Identification and Validation of SLC9A2 as A Potential Tumor Suppressor in Colorectal Cancer: Integrating Bioinformatics Analysis with Experimental Confirmation.

OBJECTIVE: To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression. METHODS: We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis. RESULTS: We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway. CONCLUSION: Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.

GSPT1 Functions as a Tumor Promoter in Human Liver Cancer.

OBJECTIVE: This study analyzed the role of G1 to S phase transition 1 protein (GSPT1) in promoting progression of liver cancer cells. METHODS: A bioinformatics database was used to analyze the expression levels of GSPT1 in liver cancer tissues and the prognosis of patients. Subsequently, Western blotting and quantitative PCR were used to verify the expression levels of GSPT1 between normal hepatocytes and hepatoma cells. We used a CRISPR/Cas9 system to construct knockouts of GSPT1 in HepG2 and HCCLM9 liver cancer cells. The effect of GSPT1 on liver cancer cell migration and invasion was analyzed using flow cytometry, migration, and tumor formation assays. RESULTS: The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset indicated that GSPT1 expression was upregulated in liver cancer cell lines, and patients with liver cancer had poor prognosis. Knockout of GSPT1 in cells significantly inhibited tumor proliferation, cell migration, and growth in vivo. CONCLUSION: In this study, we found that GSPT1 promotes the migration and invasion of liver cancer cells.

Bioinformatics Analysis and Identification of Potential Genes Associated with Pathogenesis and Prognosis of Gastric Cancer.

OBJECTIVE: Gastric cancer (GC) is a deadly cancer and a challenging public health problem globally. This study aimed to analyze potential genes associated with pathogenesis and prognosis of gastric cancer. METHODS: This work selected the overlapping differentially expressed genes (DEGs) in GC from four datasets, the GSE29272, GSE29998, GSE54129 and GSE118916 Gene Expression Omnibus databases. These DEGs were used to carry out comprehensive bioinformatic analysis to analyze the related functions and pathways enriched, the relative expression levels and immune infiltrates, the prognostic characteristics and the interaction network. RESULTS: In total, 55 DEGs increased while 98 decreased in their expression levels. For those DEGs with increased expression, they were mostly concentrated on "focal adhesion" and "ECM-receptor interaction", whereas DEGs with decreased expression were mostly associated with "gastric acid secretion" and "drug metabolism cytochrome P450". MCODE and ClueGO results were then integrated to screen 10 hub genes, which were FN1, COL1A1, COL3A1, BGN, TIMP1, COL1A2, LUM, VCAN, COL5A2 and SPP1. Survival analysis revealed that higher expression of the ten hub genes significantly predicted lower overall survival of GC patients. TIMP1 was most significantly related to neutrophils, CD8+ T cells, as well as dendritic cells, while LUM was most significantly related to macrophages. CONCLUSION: Immunohistochemistry results and functional testing showed that the expression of COL5A2 was elevated in GC and that it might be a key gene in GC tumorigenesis.

Overexpression of eRF3a Promotes Cell Proliferation and Migration in Liver Cancer.

OBJECTIVE: The eukaryotic release factor 3a (eRF3a), a member of the eukaryotic peptide chain release factor family, is overexpressed in several types of cancer. This study aims to investigate the biological role and mechanism of eRF3a in the progression of liver cancer. METHODS: Western blotting and RT-qPCR were used to detect the expression level of eRF3a in normal liver cells and liver cancer cells. The cell transfection experiments were performed to overexpress eRF3a levels in liver cancer cells HCCLM9 and Huh7, and then cell cycle and apoptosis experiments, Cell Counting Kit-8 (CCK8), plate cloning, and Transwell experiments were done to evaluate the function of eRF3a in the progression of liver cancer. The Western blotting was done to explore the mechanism of eRF3a promoting the development of liver cancer. Western blotting and RT-qPCR were used to detect the expression level of eRF3a in normal liver cells and liver cancer cells. The cell transfection experiments were performed to overexpress eRF3a levels in liver cancer cells HCCLM9 and Huh7, and then cell cycle and apoptosis experiments, Cell Counting Kit-8 (CCK8), plate cloning, and Transwell experiments were done to evaluate the function of eRF3a in the progression of liver cancer. The Western blotting was done to explore the mechanism of eRF3a promoting the development of liver cancer. RESULTS: eRF3a was significantly highly expressed in liver cancer cells, and its expression level was negatively correlated with the clinical prognosis of patients. In addition, in vitro experiments showed that eRF3a could promote the proliferation and migration of liver cancer cells through the ERK and JNK signaling pathways. CONCLUSION: This study suggests that eRF3a may be a potential prognostic marker for liver cancer and act as an oncogene by activating JNK and ERK signaling; therefore, eRF3a may be a new target for the treatment of liver cancer.

Ultrasound-targeted microbubble destruction-mediated downregulation of CD133 inhibits epithelial-mesenchymal transition, stemness and migratory ability of liver cancer stem cells.

Hepatocellular carcinoma (HCC) is an aggressive disease with a poor outcome due to the high incidence of metastasis. Cancer stem cells (CSCs) have been identified to be responsible for tumor progression and may be generated by epithelial-mesenchymal transition (EMT) characteristics. CD133 is a specific surface marker for liver cancer stem cells (LCSCs), which is also considered as an important functional factor for tumorigenesis and overall survival in HCC. Ultrasound-targeted microbubble destruction (UTMD) has recently been used as a novel, safe and effective gene transfection technology. The aim of the present study was to elucidate the regulatory mechanism of CD133 and EMT in LCSCs and whether the UTMD-based shRNA delivery system facilitated gene delivery in LCSCs. In the present study, CD133+ cells were isolated from the SMMC-7721 HCC cell line and then transfected with shCD133 mediated by UTMD and liposomes, respectively. Compared to the liposomes group, the UTMD group resulted in significantly improved transfection efficiency. The downregulation of CD133 reversed the EMT program, attenuated self-renewal, proliferation and migration of CD133+ LCSCs and suppressed the growth of CSC tumor xenografts. Additionally, the downregulation of CD133 led to downregulation of the nuclear factor-κB (NF-κB) pathway. The present study demonstrated that CD133 plays a critical role in the regulation of the EMT process, tumor-initiating properties and migratory ability of LCSCs. The UTMD technique targeted for CD133 downregulation may be examined as a potential therapeutic strategy for HCC.

Intercellular adhesion molecular-1, Fas, and Fas ligand as diagnostic biomarkers for acute allograft rejection of pancreaticoduodenal transplantation in pigs.

BACKGROUND: The early diagnosis of pancreas allograft dysfunction is crucial for the management and long-term survival of transplanted pancreases. We investigated whether intercellular adhesion molecular-1 (ICAM-1), Fas, and Fas ligand (FasL) can be used as novel biomarkers of acute pancreaticoduodenal allograft dysfunction in pigs. METHODS: Forty outbred landraces were randomly divided into three groups. In the control group (8 pigs), a sham operation was performed but no drugs were administered. In groups 1 and 2 (8 pairs each), pancreaticoduodenal transplantation was performed, with the latter administered immunosuppressive drugs and the former not administered drugs. The expression of ICAM-1, Fas, and FasL mRNA in the peripheral vein blood was assessed by flow cytometry and RT-PCR, pre-transplant and on days 1, 3, 5, and 7 after transplantation. Simultaneously, the levels of glucose, insulin, and glucagon in the serum of the recipients were evaluated. The allograft pancreas tissue was obtained to assess the pathological damage and the expression of Fas and FasL by immunohistochemistry. RESULTS: On the first 7 days after transplantation, ICAM-1, Fas, and FasL mRNA expression in the blood leukocytes of the recipient increased significantly in groups 1 and 2 compared with the control group (P < 0.01). However, the levels in group 2 were significantly lower than those in group 1 (P < 0.05). Interestingly, the FasL expression increased but the Fas expression decreased gradually in the graft pancreas tissue during the first week after transplantation in both groups 1 and 2 compared with the control group (P < 0.05). The levels of serous glucose, insulin, and glucagon in groups 1 and 2 obviously changed on day 1 after transplantation but returned to normal on day 2. The recipient's pancreas pathological sections did not exhibit any rejection changes on days 1 and 3 after transplantation but showed rejection damage on days 5 and 7. CONCLUSION: ICAM-1, Fas, and FasL were found to be sensitive biomarkers of acute pancreas allograft dysfunction after pancreaticoduodenal transplantation in pigs, and their monitoring could be used to evaluate the effectiveness of the immunosuppression therapy.

[Protective effects of agmatine on lipopolysaccharide -induced acute hepatic injury in mice].

OBJECTIVE: To observe the effect of agmatine ( AGM) on lipopolysaccharide ( LPS )-induced acute hepatic injury in mice, and to explore its related mechanism. METHODS: Sixty C57BU6 mice were randomly divided into control group ( n = 20, with intra-peritoneal injection of phosphate buffer saline 10 mg/kg), model group ( n = 20, with intra-peritoneal injection of LPS 10 mg/kg), and AGM group (n=20, with intra-peritoneal injection of LPS 10 mg/kg and AGM 200 mg/kg). Ten mice in each group were sacrificed at 6 hours and 24 hours, respectively, after modeling, blood samples were collected for the determination of tumor necrosis factor-a ( TNF -a) and interleukin ( IL-113 and IL-6) by enzyme linked immunosorbent assay (ELISA) at 6 hours after modeling , and for determination of alanine aminotransferase (ALT), aspartate transaminase (AST) and total bilirubin (TBil) by automatic biochemistry analyzer at 24 hours after modeling. Hepatic homogenate was also collected for determining the endo nuclear nuclear factor-KB ( NF -KB) p65 by Western blotting at 6 hours after modeling, and for observation of pathological changes at 24 hours after modeling. RESULTS: At 6 hours after modeling, .the mice in model group became lethargic and quiet, and their food and water assumption was reduced, but AGM was found to be able to greatly improve the general status of animals in AGM group. AGM was found to lower the contents of serum TNF-a ( IJ.g/L: 296.3 ± 42.5 vs. 627.2 ± 81.3, t=7.327, P=0.002), IL-113 ( f.Lg/L: 109.1 ± 12.3 vs. 264.2 ± 18.8, t= 11.958, P=0.001), IL-6 ( mg/L: 11.4 ± 1.9 vs. 23.6 ± 2.5, t=6.729, P=0.003), ALT (U!L: 107.9 ± 8.5 vs. 189.9 ± 13.6, t=8.856, P=0.001 ), AST (UIL: 347.4 ± 24.9 vs. 716.8 ± 60.4, t=9.793, P=0.001) and TBil ( f.Lmol!L: 8.3 ± 0.9 vs. 10.6 ± 0.5, t=3.869, P=0.018) in mice with acute hepatic injury induced by LPS. AGM also depressed TNF -a ( ng/g: 287.4 ± 32.5 vs. 461.5 ± 31.4, t=6.673, P= 0.003), IL-113 (pg/g: 146.7 ± 13.5 vs. 351.6 ± 28.7, t=11.190, P=0.001) and intranuclear NF-KB p65 level (NF-KBp65/TBP: 0.515 ± 0.060 vs. 0.853 ± 0.080, t=5.849, P=0.004) in liver tissue. Histological examination demonstrated that AGM significantly reduced liver injury caused by LPS, as manifested in amelioration of hepatocytes swelling, necrosis and neutrophil infiltration. CONCLUSION: Agmatine can reduce LPS-induced acute hepatic injury in mice via suppressing NF-κB translocation and reduction of the synthesis and release of cytokines.