The data from The Cancer Genome Atlas (TCGA) database was used to analyze the KRT8 mRNA expression in patients with LUAD and its relationship in clinicopathologic features of patients with LUAD, overall survival (OS), and disease-specific survival (DSS). Then, the aberrant expression of KRT8 was confirmed using another public database, GSE116959. Follow-up was finished on April 27, 2016, on the TCGA database.

In the present study, patients’ sample were collected with informed consent from all patients and approved by the Ethics Committee of The First Affiliated Hospital, School of Medical, Zhejiang University. Six pairs of fresh tumor tissues and adjacent normal tissues were collected and stored at −80°C or fixed in 4% paraformaldehyde overnight and paraffin-embedded for further research.

Two human lung adenocarcinoma cell lines (HCC827 and H1975) and a mice lung cancer cell line (LLC) were purchased from the Shanghai Cell Bank at the Chinese Academy of Sciences and studied in this study. Cells were cultured in alpha-Minimum Essential Medium (BI industry, Israel) supplemented with 10% fetal bovine serum (Gemini, California, USA) at 37°C in a humidified incubator with 5% CO₂.

KRT8 shRNA and vector were purchased from Sangon (Beijing, China). HCC827, H1975, and LCC cells were infected with shRNA or vector with polybrene (8 μg/ml; Sigma, USA). After 48 h after infection, KRT8 knockdown efficiency was analyzed by Western blot. The sequences of shRNA are as follows: 1#: CCGGGAGCACTTGGAGAAG and 2#: CAACAAGTTTGCCTCCTTCAT.

Western blot was performed as previously described (19). Antibodies used for Western blot are listed as follows: KRT8 was purchased from Boster (Wuhan, China) and used at 1 mg/ml. E-cadherin (1:1,000), N-cadherin (1:1,000), Vimentin (1:1,000), MMP2 (1:1,000), slug (1:1,000), and NF-kB (1:1,000) Signaling Pathway Antibody Sampler Panel were purchased from Abcam (UK). GAPDH (1:5000) or Lamin B1 (1:1000) were served as internal control.

The Cell-Counting kit-8 (CCK-8) and colony formation assay were used to measure cell viability after KRT8 shRNA transfection. For CCK-8 assay, 5 × 10³ cells were implanted into 96-well plates and cultured. Then, CCK-8 assay was performed at 24, 48, 72, and 96 h according to the manufacturer’s instructions with an ELx800 absorbance microplate reader (BioTek, USA) at 450 nm. For colony formation assay, 1 × 10³ cells were seeded into six-well plates and cultured for another 10 days. Then, the cells were stained with crystal violet, photographed, and counted.

Cell apoptosis was measured by Annexin V–FITC Apoptosis Detection Kit (Dojindo, Japan) according to the manufacturer’s instructions. After trypsinization, cells were stained with annexin V and propidium iodide at room temperature for 15 min. Then, the cells were measured by flow cytometry and analyzed using FlowJo (Tree Star).

Immunohistochemistry was performed as previously described (19). Antibodies used for Western blot are listed below: E-cadherin (1:200), N-cadherin (1:200), and Vimentin (1:200) were purchased from Abcam (UK).

Transwell migration assay was performed using Transwell chambers (12-μm pore size; Corning, USA). Cells (1 × 10⁵) in 200 μl of serum-free medium were seeded in the upper chambers, and 1,000 μl of medium containing 10% fetal bovine serum was added to the lower chambers to induce cell migration. After incubation for another 48 h, cells that migrated to the lower surface of the upper chambers were fixed and stained with crystal violet and pictured.

Transwell invasion assay was performed using Transwell chambers (12-μm pore size; Corning, USA). The Matrigel matrix (Corning, USA) and serum-free medium were mixed (volume ratio of 1: 5), and then, the mixed mixture (40 μl) was added into the upper chamber of Transwell. After solidification, 1 × 10⁵ cells in 200 μl of serum-free medium were seeded in the upper chambers, and 1,000 μl of medium containing 10% fetal bovine serum was added to the lower chambers to induce cell migration. After incubation for another 48 h, cells that migrated to the lower surface of the upper chambers were fixed and stained with crystal violet and pictured.

Six-week-old male nude mice (Shanghai SLAC Laboratory Animal Co., Ltd, China) were purchased for the xenograft assay. LCC cells with control shRNA or KRT8 shRNAs were used (six mice in each group). A total volume of 0.1 ml of PBS containing 1 × 10⁶ LCC cells was injected into intravenous tail vein. Approximately 4 weeks later, tumors were observed and collected for further research. The animal study protocol was reviewed and approved by the Animal Care Committee of Zhejiang University and designed in accordance with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Testing, and Education by the New York Academy of Sciences, Ad Hoc Animal Research Committee.

Values were presented as the mean ± SD. Experimental data were analyzed by GraphPad PRISM 6.01 software (San Diego, CA, USA). P < 0.05 indicates a statistically significant difference. * P < 0.05, ** P < 0.01, and *** P < 0.001.

The TCGA and GSE116959 databases were used to examine the expression of KRT8 in LUAD. The results from TCGA indicated that the expression of KRT8 was significantly higher in cancer tissues compared to normal tissues (P < 0.001; Figure 1A), which is similar with the results from the GSE116959 database (P < 0.001; Figure 1B). Interestingly, the expression of KRT8 was moderately increased from stage I to stage III (Figure 1C). Further, the protein level of KRT8 was measured using patients’ samples by Western blot and IHC. Consistently, the protein expression of KRT8 was obviously upregulated in cancer tissues compared to paired normal tissues (Figures 1E, F). These results suggested that the upregulated KRT8 may play a vital role in the initiation and progression of LUAD.

We then divided the TCGA cohorts into low- and high-expression subgroups according to the median expression of KRT8 to determine the significance of KRT8 in LUAD. The results from KM survival analysis indicated that both the 5-year OS and DSS rates were significantly better among patients with low KRT8 expression compared to those with high expression (P < 0.001; Figure 1D). Correlation analysis showed that KRT8 expression was significantly associated with gender (P = 0.027), advanced T stage (P = 0.001), advanced N stage (P = 0.048), and advanced pathologic stage (P = 0.025). No correlation was found between KRT8 expression and other clinicopathologic features (Table 1). Univariate Cox analysis demonstrated that KRT8 was a predictor of OS [hazard ratio (HR) = 1.526; 95% confidence interval (CI) 1.141–2.040; P = 0.004] and DSS (HR = 1.625; 95% CI 1.123-2.353; P = 0.010) in the TCGA database (Tables 2 and 3).

According to the relationship between KRT8 expression and clinicopathologic features, KRT8 may alter the ability of cell proliferation and apoptosis in LUAD. The CCK-8 and colony formation assay were used in two human lung cancer cell lines (HCC827 and H1975) to explore the effects of KRT8 knockdown on cell proliferation (Figure 2A). As shown in Figure 2B, CCK-8 assays showed that KRT8 knockdown inhibited the proliferation of LUAD cells. Meanwhile, the colony formation assay showed that the proliferation of lung cancer cells was suppressed by KRT8 knockdown (Figure 2D). Moreover, flow cytometry was applied in HCC827 and H1975 to explore the effects of KRT8 knockdown on cell apoptosis. As shown in Figure 2C, KRT8 knockdown obviously induced cell apoptosis of LUAD cells.

According to the relationship between KRT8 expression and clinicopathologic features, KRT8 may also alter the ability of cell migration and invasion in LUAD. Thus, the Transwell migration and invasion assay were applied in HCC827 and H1975 to explore the effects of KRT8 knockdown on lung cancer cell motility. The results indicated that KRT8 knockdown obviously reduced the number of migrated and invaded HCC827 and H1975 cells (Figure 3). The expression of MMP2 was then measured by Western blot in HCC827 and H1975 cells. As shown in Figure 6A, KRT8 knockdown significantly downregulated the protein expression of MMP2. Overall, these data demonstrated that KRT8 knockdown could inhibit lung cancer cell migration and invasion in vitro.

To examine the findings in vitro, an in situ lung cancer model was built by tail intravenous injection of LLC cells. LLC cells were transfected with KRT8 shRNA or vector lentivirus and enriched by puromycin selection before injection (Figure 4A). After another 30 days feeding, the mice were sacrificed, and the lung was pictured and collected for Hematoxylin and eosin staining (HE) and Immunohistochemistry staining (IHC). The results showed that KRT8 knockdown efficiently inhibited the growth of tumors measured by tumor size and number (Figure 4B). In addition, the results from IHC indicated that KRT8 knockdown significantly downregulated N-cadherin and Vimentin but upregulated E-cadherin (Figure 4C). These results indicated that KRT8 could regulate metastasis and EMT of LUAD in vivo.

EMT was measured by Western blot because of its key role in cancer cell migration and invasion. The results showed that KRT8 knockdown downregulated mesenchymal markers N-cadherin, Slug, and Vimentin but upregulated the epithelial marker E-cadherin in HCC827 and H1975 cells (Figure 5A). Collectively, these results indicated that KRT8 regulates the ability of migration and invasion by EMT in LUAD in vitro.

Studies have already demonstrated that NF-κB pathway plays a vital role in EMT and cell motility (20). We validated the role of NF-κB pathway in KRT8-induced EMT and cell motility change. As shown in Figure 5B, KRT8 knockdown had no obvious effects on p-IKKα/β but significantly downregulated the protein expression of p-IκBα and p-p65 in HCC827 and H1975 cells. Moreover, p65 nuclear translocation that reflects the NF-κB activity was examined with NF-κB inducer TNF-α or inhibitor CAPE in HCC827 cells. Indeed, TNF-α promoted the baseline of p65 nuclear translocation, whereas CAPE decreased the baseline of p65 nuclear translocation. Nevertheless, KRT8 knockdown significantly inhibited p65 nuclear translocation in both TNF-α and CAPE-treated groups (Figure 5C). Together, KRT8-induced EMT and cell motility are mediated by NF-κB signaling in LUAD (Figure 6).