Fetal bovine serum (FBS) and cell culture medium were purchased from HyClone (South Logan, UT, USA). MG132 and kanamycin were purchased from Sigma (St. Louis, MO, USA). JNKIN8 was obtained from MedChem Express (Shanghai, China). Plasmids for overexpressing HMGA1 or GRP75 and HMGA1-specific short hairpin RNA (shRNA) were designed and synthesized by GeneChem (Shanghai, China). Small interfering RNA (siRNA) oligonucleotides targeting HMGA1 or GRP75 were obtained from RiboBio (Guangzhou, China). A cell counting kit-8 (CCK8) kit was purchased from Bioss (Beijing, China). A migration assay kit was obtained from BD Biosciences (Franklin Lakes, NJ, USA). Cell invasion assay chambers (24 wells) were purchased from Corning (NY, USA). Cycloheximide (CHX) and primary antibodies against HMGA1, GRP75, Ki67, Ubiquitin, JNK, P-JNK (Thr183/Tyr185), c-JUN, P-c-JUN (Ser63) and P-c-JUN (Ser73) and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). A Pierce coimmunoprecipitation kit was obtained from Thermo Fisher Scientific (Waltham, MA, USA).
Cell lines, cell transfection and RNA interference
The lung adenocarcinoma cell lines A549 and PC9 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS at 37 °C in a humidified atmosphere with 5% CO2.
Cells were transfected with the indicated plasmids or siRNAs using Lipofectamine 3000 (Invitrogen, CA) according to the manufacturer’s protocol. The siRNA sequences against HMGA1 were as follows: 5′-GAAGTGCCAACACCTAAGA-3′ and 5′-AGCGAAGTGCCAACACCTA-3′. The siRNA sequences against GRP75 were as follows: 5′-TGCCTATGGTCTAGACAAA-3′ and 5′-GGATTGTCACTGATCTAAT-3′. The control siRNA sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′. The HMGA1-specific short hairpin RNA sequence was 5′-GATCCCCTGCTACCAGCGCCAAATGTTCTCGAGAACATTTGGCGCTGGTAGCAGTTTTTGGAT-3′, and the control sequence was 5′-TTCTCCGAACGTGTCACGT-3′. After transfection for 48 or 72 hours, the cells were subjected to further analyses.
Transwell and cell viability assays
The migratory and invasive abilities of LUAD cells were assessed with a transwell assay. The indicated cells were transfected with the indicated oligonucleotides or plasmids for 48 h. Then, 3 × 104 cells in serum-free growth medium were seeded in the upper chambers. The lower chambers were filled with the same medium supplemented with 10% FBS. After 24 h, the cells that had invaded the lower side of the chamber were fixed with 4% paraformaldehyde (PFA), stained with 0.1% crystal violet and dried. The number of invading cells was counted under a light microscope.
For cell viability assays, the indicated cells were plated in 96-well plates at 4 × 103 cells per well. At the indicated time points, cell viability was estimated by using CCK8 reagent according to the manufacturer’s instructions. Absorbance was measured at a wavelength of 450 nm by using a microplate reader.
Colony formation assays
After 48 h of transfection, cells were trypsinized and resuspended in 0.5 ml 0.35% agar in growth medium at a density of 1000 cells/well (24-well plate). Then, the agar-cell mixture was plated on top of a solid layer of 0.8% agar in the growth medium. The cells were cultured until visible clones appeared (approximately 3 weeks). The cell clones were washed twice with PBS, fixed with methanol for 20 min, stained with 0.1% crystal violet for 1 h, washed with PBS, and counted.
Western blot analysis
Western blot analysis was carried out according to a standard protocol. Cell lysates were generated from cultured cells with RIPA lysis buffer containing protease inhibitors (Roche). The protein concentration was determined by using a Bradford kit (Pierce, Thermo Fisher Scientific). Equal amounts of total protein were loaded, separated by SDS-PAGE and then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking in 5% nonfat milk, the membranes were incubated overnight at 4 °C with appropriate primary antibodies and then with secondary antibodies conjugated to horseradish peroxidase for 2 hours at room temperature. After washing, the bands of interest were analyzed by using an ECL kit. Images were acquired using the Bio-Spectrum Gel Imaging System (Bio-Rad, USA). Then, quantification of the Western blot results was performed using Image Lab software, and the bands were normalized with GAPDH as the internal control.
Immunoprecipitation and mass spectrometry
Immunoprecipitation was performed using a Thermo Fisher Scientific Pierce coimmunoprecipitation kit following the manufacturer’s protocol. Briefly, the indicated antibodies were first immobilized for 2 h using AminoLink Plus coupling resin. The resin was then washed and incubated with lysate overnight at 4 °C. After the incubation, the resin was washed, and proteins were eluted using elution buffer. A negative control that was provided with the co-IP kit to assess nonspecific binding received the same treatment as the samples with IgG. The immunoprecipitated samples were resolved by SDS-PAGE, followed by Western blotting with appropriate antibodies.
For liquid chromatography-mass spectrometry (LC-MS) analysis, samples, which included those with HMGA1 overexpression and a control, were separated using electrophoresis, and specific bands were identified using a mass spectrometry system; the molecules corresponding to the bands were identified in a human proteomic library to obtain proteins directly bound to HMGA1.
Degradation and deubiquitylation assay
To detect HMGA1 degradation mediated by GRP75 in A549 cells, cells with or without GRP75 overexpression were treated with CHX (CST, 100 µg/ml) for the indicated durations before analysis. Then, cell lysates were analyzed using Western blotting with an anti-HMGA1 antibody. To detect HMGA1 deubiquitination in A549 cells, cells were transfected with the indicated plasmids and treated with 20 µM proteasome inhibitor MG132 (Sigma) for 8 h before harvest. Then, the cells were lysed in IP lysis/wash buffer and incubated with the anti-HMGA1 antibody, which was used for IP. The immunoprecipitated samples were resolved by SDS-PAGE, followed by Western blotting with an anti-ubiquitin antibody.
Publicly available databases were used to evaluate gene expression and predict potential mechanisms. A clinical dataset of stage I LUAD patients was obtained from the TCGA database and analyzed using TIMER. mRNA-sequencing analysis was performed by Huada Genomics Institute (BGI, Guangzhou, China), and KEGG pathway analysis was conducted with the Dr. Tom program (https://biosys.bgi.com).
Animal studies were conducted at Daping Hospital, according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All animal experiments were approved by the Animal Research Committee of Army Medical University. Six-week-old female nude mice were utilized. To investigate the effects of oncogenic HMGA1 on LUAD cell tumorigenicity, the indicated cells were transfected with HMGA1 or HMGA1-specific shRNA-expressing or control plasmids. After 72 h of transfection, 2 × 106 cells in 0.1 mL PBS were injected subcutaneously (five mice per group). Tumor length (L) and width (W) were measured weekly, and tumor volume (V) was calculated using the formula V = L × W2/2. Four weeks after cell injection, these mice were sacrificed, and the tumors were harvested and weighed. To investigate the presence of Ki67 proteins in mouse tumor tissues, an immunohistochemistry assay was performed as described previously. For metastatic ability experiments, four groups of five mice each were given tail vein injections of 2 × 106 the indicated cells, respectively. Four weeks after injection, all mice were sacrificed, the tumor nodules formed on the lung surface were counted. The lungs were embedded with paraffin and sliced for HE staining.
All data are presented as the mean ± SD, and statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc., La Jo lla, CA, USA). The statistical significance of differences between treatment groups was analyzed by a two-tailed paired t-test or one-way analysis of variance (ANOVA). The survival rate was calculated using Kaplan-Meier survival analysis. P < 0.05 was considered statistically significant.