Correlation of LAGE3 with unfavorable prognosis and promoting tumor development in HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common clinical malignancies quite susceptible to recurrence and metastasis. Despite several improvements in therapeutic approaches, the prognosis remains poor due to the limited treatment options. A bioinformatics analysis based on TCGA databases revealed that the recombinant human L antigen family member 3 (LAGE3) might function as an effective prognostic and diagnostic biomarker for HCC, as LAGE3, a protein-coding gene, maintains several important biological functions and has a physiological significance in the CTAG family while simultaneously being involved in regulating the occurrence and invasion of numerous types of tumors. However, the LAGE3 gene's functional and regulatory mechanism in the progression of HCC remains unclear.

Methods: The LAGE3 level was investigated in 79 HCC tissues cases, ten HCC adjacent tissue cases, and six cases of normal liver tissues by IHC, while the LAGE3 level was evaluated in BEL-7404, SMCC-7721, Huh-7, HepG2, and MIHA cell lines by qRT-PCR and Western blot tests. Although the proliferation, migration, invasion, and apoptotic abilities of HCC cells were measured in vitro after silencing assay to probe the role of LAGE3 in HCC cells, the tumor xenograft growth experiment was used to verify the in vivo effect of LAGE3 gene knockdown on the growth of HCC tumors combined with bioinformatics analysis to study the LAGE3 mechanisms regulating HCC proliferation.

Results: Our results implied that LAGE3 was extensively expressed in HCC cell lines like BEL-7404, SMCC-7721, and Huh-7 cells as well as HCC tissues, but a lower expression was observed in HepG2 cells. Additionally, LAGE3 restrains cellular proliferation, promotes apoptotic pathways in HCC cells, and inhibits the growth of HCC tumors in vivo. Lastly, it was stated that LAGE3 might promote tumor development in HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways.

Conclusion: This study shows that the development of specific LAGE3 target drugs might become new effective treatment modalities for HCC patients.

Keywords: Apoptosis; HCC; Invasion; LAGE3; Migration; Proliferation.

Fig. 1

Overexpression of LAGE3 in HCC tissues and cell lines. A Usage of GEPIA2 tool to perform OS and DFS analyses of LIHC tumors in TCGA by LAGE3 gene expression for obtaining Kaplan-Meier curves. B The LAGE3 expression analyses in LIHC tumors as included in TCGA were performed by the GEPIA2 tool and the box plot data were supplied. * p < 0.05. C High expression of LAGE3 mRNA level in HCC cell lines while the data were expressed as the mean ± s. d., *** p < 0.001. D IHC staining images of LAGE3 expressions in HCC tumor tissue chips. OS: Overall survival; DFS: Disease-free survival; LIHC: liver hepatocellular carcinoma; IHC: Immunohistochemistry

Fig. 2

Silencing LAGE3 by infection of lentivirus-mediated siRNA to explore the effect of LAGE3 suppression on HCC progression. A and C The knockdown efficiency of LAGE3 by infection of lentivirus-mediated siRNA and the negative control siRNA in SMCC-7721 cells was verified by qRT-PCR and Western blot. Data were expressed as the mean ± s.d., *** P < 0.001. B and D The knockdown efficiency of LAGE3 by infection of lentivirus-mediated siRNA and the negative control siRNA in Huh-7 cells was verified by qRT-PCR and Western blot. Data were expressed as the mean ± s.d., *** P < 0.001, n.s: no significant difference

Fig. 3

LAGE3 knockdown suppressed HCC cell proliferation. A The proliferation of SMCC-7721 and Huh-7 cells was significantly inhibited by LAGE3 knockdown in the CCK8 assay, and the data were expressed as the mean ± s. d., ** p < 0.01. B The proliferation of SMCC-7721 and Huh-7 cells was significantly inhibited by LAGE3 knockdown in BrdU assay, and the data were expressed as the mean ± s. d., *** p < 0.001. C The cycle of SMCC-7721 cells was significantly arrested in the S phase by LAGE3 knockdown while the data were expressed as the mean ± s. d., ** p < 0.01, *** p < 0.001. D The cycle of Huh-7 cells was significantly arrested in the G1 phase by LAGE3 knockdown while the data were expressed as the mean ± s. d., *** p < 0.001

Fig. 4

LAGE3 knockdown suppressed HCC cell migration and invasion. A and B Migration of Huh-7 and SMCC-7721 cells was significantly inhibited by LAGE3 knockdown in scratch healing assay expressing the data as the mean ± s. d., * p < 0.05 and *** p < 0.001. C and D Migration of Huh-7 and SMCC-7721 cells were significantly inhibited by LAGE3 knockdown in Transwell assay expressing the data as the mean ± s. d., * p < 0.05 and ** p < 0.01. E and F while the invasion of Huh-7 and SMCC-7721 cells were significantly inhibited by LAGE3 knockdown in Transwell assay expressing the data as the mean ± s. d., ** p < 0.01 and *** p < 0.001

Fig. 5

LAGE3 knockdown promoted HCC cells apoptosis. The proportion of apoptotic cells was significantly increased in the LAGE3 knockdown group compared with control cells in Huh7 (A) and SMCC-7721 (B) cell lines as assessed by flow cytometry thus expressing the data as the mean ± s. d., *** p < 0.001

Fig. 6

LAGE3 knockdown suppressed tumor growth in vivo. A and B The tumor volume was significantly decreased in the LAGE3 knockdown group while expressing the data as the mean ± s. d., *** p < 0.001. C The tumor weight was significantly reduced in the LAGE3 knockdown group while expressing the data as the mean ± s. d., *** p < 0.001

Fig. 7

LAGE3 promotes the progression of HCC via PI3K/AKT/mTOR and Ras/RAF/MAPK pathways. A The mRNA levels of PI3CA, BRAF, AKT, MAP2K1, ERK, and KRAS in the siLAGE3 group were detected by qRT-PCR expressing the data as the mean ± s. d., n. s: no significant difference, *** p < 0.001. B The protein levels of PI3K, RAF, MUC1, p-Akt, p-MEK1, p-Erk, and Ras were significantly down-regulated in the siLAGE3 group while data were expressed as the mean ± s. d., ** p < 0.01 and *** p < 0.001. The overexpression efficiency of LAGE3 by LAGE3 gene lentivirus particles infection and the empty plasmid lentivirus particles in Huh7 cells was verified by Western blot (C) and qRT-PCR (D), where the data were expressed as the mean ± s. d., *** p < 0.001. The proliferation of Huh-7 cells was significantly enhanced in the overexpression of BRAF in (E) MTT assay and Brdu assay (F), while data were expressed as the mean ± s. d., * p < 0.05. G The Huh-7 cell cycle was significantly arrested in the S and G2/M phase in the BRAF overexpression, where the data were expressed as the mean ± s. d., * p < 0.05. H The proportion of apoptotic cells was significantly reduced in the BRAF overexpression group as assessed by flow cytometry and expressing the data as the mean ± s. d., * p < 0.05