OBJECTIVE: To explore the potential role of mRNA m7G modification in the pathogenesis of human adenocarcinoma of esophagogastric junction (AEG). METHODS: Pathological tissue specimens from four AEG patients who underwent surgical treatment at the People's Hospital Affiliated to Jiangsu University between 2018 and 2019 were selected. Tumor tissues and adjacent normal tissues were collected from these patients. RNA was extracted from both tissue types and subjected to m7G methylated RNA immunoprecipitation sequencing (m7G-MeRIP-seq) to analyze the patterns of m7G modification, the characteristics of differential m7G modification sites, the differentially expressed mRNA, and the correlation between m7G modification and mRNA expression levels. Differential m7G-modified genes (MSH6, BRCA1, and SOX9) were further validated using methylated RNA immunoprecipitation quantitative PCR (MeRIP-qPCR), while the expression of METTL1 and WDR4 genes was examined by real-time quantitative PCR (RT-qPCR). This study was approved by the Medical Ethics Committee of the People's Hospital Affiliated to Jiangsu University (Ethics No. 20150083). RESULTS: m7G-MeRIP-seq analysis revealed that m7G modifications in both AEG and adjacent normal tissues were predominantly located in the GC-rich region surrounding the internal start codon of mRNA. Differential m7G modification sites between the two groups were closely associated with cancer-related genes. mRNA library analysis showed that differentially expressed mRNA were predominantly upregulated in AEG tissues and downregulated in adjacent normal tissues. Cross-analysis indicated that genes with hypermethylation tended to exhibit upregulated expression, while genes with hypomethylation were typically downregulated in AEG tissues. MeRIP-qPCR validation confirmed that the mRNA expression of MSH6, BRCA1, and SOX9 were significantly upregulated in AEG tissues compared to adjacent normal tissues (AEG vs. normal, P < 0.05). RT-qPCR results demonstrated that the mRNA expression levels of METTL1 and WDR4 were also upregulated in AEG tissues (AEG vs. normal, P < 0.000 5). CONCLUSION These findings suggest that mRNA m7G modification plays a significant role in the development of AEG. Furthermore, proteins as METTL1 and WDR4 may facilitate AEG progression by regulating mRNA m7G modification. These results provide valuable insights into the molecular mechanisms underlying AEG and may inform future therapeutic strategies for this malignancy.
Humans ; RNA, Messenger/metabolism* ; Adenocarcinoma/pathology* ; Esophagogastric Junction/metabolism* ; Esophageal Neoplasms/metabolism* ; Gene Expression Regulation, Neoplastic ; Female ; Male ; Middle Aged ; DNA Methylation ; Methyltransferases/metabolism* ; Stomach Neoplasms/genetics*

BACKGROUND: Ferroptosis-related genes play a crucial role in regulating intracellular iron homeostasis and lipid peroxidation, and they are involved in the regulation of tumor growth and drug resistance. The expression of ferroptosis-related genes in tumor tissues can be used to predict patients' future survival times, aiding doctors and patients in anticipating disease progression. Based on the sequencing data of lung adenocarcinoma (LUAD) patients from The Cancer Genome Atlas (TCGA) database, this study identified genes involved in the regulation of ferroptosis, constructed a prognostic model, and evaluated the predictive performance of the model. METHODS: A total of 1467 ferroptosis-related genes were obtained from the GeneCards database. Gene expression profiles and clinical data from 541 LUAD patients were collected from the TCGA database. The expression data of all ferroptosis-related genes were extracted, and differentially expressed genes were identified using R software. Survival analysis was performed on these genes to screen for those with prognostic value. Subsequently, a prognostic risk scoring model for ferroptosis-related genes was constructed using LASSO regression model. Each LUAD patient sample was scored, and the patients were divided into high-risk and low-risk groups based on the median score. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was calculated. Kaplan-Meier survival curves were generated to assess model performance, followed by validation in an external dataset. Finally, univariate and multivariate Cox regression analyses were conducted to evaluate the independent prognostic value and clinical relevance of the model. RESULTS: Through survival analysis, 121 ferroptosis-related genes associated with prognosis were initially identified. Based on this, a LUAD prognostic risk scoring model was constructed using 12 ferroptosis-related genes (ALG3, C1QTNF6, CCT6A, GLS2, KRT6A, LDHA, NUPR1, OGFRP1, PCSK9, TRIM6, IGF2BP1 and MIR31HG). The results indicated that patients in the high-risk group had significantly shorter survival time than those in the low-risk group (P<0.001), and the model demonstrated good predictive performance in both the training set (1-yr AUC=0.721) and the external validation set (1-yr AUC=0.768). Risk scores were significantly associated with the prognosis of LUAD patients in both univariate and multivariate Cox regression analyses (P<0.001), suggesting that this score is an important prognostic factor for LUAD patients. CONCLUSIONS This study successfully established a LUAD risk scoring model composed of 12 ferroptosis-related genes. In the future, this model is expected to be used in conjunction with the tumor-node-metastasis (TNM) staging system for prognostic predictions in LUAD patients.
Humans ; Ferroptosis/genetics* ; Prognosis ; Adenocarcinoma of Lung/pathology* ; Lung Neoplasms/pathology* ; Male ; Female ; Gene Expression Regulation, Neoplastic ; Middle Aged ; ROC Curve

BACKGROUND: Lung cancer currently ranks first globally in both incidence and mortality. Pemetrexed (PMX) serves as a first-line treatment for lung adenocarcinoma (LUAD), but the patients often develop drug resistance during therapy. Milk exosome (mEXO) have the advantages of low immunogenicity, high tissue affinity, and low cost, and mEXO itself has anti-tumor effects. Hyaluronan (HA) naturally bind to CD44, a receptor which is highly expressed in LUAD tissues. This study aims to construct hyaluronan-modified milk exosome (HA-mEXO) and preliminarily investigate their molecular mechanisms for reversing PMX resistance through cellular experiments. METHODS: Exosomes were extracted from milk using high-speed centrifugation, and HA-mEXO was constructed. PMX-resistant A549 and PC-9 cell lines were treated with mEXO and HA-mEXO, respectively. CCK-8 assays, colony formation assays, Transwell assays, and flow cytometry were performed to evaluate proliferation, colony formation, migration, invasion, and apoptosis phenotypes in the treated resistant cell lines. Finally, transcriptomic sequencing, analysis, and cellular functional recovery experiments were conducted to investigate the mechanism by which HA-mEXO reverses PMX resistance in LUAD cells. RESULTS: The expression of CD44 in A549 and PC-9 LUAD drug-resistant cell lines was significantly higher than that in parental cells, and the uptake rate of HA-mEXO by drug-resistant cell lines was significantly higher than that of mEXO. Compared to the mEXO group, HA-mEXO-treated A549 and PC-9 resistant cells exhibited significantly reduced half maximal inhibitory concentration (IC50) values for PMX, markedly diminished clonogenic, migratory, and invasive capabilities, and a significantly increased proportion of apoptotic cells. Western blot analysis revealed that, compared to parental cells, A549 and PC-9 drug-resistant cells exhibited downregulated ZNF516 expression and upregulated ABCC5 expression. Immunofluorescence analysis revealed that HA-mEXO treatment downregulated ABCC5 expression in A549 and PC-9 drug-resistant cells compared to the PBS group, whereas co-treatment with HA-mEXO and ZNF516 knockdown showed no significant change in ABCC5 expression. CONCLUSIONS HA-mEXO carrying ZNF516 suppress ABCC5 expression, thereby enhancing the sensitivity of A549 and PC-9 LAUD drug-resistant cells to PMX.
Humans ; Hyaluronic Acid/chemistry* ; Drug Resistance, Neoplasm/drug effects* ; Exosomes/chemistry* ; Adenocarcinoma of Lung/genetics* ; Pemetrexed/pharmacology* ; Animals ; Lung Neoplasms/pathology* ; Milk/chemistry* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Hyaluronan Receptors/metabolism*

Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.
Humans ; Gefitinib/therapeutic use* ; Antineoplastic Agents/therapeutic use* ; Lung Neoplasms/pathology* ; Down-Regulation ; Quinazolines/therapeutic use* ; Drug Resistance, Neoplasm/genetics* ; ErbB Receptors/metabolism* ; Adenocarcinoma of Lung ; Protein Kinase Inhibitors/therapeutic use* ; RNA, Small Interfering/genetics* ; Cell Line, Tumor ; Amino Acid Transport System y+/metabolism*

Objective: To investigate the clinicopathological features, immunophenotype, and genetic alterations of rectal adenocarcinoma with enteroblastic differentiation. Methods: Four cases of rectal adenocarcinoma with enteroblastic differentiation were collected at the Affiliated Hospital of Qingdao University, Qingdao, China (three cases) and Yantai Yeda Hospital of Shandong Province, China (one case) from January to December 2022. Their clinical features were summarized. Hematoxylin and eosin stain and immunohistochemical stain were performed, while next-generation sequencing was performed to reveal the genetic alterations of these cases. Results: All four patients were male with a median age of 65.5 years. The clinical manifestations were changes of stool characteristics, bloody stools and weight loss. All cases showed mixed morphology composed of conventional adenocarcinoma and adenocarcinoma with enteroblastic differentiation. Most of the tumors consisted of glands with tubular and cribriform features. In one case, almost all tumor cells were arranged in papillary structures. The tumor cells with enteroblastic differentiation were columnar, with relatively distinct cell boundaries and characteristic abundant clear cytoplasm, forming fetal gut-like glands. Immunohistochemically, the tumor cells were positive for SALL4 (4/4), Glypican-3 (3/4) and AFP (1/4, focally positive), while p53 stain showed mutated type in 2 cases. The next-generation sequencing revealed that 2 cases had TP53 gene mutation and 1 case had KRAS gene mutation. Conclusions: Rectal adenocarcinoma with enteroblastic differentiation is rare. It shows embryonal differentiation in morphology and immunohistochemistry, and should be distinguished from conventional colorectal adenocarcinoma.
Humans ; Male ; Aged ; Female ; Biomarkers, Tumor/metabolism* ; Adenocarcinoma/pathology* ; Colorectal Neoplasms ; Rectal Neoplasms/genetics* ; Cell Differentiation

Humans ; Lung Neoplasms/pathology* ; Adenocarcinoma/pathology* ; Papilloma/genetics* ; Receptor, Fibroblast Growth Factor, Type 1/metabolism* ; Gene Amplification ; Carrier Proteins ; Cytoskeletal Proteins

Objective: To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor (EGFR) gene, and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11 (SLC7A11) on the gefitinib resistance of PC9/GR cells. Methods: RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells. The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis. Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells. PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA (shRNA) targeting SLC7A11 or negative control shRNA (sh-NC), respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA. Cell counting kit-8 (CCK-8) assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells. Mito-Tracker Red CMXRos probe and malondialdehyde (MDA) assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells. Immunohistochemistry (IHC) was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene. Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI) as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled. Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival (PFS) of the patients. Results: RNA array demonstrated that 2 888 genes were differently expressed between PC9 and PC9/GR cells. KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells. These ferroptosis-related gene cohort contained 13 genes, among which SLC7A11 exhibited the most significant difference. Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells (0.76±0.03 vs. 0.19±0.02, P<0.001). The 50% inhibiting concentration (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells, respectively. PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment, compared to the sh-NC group (213.77±26.50 vs. 47.88±4.55, P<0.001). In addition, PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment, compared to the sh-NC group [(15.43±1.60) μmol/mg vs. (82.18±7.77) μmol/mg, P<0.001]. The PFS of the patients with low expression of SLC7A11 (n=18) was significantly longer than the patients with high expression of SLC7A11 (n=18, 16.77 months vs. 9.14 months, P<0.001). Conclusion: Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.
Humans ; Gefitinib/therapeutic use* ; Antineoplastic Agents/therapeutic use* ; Lung Neoplasms/pathology* ; Down-Regulation ; Quinazolines/therapeutic use* ; Drug Resistance, Neoplasm/genetics* ; ErbB Receptors/metabolism* ; Adenocarcinoma of Lung ; Protein Kinase Inhibitors/therapeutic use* ; RNA, Small Interfering/genetics* ; Cell Line, Tumor ; Amino Acid Transport System y+/metabolism*

OBJECTIVE: To investigate the effects of expression levels of S100 calcium-binding protein A10 (S100A10) in lung adenocarcinoma (LUAD) on patient prognosis and the regulatory role of S100A10 in lung cancer cell proliferation and metastasis. METHODS: Immunohistochemistry was used to detect the expression levels of S100A10 in LUAD and adjacent tissues, and the relationship between S100A10 expression and clinicopathological parameters and prognosis of the patients was statistically analyzed. The lung adenocarcinoma expression dataset in TCGA database was analyzed using gene enrichment analysis (GSEA) to predict the possible regulatory pathways of S100A10 in the development of lung adenocarcinoma. Lactate production and glucose consumption of lung cancer cells with S100A10 knockdown or overexpression were analyzed to assess the level of glycolysis. Western blotting, CCK-8 assay, EdU-594 assay, and Transwell assays were performed to determine the expression level of S100A10 protein, proliferation and invasion ability of lung cancer cells. A549 cells with S100A10 knockdown and H1299 cells with S100A10 overexpression were injected subcutaneously in nude mice, and tumor growth was observed. RESULTS: The expression level of S100A10 was significantly upregulated in LUAD tissues as compared with the adjacent tissues, and an elevated S100A10 expression level was associated with lymph node metastasis, advanced tumor stage and distant organ metastasis (P < 0.05), but not with tumor differentiation or the patients' age or gender (P > 0.05). Survival analysis showed that elevated S100A10 expressions in the tumor tissue was associated with a poor outcome of the patients (P < 0.001). In the lung cancer cells, S100A10 overexpression significantly promoted cell proliferation and invasion in vitro (P < 0.001). GSEA showed that the gene sets of glucose metabolism, glycolysis and mTOR signaling pathway were significantly enriched in high expressions of S100A10. In the tumor-bearing nude mice, S100A10 overexpression significantly promoted tumor growth, while S100A10 knockdown obviously suppressed tumor cell proliferation (P < 0.001). CONCLUSION S100A10 overexpression promotes glycolysis by activating the Akt-mTOR signaling pathway to promote proliferation and invasion of lung adenocarcinoma cells.
Animals ; Mice ; Adenocarcinoma of Lung/pathology* ; Cell Proliferation ; Lung Neoplasms/pathology* ; Mice, Nude ; Proto-Oncogene Proteins c-akt ; Signal Transduction ; TOR Serine-Threonine Kinases ; Humans ; S100 Proteins/genetics*

BACKGROUND: Lung cancer prevails and induces high mortality around the world. This study provided real-world information on the evolution of clinicopathological profiles and survival outcomes of lung cancer, and provided survival information within stage I subtypes. METHODS: Patients pathologically confirmed with lung cancer between January 2009 and December 2018 were identified with complete clinicopathological information, molecular testing results, and follow-up data. Shifts in clinical characteristics were evaluated using χ2 tests. Overall survival (OS) was calculated through the Kaplan-Meier method. RESULTS: A total of 26,226 eligible lung cancer patients were included, among whom 62.55% were male and 52.89% were smokers. Non-smokers and elderly patients took increasingly larger proportions in the whole patient population. The proportion of adenocarcinoma increased from 51.63% to 71.80%, while that of squamous carcinoma decreased from 28.43% to 17.60%. Gene mutations including EGFR (52.14%), KRAS (12.14%), and ALK (8.12%) were observed. Female, younger, non-smoking, adenocarcinoma patients and those with mutated EGFR had better survival prognoses. Importantly, this study validated that early detection of early-stage lung cancer patients had contributed to pronounced survival benefits during the decade. Patients with stage I lung cancer, accounted for an increasingly considerable proportion, increasing from 15.28% to 40.25%, coinciding with the surgery rate increasing from 38.14% to 54.25%. Overall, period survival analyses found that 42.69% of patients survived 5 years, and stage I patients had a 5-year OS of 84.20%. Compared with that in 2009-2013, the prognosis of stage I patients in 2014-2018 was dramatically better, with 5-year OS increasing from 73.26% to 87.68%. Regarding the specific survival benefits among stage I patients, the 5-year survival rates were 95.28%, 93.25%, 82.08%, and 74.50% for stage IA1, IA2, IA3, and IB, respectively, far more promising than previous reports. CONCLUSIONS Crucial clinical and pathological changes have been observed in the past decade. Notably, the increased incidence of stage I lung cancer coincided with an improved prognosis, indicating actual benefits of early detection and management of lung cancer.
Humans ; Male ; Female ; Aged ; Lung Neoplasms/genetics* ; Adenocarcinoma/pathology* ; Prognosis ; Survival Rate ; Mutation ; ErbB Receptors/genetics* ; Neoplasm Staging ; Retrospective Studies

Objective To study the pathological types,expression of mismatch repair protein,human epidermal growth factor receptor 2(HER2),and Pan-TRK,and Epstein-Barr virus(EBV)infection in patients with colorectal cancer resected in Tibet. Methods A total of 79 patients with colorectal cancer resected in Tibet Autonomous Region People's Hospital from December 2013 to July 2021 were enrolled in this study.The clinical and pathological data of the patients were collected.The expression of mismatch repair protein,HER2,and Pan-TRK was detected by immunohistochemical(IHC)staining,and detection of HER2 gene by fluorescence in situ hybridization(FISH)in the patients with HER2 IHC results of 2+ or above.EBV was detected by in situ hybridization with EBV-encoded small RNA. Results A total of 79 colorectal cancer patients were included in this study,with the male-to-female ratio of 1.26:1 and the mean age of(57.06±12.74)years(24-83 years).Among them,4 patients received preoperative neoadjuvant therapy.Colonic cancer and rectal cancer occurred in 57(57/79,72.15%,including 31 and 26 in the right colon and left colon,respectively)and 22(22/79,27.85%)patients,respectively.The maximum diameter of tumor varied within the range of 1-20 cm,with the mean of(6.61±3.33)cm.Among the 79 colorectal cancer patients,75(75/79,94.94%)patients showed adenocarcinoma.Lymph node metastasis occurred in 12(12/21,57.14%)out of the 21 patients with severe tumor budding,13(13/23,56.52%)out of the 23 patients with moderate tumor budding,and 2(2/31,6.45%)out of the 31 patients with mild tumor budding,respectively.The lymph node metastasis rate showed differences between the patients with severe/moderate tumor budding and the patients with mild tumor budding(all P<0.001).The IHC staining showed that mismatch repair protein was negative in 10(10/65,15.38%)patients,including 5 patients with both MSH2 and MSH6 negative,4 patients with both MLH1 and PMS2 negative,and 1 patient with MSH6 negative.Pan-TRK was negative in 65 patients.The IHC results of HER2 showed 0 or 1+ in 60 patients and 2+ in 5 patients.FISH showed no positive signal in the 5 patients with HER2 IHC results of 2+.The detection with EBV-encoded small RNA showed positive result in 1(1/65,1.54%)patient. Conclusions Non-specific adenocarcinoma of the right colon is the most common in the patients with colorectal cancer resected in Tibet,and 15% of the patients showed mismatch repair protein defects.EBV-associated colorectal carcer is rare,Pan-TRK expression and HER2 gene amplification are seldom.The colorectal cancer patients with moderate and severe tumor budding are more likely to have lymph node metastasis.
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Adenocarcinoma ; Biomarkers, Tumor/genetics* ; Colorectal Neoplasms/pathology* ; DNA Mismatch Repair ; DNA-Binding Proteins/genetics* ; Epstein-Barr Virus Infections/diagnosis* ; Herpesvirus 4, Human/metabolism* ; In Situ Hybridization, Fluorescence ; Lymphatic Metastasis ; Tibet ; Young Adult ; Aged, 80 and over