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Introduction

The global incidence and mortality rates of lung cancer remain high and the five-year survival rate has been reported to be ~20% (1–3). Lung adenocarcinoma (LUAD) is one of the most common pathological subtypes of lung cancer, accounting for ~40% of cases (4,5). Despite considerable advances in anticancer therapy, the overall survival and prognosis of patients with LUAD has not improved significantly (6,7). Therefore, an improved understanding of the drivers of LUAD and the molecular mechanisms could improve the diagnosis and treatment of this deadly disease.

Heat shock proteins act as chaperones at the molecular level and have been investigated in numerous diseases associated with oxidative stress, including obesity (8,9). A specific subfamily of heat-shock proteins are the HSPB family of molecular chaperones, which comprises ten members (HSPB1-10, also called small HSP) (10). HSPB7 is a member of the HSPB family and has been shown to be ineffective in suppressing the amorphous aggregation of model proteins, however, it is very effective in preventing the aggregation of huntingtin fragments enriched with Gln residues (11). HSPB7 is crucial for heart development as it modulates actin filament assembly (12). HSPB7 interacts with dimerized filamin C, and its absence results in progressive myopathy in skeletal muscle (13).

Other genes in the HSPB family have been associated with glycolysis. Increased glycolysis, oxidative phosphorylation, and stem cell characteristics of esophageal cancer stem-like cells depend on the Hsp27 (HSPB1)-AKT-HK2 pathway (14). The bidirectional gene HspB2/αB-crystallin may be involved in the levels of reactive oxygen species and glycolysis in MCF7 cells (15). However, the effect of HSPB7 on the expression of glycolytic enzymes and level of glycolysis has not been previously reported. It was hypothesized that HSPB7 may indirectly affect the expression of glycolytic enzymes and the level of glycolysis, but further studies are required to elucidate the molecular mechanisms by which HSPB7 participates in glycolysis.

Increasing evidence has indicated that HSPB7 acts as a tumour suppressor in a variety of malignant tumours (16,17). However, there is no study on the role of HSPB7 in LUAD. In the present study, it was hypothesized that HSPB7 participates in LUAD progression and its potential role and mechanism of action were investigated. Preliminary analysis revealed that HSPB7 was significantly downregulated in LUAD tumour tissues and cells, suggesting that it may be a candidate tumour suppressor gene in LUAD.

Materials and methods

Bioinformatics analysis

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/).

Tissue collection

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.

Cell culture and transfection

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.

RT-qPCR

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.

Western blotting

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.

Immunohistochemistry (IHC) analysis

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).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-Htetrazolium bromide (MTT) assay

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.).

Colony formation

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).

Wound healing assay

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).

Transwell assay

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).

Glucose and lactate measurement

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.).

Co-Immunoprecipitation (Co-IP)

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.

Chromatin immunoprecipitation (ChIP)

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.

Tumorigenesis assay

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).

Statistical analysis

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.

Results

HSPB7 expression is downregulated in LUAD tissues and cells

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.

Figure 1. HSPB7 is downregulated in LUAD tissues and cells. (A) HSPB7 expression in the TCGA-LUAD cohort datasets was analysed. **P<0.01 by unpaired Student's t-test. (B) Kaplan-Meier survival analysis based on TCGA-LUAD datasets was analysed. (C) The expression of HSPB7 in 23 cases of LUAD tumour and paired normal tissues was determined through reverse transcription-quantitative PCR. Data are shown as the mean ± SD (n=3). **P<0.01 by paired Student's t-test. (D) The protein expression of HSPB7 in tumour and paired normal tissues of LUAD patients was detected by immunohistochemistry. (E) The protein expression of HSPB7 in human normal lung epithelial cells (BEAS-2B) and lung cancer cells (H1975, H1688, H1299 and A549) was detected by western blotting. Data are presented as the mean ± SD (n=3). **P<0.01 vs. BEAS-2B by one-way ANOVA test, followed by Tukey's post hoc test. HSPB7, heat shock protein B7; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas.

Table I. Clinical characteristics of the lung adenocarcinoma patients from The Cancer Genome Atlas.

Table I.

Clinical characteristics of the lung adenocarcinoma patients from The Cancer Genome Atlas.

Clinical characteristics	Number	Percentage, %
Age, years
<65	239	46.4
≥65	276	53.6
Sex
Female	277	53.8
Male	238	46.2
Stage
I	275	53.4
II	122	23.7
III	73	14.2
IV	11	2.1
T classification
T1	169	32.8
T2	277	53.8
T3	47	9.1
T4	19	3.7
TX	3	0.6
M classification
M0	346	67.2
M1	25	4.9
MX	140	27.2
Missing data	4	0.8
N classification
N0	331	64.3
N1	96	18.6
N2	74	14.4
N3	2	0.4
NX	11	2.1
Missing data	1	0.2
ICD-10
C34.0	2	0.4
C34.1	310	60.2
C34.2	20	3.9
C34.3	174	33.8
C34.8	4	0.8
C34.9	5	1.0

[i] ICD, International Classification of Diseases.

Table II. Clinical characteristics of the lung adenocarcinoma patients from our hospital.

Table II.

Clinical characteristics of the lung adenocarcinoma patients from our hospital.

Clinical characteristics	Number	Percentage, %
Age, years
<65	9	39.1
≥65	14	60.9
Sex
Female	10	43.5
Male	13	56.5
Stage
I	8	34.8
II	11	47.8
III	3	13.0
IV	1	4.3
T classification
T1	7	30.4
T2	13	56.5
T3	2	8.7
T4	1	4.3
M classification
M0	20	87.0
M1	3	13.0
N classification
N0	19	82.6
N1	3	13.0
N2	1	4.3
ICD-10
C34.1	15	65.2
C34.3	8	34.8

[i] ICD, International Classification of Diseases.

HSPB7 restricts LUAD cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT)

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).

Figure 2. HSPB7 restricts lung adenocarcinoma cell proliferation, migration, invasion and EMT. (A) The H1975 and A549 cells were transfected with si-NC, si1-HSPB7, si2-HSPB7, vector, and HSPB7. HSPB7 expression in H1975 and A549 cells was determined through reverse transcription-quantitative PCR. (B) HSPB7 protein expression in H1975 and A549 cells was measured using western blotting. The H1975 and A549 cells were transfected with si-NC, si1-HSPB7, vector and HSPB7. (C) MTT assay, (D) colony formation assay, (E) wound healing assay and (F) Transwell assay were used to evaluate H1975 and A549 cell proliferation, migration and invasion. (G) EMT-related proteins, including E-cadherin, N-cadherin and Snail were detected in H1975 and A549 cells via western blotting. Data are shown as the mean ± SD (n=3). **P<0.01 vs. si-NC or vector group by unpaired Student's t test. HSPB7, heat shock protein B7; EMT, epithelial-mesenchymal transition; si-, small interfering; NC, negative control.

HSPB7 inhibits glycolysis

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).

Figure 3. HSPB7 inhibits glycolysis. The H1975 and A549 cells were transfected with si-NC, si1-HSPB7, vector and HSPB7. (A) Glucose consumption in H1975 and A549 cells was detected using Glucose Assay kit. (B) Lactic acid production in H1975 and A549 cells was evaluated using Lactic Acid Assay kit. (C) Gene set enrichment analysis showed that HSPB7 could inhibit glycolysis. (D) Western blotting was used to detect glycolysis-associated proteins (PKM2, LDHA and HK2) in H1975 and A549 cells. Data are shown as the mean ± SD (n=3). **P<0.01 vs. si-NC or vector group by unpaired Student's t test. HSPB7, heat shock protein B7; si-, small interfering; NC, negative control; PKM2, pyruvate kinase muscle isoform 2; LDHA, lactate dehydrogenase A; HK2, hexokinase 2.

HSPB7 inhibits the proliferation, migration, and invasion of LUAD cells by regulating glycolysis

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).

Figure 4. Silencing HSPB7 promotes the proliferation, migration, and invasion of lung adenocarcinoma cells by regulating glycolysis. The H1975 cells were transfected with si-NC or si1-HSPB7, followed by treating with or without glycolysis inhibitor 2-DG. (A) Colony formation assay, (B) wound healing assay and (C) Transwell assay were used to evaluate H1975 cell proliferation, migration and invasion. (D) Glucose consumption in H1975 cells was detected using Glucose Assay kit. (E) Lactic acid production in H1975 cells was evaluated using Lactic Acid Assay kit. Data are shown as the mean ± SD (n=3). **P<0.01 vs. si-NC group; #P<0.05 vs. si1-HSPB7 group; ##P<0.01 vs. si1-HSPB7 group by one-way ANOVA test, followed by Tukey's post hoc test. HSPB7, heat shock protein B7; si-, small interfering; NC, negative control; 2-DG, 2-deoxy-D-glucose.

MECOM is a transcription factor of HSPB7

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).

Figure 5. MECOM is transcription factor of HSPB7. (A) MECOM expression in the TCGA-LUAD cohort datasets was analysed. (B) Kaplan-Meier survival analysis based on TCGA-LUAD datasets was analysed. (C) Correlation analysis between MECOM and HSPB7 based on TCGA-LUAD datasets using Pearson's correlation analysis. (D) Co-IP confirmed the binding between HSPB7 and MECOM in H1975 and A549 cells (E) Chromatin Immunoprecipitation assay was used to verify the binding ability of HSPB7 to transcription factor MECOM; Data are shown as mean ± SD (n=3). **P<0.01 vs. lgG group by unpaired Student's t-test. (F) The expression of MECOM in twenty-three cases of LUAD tumour and paired normal tissues was detected by reverse transcription-quantitative PCR; Data are presented as the mean ± SD (n=3). **P<0.01 by paired Student's t-test. (G) Correlation analysis of MECOM and HSPB7 in LUAD tumour tissues using Pearson's correlation analysis. **P<0.01 by paired Student's t-test. MECOM, myelodysplastic syndrome 1 and ecotropic viral integration site 1 complex locus; HSPB7, heat shock protein B7; TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; Co-IP, Co-Immunoprecipitation; HR, hazard ratio.

HSPB7 is regulated by the transcription factor MECOM

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).

Figure 6. HSPB7 is regulated by the transcription factor MECOM. (A) The mRNA expression of MECOM in human normal lung epithelial cells (BEAS-2B) and lung cancer cells (H1975, H1688, H1299 and A549) was detected using RT-qPCR. The H1975 cells were transfected with si-NC, si1-HSPB7, MECOM, si1-MECOM, or si2-MECOM. (B) The mRNA expression of MECOM in H1975 cells was measured using RT-qPCR. (C) HSPB7 protein expression in H1975 cells was measured using western blot analysis. (D) Colony formation assay, (E) wound healing assay and (F) Transwell assay were respectively to evaluate H1975 cell proliferation, migration and invasion. (G) Glucose consumption in H1975 cells was detected using Glucose Assay kit. (H) Lactic acid production in H1975 cells was evaluated using Lactic Acid Assay kit. Data are shown as the mean ± SD (n=3). *P<0.05 vs. BEAS-2B or si-NC group; **P<0.01 vs. BEAS-2B, si-NC, or vector group; ##P<0.01 vs. MECOM group by one-way ANOVA test with Tukey's post hoc test. HSPB7, heat shock protein B7; MECOM, myelodysplastic syndrome 1 and ecotropic viral integration site 1 complex locus; RT-qPCR, reverse transcription-quantitative PCR; si-, small interfering; NC, negative control.

HSPB7 suppresses tumour growth

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.

Figure 7. HSPB7 suppresses tumour growth. (A) Representative image of tumour xenografts harvested at day 27. (B) Tumour growth curves from the two groups. (C) Tumour weights from the two groups. (D) The Ki67 positive cell in the two groups was detected by immunohistochemistry. (E) A schematic diagram of HSPB7 hindering lung adenocarcinoma progression through inhibition of glycolysis. Data are presented as the mean ± SD (n=5). **P<0.01 by unpaired Student's t-test. HSPB7, heat shock protein B7; MECOM, myelodysplastic syndrome 1 and ecotropic viral integration site 1 complex locus; EMT, epithelial-mesenchymal transition.

Discussion

In the present study, the influence and underlying molecular mechanisms of HSPB7 on the biological behaviour of lung cancer cells were investigated. The present findings revealed that HSPB7 expression was downregulated in LUAD tissues and cells. MECOM was a transcription factor of HSPB7. Knockdown of HSPB7 promoted lung cancer cell proliferation, migration and invasion; all of which were related to glycolysis (Fig. 7E).

Late diagnosis and complex clinical features lead to poor prognosis in LUAD (23,24). Precision medicine targeting the epidermal growth factor receptor, anaplastic lymphoma kinase, and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) has contributed significantly to the effective treatment of LUAD (25,26). Although targeted therapy has made some progress, an increasing number of specific molecular biomarkers and therapeutic targets are required to guide and improve the prognosis and treatment of LUAD. A previous study reported that HSPB7 expression is downregulated by methylation and that HSPB7 acts as a tumour suppressor regulated by p53 in renal cell carcinoma (17). In the present study, it was found that HSPB7 was significantly downregulated in LUAD tissues and cells, and that its aberrant expression correlated with survival in patients with LUAD. In addition, silencing of HSPB7 promoted LUAD cell proliferation, invasion and migration. HSPB7 acted as a tumour suppressor regulated by MECOM in LUAD. Naderi (16) confirmed that knockdown of HSPB7 promotes the proliferation of human breast cancer cells. EMT is closely related to primary invasion and secondary metastasis of a variety of tumours and its inhibition could prevent cancer progression (27). E-cadherin transforms into N-cadherin during EMT, and expression of E-cadherin, an epithelial cell marker, is downregulated (28). The E-cadherin suppressor, Snail, facilitates cancer cell migration and invasion (29). In the present study, silencing of HSPB7 promoted EMT in LUAD cells, which showed a decrease in E-cadherin protein and an increase in N-cadherin and Snail proteins; whereas, overexpression of HSPB7 led to the opposite findings. Taken together, these results suggested that HSPB7 plays a role in LUAD progression.

Increasing evidence has indicated that glycolysis promotes cancer progression (30,31). Therefore, it is a new strategy to delay tumor progression by inhibiting glycolysis in cancer cells. In cancer cells, elevated glycolysis promotes glucose uptake and lactic acid production to meet metabolic needs, thereby increasing cell proliferation and metastasis (7). Zhou et al (31) demonstrated that silencing of circRNA enolase 1 inhibits glucose uptake and lactic acid production, thereby inhibiting glycolysis and ultimately LUAD progression. Consistently, the present data demonstrated that HSPB7 reduced glucose consumption and lactic acid production in LUAD cells. In addition, the inhibition of glycolysis by 2-DG weakened the promoting effect of knockdown of HSPB7 on cell proliferation, migration and invasion. LDHA, HK2 and PKM2 are important factors in glycolysis and their overexpression is associated with poor prognosis of patients with lung cancer (32,33). The present study demonstrated that HSPB7 inhibits the expression of LDHA, HK2 and PKM2 in lung cancer cells. Collectively, HSPB7 inhibited the proliferation, migration and invasion of LUAD cells, all of which are related to glycolysis.

MECOM, also known as MDS1/EVI1, is located on chromosome 3q26.2. It encodes the MDS1 and EVI1 complex locus proteins and consists of 1051 amino acids (34). MECOM encodes zinc-finger transcription factors and participates in a series of biological processes by regulating downstream gene expression (35). In addition, MECOM is a tumour suppressor that plays an important role in normal development and tumourigenesis (36). Li et al (37) reported that in LUAD, the mRNA levels of MECOM are downregulated and that it may serve as a potential prognostic biomarker. In the present study, MECOM expression was downregulated in LUAD tissues. The ChIP assay confirmed that MECOM directly regulated the transcription of HSPB7 in LUAD. Furthermore, overexpression of MECOM inhibited proliferation, migration, invasion, glucose consumption and lactic acid production in lung cancer cells; while knockdown of HSPB7 attenuated the inhibitory effect of MECOM on cell behaviour. Taken together, these findings confirmed that HSPB7 was regulated by MECOM and participated in the progression of LUAD.

There were certain limitations to the present study. First, an in vivo metastatic assay was not performed. Secondly, the effect of HSPB7 on apoptosis remains unclear. Future studies could address these limitations.

In summary, HSPB7 was expressed at low levels in LUAD tissues and cells, and overexpression of HSPB7 inhibited lung cancer cell proliferation, invasion and migration. HSPB7 was regulated by the MECOM and inhibited LUAD progression by inhibiting glycolysis. The findings of the present study provide a new insight into HSPB7 expression in patients with LUAD.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

ZTC conceived and designed the study. PPL, LGS and XYJ provided study materials. PPL, LGS and XYJ collected and assembled the data. ZTC analyzed and interpreted the data. LGS and XYJ confirm the authenticity of all the raw data. All authors participated in manuscript writing, read and approved the final version of the manuscript.

Ethics approval and consent to participate

The protocols of human studies (W202203060147) and animal experiments (W202203060148) were approved by the Ethics Committee of Jinan Central Hospital Affiliated to Shandong First Medical University (Jinan, China). Written informed consent was provided by all patients.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References