COL6A6 inhibits the proliferation and metastasis of non-small cell lung cancer through the JAK signalling pathway

Abstract

Background: Collagen type VI alpha 6 chain (COL6A6) plays a vital role in maintaining cell structural integrity and regulating cell function. It has been proven to be a tumor suppressor gene and molecular therapeutic target. However, the mechanism of COL6A6 in non-small cell lung cancer (NSCLC) has not been elucidated. The purpose of this study was to investigate the relationship between COL6A6 and NSCLC.

Methods: We analyzed the expression of COL6A6 in NSCLC using public databases and verified the findings in NSCLC tissues and cells. The protein expression of COL6A6 was evaluated by Western blot. The CCK8 and Transwell assays were used to assess the invasion ability of NSCLC cells after COL6A6 knockdown. At the same time, we discussed the role of the JAK signalling pathway that may be related to COL6A6.

Results: Bioinformatics analysis showed that COL6A6 expression was downregulated in NSCLC, and its high expression was associated with a better prognosis of NSCLC. In vitro, the expression of COL6A6 in NSCLC tissues was significantly lower than that in adjacent tissues. Furthermore, COL6A6 knockout accelerated the proliferation, invasion, and migration of NSCLC cells and activated the JAK signalling pathway.

Conclusions: Our study illustrates that COL6A6 is a tumor suppressor gene in NSCLC and is involved in NSCLC tumorigenesis by regulating the JAK signalling pathway. Therefore, COL6A6 holds promise as a molecular therapeutic target for NSCLC.

Keywords: Collagen type VI alpha 6 chain (COL6A6); JAK signalling; invasion; non-small cell lung cancer (NSCLC); proliferation.

Figure 1

The expression of COL6A6 in NSCLC tissues. (A) COL6A6 is differentially expressed in tumors compared to normal tissues according to the GEPIA database. (B) The expression of COL6A6 in LUAD and LUSC in TCGA database (P<0.001). (C) Western blotting analysis of COL6A6 in NSCLC and adjacent tissues was performed. (D) The expression of COL6A6 in the immortalized bronchial epithelial cell line (BEAS-2B) and 4 NSCLC cell lines (H520, A549, H1299, and H1975) was analyzed by Western blotting. (E) The Kaplan-Meier plotter was used to analyze the overall survival, which showed that the prognosis of patients with high COL6A6 expression (red) was better than that of patients with low COL6A6 expression (blue). Data are expressed as mean ± SE. *, P<0.05; ***, P<0.001. N, normal tissue; T, tumor tissue; COL6A6, collagen type VI alpha 6 chain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TCGA, The Cancer Genome Atlas; GEPIA, Gene Expression Profiling Interactive Analysis; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; NSCLC, non-small cell lung cancer; SE, standard error.

Figure 2

Verification of the transfection efficiency of COL6A6 knockdown in NSCLC cells. (A) Lentiviral siRNA targeting COL6A6 was transfected into H1299 cells. (B) More than 80% of the cells were green fluorescent protein (GFP) positive and were transfected successfully (×100). Scale bar: 100 µm. (C) Western blotting assays were used to detect the expression of COL6A6 in H1299 cells transfected with siRNA. NC: uninfected H1299 cells; NC-si: negative control siRNA transfected H1299 cells; COL6A6-si: COL6A6 siRNA transfected H1299 cells. Data are expressed as mean ± SE. **, P<0.01; ***, P<0.001. COL6A6, collagen type VI alpha 6 chain; NSCLC, non-small cell lung cancer; SE, standard error.

Figure 3

COL6A6 inhibits the proliferation and invasion of NSCLC cells. (A) The proliferation efficiency of NC, NC-si, and COL6A6-si in H1299 cells after transfection was detected by CCK8 analysis. (B) the wound healing assay analyzed the migration of H1299 cells transfected with COL6A6-si (×100). Scale bar, 100 µm. (C) the number of invasive H1299 cells was detected by the Transwell assay (200×). The cells were stained with 0.5% crystal violet dye. Scale bar, 50 µm. NC: uninfected H1299 cells; NC-si: negative control siRNA transfected H1299 cells; COL6A6-si: COL6A6 siRNA transfected H1299 cells. **, P<0.01; ***, P<0.001. COL6A6, collagen type VI alpha 6 chain; NSCLC, non-small cell lung cancer.

Figure 4

COL6A6 regulates the JAK signalling pathway to inhibit the progression of NSCLC. (A) GSEA demonstrated that COL6A6 is related to the JAK signalling pathway. (B,C) Western blotting assays for JAK-2, p-JAK2, STAT3, and p-STAT3 in H1299 cells following knockdown of COL6A6. NC: uninfected H1299 cells; NC-si: negative control siRNA transfected H1299 cells; COL6A6-si: COL6A6 siRNA transfected H1299 cells. ***, P<0.001. COL6A6, collagen type VI alpha 6 chain; GSEA, gene set enrichment analysis; NSCLC, non-small cell lung cancer.