PGRN exacerbates the progression of non-small cell lung cancer via PI3K/AKT/Bcl-2 antiapoptotic signaling

Abstract

Progranulin (PGRN) is a growth factor that is involved in the progression of multiple tumors. However, the effects and molecular mechanisms by which PGRN induces lung cancer remain unclear. The expression level of PGRN was analyzed by conducting immunohistochemistry of the histological sections of lung tissues from non-small-cell lung carcinoma (NSCLC) patients. The proliferation, apoptosis, migration, and invasion of NSCLC cells were assessed by the MTT assay, Western blot, degree of wound healing, and Transwell assays. A nude mouse xenograft model was used to validate the role of PGRN in vivo. The expression level of PGRN was higher in male patients with lung adenocarcinoma than in those with lung squamous cell carcinoma; by contrast, no difference was observed in female patients. The overexpression of PGRN promoted the proliferation and anti-apoptosis of H520 (derived from lung squamous cell carcinoma) cells, whereas knockdown of PGRN inhibited the proliferation and anti-apoptosis of A549 (derived from lung adenocarcinoma) cells. Copanlisib (targeting PI3K) inhibited the increase in the expression of cell anti-apoptosis marker Bcl-2 induced by rhPGRN protein; the PI3K agonist 740 Y-P partially reversed the decrease in Bcl-2 expression induced by PGRN deficiency in both A549 and H520 cells. PGRN increased the expression of Ki-67, PCNA, and Bcl-2 in vivo. PGRN inhibited cell apoptosis depending on the PI3K/Akt/Bcl-2 signaling axis; PGRN positivity correlated with lung adenocarcinoma. PGRN is a potential biomarker for the treatment and diagnosis of NSCLC, especially in lung adenocarcinoma.

Keywords: Ad, Adenovirus; Bcl-2; Cell apoptosis; DMSO, Dimethyl sulfoxide; FBS, Fetal Bovine Serum; IHC, immunohistochemistry; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; NSCLC; NSCLC, Non-small cell lung cancer; PBS, phosphate buffer saline; PGRN; PI3K/Akt.

Figure 1

The expression level of PGRN in lung cancer tissues and lung cancer cell lines. (A) The expression of PGRN in lung squamous cell cancer and lung adenocarcinoma were detected by IHC. (B) Evaluation of immunohistochemistry staining intensity. (C) Assessment of the percentage of positively stained cell. (D) Comprehensive immunohistochemistry staining score. (E) The protein level of PGRN in HBE, H520 and A549 was analyzed by western blot. All experiments were repeated three times independently. (F) Mapping of IHC SCORE and clinical stage in female NSCLC (Sankey diagram, P = 0.161). (G) Mapping of IHC SCORE and clinical stage in male NSCLC (Sankey diagram, P < 0.001).

Figure 2

The effects of PGRN on proliferation, apoptosis, migration and invasion in A549 and H520. (A) The expression of PGRN was detected by western blot in A549 cells after knocking down PGRN. (B) The protein level of PGRN was detected by western blot in H520 cells after transfecting PGRN overexpression plasmid. (C) Colony number of A549 was detected by colony formation test after inhibiting PGRN (upper), and the quantitative statistics of colony number was shown (lower, ∗∗∗P < 0.001 vs. Scramble group). (D) Colony number of H520 was detected by colony formation test after overexpressing PGRN (upper), and the quantitative statistics of colony number was shown (lower, ∗∗P < 0.005 vs. Negative control group). (E) The proliferation ability of A549 was detected by MTT after knocking down PGRN (∗∗P < 0.01 vs. Scramble group). (F) The proliferation ability of H520 was detected by MTT after overexpressing PGRN (∗∗P < 0.01 vs. Negative control group). (G) The migration of A549 was detected by wound healing assay after knocking down PGRN. (H) The statistical graph was shown (right, ∗P < 0.05 vs. scramble group). (I) The invasion and migration abilities of A549 were detected by transwell with or without matrix after knocking down PGRN. (J, K). The statistical graph was shown (∗P < 0.05,∗∗P < 0.01 vs. scramble group). (L) The migration of H520 was detected by wound healing assay after overexpressing PGRN. (M) The statistical graph was shown (∗∗P < 0.005 vs. Negative control group). (N) The invasion and migration abilities of H520 were detected by transwell with or without matrix after overexpressing PGRN. (O, P) The statistical graph was shown (∗P < 0.05, P = ns vs. Negative control group). All experiments were repeated three times independently.

Figure 3

The signaling pathway of MAPK/Erk and PI3K/Akt underlying the effect of PGRN on lung cancer cells. (A) The protein levels of p-Erk, Erk, p-Akt and Akt in A549 were analyzed by western blot. (B) The quantitative statistics of protein expression was shown (∗P < 0.05, ∗∗P < 0.005 vs. Scramble group). (C) The protein levels of CyclinD1, PCNA, Bcl-2, Bax, p-Erk, Erk, p-Akt and Akt in H520 after treating with LY294002 were analyzed by western blot. (D) The quantitative statistics of protein expression was shown (∗P < 0.05, ∗∗P < 0.005 vs. Each another group). (E) The protein levels of CyclinD1, PCNA, Bcl-2, Bax, p-Erk, Erk, p-Akt and Akt in H520 after treating with PD98059 were analyzed by western blot. (F) The quantitative statistics of protein expression was shown (∗P < 0.05, ∗∗P < 0.005 vs. Each another group). All experiments were repeated three times independently.

Figure 4

PGRN regulates cell apoptosis through AKT/Bcl-2 on A549 and H520 cell lines. (A) The protein levels of PGRN, p-Akt, Akt, CyclinD1, PCNA and Bcl-2 in A549 after treating with si-Grn and Copanlisib separately or together were analyzed by western blot. (B) The quantitative statistics of the ratio of p-Akt to Akt and the Bcl-2 expression was shown (∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.001). (C) The protein levels of PGRN, p-Akt, Akt, CyclinD1, PCNA and Bcl-2 in A549 after treating with si-Grn and 740 Y–P separately or together were analyzed by western blot. (D) The quantitative statistics of the ratio of p-Akt to Akt and the Bcl-2 expression was shown (∗P < 0.05, ∗∗P < 0.005). (E) The protein levels of PGRN, p-Akt, Akt, CyclinD1, PCNA and Bcl-2 in H520 after treating with rhPGRN and Copanlisib separately or together were analyzed by western blot. (F) The quantitative statistics of the ratio of p-Akt to Akt and the Bcl-2 expression was shown (∗P < 0.05, ∗∗P < 0.005, ∗∗∗P < 0.001). (G) The protein levels of PGRN, p-Akt, Akt, CyclinD1, PCNA and Bcl-2 in H520 after treating with rhPGRN and 740 Y–P separately or together were analyzed by western blot. (H) The quantitative statistics of the ratio of p-Akt to Akt and the Bcl-2 expression was shown (∗P < 0.05). All experiments were repeated three times independently.

Figure 5

The effect of PGRN on A549 and H520 cells in vivo. (A) The A549 tumors were took out from nude mice 2 weeks after tumor implantation (siPGRN group: n = 5; scramble group: n = 5). (B) The statistical graph of tumor weight was shown (∗P < 0.05 vs. scramble group). (C) The tumor growth curves were shown (∗∗P < 0.01 vs. scramble group). (D) The H520 tumors were took out from nude mice 2 weeks after tumor implantation (PGRN group: n = 5; Negative control group: n = 5). (E) The statistical graph of tumor weight was shown (∗∗P < 0.05 vs. Negative control group). (F) The tumor growth curves were shown (∗∗P < 0.01 vs. Negative control group). (G) The expression of PCNA, Bcl-2 and Ki67 were detected by immunohistochemistry.