The LUAD cell lines A549 and H1299, and the normal human bronchial epithelial cell line HBE were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in Dulbecco’s modiﬁed Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Invitrogen) at 37°C in a humidiﬁed atmosphere of 5% CO2.
Clinical tissue and specimens
A total of 20 fresh primary LUAD tissues and paired adjacent normal tissues were obtained from surgeries at Changhai Hospital, Second Military Medical University (Shanghai, China). Seventy-five paired paraffin-embedded LUAD specimens used in this study were collected from patients in 2013 who were diagnosed with primary LUAD and none of them received preoperative chemotherapy or radiotherapy. Overall survival (OS) was defined as the interval between surgery and death or last observation. This study was approved by the Ethics Committee of Changhai Hospital. All patients provided written informed consent upon enrolment.
Construction of reagents for gene overexpression and knockdown
We constructed lentiviral vectors encoding the human HKDC1 gene or green fluorescent protein (GFP) in the pLenti-EF1a-EGFP-P2A-Puro-CMV-MCS-3Flag vector (HeYuan Bio-technology Co., Shanghai, China), which were named ov-HKDC1 or ov-NC. Stable LUAD cell knockdown of HKDC1 was generated using lentiviral constructs expressing sh-HKDC1 (sh-HKDC1#1: GGTGGACAGGTTCCTGTAT), sh-HKDC1#2: GGTCAGTGCGAATGTACAA) or sh-NC. LUAD cells were infected for 48 h and then selected with puromycin. Stable LUAD cell lines were successfully established if the infection efficiency was >85%.
The LUAD tissue slides were incubated with anti-HKDC1 (1:200, Abcam, ab228729) primary antibody. IHC scoring was performed using a modified Histo-score (H-score) by two independent pathologists. Briefly, the proportion of positively stained cells was scored as 0–100% ( <25% scored 0, 25–50% scored 1, 50–75% scored 2, 75–100% scored 3) and the intensity score was scored as 0 (negative), 1+(weakly positive), 2+(moderately positive), or 3+ (strongly positive). A final score was then calculated by multiplying these two scores.
RNA extraction and quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from cultured LUAD cell lines or tissue specimens using TRIzol (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. The cDNA was synthesized using the PrimeScript RT Reagent Kit (TaKaRa Bio, Shiga, Japan) following the manufacturer’s instructions. Real-time PCR was performed on a Roche Light Cycler 480 (Roche) using SYBR Green PCR Master Mix (TaKaRa Bio, Shiga, Japan). The fold change relative to the mean value was determined by 2-ΔΔCt. All experiments were performed in triplicate. Primer sequences are listed as follows.
Whole cultured cells were homogenized in 0.1% SDS and 1 mM PMSF. Protein extracts were subjected to SDS-PAGE and analyzed using the following primary antibodies against the following antigens: HKDC1(Abcam, ab228729), phospho-AMPKα(Cell Signaling Technology, 2535), phospho-mTOR(Cell Signaling Technology, 5536), phospho-p70S6(Cell Signaling Technology, 9234), Vimentin(Santa Cruz, sc-6260), Snail(CST, 3879), E-cadherin (Abcam, ab40772), N-cadherin(Abcam, ab18203) and GAPDH (Abcam, ab8245). Then, the membranes were incubated with secondary antibodies (CST, 7076, 7074) at room temperature for 1 h. All experiments were performed in triplicate. The results of western blotting bands were quantified by measuring the gray values (ImageJ software, Rawak Software Inc, Stuttgart, Germany).
AICAR (CST, 9944) was used to activate AMPK signaling and rapamycin (Abcam, ab120224) was used to inhibit mTOR signaling in LUAD cells. LUAD cells were incubated at 37°C for 24 h with 3 mM AICAR and 100 μM rapamycin for subsequent experiments.
We utilized the Oncomine (https://www.oncomine.org/resource/login.html) and UALCAN (http://ualcan.path.uab.edu/analysis.html) databases to detect the expression level of HKDC1 genes.
Measurement of glucose and lactate
Transfected A549 and H1299 cells were seeded in 6-well plates (5×105) and the culture media were harvested 48 h after transfection. The glucose and lactate levels were measured using a Glucose Assay Kit (Sigma-Aldrich, USA) and a Lactic Acid Assay Kit (Sigma-Aldrich, USA), respectively, according to the manufacturer’s protocol. The values were normalized to the total protein concentration. All experiments were performed in triplicate.
Cell proliferation assays
Cell viability was measured by Cell Counting Kit-8 (CCK-8, Bimake, USA). Briefly, transfected cells were seeded in 96-well plates (5000 cells/well) and cultured for 3 days to assess proliferation. The absorbance was measured at 450 nm. All experiments were performed in triplicate.
Cell migration and invasion assay.
Cell migration and invasion ability was assessed by 24-well Transwell chambers (Corning) with or without a Matrigel (Corning, Bedford, MA, USA) coating. Briefly, approximately 1*105 cells were resuspended in 300 µL serum-free DMEM and seeded into the upper chambers, whereas the bottom chamber was filled with 500 µL 10% FBS medium. Twenty-four hours later, the migrated/invaded cells in the lower chamber were fixed with 4% paraformaldehyde and stained with 1% crystal violet. All experiments were performed in triplicate. Cell counting was accomplished by ImageJ software (Rawak Software Inc, Stuttgart, Germany).
Five-week-old male BALB/c nude mice were purchased from Shanghai Experimental Center (Shanghai, China). Xenograft tumor models were established by subcutaneous injection of ovHKDC1 or shHKDC1 (5×106) transfected A549 cells into the right dorsal flank. Three mice were randomly assigned to each group. One week after injection, the mice were measured once a week for a total of 4-5 weeks. Tumor volume (V, cm 3) was evaluated by a slide caliper (Shanghai, China) based on tumor length (L) and width (W) with the following formula: V= 1/2×L×W2. All manipulations were performed in accordance with currently prescribed guidelines and under a protocol approved by the SMMU Ethical Review Committee (Shanghai, China).
Data analysis was carried out using IBM SSPS 24 (IBM Corp., Armonk, NY, USA) and data were reported as the mean ± standard deviation (mean ± SD). Student’s t-test was used to determine differences between groups and two-tailed ANOVA was performed in cases of multiple groups. The association between genes and clinicopathological features was analyzed by the chi- square test or Fisher’s exact test. Kaplan-Meier curves were used to compare the OS between groups. Multivariate analysis was performed to determine independent factors affecting the prognosis of patients. Differences were considered statistically significant when P < 0.05.