lncRNA PRR34-AS1 promotes HCC development via modulating Wnt/ß-catenin pathway by absorbing miR-296-5p and upregulating E2F2 and SOX12.

Hepatocellular carcinoma (HCC) belongs to the most frequent cancer with a high death rate worldwide. Thousands of long non-coding RNAs (lncRNAs) have been confirmed to influence the development of human cancers, including HCC. Nevertheless, the biological role of PRR34 antisense RNA 1 (PRR34-AS1) in HCC remains obscure. Here, we observed via quantitative real-time reverse transcriptase polymerase chain reaction (quantitative real-time RT-PCR) that PRR34-AS1 was highly expressed in HCC cells. Functional assays revealed that PRR34-AS1 promoted HCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process in vitro and facilitated tumor growth in vivo. In addition, western blot analysis and TOP Flash/FOP Flash reporter assays verified that PRR34-AS1 stimulated Wnt/ß-catenin pathway in HCC cells. Furthermore, RNA immunoprecipitation (RIP), RNA pull-down, and luciferase reporter assays uncovered that PRR34-AS1 sequestered microRNA-296-5p (miR-296-5p) to positively modulate E2F transcription factor 2 (E2F2) and SRY-box transcription factor 12 (SOX12) in HCC cells. Importantly, chromatin immunoprecipitation (ChIP) and luciferase reporter assays uncovered that E2F2 transcriptionally activated PRR34-AS1 in turn. Further, rescue experiments reflected that PRR34-AS1 affected HCC progression through targeting miR-296-5p/E2F2/SOX12/Wnt/ß-catenin axis. Our findings found that PRR34-AS1 elicited oncogenic functions in HCC, which indicated that PRR34-AS1 might be a novel therapeutic target for HCC.

Tacrolimus- and Nerve Growth Factor-Treated Allografts for Neural Tissue Regeneration.

Treatment of injured peripheral nerves, especially long-distance nerve defects, remains a significant challenge in regenerative medicine due to complex biological conditions and a lack of biomaterials for effective nerve reconstruction. Without proper treatment, nerve injury leads to motor and sensory dysfunction. Here, we have developed an efficacious nerve allograft treated with a dual drug containing acrolimus and nerve growth factor to bridge the nerve gap and achieve rapid neural tissue recovery without immunological rejection. The recovery of the structure, activity, and function of rats treated with the dual drug-treated allograft was investigated by walking track analysis and electrophysiological measurement. The sciatic functional index was measured to be -3.0 after a 12-week treatment. The nerve conduction velocity, peak latency, and peak amplitude of the nerve action potentials demonstrate the functional recovery of the nerve. To study the synergistic effect of the dual drug on the growth of neurites, a neural cell hypoxia model was created. The dual drug exhibited a high efficiency in promoting the growth of nerve cells under the nerve injury-induced hypoxic condition. The dual drug could protect the cells against antioxidative damage from hypoxia by the expression of heat shock protein, hypoxia-inducible factor, ß-tubulin, and vimentin.

Aqueous Two-Phase Emulsion Bioink-Enabled 3D Bioprinting of Porous Hydrogels.

3D bioprinting technology provides programmable and customizable platforms to engineer cell-laden constructs mimicking human tissues for a wide range of biomedical applications. However, the encapsulated cells are often restricted in spreading and proliferation by dense biomaterial networks from gelation of bioinks. Herein, a cell-benign approach is reported to directly bioprint porous-structured hydrogel constructs by using an aqueous two-phase emulsion bioink. The bioink, which contains two immiscible aqueous phases of cell/gelatin methacryloyl (GelMA) mixture and poly(ethylene oxide) (PEO), is photocrosslinked to fabricate predesigned cell-laden hydrogel constructs by extrusion bioprinting or digital micromirror device-based stereolithographic bioprinting. The porous structure of the 3D-bioprinted hydrogel construct is formed by subsequently removing the PEO phase from the photocrosslinked GelMA hydrogel. Three different cell types (human hepatocellular carcinoma cells, human umbilical vein endothelial cells, and NIH/3T3 mouse embryonic fibroblasts) within the 3D-bioprinted porous hydrogel patterns show enhanced cell viability, spreading, and proliferation compared to the standard (i.e., nonporous) hydrogel constructs. The 3D bioprinting strategy is believed to provide a robust and versatile platform to engineer porous-structured tissue constructs and their models for a variety of applications in tissue engineering, regenerative medicine, drug development, and personalized therapeutics.

Painful Terminal Neuroma Prevention by Capping PRGD/PDLLA Conduit in Rat Sciatic Nerves.

Neuroma formation after amputation as a long-term deficiency leads to spontaneous neuropathic pain that reduces quality of life of patients. To prevent neuroma formation, capping techniques are implemented as effective treatments. However, an ideal, biocompatible material covering the nerves is an unmet clinical need. In this study, biocompatible characteristics presented by the poly(D,L-lactic acid)/arginylglycylaspartic acid (RGD peptide) modification of poly{(lactic acid)-co- [(glycolic acid)-alt-(L-lysine)]} (PRGD/PDLLA) are evaluated as a nerve conduit. After being capped on the rat sciatic nerve stump in vivo, rodent behaviors and tissue structures are compared via autotomy scoring and histological analyses. The PRGD/PDLLA capped group gains lower autotomy score and improves the recovery, where inflammatory infiltrations and excessive collagen deposition are defeated. Transmission electron microscopy images of the regeneration of myelin sheath in both groups show that abnormal myelination is only present in the uncapped rats. Changes in related genes (MPZ, MBP, MAG, and Krox20) are monitored quantitative real-time polymerase chain reaction (qRT-PCR) for mechanism investigation. The PRGD/PDLLA capping conduits not only act as physical barriers to inhibit the invasion of inflammatory infiltration in the scar tissue but also provide a suitable microenvironment for promoting nerve repairing and avoiding neuroma formation during nerve recovery.

A conductive sodium alginate and carboxymethyl chitosan hydrogel doped with polypyrrole for peripheral nerve regeneration.

Polymer materials with electrically conductive properties have good applications in their respective fields because of their special properties. However, they usually exhibited poor mechanical properties and biocompatibility. In this work, we present a simple approach to prepare conductive sodium alginate (SA) and carboxymethyl chitosan (CMCS) polymer hydrogels (SA/CMCS/PPy) that can provide sufficient help for peripheral nerve regeneration. SA/CMCS hydrogel was cross-linked by calcium ions provided by the sustained release system consisting of d-glucono-δ-lactone (GDL) and superfine calcium carbonate (CaCO3), and the conductivity of the hydrogel was provided by doped with polypyrrole (PPy). Gelation time, swelling ratio, porosity and Young's modulus of the conductive SA/CMCS/PPy hydrogel were adjusted by polypyrrole content, and the conductivity of it was within 2.41 × 10-5 to 8.03 × 10-3 S cm-1. The advantages of conductive hydrogels in cell growth were verified by controlling electrical stimulation of cell experiments, and the hydrogels were also used as a filling material for the nerve conduit in animal experiments. The SA/CMCS/PPy conductive hydrogel showed good biocompatibility and repair features as a bioactive biomaterial, we expect this conductive hydrogel will have a good potential in the neural tissue engineering.

Umbilical cord blood-derived non-hematopoietic stem cells retrieved and expanded on bone marrow-derived extracellular matrix display pluripotent characteristics.

BACKGROUND: Umbilical cord blood (UCB) not only contains hematopoietic stem cells (HSCs), but also non-hematopoietic stem cells (NHSCs) that are able to differentiate into a number of distinct cell types. Based on studies published to date, the frequency of NHSCs in UCB is believed to be very low. However, the isolation of these cells is primarily based on their adhesion to tissue culture plastic surfaces. METHODS AND RESULTS: In the current study, we demonstrate that this approach overlooks some of the extremely immature NHSCs because they lack the ability to adhere to plastic. Using a native extracellular matrix (ECM), produced by bone marrow (BM) stromal cells, the majority of the UCB-NHSCs attached within 4 h. The colony-forming unit fibroblast frequency of these cells was 1.5 × 104/108 mononuclear cells, which is at least 4000-fold greater than previously reported for UCB-NHSCs. The phenotype of these cells was fibroblast-like and different from those obtained by plastic adhesion; they formed embryonic body-like clusters that were OCT4-positive and expressed other human embryonic stem cell-related markers. Importantly, when implanted subcutaneously for 8 weeks into immunocompromised mice, these ECM-adherent and expanded NHSCs generated three germ layer-derived human tissues including muscle, fat, blood vessel, bone, gland, and nerve. Moreover, injection of these cells into muscle damaged by cryoinjury significantly accelerated muscle regeneration. CONCLUSIONS: These results indicate that UCB may be a virtually unlimited source of NHSCs when combined with isolation and expansion on ECM. NHSCs may be a practical alternative to embryonic stem cells for a number of therapeutic applications.

A Supramolecular Gel Approach to Minimize the Neural Cell Damage during Cryopreservation Process.

The storage method for living cells is one of the major challenges in cell-based applications. Here, a novel supramolecular gel cryopreservation system (BDTC gel system) is introduced, which can observably increase the neural cell viability during cryopreservation process because this system can (1) confine the ice crystal growth in the porous of BDTC gel system, (2) decrease the amount of ice crystallization and cryopreservation system's freezing point, and (3) reduce the change rates of cell volumes and osmotic shock. In addition, thermoreversible BDTC supramolecular gel is easy to be removed after thawing so it does not hinder the adherence, growth, and proliferation of cells. The results of functionality assessments indicate that BDTC gel system can minimize the neural cell damage during cryopreservation process. This method will be potentially applied in cryopreservation of other cell types, tissues, or organs and will benefit cell therapy, tissue engineering, and organs transplantation.

Effect of Hydroxyapatite Nanoparticles on the Growth Potential of Hepatoma Cells in Nude Mice.

AIM: To evaluate the activity of hydroxyapatite (HAP) nanoparticles against the proliferation of hepatoma cells. METHODS: HAP nanoparticles were prepared by homogeneous precipitation. The size distribution and morphology of these nanoparticles were determined by laser particle analysis and transmission electron microscopy, respectively. Xenograft tumor models of human hepatoma cells (Bel-7402) implanted in nude mice under the right scruff skin were established and divided into two groups: treatment and control. Once the xenograft tumor grew to a diameter of 0.8 cm, 0.2 ml HAP nanoparticle suspension was injected into the tumor every day for 2 weeks. The long and short diameters of the tumors were measured before and after HAP injection, and the inhibition rate of tumor growth was calculated. Paraffin tissue sections were prepared from xenograft tumors treated as above for 2 weeks, histologically stained for DNA and agyrophilic nucleolar organizer region (AgNORs), and immuno-histologically stained for proliferating cell nuclear antigens (PCNAs). The stained sections were examined by microscopy. Images of these sections were recorded and analyzed by image analysis system and relevant software for DNA content, AgNOR intensity, and PCNA expression in the nucleus, nucleoli, and hepatoma cells, respectively. RESULTS: The HAP nanoparticles were uniformly distributed, with a size of 44.6 nm to 86.8 nm. Upon the local injection of the tumor with the HAP nanoparticles, the average volumes of the tumors were significantly reduced compared with those of the control group, which had a tumor inhibition rate of 51.32%. The DNA content, AgNOR intensity, and PCNA expression in the hepatoma cells were all significantly reduced (P < 0.01) compared with those in the control group. CONCLUSION: HAP nanoparticles inhibit the proliferation of hepatoma cells in vivo.

Calcium Carbonate Nanoplate Assemblies with Directed High-Energy Facets: Additive-Free Synthesis, High Drug Loading, and Sustainable Releasing.

Developing drug delivery systems (DDSs) with high drug-loading capacity and sustainable releasing is critical for long-term chemotherapeutic efficacy, and it still remains challenging. Herein, vaterite CaCO3 nanoplate assemblies with exposed high-energy {001} facets have been synthesized via a novel, additive-free strategy. The product shows a high doxorubicin-loading capacity (65%); the best of all the CaCO3-based DDSs so far. Also, the product's sustainable releasing performance and its inhibition of the initial burst release, together, endow it with long-term drug efficacy. The work may shed light on exposing directed high-energy facets for rationally designing of a drug delivery system with long-term efficacy.

PRGD/PDLLA conduit potentiates rat sciatic nerve regeneration and the underlying molecular mechanism.

Peripheral nerve injury requires optimal conditions in both macro-environment and micro-environment for reestablishment. Though various strategies have been carried out to improve the macro-environment, the underlying molecular mechanism of axon regeneration in the micro-environment provided by nerve conduit remains unclear. In this study, the rat sciatic nerve of 10 mm defect was made and bridged by PRGD/PDLLA nerve conduit. We investigated the process of nerve regeneration using histological, functional and real time PCR analyses after implantation from 7 to 35 days. Our data demonstrated that the ciliary neurotrophic factor highly expressed and up-regulated the downstream signaling pathways, in the case of activated signals, the expressions of axon sprout relative proteins, such as tubulin and growth-associated protein-43, were strongly augmented. Taken together, these data suggest a possible mechanism of axon regeneration promoted by PRGD/PDLLA conduit, which created a micro-environment for enhancement of diffusion of neurotrophic factors secreted by the injured nerve stumps, and activation of molecular signal transduction involved in growth cone, to potentiate the nerve recovery.

Promotion of peripheral nerve regeneration and prevention of neuroma formation by PRGD/PDLLA/ß-TCP conduit: report of two cases.

In the field of nerve repair, one major challenge is the formation of neuroma. However, reports on both the promotion of nerve regeneration and prevention of traumatic neuroma in the clinical settings are rare in the field of nerve repair. One of the reasons could be the insufficiency in the follow-up system. We have conducted 33 cases of nerve repair using PRGD/PDLLA/ß-TCP conduit without any sign of adverse reaction, especially no neuroma formation. Among them, we have selected two cases as representatives to report in this article. The first case was a patient with an upper limb nerve wound was bridged by PRGD/PDLLA/ß-TCP conduit and a plate fixation was given. After nearly 3-years' follow-up, the examination results demonstrated that nerve regeneration effect was very good. When the reoperation was performed to remove the steel plate we observed a uniform structure of the regenerated nerve without the formation of neuroma, and to our delight, the implanted conduit was completely degraded 23 months after the implantation. The second case had an obsolete nerve injury with neuroma formation. After removal of the neuroma, the nerve was bridged by PRGD/PDLLA/ß-TCP conduit. Follow-up examinations showed that the structure and functional recovery were improved gradually in the 10-month follow-up; no end-enlargement and any other abnormal reaction associated with the characteristic of neuroma were found. Based on our 33-case studies, we have concluded that PRGD/PDLLA/ß-TCP nerve conduit could both promote nerve regeneration and prevent neuroma formation; therefore, it is a good alternative for peripheral nerve repair.

Different inhibitory effect and mechanism of hydroxyapatite nanoparticles on normal cells and cancer cells in vitro and in vivo.

Hydroxyapatite (HAP), similar to inorganic phase in bones, shows good biocompatibility and bioactivity as bone defect repairing material. Recently, nanoscaled HAP shows the special properties differing from bulk HAP in physics, chemistry and biology. This paper demonstrates that HAP nanoparticle (nHAP) possesses the ability for inhibiting cancer cell growth in vitro and in vivo. In vitro, after treatment with nHAP for 3 days, proliferation of human cancer cells are inhibited by more than 65% and by less than 30% for human normal cells. In vivo, injection of nHAP in transplanted tumor results in significant reduction (about 50%) of tumor size. The anticancer effect of nHAP is mainly attributed to high amount by endocytosis in cancer cells and inhibition on protein synthesis in cells. The abundant nHAP internalized in cancer cells around endoplasmic reticulum may inhibit the protein synthesis by decreasing the binding of mRNA to ribosome due to its high adsorption capacity for ribosome and arrest cell cycle in G0/G1 phase. nHAP shows no ROS-involved cytotoxicity and low cytotoxicity to normal cells. These results strongly suggest that nHAP can inhibit cancer cell proliferation and have a potential application in cancer treatment.

Conductive PPY/PDLLA conduit for peripheral nerve regeneration.

The significant drawbacks and lack of success associated with current methods to treat critically sized nerve defects have led to increased interest in neural tissue engineering. Conducting polymers show great promise due to their electrical properties, and in the case of polypyrrole (PPY), its cell compatibility as well. Thus, the goal of this study is to synthesize a conducting composite nerve conduit with PPY and poly(d, l-lactic acid) (PDLLA), assess its ability to support the differentiation of rat pheochromocytoma 12 (PC12) cells in vitro, and determine its ability to promote nerve regeneration in vivo. Different amounts of PPY (5%, 10%, and 15%) are used to synthesize the conduits resulting in different conductivities (5.65, 10.40, and 15.56 ms/cm, respectively). When PC12 cells are seeded on these conduits and stimulated with 100 mV for 2 h, there is a marked increase in both the percentage of neurite-bearing cells and the median neurite length as the content of PPY increased. More importantly, when the PPY/PDLLA nerve conduit was used to repair a rat sciatic nerve defect it performed similarly to the gold standard autologous graft. These promising results illustrate the potential that this PPY/PDLLA conducting composite conduit has for neural tissue engineering.

[Detection of FasL mRNA, sFasL and their regulatory effect on T lymphocyte subsets in patients with severe acute pancreatitis].

OBJECTIVE: To investigate the levels of FasL mRNA in peripheral blood mononualear cells (PBMCs), serum soluble Fas ligand (sFasL) and their regulatory effect on T lymphocyte subsets in patients with severe acute pancreatitis (SAP). METHODS: Forty-eight patients with pancreatitis were randomly divided into two groups: 20 cases with SAP and 28 cases with mild acute pancreatitis (MAP). Twenty-eight healthy volunteers were selected as control group. The expression of FasL mRNA in PBMCs was detected by real-time quantitative PCR(qRT-PCR), and serum sFasL was measured by ELISA. T lymphocyte subsets in peripheral blood were detected by flow cytometry. RESULTS: Compared with control group and MAP group, FasL mRNA of PBMCs and serum sFasL increased significantly in SAP group (P<0.05), a little increase in MAP group, and there was no significant difference between MAP group and control group (P>0.05). The CD4(+) T cell ratio, CD4(+)/CD8(+) ratio decreased significantly in SAP group (P<0.05) vs control group and MAP group), and they were found negatively related to FasL mRNA, serum sFasL level. CONCLUSION: The SAP patients showed the significantly increased FasL mRNA of PBMCs and serum sFasL and decreased CD4(+) T-cell ratio, CD4(+)/CD8(+) ratio. FasL may mediate the apoptosis of T lymphocytes.

The role of Fas expression on the occurrence of immunosuppression in severe acute pancreatitis.

BACKGROUND: Severe acute pancreatitis (SAP) is a dangerous illness with high mortality where most patients do not die of excessive inflammation, but die of immunosuppression and multiple infections at a later stage. The mechanism of immunosuppression in SAP is unknown. AIM: The purpose of this study was to analyze the role of Fas expression on the occurrence of immunosuppression in patients with SAP. METHODS: Forty-eight patients with pancreatitis were divided into two groups: 20 cases with SAP (7 cases with sepsis, 13 cases without sepsis) and 28 cases with mild acute pancreatitis (MAP). Twenty-eight healthy volunteers were selected as controls. Fas mRNA expression in peripheral blood was detected by qPCR and Fas protein of lymphocyte membranes; T lymphocyte subsets and expression of monocyte Human leukocyte antigen DR (HLA-DR) in peripheral blood were detected by flow cytometry. RESULTS: Compared with MAP and control groups, expression level of Fas mRNA and lymphocyte Fas protein in peripheral blood were significantly increased in the SAP group (all P < 0.01). There was a further significant increase in the SAP group with sepsis compared to those without sepsis (all P < 0.01). The CD4(+) T cell ratio, CD4(+)/CD8(+) ratio and monocyte HLA-DR expression in the SAP group were decreased significantly compared with MAP and control groups (all P < 0.01). Significant negative relationships were observed between Fas mRNA expression and CD4(+) T-cell ratio, CD4(+)/CD8(+) ratio, and monocyte HLA-DR expression in SAP patients with sepsis (all P < 0.05). CONCLUSIONS: The results suggest that expression level of Fas is related to severity and immune status of pancreatitis. Overexpression of Fas may lead to the occurrence of immunosuppression and sepsis.

Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration.

BACKGROUND: Nerve conduits provide a promising strategy for peripheral nerve injury repair. However, the efficiency of nerve conduits to enhance nerve regeneration and functional recovery is often inferior to that of autografts. Nerve conduits require additional factors such as cell adhesion molecules and neurotrophic factors to provide a more conducive microenvironment for nerve regeneration. METHODS: In the present study, poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]} (PLGL) was modified by grafting Gly-Arg-Gly-Asp-Gly (RGD peptide) and nerve growth factor (NGF) for fabricating new PLGL-RGD-NGF nerve conduits to promote nerve regeneration and functional recovery. PLGL-RGD-NGF nerve conduits were tested in the rat sciatic nerve transection model. Rat sciatic nerves were cut off to form a 10 mm defect and repaired with the nerve conduits. All of the 32 Wistar rats were randomly divided into 4 groups: group PLGL-RGD-NGF, group PLGL-RGD, group PLGL and group autograft. At 3 months after surgery, the regenerated rat sciatic nerve was evaluated by footprint analysis, electrophysiology, and histologic assessment. Experimental data were processed using the statistical software SPSS 10.0. RESULTS: The sciatic function index value of groups PLGL-RGD-NGF and autograft was significantly higher than those of groups PLGL-RGD and PLGL. The nerve conduction velocities of groups PLGL-RGD-NGF and autograft were significantly faster than those of groups PLGL-RGD and PLGL. The regenerated nerves of groups PLGL-RGD-NGF and autograft were more mature than those of groups PLGL-RGD and PLGL. There was no significant difference between groups PLGL-RGD-NGF and autograft. CONCLUSIONS: PLGL-RGD-NGF nerve conduits are more effective in regenerating nerves than both PLGL-RGD nerve conduits and PLGL nerve conduits. The effect is as good as that of an autograft. This work established the platform for further development of the use of PLGL-RGD-NGF nerve conduits for clinical nerve repair.

[Effects of matrine on proliferation and telomerase activity of colon cancer SW1116 cells].

OBJECTIVE: To investigate the effects of matrine on proliferation and telomerase activity of colon cancer SW1116 cells. METHODS: The proliferation inhibitory rate was evaluated by MTT assay. The telomerase activity was analyzed by TRAP-ELISA, and the expression of hTERT mRNA was determined by semi-quantitative RT-PCR. RESULTS: Matrine displayed strong proliferation inhibitory effect in a dose-and-time-dependent manner against SW1116 cells. Compared with control group, the telomerase activity and the expression of hTERT mRNA decreased significantly (P < 0.05 or P < 0.01) in matrine group. CONCLUSION: Matrine can inhibit the telomerase activity by depressing the expression of hTERT in SW1116 cells and inhibiting cell proliferation.