Expression Of Raf-1 Research Articles

MicroRNA-503 Suppresses Oral Mucosal Fibroblast Differentiation by Regulating RAS/RAF/MEK/ERK Signaling Pathway

The abnormal proliferation and differentiation of oral mucosal fibroblasts (FBs) is the key to the progression of oral submucosal fibrosis. To clarify the mechanism of platelet-derived growth factor (PDGF-BB)-induced FBs fibrosis in oral mucosa, real-time quantitative polymerase chain reaction and Western blot were used in this study to detect the expression of miR-503 and the expression of p-MEK, p-ERK, miR-503, RAF, smooth actin and type I collagen under different time and concentration stimulation of PDGF-BB. The effects of overexpression of miR-503 or RAF on the proliferation and migration of FBs were detected by cell counting kit 8 and cell scratch assay, respectively. A dual luciferase reporter gene assay was used to verify the targeting effect of miR-503 on RAF. The results showed that miR-503 was downregulated in a dose- and time-dependent manner in PDGF-BB-induced FBs. In addition, RAF is a direct target of miR-503 and can be negatively regulated. Overexpression of RAF can promote FB proliferation, migration, differentiation, collagen synthesis, and activation of downstream molecules (MEK/ERK), while overexpression of miR-503 can partially reverse the effects of RAF. Therefore, miR-503 regulates the biological behavior of PDGF-BB-induced oral mucosal FBs by influencing the activation of the RAS/RAF/MEK/ERK signaling pathway.

MicroRNA-605-3p Inhibited the Growth and Chemoresistance of Osteosarcoma Cells via Negatively Modulating RAF1.

Osteosarcoma (OS) is the leading cancer-associated mortality in childhood and adolescence. Increasing evidence has demonstrated the key function of microRNAs (miRNAs) in OS development and chemoresistance. Among them, miRNA-605-3p acted as an important tumor suppressor and was frequently down-regulated in multiple cancers. However, the function of miR-650-3p in OS has not been reported. The aim of this work is to explore the novel role of miR-605-3p in osteosarcoma and its possible involvement in OS chemotherapy resistance. The expression levels of miR-605-3p in OS tissues and cells were assessed by reverse transcription quantitative PCR (RT-qPCR). The relevance of miR-605-3p with the prognosis of OS patients was determined by the Kaplan-Meier analysis. Additionally, the influence of miR-605-3p on OS cell growth was analyzed using the cell counting kit-8, colony formation assay, and flow cytometry. The mRNA and protein expression of RAF1 were detected by RT-qPCR and western blot. The binding of miR-605-3p with the 3'-UTR of RAF1 was confirmed by dual-luciferase reporter assay. Our results showed that miR-605-3p was markedly decreased in OS tissues and cells. A lower level of miR-605-3p was strongly correlated with lymph node metastasis and poor 5-year overall survival rate of OS patients. In vitro assay found that miR-605-3p suppressed OS cell proliferation and promoted cell apoptosis. Mechanistically, the proto-oncogene RAF1 was seen as a target of miR-605-3p and strongly suppressed by miR-605-3p in OS cells. Restoration of RAF1 markedly eliminated the inhibitory effect of miR-605-3p on OS progression, suggesting RAF1 as a key mediator of miR-605-3p. Consistent with the decreased level of RAF1, miR-605-3p suppressed the activation of both MEK and ERK in OS cells, which are the targets of RAF1. Moreover, lower levels of miR-605-3p were found in chemoresistant OS patients, and downregulated miR-605-3p increased the resistance of OS cells to therapeutic agents. Our data revealed that miR-605-3p serves as a tumor suppressor gene by regulating RAF1 and increasing the chemosensitivity of OS cells, which provided the novel working mechanism of miR-605-3p in OS. Engineering stable nanovesicles that could efficiently deliver miR-605-3p with therapeutic activity into tumors could be a promising therapeutic approach for the treatment of OS.

Vitamin D Relieves Epilepsy Symptoms and Neuroinflammation in Juvenile Mice by Activating the mTOR Signaling Pathway via RAF1: Insights from Network Pharmacology and Molecular Docking Studies.

Epilepsy is a common neurological disorder, and the exploration of potential therapeutic drugs for its treatment is still ongoing. Vitamin D has emerged as a promising treatment due to its potential neuroprotective effects and anti-epileptic properties. This study aimed to investigate the effects of vitamin D on epilepsy and neuroinflammation in juvenile mice using network pharmacology and molecular docking, with a focus on the mammalian target of rapamycin (mTOR) signaling pathway. Experimental mouse models of epilepsy were established through intraperitoneal injection of pilocarpine, and in vitro injury models of hippocampal neurons were induced by glutamate (Glu) stimulation. The anti-epileptic effects of vitamin D were evaluated both in vivo and in vitro. Network pharmacology and molecular docking analysis were used to identify potential targets and regulatory pathways of vitamin D in epilepsy. The involvement of the mTOR signaling pathway in the regulation of mouse epilepsy by vitamin D was validated using rapamycin (RAPA). The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were assessed by enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining was used to analyze the apoptosis of hippocampal neurons. In in vivo experiments, vitamin D reduced the Racine scores of epileptic mice, prolonged the latency of epilepsy, and inhibited the production of TNF-α, IL-1β, and IL-6 in the hippocampus. Furthermore, network pharmacology analysis identified RAF1 as a potential target of vitamin D in epilepsy, which was further confirmed by molecular docking analysis. Additionally, the mTOR signaling pathway was found to be involved in the regulation of mouse epilepsy by vitamin D. In in vitro experiments, Glu stimulation upregulated the expressions of RAF1 and LC3II/LC3I, inhibited mTOR phosphorylation, and induced neuronal apoptosis. Mechanistically, vitamin D activated the mTOR signaling pathway and alleviated mouse epilepsy via RAF1, while the use of the pathway inhibitor RAPA reversed this effect. Vitamin D alleviated epilepsy symptoms and neuroinflammation in juvenile mice by activating the mTOR signaling pathway via RAF1. These findings provided new insights into the molecular mechanisms underlying the anti-epileptic effects of vitamin D and further supported its use as an adjunctive therapy for existing anti-epileptic drugs.

Transgenic expression of Rubisco accumulation factor2 and Rubisco subunits increases photosynthesis and growth in maize.

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown.

Network pharmacology analysis and experimental validation to explore the effect and mechanism of tetramethylpyrazine for spinal cord injury

ObjectiveTo investigate the effect and mechanism of Tetramethylpyrazine (TMP) in treating Spinal Cord Injury (SCI) using network pharmacology analysis and animal experiments. MethodsThis study was based on public databases, including PharmMapper, BATMAN-TCM, and STRING, as well as KEGG pathway analysis and other methods of network pharmacology were used to preliminarily explore the molecular mechanism of TMP in the treatment of SCI. Using a mouse SCI compression injury model, the efficacy of TMP was evaluated, and the expression of predictive targets on the PI3K/AKT and MAPK signaling pathways was measured using Western blotting and q-PCR. ResultsNetwork pharmacology analysis showed that TMP may exert therapeutic effects through the MAPK and PI3K/AKT signaling pathways. In animal experimental validation studies, it was shown that after treatment with TMP, the hind limb motor function scores and ramp test scores of the TMP-treated mice improved significantly. HE staining showed that after treatment with TMP, cavities decreased, fewer glial cells proliferated, and fewer inflammatory cells infiltrated; Nielsen staining showed less neuronal loss. Western blot studies showed that compared with the model group, expression of RAS, ERK1/2, RAF1, PI3K, and p-AKT proteins in the spinal cord tissue of mice treated with high-dose TMP was significantly lower. Accordingly, q-PCR studies showed that compared with the model group, the expression levels of RAS, ERK1/2, RAF1, PI3K, and p-AKT genes in the spinal cords of mice in the high-dose TMP group were significantly lower. ConclusionTMP exhibits a good neuroprotective effect after SCI, which may be related to inhibition of the MAPK and PI3K/AKT signaling pathways.

Hsa-miR-34a-5p enhances temozolomide anti-tumoral effects on glioblastoma: in-silico and in-vitro study.

Glioblastoma multiform (GBM) is a commonly diagnosed brain neoplasm with a poor prognosis. Accumulating evidence has highlighted the significance of microRNA (miR) dysregulation in tumor development and progression. This study investigated the effect of hsa-miR-34a-5p and its combination with temozolomide on GBM, the related molecular mechanisms, and the signaling pathway using in-silico and in-vitro approaches. The in-silico tumor bulk and single-cell RNA sequencing analyses were done on TCGA-GTEx, CGGA, GSE13276, GSE90603, and GSE182109 datasets. After selecting the A172 cell line, hsa-miR-34a-5p mimics were transfected, and the cell viability, migration, cell cycle, clonogenicity, and apoptosis of studied groups were studied using MTT, scratch, flow cytometry, colony formation, and Annexin V/PI assays. The mRNA expression of CASP9, CASP3, CASP8, MMP2, CD44, CDK6, CDK4, CCND1, RAF1, MAP2K1, MET, SRC, and CD274 was studied using qRT-PCR method. hsa-miR-34a-5p downregulated RAF1 expression, as the signaling factor of the MAPK pathway. The combined treatment significantly downregulated the expression of MET, SRC, and MAP2K1, leading to the inhibition of the MET/MAPK pathway compared to temozolomide. Besides exerting anti-tumoral effects on the cell viability, migration, cell cycle, apoptosis, and clonogenicity of A172 cells, its combination with temozolomide enhanced temozolomide anti-tumoral effect. Compared to temozolomide, the combined treatment significantly decreased CDK4, CDK6, CCND1, and MMP2 expression. hsa-miR-34a-5p targets RAF1, as the signaling factor of the MAPK pathway, and potentiates the temozolomide anti-tumoral effect on A172 cells.

METTL3/YTHDC1-medicated m6A modification of circRNA3634 regulates the proliferation and differentiation of antler chondrocytes by miR-124486-5-MAPK1 axis

BackgroundThe deer antler, a remarkable mammalian appendage, has a growth rate surpassing that of any other known osseous organ. Emerging evidence indicates that circRNA and MAPK1 play critical roles in chondrocytes. Thus, exploration of their functions in antler chondrocytes will help us to understand the mechanism regulating the rapid antler growth.MethodsqRT-PCR, western blot, and immunohistochemistry were used to assess the expression of mRNAs and proteins. CCK-8, EdU, Cell migration, ALP activity detection, and ALP staining examined the effects of MAPK1 in antler chondrocytes. FISH, RIP, and luciferase assays were performed to evaluate the interactions among circRNA3634/MAPK1 and miR-124486-5. RIP and RAP assays proved the binding interaction between circRNA3634 and RBPs. Me-RIP was used to determine the m6A methylation modification of circRNA3634.ResultsThis study revealed high MAPK1 expression in antler cartilage tissue. Overexpression of MAPK1 promoted the proliferation, migration, and differentiation of antler chondrocytes and increased the expression of MAPK3, RAF1, MEK1, RUNX2, and SOX9. The silencing of MAPK1 had the opposite effect. CircRNA3634 was found to act as a molecular sponge for miR-124486-5, leading to increased MAPK1 expression and enhanced proliferation and migration of antler chondrocytes through competitive miR-124486-5 binding. We discovered that METTL3 mediates m6A modification near the splicing site of circRNA3634 and is involved in the proliferation and differentiation of antler chondrocytes. The m6A reader YTHDC1 facilitated the nuclear export of circRNA3634 in an m6A-dependent manner. Our results indicate that m6A-modified circRNA3634 promotes the proliferation of antler chondrocytes by targeting MAPK1 and show that the nuclear export of circRNA3634 is related to the expression of YTHDC1, suggesting that circRNA3634 could represent a critical regeneration marker for the antler.ConclusionsOur results revealed a novel m6A-modified circRNA3634 promoted the proliferation and differentiation of antler chondrocytes by regulating MAPK1. The nuclear export of circRNA3634 was related to the expression of YTHDC1.

The ASH1L-AS1-ASH1L axis controls NME1-mediated activation of the RAS signaling in gastric cancer.

Gastric cancer (GC) is one of the most leading cause of malignancies. However, the molecular mechanisms underlying stomach carcinogenesis remain incompletely understood. Dysregulated genetic and epigenetic alternations significantly contribute to GC development. Here, we report that ASH1L and its antisense lncRNA ASH1L-AS1, which are transcribed from the most significant GC-risk signal at 1q22, act as novel oncogenes. The high levels of ASH1L or lncRNA ASH1L-AS1 expression in GC specimens are associated with worse prognosis of patients. In line with this, ASH1L and ASH1L-AS1 are functionally important in promoting GC disease progression. LncRNA ASH1L-AS1 up-regulates ASH1L transcription, increases histone methyltransferase ASH1L expression and elevates genome-wide H3K4me3 modification levels in GC cells. Furthermore, ASH1L-AS1 directly interacts with transcription factor NME1 protein to form the ASH1L-AS1-NME1 ribonucleoprotein, which transcriptionally promotes expression of ASH1L, ASH1L-AS1, KRAS and RAF1, and activates the RAS signaling pathway in GC cells. Taken together, our data demonstrated that the ASH1L-AS1-ASH1L regulatory axis controls histone modification reprogram and activation of the RAS signaling in cancers. Thus, ASH1L-AS1 might be a novel targets of GC therapeutics and diagnosis in the clinic.

Targeting the Mutant Region in K-RAS by RNA Interference Controlling Cell Proliferation and Programmed Cell Death in Liver Cancer Cells.

Background Hepatocellular carcinoma (HCC) is a high-incidence and significant cause of cancer globally. HCC is the world's third most common death-related cancer and the sixth most common tumor. In HCC, various cellular signaling is activated to ensure malignancy transformation, angiogenesis, and metastasis. The most efficient signaling pathway in cancer is mitogen-activated protein kinase (MAPK), which controls malignancy and regulates apoptosis. The mutant k-ras gene led to the activation of the RAS protein with GTPase activity, which stimulated hepatocellular proliferation and transformation. Here we aim to investigate the correlation between the knock-down of k-ras and HCC evaluation in-vitro. 
 Material and Methods We used the HepG2 cell line to investigate the direct connection between the k-ras expression profile and RAF/MEK pathway using a respective siRNA antagonist k-ras. 
 Results Interestingly, the siRNA antagonist k-ras altered cell morphology and a number of the living HepG2 cells. In addition, the transfection of HepG2 cells with the firstly designed siRNA successfully reduced the expression profile k-ras and the relative gene expression of Raf-1 and Mek1, the downstream targets of RAS signaling. Furthermore, interleukin 8 (IL-8) and interleukin 6 (IL-6) were monitored in the fluids media at different time points following transfection. Both IL-6 and Il-8 production significantly increased in cells transfected with siRNA targeting k-ras. 
 Conclusion Our findings provide evidence for the influential role of the k-ras mutated gene in HCC development and suggest the possible regulation of this gene as a potential treatment for HCC.

Molecular insights into the lipid-carbohydrates metabolism switch under the endurance effort in Arabian horses.

Recent studies have shown that in Arabian horse muscle, long-term exercise-induced expression of genes related to fatty acid degradation and the downregulation of genes belonging to the glycolysis/gluconeogenesis and insulin signalling pathways. Long-lasting physical exertion may trigger the metabolism to switch the main energy source from carbohydrates to lipids due to higher caloric content. To describe the metabolism adaptation at the whole transcriptome of blood to endurance effort in Arabian horses. In vivo experiment. Venous blood samples from 10 Arabian horses were taken before and after a 120 km long endurance ride to isolate the RNA and perform the high-throughput NGS transcriptome sequencing. The results, including KEGG (Kyoto Encyclopaedia of Genes and Genomes) and GO (Gene Ontology) analyses, allowed us to describe the most significantly upregulated-ARV1, DGAT2, LIPE, APOA2, MOGAT1, MOGAT2, GYS1, GYS2 and downregulated-ACACA, ACACB, FADS1, FADS2 genes involved in carbohydrate and lipid metabolism. Also, the increased expression of RAF1, KRAS and NRAS genes involved in the Insulin pathway and PI3K-Akt was shown. Limited sample size, Arabians used for endurance racing were not compared to Arabians from other equestrian disciplines. This general insight into the processes described supports the thesis of the lipid-carbohydrates metabolism switch in endurance Arabian horses and provides the basis for further research.

Plasma microRNA-195, −34c, and − 1246 as novel biomarkers for the diagnosis of trastuzumab-resistant HER2-positive breast cancer patients

Recently, miRNAs have been regarded as potential candidates for mediating therapeutic functions by targeting genes related to drug response. In this study, we suggested that plasma miRNAs may be correlated with response to trastuzumab in HER2-positive breast cancer patients. To determine whether miR-195, miR-23b-3p, miR-1246, and miR-34c-3p are involved in trastuzumab resistance, we screened their expressions in the BT-474 cell line, which was followed by plasma analysis from 20 trastuzumab-resistant HER2-positive breast cancer patients and 20 nonresistance subjects. Then, TargetScan, Pictar, and miRDB were applied to find the possible targets of the selected miRNAs. In addition, the expression status of admitted targets was evaluated. Our results showed that in resistant BT-474 cells, miR-1246, and miR-23b-3p were significantly upregulated, and miR-195-5p and miR-34c-3p were downregulated. In plasma analysis, we found miR-195-5p, miR-34c-3p, and miR-1246 meaningfully diminished in the resistant group, while the expression of miR-23b-3p was not statistically different. The expression levels of confirmed targets by qRT-PCR showed that the expression of RAF1, AKT3, c-MET, CCND1, PHLPP2, MYB, MAP2K1, and PTEN was significantly upregulated, while the expression of CCNG2 was significantly downregulated. The networks of miRNAs with their confirmed targets improved comprehension of miRNA-mediated therapeutic responses to trastuzumab and might be proposed for more characterization of miRNA functions. Moreover, these data indicated that miR-195-5p, miR-34c-3p, and miR-1246 could be possible biomarkers for prognosis and early detection of the trastuzumab-resistant group from the sensitive group of HER2-positive breast cancer patients.

Sanjin Paishi Decoction improves the imbalance of gut microbiota and regulates MAPK signaling pathway to inhibit calcium oxalate stones in rats.

Sanjin Paishi Decoction (SJPSD) has positive effects on stone prevention; however, there is a lack of convincing evidence in the prevention of calcium oxalate stones. This study aimed investigates the effect of SJPSD on calcium oxalate stones and to explore its mechanism. The rat model of calcium oxalate stones was established and rats were treated with different doses of SJPSD. The pathological damage of kidney tissues was observed by HE staining, the deposition of calcium oxalate crystals in kidney tissues was examined by Von Kossa staining, and the levels of creatinine (CREA), urea (UREA), calcium (Ca), phosphorus (P), and magnesium (Mg) in serum were analyzed biochemically, the levels of IL-1β, IL-6, and TNF-α in serum were measured by ELISA, and the protein expression of Raf1, MEK1, p-MEK1, ERK1/2, p-ERK1/2, and Cleaved caspase-3 in kidney tissues was analyzed by Western blot. Moreover, the changes in gut microbiota were analyzed by 16S rRNA sequencing. SJPSD attenuated the pathological damage of renal tissues, reduced the levels of CREA, UREA, Ca, P, and Mg, and inhibited the expression of Raf1, p-MEK1, p-ERK1/2, and Cleaved caspase-3 in renal tissues (P < 0.05). SJPSD treatment affected the composition of intestinal microbiota in rats with calcium oxalate stones. The mechanism of SJPSD inhibition of calcium oxalate stone injury in rats may be related to the inhibition of the MAPK signaling pathway and regulation of gut microbiota imbalance.

Patchouli alcohol against renal fibrosis of spontaneously hypertensive rats via Ras/Raf-1/ERK1/2 signalling pathway.

The present study was designed to obverse the protection of patchouli alcohol (PA) ameliorates hypertensive nephropathy in spontaneously hypertensive rats (SHR) and reveals potential mechanism. Briefly, the adult spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats (half male and half female) were intragastric gavaged or not with PA (80, 40 and 20 mg/kg) for 8 weeks. Body weight, blood pressure (BP), renal weight, renal function and renal morphology were measured. Further, western blotting and immunohistochemical analysis were used to study the underlying mechanism. Compared with the WKY group, plasmatic levels of renin, angiotensin II (Ang-II), transforming growth factor beta 1(TGF-β1), plasminogen activator inhibitor-1(PAI-1), creatinine (Cr), blood urea nitrogen (BUN), renal index, mRNA levels of ERK1/2 and α-SMA were significantly increased in SHR. Histology results showed that renal tubular injury and tubulointerstitial fibrosis occurred in SHR. After administration, SBP of captopril group decreased at each week after administration, especially at 3, 5, 6 7 and 8 weeks (P < 0.05 or P < 0.01). There is no significant effect was assessed in the olive oil group. Decreased plasma Cr, Renin, Ang-II, TGF-β1, PAI-1, SCFAs and Renin, TGF-β1, PAI-1 in renal tissues were observed significantly in captopril (P <0.05 or P < 0.01). Plasma BUN, Ang-II, TGF-β1 and PAI-1 in renal tissues decreased in the olive oil group significantly (P <0.05 or P < 0.01). PA (80, 40 and 20 mg/kg) lowered BP and plasmatic levels of Renin, Ang-II, TGF-β1 and PAI-1. Treatment with PA (40, 20 mg/kg) decreased levels of Cr, BUN and suppressed of activation of pro-fibrosis cytokines including TGF-β1 in kidney. There is no ameliorative change in the olive oil group and the captopril group (P > 0.05) while PA treatment alleviated renal tubular injury and produced dramatic collagen fibre area reductions in mesangial membrane, basement membrane, and renal interstitium obviously (P < 0.05 or P < 0.01). Treatment of SHR with PA-inhibited MFB activation and downregulated mRNA of α-SMA. Treatment with PA suppressed excessive production of the extracellular matrix (ECM) via decreasing Col I, III and FN, downregulating mRNA of tissue inhibitor of TIMP-1 along with upregulating mRNA of MMP-9. The expression of Col III and MMP-9 mRNA-reduced in the captopril group (P < 0.05). In addition, the expression of ERK1/2 and pERK1/2 also reduced in the captopril group significantly (P < 0.05 or P < 0.01). Treatment with PA (20 mg/kg) downregulated proteins expression of Raf-1, ERK1/2 and pERK1/2 and mRNA expression of Ras, Raf-1 and ERK1/2. Overall, PA restored normal BP, alleviated renal dysfunction and renal fibrosis, possibly by suppressing Ang II and TGF-β1-mediated Ras/Raf-1/ERK1/2 signalling pathway.

Cardiac angiogenesis enhances by activating Mir-126 and related target proteins in type 2 diabetic rats: Rescue combination effect of Sodium butyrate and voluntary exercise therapy.

type 2 diabetes, metabolic disorder, is one of the main risk factors for cardiovascular disease, leading to angiogenesis injury. The present study wanted to discover the effect of sodium butyrate (NaB) and voluntary exercise, alone or together, on miR-126 and related proteins in rats with type 2 diabetes. thirty-five male Wistar rats (200-250g) were randomly divided into five groups: control, diabetes, diabetes-NaB, diabetes-exercise, and diabetes-NaB-exercise. Type 2 diabetes was induced by intraperitoneal injection of streptozotocin (35mg/kg) and high-fat diet. The rats were then administrated NaB (200mg/kg. ip) or were subjected to voluntary exercise, or combined NaB and voluntary exercise for 8weeks. MiR-126 expression in the cardiac tissue was determined by real-time PCR, and the SPRED-1 and RAF proteins expression levels were measured by western blot. NaB and voluntary exercise up-regulated cardiac miR-126 and RAF expression levels and down-regulated SPRED-1 in cardiac tissue of type 2 diabetic rats. Moreover, the combination of NaB and voluntary exercise amplified their effects on those parameters. Both NaB and voluntary exercise or together markedly modulated serum glucose and HbA1c. The present findings demonstrated that NaB combined with exercise could improve cardiac angiogenesis by increasing miR-126 and affecting related proteins. Thus, NaB together with voluntary exercise might be a promising intervention for the treatment and prevention of type 2 diabetes.

Prevention of Cell Proliferation Signaling Via RNA Interference of c-myc Gene Expression in Colon Cancer In-vitro

Colon cancer is a cancer begins in the large intestine (colon). Its aggression is due to late diagnosis, so poor prognosis and higher mortality rates are reported. Colon cancer has become a thorny research area that requires more examination of cellular pathways involved in its emergency. Based on this, we sought to check the biological role of cMyc in colon cancer cell proliferation through targeting its expression by a respective siRNA using CaCo-2 cell lines. Notably, the cytotoxic potential of cMyc knockdown was monitored by cell imaging using an inverted microscope, the production of lactate dehydrogenase (LDH) and viability of the transfected cells using MTT assay. The relative gene expression of c-myc and other related factors such as Raf-1 was investigated using quantitative real-time PCR (qRT-PCR). ELISA assay has been used to achieve the production of pro and anti-inflammatory cytokines released from transfected cells. Interestingly, the transfection with a new designed siRNA antagonist cMyc markedly decreased CaCo-2 cell viability rate in a dose-dependent manner compared with the transfection with specific siRNA antagonist Luciferase gene, which served as control. Likewise, number of survived cells significantly reduced following transfection with siRNA against c-myc, while the relative production of LDH significantly increased. Importantly, the cytotoxic effect of the transfection protocol showed neglected influence in normal colon epithelial cells (NCM-460 cell lines) indicating the selective regulation of colon cancer cell proliferation by transfection with siRNA targeting c-myc. Noteworthy, the knockdown efficiency of c-myc showed more than 80% inhibition of its expression in CaCo-2 cells transfected with the siRNA antagonist c-myc indicated by qRT-PCR. The relative gene expression of Raf-1 significantly decreased in c-myc knockdown cells, while the relative expression of both P53 and Caspase 3, as cell death indicators, significantly upregulated compared with control-transfected cells. Furthermore, the transfection with siRNA targeting c-myc markedly increased the production of interleukin 1 alpha (IL-1α ) and IL-1β, as pro-inflammatory cytokines, while decreased the production of IL- 4 and IL-10, as anti-inflammatory cytokines. These data provide evidence for the involvement of c-myc gene expression in colon cancer cell proliferation and immune response during cancer development.

Anti-Cancer Properties of Clove Bud Essential Oil in Colon Cancer Cell Line

Colon cancer is a type of cancer that begins in the large intestine (colon). Its aggression is owing to late diagnosis so poor prognosis and higher mortality rate are reported. Colon cancer has become a thorny research region that requires more examination in cellular pathways involved in its emergency. Here we aim to investigate the possible anticancer properties of the clove oil as natural and popular oil in colon cancer CaCo-2 cells. Accordingly, cell viability rate and number of survived cells were monitored in response to the clove oil treatment compared with the black seed oil and DMSO-treatment. Lactate dehydrogenase (LDH) production was also considered as an indicator for necrotic event following treatment. The relative gene expression of Raf-1, MEK, and ERK was detected as proliferation effectors that linked to the expression profile of the tumor suppressor genes PTEN and TP53 using quantitative real-time PCR (qRT-PCR). Interestingly, here we identified Raf-1 pathway as a potential targeted singling by the clove oil resulted in controlling colon cancer cell proliferation. The current study elucidates the anticancer activities of the clove oil in colon cancer division through restoring the relative gene expression of PTEN and TP53 accompanied by over-production of interleukin 1 alpha (IL-1α) and IL-1β. Accordingly, treatment of the CaCo-2 cells with different concentrations of the clove oil increased programmed cells cell death (PCD) and inhibited the proliferation singling when compared with the black seed oil. Collectively, our results demonstrate that controlling of Raf-1 activity and PCD by clove oil treatment provide evidence for its anticancer effect in colon cancer cells.

Saffron essential oil ameliorates CUMS-induced depression-like behavior in mice via the MAPK-CREB1-BDNF signaling pathway

Ethnopharmacological relevanceSaffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect.Aim of the study: This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS). Materials and methodsIn this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB). ResultsAccording to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group. ConclusionOverall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway.

Direct Targeting of the Raf-MEK-ERK Signaling Cascade Inhibits Neuroblastoma Growth.

The Raf-MEK-ERK signaling network has been the subject of intense research due to its role in the development of human cancers, including pediatric neuroblastoma (NB). MEK and ERK are the central components of this signaling pathway and are attractive targets for cancer therapy. Approximately 3–5% of the primary NB samples and about 80% of relapsed samples contain mutations in the Raf-MEK-ERK pathway. In the present study, we analyzed the NB patient datasets and revealed that high RAF and MEK expression leads to poor overall survival and directly correlates with cancer progression and relapse. Further, we repurposed a specific small-molecule MEK inhibitor CI-1040 to inhibit the Raf-MEK-ERK pathway in NB. Our results show that CI-1040 potently inhibits NB cell proliferation and clonogenic growth in a dose-dependent manner. Inhibition of the Raf-MEK-ERK pathway by CI-1040 significantly enhances apoptosis, blocks cell cycle progression at the S phase, inhibits expression of the cell cycle-related genes, and significantly inhibits phosphorylation and activation of the ERK1/2 protein. Furthermore, CI-1040 significantly inhibits tumor growth in different NB 3D spheroidal tumor models in a dose-dependent manner and by directly inhibiting spheroidal tumor cells. Overall, our findings highlight that direct inhibition of the Raf-MEK-ERK pathway is a novel therapeutic approach for NB, and further developing repurposing strategies using CI-1040 is a clinically tractable strategy for effectively treating NB.

The Role of Interventional Irisin on Heart Molecular Physiology.

Irisin, encoded by the FNDC5 (fibronectin type III domain containing 5) gene, is a novel myokine that has been implicated as an essential mediator of exercise benefits. Effects of irisin on heart physiology is still ambiguous. This study aimed to evaluate the impact of exogenous administration of irisin on heart physiology and the pharmacokinetic profile of pump-administered irisin. To do so, Sprague Dawley rats were implanted with an irisin-loaded osmotic pump (5 μg/kg/day) for 42 days, and other animals were administered with single bolus subcutaneous injections of irisin (5 µg/kg). Body weights and blood samples were collected weekly for 42 days for serum irisin quantification and histopathology. Clinical biochemistry analyses were performed. Heart mRNA expression was assessed in 26 selected genes. Chronic interventional exogenous irisin significantly reduced body weight without affecting the heart myocyte size and significantly reduced creatine kinase enzyme level. Blood CBC, serum biochemistry, and heart morphology were normal. Gene expression of FNCD5, Raf1, CPT1, IGF-1, and CALCIN, encoding for heart physiology, increased while PGC1, Nox4, and Mfn1 significantly decreased. Nevertheless, irisin increased the expression of cardioprotective genes and inhibited some genes that harm heart physiology. Administration of irisin promotes myocardial functions and could be translated into clinical settings after preclinical profiling.

RAF1 promotes lymphatic metastasis of hypopharyngeal carcinoma via regulating LAGE1: an experimental research

BackgroundLymphatic metastasis was an independent prognostic risk factor for hypopharyngeal carcinoma and was the main cause of treatment failure. The purpose of this study was to screen the differential genes and investigate the mechanism of lymphatic metastasis in hypopharyngeal carcinoma.MethodsTranscriptome sequencing was performed on primary tumors of patients, and differential genes were screened by bioinformatics analysis. The expression of differential genes was verified by qRT-PCR, western-blotting and immunohistochemical, and prognostic value was analyzed by Kaplan–Meier and log-rank test and Cox’s test. Next, FADU and SCC15 cell lines were used to demonstrate the function of differential genes both in vitro by EdU, Flow cytometry, Wound Healing and Transwell assays and in vivo by a foot-pad xenograft mice model. Proteomic sequencing was performed to screen relevant targets. In addition, in vitro and in vivo experiments were conducted to verify the mechanism of lymphatic metastasis.ResultsResults of transcriptome sequencing showed that RAF1 was a significantly differential gene in lymphatic metastasis and was an independent prognostic risk factor. In vitro experiments suggested that decreased expression of RAF1 could inhibit proliferation, migration and invasion of tumor cells and promote apoptosis. In vivo experiments indicated that RAF1 could promote tumor growth and lymphatic metastasis. Proteomic sequencing and subsequent experiments suggested that LAGE1 could promote development of tumor and lymphatic metastasis, and was regulated by RAF1.ConclusionsIt suggests that RAF1 can promote lymphatic metastasis of hypopharyngeal carcinoma by regulating LAGE1, and provide a basis for the exploring of novel therapeutic target and ultimately provide new guidance for the establishment of intelligent diagnosis and precise treatment of hypopharyngeal carcinoma.