Patients and Gastric Tissue Sample
The study was primarily based on 2 independent patient cohorts. Cohort 1 included 349 gastric cancer tissues collected from January 2010 to April 2014 at Fujian Medical University Union Hospital. Gastric tissue specimens included tumor tissues of the stomach and adjacent non-tumor tissues. Cohort 2 included 93 gastric cancer tissues collected from January 2010 to April 2014 at Qinghai University Hospital for external validation. The inclusion criteria were as follows: (a) histological identification of gastric cancer; (b) availability of follow-up data and clinicopathological characteristics; (c) TNM staging of gastric cancer tumors according to the 2010 International Union Against Cancer (UICC) guidelines. The exclusion criteria were as follows: (1) patients with no formalin-fixed paraffin-embedded (FFPE) tumor sample (CT and IM) from initial diagnosis; (2) patients who received chemotherapy or radiotherapy before surgery. All participating patients with advanced GC routinely received fluorine-based chemotherapy (Table 1).
Construction of Tissue microarray (TMA)
From January 2010 to April 2014, a total of 123 gastric cancer tissue samples were selected. Briefly, the pathologist examined all gastric cancer tissues, and marked the paraffin blocks based on the tumor position of the HE stained section and immunohistochemical slides, and selected more areas of the tumor tissue without the representation of necrosis and hemorrhagic material Area to prepare tissue chips for experiments. Mix paraffin wax and an equal amount of beeswax to make 2 blank wax blocks. Create a puncture hole with a diameter of 1 mm in blank paraffin to separate the two holes, and perform 80 punches. For each sample, a 1.5 mm core was punched from the donor block using a tissue microarray instrument. Use a tissue analyzer to sample the tumor-marked wax block, put the sampled tissue into the corresponding channel of the blank wax block, and transfer the determined array position to the recipient paraffin block. Several serial sections (4 μm thick) were cut from all TMAs, and one section of each TMA was stained with hematoxylin-eosin to ensure that the TMA was constructed correctly. The intratumor dot was derived from the center of the tumor, while the peritumor dot was punched out from the area ≥2 cm from the tumor margin. The prepared TMA slides are used for immunohistochemistry (IHC).
Follow-up
All patients were systematically followed up by trained doctors who abided by the institutional follow-up protocol; options for follow-up included outpatient services, letters, telephone, mail or visits. Follow-up was conducted every 3–6 months for the first 2 years, every 6–12 months for the 3–5 years, and annually thereafter. Survival time was defined as the time from the date of surgery until the date of last follow-up or death. All 565 patients involved in the IHC analysis completed the follow-up.
Immunohistochemistry (IHC)
The serial sections of the FFPE sample were 4μm and mounted on a glass slide for IHC analysis. The sections were deparaffinized with xylene and rehydrated with alcohol. We blocked the endogenous peroxidase by immersing the slices in a 3% H2O2 aqueous solution for 10 minutes and microwaved them in 0.01 mol/L sodium citrate buffer (pH 6.0) for 10 minutes for antigen retrieval. The slides were then washed in phosphate-buffered saline (PBS) and then incubated with 10% normal goat serum (Zhongshan Biotechnology Co., Ltd., China) to eliminate non-specific reactions. Subsequently, the primary antibody was incubated with the antibody overnight at 4°C. The treatment of the negative control is the same, but the primary antibody is omitted. After rinsing 3 times with PBS, dilute the slide and secondary antibody for 30 minutes at room temperature, and develop with diaminobenzidine (DAB) solution. Finally, the slides were counter-stained with hematoxylin, dehydrated, and fixed with a cover glass and neutral resin.
We performed LHPP (NBP1-83273, Novus, 1:300), METTL14 (ab220030, abcam, 1:1000), HIF-1α (ab243861, abcam, 1:500), GSK-3β (phospho S9) (ab75814, abcam, 1:200) immunohistochemical staining on the tumor tissue of gastric cancer patients. The staining intensity and average percentage of positive cells in 5 randomly selected regions were evaluated to represent the protein expression level. The scoring criteria are as follows: staining intensity is divided into 0 (negative staining), 1 (weak staining, light yellow), 2 (medium staining, yellow-brown) or 3 (strong staining, brown), and positive staining of tumor cells The proportion is divided into 0 (≤5% positive cells), 1 (6%-25% positive cells), 2 (26%-50% positive cells) or 3 (≥51% positive cells) cells). The final expression is calculated by multiplying the staining intensity score by the proportional staining score (total 0 to 9). Patients with final scores of 0, 1, 2, and 3 were classified as low expression group, and patients with scores 4, 6, and 9 were classified as high expression group.
The IHC results were evaluated by two independent gastroenterology pathologists who were blinded to the clinical data prognosis of the patients. Approximately 90% of the scoring results are the same. When the scores of the two independent pathologists diverged, another pathologist checked the results again and chose one of the scores of the first two doctors, or the three pathologists discussed the decision together.
Gene set enrichment analysis
Gene set enrichment analysis (GSEA) performed by the Molecular Signature Database (MSigDB) was used to identify the pathways and functions that were significantly enriched in LHPP low tumor samples. If a gene set had a positive enrichment score, the majority of its members had higher expression accompanied with higher risk score, and the set was termed ‘enriched’.
Statistical analysis
All data were processed using SPSS 25.0 (SPSS Inc. Chicago, IL) and R software (version 4.0.0). The Kaplan-Meier method was used to estimate median survival. We defined the survival time of patients who were lost to follow-up as the time from surgery to the last follow-up time, and the survival time of patients who were still alive in the end was defined as the time from surgery to the database deadline. A two-tailed P values < 0.05 were considered significant differences.
RNA-sequencing analysis
Total RNA extraction was performed with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA-sequencing analysis was performed at KangChen Bio-tech Inc. (Shanghai, China).
Establishment of cell lines
Overexpression and knockdown lentiviruses for LHPP (NM_022126.4) as well as control lentivirus were purchased from GeneChem Corporation (Shanghai, China). Transfection was performed according to the manufacturer’s instructions. Puromycin (2 μg/ml, Sigma) was used to select stable clones for at least 1 week. At the indicated time points, the cells were harvested for mRNA and protein analysis as well as for other assays.
RNA immunoprecipitation (RIP)
LHPP m6A immunoprecipitation was performed using a Magna MeRIP m6A Kit (17–10499, Merck Millipore, USA) according to the manufacturer’s protocol, and the immunoprecipitated RNA extracts were reversetranscribed and analyzed by qRT-PCR.
Human Phospho-kinase Array
The relative levels of protein phosphorylation were tested using the Human Phospho-Kinase Array Kit (ARY003B, R&D Systems, Inc. USA & Canada) according to the manufacturer’s protocol. An equal amount of protein (600 mg) was extracted from stable cells (Overexpression LHPP HGC-27 and Control HGC-27) and used to compare the kinase activity with or without LHPP overexpression.
Tumor formation and metastasis assays
All male BALB/c nude mice (4–5 weeks old) used in our study were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. A total of 5 × 106 stably transfected MGC-803 cells were subcutaneously injected into the right axillary fossa of nude mice. Tumor volume was measured every 3 days and calculated with the following formula: V = (L ×W2)/2 cm2 (V, tumor volume; L, length; W, width). The mice were sacrificed at 3–4 weeks after injection, and the tumors were weighed. For the lung or liver metastasis model, 5 × 106 stably transfected MGC-803 cells were injected into the tail veins or the spleen of nude mice. Forty-five days later, the mice were sacrificed, and the lungs or the livers were dissected to examine the histopathological metastatic loci. The peritoneal dissemination ability of GC cells was evaluated via intraperitoneal injection. A total of 5 × 106 stably transfected MGC-803 cells in 500 μl of PBS were injected into the peritoneal cavity of BALB/c nude mice. Mice were carefully monitored until they were killed at 4 weeks, at which point peritoneal metastases were examined and recorded. All animal experiments were performed according to the Animal Protection Committee of Fujian Medical University (Fuzhou, China) and approved by the Ethics Committee of Fujian Medical University/Laboratory Animal Center (Fuzhou, China).
Western blot assay
Samples and cells were collected for Western blotting as previously described. Western blot analysis was performed using the following antibodies: LHPP (NBP1-83273, Novus, 0.2 ug/ml), METTL14 (ab220030, abcam, 1:1000), HIF-1α (ab243861, abcam, 1:1000), β-ACTIN (ab8226, abcam, 1:2000), Acetyl Lysine (ab190479, abcam, 1:1000), P300 (ab10485, abcam, 1:5000), TIP60 (ab151432, abcam, 1:1000), GCN5 (ab282176, abcam, 1:1000), PCAF (ab176316, abcam, 1:1000), GLUT1 (ab115730, abcam, 1:5000), c-Myc (ab32072, abcam, 1:1000), PKM2 (ab137852, abcam, 1:1000), ALDOLASE (ab252953, abcam, 1:1000), ENOLASE1 (H00002023-M01, Novus, 1:500), GLS1 (H00002744-M01, Novus, 1:500), GSK-3β (phospho S9) (ab75814, abcam, 1:5000), GSK-3β (ab32391, abcam, 1:5000), β-CATENIN (phospho S37) (ab75777, abcam, 1:500).
Table 1. Relationship Between LHPP Expression and Baseline Characteristics of Patients
variables
|
Internal Set
|
External Set
|
LHPPlow
|
LHPPhigh
|
P
|
LHPPlow
|
LHPPhigh
|
P
|
All patients
|
214
|
135
|
|
53
|
40
|
|
Gender
|
|
|
0.581
|
|
|
0.816
|
Female
|
53
|
35
|
|
15
|
12
|
|
Male
|
161
|
100
|
|
38
|
28
|
|
Age at surgery(yr)
|
|
|
0.025
|
|
|
0.454
|
<65
|
126
|
77
|
|
39
|
33
|
|
≥65
|
88
|
58
|
|
14
|
7
|
|
BMI
|
|
|
0.458
|
|
|
0.555
|
<25
|
186
|
113
|
|
44
|
37
|
|
≥25
|
28
|
22
|
|
9
|
3
|
|
TNM stage
|
|
|
0.000
|
|
|
0.001
|
I
|
24
|
23
|
|
11
|
9
|
|
II
|
74
|
43
|
|
19
|
15
|
|
III
|
116
|
69
|
|
23
|
16
|
|
Chemotherapy*
|
|
|
0.004
|
|
|
0.013
|
No
|
100
|
77
|
|
31
|
16
|
|
Yes
|
114
|
58
|
|
22
|
24
|
|
Tumor size (mm)
|
|
|
0.000
|
|
|
0.030
|
≤40
|
77
|
57
|
|
26
|
25
|
|
>40
|
137
|
78
|
|
27
|
15
|
|
Resection type
|
|
|
0.336
|
|
|
0.167
|
Part gastrectomy
|
98
|
58
|
|
40
|
36
|
|
Total gastrectomy
|
116
|
77
|
|
13
|
4
|
|
Pathological type
|
|
|
0.973
|
|
|
0.175
|
Adenocarcinoma
|
176
|
113
|
|
44
|
38
|
|
mix
|
32
|
12
|
|
8
|
1
|
|
non Adenocarcinoma
|
6
|
10
|
|
1
|
1
|
|
|
|
|
|
|
|
|
|
|
|
|
P < 0.05 marked in bold font shows statistical significance.
*Adjuvant chemotherapy after surgery, no radiotherapy was administered to anyone of the patients enrolled.