The expression profiles of myelodysplastic syndrome 1 (MDS1) and ecotropic viral integration site 1 (EVI1) complex locus (MECOM) of patients with LUAD was analysed using the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia2.cancer-pku.cn/#index) (18). In GEPIA website, it was not possible to acquire clinical characteristics of 483 LUAD patients. To show clinical characteristics, HSPB7 expression profiles and clinical characteristics of patients with LUAD [515 cases, International Classification of Diseases (ICD-10) (19), C34] were downloaded from TCGA (https://portal.gdc.cancer.gov/), and then analyzed via R software (version 3.6.1, http://www.r-project.org/). A survival curve for HSPB7 expression was obtained using GEPIA. The survival curve of MECOM expression was obtained from the Kaplan Meier plot (http://kmplot.com/analysis/). Survival analysis was performed using the Kaplan-Meier method, and the log-rank test was used to compare survival times between the low and high expression groups. Gene set enrichment analysis (GSEA) was used to identify HSPB7 related signaling pathways in the LUAD dataset using the following parameters: Number of permutations=1,000; permutation type=gene_set; enrichment statistic=weighted; and metric for ranking genes=Signal2Noise. The binding site of HSPB7 and MECOM was predicted using the JASPAR website (http://jaspar.genereg.net/).

Between September 2020 and December 2021, 23 tumour tissues and paired normal tissues from patients with LUAD (13 males and 10 females; age range: 28–75 years old; TNM stage: I–IV; ICD-10: C34.1 and C34.3) were collected from Jinan Central Hospital Affiliated to Shandong University (Jinan, China) and stored immediately at −80°C for subsequent analysis. Pathologists confirmed the correct identification of the tumour tissues and paired normal tissues. The present study was approved (approval no. W202203060147) by the Ethics Committee of Jinan Central Hospital Affiliated to Shandong First Medical University (Jinan, China). Written informed consent was obtained from all enrolled patients.

Normal human lung epithelial cells (BEAS-2B; cat. no. CRL-3588) and lung cancer cells [H1975 (cat. no. CRL-5908), H1688 (cat. no. CCL-257), H1299 (cat. no. CRL-5803) and A549 (cat. no. CRM-CCL-185)] were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO₂. Small interfering (si)RNAs si1-HSPB7 and si2-HSPB7, which specifically targeted HSPB7 were synthesized and purified by Guangzhou RiboBio Co., Ltd. The sequences were as follows: si1-HSPB7 sense, 5′-GGUGCUGUGGGAGGACAAAGA-3′ and antisense, 5′-UUUGUCCUCCCACAGCACCUG-3′; si2-HSPB7 sense, 5′-GGAAGACUAUGUCACACUGCC-3′, and antisense, 5′-CAGUGUGACAUAGUCUUCCUG-3′; si1-MECOM sense, 5′-GGAUGAUGAAGAAGUUGAAGA-3′ and antisense, 5′-UUCAACUUCUUCAUCAUCCAG-3′; si2-MECOM sense, 5′-CCUGCUAGUUCUCCUGUUAAA-3′ and antisense, 5,-UAACAGGAGAACUAGCAGGUA-3′ and si-NC sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5,′-ACGUGACACGUUCGGAGAATT-3′. HSPB7 and MECOM were cloned into the pcDNA3.1 eukaryotic expression vector to allow their overexpression. All the vectors were purchased from Guangzhou RiboBio Co., Ltd. When the fusion rate of H1975 and A549 cells reached 70–80%, Cells were transfected with 40 nM vectors using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.) for 10 h at 37°C. After 48 h, the transfection efficiency was tested by quantitative reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Before transfection, H1975 cells were pretreated with 5 mM 2-deoxy-D-glucose (2-DG; Sigma-Aldrich; Merck KGaA) for 8 h.

Total RNA was isolated from tissues and cells by homogenization using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized using a PrimeScript RT Reagent kit (Takara Bio, Inc.) according to the manufacturer's protocols. The SYBR Green System (Takara Bio, Inc.) was used to assess gene expression. The sequences of primers used for amplification were as follows: HSPB7 forward, 5′-AACCACATCGAGCTGGCG-3′ and reverse, 5′-GAAAGGGAAGGGAGAGGCAC-3′; MECOM forward, 5′-CTTCTTGACTAAAGCCCTTGGA-3′ and reverse, 5′-GTACTTGAGCCAGCTTCCAACA-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-AATGGGCAGCCGTTAGGAAA-3′ and reverse, 5′-GCCCAATACGACCAAATCAGAG-3′. The thermocycling conditions involved initial denaturation at 95°C for 4 min; 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 35 sec, elongation at 72°C for 30 sec; and final extension at 72°C for 10 min. The 2^−ΔΔCq method (20) was used to calculate the mRNA expression level. Expression levels were normalized to the internal reference gene, GAPDH.

Total protein from the cells was isolated and quantified using radioimmunoprecipitation (RIPA) assay lysis buffer and a bicinchoninic acid kit (both from Beyotime Institute of Biotechnology), respectively. Samples (30 µg) were separated by 12% polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: HSPB7 monoclonal antibody (1:1,000; cat. no. ab248960; Abcam), E-cadherin polyclonal antibody (1:1,000; cat. no. GB11868; Wuhan Servicebio Technology Co., Ltd.), N-cadherin polyclonal antibody (1:1,000; cat. no. GB111009; Wuhan Servicebio Technology Co., Ltd.), Snail polyclonal antibody (1:1,000; cat. no. 13099-1-AP; Proteintech Group, Inc.), lactate dehydrogenase A (LDHA) polyclonal antibody (1:1,000; cat. no. 19987-1-AP; Proteintech Group, Inc.), hexokinase 2 (HK2) monoclonal antibody (1:1,000; cat. no. 2867; Cell Signaling Technology, Inc.); pyruvate kinase muscle isoform 2 (PKM2) polyclonal antibody (1:500; cat. no. SAB4200094; Sigma-Aldrich; Merck KGaA) and GAPDH polyclonal antibody (1:1,000; cat. no. AC001; ABclonal Biotech Co., Ltd.). The PVDF membranes were then incubated with the HRP-conjugated secondary antibody goat anti-rabbit IgG (1:5,000; cat. no. AS014; ABclonal Biotech Co., Ltd.) at room temperature for 1 h. The bands were visualized using enhanced chemiluminescence (Beijing Solarbio Science & Technology Co., Ltd.) and quantified using Quantity One^® 4.1.1 gel analysis software (Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control.

The lung tissue and xenograft tumours sections were deparaffinized with xylene and rehydrated in gradient ethanol at room temperature. To eliminate endogenous peroxidase activity, the sections (4 µm) were incubated with 3% H₂O₂ at room temperature for 5–10 min. Subsequently, the sections were rinsed three times with distilled water and soaked in phosphate buffered saline for 5 min. After blocking in 5–10% normal goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.) for 10 min, the sections were incubated with primary antibodies against HSPB7 (1:200; cat. no. ab150390; Abcam) and Ki67 (1:200; cat. no. ab15580; Abcam) overnight at 4°C, followed by HRP-conjugated secondary antibody (1:200) at 37°C for 1 h. Finally, the sections were stained with the developer 3,3′-diaminobenzidine (Beijing Solarbio Science & Technology Co., Ltd.) for 3–15 min and observed under a light microscope (DMI3000 B; Leica Microsystems GmbH).

After transfection, H1975 and A549 cells (1×10³ cells/well) were seeded into a 96-well plate and cultured at 37°C for 24, 48 and 72 h. Next, cells were treated with 10 µl MTT reagent (Beyotime Institute of Biotechnology) at 37°C for 4 h, dimethyl sulfoxide (Thermo Fisher Scientific, Inc.) was applied to treat cells at room temperature for 10 min, and the value of optical density was measured at 570 nm using a microplate reader (BioTek Instruments, Inc.).

After transfection, H1975 and A549 cells (5×10² cells/well) were seeded into six-well plates. After ~14 d, visible clones appeared in the six-well plates. Colonies (>50 cells) were fixed with 4% formaldehyde for 15 min and stained with 0.1% crystal violet (both from Sigma-Aldrich; Merck KGaA) for 25 min. Finally, the number of effective clones was calculated using a microscope (DMI3000 B; Leica Microsystems GmbH).

H1975 and A549 cells (4×10⁵ cells/well) were seeded in six-well cell culture plates. When the confluence of the monolayer cells reached 70–80%, a new 200-µl pipette tip was used to gently scratch a wound on the monolayer cells. Cells were cultured in serum-free medium for 48 h. Images were captured by a microscope (DMI3000 B; Leica Microsystems GmbH) at 0 and 48 h, and the wound width was analyzed using ImageJ software v1.51 (National Institutes of Health).

A 6.5 mm Transwell with 8.0 µm Pore Polycarbonate Membrane Insert, Sterile (Corning, Inc.) was used in invasion assay. The transfected H1975 and A549 cells (1×10⁵ cells/ml) were resuspended in serum-free DMEM in the upper chamber with Matrigel (BD Biosciences) at 37°C for 30 min, while the lower chamber was filled with 150 µl DMEM containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). After culturing at 37°C for 48 h, 800 µl methanol and 800 µl crystal violet were used to fix for 15 min at room temperature and stain for 30 min at room temperature cells. A total of five randomly selected fields were observed under microscope (Leica Microsystems GmbH).

A Glucose Assay kit (cat. no. ab65333; Abcam) and a Lactic Acid Assay kit (cat. no. MAK064, Sigma-Aldrich; Merck KGaA) were used to measure glucose consumption and lactic acid production, respectively, in H1975 and A549 cells. Glucose enzyme mix specifically oxidizes glucose, to generate a product which reacts with a dye to generate color (OD 570 nm). Glucose content in the cell culture medium was determined and cells in each well were counted to normalize glucose concentration. Glucose uptake was determined indirectly by determining the remained glucose content in cell culture medium. Lactate specifically reacts with an enzyme mix to generate a product, which interacts with lactate probe to produce color (OD 570 nm). Lactate production in the cell culture medium was detected and cells in each well were counted to normalize lactate concentration. Absorbance at OD 570 nm was detected using a microplate reader (BioTek Instruments, Inc.).

Whole-cell extracts were lysed in RIPA buffer (Beyotime Institute of Biotechnology). Lysates were clarified by centrifugation at 14,000 g for 15 min at 4°C. Supernatant was incubated with 20 µl/ml protein A/G sepharose beads (Beyotime Institute of Biotechnology) at 4°C for 1 h to remove non-specific hybrid proteins. A total of 5 µg IgG (cat. no. ab37415; Abcam), anti-HSPB7 (cat. no. ab150390; Abcam) or anti-MECOM (cat. no. 23201-1-AP; Proteintech Group, Inc.) antibodies were added to pre-cleared cell lysate and incubated at 4°C overnight and then rotated at 4°C with a mixture of protein A/G sepharose beads (20 µl/ml) for 4 h. The beads were then washed 3 times with RIPA buffer to obtain protein samples for western blot analysis.

In brief, H1975 and A549 cells were first cross-linked with formaldehyde at room temperature for 15 min, and then cleaved by the Magna ChIP^™ Protein G Kit (Sigma-Aldrich; Merck KGaA) to obtain chromatin, followed by ultrasonic fragmentation. MECOM antibody (cat. no. HPA046537; Sigma-Aldrich; Merck KGaA) was used to recruit HSPB7 DNA overnight at 4°C, and the protein-DNA complex was precipitated with protein G magnetic beads for 2 h. After immunoprecipitation, the DNA in the complex was purified, and enriched HSPB7 was quantified by PCR.

Male nude mice aged 4–6 weeks (weight, 14–15 g; GemPharmatech Biotechnology Co., Ltd.) were used, with five mice per experimental group. All mice were maintained under controlled temperature (22±1°C) and humidity (50±5%) in a 12/12-h light/dark cycle with food and water available ad libitum. A total of 1×10⁶ control cells and A549 cells with overexpression of HSPB7 were resuspended in phosphate-buffered saline and injected subcutaneously into the back flank of mice, and tumour volume was recorded every 3 days. Tumor volume >2,000 mm³ was considered the humane endpoint. The tumor volume was calculated as follows: Volume=0.5× length × width². At the end of the experiments, the weight of the mice was 18–19 g. After 27 days, mice were sacrificed by cervical dislocation under anesthesia (pentobarbital sodium, 50 mg/kg, intraperitoneal injection) and confirmed the sacrifice by cessation of heartbeat. Animal experiments were approved (approval no. W202203060148) by the Animal Ethics Committee of Jinan Central Hospital Affiliated to Shandong First Medical University (Jinan, China).

All statistical analyses were performed using GraphPad Prism 6 software (Dotmatics). All data are presented as the mean ± SD. Each experiment was performed in triplicate. Survival curves were analysed by the Kaplan-Meier method and compared by the log-rank test. Comparisons were analysed using the paired or unpaired Student's t-test or one-way ANOVA, followed by Tukey's post hoc test. Pearson's correlation analysis was performed to assess the correlation between MECOM and HSPB7 expression levels in the tumour tissues. P<0.05 was considered to indicate a statistically significant difference.

A total of 515 patients with LUAD were acquired from TCGA-LUAD cohort datasets and analyzed to identify the HSPB7 expression in LUAD. The detailed clinical features of the patients are listed in Table I. As revealed in Fig. 1A, HSPB7 expression was downregulated in LUAD tumour tissues compared with non-tumour tissues. Survival analysis demonstrated that patients with high HSPB7 expression had a higher survival rates (Fig. 1B). The detailed clinical features of the 23 tumor tissues and paired normal tissues are presented in Table II. The RT-qPCR and IHC data indicated that HSPB7 was expressed at low levels in LUAD tumour tissues compared with the paired normal tissues (Fig. 1C and D). In addition, the expression of HSPB7 protein was detected in lung cancer cell lines. HSPB7 expression showed different degrees of reduction in the H1975, H1688, H1299 and A549 cells compared with that in BEAS-2B cells (Fig. 1E). To further clarify the role of HSPB7 in LUAD, H1975 cells with the highest HSPB7 expression and A549 cells with the lowest expression were selected among the four cell lines for a follow-up study.

To improve understanding of the effects of HSPB7 on LUAD cell behaviour, cell proliferation, migration, invasion and EMT were evaluated. First, HSPB7 low expressing and overexpressing cells were constructed, and RT-qPCR and western blotting results revealed that the construction was successful (Fig. 2A and B). In the MTT and colony formation assays, it was observed that knockdown of HSPB7 promoted H1975 cell proliferation, while overexpression of HSPB7 inhibited A549 cell proliferation (Fig. 2C and D). In wound healing and Transwell experiments, the data also revealed that low expression of HSPB7 promoted H1975 cell migration and invasion, whereas overexpression of HSPB7 inhibited A549 cell migration and invasion (Fig. 2E and F). As western blotting results demonstrated, HSPB7 knockdown significantly reduced E-cadherin protein expression and increased the protein expression of N-cadherin and Snail in H1975 cells. Conversely, overexpression of HSPB7 increased the expression of E-cadherin protein and inhibited the protein expression of N-cadherin and Snail in A549 cells (Fig. 2G).

To support uncontrolled proliferation, migration and invasion, LUAD cells can shift their glucose metabolism pattern to aerobic glycolysis (21). It was hypothesized that HSPB7 participates in the regulation of aerobic glycolysis, thereby affecting LUAD progression. As revealed in Fig. 3A and B, low HSPB7 expression increased glucose consumption and lactic acid production in H1975 cells. On the contrary, when HSPB7 was overexpressed, A549 cells reduced glucose consumption and lactic acid production. Furthermore, GSEA revealed that HSPB7 was involved in glycolysis in LUAD cells (Fig. 3C). Therefore, it was hypothesized that HSPB7 participates in the regulation of LUAD by inhibition of glycolysis. To test this hypothesis, the levels of the glycolysis-associated proteins (LDHA, HK2 and PKM2) were examined in the H1975 and A549 cells. The results of western blotting indicated that HSPB7 knockdown increased the expression of LDHA, HK2 and PKM2, whereas HSPB7 overexpression inhibited the expression of LDHA, HK2 and PKM2 (Fig. 3D).

To analyze the relationship among HSPB7, glycolysis and tumour progression, the glycolysis inhibitor, 2-DG, was added into the H1975 cells that silenced HSPB7 expression. 2-DG is a glucose analogue that differs from glucose only by the removal of an oxygen atom at the C-2 position, which prevents the isomerization of glucose-6-phosphate to fructose-6-phosphate, thereby inhibiting glycolysis (22). As shown in Fig. 4A, silencing of HSPB7 promoted H1975 proliferation, but the addition of 2-DG weakened the promoting effect of si1-HSPB7 on cell proliferation. The cell migration and invasion abilities were significantly reduced in the si1-HSPB7 + 2-DG group compared with si1-HSPB7 group (Fig. 4B and C). In addition, it was observed that 2-DG significantly reduced the consumption of glucose and production of lactic acid induced by HSPB7 knockdown in H1975 cells (Fig. 4D and E).

TCGA dataset analysis identified that MECOM was poorly expressed in LUAD tumour tissues, and the survival rate of patients with low MECOM expression was also relatively low (Fig. 5A and B). Furthermore, MECOM levels were positively correlated with HSPB7 expression (Fig. 5C). Co-IP assays were conducted in H1975 and A549 cells. The results of Co-IP revealed that HSPB7 could interact with MECOM (Fig. 5D). According to JASPAR website analysis, the binding site of MECOM is located within 1 kb upstream of HSPB7. To verify this, ChIP analysis was performed and it was found that HSPB7 was significantly enriched in the MECOM group (Fig. 5E). The RT-qPCR results revealed that MECOM was downregulated in tumour tissues, and there was a positive correlation between the levels of MECOM and HSPB7 (Fig. 5F and G).

The expression levels of MECOM mRNA in the lung cancer cell lines were also measured. MECOM mRNA expression was decreased in H1975, H1688, H1299 and A549 cells compared with BEAS-2B cells (Fig. 6A). To clarify the roles of HSPB7 and MECOM in LUAD, rescue experiments were conducted in H1975 cells. As demonstrated in Fig. 6B, MECOM expression was increased in H1975 cells transfected with MECOM overexpression plasmid, whereas was decreased in H1975 cells transfected with si-MECOM. Overexpression of MECOM increased HSPB7 protein expression, while silencing of MECOM decreased HSPB7 protein expression (Fig. 6C). In terms of proliferation, overexpression of MECOM decreased the colony number of H1975 cells, but co-transfection of si1-HSPB7 reversed the inhibitory effect of MECOM on cell proliferation (Fig. 6D). The migration and invasion abilities of H1975 cells in the si1-HSPB7 + MECOM group were significantly higher than those in the MECOM group (Fig. 6E and F). In addition, the consumption of glucose and production of lactic acid by the H1975 cells in the si1-HSPB7+MECOM group were increased compared with MECOM group (Fig. 6G and H).

The effects of HSPB7 overexpression on the tumorigenic ability of A549 cells derived from nude mice were determined. Tumours derived from injected HSPB7 overexpression cells were significantly smaller than those derived from vector-transfected mice (Fig. 7A and B). The average weight of tumours from nude mice injected with HSPB7 overexpression cells was lower than that of tumours from nude mice injected with control A549 cells (Fig. 7C). The number of Ki67 positive cells, a marker of proliferation, in the HSPB7 overexpression group decreased significantly (Fig. 7D). These results suggested that overexpression of HSPB7 in A549 cells inhibited their tumorigenic ability.