Clinical significance and biological function of fucosyltransferase 2 in lung adenocarcinoma

Abstract

Fucosylation, which is catalyzed by fucosyltransferases (FUTs), is one of the most important glycosylation events involved in cancer. Studies have shown that fucosyltransferase 8 (FUT8) is overexpressed in NSCLC and promotes lung cancer progression. However, there are no reports about the pathological role of fucosyltransferase 2 (FUT2) in lung cancer. To identify FUT2 associated with lung cancer, the expression and clinical significance of FUT2 in lung cancer was investigated by Real-Time PCR, Immunohistochemistry and Western Blot. In addition, we investigated the effect of knockdown FUT2 in lung adenocarcinoma cells. The results showed that the expression of FUT2 in lung adenocarcinoma is higher than that in adjacent noncancerous tissues. Knocking down FUT2 in A549 and H1299 cells decreased cell proliferation, migration and invasion, and increased cell apoptosis compared to corresponding control cells. Furthermore, Western Blot showed that knockdown FUT2 can impact the expression of migration-associated and apoptosis-associated proteins in A549 cells. Our results suggest that FUT2 may be associated with lung adenocarcinoma development and thus is a potential biomarker or/and therapeutic target in lung adenocarcinoma.

Keywords: FUT2; fucosyltransferases; lung adenocarcinaoma; metastasis; proliferation.

Figure 1. The mRNA expression levels of FUTs in lung cancer

(A) (C) (E) (G) The FUT2/FUT4/FUT7/FUT8 mRNA expression level in lung cancer and adjacent noncancerous tissue. (B) (D) (F) (H) The FUT2/FUT4/FUT7/FUT8 mRNA expression level in lung adenocarcinoma and adjacent noncancerous tissue. N: adjacent noncancerous tissue; C: cancer tissue; n: number of cases. ^*P < 0.05, ^**P < 0.01, significant difference between groups as indicated.

Figure 2. The protein expression levels of FUT2 and FUT8 in lung cancer

(A) Western Blot analysis of FUT2 and FUT8 levels in tumor tissues. (B) (D) The protein expression levels of FUT2/FUT8 in lung cancer. (C) (E) The protein expression levels of FUT2/FUT8 in lung adenocarcinoma. Tubulin was acted as a loading control. N: adjacent noncancerous tissues; C: cancer tissue. ^*P < 0.05, ^**P < 0.01, significant difference between groups as indicated.

Figure 3. The expression levels of FUT2 protein in lung adenocarcinoma

(A) Tissue histology revealed tumor’s morphological characteristics: ×200. (B) Representative images of FUT2 staining using IHC assay in normal adjacent tissue and 74 cases of archived lung adenocarcinoma specimens with different clinical stages: ×200. (C) (D) Quantitative analysis of the average MOD of FUT2 staining in normal adjacent tissue and lung adenocarcinoma specimens. (E) The statistics of protein expression of FUT2 in cancer and normal tissue. N: Normal adjacent tissue; C: Cancer tissue; n: number of cases; CI: Cancer stage I, CII: Cancer stage II, CIII: Cancer stage III. ^****P < 0.0001, significant difference between groups as indicated.

Figure 4. FUT2 knockdown inhibits migration and invasion of lung adenocarcinoma cells

(A) FUT2 and GAPDH amplification curve and melting curve, and the relative FUT2 mRNA level. (B) The FUT2 protein expression in the A549 cells by Western Blot analysis, GAPDH was acted as a loading control, the intensity was evaluated using Image J computer software. (C) The expression of FUT2 protein in the H1299 cells by Western Blot analysis, GAPDH was acted as a loading control, the intensity was evaluated using Image J computer software. (D) Cell migration was evaluated by transwell assay. (E) Cell invasion ability was evaluated by matrigel transwell assay. A549: untreated group, NC: transfected scramble vector into lung adenocarcinoma cells, RNAi-FUT2: transfected sh-FUT2 vector into lung adenocarcinoma cells, H1299: untreated group, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Data are presented as means ± SEM, significant difference between groups as indicated.

Figure 5. Effect of down-regulation of FUT2 on cell proliferation and apoptosis of lung adenocarcinoma cells

(A) The colony formation abilities of A549 and H1299 cells. (B) The flow cytometry analysis cell DNA content distribution of A549 cells. (C) The flow cytometry analysis cell apoptosis of A549 cells. (D) The CCK8 assay analysis cell proliferation of A549 cells and H1299. A549: untreated A549 cells, NC: transfected scramble vector into adenocarcinoma cells, RNAi-FUT2: transfected sh-FUT2 vector into adenocarcinoma cells, H1299: untreated H1299 cells, ^*P < 0.05, ^**P < 0.01. Data are presented as means ± SEM, significant difference between groups as indicated.

Figure 6. Effects of down-regulation of FUT2 on the expression of migration-associated and apoptosis-associated proteins in A549 cells

(A-B) Western Blot analysis of migration-associated protein expression, Tubulin was acted as a loading control. (C-D) Western Blot analysis of apoptosis-associated protein expression, GAPDH was acted as a loading control. The intensity was evaluated using Image J computer software. A549: untreated A549 cells, NC: transfected scramble vector into A549 cells, RNAi-FUT2: transfected sh-FUT2 vector into A549 cells, ^*P < 0.05, ^**P < 0.01. Data are presented as means ± SEM, significant difference between groups as indicated.

Figure 7. Effect of down-regulation of FUT2 on tumor growth in vivo

(A) Tumor growth curve. (B) Photographs of tumors dissected from mice injected with A549 cells. (C) Average body weight of mice. (D) The volume of tumor. (E) The tumor weight. (F) Immunohistochemical examination of FUT2 in tumor sections from nude mice. A549: untreated group, NC: transfected scramble vector into A549, RNAi-FUT2: transfected sh-FUT2 vector into A549, ^*P < 0.05, ^**P < 0.01. Data are presented as means ± SEM, significant difference between groups as indicated.