A systematic analysis of molecular mechanisms in non-metastatic renal cancer delineates affected regulatory pathways and genes in tumor growth.

In the recent century, Kidney cancer has emerged as one of the critical renal diseases. Therefore, we analyzed gene expression profiles of non-metastatic kidney cancer to find mechanisms associated with the early-stage pathogenesis of the disease. We concentrated on the most dysregulated genes in expression to discover possible unknown proliferative molecular mechanisms and oncogenic pathways promoting kidney renal cancer growth. Survival analysis, expression profiling, and gene set over-representation analysis were conducted on the most upregulated and most down-regulated genes alongside the hub genes. Our results demonstrated that pathways engaged in the metabolism of amino acids and carbohydrates and those involved in peroxisome organization were shown to be important in developing benign tumors. Furthermore, upregulation of genes such as CXCL9 and 10 genes and CXCR4 in chemokine response pathways would bolster differentiation and engagement of immune cells in the tumor microenvironment. C3, one of the essential members of the complement system, with a high degree and betweenness centrality in the PPI network, upregulated significantly not only in our analysis but also in the validation expression profiling results and survival analysis. We also identified genes such as TYROBP, ITGB2, and EGFR to be engaged in both immunological pathways and superoxide pathways. Furthermore, we found that downregulation of Aldolase B engaged in Glycolysis and Gluconeogenesis pathways would help develop benign tumors. Finally, many top hub genes, including TYMS, PTPRC, AURKA, FN1, UBE2C, and CD53 were proposed to be engaged in the progression of non-metastatic renal tumors. This holistic interrogation calls attention to investigate further and experimentally validate the proposed molecular mechanisms.

Long non-coding RNA H19 acts as a microRNA-194 sponge to inhibit the apoptosis and promote the proliferation of hypertrophic scar fibroblasts.

The effects of long non-coding RNAs (lncRNAs) on the proliferation of hypertrophic scars have been described, however, the underlying mechanisms are not well characterized. The present study aimed to investigate the mechanisms of lncRNA H19 in hypertrophic scars. The effects of the lncRNA H19 on the proliferation and apoptosis of hypertrophic scar fibroblasts (HSFs) were analyzed using 5'-ethynyl-2'-deoxyuridine staining, flow cytometry, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). The results revealed H19 promoted the proliferation and inhibited the apoptosis in HSF. In addition, the binding associations between H19 and microRNA-194 (miR-194), and miR-194 and insulin-like growth factor I receptor (IGF1R) were identified using bioinformatics screening and verified using dual-luciferase assays. Furthermore, the effects of the IGF1R knockdown on H19-induced HSF phenotypes and regulation over the p38 MAPK pathway were determined. Mechanistically, miR-194 was identified as the downstream effector of the H19-mediated phenotypes of HSFs through its ability to directly target IGF1R, thus modulating the p38 MAPK signaling pathway. In conclusion, the findings suggested that H19 may inhibit the apoptosis and promote the proliferation of HSFs through the miR-194/IGF1R/p38 MAPK signaling axis, thereby contributing to the progression of hypertrophic scars. These findings may provide novel targets for the treatment of hypertrophic scars.

Heparanase-1-induced shedding of heparan sulfate from syndecan-1 in hepatocarcinoma cell facilitates lymphatic endothelial cell proliferation via VEGF-C/ERK pathway.

Heparanase-1/syndecan-1 axis plays critical roles in tumorigenesis and development. The main mechanism includes heparanase-1 (HPA-1) degrades the heparan sulfate chain of syndecan-1 (SDC-1), and the following shedding of heparan sulfate from tumor cell releases and activates SDC-1 sequestered growth factors. However, the significance of Heparanase-1/syndecan-1 axis and its effects on the microenvironment of lymphatic metastasis in hepatocellular carcinogenesis (HCC) procession have not been reported. Herein, we found that HPA-1 could degrade the heparan sulfate on hepatocarcinoma cell surface. Importantly, HPA-1-induced shedding of heparan sulfate chain from SDC-1 facilitated the release of vascular endothelial growth factor C (VEGF-C) from SDC-1/VEGF-C complex into the medium of hepatocarcinoma cell. Further studies indicated that VEGF-C secretion from hepatocarcinoma cell promoted lymphatic endothelial cell growth through activating extracellular signal-regulated kinase (ERK) signaling. Taken together, this study reveals a novel existence of Heparanase-1/syndecan-1 axis in hepatocarcinoma cell and its roles in the cross-talking with the microenvironment of lymphatic metastasis.

GATA6 inhibits the biological function of non-small cell lung cancer by modulating glucose metabolism.

PURPOSE: This study aims to explore the role of GATA6 in lung cancer, with a focus on its impact on metabolic processes. METHODS: We assessed GATA6 expression in lung cancer tissues and its association with patient prognosis. In vitro cell function experiments were conducted to investigate the effects of altered GATA6 levels on lung cancer cell proliferation and migration. Mechanistic insights were gained by examining GATA6's influence on glucose metabolism-related genes, particularly its effect on c-Myc mRNA expression. RESULTS: Our study revealed significant down-regulation of GATA6 in lung cancer tissues, and this down-regulation was strongly correlated with unfavorable patient prognosis. Elevating GATA6 levels effectively inhibited the proliferation and migration of lung cancer cells in our cell function experiments. Mechanistically, we found that GATA6 suppressed the expression of c-Myc mRNA, impacting genes related to glucose metabolism. As a result, glucose uptake and metabolism in lung cancer cells were disrupted, ultimately impeding their malignant behaviors. CONCLUSION: Our study provides crucial insights into the metabolic regulation of GATA6 in lung cancer cells. These findings have the potential to offer a solid theoretical foundation for the development of novel clinical treatments for lung cancer.

LncRNA CAI2 contributes to poor prognosis of glioma through the PI3K-AKt signaling pathway.

AIMS: We aim to explore new potential therapeutic targets and markers in human glioma. BACKGROUND: Gliomas are the most common malignant primary tumor in the brain. OBJECTIVE: In the present research, we evaluated the effect of CAI2, a long non-coding RNA, on the biological behaviors of glioma and explored the related molecular mechanism. METHOD: The expression of CAI2 was analyzed using qRT-PCR in 65 cases of glioma patients. The cell proliferation was determined with MTT and colony formation assays, and the PI3K-AKt signaling pathway was analyzed using western blot. RESULT: CAI2 was upregulated in human glioma tissue compared with the matched, adjacent nontumor tissue and was correlated with WHO grade. Survival analyses proved that the overall survival of patients with high CAI2 expression was poor compared to that of patients with low CAI2 expression. High CAI2 expression was an independent prognostic factor in glioma. The absorbance values in the MTT assay after 96 h were .712 ± .031 for the si-control and .465 ± .018 for the si-CAI2-transfected cells, and si-CAI2 inhibited colony formation in U251 cells by approximately 80%. The levels of PI3K, p-AKt, and AKt in si-CAI2-treated cells were decreased. CONCLUSION: CAI2 may promote glioma growth through the PI3K-AKt signaling pathway. This research provided a novel potential diagnostic marker for human glioma.

Caveolin-1 up-regulates ST6Gal-I to promote the adhesive capability of mouse hepatocarcinoma cells to fibronectin via FAK-mediated adhesion signaling.

Caveolin-1 is a major structural protein of caveolae and plays important functions in tumorigenesis and development. Hca-F and Hepa1-6 are mouse hepatocarcinoma cell lines with high and low malignant potential, respectively. Our previous studies revealed that caveolin-1 promoted cell invasion by up-regulating the glycosylation of matrix metalloproteinase inducer CD147 of Hepa1-6 and Hca-F cells. However, the roles of caveolin-1 in cell-ECM adhesion and the mechanisms involved remain unknown. This study showed that caveolin-1 overexpression in Hepa1-6 cells up-regulated sialyltransferase ST6Gal-I expression and activated FAK-mediated adhesion signaling, and down-regulation of ST6Gal-I attenuated caveolin-1-induced increase in the adhesive ability of Hepa1-6 cells to fibronectin. Conversely, caveolin-1 knockdown in Hca-F cells inhibited ST6Gal-I expression and FAK signaling-mediated cell adhesion to fibronectin. Re-expression of wild-type caveolin-1 or ST6Gal-I rescued the decreased ST6Gal-I expression and adhesion of Hca-F cells caused by caveolin-1 silencing. Further studies indicated that caveolin-1 might regulate ST6Gal-I expression through caveolin-1 scaffolding domain. Taken together, these results demonstrate for the first time that caveolin-1 can up-regulate ST6Gal-I expression and further contribute to promoting mouse hepatocarcinoma cell adhesion to fibronectin by activating FAK-mediated adhesion signaling.

N-acetylglucosaminyltransferase IVa regulates metastatic potential of mouse hepatocarcinoma cells through glycosylation of CD147.

N-acetylglucosaminyltransferase (GnT)-IV a is a key enzyme that catalyzes the formation of the GlcNAC ß1-4 branch on the core structure of complex N-Glycans, which is the common substrate for other N-acetylglucosaminyltransferases, such as GnT-III and GnT-V. Our recent study indicates that the expression of GnT-IVa in Hca-F cells was much higher than that in Hepa1-6 cells, these two mouse hepatocarcinoma cell lines have high and no metastatic potential in lymph nodes respectively. To investigate the effects of GnT-IVa on the metastasis of hepatocarcinoma, exogenous GnT-IVa was introduced into Hepa1-6 cells, and on the other hand, the expression of GnT-IVa was down-regulated in Hca-F cells. The engineered overexpression of GnT-IVa in Hepa1-6 cells increased the antennary branches of complex N-glycans and reduced bisecting branches in vitro and in vivo, which leads to the increase in migration and metastatic capability of hepatocarcinoma cells. Conversely, down-regulated expression of GnT-IVa in Hca-F cells showed reduced tetra-antennary branches of N-Glycans, and significantly decreased the migration and metastatic capability. Furthermore, we found that the regulated GnT-IVa converts the heterogeneous N-glycosylated forms of CD147 in Hepa1-6 and Hca-F cells, and significantly changed the antennary oligosaccharide structures on CD147. These results suggest that GnT-IVa could be acting as a key role in migration and metastasis of mouse hepatocarcinoma cells through altering the glycosylation of CD147. These findings should be valuable in delineating the important function of GnT-IVa during the process of hepatocarcinoma growth and metastasis.

Immune cell infiltration and immunotherapy in hepatocellular carcinoma.

Hepatocellular carcinoma is a highly malignant tumor and patients yield limited benefits from the existing treatments. The application of immune checkpoint inhibitors is promising but the results described in the literature are not favorable. It is therefore urgent to systematically analyze the immune microenvironment of HCC and screen the population best suited for the application of immune checkpoint inhibitors to provide a basis for clinical treatment. In this study, we collected The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC)-related data sets to evaluate the immune microenvironment and immune cell infiltration (ICI) in HCC. Three independent ICI subtypes showing significant differences in survival were identified. Further, TCGA-LIHC immunophenoscore (IPS) was used to identify the differentially expressed genes between high- and low-IPS in HCC, so as to identify the immune gene subtypes in HCC tumors. The ICI score model for HCC was constructed, whereby we divided HCC samples into high- and low-score groups based on the median ICI score. The differences between these groups in genomic mutation load and immunotherapy benefit in HCC were examined in detail to provide theoretical support for accurate immunotherapy strategy in HCC. Finally, four genes were screened, which could accurately predict the subtype based on the tumor immune infiltration score. The findings may provide a basis and simplify the process for screening clinical drugs suitable for relevant subgroups.

Curcumin inhibits the migration and invasion of mouse hepatoma Hca-F cells through down-regulating caveolin-1 expression and epidermal growth factor receptor signaling.

Mouse hepatoma cellular carinoma cell line (Hca-F) cells have highly invasive and lymphatic metastasis potential in vitro and in vivo. Curcumin (diferuloylmethane) is an active component of the spice turmeric and has a diversity of antitumor activities. However, there is no available information to address the effects of curcumin on migration and invasion of mouse hepatoma Hca-F cells. In this study, we found that curcumin exerted a concentration- and time-dependent inhibitory effect on the migration and invasion of Hca-F cells in vitro. Curcumin inhibited the expression of the tumor promoter caveolin-1 (Cav-1) in Hca-F cells. Up-regulation of Cav-1 expression by pcDNA3.1/Cav-1 plasmid was able to reverse the curcumin-induced antimigration and anti-invasion effects in vitro. Curcumin down-regulated the expression of cluster of differntiation (CD)147, matrix metalloproteinase 2, and matrix metalloproteinase 9 and inhibited the phosphorylation of epidermal growth factor receptor (EGFR), the phosphoinositilde 3-kinase (PI3K)/protein kinase B (Akt), p38 mitogen-activated protein kinase (MAPK), and p44/42MAPK in Hca-F cells. Taken together, our findings suggest that curcumin can suppress the migratory and invasive ability of mouse hepatoma Hca-F cells, and this action is mediated through a novel mechanism involving inactivation of Cav-1 and EGFR signaling pathways.

MiR-34a inhibits lymphatic metastasis potential of mouse hepatoma cells.

MicroRNAs are small non-coding RNAs that regulate the expression of other genes in a post-transcriptional manner. MiR-34a can induce apoptosis, cell cycle arrest, and senescence. However, its role in tumor progress remains to be fully elucidated. In the present study, the role of miR-34a in lymphatic metastasis was investigated using mouse hepatocarcinoma cell lines Hca-F and Hepa1-6. MicroRNA profiling and Hairpin-RT-PCR analysis showed that the expression level of miR-34a was higher in Hepa1-6 cells (of no metastatic ability) than that in Hca-F cells (of high metastatic ability). Ectopic expression of miR-34a can inhibit cell growth and cell invasion in Hepa1-6 and Hca-F cells. Moreover, miR-34a triggers G1 arrest and down-regulates CyclinD1 and CDK6 in Hepa1-6 cells. Furthermore, we proved that miR-34a decreased adhesion of Hca-F cells to regional lymph node in vitro, reduced lymph nodes-metastasized burden, and inhibited tumor lymph node metastases in vivo. All these results suggest that miR-34a plays multiple tumor suppressive roles in murine hepatocarcinoma, not only inhibiting cell growth by cell cycle arrest, but also repressing metastasis, and may serve as a novel therapeutic target for hepatocarcinoma.

Effects of PI3K/AKT/mTOR pathway regulation of HIF-1α on Lanthanum-induced neurotoxicity in rats.

This study examined the effects of the AKT/mTOR/HIF-1α signaling pathway on learning and memory in offspring rats induced by lanthanum from neuroethology and molecular biology perspectives. 32 pregnant adult Wistar rats were divided into four groups randomly: control group (NC), 0.25%, 0.5% and 1.0% LaCl3 groups (n = 8). All rats were poisoned through free drinking from day 0 of pregnancy to postnatal day 21 (suckling period). All offspring rats were poisoned through free drinking from delactation to postnatal day 48. Offspring rats aged 49-days-old were used as sampling objects to construct an LaCl3 poisoning model of offspring rats. Changes in hippocampal neurons, apoptosis of hippocampal neurons, learning and memory abilities of LaCl3-poisoned animals were measured by Nissl staining, TUNEL method and the shuttle box test, respectively. Expressions of PI3K, AKT, and mTOR, HIF-1α, and VEGF in the hippocampus were tested by qPCR and Western blot. Distributions of PI3K and p-AKT in hippocampal neurons were observed through the immunohistochemical method. With increasing LaCl3 dose, lightning strike time and active avoidance incubation period of offspring rats in the different LaCl3 groups were significantly prolonged. The Nissl body positive neurons of hippocampal neurons gradually declined while apoptosis in cells increased. The expressions of both mRNA (PI3K, AKT, mTOR, HIF-1α, VEGF) and proteins (PI3K, p-AKT, p-mTOR, HIF-1α, VEGF) in the hippocampus of the LaCl3 groups were significantly lower than those of NC group (p < 0.05). LaCl3 poisoning can induce developmental injuries in hippocampal neurons and can increase cell apoptosis. As a result, learning and memory abilities of offspring rats, as well as the expressions of PI3K/AKT/mTOR, are decreased, thus inhibiting activation of HIF-1α and influencing the expression of the downstream VEGF gene.

ST8SIA1 inhibits the proliferation, migration and invasion of bladder cancer cells by blocking the JAK/STAT signaling pathway.

Bladder cancer (BLCA) is the most common malignant tumor of the urinary system, with distant metastasis of the tumor being the main cause of death. The identification of an effective biomarker may provide a novel direction for BLCA diagnosis and treatment. The aim of the present study was to screen the BLCA-related genes involved in sialyl transferase (ST) dysregulation and to investigate the functional mechanisms of α-2,8-ST1 (ST8SIA1) in BLCA cells. Data from The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis databases suggested that the mRNA expression levels of ST8SIA1 were decreased in BLCA tissues compared with normal tissues, which was also demonstrated using immunohistochemistry and western blot analysis. The expression levels of ST8SIA1 were negatively associated with the pathological grade and invasiveness of BLCA. Western blot analysis revealed that the expression levels of ST8SIA1 were lower in BLCA cell lines than in a normal urothelial cell line. CCK-8, flow cytometry, wound healing, colony formation and Transwell assays indicated that ST8SIA1 overexpression attenuated the proliferation, migration and invasion of T24 and 5637 BLCA cells. Further experiments revealed that ST8SIA1 could inhibit the phosphorylation of Janus kinase (JAK)2 and STAT3, as well as decrease the expression levels of JAK/STAT pathway-targeting signal molecules, including MMP2, proliferating cell nuclear antigen, cyclin D1 and Bcl2 in two BLCA cell lines. In conclusion, to the best of our knowledge, the present study was the first to indicate that the antitumor effect of ST8SIA1 in BLCA cells was mediated by the JAK/STAT signaling pathway, and the results provided a novel target for the diagnosis and treatment of BLCA.

Effects of small interfering RNA targeting heparanase-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cell lines.

BACKGROUND AND OBJECTIVE: Heparanase-1 (HPA-1) can promote angiogenesis and metastasis of malignant tumors and plays an important role in the genesis and development of tumors. This study was to explore the effects of specific small interfering RNA (siRNA) targeting HPA-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cells. METHODS: The expression of HPA-1 in Hca-F, Hca-P, and Hepa1-6 cells, which have high, low, and no metastatic potential, respectively, was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and enzyme-linked immunosorbent assay (ELISA). After transfection with two specific siRNAs targeting HPA-1, siRNA-1 and siRNA-2, and treatment with heparin, invasiveness of Hca-F cells was observed by Matrigel invasion assay. RESULTS: HPA-1 was negative in Hepa1-6 cells while positive in both Hca-F and Hca-P cells. The expression levels of both HPA-1 mRNA and protein were obviously higher in Hca-F cells than in Hca-P cells. HPA-1 proteins could be secreted into culture supernatant of Hca-F and Hca-P cells, and the amount of secreted HPA-1 detected by Western blot analysis was larger in Hca-F cells than in Hca-P cells (1.34 ± 0.02 vs. 0.60 ± 0.01, P < 0.001), which was consistent with the results of ELISA. Both siRNA-1 and siRNA-2 downregulated the expression of HPA-1 and the siRNA-2 did more efficiently. The number of invasive Hca-F cells treated with siRNA-2 or heparin alone was larger than that of Hca-F cells treated with combination of them (9 ± 1 vs. 4 ± 1, P = 0.013; 15 ± 2 vs. 4 ± 1, P = 0.008), but smaller than that of untreated Hca-F cells (9 ± 1 vs. 22 ± 2, P = 0.006; 15 ± 2 vs. 22 ± 2, P = 0.026). CONCLUSION: The combined application of specific siRNA targeting HPA-1 and heparin is more effective in inhibiting the invasiveness of mouse hepatoma cells.

Lanthanum Chloride Causes Neurotoxicity in Rats by Upregulating miR-124 Expression and Targeting PIK3CA to Regulate the PI3K/Akt Signaling Pathway.

BACKGROUND: Lanthanum (La) exposure can cause central nervous system (CNS) damage and dysfunction in children, seriously affecting intellectual development. miR-124 plays an important role in the development of the nervous system. We exposed rats to a La environment then observed the rats' learning and memory damage and neurotoxicity and the relationship with miR-124. METHODS: Rats were exposed to LaCl3 via drinking water. The rats' offspring were exposed to LaCl3 from their mother before weaning, then from La water for 28 days. A Morris water maze was used to observe spatial memory capabilities. H&E staining and TUNEL assays were used to observe pathological changes and apoptosis in the hippocampus. miR-124 was detected by RT-qPCR, and its targeting was confirmed by luciferase assay. The HT22 cell line was cultured with LaCl3 and treated with miR-124 mimics or inhibitors; then, expression of PI3K/Akt-related proteins was detected by western blot. RESULTS: La exposure can lead to impaired learning and memory ability in offspring. Offspring with La accumulations in the hippocampus showed severe damage, disordered cells, and increased neurocyte apoptosis. In vitro, the postsynaptic density protein 95 was downregulated under La exposure and apoptosis increased. This effect of La can be attenuated by miR-124 inhibitors and enhanced by miR-124 mimics. LaCl3 exposure increased miR-124 expression and targeting on PIK3CA, downregulating PI3K, p-Akt, and p-NF-κB p65. CONCLUSION: La causes neurotoxicity by upregulating miR-124 expression and targeting PIK3CA through the PI3K/Akt signaling pathway.

Overexpression of DEK is an indicator of poor prognosis in patients with gastric adenocarcinoma.

Increased expression of the human DEK proto-oncogene (DEK) gene has been associated with numerous human malignancies. The DEK protein is associated with chromatin reconstruction and gene transcription, and is important in cell apoptosis. The present study aimed to elucidate the role of DEK with regard to gastric adenocarcinoma tumor progression and patient prognosis. DEK protein expression was analyzed using immunohistochemistry in 192 tumors paired with adjacent non-cancerous gastric mucosa that had been surgically resected from patients with primary gastric adenocarcinoma. The association between DEK expression and the clinicopathological characteristics of the patients was evaluated using the χ2 test and Fisher's exact test. The survival rates of the patients were calculated using the Kaplan-Meier method. Cox analysis evaluated the association between the expression of DEK and the survival rate of the patients. The DEK protein was expressed in 84 patients with gastric adenocarcinoma (43.8%) and in 20 of the paired normal gastric mucosa tissues (11.5%). The DEK expression rate was found to be associated with tumor size (P=0.006), tumor grade (P=0.023), lymph node metastasis (P=0.018), serous invasion (P=0.026), tumor stage (P=0.001) and Ki-67 expression (P=0.003). Furthermore, patients with gastric adenocarcinoma that expressed DEK had decreased disease-free (log-rank, 16.785; P<0.0001) and overall (log-rank, 15.759; P<0.0001) survival rates compared with patients without DEK expression. Patients with late-stage gastric adenocarcinoma that expressed DEK exhibited a lower overall survival rate compared with patients without DEK expression (P=0.002). Additional analysis revealed that DEK expression was an independent prognostic factor for the prognosis of gastric adenocarcinoma (hazard ratio, 0.556; 95% confidence interval, 0.337-0.918; P=0.022). From the results of the present study, it can be concluded that the detection of DEK protein expression in gastric adenocarcinoma tissues may be important for the diagnosis and prognosis of patients, and may be a targeted therapy for the treatment of gastric adenocarcinoma.

Caveolin-1 up-regulates integrin α2,6-sialylation to promote integrin α5ß1-dependent hepatocarcinoma cell adhesion.

The alterations of integrin glycosylation play a crucial role in tumor metastasis. Our previous studies indicated that caveolin-1 promoted the expression of the key α2,6-sialytransferase ST6Gal-I and fibronectin-mediated adhesion of mouse hepatocarcinoma cell. Herein, we investigated the role of α2,6-sialylated α5-integrin in the adhesion of mouse hepatocarcinoma H22 cell. We demonstrated that caveolin-1 up-regulated cell surface α2,6-linked sialic acid via stimulating ST6Gal-I transcription. Cell surface α2,6-sialylation was required for integrin α5ß1-dependent cell adhesion to fibronectin, and an increase in α2,6-linked sialic acid on α5-subunit facilitated fibronectin-mediated focal adhesion kinase phosphorylations, suggesting that α2,6-sialylated α5-subunit promoted integrin α5ß1-dependent cell adhesion.