2.1 Retrieval of data
The Transcripts Per Million(TPM) sequencing data from the ESCC dataset were processed after being retrieved through the TCGA database (https://www.example.com). Sample clinical information from 93 cases of ESCC was obtained for further analysis, following the exclusion of data samples lacking prognosis information and duplicates (Table S1). The datasets, such as GSE161533 (comprising 28 normal esophageal squamous epithelial samples and 28 ESCC samples) [14] and GSE53624 (encompassing 119 normal esophageal epithelial and 119 ESCC samples) were accessed at the GEO database (https://www.ncbi.nlm.nih.gov/geo/) [15]. Furthermore, patient clinical information included in the GSE53624 dataset was retrieved (Table S2).
2.2 Survival analysis
The Kaplan-Meier plots were drawn on the basis of TCGA-ESCC data by using the survivalROC package to assess whether MIR210HG expression was associated with survival among patients with ESCC [16]. Validation was conducted using the external dataset GSE53624. Additionally, MIR210HG localization on chromosomes was performed using the RCircos package [17].
2.3 Functional enrichment analysis
The most relevant 200 genes of MIR210HG were transferred to the Database for Annotation, Visualization and Integrated Discovery(DAVID) (https://david.ncifcrf.gov/) database for analysis and annotation using the GSE53624 and TCGA-ESCC datasets. Enrichment analysis results for GO and KEGG were obtained using the official gene names as identifiers and Homo sapiens as the species. Finally, the top 7 enrichment results were presented in descending order of P-value (P < 0.05).
2.4 Gene set enrichment analysis
Utilizing the TCGA-ESCC dataset, the samples were categorized into low-expression and high-expression groups to assess the impact of gene expression on tumors. Subsequently, GSEA was conducted to analyze gene expression patterns [18]. GSEA, a computational approach, is commonly utilized to assess statistical differences between two biological states exhibited by a specific gene set. It estimates pathway changes and bioprocess activity within expression dataset samples. Additionally, the GSE53624 dataset was employed for validation.
2.5 ESTIMATE immunoreactivity analysis
Estimation of STromal and Immune cells in MAlignant Tumour tissues using Expression data (ESTIMATE) determines the content of tumor cells along with infiltrating stromal and immune cells by transcriptional profiling of cancer samples. It can also calculate the abundance or proportion of immune, tumor, and stromal cells in tumor tissues associated with the TME. Based on the TCGA-ESCC dataset, immune and stromal scores in ESTIMATE were obtained for all patients using the ESTIMATE package for R [19]. Additionally, the GSE53624 dataset was used for validation.
2.6 Immune cell infiltration analysis
Immune infiltration analysis was carried out utilizing CIBERSORTx to assess the association between immune cells and MIR210HG. The CIBERSORT package [20] utilizes the linear support vector regression principle for the deconvolution of the transcriptome expression matrix, thus evaluating the abundance and composition of immune cells within mixed cells. Utilizing the CIBERSORT algorithm, samples with an output P-value < 0.05 were filtered based on the gene expression feature set of 22 immune cell subtypes, thereby estimating the abundance and composition of immune cells within mixed cells. Considering the growing significance of immunotherapy in oncology, several common immune checkpoints were analyzed.
2.7 Drug sensitivity testing
For further exploration of the potential guidance provided by high and low MIR210HG expression for drug therapy, IC50 values of drugs were accessed from the Genomics of Cancer Drug Sensitivity (GDSC) using the R package oncoPredict [21]. Spearman correlation analysis was utilized to determine the correlation between MIR210HG high/low expression and drug IC50 values, enabling the screening of drugs. Then, bar graphs were generated for the comparison of the variances in IC50 values between patients with low and high expression of MIR210HG.
2.8 Cell line culture
Four human ESCC cell lines (Eca9706, KYSE150, KYSE510, and KYSE30) with different degrees of differentiation along with a normal esophageal epithelial cell line (HEEC) were acquired from the cell bank of the Shanghai Research Institute of the People's Republic of China. In a humidified incubator, Eca9706, KYSE150, KYSE510, and KYSE30 cells underwent cultivation at 37°C with 5% CO2 in RPMI-1640 medium (Kibbutz Beit HaEmek BI) enriched with 10% fetal bovine serum (FBS). HEEC cells were then cultivated in a humidified incubator at 37 ℃ with 5% CO2 in a DMEM medium (Gibco) containing 10% FBS. All cells were passaged no more than 15 times.
2.9 Cellular lentiviral transduction and processing
Negative control lentiviral vectors(Vector) and MIR210HG-overexpressing lentiviral vectors(OE-MIR210HG) were obtained from GenePharma. Lentiviral vectors were transduced into KYSE30 and KYSE150 cells, respectively, and subsequently screened utilizing a medium containing 5 µg/mL puromycin for obtaining stably transfected cell lines. Cells in the respective subgroups were treated with PFT-α (p53 inhibitor; MedChemExpress 40 µmol/L) following the provided protocol.
2.10 Quantitative real-time fluorescence polymerase chain reaction
Cells underwent total RNA extraction utilizing RNAiso Plus (TaKaRa, Dalian, China). Subsequently, reverse transcription was conducted utilizing the RT Reagent Kit (TaKaRa, Dalian, China) to produce cDNA. Subsequently, SYBR Green Real-Time PCR Master Mix (TaKaRa, Dalian, China) was employed to perform quantitative PCR (qPCR), with GAPDH serving as the endogenous reference gene. The following are the PCR primers employed in this research:
MIR210HG:
forward primer: 5'-TGTTCCCTTTGTGTGCTCCAG-3'.
reverse primer: 5'-GCCCTAGATCATGGGGGTCTT-3'.
GAPDH:
forward primer: 5'-AGAGCCTCGCCTTTGCCGATCC-3'.
reverse primer: 5'-ATACACCCGCTGCTCCGGGGTC-3'.
2.11 Partial separation of nucleocytoplasmic RNA
The Cytoplasmic and nuclear rna purification kit (Norgen, Cat. No 21000, Canada) was employed to separate cytoplasmic and nuclear RNA from KYSE30 and KYSE150 cells. qPCR was conducted to assess the relative amount of MIR210HG in the cytoplasm and nucleus of the cells. U6 and GAPDH were utilized as the endogenous controls in the nucleus and cytoplasm, respectively, to elucidate the corresponding proportion of MIR210HG expression in the nucleus and cytoplasm. The U6 primer sequences are mentioned below:
forward primer: 5'-CTCGCTTCGGCAGCACA-3'.
reverse primer: 5'-AACGCTTCACGAATTTGCGT-3'.
2.12 Cell invasion and migration assays
The Transwell chamber (24-well) is composed of lower and upper compartments, divided by a polycarbonate membrane featuring an 8 µm opening. Cell invasion was detected using a matrigel (Corning)-coated polycarbonate membrane, while cell migration was assessed by utilizing an uncoated polycarbonate membrane. A solution of exponential phase lentiviral vectors-transfected cells was introduced into the upper compartment of the aforementioned chamber, whereas a medium enriched with 20% FBS was introduced to the lower compartment. After 12 hours of incubation with 5% CO2 at 37°C, cells were allowed to traverse the polycarbonate membrane, underwent fixation with 4% paraformaldehyde, and then subjected to crystal violet staining. The number of stained cells passing through the polycarbonate membrane into the lower compartment was counted under an inverted microscope.
2.13 Colony formation assay
Different groups of stably transfected cells were seeded into six-well plate cell culture plates (1000 per well) and incubated for 12 days. The formed cell colonies were subjected to fixation by 4% paraformaldehyde and subsequently underwent 0.1% crystal violet staining. Ultimately, the number of colonies exceeding 50 cells was counted.
2.14 Wound healing assay
Each group of cells was inoculated uniformly (density: 4×105 cells/well) in 6-well plates and subsequently cultured in a cell culture incubator. Once the cells adhered to the wall and reached a density of 90–95%, a 200 µL tip was employed for the creation of a straight linear scratch on the cell monolayer. After gently washing the detached cells with PBS, a serum-free medium was introduced, and the scratches were photographed at 0 hours under a microscope to calculate the initial wound area. Subsequently, the cells were put back into the incubator to continue incubation for 24 hours. After this period, the wound area was photographed under the microscope and then measured. ImageJ was utilized to quantify the wound area at the same location. The following formula was utilized to assess the cell migration rate:
cell migration rate (%) = (initial wound area - wound area after 24 hours) ÷ initial wound area × 100%.
2.15 Western blotting analysis
The lysis of cells and tissues was carried out using radioimmunoprecipitation assay(RIPA) lysis buffer enriched with phenylme thylsulfonyl fluoride(PMSF) and phosphatase inhibitors for the extraction of proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was employed for the separation of the protein samples. Subsequently, they were transferred onto a polyvinylidene difluoride(PVDF) membrane. After that, 5% BSA was employed to block the membrane for 1 hour. Afterward, the membrane underwent overnight incubation at 4°C with primary antibodies, including anti-Bax(HUABIO Catalog# ET1603-34 Dilution ratio1:20000), anti-Bcl-2(HUABIO Catalog# ET1702-53 Dilution ratio1:2000), anti-Beclin-1(Proteintech Cat No : 66665-1-Ig Dilution ratio1:2000), anti-C-caspase3(Abcam ab2302 Dilution ratio1:100), anti-GAPDH(Abcam ab181602 Dilution ratio1:10000), anti-LC3B-I/II(Abcam ab192890 Dilution ratio1:2000), anti-P53(HUABIO Catalog# ET7107-33 Dilution ratio1:1000), anti-P62(HUABIO Catalog# HA721171 Dilution ratio1:5000), anti-AMPKa(Abcam ab207442 Dilution ratio1:1000), and anti-P-AMPKa(Abcam ab314032 Dilution ratio1:1000). Subsequently, the membrane was incubated again for 1 hour at room temperature with the specified secondary antibodies and enhanced chemiluminescence (ECL) was utilized for visualizing the membrane. The relative expression of target proteins was assessed with GAPDH serving as an internal reference.
2.16 EdU cell proliferation assay
Stably transfected ESCC cells in the logarithmic growth phase were placed into 96-well plates (5000 cells/well). Following this, the plates underwent incubation for 24 hours. Subsequently, the cells were exposed to EdU for a duration of 3 hours. After that, they were labeled, washed, and fixed with paraformaldehyde for 30 minutes. Following fixation, they underwent destaining through osmotic decolorization for 10 minutes with 0.5% Triton X-100. Ultimately, 4',6-diamidino-2-phenylindole (DAPI) was employed for 30 minutes for staining the nuclei of the cells. Using a fluorescence microscope, the total number of cells and the number of EdU-positively stained cells were identified, and corresponding images were captured. The formula to calculate the cell proliferation rate is mentioned below:
cell proliferation rate = number of EdU-positive cells/total number of cells × 100%.
2.17 Monodansylcadaverine staining
Cellular autophagy was assessed through MDC staining. The cells from various treatment groups were washed once with 1xWash buffer, followed by incubation with MDC staining reagent (Solarbio) for 30 minutes in the dark. Subsequently, the cells were rinsed thrice with 1xWash buffer. Finally, the cells were covered with a collection buffer. MDC staining was observed, photographed, and counted using a fluorescence microscope. The formula to calculate the autophagy rate is mentioned below:
autophagy rate = number of autophagic vacuoles/total number of cells × 100%.
2.18 Apoptosis detection via flow cytometry
Apoptosis was detected via flow cytometry utilizing the PE Annexin V Apoptosis Detection Kit I (BD Biosciences). Test cells were prepared into cell suspensions by trypsin digestion. Samples were assessed by flow cytometry after double staining with P-phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD).
2.19 ESCC xenograft model
A total of 10 female BALB/C mice (weight: 18–22 g and age: 4–6 weeks) were procured from Beijing Charles River Laboratory Animal Technology Co., Ltd. These mice underwent random allocation into two groups (5 mice/group). KYSE150 cells (100µL, 7.5×105 cells), transfected with lentivirus from Vector and OE-MIR210HG, were administered into the axilla (right) of the mice by subcutaneous injection. The mice were kept at 20–26°C in a specific pathogen-free (SPF) facility with a humidity range between 40 and 60%.When the transplanted tumor reached 100 mm³, its volume and size were assessed and recorded every 2 days. Eighteen days post-inoculation, all mice were euthanized by cervical dislocation. Subsequently, the subcutaneous grafted tumors were completely excised, and their weights were measured. Furthermore, the expression levels of relevant genes and proteins in the isolated tumor tissues were assessed using Western blotting analysis, qRT-PCR, and immunohistochemistry. Animal experiments were conducted in accordance with the ARRIVE guidelines, and the study protocols for animal experiments were reviewed and approved by the Ethical Review Committee of Shandong Provincial hospital affiliated to Shandong First Medical University.
2.20 Statistical analyses
All data processing and analyses were done through R software (version 4.1.1). For comparisons between two groups of continuous variables, statistical significance of normally distributed variables was estimated by independent Student t-tests and differences between non-normally distributed variables were analysed by Mann-Whitney U-tests (i.e., Wilcoxon rank sum tests). The chi-square test or Fisher exact test was used to compare and analyse the statistical significance between categorical variables in the two groups.The survival package of R was used to perform survival analyses, the Kaplan-Meier survival curves were used to show differences in survival, and the log-rank test (log-rank test) was used to assess the significance of the differences in the survival times of the two groups of patients. Both univariate and multivariate Cox analyses were based on the survival R package, and lasso analysis was based on the glmnet R package. All statistical p-values were two-sided, with p < 0.05 considered statistically significant.