2.1 Bioinformatics Analysis
2.1.1 Data collection
The RNA sequencing (RNA-seq) dataset (including FPKM and read count) and clinical data of 622 CRC (including COAD and READ) patients were retrieved from the TCGA Project in UCSC (https://xenabrowser.net/datapages/). Seven independent datasets including GSE15960 (6 patients), GSE9348 (32 patients), GSE8671 (32 patients), GSE37364 (38 patients), GSE33133 (90 patients), GSE21510 (123 patients) and GSE39582 (443 patients) were downloaded from Gene Expression Omnibus (GEO), and gene expression was detected by the GPL570 platform of U133 plus 2 array.
2.1.2 Identify novel differentially expressed LncRNAs
The differential expression analysis was performed by comparing the expression profiles between tumor samples and normal samples for the expression data (read counts) detected by RNA-seq in TCGA, differentially expressed genes (DEGs) were identified using R package EdgeR and Deseq2 [33] with FDR ≤ 0.05 and fold change (FC) ≥ 2 or ≤ 0.5. For subsequent validation, only DEGs with high-expression level (median FRKM expression > 1) were retained, and probes were annotated on the GPL570 platform. For the expression data detected by GPL570 platform, the DEGs were identified using Limma [34] with FDR ≤ 0.05 and FC ≥ 1.2 or ≤ 1/1.2 in GEO datasets.
2.1.3 Evaluation of the LncRNA related prognostic model
Except for differential expression analysis, log2 (FPKM + 1) was conducted for other analysis in TCGA. In TCGA and GEO datasets, CRC patients were stratified into two subgroups based on the median level of TRG-AS1 expression. The survival curves (including overall survival and disease-free survival) were estimated using the Kaplan-Meier method, and the log-rank test was utilized to analyze differences in survival time. The p value < 0.05 was considered as statistically significant.
2.1.4 Functional enrichment analysis
Pearson correlation analysis was adopted to estimate expression correlation of TRG-AS1 and the key enzyme of glycolysis. In TCGA, CRC patients were stratified into two subgroups based on the median level of TRG-AS1 expression. Differential expression analysis was used to obtain the gene list associated with TRG-AS1 GO enrichment analysis and GSEA analysis were implemented using ClusterProfiler package based on MsigDB datasets [35].
2.2 Tissue specimens
Participants were recruited from subjects who underwent endoscopy and were pathologically diagnosed with colorectal cancer at Zhengzhou central Hospital Affiliated to Zhengzhou University during the period 2020–2021. Patients were excluded if they were confirmed or highly suspicious for recurrent gastric cancer, secondary malignant disease, received chemotherapy, immunotherapy, or radiotherapy [36]. Finally, 66 patients with complete information were included in this cohort. Detailed clinical characteristics were presented in Table 1. The procedures used in this study adhere to the tenets of the Declaration of Helsinki. With institutional review board approval (Ethical Batch Number:202021), and following informed consent, tumor tissue and paired normal tissue were collected and promptly frozen in a -80°C refrigerator.
Table 1
Detailed clinical characteristics
clinical characteristics
|
|
n = 66
|
%(n = 66)
|
Age(years)
|
|
|
|
|
≤ 60
|
20
|
30.30%
|
|
> 60
|
46
|
69.70%
|
Gender
|
|
|
|
|
Male
|
39
|
59.09%
|
|
Female
|
27
|
40.91%
|
Tumor volume
|
|
|
|
|
≤ 5cm3
|
21
|
31.82%
|
|
> 5cm3
|
45
|
68.18%
|
Depth of invasion
|
|
|
|
|
T1-T2
|
6
|
9.09%
|
|
T3-T4
|
60
|
90.91%
|
Metastasis
|
|
|
|
|
Yes
|
35
|
53.03%
|
|
NO
|
31
|
46.97%
|
TNM stage
|
|
|
|
|
I + II
|
29
|
43.94%
|
|
III + IV
|
37
|
56.06%
|
Serum CEA level
|
|
|
|
|
≤ 10 ng/ml
|
51
|
77.27%
|
|
10 ng/ml
|
15
|
22.73%
|
Grade
|
|
|
|
|
G1 + G2
|
46
|
69.70%
|
|
G3 + G4
|
20
|
30.30%
|
Smoking history
|
|
|
|
|
Yes
|
11
|
16.67%
|
|
NO
|
55
|
83.33%
|
Drinking history
|
|
|
|
|
Yes
|
10
|
15.15%
|
|
NO
|
56
|
84.85%
|
2.3 Cell culture
All colorectal cancer cell lines including NCM460 (RRID:CVCL_0460), HCT8 (RRID:CVCL_2478), HCT116 (RRID:CVCL_0291), HT29 (RRID:CVCL_0320), SW480 (RRID:CVCL_0546), CaCo2 (RRID:CVCL_0025), RKO (RRID:CVCL_HE15) and Lovo (RRID:CVCL_0399) were purchased from Procell Life Science Technology, routinely monitored by PCR to ensure they were mycoplasma free and authenticated by STR profiling. NCM460 and HCT8 cells were cultured in RPMI 1640 medium. HCT116 and HT29 cells were cultured in McCoy’s 5A medium. SW480 cells were cultured in IMDM medium. CaCo2 cells were cultured in DMDM medium. All culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Carlsbad, CA), 100U/ml penicillin, and 100 mg/ml streptomycin (Beijing Solarbio Science & Technology). All cells were cultured under standard incubator conditions (37°C, 5% CO2) and passaged at approximately 90% confluence.
2.4 Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from tissues and cells using reagent (Invitrogen, USA) and RNeasy Mini Kit according to the manufacturer’s instructions. RNA concentration and purity were measured on the Nanodrop 2000, and the RNA integrity was verified by gel electrophoresis. First-strand cDNA was synthesized from the total RNA using a reverse transcriptional kit (Invitrogen, USA). Quantitative RT-PCR was performed on an Applied Biosciences 7500 Real-Time PCR system following the QuantiTect SYBR Green PCR kit protocols (Qiagen). A list of primer sequences is reported in Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization and relative abundance of lncRNA and mRNA. The 2 − ΔΔCTmethod was used to calculate the expression of LncRNA [37].
Table 2
The primers and siRNAs used in this study.
Human Gene
|
Sequences (5′- 3′)
|
GAPDH
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Forward primer: CCGGGAAACTGTGGCGTGATGG
|
|
Reverse primer: AGGTGGAGGAGTGGGTGTCGCTGTT
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LncRNA TRG-AS1
|
Forward primer: GCCTAGGCTAATGTGTGGCT
|
|
Reverse primer: AGGCAGAAGAGTCGCTTGAC
|
Si RNA-NC
|
Sense: UUCUCCGAACGUGUCACGUTT
|
|
Antisense: ACGUGACACGUUCGGAGAATT
|
Si RNA TRG-AS1#1
|
Sense: GGACUACAGUGAUAUUGAAGA
|
|
Antisense: UUCAAUAUCACUGUAGUCCAG
|
Si RNA TRG-AS1#2
|
Sense: GGUUUAUGUAAUUACUAUAUU
|
|
Antisense: UAUAGUAAUUACAUAAACCAG
|
Si RNA TRG-AS1#3
|
Sense: GGUUGAAUGUUCUUUAUAAA
|
|
Antisense: UAAUAAAGAACAUUCAACCCU
|
2.5 Cell Transfection
When 50% confluent, transient transfection of cells were performed using interferin transfection reagent (Polyplus-transfection), according to manufacturer’s instructions. The sequences of negative control small interfering RNA (siRNA) and on-target individual siRNAs were described in Table 2 and were synthesized by Shanghai GenePharma. siRNA oligonucleotides were transfected at a final working concentration of 20 nmol and kept at − 20°C. Six hours after transfection, the culture medium was replaced. The knockdown and transfect efficiency of siRNA was determined 48 hours post transfection by qRT–PCR.
2.6 Lentivirus packaging and stable cell lines
Lentiviral vectors that stably overexpressing lncRNA TRG-AS1 (OE-TRG-AS1) and negative control lentiviral vectors (OE-NC) were designed and packaged by Shanghai Jikai Gene Company. HCT116 and SW480 cells were transfected with lentivirus /medium at a ratio of 1:50. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas lncRNA TRG-AS1 levels were verified by real-time PCR.
2.7 Glucose uptake assay
Cells were collected and re-suspended in distilled water, ultrasonic wave disruption the cells in ice bath with 120W for 7min. The mixture was placed in a boiling water bath for 10min, cooled at room temperature, and then centrifuged after cooling at 8000g for 10min. After add 180µL mix reagent to 20µL supernatant and incubation at 37°C for 15min, the absorbance values at 505 nm were determined.
2.8 Pyruvate assay
Cells were collected and re-suspended in extraction buffer, ultrasonic wave disruption the cells in ice bath with 120W for 7min. The homogenate was left to stand for 30min, centrifuged for 10min at 8000g and the supernatant was removed. Standard solution in the kit was used to prepare the standard curve. The working solution was added to supernatant, which was left for 2min and then added to working solution Ⅱ. Absorbance was measured at a wavelength of 520 nm.
2.9 CCK-8 cell viability assays
Cell counting kit 8 (CCK8) experiment for in vitro proliferation assay were performed using the CCK8 Kit (Sigma-Aldrich) according to the manufacturer’s protocol. The transfected cells were seeded into 96-well plates at a density of 2000 cells per well with 6 replicates. The medium was replaced with 100µl fresh complete medium containing 10µl CCK8 solution following a culture of 0, 24, 48 and 72h, respectively. Then, the plates were incubated at 37°C and 5% CO2 for 2h, and the absorbance was measured at a wavelength of 450nm.
2.10 Colony formation assays
1000 cells/well were plated in 6-well plates, and pools of cells were used to assess growth and clonogenic ability. Ten days after plating the cells, clonogenic progenitors were determined and cells in each group were replated for 6 times. Colonies were rinsed twice with ice-cold PBS, fixed with 4% paraformaldehyde for 20 min on ice and washed twice with PBS. After fixed with methanol, cells were stained with 0.1% crystal violet solution 30 min and then the colonies were imaged and counted.
2.11 Ethynyl deoxyuridine (EdU) Staining
Transfected cells were inoculated at a density of 2 × 104/ml per well in 96-black bottom. A total of 6 reduplicate wells were set up for each group. EdU staining using the Click EdU kit (Abbkine, USA) was performed according to protocol. Cells were incubated with basal medium containing 10µM EdU at 37°C for 2h. Subsequently, cells were fixed with 4% paraformaldehyde for 30min followed by washing with 3% BSA and permeabilization with 0.1% Triton X-100. Afterwards the cells were stained with 50µL of fresh reaction cocktail, and the nucleus was stained with 50µL of DAPI, followed by viability determination with a fluorescence microscope (LEICA DFC450C). The EDU-positive cells were counted in five random visual fields from each well and percentages were calculated.
2.12 Cell migration and invasion assays
Transfected cells resuspended in serum-free medium were seeded into the upper chamber of 24‐well inserts with or without Matrigel (BD, USA). Then, 600ul of medium supplemented with 20% FBS was added into the lower chamber. After incubation for 24 hours (SW480) or 36 hours (HCT116), non-invading cells in the upper chamber were scraped off. The cells on the bottom surface of the chambers were fixed using 4% paraformaldehyde, and stained with crystal violet solution. Invading cells or migrating cells were photographed and counted under a light microscope (LEICA DFC450C).
2.13 In vivo tumor xenograft experiments
Ten 6-week-old BALB/c male nude mice were used for the tumor xenograft experiment. The cell suspensions of HCT116-OE-NC and OE-TRG-AS1 were inoculated subcutaneously on the upper right forelimb of nude mouse, respectively. The body weight and tumor morphology were recorded. Tumor volume was measured every 3d with a vernier caliper and calculated as 0.5 × width2 (cm2) × length (cm). All experimental procedures have been approved by the Ethics Committee of Zhengzhou Central Hospital Affiliated to Zhengzhou University.
2.14 Statistical Analysis
The data were presented as mean ± standard deviation. Statistical analysis between two groups was performed using the Student's t-test. The expression correlation was determined by Pearson coefficient analysis. The correlation between TRG-AS1 expression and clinicopathological features of CRC patients was evaluated by Chi-square test. The P < 0 .05 was considered significant. Statistical analysis was performed on SPSS 24.0.