A total of 15 pairs of lung cancer and adjacent non-cancer tissues were obtained from patients with lung cancer (age range, 45-76 years; sex, 9 males and 6 females) between January, 2019 and February, 2020 from the Shandong Provincial Hospital (Jinan, China). The samples were stored at −80°C until required for subsequent experimentation. Written informed consent was obtained from each patient prior to participation and the clinical study was approved by the Ethics Committee of Clinical Research of Shandong Provincial Hospital.

For the analysis using public data, 57 pairs of lung cancer and adjacent non-cancer tissues were analyzed using a dataset from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases. In addition, the expression levels of DNAJC12 were also analyzed in 54 control and 498 cancer unpaired tissues.

Lung cancer cell lines (A549, NCI-H1299, NCI-H1975 and 95D) and 293T cells were purchased from the American Type Culture Collection (ATCC). Mycoplasma testing was performed for all cell lines and all cell lines were authenticated using STR profiling. Cells were cultured in DMEM (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Total RNA was extracted from the cancer tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 μg) was reverse transcribed into cDNA using a cDNA synthesis kit (cat. no. 6130, Takara Bio, Inc.). qPCR was subsequently performed using an SYBR-Green II reagent kit (cat. no. RR820A; Takara Bio, Inc.) as previously described (20). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 5 min, followed by 40 cycles at 95°C for 15 sec, 60°C for 30 sec and 70°C for 10 sec. The following primer pairs were used for the qPCR: DNAJC12 forward, 5′-AATGGTTGGCACCTTCGTTTC-3′ and reverse, 5′-GTTGGCAGCATAGGGGACAG-3′; CTNNB1 forward, 5′-AGCTTCCAGACACGCTATCAT-3′ and reverse, 5′-CGGTACAACGAGCTGTTTCTAC-3′; VIM forward, 5′-AGTCCACTGAGTACCGGAGAC-3′ and reverse, 5′-CATTTCACGCATCTGGCGTTC-3′; RELA forward, 5′-GTGGGGACTACGACCTGAATG-3′ and reverse, 5′-GGGGCACGATTGTCAAAGATG-3′; and GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. The mRNA expression levels were quantified using the 2^−ΔΔCq method as previously described (21).

Short hairpin RNA (shRNA/sh) targeting DNAJC12 (shDNAJC12; 5′-GGATGTGATGAACTATCTT-3′) and control shRNA (shCtrl; 5′-TTCTCCGAACGTGTCACGT-3′) were cloned into GV115 plasmids (Shanghai GeneChem Co., Ltd.). For overexpression, the coding sequences of CTNNB1 (NM_001904.4), p65 (NM_021975.4), VIM (NM_003380.5) were cloned into GV610 vectors (Shanghai GeneChem Co., Ltd.). The GV115 or GV610 plasmids (20 μg) were subsequently co-transfected into 293T cells alongside pHelper1.0 (15 μg) and pHelper2.0 (10 μg) packaging vectors using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) as previously described (22). Following transfection with the lentiviruses for 48 h (MOI=10), the transduced cells were selected with 5 μg/ml puromycin (Sigma-Aldrich; Merck KGaA).

A HCS assay was performed to determine the cell number by counting the GFP-expressing cells, as previously described (22), following the culture of 2×10³ A549 cells in 96-well plates. ArrayScan™ HCS software (Cellomics Inc.) was used to analyze the cell proliferation of each well every 24 h.

The proliferation of the A549 and NCI-H1975 cells seeded into 96-well plates at a density of 2×10³ cells/well was analyzed using an MTT assay kit (Gen-view Scientific, Inc.). MTT solution was added to each well and incubated for 4 h at 37°C. Subsequently, the solution was replaced with 100 μl/well DMSO, and the plates were agitated for 5 min. The optical density of each well was read at 490 nm using a microplate spectrophotometer (Tecan infinite, M2009PR; Tecan Group, Ltd.).

A total of 1×10³ A549 and NIC-H1975 cells with or without DNAJC12 knockdown were plated into 6-well plates and cultured for 2 weeks. Colonies were subsequently stained with crystal violet (Sangon Biotech Co., Ltd.) and stained colonies were visualized, followed by evaluation of colony number and size.

A549 and NCI-H1975 cell migration was determined in 6-well plates (10⁵ cells/well) using a wound healing assay, as previously described (23). Briefly, the cells were used in the wound healing assay when the confluency of the cells was closed to 100%. After the cells were scratched using 10 μl pipette tips, the medium was replaced with FBS-free medium. The images were captured at 0, 24 and 48 h using an inverted fluorescence microscope (Olympus Corporation; magnification, ×100).

A Transwell migration and Matrigel invasion assay was used to determine the migration and invasive ability of the A549 and NCI-H1975 cells. For migration, 100 μl FBS free medium were added onto the upper surface of the migration chambers (cat. no. 3422; Corning, Inc.) and incubated at 37°C for 30 min. Subsequently, the FBS-free medium was discarded and a total of 1×10⁵ cells in 100 μl FBS-free medium was added onto the upper surface of the migration chambers. The lower chambers were filled with 30% FBS medium. For invasion assay, a total of 500 μl of the mixture was added onto the upper surface of the invasion chambers (cat. no. 354480; Corning, Inc.), while the lower chambers were filled with 30% FBS medium. Following incubation for 24 or 48 h at 37°C, the culture medium and the cells attached on the upper surface were removed. Cells attached on the lower surface were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with Giemsa stain (cat. no. 32884; Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. The images of migratory and invasive cells were collected using a light microscope (Olympus Corporation; magnification, ×100).

The apoptosis of the lung cancer cells was analyzed using an Annexin V-allophycocyanin (APC) apoptosis detection kit (eBioscience; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, the A549 and NCI-H1975 cells were infected with lentiviruses expressing shCtrl or shDNAJC12 for 24 h. The cells were then harvested (1,000 × g, 5 min, room temperature) and washed with PBS. The cells were subsequently resuspended in a staining buffer to a final density of 1×10⁶ cells/ml. Finally, 100 μl cell suspension was incubated with 5 μl Annexin V-APC at room temperature for 15 min. The stained cells were subjected to flow cytometry using a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (version 7.6.1; FlowJo LLC).

Female BALB/c athymic nude mice (age, 4 weeks; weight, 15-17 g) were obtained from Charles River Laboratories, Inc. and were randomly divided into the following two groups (10 mice/group): i) ShCtrl; and ii) shDNAJC12. Mice were healthy and housed under SPF-grade conditions. A total of 1×10⁷ shCtrl- or shDNAJC12-transfected A549 cells were subcutaneously implanted into the right flanks of the nude mice, according to the protocol as described previously (24-26). The tumor volume was measured every 3 days starting at 30 days following transplantation and the mice were sacrificed by cervical dislocation at day 42. The animal experiments were performed according to the indicated protocols and were approved by the Animal Research Ethics Committee of Shandong Provincial Hospital.

Total protein was extracted from the lung cancer tissues and A549 and NCI-H1975 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitor cocktail. The protein concentration was determined using a BCA assay kit (Thermo Fisher Scientific, Inc.) and 40 μg protein/lane was separated via 12% SDS-PAGE. The separated proteins were subsequently transferred onto PDVF membranes and blocked with 5% fat-free milk in TBS-Tween-20 (TBST) for 1 h at room temperature. The membranes were then washed with TBST and incubated with the following primary antibodies at 4°C overnight: Anti-DNAJC12 (Abcam; cat. no. ab167425), anti-GAPDH (Santa Cruz Biotechnology, Inc.; cat. no. sc-32233), anti-p65 (Cell Signaling Technology, Inc.; cat. no. 8242), anti-phosphorylated (p)-p65 (Cell Signaling Technology, Inc.; cat. no. 3033), anti-β-catenin (Cell Signaling Technology, Inc.; cat. no. 8480), anti-p-β-catenin (Cell Signaling Technology, Inc.; cat. no. 2009) and anti-vimentin (Cell Signaling Technology, Inc.; cat. no. 3932). Following the primary antibody incubation, the membranes were washed with TBST and incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology, Inc.; cat. nos. 7076 and 7074) at room temperature for 2 h. The membranes were subsequently washed with TBST and protein bands were visualized using Pierce™ ECL Western Blotting substrate (Thermo Fisher Scientific, Inc.).

All experiments were performed at least in triplicate and data are presented as the mean ± SEM. Statistical differences between two groups were determined using a Student's t-test, while statistical differences between ≥3 groups were determined using a one-way ANOVA followed by a Tukey's post hoc test. Categorical data were analyzed using a χ² test. P<0.05 was considered to indicate a statistically significant difference.

To determine the potential roles of DNAJC12 in lung cancer, the expression levels of DNAJC12 in lung cancer and adjacent non-cancer tissues were analyzed by RT-qPCR. The expression levels of DNAJC12 were significantly upregulated in the lung cancer tissues compared with the adjacent non-cancer tissues (Fig. 1A and B). In addition, the expression levels of DNAJC12 in 57 pairs of lung cancer and control tissues from TCGA database were analyzed. The results also revealed that the expression levels of DNAJC12 were upregulated in lung cancer tissues compared with non-cancer control lung tissues (Fig. 1C and D). Furthermore, analysis of a large unpaired TCGA dataset (54 control and 498 cancer tissues) also demonstrated that the expression levels of DNAJC12 were upregulated in lung cancer tissues (Fig. 1E). In addition, the association between patient clinicopathological features and DNAJC12 expression in 498 lung cancer tissues from TCGA database is presented in Table I. The expression of DNAJC12 was not associated with age, sex, T stage, N metastasis and M metastasis. Therefore, these findings suggested that DNAJC12 may be involved in the development of lung cancer.

To determine the potential roles of DNAJC12 in lung cancer cells, the expression levels of DNAJC12 were first analyzed in four lung cancer cell lines. The results revealed that the A549 and NCI-H1975 cells expressed higher expression levels of DNAJC12 compared with the NCI-H1299 and 95D cells (Fig. 2A). Therefore, the A549 and NCI-H1975 cells were selected for use in further experiments. Lentiviruses expressing shRNA targeting DNAJC12 (shDNAJC12) and control shRNA (shCtrl) were subsequently successfully infected into A549 and NCI-H1975 cells (Fig. 2B and C). The results of RT-qPCR and western blot analysis demonstrated that the lentivirus-mediated knockdown of DNAJC12 significantly downregulated the expression levels of DNAJC12 in the A549 and NCI-H1975 lung cancer cells (Fig. 2D and E). Thus, A549 and NCI-H1975 cells with/without stable DNAJC12 knockdown were successfully generated for use in further experiments.

The present study first determined the effects of the knockdown of DNAJC12 on the proliferation rate of A549 and NCI-H1975 lung cancer cells using HCS and MTT assays. The results determined that the knockdown of DNAJC12 significantly reduced the proliferation rate of the A549 and NCI-H1975 cells (Fig. 3). Colony formation is a key feature of cancer cells (27); therefore, the present study also analyzed the effects of DNAJC12 on the colony formation of lung cancer cells. The results revealed that DNAJC12 knockdown reduced the number and size of colonies formed from the A549 and NCI-H1975 lung cancer cells (Fig. 4). Taken together, these findings suggest that the knockdown of DNAJC12 may suppress the proliferation of lung cancer cells.

To determine whether DNAJC12 regulates lung cancer cell growth in vivo, xenograft experiments were performed. A549 cells expressing shCtrl and shDNAJC12 lentiviruses were subcutaneously transplanted into the right flanks of 4-week-old female nude mice. Tumor growth was monitored for 42 days following implantation. The results revealed that the knockdown of DNAJC12 reduced the growth rate, size and weight of the lung cancer tumors (Fig. 5). Thus, these results indicate that DNAJC12 may contribute to lung cancer growth in vivo.

Metastasis is a feature of human lung cancer cells (27). To determine whether DNAJC12 regulates the invasion of lung cancer cells, the Transwell assay and Transwell Matrigel assay was performed using the A549 and NCI-H1975 cells. The results revealed that A549 and NCI-H1975 cell invasion was significantly suppressed following the knockdown of DNAJC12 (Fig. 6A and B). Matrigel invasion assay also demonstrated that DNAJC12 knockdown suppressed the invasion of A549 and NCI-H1975 cells (Fig. 6C and D). The present study subsequently analyzed the effects of DNAJC12 on the migration of lung cancer cells by performing wound healing assays. The results revealed that the migration of the A549 and NCI-H1975 cells, which was observed within 48 h, was inhibited by DNAJC12 knockdown (Fig. 6E and F). Taken together, these findings suggest that DNAJC12 regulates the invasion and migration of lung cancer cells.

The suppression of cancer growth and metastasis may result from cancer cell apoptosis induced by clinical drug treatment (27). Thus, the present study aimed to determine whether the DNAJC12-induced effects on the proliferation, migration and invasion of lung cancer cells were associated with the apoptosis of lung cancer cells. Cell apoptosis was analyzed by flow cytometry in the A549 and NCI-H1975 cells. The results revealed that the knockdown of DNAJC12 induced the apoptosis of A549 and NCI-H1975 cells (Fig. 7), which may be associated with the effects of DNAJC12 on the proliferation, migration and invasion of lung cancer cells.

The biological pathways regulated by DNAJC12 were subsequently investigated. The growth and metastasis of lung cancer cells are regulated by pivotal intracellular transcriptional regulators, such as NF-κB and β-catenin, as well as key extracellular matrix regulators, such as matrix metalloproteinase (MMP) and vimentin (14,28,29). The present study analyzed the effects of DNAJC12 on the expression levels of these proteins by western blotting. The knockdown of DNAJC12 reduced the phosphorylation of NF-κB p65 and downregulated the expression levels and activation of β-catenin in A549 cells (Fig. 8A and B). The knockdown of DNAJC12 also downregulated the expression levels of vimentin (Fig. 8C), which is a factor of the extracellular matrix. Therefore, these findings suggest that DNAJC12 may target key regulators of lung cancer growth and metastasis.

To determine whether p65, β-catenin and vimentin are involved in the effects of DNAJC12 on lung cancer cells, p65, β-catenin and vimentin were overexpressed in A549 cells in which DNAJC12 expression was knocked down. RT-qPCR analysis indicated that DNAJC12 was successfully knockdown, while p65, β-catenin and vimentin were effectively overexpressed in the A549 cells (Fig. 9A). An HCS assay was subsequently performed and the results revealed that the overexpression of β-catenin, but not that of p65 or vimentin, reversed the suppressive effects of DNAJC12 knockdown on the proliferation of A549 cells (Fig. 9B). The involvement of β-catenin in the regulatory role of DNAJC12 over A549 cell proliferation was also validated using an MTT assay (Fig. 9C). Furthermore, the reduced invasive capacity of the A549 cells induced by DNAJC12 knockdown was also reversed by the overexpression of β-catenin (Fig. 9D). Taken together, these findings suggest that DNAJC12 may regulate the proliferation and invasion of lung cancer cells in a β-catenin-dependent manner.