The cell lines used in this study (16HBE, A549, SPCA-1, HA579, H520, H460 and H1299) were purchased from American Type Culture Collection (ACTT) and incubated in RPMI-1640 medium containing 10% feral bovine serum (FBS) (Gibco, Carlsbad, CA, USA), at 37°C, 5% CO₂-95% O₂ humid incubator.

The lentivirus expression vectors were purchased from GeneChem Co., Ltd. (Shanghai, China). To obtain stable infection, cells seeded in 96-well plates were infected with 2 µl/well lentivirus expression vectors (1×10⁸ TU/ml) using 10 µl/well polybrene (5 µg/ml) reagent and 90 µl/well RPMI-1640. After 10 h incubation in serum and antibiotic free condition, the medium with free FBS was replaced by the medium containing 10% FBS. Cells were incubated with puromycin-containing medium (2 µg/ml) or selected by means of limited dilution to select green fluorescent monoclonal cells to select stable cell lines.

The cased slides were washed three times with PBS in a plate and fixed with 4% paraformaldehyde for 15 min. After immobilization, the dishes were wash with PBS three times. Normal goat serum was added to the slide and closed at room temperature for 30 min. The blocking solution was removed and a sufficient amount of resistance was added and incubated overnight at 4°C. The next day, tablets were washed 3 times with PBST, and then fluorescent secondary antibody was added. Incubated at room temperature for 1 h and then washed with PBST 3 times, DAPI was added for 5 min in the dark. The plates were again washed with PBST 4 times and then sealed with a sealant containing a fluorescent quencher. Finally, images were observed under a laser confocal microscope.

Cells were washed with cold phosphate buffered saline (PBS) and lysed in RIPA buffer with protease inhibitor cocktail (CWBIO, Beijing, China) for 20 min. Cell extracts were separated by 10% SDS-PAGE gel, and transferred to nitrocellulose membrane (0.45 µm HATF, 10TF of filter; Millipore, Carrigtwohill, Ireland). After blocking in 0.5% milk for 1 h, the blots were incubated with rabbit anti-DAL-1 mAb (Abcam, Cambridge, MA, USA), rabbit anti-HSPA5 mAb (Abcam), rabbit anti-E-cadherin mAb (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-β-catenin mAb (Cell Signaling Technology), rabbit anti-N-cadherin mAb (Cell Signaling Technology), rabbit anti-vimentin mAb (Cell Signaling Technology), rabbit anti-PI3K mAb (Cell Signaling Technology, rabbit anti-p-PI3K mAb (Cell Signaling Technology), rabbit anti-Akt mAb (Cell Signaling Technology), rabbit anti-p-Akt (Ser473) mAb (Cell Signaling Technology), rabbit anti-Mdm2 mAb (Abcam), rabbit anti-p-Mdm2 mAb (Abcam), rabbit anti-p53 mAb (Abcam), rabbit anti-β-actin mAb (Cell Signaling Technology). After being washed three times, blots were further incubated with 1/1000 anti-rabbit IgG (Cell Signaling Technology). Chemiluminescence was measured in a Fusion Solo 2M instrument (Fusion Solo; Vilber Lourmat, Paris, France).

Total RNA was purified using TRIzol reagent (Takara, Shiga, Japan) according to the manufacturer's instructions. Quantification was performed with a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). RNA (500 ng) was reverse-transcribed using Prime Script TR Reagent kit (RR037A; Takara). The product was used in subsequent RT-qPCR using three duplicate PCR reactions containing 1X SYBR-Green Master Mix (SYBR Premix Ex Taq II; RR820A; Takara) with the primers in Table I. Relative expression levels of target genes were determined by the ∆∆Cq method, using GAPDH gene expression to normalize all samples.

The Transwell insert was coated with 30 µl (1 µg/ml) Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and 2×10⁵ cells in 200 µl serum-free media were transferred to the top chamber (6.5 mm; Corning Inc., Corning, NY, USA). The lower chamber was filled with 700 µl medium containing 5% FBS. The cells in the lower chamber were fixed by methanol and stained with Giemsa after culturing for 24 h. For Transwell migration assays, 2×10⁵ cells were plated in the top chamber onto the non-coated membrane. The invasion and migrating cells were counted under a microscope from 5 randomly selected fields (magnification, ×200).

Cells (1×10³) per well were seeded in triplicate into the 96-well plats. After incubation for 24 h, the CCK-8 reagent (10 µl/well; CCK-8; Dijindo, Kumamoto, Japan) was added to each well and then incubated for a further 2 h. The quantity of absorbance on the plat was measured at a wavelength of 450 nm by microplate reader to detect the survival of cells. All experiments were independently repeated at least three times.

All analyses were performed by SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± SD from at least three independent experiments. The difference between different groups was analyzed using a factorial model one-way analysis of variance and P-value <0.05 was considered to indicate a statistically significant difference.

The Oncomine database (http://www.oncomine.org) was utilized to analyze the mRNA expression of DAL-1 and HSPA5 in lung cancer patients. Data from ten databasesdemonstrated that, compared with normal tissues, the mRNA expression of DAL-1 in lung cancer tissues was downregulated (P<0.05; Fig. 1A) while the mRNA expression of HSPA5 was upregulated (P<0.05; Fig. 1B). The western blot assay was used to investigate the protein expression level of DAL-1 and HSPA5 in NSCLC cells (A549, SPCA-1, H1975, H520, H460 and H1299). As shown in Fig. 1C, compared with the immortalized bronchial epithelial cells (16HBE), the protein expression of DAL-1 was lower in A549, SPCA-1 and H1299 cells (P<0.05) while higher in H520 and H460 cells (P<0.05). The protein expression of HSPA5 was higher in all the NSCLC cells (P<0.05). Fig. 1D showed a statistical analysis of the gray scale of the protein.

Knowing that DAL-1 protein and HSPA5 protein can be combined directly in NSCLC cells, we further observed their co-localization in H460 cells which express the two proteins simultaneously. The DAL-1 protein and HSPA5 protein were stained by immunofluorescence and the co-localization of them was observed by laser confocal scanning microscope. The nucleus labeled by DAPI showed blue color (Fig. 2A), DAL-1 proteins were marked by green fluorescence (Fig. 2B) and HSPA5 proteins were labeled by red fluorescence (Fig. 2C). After merging, we can see these two proteins co-localized in cytoplasm and nucleus (Fig. 2D).

In order to investigate the functional role of DAL-1 in NSCLC cells, a lentivirus vector LV-DAL-1 was established to functionally overexpress DAL-1 in A549 cells. The protein expression of DAL-1 in stably transfected cells (A549/LV-DAL-1) was confirmed by western blot assay (Fig. 3A). The mRNA expression of E-cadherin and β-catenin were increased while the mRNA expression of N-cadherin and vimentin were decreased in A549/LV-DAL-1 cells compared with control cells (A549/LV-CON1) (P<0.05; Fig. 3C). Similarly, the protein expression of E-cadherin and β-catenin were upregulated while the protein of N-cadherin and vimentin were downregulated in A549/LV-DAL-1 cells (Fig. 3D). Transwell migration and invasion assays revealed the suppression role of DAL-1 on migration and invasion abilities in A549 cells (P<0.05; Fig. 3E and F), and CCK-8 cell proliferation assay indicated the suppression role of DAL-1 on proliferation (P<0.05; Fig. 3B). Collectively, these data suggest that DAL-1 plays a role in suppressing EMT, migration, invasion and proliferation in NSCLC cells.

To investigate the functional role of HSPA5 in NSCLC cells, the lentivirus vectors LV-HSPA5 RNAi-(1/2/3) were established to functionally silence HSPA5 expression in A549 cells. The western blot assay indicated that the protein level of HSPA5 in A549/LV-HSPA5 RNAi-3 decreased most obviously and was used in subsequent experiments (Fig. 4A). The qRT-PCR assay showed that the mRNA expression of E-cadherin and β-catenin were increased while the mRNA expression of N-cadherin and vimentin were decreased in silenced-HSPA5 cells (A549/LV-HSPA5 RNAi-3) compared with silenced-control cells (A549/LV-CON2) (P<0.05; Fig. 4C). The western blot assay demonstrated that silence of HSPA5 upregulated the protein expression of E-cadherin and β-catenin, while downregulated the protein expression of N-cadherin and vimentin of A549 cells (Fig. 4D). Transwell migration and invasion assays revealed that HSPA5-silence significantly decreased the migration and invasion abilities of A549 cells (P<0.05; Fig. 4E and F). CCK-8 cell proliferation assay indicated that the proliferation ability was suppressed by silence of HSPA5 (P<0.05; Fig. 4B). These results demonstrate that HSPA5 plays a metastasis and growth promoting role in NSCLC cells.

To determine whether DAL-1 could influence the expression of HSPA5, we examined the expression of HSPA5 in A549/LV-DAL-1 cells and A549/LV-CON1 cells. The result obtained in the qRT-PCR assay demonstrated that the mRNA expression of HSPA5 was significantly reduced by overexpressing DAL-1 (P<0.001; Fig. 5A) and western blot assay indicated that the protein expression of HSPA5 was also suppressed by DAL-1 (Fig. 5B). The results suggest that DAL-1 plays a role in suppressing HSPA5 expression.

Knowing that DAL-1 can attenuate while HSPA5 can promote EMT, migration, invasion and proliferation in NSCLC cells, and overexpression of DAL-1 can reduce the mRNA and protein expression of HSPA5, we have reason to believe that DAL-1 can inhibit metastasis and proliferation by suppressing HSPA5 expressing. To elucidate whether HSPA5 is involved in DAL-1-suppressed EMT, migration, invasion and proliferation, A549/LV-DAL-1 cells were stably infected with HSPA5-overexpressed vector LV-HSPA5 (Fig. 6A), and the role of overexpressed HSPA5 in EMT, migration, invasion and proliferation was detected. The results of qRT-PCR and western blot assays in Fig. 6C (P<0.05) and Fig. 6D revealed that the overexpression of HSPA5 downregulated the mRNA and protein expression levels of E-cadherin and β-catenin while upregulated the mRNA and protein expression levels of N-cadherin and vimentin. The results of Transwell migration and invasion assays indicated that the migration and invasion abilities in A549/LV-DAL-1 cells were obviously increased by overexpressing HSPA5 (P<0.05; Fig. 6C and D). The CCK-8 assay indicated the proliferation ability of A549/LV-DAL-1 cells could be promoted by infected LV-HSPA5 (Fig. 6B). These findings support that HSPA5 is a potential target for DAL-1 and involves in DAL-1-mediated EMT, migration, invasion and proliferation in NSCLC cells.

A549/LV-HSPA5 RNAi-3 cells were infected with the DAL-1 overexpression lentiviral vector LV-DAL-1 (Fig. 7A) to investigate whether the suppression of HSPA5 is necessary for DAL-1 to inhibit the metastasis and growth in NSCLC cells. The results of qRT-PCR and western blot assays shown in Fig. 7C (P<0.05) and Fig. 7D demonstrated that, after overexpressing DAL-1 in HSPA5-slienced A549 cells, the mRNA and protein expression of E-cadherin and β-catenin were increased, while the mRNA and protein expression of N-cadherin and vimentin were decreased. Transwell migration and invasion assays shown in Figs. 6E and 7F (P<0.05), and CCK-8 assay shown in Fig. 7B (P<0.05) revealed that overexpression of DAL-1 decreased the migration, invasion and proliferation ability in A549/LV-HSPA5 RNAi-3 cells. These data indicate that DAL-1 does not depend on HSPA5 to suppress EMT, migration, invasion and proliferation.

Studies have shown that the abnormal activation of PI3K/Akt/Mdm2/p53 signal pathway was closely related to the progression of cancer. It has been reported that HSPA5 can promote lung cancer metastasis by activating PI3K/Akt signaling pathway. However, there is still no report on the effects of DAL-1 on PI3K/Akt/Mdm2/p53 signaling pathway. In this study, western blot assay was utilized to explore the potential roles of DAL-1 and HSPA5 on PI3K/Akt/Mdm2/p53 signaling pathway in NSCLC cells. The LV-DAL-1 lentivirus vectors transfected into A549 cells displayed an inhibition role on expression of PI3K, p-PI3K, Akt, p-Akt, Mdm2 and p-Mdm2 while played a role on upregulating p53 expression (Fig. 8A). Similarly, the lentivirus vectors LV-HSPA5 RNAi3 also functioned reducing the protein expression of PI3K, p-PI3K, Akt, p-Akt, Mdm2 and p-Mdm2 while increased the expression of p53 protein. These results suggested that DAL-1 could suppress EMT, migration, invasion and proliferation by inhibiting PI3K/Akt/Mdm2/p53 signaling pathway through suppressing HSPA5 expression.

To determine the role of HSPA5 in DAL-1 inhibiting PI3K/Akt/Mdm2/p53 signaling pathway, the effect of LV-HSPA5 on the protein expression of PI3K, p-PI3K, Akt, p-Akt, Mdm2, p-Mdm2 and p53 in A549/LV-DAL-1 cells was investigated. The results shown in Fig. 9A confirmed that overexpressing HSPA5 increased the protein expression of PI3K, p-PI3K, Akt, p-Akt, Mdm2 and p-Mdm2 while decreased the protein expression of p53 in A549/LV-DAL-1 cells, which told us that HSPA5 is involved in DAL-1-inhibited PI3K/Akt/Mdm2/p53 signaling pathway. To further study if HSPA5 is necessary for DAL-1 to regulate the PI3K/Akt/Mdm2 signaling pathway, we examined the protein expression of PI3K, p-PI3K, Akt, p-Akt, Mdm2, p-Mdm2 and p53 in A549/LV-HSPA5 RNAi-3 cells after infected with LV-DAL-1. The results in Fig. 9B show that, after overexpressing DAL-1, the protein expression of PI3K, p-PI3K, Mdm2, p-Mdm2, Akt, p-Akt and p53 in HSPA5-silenced cells were all increased, which suggested that the suppression of HSPA5 is necessary for DAL-1 to inhibit PI3K/Akt/Mdm2 signaling pathway.