (LUAD) is a heavy social burden worldwide. Because the mechanisms involved in LUAD remain unclear, the prognosis of LUAD remains poor. Consequently, it is urgent to investigate the potential mechanisms of LUAD. Junctional adhesion molecule-like protein (JAML), is recognized as a tumorigenesis molecule in gastric cancer. However, the role of JAML in LUAD is still unclear. Here we aimed to evaluate the role of JAML in LUAD.

Methods: qRT-PCR, Western blotting and immunohistochemistry were conducted to investigate the expression of JAML in LUAD tissues. JAML was knocked down and overexpressed in LUAD cells using transient transfection by siRNA and plasmids or stable transfection by lentivirus. Proliferation potential of LUAD cells were detected by Cell Counting Kit-8, EdU incorporation and Colony formation assay. Migration and invasion abilities of LUAD cells were determined by wound healing, transwell migration and invasion assays. Cell cycle and cell apoptosis were detected by flow cytometry. The effects of JAML in vivo were studied in xenograft tumor models. Western blotting was used to explore the molecular mechanisms of JAML function. In addition, rescue experiments were performed to verify the possible mechanisms.

Results: JAML expression was elevated in LUAD tissues compared with peritumor tissues, and this upregulation was positively related to pT and pTNM. Furthermore, both in vitro and in vivo, JAML silencing markedly repressed malignant behaviors of LUAD cells and vice versa. Knockdown of JAML also mediated cell cycle arrest at G₀/G₁ phase and promoted apoptosis in LUAD cells. Mechanistically, silencing JAML repressed the process of epithelial-mesenchymal transition by inactivating the Wnt/β-catenin pathway in LUAD cells. Effects of JAML can be rescued by Wnt/β-catenin pathway activator in A549 cells.

Conclusions: Our data reveal the oncogenic role of JAML in LUAD, indicating that JAML may be a predictive biomarker and novel therapeutic target for LUAD.

Keywords: Invasion; Junctional adhesion molecule-like protein; Lung adenocarcinoma; Migration; Tumor progression.

Fig. 1

JAML expression is elevated in human LUAD tissues. a, b The expression of JAML was elevated in 15 pairs of LUAD tissues detected by qRT-PCR (a) and Western blotting (b). c Representative IHC images of JAML expression in 30 pairs of LUAD tissues. d Mean of IOD for JAML in 30 pairs of LUAD tissues. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

JAML overexpression promotes LUAD cell proliferation. a–d The efficiency of JAML knockdown in A549 (a) and H1299 cells (b) or overexpression in PC9 (c) and H1975 cells (d) were confirmed by Western blotting. The internal reference gene was β-actin. e–h Cells growth of A549 (e), H1299 (f), PC9 (g) and H1975 cells (h) with JAML knockdown or overexpression was analyzed with CCK8 assay. *P < 0.5, **P < 0.01, ***P < 0.001, compared with SiNC or Vector group. i–l Cell proliferation in A549 (i) and H1299 cells (j) after JAML knockdown and in PC9 (k) and H1975 cells (l) after JAML overexpression was observed through EdU assay, quantitative analysis n = 3, unpaired t test, **P < 0.01, ***P < 0.001, compared with SiNC or Vector group. m, n The colony formation showed that cell growth potential was inhibited in A549 cells (m) following JAML knockdown, and that cell growth potential was increased in PC9 cells (n) following JAML overexpression (top panels). The number of foci was shown in the bottom panels. Quantitative analysis n = 3, unpaired t test, *P < 0.05, compared with the ShNC group, **P < 0.01, compared with the vector group

Fig. 3

JAML overexpression promotes migration and invasion in LUAD cells. a–d The wound healing assay indicated that JAML knockdown suppressed the migratory capabilities of A549 (a) and H1299 cells (b), and that JAML overexpression promoted the migratory capabilities of PC9 (c) and H1975 cells (d). Representative images of wound healing assays were obtained (left panel). ImageJ software was used to measure the migration rate (right panel). n = 3, unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the SiNC or vector groups. e–h The transwell migration assay confirmed that JAML knockdown suppressed the migratory capabilities of A549 e and H1299 cells (f), and that JAML overexpression promoted the migratory capabilities of PC9 (g) and H1975 cells h. Representative images of transwell migration assays were obtained (left panel). ImageJ software was used to count the number of migrated cells (right panel). n = 3, unpaired t test, ***P < 0.01, compared with the SiNC or vector groups. i–l Transwell invasion assays indicated that the invasion ability was downregulated in A549 (i) and H1299 (j) cells when JAML was knocked down, and that the invasion ability was upregulated in PC9 (k) and H1975 cells (l). Representative images of transwell invasion were obtained (left panel). ImageJ software was used to count the number of invaded cells from 3 randomly selected fields (right panel). n = 3, unpaired t test, ***P < 0.001, compared with the SiNC or vector groups

Fig. 4

JAML knockdown mediates cell cycle arrest and promotes cell apoptosis in LUAD cells. a–e The percentages of cells in the different cycle phases were showed after JAML knockdown in A549 (a) and H1299 cells (b) and JAML overexpression in PC9 (c) and H1975 cells (d). Superimposed histograms of different cycle phases are shown in (e). f–j Cell apoptosis was analyzed by staining with Annexin V-FITC and PI after JAML knockdown in A549 (f) and H1299 cells (g) and JAML overexpression in PC9 (h) and H1975 cells (i). Histograms of the results from different apoptotic cells is displayed on (j)

Fig. 5

JAML overexpression promotes LUAD cell proliferation in vivo. a, b Knockdown or overexpression efficiency of JAML was confirmed in A549 (a) or PC9 cells (b) after stable transfection of lentivirus by Western blotting. The internal reference gene was β-actin. c, d Representative images of tumors formed by A549 (c) or PC9 cells (d) 5 weeks after subcutaneous injection, quantitative analysis n = 6, unpaired t test, *P < 0.05, ***P < 0.001, compared with the ShNC or vector groups. e, f Representative images of IHC staining for JAML in xenograft tumors caused by A549 (e) or PC9 cells (f). g, h Representative images of IHC staining for Ki67 in xenograft tumors caused by A549 (g) and PC9 cells (h) 

Fig. 6

Effects of JAML on MMPs, Bax/Bcl2, and EMT in LUAD cells. a, b Expression of MMP2, MMP9, Bax, Bcl2 and EMT markers when JAML was knockdown in LUAD cells (A549, H1299). c, d Expression of MMP2, MMP9, Bax, Bcl2 and EMT markers when JAML was overexpressed in LUAD cells (PC9, H1975). The internal reference gene was β-actin

Fig.7

JAML facilitates EMT partially by activating the Wnt/β-catenin signaling pathway in LUAD cells. a Changes of key molecules in Wnt/β-catenin pathway when JAML was knockdown in LUAD cells (A549, H1299). b Changes of key molecules in Wnt/β-catenin pathway when JAML was overexpressed in LUAD cells (PC9, H1975). The internal reference gene was β-actin

Fig. 8

Effects of JAML can be rescued by Wnt/β-catenin activator in A549 cells. A549 cells with JAML knockdown was treated with Wnt/β-catenin activator (CHIR99021, 5 μM/ml) for 24 h. a The protein levels of MMP2, MMP9, EMT markers and key molecules of Wnt/β-catenin pathway (β-catenin, cyclin D1, survivin) were detected by Western blotting. The internal reference gene was β-actin. b, c EdU assay showed that repression of cell proliferation caused by JAML knockdown in A549 cells can be rescued by CHIR99021, quantitative analysis n = 3, unpaired t test, *P < 0.05, compared with CHIR99021(-) group. d, e Transwell assays showed that inhibition of migration (d) and invasion (e) abilities caused by JAML knockdown in A549 cells can be rescued by CHIR99021. Representative images of transwell were obtained (top panel). ImageJ software was used to count the number of migrated and invaded cells from 3 randomly selected fields (bottom panel), n = 3, unpaired t test, **P < 0.01, ***P < 0.001, compared with the CHIR99021(-) group