[Anti-glioma mechanism of pterostilbene by regulating apoptosis and GSDME-mediated pyroptosis pathways: a study based on network pharmacology and experimental research].

This study aimed to explore the anti-glioma effect of natural compound pterostilbene(PTE) through regulating pyroptosis and apoptosis pathways, and to analyze the possible anti-glioma pathways and targets of PTE by network pharmacology and molecular docking. In this study, the action targets of PTE and the glioma targets were obtained by network pharmacology to construct a target network and a protein-protein interaction(PPI) network to predict the possible action targets of PTE against glioma. Molecular docking was performed on the core targets by AutoDock and the action pathways of PTE against glioma were predicted by enrichment analysis. In addition, the effect of PTE on the viability of U87MG and GL261 glioma cells was detected by CCK-8 assay. Clone formation assay and cell scratching assay were used to explore the effect of different concentrations of PTE on the proliferation and migration, respectively of glioma cells. Hoechst staining was used to observe PTE-induced apoptosis in glioma cells. The changes in mitochondrial membrane potential were detected by JC-1 staining. The pyroptosis-inducing effect of PTE on glioma cells was observed by inverted microscopy and lactate dehydrogenase(LDH) assay. Hoechst 33342/PI dual staining assay was performed to detect the integrity of glioma cell membranes. The expressions of pyroptosis and apoptosis-related proteins in glioma cells after PTE induction were determined by Western blot. In this study, 37 anti-glioma targets of PTE were obtained, and enrichment analysis suggested that PTE exerted anti-glioma effects through various signaling pathways including cancer pathway, proteoglycan in cancer, PI3K/AKT pathway, and apoptosis regulatory pathway. Molecular docking revealed that PTE had good binding activity with the main targets. Compared with the control group, PTE significantly reduced the viability as well as the proliferation, migration and adhesion abilities of U87MG and GL261 cells; it induced the apoptosis of the two glioma cells and the decrease of mitochondrial membrane potential in U87MG cells, and the effects increased with the increase of drug concentration. Compared with the conditions in the control group, glioma cells in the PTE group had increased pyroptosis-specific appearance and gradually increased LDH release; the number of PI positive cells was significantly elevated with the increase of PTE concentration as revealed by Hoechst 33342/PI staining; the expression levels of apoptosis-related factors cleaved PARP1 and B-cell lymphoma-2(Bcl-2) associated X(BAX) in the PTE group were markedly up-regulated, while the expression level of Bcl-2 was markedly down-regulated; the activation levels of pyroptosis-related proteins cleaved caspase-3 and gasdermin E-N(GSDME-N) had a remarkable rise in the PTE group, while no significant changes were found in the activation levels of gasdermin D-N(GSDMD-N) and cleaved caspase-1. In summary, PTE plays an anti-glioma role by inhibiting cell viability, proliferation, and migration and activating the caspase-3/GSDME-mediated pyroptosis pathway and mitochondrial apoptosis pathway.

Intravital biobank and personalized cancer therapy: the correlation with omics.

Biobanks have played a decisive role in all aspects of the field of cancer, including pathogenesis, diagnosis, prognosis and treatment. The significance of cancer biobanks is epitomized through the appropriate application of various "-omic" techniques (omics). The mutually motivated relationship between biobanks and omics has intensified the development of cancer research. Human cancer tissues that are maintained in intravital biobanks (or living tissue banks) retain native tumor microenvironment, tissue architecture, hormone responsiveness and cell-to-cell signalling properties. Intravital biobanks replicate the structural complexity and heterogeneity of human cancers, making them an ideal platform for preclinical studies. The application of omics with intravital biobanks renders them more active, which makes it possible for the cancer-related evaluations to be dynamically monitored on a real-time basis. Integrating intravital biobank and modern omics will provide a useful tool for the discovery and development of new drugs or novel therapeutic strategies. More importantly, intravital biobanks may play an essential role in the creation of meaningful patient-tailored therapies as for personalized medicine.

Reprogramming human umbilical cord mesenchymal stromal cells to islet-like cells with the use of in vitro-synthesized pancreatic-duodenal homebox 1 messenger RNA.

BACKGROUND AIMS: Human umbilical cord mesenchymal stromal cells (hUC-MSCs) hold great potential as a therapeutic candidate to treat diabetes, owing to their unlimited source and ready availability. METHODS: In this study, we differentiated hUC-MSCs with in vitro-synthesized pancreatic-duodenal homebox 1 (PDX1) messenger (m)RNA into islet-like cell clusters. hUC-MSCs were confirmed by both biomarker detection and functional differentiation. In vitro-synthesized PDX1 messenger RNA can be transfected into hUC-MSCs efficiently. The upregulated expression of PDX1 protein can be detected 4 h after transfection and remains detectable for 36 h. RESULTS: The induction of islet-like structures was confirmed by means of morphology and dithizone staining. Reverse transcriptase-polymerase chain reaction results revealed the expression of some key pancreatic transcription factors, such as PDX1, NeuroD, NKX6.1, Glut-2 and insulin in islet-like cell clusters. Immunofluorescence analysis showed that differentiated cells express both insulin and C-peptide. Enzyme-linked immunosorbent assay analysis validated the insulin secretion of islet-like cell clusters in response to the glucose stimulation. CONCLUSIONS: Our results demonstrate the use of in vitro-synthesized PDX1 messenger RNA to differentiate hUC-MSCs into islet-like cells and pave the way toward the development of reprogramming and directed-differentiation methods for the expression of encoded proteins.

Circulating Expression Level of LncRNA Malat1 in Diabetic Kidney Disease Patients and Its Clinical Significance.

BACKGROUND: Long noncoding RNA MALAT1 is closely related to diabetes and kidney diseases and is expected to be a new target for the diagnosis and treatment of diabetic nephropathy. OBJECTIVE: This study aimed to explore the circulating expression level and significance of lncRNA Malat1 in patients with type 2 diabetes mellitus (T2DM) and diabetic kidney disease (DKD). METHODS: Quantitative real-time PCR (qPCR) was conducted to assess the expression of lncRNA Malat1 in 20 T2DM patients, 27 DKD patients, and 14 healthy controls, and then, the clinical significance was analyzed. RESULTS: LncRNA MALAT1 expression in peripheral blood mononuclear cells (PBMC) was significantly upregulated in T2DM and DKD groups when compared to control. Pearson's correlation analysis showed correlation of lncRNA MALAT1 levels with ACR, urine ß2-microglobulin (ß2-MG), urine α1-microglobulin (α1-MG), creatinine (Cr), and glycosylated hemoglobin (HbA1c), while negative with superoxide dismutase (SOD) (r = -0.388, P < 0.05). Binary regression analysis showed that ACR, creatinine, α1-MG, and LncRNA Malat1 were the risk factors for diabetic nephropathy with OR value of 1.166, 1.031, 1.031, and 2.019 (P < 0.05). The area under ROC curve (AUC) of DKD identified by the above indicators was 0.914, 0.643, 0.807, and 0.797, respectively. The AUC of Joint prediction probability of DKD recognition was 0.914, and the sensitivity and specificity of DKD diagnosis were 1.0 and 0.806, respectively. (Take ≥0.251 as the diagnostic cutoff point). CONCLUSION: LncRNA Malat1 is highly expressed in DKD patients, and the combined detection of ACR, creatinine, α1-MG, and LncRNA Malat1 with diabetes mellitus may be the best way to diagnose diabetic nephropathy.

The sterol carrier protein 2/3-oxoacyl-CoA thiolase (SCPx) is involved in cholesterol uptake in the midgut of Spodoptera litura: gene cloning, expression, localization and functional analyses.

BACKGROUND: Sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCPx) gene has been suggested to be involved in absorption and transport of cholesterol. Cholesterol is a membrane component and is a precursor of ecdysteroids, but cannot be synthesized de novo in insects. However, a direct association between SCPx gene expression, cholesterol absorption and development in lepidopteran insects remains to be experimentally demonstrated. RESULTS: An SCPx cDNA (SlSCPx) cloned from the common cutworm, Spodoptera litura, was characterized. The SlSCPx cDNA encoded a 535-amino acid protein consisting of a 3-oxoacyl-CoA thiolase (SCPx-t) domain and a SCP-2 (SCPx-2) domain. SlSCPx mRNA was expressed predominately in the midgut, while SlSCPx-2 mRNA was detected in the midgut, fat body and epidermis and no SlSCPx-t mRNA was detected. A 58-kDa full-length SCPx protein and a 44-kDa SCPx-t protein were detected in the midgut of sixth instar larvae when the anti-SlSCPx-t antibody was used in western blotting analysis; a 16-kDa SCP-2 protein was detected when anti-SlSCPx-2 antibody was used. SlSCPx protein was post-translationally cleaved into two smaller proteins, SCPx-t and SCPx-2. The gene appeared to be expressed into two forms of mRNA transcripts, which were translated into the two proteins, respectively. SlSCPx-t and SlSCPx-2 proteins have distinct and different locations in the midgut of sixth instar larvae. SlSCPx and SlSCPx-t proteins were detected predominately in the cytoplasm, whereas SlSCPx-2 protein was detected in the cytoplasm and nuclei in the Spli-221 cells. Over-expression of SlSCPx and SlSCPx-2 proteins enhanced cholesterol uptake into the Spli-221 cells. Knocking-down SlSCPx transcripts by dsRNA interference resulted in a decrease in cholesterol level in the hemolymph and delayed the larval to pupal transition. CONCLUSION: Spatial and temporal expression pattern of this SlSCPx gene during the larval developmental stages of S. litura showed its specific association with the midgut at the feeding stage. Over-expression of this gene increased cholesterol uptake and interference of its transcript decreased cholesterol uptake and delayed the larval to pupal metamorphosis. All of these results taken together suggest that this midgut-specific SlSCPx gene is important for cholesterol uptake and normal development in S. litura.

Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

BACKGROUND: FAM92A1-289(abbreviated FAM289) is recognized as one of the newly-discovered putative oncogenes. However, its role and molecular mechanisms in promoting cancer progression has not yet been elucidated. This study was performed to reveal its oncogenic functions and molecular mechanisms in human glioblastoma multiforme (GBM) cell models with knockdown or overexpression of FAM289 in vitro and in vivo. METHODS: To elucidate the molecular mechanisms underlying FAM289-mediated tumor progression, the protein-protein interaction between FAM289 and Galectin-1 was verified by co-immunoprecipitation, followed by an analysis of the expression and activity of Galectin-1-associated signaling molecules. Knockdown and overexpression of FAM289 in glioma cells were applied for investigating the effects of FAM289 on cell growth, migration and invasion. The determination of FAM289 expression was performed in specimens from various stages of human gliomas. RESULTS: FAM289-galectin-1 interaction and concomitant activation of the extracellular signal-regulated kinase (ERK) pathway participated in FAM289-mediated tumor-promoting function. Since the expression of DNA methyl transferase 1 (DNMT1) and DNA methyl transferase 3B (DNMT3B) was regulated by FAM289 in U251 and U87-MG glioma cells, Galectin-1 interaction with FAM289 may promote FAM289 protein into the cell nucleus and activate the ERK pathway, thereby upregulating DNMTs expression. Drug resistance tests indicated that FAM289-mediated TMZ resistance was through stem-like property acquisition by activating the ERK pathway. The correlation between FAM289, Galectin-1 expression and the clinical stage of gliomas was also verified in tissue samples from glioblastoma patients. CONCLUSIONS: Our results suggest that high expression of FAM289 in GBM tissues correlated with poor prognosis. FAM289 contributes to tumor progression in malignant glioma by interacting with Galectin-1 thereby promoting FAM289 protein translocation into the cell nucleus. FAM289 in the nucleus activated the ERK pathway, up regulated DNMTs expression and induced stem-like property gene expression which affects drug resistance of glioma cells to TMZ. This study provided functional evidence for FAM289 to be developed as a therapeutic target for cancer treatment.

Forced expression of Nanog with mRNA synthesized in vitro to evaluate the malignancy of HeLa cells through acquiring cancer stem cell phenotypes.

Nanog is a pluripotency-related factor. It was also found to play an important role in tumorigenesis. To date, the mechanisms underlying cervical tumorigenesis still need to be elucidated. In the present study, Nanog mRNA was synthesized in vitro and transfected into HeLa cells. After mRNA transfection, the forced expressed of Nanog in HeLa cells led to markedly increased invasion, migration, resistance to chemotherapeutic agents and dedifferentiation. In a subcutaneous xenograft assay, these cells had significantly increased tumorigenic capacity. Real-time PCR indicated that Nanog­induced dedifferentiation was associated with increased expression of endogenous Oct4, Sox2 and FoxD3. In addition, the dedifferentiated HeLa cells acquired features associated with cancer stem cells (CSCs), such as multipotent differentiation capacity, and expression of CSC markers such as CD133. These data imply that Nanog is a positive regulator of cervical cancer dedifferentiation.

The Tumor-promoting Effects of FAM92A1-289 in Cervical Carcinoma Cells.

BACKGROUND/AIM: FAM92A1-289 is recognized as one of the newly-discovered putative oncogenes. This study was performed to reveal its oncogenic functions in human cervical carcinoma cells. MATERIALS AND METHODS: The FAM92A1-289+ cell line was established with knock-in technique and selected by puromycin-resistance screening. Scratch assay, methylthiazol tetrazolium assay, colony forming assay and xenograft test were used to examine cell migration, cell proliferation, cell viability and tumor formation, respectively. RESULTS: FAM92A1-289+ cells showed higher migration rate (p<0.05), higher cell viability (p<0.01), higher colony formation and tumor growth. The FAM92A1-289 protein was pulled-down by antibodies against proliferating cell nuclear antigen (PCNA) in the co-immunoprecipitation assay. CONCLUSION: The up-regulated expression of FAM92A1-289 could facilitate cell migration, boost cell proliferation and promote colony formation in vitro and tumor growth in vivo. The interaction between FAM92A1-289 and PCNA was verified by co-immunoprecipitation. This study provided functional evidence for FAM92A1-289 to be developed as a therapeutic target for cancer treatment.

NaHCO3 enhances the antitumor activities of cytokine-induced killer cells against hepatocellular carcinoma HepG2 cells.

The extracellular pH is lower inside solid tumors than in normal tissue. The acidic environment inhibits the cytotoxicity of lymphocytes in vitro and promotes tumor cell invasion. In the present study, both in vitro and in vivo experiments were conducted to investigate how NaHCO3 would affect the antitumor activities of cytokine-induced killer (CIK) cells against hepatocellular carcinoma (HCC) cells. For the in vitro experiments, HepG2 cells were cultured at pH 6.5 and 7.4 in the presence of CIK cells or CIK cell-conditioned medium (CMCIK). For the in vivo experiments, nude mice were xenografted with HepG2-luc cells and divided into four groups: i) CIK cells injection plus NaHCO3 feeding; ii) CIK cells injection plus drinking water feeding; iii) normal saline injection plus NaHCO3 feeding; and iv) normal saline injection plus drinking water feeding. The results indicated that the viability and growth rate of HepG2 cells were remarkably suppressed when co-cultured with CIK cells or CMCIK at pH 7.4 compared with those of HepG2 cells cultured under the same conditions but at pH 6.5. In the xenograft study, a marked synergistic antitumor effect of the combined therapy was observed. NaHCO3 feeding augmented the infiltration of cluster of differentiation 3-positive T lymphocytes into the tumor mass. Taken together, these data strongly suggest that the antitumor activities of CIK cells against HepG2 cells were negatively affected by the acidic environment inside the tumors, and neutralizing the pH (for example, via NaHCO3 administration), could therefore reduce or eliminate this influence. In addition, it should be recommended that oncologists routinely prescribe soda water to their patients, particularly during CIK cell therapy.

PTEN-mRNA engineered mesenchymal stem cell-mediated cytotoxic effects on U251 glioma cells.

Mesenchymal stem cells (MSCs) have been considered to have potential as ideal carriers for the delivery of anticancer agents since the capacity for tumor-oriented migration and integration was identified. In contrast to DNA-based vectors, mRNA synthesized in vitro may be readily transfected and is mutagenesis-free. The present study was performed in order to investigate the effects of phosphatase and tensin homolog (PTEN) mRNA-engineered MSCs on human glioma U251 cells under indirect co-culture conditions. PTEN-bearing mRNA was generated by in vitro transcription and was transfected into MSCs. The expression of PTEN in transfected MSCs was detected by immunoblotting, and the migration ability of MSCs following PTEN-bearing mRNA transfection was verified using Transwell co-cultures. The indirect co-culture was used to determine the effects of PTEN-engineered MSCs on the viability of U251 glioma cells by luminescence and fluorescence microscopy. The synthesized PTEN mRNA was expressed in MSCs, and the expression was highest at 24 h subsequent to transfection. An enhanced migration rate was observed in MSCs transfected with PTEN mRNA compared with non-transfected MSCs (P<0.05). A significant inhibition of U251 cells was observed when the cells were cultured with conditioned medium from PTEN mRNA-engineered MSCs (P<0.05). The results suggested that anticancer gene-bearing mRNA synthesized in vitro is capable of being applied to a MSC-mediated anticancer strategy for the treatment of glioblastoma patients.

The application of mRNA-based gene transfer in mesenchymal stem cell-mediated cytotoxicity of glioma cells.

Since the tumor-oriented homing capacity of mesenchymal stem cells (MSCs) was discovered, MSCs have attracted great interest in the research field of cancer therapy mainly focused on their use as carries for anticancer agents. Differing from DNA-based vectors, the use of mRNA-based antituor gene delivery benefits from readily transfection and mutagenesis-free. However, it is essential to verify if mRNA transfection interferes with MSCs' tropism and their antitumor properties. TRAIL- and PTEN-mRNAs were synthesized and studied in an in vitro model of MSC-mediated indirect co-culture with DBTRG human glioma cells. The expression of TRAIL and PTEN in transfected MSCs was verified by immunoblotting analysis, and the migration ability of MSCs after anticancer gene transfection was demonstrated using transwell co-cultures. The viability of DBTRG cells was determined with bioluminescence, live/dead staining and real time cell analyzer. An in vivo model of DBTRG cell-derived xenografted tumors was used to verify the antitumor effects of TRAIL- and PTEN-engineered MSCs. With regard to the effect of mRNA transfection on MSCs' migration toward glioma cells, an enhanced migration rate was observed with MSCs transfected with all tested mRNAs compared to non-transfected MSCs (p<0.05). TRAIL- and PTEN-mRNA-induced cytotoxicity of DBTRG glioma cells was proportionally correlated with the ratio of conditioned medium from transfected MSCs. A synergistic action of TRAIL and PTEN was demonstrated in the current co-culture model. The immunoblotting analysis revealed the apoptotic nature of the cells death in the present study. The growth of the xenografted tumors was significantly inhibited by the application of MSCPTEN or MSCTRAIL/PTEN on day 14 and MSCTRAIL on day 28 (p<0.05). The results suggested that anticancer gene-bearing mRNAs synthesized in vitro are capable of being applied for MSC-mediated anticancer modality. This study provides an experimental base for further clinical anticancer studies using synthesized mRNAs.

Gene Manipulation of Human Embryonic Stem Cells by In Vitro-Synthesized mRNA for Gene Therapy.

The difficulty in producing genetically modified human embryonic stem cells (hESCs) limits research on their applications. Virus-based gene transfer is not safe for clinical use, whereas DNAbased non-viral methods are not efficient or safe, and mRNA-based methods are useful for genetic manipulation. In this study, we easily obtained multiple types and large amounts of in vitro-synthesized mRNA by PCR. The efficiency of different transfection methods was studied by flow cytometry. The effect of different mRNA modifications on protein translation efficiency and dynamics of luciferase mRNA expression in hESCs were studied using a bioluminescence imaging system. The pluripotency of hESCs after transfection was studied by immunofluorescence. In vitro-synthesized pancreatic-duodenal homeobox 1 (PDX1) mRNA was used to induce the differentiation of hESCs into insulin-producing cells. We found that electroporation is the most efficient transfection method, and it produces more than 95% transgene expression in multiple hESC lines. Synthesized mRNA with a combination of a polyA tail, cap and base analogues is more efficiently translated into protein in hESCs compared with single-modified mRNA. Transfection of mRNA into hESCs by trypsinizing the cells into single-cell suspensions did not affect their pluripotency, and multiple types of mRNAs can be transfected into hESCs efficiently. We found that PDX-1 mRNA transfection significantly improved the expression level of genes related to beta cells and differentiated cells that express insulin and C-peptide. ELISA analysis validate the insulin secretion of islet-like cell clusters in response to glucose stimulation. Our results indicate that electroporation of in vitro-synthesized mRNA is useful for genetic manipulation of hESCs and differentiation of hESCs into particular cell types, and this finding will pave the way for clinical applications of this method.

PDX-1 mRNA-induced reprogramming of mouse pancreas-derived mesenchymal stem cells into insulin-producing cells in vitro.

Pancreatic islet transplantation has remained an effective therapy for type 1 diabetes since 2000. Its widespread use has been prohibited by the shortage of suitable donors. It is critical to explore an applicable alternative for ß-cell replacement. This study was performed to generate insulin-producing cells (IPCs) from pancreas-derived mesenchymal stem cells (pMSCs). pMSCs were isolated from discarded pancreatic tissue in the filter liquor during islet isolation procedure in mice and ex vivo expanded in culture. IPCs were induced by transfection of pancreas and duodenal transcription factor 1 (PDX-1) mRNA in vitro. Some islet characteristics were identified on pMSC-derived IPCs in mRNA and protein levels. Our results demonstrated that mouse pMSCs can be transdifferentiated into effective glucose-responsive insulin-producing cells through transfecting synthetic modified PDX-1 mRNA in vitro. The study of PDX-1 mRNA-induced pMSC reprogramming may pave the way toward the development of a novel ß-cell source for the treatment of diabetes.

Cancer cell-oriented migration of mesenchymal stem cells engineered with an anticancer gene (PTEN): an imaging demonstration.

BACKGROUND: Mesenchymal stem cells (MSCs) have been considered to hold great potential as ideal carriers for the delivery of anticancer agents since the discovery of their tumor tropism. This study was performed to demonstrate the effects of phosphatase and tensin homolog (PTEN) engineering on MSCs' capacity for cancer cell-oriented migration. METHODS: MSCs were engineered with a PTEN-bearing plasmid and the expression was confirmed with Western blotting. A human glioma cell line (DBTRG) was used as the target cell; DBTRG cell-oriented migration of MSCs was monitored with a micro speed photographic system. RESULTS: The expression of transfected PTEN in MSCs was identified by immunoblotting analysis and confirmed with cell viability assessment of target cells. The DBTRG cell-oriented migration of PTEN-engineered MSCs was demonstrated by a real-time dynamic monitoring system, and a phagocytosis-like action of MSCs was also observed. CONCLUSION: MSCs maintained their capacity for cancer cell-directed migration after they were engineered with anticancer genes. This study provides the first direct evidence of MSCs' tropism post-anticancer gene engineering.