miR-143-3p inhibits proliferation and invasion of hepatocellular carcinoma cells by regulating its target gene FGF1.

PURPOSE: To explore FGF1 and miR-143-3p expression in hepatocellular carcinoma (HCC) cells and its related mechanisms. METHODS: Eighty-two HCC patients treated at our hospital from January 2018 to January 2019 were enrolled as Group A, while further 80 healthy people undergoing physical examinations during the same time period were enrolled as Group B. HCC cells and normal human liver cells were purchased, with HepG2 and SMMC-7721 cells transfected with pcDNA3.1-FGF1, si-FGF1, NC, miR-143-3p-inhibitor and miR-143-3p-mimics. FGF1 and miR-143-3p expression was detected by qRT-PCR. The expression of N-cadherin, vimentin, Snail, Slug, E-cadherin and γ-catenin was detected by Western Blotting (WB). Cell proliferation was detected by MTT assay. Cell invasion was detected by Transwell. Cell apoptosis was detected by flow cytometry (FCM). RESULTS: FGF1 was highly expressed but miR-143-3p was poorly expressed in HCC cells. Areas under the curves (AUCs) of the two indicators were > 0.8. The indicators were correlated with the age, gender, tumor invasion, degree of differentiation, tumor location and TNM staging of the patients. Silencing FGF1 and overexpressing miR-143-3p could promote cell apoptosis, inhibit cell growth, cell epithelial-mesenchymal transition (EMT) and the expression of N-cadherin, vimentin, Snail and Slug, and increase the expression of E-cadherin and γ-catenin. Dual luciferase reporter gene assay (DLRGA) confirmed that FGF1 and miR-143-3p had a targeted relationship. The rescue experiment showed that the proliferation, invasion and apoptosis of HepG2 and SMMC-7721 cells in the miR-143-3p-mimics+pcDNA3.1-FGF1 and miR-143-3p-inhibitor+Si-FGF1 groups were not different from those in the miR-NC group. CONCLUSION: Inhibiting FGF1 can upregulate miR-143-3p-mediated Hedgehog signaling pathway, and affect cells' EMT, proliferation and invasion, so FGF1 is expected to become a potential therapeutic target for HCC.

Mast cells and immunoexpression of FGF-1 and Ki-67 in rat subcutaneous tissue following the implantation of Biodentine and MTA Angelus.

AIM: To compare the formation of fibrous capsules around Biodentine and MTA Angelus implants as well as the participation of fibroblast growth factor-1 (FGF-1) and mast cells in the tissue response to these endodontic materials. METHODOLOGY: Sixty polyethylene tubes filled with Biodentine or MTA, and empty tubes (control group) were implanted into the dorsal subcutaneous tissues of male rats. After 7, 15, 30 and 60 days, the specimens were embedded in paraffin and the number of fibroblasts and mast cells was quantified in the sections stained with Masson's trichrome or Alcian Blue, respectively. FGF-1 and Ki-67 were detected by immunohistochemistry, and the number of immunolabelled cells was computed. The collagen content was estimated in the picrosirius red-stained sections. The data were subjected to two-way ANOVA followed by Tukey's test (P ≤ 0.05). RESULTS: The capsules were associated with a significant increase (P < 0.0001) in the number of fibroblasts and mast cells, and in the collagen content over time. A significant decrease (P < 0.0001) in the immunoexpression of FGF-1 and Ki-67 was observed in all groups from the 7th-60th day. At 60 days, the number of fibroblasts (P = 0.0226) and the collagen content (P < 0.0001) were significantly greater in MTA than Biodentine specimens, while the greatest number of mast cells and FGF-1-immunolabelled cells was observed in Biodentine specimens (P < 0.0001). A significant difference in Ki-67 immunoexpression was not detected between specimens of Biodentine and MTA. CONCLUSIONS: The collagen-rich capsule formed slowly around Biodentine in comparison with MTA. FGF-1 and mast cells participated in capsule remodelling, stimulating fibroblast proliferation and subsequent collagen production, in response to subcutaneous implants.

Acidic and basic fibroblast growth factor expression levels in cervical cancer and their effects on tumor cell proliferation.

Fibroblast growth factors (FGFs) play important roles in angiogenesis, wound healing, embryonic development, and endocrine signaling pathways. Increasingly, recent studies have reported aberrant FGF expression in various malignancies. However, the involvement of FGFs in cervical carcinoma pathogenesis remains unclear. We aimed to investigate expression of acidic (aFGF) and basic FGF (bFGF) in patients with this disease, and assess their effects on cervical cancer cell proliferation. Twenty cervical cancer patients and 10 cervical intraepithelial neoplasia (CIN) patients were recruited, and 10 cancer-free individuals were included as controls. Reverse transcription-polymerase chain reaction and western blotting were employed to detect FGF mRNA and protein levels, respectively. Furthermore, HeLa cells were treated with FGFs and subjected to thiazolyl blue tetrazolium bromide assays to quantify proliferation. Compared with CIN and normal cervical tissues, aFGF and bFGF mRNA and protein levels were significantly elevated in cervical carcinomas (P < 0.05). CIN tissues exhibited higher expression of these FGFs than normal tissues (P < 0.05). Moreover, their mRNA levels were increased in advanced cancer stages (P < 0.05), although no significant difference was detected between tumors of different differentiation grades in this regard (P > 0.05). HeLa cell proliferation increased in an aFGF- and bFGF-dose-dependent manner (P < 0.05), the latter exerting a more potent proliferative influence, with its effect peaking at 75 ng/mL. aFGF and bFGF were highly expressed in cervical cancer tissues and their levels positively correlated with clinical stage. Both facilitate proliferation of cervical carcinoma cells and are implicated in cancer pathogenesis and progression.

Cytokine and Growth Factor Activation In Vivo and In Vitro after Spinal Cord Injury.

Spinal cord injury results in a life-disrupting series of deleterious interconnected mechanisms encompassed by the primary and secondary injury. These events are mediated by the upregulation of genes with roles in inflammation, transcription, and signaling proteins. In particular, cytokines and growth factors are signaling proteins that have important roles in the pathophysiology of SCI. The balance between the proinflammatory and anti-inflammatory effects of these molecules plays a critical role in the progression and outcome of the lesion. The excessive inflammatory Th1 and Th17 phenotypes observed after SCI tilt the scale towards a proinflammatory environment, which exacerbates the deleterious mechanisms present after the injury. These mechanisms include the disruption of the spinal cord blood barrier, edema and ion imbalance, in particular intracellular calcium and sodium concentrations, glutamate excitotoxicity, free radicals, and the inflammatory response contributing to the neurodegenerative process which is characterized by demyelination and apoptosis of neuronal tissue.

Upregulation of fibroblast growth factor 1 in the synovial membranes of patients with late stage osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of the systemic joint that involves multiple cytokines and growth factors. Fibroblast growth factor 1 (FGF-1) is increased in patients with rheumatic arthritis. The aim of this study was to determine whether the expression and secretion of FGF-1 differed in synovial tissue from patients with late stage OA from that in normal tissues. We selected eight patients with late stage OA and eight healthy donors for this study. An enzyme-linked immunosorbent assay was used to determine the amount of FGF-1 in the synovial fluid and in the culture medium of synovial fibroblasts. Real time quantitative polymerase chain reaction (qPCR) analysis was performed to examine the expression levels of FGF-1 and FGF receptor 2 (FGFR2) in synovial and cartilage tissues. We detected FGF-1 in the synovial fluid from all eight donors, as well as in the culture medium of synovial fibroblasts. Synovial fluid from patients with OA and culture medium of OA synovial fibroblasts contained significantly more FGF-1 than those from controls. FGF-1 expression was also lower in the synovial membranes of normal donors than in those of OA patients. FGFR2 expression was also higher in OA cartilage than in normal cartilage. Overall, these results demonstrated that FGF-1 synthesis and secretion by synovial fibroblasts were significantly increased in OA. FGFR2 expression was also shown to be upregulated in patients with OA. These findings suggest that increased FGF-1 signaling correlates with an OA pathological condition.

Modulation of gap junction channels and hemichannels by growth factors.

Gap junction hemichannels and cell-cell channels have roles in coordinating numerous cellular processes, due to their permeability to extra and intracellular signaling molecules. Another mechanism of cellular coordination is provided by a vast array of growth factors that interact with relatively selective cell membrane receptors. These receptors can affect cellular transduction pathways, including alteration of intracellular concentration of free Ca(2+) and free radicals and activation of protein kinases or phosphatases. Connexin and pannexin based channels constitute recently described targets of growth factor signal transduction pathways, but little is known regarding the effects of growth factor signaling on pannexin based channels. The effects of growth factors on these two channel types seem to depend on the cell type, cell stage and connexin and pannexin isoform expressed. The functional state of hemichannels and gap junction channels are affected in opposite directions by FGF-1 via protein kinase-dependent mechanisms. These changes are largely explained by channels insertion in or withdrawal from the cell membrane, but changes in open probability might also occur due to changes in phosphorylation and redox state of channel subunits. The functional consequence of variation in cell-cell communication via these membrane channels is implicated in disease as well as normal cellular responses.

Connexin hemichannel composition determines the FGF-1-induced membrane permeability and free [Ca2+]i responses.

Cell surface hemichannels (HCs) composed of different connexin (Cx) types are present in diverse cells and their possible role on FGF-1-induced cellular responses remains unknown. Here, we show that FGF-1 transiently (4-14 h, maximal at 7 h) increases the membrane permeability through HCs in HeLa cells expressing Cx43 or Cx45 under physiological extracellular Ca(2+)/Mg(2+) concentrations. The effect does not occur in HeLa cells expressing HCs constituted of Cx26 or Cx43 with its C-terminus truncated at aa 257, or in parental nontransfected HeLa cells. The increase in membrane permeability is associated with a rise in HC levels at the cell surface and a proportional increase in HC unitary events. The response requires an early intracellular free Ca(2+) concentration increase, activation of a p38 MAP kinase-dependent pathway, and a regulatory site of Cx subunit C-terminus. The FGF-1-induced rise in membrane permeability is also associated with a late increase in intracellular free Ca(2+) concentration, suggesting that responsive HCs allow Ca(2+) influx. The cell density of Cx26 and Cx43 HeLa transfectants cultured in serum-free medium was differentially affected by FGF-1. Thus, the FGF-1-induced cell permeabilization and derived consequences depend on the Cx composition of HCs.

Adrenalectomy counteracts the local modulation of astroglial fibroblast growth factor system without interfering with the pattern of 6-OHDA-induced dopamine degeneration in regions of the ventral midbrain.

The present study investigated the effects of bilateral adrenalectomy (ADX) on the synthesis of basic fibroblast growth factor (bFGF, FGF-2) mRNA and on the expression of its FGF receptor subtype-2 (FGFR2) mRNA after a 6-hydroxydopamine (6-OHDA)-induced lesion of nigrostriatal dopamine system. In previous papers we have demonstrated that corticosterone increases FGF-2 immunoreactivity mainly in the astrocytes of the substantia nigra [Chadi, G., Rosen, L., Cintra, A., Tinner, B., Zoli, M., Pettersson, R.F., Fuxe, K., 1993b. Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. Neuroreport 4, 783-786.] and that 6-OHDA injected in the ventral midbrain upregulates FGF-2 synthesis in reactive astrocytes in the ascending dopamine pathways [Chadi, G., Cao, Y., Pettersson, R.F., Fuxe, K., 1994. Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience 61, 891-910.]. Rats were adrenalectomized and received a 6-OHDA stereotaxical injection in the ventral midbrain 2 days later. Seven days after the dopamine lesion, Western blot analysis showed a decreased level of tyrosine hydroxylase in the lesioned side of the midbrain, an event that was not altered by ADX or corticosterone replacement. Moreover, the degeneration of nigral dopamine neurons, which was confirmed by the disappearance of acidic FGF (FGF-1) mRNA and the decrement of tyrosine hydroxylase mRNA labeled nigral neurons, was not altered by ADX. The FGF-2 protein (23 kDa isoform but not 21 kDa fraction) levels increased in the lesioned side of the ventral midbrain. This elevation was counteracted by ADX, an effect that was fully reversed by corticosterone replacement. In situ hybridization revealed that ADX counteracted the elevated FGF-2 mRNA levels in putative glial cells of the ipsilateral pars compacta of the substantia nigra and in the ventral tegmental area. The ADX also counteracted the increased density and intensity of the astroglial FGF-2 immunoreactive profiles within the lesioned pars compacta of the substantia nigra and the ventral tegmental area as determined by stereology. The stereotaxical mechanical needle insertion triggered the expression of FGFR2 mRNA in putative glial cells, spreading to the entire ipsilateral ventral midbrain from the region of needle track, an occurrence that was partially reversed by ADX. In conclusion, bilateral ADX counteracted the increased astroglial FGF-2 synthesis in the dopamine regions of the ventral midbrain following a 6-OHDA-induced local lesion and interfered with FGF receptor regulation around injury. These findings give further evidence that adrenocortical hormones may regulate the astroglial FGF-2-mediated trophic mechanisms and wound repair events in the lesioned central nervous system.

Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis.

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.

Complexity of astrocyte-motor neuron interactions in amyotrophic lateral sclerosis.

Neurons and surrounding glial cells compose a highly specialized functional unit. In amyotrophic lateral sclerosis (ALS) astrocytes interact with motor neurons in a complex manner to modulate neuronal survival. Experiments using chimeric mice expressing ALS-linked mutations to Cu,Zn superoxide dismutase (SOD-1) suggest a critical modulation exerted by neighboring non-neuronal cell types on disease phenotype. When perturbed by primary neuronal damage, e.g. expression of SOD-1 mutations, neurons can signal astrocytes to proliferate and become reactive. Fibroblast growth factor-1 (FGF-1) can be released by motor neurons in response to damage to induce astrocyte activation by signaling through the receptor FGFR1. FGF-1 stimulates nerve growth factor (NGF) expression and secretion, as well as activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor. Nrf2 leads to the expression of antioxidant and cytoprotective enzymes such as heme oxygenase-1 and a group of enzymes involved in glutathione metabolism that prevent motor neuron degeneration. However, prolonged stimulation with FGF-1 or SOD-mediated oxidative stress in astrocytes may disrupt the normal neuron-glia interactions and lead to progressive neuronal degeneration. The re-expression of p75 neurotrophin receptor and neuronal NOS in motor neurons in parallel with increased NGF secretion by reactive astrocytes may be a mechanism to eliminate critically damaged neurons. Consequently, astrocyte activation in ALS may have a complex pathogenic role.

Thyroid hormone induces cerebellar astrocytes and C6 glioma cells to secrete mitogenic growth factors.

In this study, the effect of thyroid hormone (triiodothyronine, T(3)) on the secretion of mitogenic growth factors in astrocytes and C6 glioma cells was examined. The proliferating activity of T(3) could be due, at least in part, to the astrocyte secretion of acidic and basic fibroblast growth factor (aFGF and bFGF), tumor necrosis factor-beta, and transforming growth factor-beta. In contrast, the conditioned medium (CM) of T(3)-treated C6 cells was mitogenic to this cell line only after hyaluronidase digestion, suggesting the impairment of growth factor mitogenic activity by hyaluronic acid. Furthermore, the presence of bFGF was significantly greater in the CM of both T(3)-treated astrocytes and T(3)-treated C6 cells than in the corresponding control CM. These data show that T(3) induces cerebellar astrocytes to secrete mitogenic growth factors, predominantly bFGF, that could influence astrocyte and neuronal proliferation via autocrine and paracrine pathways.

Structure and function of fibroblast growth factor.

We describe some structural requirements of the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1) to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50 = 10-20 microM) and 3) to compete with human FGF-1 for cellular binding (ID50 = 30-50 microM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, we propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

Structure and function of fibroblast growth factor

We describe some structural requirements od the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1)to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50=10-20 muM) and 3)to compete with human FGF-1 for cellular binding (ID50=30-50 muM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, ee propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

Helicobacter pylori interacts with heparin and heparin-dependent growth factors.

The pathogenic bacterium Helicobacter pylori, which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 x 10(-12)M), and also heparan sulfate and heparin with higher affinity (Kd 9 x 10(-9)M) than FGFs bind to heparin (10(-8) - 10(-9)M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori, which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanisms. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.