RNA adenosine modifications related to prognosis and immune infiltration in osteosarcoma.

BACKGROUND: RNA adenosine modifications, which are primarily mediated by "writer" enzymes (RMWs), play a key role in epigenetic regulation in various biological processes, including tumorigenesis. However, the expression and prognostic role of these genes in osteosarcoma (OS) remain unclear. METHODS: Univariate and multivariate Cox analyses were used to construct the RMW signature for OS using Target datasets. RMW expression in OS tissue was detected by qPCR analysis. Xcell and GSVA were used to determine the relationship between RMWs and immune infiltration. The DGIdb and CMap databases were used for drug prediction. In vivo and in vitro experiments showed that strophanthidin elicited antitumor activity against OS. RESULTS: A 3-RMW (CSTF2, ADAR and WTAP) prognostic signature in OS was constructed using the Target dataset and verified using GEO datasets and 63 independent OS tissues via qPCR analysis. High-risk OS patients had poor overall survival, and the prognostic signature was an independent prognostic factor for OS. Functional studies showed that tumour-, metabolism-, cell cycle- and immune-related pathways were related to high risk. Next, we found that RMW-derived high-risk patients exhibited increased infiltration of M2 macrophages and cDCs. Furthermore, we predicted the potential drugs for OS using the DGIdb and CMap databases. In vivo and in vitro experiments showed that strophanthidin elicited antitumor activity against OS by repressing cell growth and inducing cell cycle arrest at the G1 phase. CONCLUSION: The 3-RWM-based prognostic signature established in this study is a novel gene signature associated with immune infiltration, and strophanthidin was identified as a candidate therapy for OS by repressing OS cell growth and the cell cycle.

Correction: MicroRNA-126 inhibits tumor proliferation and angiogenesis of hepatocellular carcinoma by down-regulating EGFL7 expression.

[This corrects the article DOI: 10.18632/oncotarget.11877.].

The function and mechanism of the miR-210-3p/KRAS axis in bone marrow-derived mesenchymal stem cell from patients with osteoporosis.

The disturbance of homeostasis in bone marrow-derived mesenchymal stem cell (BMSC) adipogenesis and osteogenesis could result in pathologic consequences that plays a critical role in osteoporosis pathogenesis. In the present study, we demonstrated that miR-210-3p was abnormally upregulated within the femur of osteoporosis patients and abnormally downregulated in osteogenically differentiated BMSCs. The predicted targets of candidate miRNAs were enriched in the Pluripotent stem cell differentiation signaling. KRAS, as a vital factor of the KRAS/MAPK/ERK signaling, was upregulated in osteogenically differentiated BMSCs. In osteoporosis-BMSCs, the expression level of KRAS showed to be decreased, whereas the expression level of miR-210-3p showed to be increased. Within normal-BMSCs, miR-210-3p overexpression or KRAS silencing significantly inhibited the osteogenic differentiation and the activation of the MAPK signaling. miR-210-3p directly targeted KRAS and inhibited KRAS expression. The effects of miR-210-3p overexpression upon KRAS expression, MAPK signaling, and BMSC osteogenic differentiation were significantly reversed by KRAS overexpression. Altogether, miR-210-3p suppresses normal BMSC osteogenic differentiation through targeting KRAS and suppressing the MAPK signaling; KRAS overexpression could reverse the suppressive effects of miR-210-3p overexpression.

LncRNA-ROR/microRNA-185-3p/YAP1 axis exerts function in biological characteristics of osteosarcoma cells.

AIM: The co-expression network of long non-coding RNA ROR (lncRNA-ROR) and microRNA-185-3p (miR-185-3p) has not been focused on osteosarcoma. Therein, this work was initiated to uncover lncRNA-ROR and miR-185-3p functions in osteosarcoma. METHODS: LncRNA-ROR, miR-185-3p and Yes-associated protein 1 (YAP1) expression in osteosarcoma tissues and cells were detected. The screened cells (MG63 and U2OS) were transfected with decreased and/or increased lncRNA-ROR and miR-185-3p to explore osteosarcoma progression. Tumor growth was detected by tumor xenografts in mice. RESULTS: Up-regulated lncRNA-ROR and YAP1 and down-regulated miR-185-3p were found in osteosarcoma. LncRNA ROR knockdown or miR-185-3p overexpression inhibited osteosarcoma cell progression while lncRNA ROR elevation or miR-185-3p inhibition presented the opposite effects. Function of lncRNA ROR was rescued by miR-185-3p and regulated the growth and metastasis of osteosarcoma cells via modulating YAP1, the target gene of miR-185-3p. CONCLUSION: This work illustrates that lncRNA-ROR down-regulation or miR-185-3p up-regulation inhibits osteosarcoma progression via YAP1 repression.

Network Pharmacology to Uncover the Biological Basis of Spleen Qi Deficiency Syndrome and Herbal Treatment.

Spleen qi deficiency (SQD) syndrome is one of the basic traditional Chinese medicine (TCM) syndromes related to various diseases including chronic inflammation and hypertension and guides the use of many herbal formulae. However, the biological basis of SQD syndrome has not been clearly elucidated due to the lack of appropriate methodologies. Here, we propose a network pharmacology strategy integrating computational, clinical, and experimental investigation to study the biological basis of SQD syndrome. From computational aspects, we used a powerful disease gene prediction algorithm to predict the SQD syndrome biomolecular network which is significantly enriched in biological functions including immune regulation, oxidative stress, and lipid metabolism. From clinical aspects, SQD syndrome is involved in both the local and holistic disorders, that is, the digestive diseases and the whole body's dysfunctions. We, respectively, investigate SQD syndrome-related digestive diseases including chronic gastritis and irritable bowel syndrome and the whole body's dysfunctions such as chronic fatigue syndrome and hypertension. We found innate immune and oxidative stress modules of SQD syndrome biomolecular network dysfunction in chronic gastritis patients and irritable bowel syndrome patients. Lymphocyte modules were downregulated in chronic fatigue syndrome patients and hypertension patients. From experimental aspects, network pharmacology analysis suggested that targets of Radix Astragali and other four herbs commonly used for SQD syndrome are significantly enriched in the SQD syndrome biomolecular network. Experiments further validated that Radix Astragali ingredients promoted immune modules such as macrophage proliferation and lymphocyte proliferation. These findings indicate that the biological basis of SQD syndrome is closely related to insufficient immune response including decreased macrophage activity and reduced lymphocyte proliferation. This study not only demonstrates the potential biological basis of SQD syndrome but also provides a novel strategy for exploring relevant molecular mechanisms of disease-syndrome-herb from the network pharmacology perspective.

Cinnamaldehyde Treatment of Prostate Cancer-Associated Fibroblasts Prevents Their Inhibitory Effect on T Cells Through Toll-Like Receptor 4.

INTRODUCTION: Cancer-associated fibroblasts (CAFs) promote tumor progression; thus, drugs that can modify CAFs need to be identified. METHODS: To test the effect of cinnamaldehyde on prostate CAFs, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide assay was used to determine their survival. When spleen cells were treated with CAF supernatant, the proliferation of T cells was inhibited as determined by flow cytometry. After cinnamaldehyde treatment, this immunosuppressive function of CAFs was partially reversed. To explore the molecular mechanism, Western blotting and the quantitative real-time polymerase chain reaction were applied, and TLR4-dependent signaling pathway-related protein and mRNA levels were quantified. RESULTS: Cinnamaldehyde acted on the TLR4-dependent signaling pathway, altering the function of CAFs such that its supernatant no longer inhibited the proliferation of T cells. CONCLUSION: These data indicate that cinnamaldehyde can modify the functions of CAFs, which may be helpful for treating tumors. Cinnamaldehyde can suppress CAF T-cell inhibition.

WTAP promotes osteosarcoma tumorigenesis by repressing HMBOX1 expression in an m6A-dependent manner.

N6-methyladenosine (m6A) regulators are involved in the progression of various cancers via regulating m6A modification. However, the potential role and mechanism of the m6A modification in osteosarcoma remains obscure. In this study, WTAP was found to be highly expressed in osteosarcoma tissue and it was an independent prognostic factor for overall survival in osteosarcoma. Functionally, WTAP, as an oncogene, was involved in the proliferation and metastasis of osteosarcoma in vitro and vivo. Mechanistically, M6A dot blot, RNA-seq and MeRIP-seq, MeRIP-qRT-PCR and luciferase reporter assays showed that HMBOX1 was identified as the target gene of WTAP, which regulated HMBOX1 stability depending on m6A modification at the 3'UTR of HMBOX1 mRNA. In addition, HMBOX1 expression was downregulated in osteosarcoma and was an independent prognostic factor for overall survival in osteosarcoma patients. Silenced HMBOX1 evidently attenuated shWTAP-mediated suppression on osteosarcoma growth and metastasis in vivo and vitro. Finally, WTAP/HMBOX1 regulated osteosarcoma growth and metastasis via PI3K/AKT pathway. In conclusion, this study demonstrated the critical role of the WTAP-mediated m6A modification in the progression of osteosarcoma, which could provide novel insights into osteosarcoma treatment.

Spectrum-effect relationship between fingerprints and hemopoietic effects of small molecular fraction of Polygoni Multiflori radix praeparata.

Polygoni multiflori Radix Praeparata (PMRP) is a traditional medicine used for nourishing essence and blood in China. However, it is unclear which PMRP compounds are responsible for its hematopoietic effect. In this study, spectrum-effect relationship was used to discovery potential hematopoietic compounds. The fingerprints of 20 PMRP batches were established by HPLC and the hematopoietic effect was determined using red blood cell, hemoglobin, hematocrit, and platelet indexes in aplastic anemia model mice. The spectrum-effect relationship between common peaks and hematopoietic efficacy values was established using gray relational analysis and partial least squares analysis. Spectrum-effect relationship results showed that peaks 21 (emodin-8-O-(6´-O-acetyl)-ß-D-glucoside), 15 (2, 3, 5, 4'-tetrahydroxystilbene-2-O-di-glucoside), 16 (cis-2,3,5,4'-tetrahydroxy-stilbene-2-O-ß-D-glucoside), 11 (unknown), 20(unknown, 12 (epicatechin), 29 (carboxyl emodin), and 31 (emodin) in the fingerprints were closely related to the hematopoietic effect. This work successfully established the spectrum-effect relationship between PMRP hematopoietic effect and its fingerprints, which can be used to explain the material basis for the PMRP hematopoietic effect.

Ligustilide promotes apoptosis of cancer-associated fibroblasts via the TLR4 pathways.

The goal of this research was to study the selective pro-apoptotic effect of ligustilide on prostate-cancer-associated fibroblast in the tumor microenvironment and the related molecular mechanisms. The effects of ligustilide on cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) isolated from the prostate were determined by MTT assay. Flow cytometry and cellular immunofluorescence were used to detect the effects of ligustilide on the cell cycle and apoptosis. Western blotting was used to detect the expression of apoptosis-related proteins after the action of ligustilide on CAFs. In the investigation, ligustilide had a selective pro-apoptotic effect on prostate-CAFs. After ligustilide treatment, the proportion of CAFs in the G2-M phase of the cell cycle increased, and the expression of apoptosis-related proteins (p-P53, Bcl-2, Caspase9 and Cytochrome C) changed. Ligustilide blocks the CAF cell cycle and induces the apoptosis of CAFs.

Hematopoietic effect of small molecular fraction of Polygoni multiflori Radix Praeparata in cyclophosphamide-induced anemia mice.

The aim of this study is to investigate the protective effects of a small molecular fraction (SMF) of Polygoni multiflori Radix Praeparata (PMRP) in a cyclophosphamide (CTX) induced anemia mouse model. Small molecular fraction of PMRP was prepared and identified by high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). In pharmacology, we examined the peripheral hemogram and thymus and spleen index. The content of granulocyte-macrophage colony-stimulating factor (GM-CSF) in serum was mensurated by enzyme-linked immunosorbent assay (ELISA); The level of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in serum and spleen tissue homogenate were detected, and glutathione peroxidase (GSH-PX) was assayed in spleen. The results show that SMF can significantly accelerate the recovery of peripheral hemogram, increase the activity of antioxidant enzymes and GM-CSF in serum and spleen. SMF also increases the number of spleen cells, improves bone marrow pathology. In conclusion, the SMF of PMRP promoted the recovery of hematopoietic function in a CTX-induced anemia mouse, which can support SMF to be used as an adjunct to chemotherapy to counteract its side effects.

Cinnamaldehyde causes apoptosis of myeloid-derived suppressor cells through the activation of TLR4.

Malignant tumors are among the most life-threatening diseases in the world. Although many different types of antitumor agents are available, severe side effects and toxicity limit their applications. Myeloid-derived suppressor cells (MDSCs) inhibit the antitumor immune response by suppressing the proliferation of T cells, the production of cytokines and the killing of tumor cells. As MDSCs have become novel targets in cancer therapy, this research focused on the anti-MDSC function of cinnamaldehyde (CA), which is extracted from cinnamon, a traditional Chinese spice. In the present study, MDSCs isolated from the spleens of mice with colon cancer were used as an in vitro model to assess the efficacy of CA. Treatment of MDSCs with CA significantly decreased cell proliferation and induced apoptotic cell death. Subsequent experiments demonstrated that CA treatment enhanced the expression of Bax and caspase-9 and inhibited the expression of Bcl-2, suggesting that CA induced apoptosis in the MDSCs via the intrinsic pathway. Taken together, the results demonstrated that CA exhibited significant anti-MDSC activity and attenuated the suppression of the antitumor immune response, indicating a potential use for CA in cancer therapy.

MPSSS impairs the immunosuppressive function of cancer-associated fibroblasts via the TLR4-NF-κB pathway.

The polysaccharides MPSSS was extracted from Lentinus edodes and has been reported to effectively inhibit tumor growth and eliminate the function of myeloid-derived immune suppressor cell-mediated T cell inhibition, thus improving the efficacy of cancer therapy. The exploration of how MPSSS affects the functions of cancer-associated fibroblasts (CAFs) will provide a new perspective for understanding the antitumor effects of MPSSS. In the present study, prostate CAFs were selected as target cells to study whether MPSSS affected cell proliferation and function. The results showed that MPSSS did not directly inhibit the growth of prostate CAFs but interfered with CAF-mediated T cell inhibition and affected the immunosuppressive function of prostate CAFs. Mechanistic studies were further performed and showed that MPSSS activated key node proteins in the NF-κB pathway that were dependent on MyD88, and a TLR4 inhibitor blocked the changes in these proteins and the effect of MPSSS. We hypothesize that MPSSS can activate the MyD88-dependent TLR4-NF-κB signaling pathway to change the function of CAFs. In conclusion, these results demonstrate that MPSSS can not only effectively inhibit the growth of prostate cancer as we previously reported but also alter the function of prostate CAFs by activating the TLR4-NF-κB pathway, providing a new strategy for the comprehensive treatment of tumors.

Physicochemical properties, antioxidant activity and immunological effects in vitro of polysaccharides from Schisandra sphenanthera and Schisandra chinensis.

Schisandra sphenanthera and Schisandra chinensis are widely consumed either as food or for medicinal purposes. Nevertheless, no detailed comparative assessments of their physicochemical properties and biological activity have been reported. In this paper, using hot-water extraction, alcohol precipitation, and deproteinization, we obtained polysaccharidic extracts from Schisandra sphenanthera and Schisandra chinensis (denoted as SSP and SCP, respectively) and investigated their antioxidant and immunological activities. The extracts were different from each other with regard to sugar, protein, and uronic acid contents. Both extracts were mainly composed of arabinose, glucose, and galactose, but their contents varied greatly; SSP had more galacturonic acid. Compared with SCP, SSP had stronger free radical scavenging ability, protective effects on biomolecules, cellular antioxidant activity, owing to its higher protein (35.35 ±â€¯1.73%) and uronic acid (12.81 ±â€¯1.15%) contents. With respect to cell viability, neutral red phagocytosis, NO production, and acid phosphatase activity, SCP had stronger effects than SSP; this was largely due to its high levels of mannose, galactose, arabinose, and glucose. These results provide evidence to support the use Schisandra-derived polysaccharides for several purposes, including clinical, agricultural, and industrial applications.

Ligustilide inhibits the activation of cancer-associated fibroblasts.

The purpose of this work was to study the effects and underlying molecular mechanisms of ligustilide on cancer-associated fibroblasts (CAFs). The effects of ligustilide on the growth of CAFs and splenocytes were detected by MTT assay, and flow cytometry was used to detect effects on T-cell proliferation. Western blotting was used to detect the expression levels of CAF-related proteins after ligustilide treatment. This study found that ligustilide had no effect on the growth of splenocytes but that it could change the immunosuppressive function of CAFs through the TLR4-NF-κB pathway and restore T-cell proliferation previously inhibited by the CAF supernatant. Thus, ligustilide is expected to be a candidate for new antitumor drugs.

MicroRNA-9 modified bone marrow-derived mesenchymal stem cells (BMSCs) repair severe acute pancreatitis (SAP) via inducing angiogenesis in rats.

BACKGROUND: Severe acute pancreatitis (SAP) is an acute abdominal disease characterized by pancreatic necrosis and systemic disease. In a previous study, we showed that bone marrow-derived mesenchymal stem cells (BMSCs) can reduce SAP by secreting microRNA (miR)-9; however, the underlying mechanism remains unclear. The present study investigated the mechanism underlying BMSC-induced pancreatic regeneration. METHODS: BMSCs were isolated, and miR-9 modified/antagonized BMSCs (pri-miR-9-BMSCs/TuD-BMSCs) were generated and injected into SAP rats. The levels of inflammatory cytokines and histopathologic changes were examined using ELISA and H&E staining. Angiogenesis was analyzed by qRT-PCR, western blotting, and immunohistochemistry. Cell function tests, dual luciferase reporter assays, cell co-culture, western blotting, and cell tracing were used to explore the mechanisms underlying miR-9 induced angiogenesis. RESULTS: Pri-miR-9-BMSCs induced angiogenesis in SAP rats (Ang-1↑, TIE-2↑, and CD31↑) and repaired damaged vascular endothelial cells (VECs) in vitro, promoting angiogenesis (Ang-1↑, TIE-2↑, PI3K↑, AKT↑, p-AKT↑, CD31↑, and CD34↑). Pri-miR-9-BMSCs released miR-9 into VECs or injured pancreatic tissue, targeting the VE-cadherin gene and promoting PI3K/AKT signaling to treat SAP (VE-cadherin↓, ß-catenin↓, PI3K↑, p-AKT↑), whereas antagonizing miR-9 in BMSCs did not alleviate or aggravated SAP. CONCLUSIONS: Pri-miR-9-BMSCs can repair injured pancreatic tissue by secreting miR-9 and promoting angiogenesis.

Polysaccharides from Chinese Herbal Lycium barbarum Induced Systemic and Local Immune Responses in H22 Tumor-Bearing Mice.

Lycium barbarum polysaccharide (LBP) is isolated from the fruit of Chinese herbal Lycium barbarum. Previous studies had demonstrated that LBP could inhibit tumor growth and enhance the immunity in mice. However, the effect of LBP on systemic and local immune responses in vivo, especially on phenotypic and functional changes of T cells, is still largely unknown. In the present study, we investigated the effects of LBP on systemic and local T cell-dependent antitumor immune responses in H22 tumor-bearing mice. The results showed that LBP could inhibit the solid tumor growth in mice, but showed little effect on the body weight or spleen index. Furthermore, LBP could maintain high levels of T cells in peripheral blood (PB), tumor draining lymph node (TDLN), and tumor tissue, prevent the increase of Tregs while promote infiltration of CD8+ T cells in tumor tissue, inhibit the production of TGF-ß1 and IL-10 in serum, decrease the exhaustion phenotype of T cells, and maintain cytotoxicity of lymphocytes. Taken together, our results demonstrated that LBP simultaneously induced systemic and local immune responses in H22 tumor-bearing mice by alleviating immunosuppression and maintaining antitumor immune responses in mice.

An Asparagus polysaccharide fraction inhibits MDSCs by inducing apoptosis through toll-like receptor 4.

Despite decades of research, malignant tumors are extremely difficult to eliminate with conventional methods. Although surgical resection potentially eradicates the problem, only a few cases are suitable for operation, and other approaches often involve harmful consequences. Revolutionary methods are desperately needed to improve patient outcomes and diminish harmful side effects. Myeloid-derived suppressor cells (MDSCs), downregulators of the innate and adaptive immune systems, have been widely studied over the past 2 decades. MDSCs inhibit the antitumor immune response by suppressing T cell proliferation, cytokine production, and tumor cell killing. With MDSCs becoming novel targets in cancer therapy, our research has focused on the anti-MDSC function of Asparagus polysaccharide (AP), extracted from asparagus, a traditional Chinese herb. In this study, we have used MDSCs isolated from the spleen of mice with colon cancer as an in vitro model to assess the efficacy of AP. Treatment of MDSCs with AP significantly decreased cell proliferation and induced apoptotic cell death through a toll-like receptor 4 dependent way. Subsequent studies showed that the AP treatment enhanced the expression of Bax and Caspase-9 and inhibited the expression of Bcl-2, suggesting that AP induced apoptosis in the MDSCs via the intrinsic pathway. Altogether, the results showed that AP exhibited a significant anti-MDSC activity and attenuated suppression of the antitumor immune response, thereby indicating its potential use in cancer therapy.

miR-194 Suppresses Proliferation and Migration and Promotes Apoptosis of Osteosarcoma Cells by Targeting CDH2.

BACKGROUND/AIMS: Studies have shown that miR-194 functions as a tumour suppressor and is associated with tumour growth and metastasis. This study intends to uncover the mechanism of tumour suppression by miR-194. The expression of miR-194 in osteosarcoma cell lines and tissues were monitored by real-time PCR. METHODS: The proliferation ability was examined by MTT assay. Migration and apoptosis of cells were monitored by migration assay and flow cytometry, respectively. The regulation of miR-194 on CDH2 was determined by luciferase assays and western blot assays. RESULTS: The results showed that miR-194 was significantly reduced in osteosarcoma compared with that in normal bone tissue. Overexpression of miR-194 significantly attenuated the proliferation and migration and induced the apoptosis of osteosarcoma cells. Furthermore, we demonstrated that miR-194 has inhibited the malignant behaviour of osteosarcoma by downregulating CDH2 expression. CONCLUSIONS: These findings suggested that miR-194 may act as a tumour suppressor in osteosarcoma. miR-194/CDH2 may be a novel therapeutic target in the treatment of osteosarcoma.

Fraction From Lycium barbarum Polysaccharides Reduces Immunotoxicity and Enhances Antitumor Activity of Doxorubicin in Mice.

The aim of the present study was to investigate whether fraction from Lycium barbarum polysaccharide (LBP) could reduce immunotoxicity and enhance antitumor activity of doxorubicin (Dox) in mice. A water-soluble LBP fraction, designated LBP3, was isolated from edible Chinese herbal Lycium barbarum and used in this study. To investigate the effect of LBP3 on Dox-induced immunotoxicity, tumor-free mice were used and treated with either normal saline, Dox, or Dox plus LBP3. To investigate the effect of LBP3 on antitumor activity of Dox, H22 tumor-bearing mice were used and treated with either normal saline, Dox, LBP3, or Dox plus LBP3. The results showed that LBP3 did not protect against the body weight loss caused by Dox, but it promoted the recovery of body weight starting at day 5 after Dox treatment in tumor-free mice. LBP3 also improved peripheral blood lymphocyte counts, promoted cell cycle recovery in bone marrow cells, and restored the cytotoxicity of natural killer cells. Furthermore, in H22 tumor-bearing mice, LBP3 enhanced antitumor activity of Dox and improved peripheral blood lymphocyte counts and the cytotoxicity of splenocytes. In brief, our results demonstrated that LBP3 could reduce the immunotoxicity and enhance antitumor activity of Dox.