Actin-like protein 8 promotes cell proliferation, colony-formation, proangiogenesis, migration and invasion in lung adenocarcinoma cells

Abstract

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated mortality worldwide of which lung adenocarcinoma (LUAD) is the most common. The identification of oncogenes and effective drug targets is the key to individualized LUAD treatment. Actin-like protein 8 (ACTL8), a member of the cancer/testis antigen family, is associated with tumor growth and patient prognosis in various types of cancer. However, whether ACTL8 is involved in the development of LUAD remains unknown. The aim of the present study was to demonstrate the role of ACTL8 in human LUAD cells.

Methods: The expression of ACTL8 in LUAD tissues and cell lines was assessed using immunohistochemistry and western blotting. Additionally, plasmids expressing ACTL8-specific short hairpin RNAs were used to generate lentiviruses which were subsequently used to infect A549 and NCI-H1975 human LUAD cells. Cell proliferation, migration, invasion and apoptosis, as well as cell cycle progression and the expression of protein markers of epithelial to mesenchymal transition were investigated. A549 cell tumor growth in nude mice was also examined.

Results: The results showed that ACTL8 was highly expressed in A549 and NCI-H1975 LUAD cell lines. Additionally, ACTL8-knockdown inhibited proliferation, colony formation, cell cycle progression, migration and invasion, and increased apoptosis in both cell lines. Furthermore, in vivo experiments in nude mice revealed that ACTL8-knockdown inhibited A549 cell tumor growth.

Conclusion: These results suggest that ACTL8 serves an oncogenic role in human LUAD cells, and that ACTL8 may represent a potential therapeutic target for LUAD.

Key points: Our results suggest that ACTL8 serves an oncogenic role in human LUAD cells, and that ACTL8 may represent a potential therapeutic target for LUAD.

Keywords: ACTL8; angiogenesis; cell proliferation; lung adenocarcinoma; migration.

Figure 1

High expression levels of ACTL8 in LUAD tissues and lung cancer cells. (a) LUAD (n = 45), PC (n = 45), and normal (n = 10) tissues were prepared for IHC. (b) IHC analysis was scored according to the above mentioned methods. The data are presented as the mean ± standard deviation; ***P < 0.001. (c and d) Protein expression levels of ACTL8 were determined using western blotting in HCC827, A549, H1299, NCI‐H1975, 95‐D and PC‐9 lung cancer cell lines. 10HBE and Beas‐2B normal human bronchial epithelial cells were used as the controls. ACTL8, Actin‐like protein 8; LUAD, lung adenocarcinoma; PC, paracancerous; IHC, immunohistochemistry.

Figure 2

shACTL8 lentivirus decreases the mRNA and protein expression levels of ACTL8. Expression of the ACTL8 gene at the mRNA and protein level in A549 (a) and NCI‐H1975 (b) cells. **P < 0.01. ACTL8, Actin‐like protein 8; sh, short‐hairpin. () shCtrl and () shACTL8.

Figure 3

ACTL8 promotes human LUAD cell proliferation. (a) A549 and NCI‐H1975 cell counts using the Celigo cell counting system. (b) Cells were infected with lenti‐shACTL8 and lenti‐shCtrl for three days, and proliferation was assessed every day for five consecutive days. () shCtrl and () shACTL8. (c) Flow cytometric analysis of A549 and NCI‐H1975 cells (d) three days after infection; the number of cells in the G₀/G₁ phase was increased, the number in the S phase was decreased, and the number in the G₂/M phase was increased in the lenti‐shACTL8 group, compared with the control group. () shCtrl and () shACTL8. The data are presented as the mean ± standard deviation; n = 3. *P < 0.05 and **P < 0.01 versus lenti‐shCtrl. ACTL8, Actin‐like protein 8; LUAD, lung adenocarcinoma; sh, short‐hairpin; lenti, lentivirus; Ctrl, control.

Figure 4

ACTL8 regulates human LUAD cell apoptosis. (a) The percentage of apoptotic A549 and NCI‐H1975 cells was measured using flow cytometry, and the number of apoptotic cells was significantly increased in the lenti‐shACTL8 groups. The apoptotic rate was calculated as the percentage of FITC‐positive cells. The data are presented as the mean ± standard deviation; n = 3. **P < 0.01 versus lenti‐shCtrl. () shCtrl and () shACTL8. (b) Apoptotic A549 and NCI‐H1975 cells were assessed using a TUNEL assay (magnification, 200x). (c) The expression of apoptosis‐related proteins apoptosis regulator BAX and Caspase 3 was determined by western blotting, and GAPDH was used as the loading control. ACTL8, Actin‐like protein 8; LUAD, lung adenocarcinoma; sh, short‐hairpin; lenti, lentivirus; Ctrl, control.

Figure 5

ACTL8 regulates the colony‐forming ability of human LUAD cells. (a) Colony‐formation assays were performed using A549 cells following infection with lenti‐shACTL8 or lenti‐shCtrl. (b) The colony‐forming ability of NCI‐H1975 cells following transfection with lenti‐shACTL8 or lenti‐shCtrl. The data are presented as the mean ± standard deviation; n = 3. *P < 0.05 and ***P < 0.001 vs. lenti‐shCtrl. ACTL8, Actin‐like protein 8; LUAD, lung adenocarcinoma; sh, short‐hairpin; lenti, lentivirus; Ctrl, control. () shCtrl and () shACTL8.

Figure 6

ACTL8 regulates angiogenesis in human LUAD cells. (a) Infection with lenti‐shACTL8 for 24 hours abolished the A549 cell‐induced increase in tube formation. (b) Infection with lenti‐shACTL8 for 24 hours had no effect on the NCI‐H1975‐induced increase in tube formation. The area of the tube branches was calculated and quantified. The data are presented as the mean ± standard deviation; n = 3. *P < 0.05 and ***P < 0.001 versus lenti‐shCtrl. ACTL8, Actin‐like protein 8; LUAD, lung adenocarcinoma; sh, short‐hairpin; lenti, lentivirus; Ctrl, control. () shCtrl and () shACTL8.

Figure 7

ACTL8 regulates the migration and invasion of human LUAD cells. Wound‐closure experiments were conducted using A549 and NCI‐H1975 cells infected with lenti‐shACTL8 or lenti‐shCtrl. (a and b) Images were captured at 0 and 24 hours (left), and the relative cell migration was analyzed by comparison to the relative change in the wound‐space distance (right) of A549 and NCI‐H1975 cells. Invaded (c and e) A549 and (d and f) NCI‐H1975 cells were assessed using Transwell assays with Matrigel (c and d) or without Matrigel (e and f), stained with Giemsa, and relative cell invasion was determined. The data are presented as the mean ± standard deviation; n = 3. *P < 0.05 and **P < 0.01 versus lenti‐shCtrl. ACTL8, Actin‐like protein 8; LUAD, lung adenocarcinoma; sh, short‐hairpin; lenti, lentivirus; Ctrl, control. () shCtrl and () shACTL8.

Figure 8

ACTL8 regulates the epithelial to mesenchymal transition of human LUAD cells. A549 and NCI‐H1975 cells were infected with lenti‐shACTL8 or shCtrl for 72 hours, and the expression levels of N‐cadherin, Vimentin and β‐Catenin were determined using western blotting. ACTL8, Actin‐like protein 8; LUAD, lung adenocarcinoma; sh, short‐hairpin; lenti, lentivirus; Ctrl, control.

Figure 9

shRNA inhibition of ACTL8 reduces tumor growth in vivo. Six‐weeks‐old female BALB/c nu/nu mice were subcutaneously injected with lentivirus‐infected A549 cells. (a) Representative live images of the mice treated with A549 cells four weeks after inoculation. (b) Bioluminescence signals were analyzed and quantified using the Living Image 3.0 system four weeks post‐inoculation. (c) Tumor weight change after cell injection. The data are presented as the mean ± standard deviation; n = 10. *P < 0.05 and **P < 0.01 versus lenti‐shCtrl. () shCtrl and () shACTL8. (d) H&E staining of the excised tumors (magnification, x200). ACTL8, Actin‐like protein 8; sh, short‐hairpin; lenti, lentivirus; Ctrl, control.