Expression Levels Of VEGF Research Articles

Intramuscular injection of sotagliflozin promotes neovascularization in diabetic mice through enhancing skeletal muscle cells paracrine function.

Diabetes mellitus is associated with series of macrovascular and microvascular pathological changes that cause a wide range of complications. Diabetic patients are highly susceptible to hindlimb ischemia (HLI), which remains incurable. Evidence shows that skeletal muscle cells secrete a number of angiogenic factors to promote neovascularization and restore blood perfusion, this paracrine function is crucial for therapeutic angiogenesis in diabetic HLI. In this study we investigated whether sotagliflozin, an anti-hyperglycemia SGLT2 inhibitor, exerted therapeutic angiogenesis effects in diabetic HLI in vitro and in vivo. In C2C12 skeletal muscle cells, we showed that high glucose (HG, 25 mM) under hypoxia markedly inhibited cell viability, proliferation and migration potentials, which were dose-dependently reversed by pretreatment with sotagliflozin (5-20 μM). Sotagliflozin pretreatment enhanced expression levels of angiogenic factors HIF-1α, VEGF-A and PDGF-BB in HG-treated C2C12 cells under hypoxia as well as secreted amounts of VEGF-A and PDGF-BB in the medium; pretreatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME2, 10 μM) or HIF-1α knockdown abrogated sotagliflozin-induced increases in VEGF-A and PDGF-BB expression, as well as sotagliflozin-stimulated cell proliferation and migration potentials. Furthermore, the conditioned media from sotagliflozin-treated C2C12 cells in HG medium enhanced the migration and proliferation capabilities of vascular endothelial and smooth muscle cells, two types of cells necessary for forming functional blood vessels. In vivo study was conducted in diabetic mice subjected to excising the femoral artery of the left limb. After the surgery, sotagliflozin (10 mg/kg) was directly injected into gastrocnemius muscle of the left hindlimb once every 3 days for 3 weeks. We showed that intramuscular injection of sotagliflozin effectively promoted the formation of functional blood vessels, leading to significant recovery of blood perfusion in diabetic HLI mice. Together, our results highlight a new indication of SGLT2 inhibitor sotagliflozin as a potential therapeutic angiogenesis agent for diabetic HLI.

Potential biomarkers and the molecular mechanism associated with DLL4 during renal cell carcinoma progression

BackgroundDelta-like canonical notch ligand 4 (DLL4) is considered a potential prognostic gene for renal cell carcinoma (RCC). We assessed the molecular mechanisms and novel biomarkers associated with DLL4 during RCC development. MethodsFour gene expression profiles were downloaded from the GEO database. Differentially expressed genes (DEGs) were identified between RCC and normal renal samples, including common DEGs (co-DEGs). Thereafter, RCC-associated gene exploration was performed and a PPI network was constructed to identify the core genes. Survival analysis of core genes in the high expression group (H group) and low expression group (L group) was also performed. The key genes related to the core genes were investigated, and the miRNA-target genes and TFs-target genes were analyzed. Finally, the expression levels of VEGFA, FLT1, EGLN3, and DLL4 in RCC and paracancerous tissues were determined. ResultsA total of 11,867 DEGs and 622 co-DEGs were identified in this study, and 67 RCC-associated genes that were mainly enriched in signal transduction and angiogenesis function were further explored. VEGFA was identified as the core gene. Further, 30 DEGs and 9 DE-miRNAs were identified between the H and L groups. VEGFA was positively correlated with 19 genes, including EGLN3, FLT1, and DLL4. A total of 18 miRNA-target interactions, including miR-134-5p-DLL4, were obtained. VEGFA, FLT1, EGLN3, and DLL4 were significantly expressed in RCC tissues compared with paracancerous tissues. ConclusionsDLL4 may contribute to the development of RCC by participating in signal transduction and angiogenesis. VEGFA, FLT1, EGLN3, DLL4, and miR-134-5p may be novel biomarkers for RCC.

Mechanism of the inhibitory effects of thalidomide on expressions of VEGF and bFGF

Objective: To investigate the inhibitory effects of thalidomide on the expressions of VEGF and bFGF in human lung adenocarcinoma A549 cells and human hepatocellular carcinomas HepG2 cells mediated by cereblon (CRBN). Methods: shRNA technology was used to construct the A549 cell line (A549CRBN) and HepG2 cell line (HepG2CRBN) with stable knockdown of CRBN, which was verified by real-time PCR and Western blot. A549 cells were divided into negative control group (A549luciferase) and CRBN down-regulation group (A549CRBN); HepG2 cells were divided into negative control group (HepG2luciferase) and CRBN down-regulation group (HepG2CRBN). The above cells were seeded into 6-well plates at 3×105 cells/well, and cultured in a 37℃, 5% CO2 incubator for 24 h. Then, 1 ml medium containing 100 μmol/L thalidomide (thalidomide group) and 1 ml medium containing 1‰ DMSO (control group) were added respectively, and the culture was continued for 24 hours before subsequent experiments. Each group was designed with three replicate wells. The effect of thalidomide on the activity of A549 cell line was detected by MTS assay. Real-time PCR was performed to detect mRNA expression levels of VEGF, bFGF and c-jun. ELISA assay was performed to detect protein expressions of VEGF and bFGF. Results: Compared with the control group, thalidomide at the concentrations of 1, 10, 50 and 100 μmol/L had no significant effects on the proliferation of A549 and HepG2 cells (P＞0.05). VEGF and bFGF levels in the A549CRBN or HepG2luciferase groups were significantly lower than those in the A549CRBN or HepG2CRBN groups (P＜0.05). Compared with the control group of the A549luciferase or HepG2luciferase, thalidomide inhibited the expressions of VEGF and bFGF in A549luciferase and HepG2luciferase cells (P＜ 0.05), but did not inhibit the expressions of VEGF and bFGF in A549CRBN and HepG2CRBN cells. Compared with the control group of the HepG2luciferase, thalidomide inhibited c-Jun expression in HepG2luciferase cells (P＜0.01), but did not significantly inhibit c-Jun expression in HepG2CRBN cells. Conclusion: The inhibitory effects of thalidomide on VEGF and bFGF expressions may be mediated by CRBN in A549 and HepG2 cells, and c-Jun may be one of the key transcription factors responsible for this inhibition.

The effect of miR-138 on the proliferation and apoptosis of breast cancer cells through the NF-κB/VEGF signaling pathway.

The analyze the effect of miR-138 on the proliferation and apoptosis of breast cancer cells through the NF-κB/VEGF signaling pathway is the Objective of this experiment. For this aim, the endometrial stem breast cancer cell line MCF-7 was cultured in vitro, and the overexpression mimic miR-138 mimics and the inhibitor anti-miR-138 were transfected into the endometrial stem breast cancer cell line MCF-7, which was set to overexpress miR-138 group and interfere with miR-138, and set up negative control of overexpression and negative control of inhibitor. Observe the cell proliferation and apoptosis ability of each group, and the changes in tumor necrosis factor-α (TNF-α), interleukin 1β, 6, 18 (IL-1β, IL-6, IL-18) levels, and compare the Bax of each group, NF-κB, VEGF protein expression level. Results showed that the proliferation ability of the miR-138 overexpression group was significantly lower than that of the miR-138 overexpression control group (P<0.05); the proliferation ability of the miR-138 interference group was significantly higher than that of the miR-138 interference control group (P<0.05). The apoptosis rate, caspase-3 and caspase-9 expression levels of the miR-138 overexpression group were significantly higher than those of the miR-138 overexpression control group (P<0.05); the apoptosis rate, caspase-3 and caspase-9 expression levels of the miR-138 interference group were significantly lower than those of the miR-138 interference control group (P<0.05). The expression levels of IL-1 β, IL-6, IL-18 and TNF - α in the miR-138 overexpression group were significantly lower than those in the miR-138 overexpression control group (P < 0.05). The protein expression levels of Bax, NF-κB and VEGF in the miR-138 overexpression group were significantly lower than those in the miR-138 overexpression control group (P < 0.05); the protein expression levels of Bax, NF-κB and VEGF in the miR-138 interference group were significantly higher than those in the miR-138 interference control group (P <0.05). The proliferation ability of the miR-138 overexpression group was significantly lower than that of the miR-138 overexpression control group (P < 0.05); the proliferation ability of the miR-138 + NF-κB overexpression group was significantly higher than that of the miR-138 overexpression group (P<0.05). The apoptosis rate of the miR-138 + NF-κB overexpression group was significantly lower than that of the miR-138 overexpression group (P < 0.05). Then MiR-138 can significantly inhibit the proliferation of breast cancer cells, promote apoptosis, and regulate the expression of inflammatory factors in the cells. It is speculated that the related mechanism may be related to the negative regulation of the NF-κB/VEGF signaling pathway.

Extracts of Portulaca oleracea promote wound healing by enhancing angiology regeneration and inhibiting iron accumulation in mice

ObjectiveTo investigate the role of Portulaca oleracea (POL) in promoting revascularization and re-epithelization as well as inhibiting iron aggregation and inflammation of deep tissue pressure injury (DTPI). MethodsThe hydroalcoholic extract of POL (P) and aqueous phase fraction of POL (PD) were prepared based on maceration and liquid–liquid extraction. The number of new blood vessels and VEGF-A expression level were assessed using H&E stain and Western blot on injured muscle to examine the role of POL different extracts in vascularization. The iron distribution and total elemental iron of injured muscle were detected using laser ablation inductively coupled plasma mass spectrometry (ICP-MS) and Perls’ staining to determine whether POL extracts can inhibit the iron accumulation. Besides, the ability of POL extracts to promote wound healing by combining re-epithelization time, inflammation degree and collagen deposition area were comprehensively evaluated. ResultsIn vitro, we observed a significant increase in HUVEC cell viability, migration rate and the number of the tube after P and PD treatment (P < 0.05). In vivo, administration of P and PD impacted vascularization and iron accumulation on injured tissue, evident from more new blood vessels, higher expression of VEGF-A and decreased muscle iron concentration of treatment groups compared with no-treatment groups (P < 0.05). Besides, shorter re-epithelization time, reduced inflammatory infiltration and distinct collagen deposition were associated with administration of P and PD (P < 0.05). ConclusionPOL extract administration groups have high-quality wound healing, which is associated with increased new blood vessels, collagen deposition and re-epithelization, along with decreased iron accumulation and inflammatory infiltration. Our results suggest that that POL extract is beneficial to repair injured muscle after ischemia–reperfusion, highlighting the potential of POL in the DTPI treatment.

Breast cancer cells-derived Von Willebrand Factor promotes VEGF-A-related angiogenesis through PI3K/Akt-miR-205-5p signaling pathway

The metastasis and angiogenesis of breast cancer has always been a difficult problem for treatment. It has recently been discovered that Von Willebrand Factor (vWF), in addition to hemostasis, also plays a role in tumor metastasis and angiogenesis. We noticed that besides endothelial cells, breast cancer cells (MDA-MB-231 and MCF-7) could also express vWF. In vitro experiments showed that knocking down vWF inhibited breast cancer cell metastasis. And we found that overexpression of vWF significantly promoted VEGF-A-dependent vascular proliferation in vitro by activating the PI3K/Akt signaling pathway. Further studies indicated that inhibition of PI3K/Akt signaling pathway up-regulated the expression of miR-205-5p, and miR-205-5p could bind to the 3′UTR region of VEGF-A to hinder the production of VEGF-A. Furthermore, when a spontaneous lung metastasis model was established in Balb/c female mice, knockdown of vWF in 4 T1 cells resulted in a decrease in tumor blood vessel density and effectively inhibited lung metastasis, accompanied by a decrease in the expression level of VEGF-A and an increase in the expression level of miR-205-5p. In summary, our findings provide experimental evidence that overexpression of vWF in breast cancer cells down-regulates the expression of miR-205-5p and up-regulates the expression of VEGF-A through the PI3K/Akt signaling pathway, thereby promoting tumor angiogenesis and metastasis.

NR3C2 suppresses the proliferation, migration, invasion and angiogenesis of colon cancer cells by inhibiting the AKT/ERK signaling pathway.

Nuclear receptor subfamily 3, group C, member 2 (NR3C2) serves an antitumorigenic role in several types of cancer; however, its role and mechanisms of action in colon cancer remains to be elucidated. The aim of the present study was to explore the effects of NR3C2 on the proliferation, migration, invasion and angiogenesis of colon cancer cells. The expression levels of NR3C2 in human colon epithelial NCM460 cells (spontaneously immortalized cell line) and colon cancer cell lines was detected using reverse transcription-quantitative PCR and western blotting. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to assess cell viability and wound healing and Transwell assays were used to detect cell invasion and migration. ELISA was used to detect the expression levels of VEGF and tube formation assays were used to assess angiogenesis. The expression levels of angiogenesis-related proteins and AKT/ERK signaling pathway-related proteins were detected by western blotting. NR3C2 expression was downregulated in colon cancer cells and overexpression of NR3C2 inhibited proliferation, colony formation, migration and invasion of colon cancer cells. Overexpression of NR3C2 inhibited angiogenesis and activity of the AKT/ERK signaling pathway in colon cancer cells. Thus, it was demonstrated that NR3C2 inhibited the proliferation, colony formation, migration, invasion and angiogenesis of colon cancer cells through the AKT/ERK signaling pathway. These results may highlight novel targets for the treatment of colon cancer.

A comparative study of the effects of crab derived exosomes and doxorubicin in 2 & 3-dimensional in vivo models of breast cancer

IntroductionUsing tissue engineering and modifying the tumor microenvironment, three-dimensional (3D) in vitro and in vivo cancer modeling can be performed with appropriate similarity to native. Exosomes derived from different sources have recently been used in cancer studies due to their anticancer effects. In this study, the effect of crab derived exosomes in 2 & 3-dimensional (2& 3D) in vivo models of breast cancer (BC) were investigated and compared with the doxorubicin (DOX). Methods2D and 3D models of BC were induced using the chitosan/β-glycerol phosphate hydrogel (Ch/β-GP) and 1 × 106 4T1 cells in the female mice aged 6–8 weeks. 1 mg/ml exosome and 5 mg/kg DOX were injected by intratumoral (IT), intravenous (IV), and intraperitoneal (IP) methods into mice on day 9, 13, and 17 with and without hydrogel as a drug delivery system. After 21 days, the mice were sacrificed, and the tissues (lung, liver, and tumor) were removed. The weight and size of the tumor were measured. Real-time PCR assessed changes of VEGF, Bcl2, and P53 genes expression levels. Nitric oxide (NO) secretion from the cancer 3D model was evaluated by Griess assay. Results and ConclusionBased on the results, the size and weight of tumors in treated groups with exosomes and DOX were reduced significantly (P ≤ 0.001, P ≤ 0.002, P ≤ 0.02) in 2D and 3D models. Changes in VEGF, Bcl2 and P53 gene expression levels were less in the 3D model than in the 2D model. Drug delivery with hydrogel increased tumor inhibition compared to drug injection without hydrogel. Decreased NO secretion was observed in all treatment groups compared to the control group (untreated). Crab exosomes showed anti cancer effects on 2&3D models of BC. 3D model of BC showed greater drug resistance than the 2D model after treating with crab derived exosomes and DOX. 3D model of BC mimics native tumor better than 2D and can be used in cancer studies and for drug screening with greater confidence than 2D model. Also, the use of slow release drug delivery system reduced drug resistance in both models.

Therapeutic effect of adipose stromal vascular fraction spheroids for partial bladder outlet obstruction induced underactive bladder

BackgroundUnderactive bladder (UAB) is a common clinical problem but related research is rarely explored. As there are currently no effective therapies, the administration of adipose stromal vascular fraction (ad-SVF) provides a new potential method to treat underactive bladder.MethodsMale Sprague–Dawley rats were induced by partial bladder outlet obstruction (PBOO) for four weeks and randomly divided into three groups: rats treated with PBS (Sham group); rats administrated with ad-SVF (ad-SVF group) and rats performed with ad-SVF spheroids (ad-SVFsp group). After four weeks, urodynamic studies were performed to evaluate bladder functions and all rats were sacrificed for further studies.ResultsWe observed that the bladder functions and symptoms of UAB were significantly improved in the ad-SVFsp group than that in the Sham group and ad-SVF group. Meanwhile, our data showed that ad-SVF spheroids could remarkably promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation in bladder tissue than that in the other two groups. Moreover, ad-SVF spheroids increased the expression levels of bFGF, HGF and VEGF-A than ad-SVF. IVIS Spectrum small-animal in vivo imaging system revealed that ad-SVF spheroids could increase the retention rate of transplanted cells in bladder tissue.ConclusionsAd-SVF spheroids improved functions and symptoms of bladder induced by PBOO, which contributes to promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation. Ad-SVF spheroids provide a potential treatment for the future patients with UAB.

VEGF alleviates lower limb ischemia in diabetic mice by altering muscle fiber types.

Lower limb ischemia caused by diabetic foot (DF) is one of the most serious complications of diabetes. The therapeutic role of VEGF in DF is well documented. However, the mechanism for action of VEGF is still not clear. The present study aimed to explore the effects of VEGF-mediated skeletal muscle fiber type switch in angiogenesis and the treatment of DF. C57BL/6 mice housed in cages equipped with a voluntary running wheel were used to access VEGF protein level and citrate synthase activity (by ELISA) as well as muscle fiber type changes (by immunofluorescence) in the gastrocnemius muscle. C57BL/6 mice were fed on a high-fat diet for 6 weeks and then injected with streptozocin to induce diabetic lower limb ischemia model. Control adenovirus (Ad-GFP) or Ad-VEGF-GFP were then injected into the left gastrocnemius of the ischemic diabetic mice. Blood flow perfusion was examined by laser Doppler imaging at 1, 3, 7 and 14 days after adenovirus transduction. On day 14, all mice were anesthetized and sacrificed. VEGF expression levels, citrate synthase activity and muscle fiber type changes in the gastrocnemius muscle were assayed by ELISA and immunofluorescence analysis of myosin heavy chain IIa (MHCIIa) expression, respectively. Transwell assays were performed to determine whether VEGF-treated C2C12 myotubes played a role on tubule formation and migration of HUVECs. It was found that VEGF levels and citrate synthase activity were upregulated after voluntary exercise, along with the increased frequency of oxidized muscle fibers. Notably, adenovirus-mediated VEGF overexpression in the muscle also increased the frequency of oxidized (MHCIIa-positive) muscle fibers, enhanced citrate synthase activity and ameliorated lower limb ischemia in diabetic mice. VEGF treatment enhanced the phosphorylation of PI3K, Akt and AMPK (assayed by western blotting), as well as glucose consumption and metabolism (assayed by western blotting and glucose uptake assay), in the C2C12 myotubes. Interestingly, VEGF-treated C2C12 myotubes promoted the migration and tubule formation of HUVEC cells. The present findings suggest that skeletal muscle fiber conversion might be a potential approach for VEGF-mediated angiogenesis and disease treatment, which may provide new options for the prevention and treatment of DF.

Ischemic preconditioning-induced protective effect for promoting angiogenesis in renal ischemia-reperfusion injury by regulating miR-376c-3p/HIF-1α/VEGF axis in male rats

ObjectiveIschemic preconditioning (IPC) is defined as a well-established phenomenon in which brief exposure to sublethal episodes of ischemia and reperfusion induces a tolerance to injurious effects of prolonged ischemia by exploiting intrinsic defence mechanisms. The present study was performed to determine the protective effect of IPC on the rat renal ischemia-reperfusion injury (IRI) via miR-376c-3p/HIF-1α/VEGF axis. MethodsIn vivo, these male Sprague-Dawley rats were treated by IRI and IPC. Meanwhile, these rats from different treatment groups were also injected subcutaneously with 2 nmol agomir-376c-3p and/or 10 nmol recombinant rat HIF-1α. At 72 h after reperfusion, serum samples were respectively collected for renal function. Besides, kidney tissues were harvested to observe renal morphology changes. Subsequently, the expression levels of CD31, HIF-1α and VEGF in the kidney tissues were measured using immunohistochemical staining, quantitative real-time PCR, as well as Western blotting analysis at the indicated time points after reperfusion. In vitro, HK-2 cells were used to detect the cell activity by CCK-8 and transfection of mir-376c-3p mimic in Hypoxia/Reoxygenation (H/R) group. ResultsIn vivo, the pathological changes were significantly relieved in the rats with IPC group, compared to the IRI group. Rats which were treated IPC significantly reduced the levels of blood urea nitrogen (BUN), serum creatinine (Scr) at 24 h after operation, compared to IRI group. After IPC treatment, the expression level of CD31 was obviously decreased, compared to IRI group. A total of differently expressed microRNAs were screened out by microRNA microarray assay in rat renal ischemia tissue, especially showing that miR-376c-3p was selected as the target miRNA. Compared to IRI group, the expression level of miR-376c-3p were obviously higher in IPC-treated group. Double-luciferase reporter assay demonstrated that miR-376c-3p directly targeted HIF-1α. In vitro, IPC significantly increased cell viability of HK-2, and promoted the angiogenesis via up-regulating miR-376c-3p/HIF-1α/VEGF axis. Furthermore, the expression level of HIF-1α was apparently decreased in HK-2 treated with H/R after miR-376c-3p mimic transfection respectively, as well as the increased expression level of VEGF. ConclusionsOur study provided a novel insight for investigating the protective effect of IPC on renal IRI. Consequently, miR-376c-3p played an important role in renal IRI by promoting angiogenesis via targeting HIF-1α/VEGF pathway in male rats.

Healing effect of carboxymethyl chitosan-plantamajoside hydrogel on burn wound skin

BackgroundIt is known that hydrogels based on carboxymethyl chitosan (CMCS) have properties controling microbial growth, reducing inflammatory cell infiltration, and promoting collagen deposition. Plantamajoside (PMS), a natural Chinese herbal medicine with biological activity, has the properties of reducing inflammation, anti-oxidation, and promoting wound healing. However, the effects of carboxymethyl chitosan/plantamajoside hydrogel on partial thickness burn wounds remain unclear. MethodsThe healing effect of carboxymethyl chitosan/plantamajoside hydrogel was evaluated by in vitro cell viability assay, cell migration assay, and further evaluated in a rat model of partial-thickness burn wounds. ResultsThe hydrogels were highly porous with a pore size of about 250 μm, and these pores were interconnected. After adding plantamajoside, a dense microstructure was further formed. The hydrogels containing 0.25% plantamajoside significantly increased the viability and migration of L929 cells (P < 0.05). Carboxymethyl chitosan/plantamajoside hydrogel significantly improved wound healing, granulation tissue proliferation and re-epithelialization, and promoted collagen deposition (P < 0.05). Carboxymethyl chitosan/plantamajoside hydrogel also significantly decreased IL (interleukin)-1β, IL-6 and TNF-α expression, and increased IL-10 expression (P < 0.05). Furthermore, carboxymethyl chitosan/plantamajoside hydrogel significantly promoted the expression levels of VEGF, CD31, α-SMA (α-smooth muscle actin) and collagen III, and reduced the expression level of collagen Ⅰ (P < 0.05). Our data suggest that carboxymethyl chitosan/plantamajoside hydrogel promotes burn wound healing by accelerating angiogenesis and collagen deposition and reducing the inflammatory response.

Predictive Value of the Prognostic Nutritional Index in Neoadjuvant Chemoradiotherapy for Rectal Cancer.

Prognostic nutritional index (PNI) and neutrophil-to-lymphocyte ratio (NLR) indicate nutritional status and host immunity. We used immunohistochemistry and apparent diffusion coefficient (ADC) values calculated using diffusion-weighted imaging (DWI) to investigate relationships of these factors with pathological and radiological characteristics in rectal cancer treated with neoadjuvant chemoradiotherapy (nCRT). We evaluated expression levels of VEGFA, CD8, CD33, and ADC values in tumors pre/post nCRT; and analyzed the relationships between those factors and PNI, NLR in 32 patients. Pretreatment PNI negatively correlated with change in tumor stromal CD8 + T cells and positively correlated with ADC values. Pretreatment NLR and PNI change correlated with recurrence-free survival (RFS). Patients with higher pretreatment PNI had greater changes in ADC values and stromal CD8 + T-cell counts, and those with greater PNI reduction from nCRT had a worse prognosis. Proper nutritional management during nCRT benefits patients and may lead to better prognosis in rectal cancer.

Calbindin S100A16 Promotes Renal Cell Carcinoma Progression and Angiogenesis via the VEGF/VEGFR2 Signaling Pathway.

Purpose Recent research has indicated that the calcium-binding protein S100A16 promotes carcinogenesis and tumor growth in several forms of cancer. The objective of this study was to examine the relationship between S100A16 and renal cell cancer. Methods By using The Cancer Genome Atlas (TCGA) database, the differentially expressed gene S100A16 was identified, and its appearance and link to the prognosis of persons with renal cancer were confirmed. Cox regression was used in multivariate analysis, and a nomogram was developed for internal validation. The correlation between S100A16 and immune cells was analyzed in the TIMER database. Moreover, the potential mechanism of action was investigated utilizing GO and KEGG enrichment analyses. Proliferation, migration, and angiogenesis were investigated in vitro, and the involvement of S100A16 in the undesirable biological events of renal cell carcinoma (RCC) was further explored. Results S100A16 was the differentially expressed molecule identified through database screening. Malignant tissues showed higher S100A16 expression than noncancerous tissues, and S100A16 expression was mostly localized in the cytoplasm. According to the TCGA and KM-plotter datasets, patients with RCC and low S100A16 expression had superior OS, PFI, and DSS. The C-index of the nomogram was 0.754 (0.726–0.782), and the accuracy of the prediction model was high. The TIMER database shows that the expression of S100A16 is associated with immune infiltration and may play an important role in promoting tumor cell immune escape in the RCC tumor microenvironment. S100A16 may influence the biological processes of RCC via the VEGF/VEGFR2 signaling route and PI3K-Akt signaling pathway and through P53 alteration and cell cycle according to the gene enrichment technique. In vitro cytological experiments demonstrated that S100A16 knockdown can inhibit the proliferation and migration of renal cancer cells and the expression levels of VEGF, VEGFR2, and phosphorylated AKT within renal cancer cells, thereby inhibiting angiogenesis in renal cancer cells and resulting in a poor prognosis of RCC. Conclusion A decrease in S100A16 expression may dramatically increase the OS, PFI, and DSS of patients with RCC and may thus be used as a biomarker for predicting RCC. It may be associated with the immune infiltration of RCC and play a crucial role in the immune evasion of tumor cells within the RCC microenvironment. Intervention of s100a16 can promote the progression and angiogenesis of renal cell carcinoma through the VEGF/VEGFR2 signal transduction pathway and lead to poor prognosis of renal cell carcinoma. These findings suggest a potential target for the development of anticancer strategies for renal cancer.

The dual role of interleukin-17 in the growth of human papillomavirus-negative cervical cancer cells

To explore the relationship between IL-17 mediated immune response and HPV-negative cervical cancer, we established the co-culture system of cervical cancer C-33A cells and PBMC, and treated with different concentrations of rhIL-17. PBMC were extracted from 10ml anticoagulant blood provided by healthy female volunteers and colleagues in our hospital, which were co-cultured with cervical cancer C-33A cells. After treatment with different concentrations of rhIL-17, the proliferation, apoptosis, invasion behavior and VEGF expression level of cancer cells in each group, were determined by CCK-8, flow cytometry, transwell invasion assay and ELISA, respectively. The proliferation rate of C-33A cells slightly increased with the ascending of IL-17 concentration, but no statistical significance was found among each group ( P &gt; 0.05). When at low concentration of IL-17, the cell apoptosis rate seems to slightly decline. With the increase of concentration, the decrease of apoptosis rate became more obvious ( P &lt; 0.05). However, differing from those shown above, we found that IL-17 slightly inhibited the cancer cell invasion at high concentration, while it was more obvious at low concentration ( P &lt; 0.05). Furthermore, we found that there was no significant correlation between IL-17 and VEGF ( P &gt; 0.05). In summary, there is a close correlation between IL-17 mediated immune response and HPV-negative cervical cancer. Also, we think that IL-17 may play a dual role in tumor progression and could be a possible significant influence on tumor proliferation, apoptosis, and metastasis, which provides a basic theoretical basis for the further study of immunotherapy measures for cervical cancer.

Role of Micro-RNA in the Regulation of Cell Polarization in Hepatocellular Carcinoma

The occurrence of tissue scarring and architecture-modifying signaling led to a tumorigenic microenvironment. Targeting specifically the biological mediators responsible for the physiological and morphological changes accommodating hepatocellular carcinoma (HCC) growth may be the key for identifying a future HCC cure. Morphological and physiological features of cultured HepG2 cells in both stimulated recombinant human vascular endothelial growth factor (VEGF165) and unstimulated (control) conditions were assessed. Quantitative reverse transcription (RT)-PCR measured endogenous VEGF expression levels. The assessment of proangiogenic biological mediator (micro RNA [miR]-296, miR-31, and miR-17) profiles was achieved by polarization-inducing VEGF165 stimulation followed by quantitative RT-PCR. In vitro conditions reproduced successfully the physiological environment leading to the occurrence of HCC, including the successful HepG2 polarization following VEGF stimulation. While endogenous VEGF production only occurs if complete polarization has been reached, the quantified biological mediator profiles determined here pointed at either possible early stages of depolarization or at the lack of tumorigenic potential of the HepG2 cells. All tested miRs displayed upregulated profiles, although miR-296 was less amplified (3.78-fold compared with control) than miR-31 or miR-17 (6.5- and 6.6-fold, respectively). The findings surrounding miR-17 reproduce similar data reported in the literature; the unexpected high miR-31 expression was intriguing. Given HepG2 cells' minimal tumorigenic potential, the unexpected multifold upregulation of miR-31 may be a cause or a consequence of HepG2 cells' low tumorigenic potential. The exploration of miR-31 therapeutic potential may be a future rewarding endeavor.

BRAFV600E mutant colorectal cancer cells mediate local immunosuppressive microenvironment through exosomal long noncoding RNAs.

BACKGROUNDBRAFV600E mutated colorectal cancer (CRC) is prone to peritoneal and distant lymph node metastasis and this correlates with a poor prognosis. The BRAFV600E mutation is closely related to the formation of an immunosuppressive microenvironment. However, the correlation between BRAFV600E mutation and changes in local immune microenvironment of CRC is not clear.AIMTo explore the effect and mechanism of BRAFV600E mutant on the immune microenvironment of CRC.METHODSThirty patients with CRC were included in this study: 20 in a control group and 10 in a treatment group. The density of microvessels and microlymphatic vessels, and M2 subtype macrophages in tumor tissues were detected by immunohistochemistry. Screening and functional analysis of exosomal long noncoding RNAs (lncRNAs) were performed by transcriptomics. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were detected by CCK-8 assay and scratch test, respectively. The tube-forming ability of endothelial cells was detected by tube formation assay. The macrophage subtypes were obtained by flow cytometry. The expression of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-β1, VEGF-C, claudin-5, occludin, zonula occludens (ZO)-1, fibroblast activation protein, and α-smooth muscle actin was assessed by western blot analysis. The levels of cytokines interleukin (IL)-6, TGF-β1, and VEGF were assessed by enzyme-linked immunosorbent assay.RESULTSBRAFV600E mutation was positively correlated with the increase of preoperative serum carbohydrate antigen 19-9 (P < 0.05), and with poor tumor tissue differentiation in CRC (P < 0.01). Microvascular density and microlymphatic vessel density in BRAFV600E mutant CRC tissues were higher than those in BRAF wild-type CRC (P < 0.05). The number of CD163+ M2 macrophages in BRAFV600E mutant CRC tumor tissue was markedly increased (P < 0.05). Compared with exosomes from CRC cells with BRAF gene silencing, the expression of 13 lncRNAs and 192 mRNAs in the exosomes from BRAFV600E mutant CRC cells was upregulated, and the expression of 22 lncRNAs and 236 mRNAs was downregulated (P < 0.05). The biological functions and signaling pathways predicted by differential lncRNA target genes and differential mRNAs were closely related to angiogenesis, tumor cell proliferation, differentiation, metabolism, and changes in the microenvironment. The proliferation, migration, and tube formation ability of HUVECs and HLECs induced by exosomes in the 1627 cell group (HT29 cells with BRAF gene silencing) was greatly reduced compared with the HT29 cell group (P < 0.05). Compared with the HT29 cell group, the expression levels of VEGF-A, bFGF, TGF-β1, and VEGF-C in the exosomes derived from 1627 cells were reduced. The expression of ZO-1 in HUVECs, and claudin-5, occludin, and ZO-1 in HLECs of the 1627 cell group was higher. Compared with the 1627 cell group, the exosomes of the HT29 cell group promoted the expression of CD163 in macrophages (P < 0.05). IL-6 secretion by macrophages in the HT29 cell group was markedly elevated (P < 0.05), whereas TGF-β1 was decreased (P < 0.05). The levels of IL-6, TGF-β1, and VEGF secreted by fibroblasts in the 1627 cell group decreased, compared with the HT29 cell group (P < 0.05).CONCLUSIONBRAFV600E mutant CRC cells can reach the tumor microenvironment by releasing exosomal lncRNAs, and induce the formation of an immunosuppressive microenvironment.

HMGA2 promotes the migration and invasion of gallbladder cancer cells and HMGA2 knockdown inhibits angiogenesis via targeting VEGFA.

The high mobility group AT-hook 2 (HMGA2) protein has been found to be upregulated in the majority of tumor types and is associated with a poor prognosis. Previous studies have suggested the oncogenic role of HMGA2 in gallbladder cancer (GBC). The present study aimed to investigate the effects of HMGA2 on the invasion, migration and angiogenesis of GBC cells. To achieve this aim, HMGA2 was overexpressed or silenced in the GBC cell line, EH-GB1, and then the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) abilities of EH-GB1 cells were investigated using Cell Counting Kit-8, wound healing, Transwell and western blotting assays. In addition, the expression levels of VEGFA were determined in EH-GB1 cells using western blotting and reverse transcription-quantitative PCR following HMGA2 overexpression or silencing. Furthermore, HMGA2-silenced EH-GB1 cells were transfected with VEGFA overexpression plasmids to evaluate the tube formation ability of HUVECs using tube formation assay. The results demonstrated that HMGA2 silencing inhibited GBC cell proliferation, migration, invasion and EMT, as evidenced by the downregulated expression of Ki67, proliferating cell nuclear antigen, MMP2, MMP9, N-cadherin, snail family transcriptional repressor 2 and zinc finger E-box-binding homeobox 1, and attenuated cell migration and invasion. However, the opposite results were obtained following HMGA2 overexpression. Moreover, HMGA2 knockdown and overexpression downregulated and upregulated VEGFA expression, respectively. In addition, the tube formation ability of HUVECs and the expression levels of CD31, VEGFR1 and VEGFR2 were downregulated following HMGA2 silencing. However, these effects were partially rescued by simultaneous VEGFA overexpression. In conclusion, the findings of the present study revealed that HMGA2 may promote GBC cell migration, invasion, EMT and angiogenesis. Therefore, inhibiting HMGA2 expression could be considered as a possible therapeutic approach for GBC.

Membrane fixation enhances guided bone regeneration in standardized calvarial defects: A pre-clinical study.

To determine whether collagen membrane (CM) fixation enhances guided bone regeneration in standardized defects. Four 8-mm-diameter defects were surgically made in eight rabbit calvaria, and randomly allocated into four groups: control (empty), unfixed-CM, fixed-CM, and unfixed-CM with bone graft (BG + CM) (positive control). After 1- and 4-week healing periods, the animals were sacrificed and quantitative reverse transcription polymerase chain reaction, micro-computed tomography, and histological outcomes were assessed. At week 1, the expression levels of BMP-2, FGF-2, VEGF, and osteocalcin were significantly higher in the fixed-CM group than in the unfixed-CM and control groups (p< .05). Conversely, cathepsin-K was significantly expressed in the unfixed-CM group. No significant differences in expression markers were observed between the fixed-CM and BG + CM groups (p> .05). At week 4, new bone formation was significantly higher in the fixed-CM group than the unfixed-CM and control groups (p< .05), but similar to the BG + CM group (p> .05). CM fixation enhances the expression of osteogenic factors similar to BG + CM, leading to significantly more new bone formation. This suggests that the osteogenic potential is greater when membranes are fixed, thereby limiting the necessity of membrane-supporting materials to enhance bone formation.

Pyruvate kinase M2 (PKM2) improve symptoms of post-ischemic stroke depression by activating VEGF to mediate the MAPK/ERK pathway.

PurposeTo evaluate and identify the effects and explore the mechanisms of pyruvate kinase M2 (PKM2) on stroke‐induced post stroke depression (PSD).MethodsRats were separated into six different groups, including sham + saline, Stroke + saline, PSD + saline, PSD + recombinant pyruvate kinase M2 (rPKM2) (112 ng/kg), PSD + rPKM2 (224 ng/kg), and PSD + rPKM2 (224 ng/kg) + bevacizumab. Then, the body weight, sucrose preference rate, immobility time, horizontal movement, and vertical movement were determined to evaluate the effect of PKM2 on improving the depressive behavior of PSD rats. Subsequently, the proliferation of oligodendrocytes in subventricular zone (SVZ) of rats in each group was examined by western blot and immunofluorescent staining. Furthermore, the mRNA and protein expression levels of TNF‐α, IL‐6, and IL‐1β were also detected by qPCR and ELISA to verify the anti‐inflammatory effects of PKM2 on PSD rats. In addition, the protein expression levels of MDA, LDH, and NO were tested to reveal that PKM2 can reduce oxidative stress in PSD rats. The western blot and IHC assays were employed to examine the protein expression levels of VEGF, PKM2, and ERK in PSD rats.ResultsIn this study, the results showed that PKM2 can improve the depressive behavior and proliferation of oligodendrocytes in PSD rats. In addition, PKM2 has anti‐inflammatory and anti‐oxidative stress effects on PSD rats. Meanwhile, PKM2 activated the expression level of VEGF/MAPK/ERK pathway.ConclusionPKM2 improves symptoms of post‐ischemic stroke depression by activating VEGF‐mediated MAPK/ERK pathway.