KRAS Expression Levels Research Articles

Expression of Five Important Biomarkers in Non-small Cell Lung Cancer: MicroRNAs-128, miR-335-5p, miR-1254, VEGF, and K-RAS

Background: Lung cancers, such as non-small cell lung cancer (NSCLC), are the most common malignancy and leading cause of cancer-related death worldwide. MicroRNAs (miRNAs) play a role in the occurrence of lung cancer by targeting both tumor suppressor genes and oncogenes. Among them, miR-128, miR-335-5p, and miR-1254 are mainly reported to function in regulating lung cancer cell behavior. Objectives: The aim of the study was to investigate the existence and expression levels of miRNA-128, miR-335-5p, and miR-1254 as well as mRNA and protein expression levels of VEGF and mRNA expression levels of K-RAS in both peripheral blood and cancerous tissue of NSCLC patients compared to the healthy subjects. Methods: Blood and tissue samples were collected from 50 healthy donors and 50 NSCLS patients. The serum level of VEGF was measured using the commercial enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of K-RAS and VEGF, as well as the expression of miRNA-128, miR-335-5p, and miR-1254, were determined, using real-time PCR. Results: Lower mRNA expression levels of miRNA-128, miR-335-5p, and miR-1254, as well as higher mRNA expression levels of VEGF and K-RAS, were detected in both peripheral blood and tissue samples from NSCLC patients compared to those of the healthy subjects. The VEGF protein levels in the peripheral blood of the patients were also found to be significantly lower than those in the healthy subjects. Conclusions: The findings from this study suggest that miR-335-5p, miR-1254, and miR-128, may contribute to the pathogenesis and tumorigenesis of NSCLC at least in part by modulation of proliferation, migration, and angiogenesis via targeting VEGF and K-RAS signaling pathways. K-RAS and VEGF may be target genes for miR-335-5p, miR-1254, and miR-128 in NSCLC patients.

Identification and validation of oxidative stress-related diagnostic markers for recurrent pregnancy loss: insights from machine learning and molecular analysis.

It has been recognized that oxidative stress (OS) is implicated in the etiology of recurrent pregnancy loss (RPL), yet the biomarkers reflecting oxidative stress in association with RPL remain scarce. The dataset GSE165004 was retrieved from the Gene Expression Omnibus (GEO) database. From the GeneCards database, a compendium of 789 genes related to oxidative stress-related genes (OSRGs) was compiled. By intersecting differentially expressed genes (DEGs) in normal and RPL samples with OSRGs, differentially expressed OSRGs (DE-OSRGs) were identified. In addition, four machine learning algorithms were employed for the selection of diagnostic markers for RPL. The Receiver Operating Characteristic (ROC) curves for these genes were generated and a predictive nomogram for the diagnostic markers was established. The functions and pathways associated with the diagnostic markers were elucidated, and the correlations between immune cells and diagnostic markers were examined. Potential therapeutics targeting the diagnostic markers were proposed based on data from the Comparative Toxicogenomics Database and ClinicalTrials.gov. The candidate biomarker genes from the four models were further validated in RPL tissue samples using RT-PCR and immunohistochemistry. A set of 20 DE-OSRGs was identified, with 4 genes (KRAS, C2orf69, CYP17A1, and UCP3) being recognized by machine learning algorithms as diagnostic markers exhibiting robust diagnostic capabilities. The nomogram constructed demonstrated favorable predictive accuracy. Pathways including ribosome, peroxisome, Parkinson's disease, oxidative phosphorylation, Huntington's disease, and Alzheimer's disease were co-enriched by KRAS, C2orf69, and CYP17A1. Cell chemotaxis terms were commonly enriched by all four diagnostic markers. Significant differences in the abundance of five cell types, namely eosinophils, monocytes, natural killer cells, regulatory T cells, and T follicular helper cells, were observed between normal and RPL samples. A total of 180 drugs were predicted to target the diagnostic markers, including C544151, D014635, and CYP17A1. In the validation cohort of RPL patients, the LASSO model demonstrated superiority over other models. The expression levels of KRAS, C2orf69, and CYP17A1 were significantly reduced in RPL, while UCP3 levels were elevated, indicating their suitability as molecular markers for RPL. Four oxidative stress-related diagnostic markers (KRAS, C2orf69, CYP17A1, and UCP3) have been proposed to diagnose and potentially treat RPL.

Effects of different KRAS mutants and Ki67 expression on diagnosis and prognosis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a prevalent form of non-small cell lung cancer with a rising incidence in recent years. Understanding the mutation characteristics of LUAD is crucial for effective treatment and prediction of this disease. Among the various mutations observed in LUAD, KRAS mutations are particularly common. Different subtypes of KRAS mutations can activate the Ras signaling pathway to varying degrees, potentially influencing the pathogenesis and prognosis of LUAD. This study aims to investigate the relationship between different KRAS mutation subtypes and the pathogenesis and prognosis of LUAD. A total of 63 clinical samples of LUAD were collected for this study. The samples were analyzed using targeted gene sequencing panels to obtain sequencing data. To complement the dataset, additional clinical and sequencing data were obtained from TCGA and MSK. The analysis revealed significantly higher Ki67 immunohistochemical scores in patients with missense mutations compared to controls. Moreover, the expression level of KRAS was found to be significantly correlated with Ki67 expression. Enrichment analysis indicated that KRAS missense mutations activated the SWEET_LUNG_CANCER_KRAS_DN and CREIGHTON_ENDOCRINE_THERAPY_RESISTANCE_2 pathways. Additionally, patients with KRAS missense mutations and high Ki67 IHC scores exhibited significantly higher tumor mutational burden levels compared to other groups, which suggests they are more likely to be responsive to ICIs. Based on the data from MSK and TCGA, it was observed that patients with KRAS missense mutations had shorter survival compared to controls, and Ki67 expression level could more accurately predict patient prognosis. In conclusion, when utilizing KRAS mutations as biomarkers for the treatment and prediction of LUAD, it is important to consider the specific KRAS mutant subtypes and Ki67 expression levels. These findings contribute to a better understanding of LUAD and have implications for personalized therapeutic approaches in the management of this disease.

KRAS modulates immune infiltration levels and survival outcomes in patients with lung adenocarcinoma

The murine sarcoma virus oncogene (KRAS) is a key gene associated with tumorigenesis and chemotherapy resistance. However, little is known about the molecular mechanisms and immune infiltration of RASs in lung adenocarcinoma. Gene Expression Profiling Interaction Analysis was used for RASs expression analysis, and Kaplan–Meier analysis was used to analyze the potential of RASs in clinical prognosis. The effect of KRAS on immune infiltration was analyzed by TIMER. In addition, the correlation between KRAS expression and molecular mechanisms was investigated by TIMER and Cancer Single-cell State Atlas (Cancer SEA). KRAS expression levels were associated with good prognosis and tumor progression. Furthermore, KRAS expression correlates with several immune cell markers and regulates tumorigenesis. KRAS expression is involved in the regulation of multiple oncogenes and tumorigenesis, especially in the prognosis and immune infiltration of lung adenocarcinoma.

MiR-204-3p overexpression inhibits gastric carcinoma cell proliferation by inhibiting the MAPK pathway and RIP1/MLK1 necroptosis pathway to promote apoptosis.

Gastric carcinoma (GC) is the third most frequent cause of cancer-related death, highlighting the pressing need for novel clinical treatment options. In this regard, microRNAs (miRNAs) have emerged as a promising therapeutic strategy. Studies have shown that miRNAs can regulate related signaling pathways, acting as tumor suppressors or tumor promoters. To explore the effect of miR-204-3p on GC cells. We measured the expression levels of miR-204-3p in GC cells using quantitative real-time polymerase chain reaction, followed by the delivery of miR-204-3p overexpression and miR-204-3p knockdown vectors into GC cells. CCK-8 was used to detect the effect of miR-204-3p on the proliferation of GC cells, and the colony formation ability of GC cells was detected by the clonal formation assay. The effects of miR-204-3p on GC cell cycle and apoptosis were detected by flow cytometry. The BABL/c nude mouse subcutaneous tumor model using MKN-45 cells was constructed to verify the effect of miR-204-3p on the tumorigenicity of GC cells. Furthermore, the study investigated the effects of miR-204-3p on various proteins related to the MAPK signaling pathway, necroptosis signaling pathway and apoptosis signaling pathway on GC cells using Western blot techniques. Firstly, we found that the expression of miR-204-3p in GC was low. When treated with the lentivirus overexpression vector, miR-204-3p expression significantly increased, but the lentivirus knockout vector had no significant effect on miR-204-3p. In vitro experiments confirmed that miR-204-3p overexpression inhibited GC cell viability, promoted cell apoptosis, blocked the cell cycle, and inhibited colony formation ability. In vivo animal experiments confirmed that miR-204-3p overexpression inhibited subcutaneous tumorigenesis ability in BABL/c nude mice. Simultaneously, our results verified that miR-204-3p overexpression can inhibit GC cell proliferation by inhibiting protein expression levels of KRAS and p-ERK1/2 in the MAPK pathway, as well as inhibiting protein expression levels of p-RIP1 and p-MLK1 in the necroptosis pathway to promote the BCL-2/BAX/Caspase-3 apoptosis pathway. MiR-204-3p overexpression inhibited GC cell proliferation by inhibiting the MAPK pathway and necroptosis pathway to promote apoptosis of GC cells. Thus, miR-204-3p may represent a new potential therapeutic target for GC.

In silico and in vitro studies of novel cyanoacrylamides incorporating pyrazole moiety against breast and prostate carcinomas

We report novel cyanoacrylamide derivatives bearing the pyrazole moiety. The molecular structures of the prepared cyanoacrylamides were confirmed by the different spectral tools such as NMR, IR, and elemental analyses. The anticancer effect of all the newly prepared cyanoacrylamides was studied against four cancer cell lines (HEPG2, MCF7, PACA2, and PC3) as well as the normal cell line (BJ1). The best cytotoxic effect was shown against PC3, where compounds 5f and 5i revealed promising IC50 values (11.7 and 66.8 µM) respectively compared to doxorubicin (43.8 µM). In addition, the effective compounds were screened against a normal BJ1 cell line, which showed promising selectivity against PC3 and moderate selectivity toward MCF7 cells. The molecular docking study showed the affinities of compounds 5c and 5d toward STAT1 protein and compound 5i toward KRAS with promising energy scores. The subsequent molecular experiments were studied on compounds 5b, 5c, 5d, 5f, and 5i. Quantitative Real-time-PCR revealed that the expression of RBL2 and STAT2 genes were down-regulated in 5c and 5d treated MCF7 cells much lower than the other treated MCF7 samples. Also, the expression level of KRAS and SMAD genes was determined, which revealed the significant down-regulation of them in compounds 5f and 5i treated PC3 cells. The percentages of DNA damage were raised significantly in all treated MCF7 and PC3 samples as compared to the negative control, and the highest percentages were for compounds 5c and 5d treated MCF7 cells.Graphical

KRAS is a prognostic biomarker associated with diagnosis and treatment in multiple cancers.

KRAS encodes K-Ras proteins, which take part in the MAPK pathway. The expression level of KRAS is high in tumor patients. Our study compared KRAS expression levels between 33 kinds of tumor tissues. Additionally, we studied the association of KRAS expression levels with diagnostic and prognostic values, clinicopathological features, and tumor immunity. We established 22 immune-infiltrating cell expression datasets to calculate immune and stromal scores to evaluate the tumor microenvironment. KRAS genes, immune check-point genes and interacting genes were selected to construct the PPI network. We selected 79 immune checkpoint genes and interacting related genes to calculate the correlation. Based on the 33 tumor expression datasets, we conducted GSEA (genome set enrichment analysis) to show the KRAS and other co-expressed genes associated with cancers. KRAS may be a reliable prognostic biomarker in the diagnosis of cancer patients and has the potential to be included in cancer-targeted drugs.

Forkhead Box q1 promotes invasion and metastasis in colorectal cancer by activating the epidermal growth factor receptor pathway

BACKGROUNDColorectal cancer (CRC) is an extremely malignant tumor with a high mortality rate. Little is known about the mechanism by which forkhead Box q1 (FOXQ1) causes CRC invasion and metastasis through the epidermal growth factor receptor (EGFR) pathway.AIMTo illuminate the mechanism by which FOXQ1 promotes the invasion and metastasis of CRC by activating the heparin binding epidermal growth factor (HB-EGF)/EGFR pathway.METHODSWe investigated the differential expression and prognosis of FOXQ1 and HB-EGF in CRC using the Gene Expression Profiling Interactive Analysis (GEPIA) website (http://gepia.cancer-pku.cn/index.html). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of FOXQ1 and HB-EGF in cell lines and tissues, and we constructed a stable low-expressing FOXQ1 cell line and verified it with the above method. The expression changes of membrane-bound HB-EGF (proHB-EGF) and soluble HB-EGF (sHB-EGF) in the low-expressing FOXQ1 cell line were detected by flow cytometry and ELISA. Western blotting was used to detect changes in the expression levels of HB-EGF and EGFR pathway-related downstream genes when exogenous recombinant human HB-EGF was added to FOXQ1 knockdown cells. Proliferation experiments, transwell migration experiments, and scratch experiments were carried out to determine the mechanism by which FOXQ1 activates the EGFR signaling pathway through HB-EGF, and then to evaluate the clinical relevance of FOXQ1 and HB-EGF.RESULTSGEPIA showed that the expression of FOXQ1 in CRC tissues was relatively high and was related to a lower overall survival rate. PCR array results showed that FOXQ1 is related to the HB-EGF and EGFR pathways. Knockdown of FOXQ1 suppressed the expression of HB-EGF, and led to a decrease in EGFR and its downstream genes AKT, RAF, KRAS expression levels. After knockdown of FOXQ1 in CRC cell lines, cell proliferation, migration and invasion were attenuated. Adding HB-EGF restored the migration and invasion ability of CRC, but not the cell proliferation ability. Kaplan–Meier survival analysis results showed that the combination of FOXQ1 and HB-EGF may serve to predict CRC survival.CONCLUSIONBased on these collective data, we propose that FOXQ1 promotes the invasion and metastasis of CRC via the HB-EGF/EGFR pathway.

Relevance of BRAF Subcellular Localization and Its Interaction with KRAS and KIT Mutations in Skin Melanoma.

Although skin melanoma (SKM) represents only one-quarter of newly diagnosed skin malignant tumors, it presents a high mortality rate. Hence, new prognostic and therapeutic tools need to be developed. This study focused on investigating the prognostic value of the subcellular expression of BRAF, KRAS, and KIT in SKM in correlation with their gene-encoding interactions. In silico analysis of the abovementioned gene interactions, along with their mRNA expression, was conducted, and the results were validated at the protein level using immunohistochemical (IHC) stains. For IHC expression, the encoded protein expressions were checked on 96 consecutive SKMs and 30 nevi. The UALCAN database showed no prognostic value for the mRNA expression level of KRAS and BRAF and demonstrated a longer survival for patients with low mRNA expression of KIT in SKMs. IHC examinations of SKMs confirmed the UALCAN data and showed that KIT expression was inversely correlated with ulceration, Breslow index, mitotic rate, and pT stage. KRAS expression was also found to be inversely correlated with ulceration and perineural invasion. When the subcellular expression of BRAF protein was recorded (nuclear vs. cytoplasmatic vs. mixed nucleus + cytoplasm), a direct correlation was emphasized between nuclear positivity and lymphovascular or perineural invasion. The independent prognostic value was demonstrated for mixed expression of the BRAF protein in SKM. BRAF cytoplasmic predominance, in association with KIT’s IHC positivity, was more frequently observed in early-stage nonulcerated SKMs, which displayed a low mitotic rate and a late death event. The present study firstly verified the possible prognostic value of BRAF subcellular localization in SKMs. A low mRNA expression or IHC cytoplasmic positivity for KIT and BRAF might be used as a positive prognostic parameter of SKM. SKM’s BRAF nuclear positivity needs to be evaluated in further studies as a possible indicator of perineural and lymphovascular invasion.

MiR-622 acts as a tumor suppressor to induce cell apoptosis and inhibit metastasis in human prostate cancer.

Growing evidence indicating the critical modulator roles of microRNAs (miRNAs) involved in prostate cancer (PCa) metastasis that holds great promise as therapeutic targets. Herein, we transfected the miR-622 mimic into PC3 cells and evaluated the effects of this interference on these tumour cells' growth and the expression of specific metastatic genes. Transfecting of miR-622 mimic and inhibitor, negative control (NC) inhibitor and NC was established using Lipofectamine 2000. The mRNA levels of miR-622 and metastatic genes were evaluated using the qRT-PCR and Western blot. Cytotoxic effects of miR-622 were assessed by MTT. Apoptosis was detected using an ELISA cell death assay kit. miR-622 is down-regulated in PC3 cells. As expected, cell viability effects after transfection were described as miR-622 inhibitor >NC and NC inhibitor >miR-622 mimic (p<.01). Importantly, we showed that transfected miR-622 mimic could enhance the apoptosis of PC3 cells, while transfected miR-622 inhibitor could decrease cell apoptosis (p<.01). Furthermore, miR-622 overexpression could increase significantly down-regulated the MMP2, MMP9, CXCR-4, c-Myc and K-Ras expression levels. Findings demonstrate a novel mechanism by which miR-622 modulates PCa cells' metastasis by targeting metastatic genes. These data confirm the tumour-suppressive function of miR-622 in PCa cells by enhancing apoptosis and reducing metastasis.

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.

Overexpression of miRNA-145 induces apoptosis and prevents proliferation and migration of MKN-45 gastric cancer cells.

MiR-145 is a tumor suppressor miRNA that its ubiquitously expressed in the body but in numerous types of cancers such as GC, its expression became reduced or sometimes ceased in many subjects. This study aimed at restoring the function of the miR-145 in MKN-45 cells and investigating the function of this miRNA in proliferation, apoptosis, and migration of GC cells. MKN-45 cells were transfected using the PCMV-miR-145 plasmid vector. The MTT, DAPI staining, and wound healing assays were applied to estimate the impacts of ectopic expression of miR-145 in vitro. Moreover, alterations in the expression levels of K-Ras, c-Myc, caspase-3, caspase-9, Bax, Bcl-2, and MMP-9 mRNA were measured by qRT-PCR analysis. The findings designated that high expression of miR-145 reduced the proliferation and migration and increased the apoptosis of the MKN-45 cells. These effects occur with concurrent suppression of c-Myc, K-Ras, Bcl-2, and MMP-9 as well as induction of caspase-3, caspase-9, and Bax expression. Exogenous miR-145 influences multiple oncogenic pathways and can be regarded as a promising avenue of future therapeutic interventions for GC therapy.

Elevated protein expression levels of KRAS in Pancreatic Ductal Adenocarcinoma (PDAC) are associated with better prognosis

Elevated protein expression levels of KRAS in Pancreatic Ductal Adenocarcinoma (PDAC) are associated with better prognosis

KRAS expression is a prognostic indicator and associated with immune infiltration in breast cancer.

Breast cancer is the most common cancer and the leading cause of death among women. KRAS is known as an oncogene, its expression also associates with cancer prognosis. The purpose of this study was to investigate the prognostic value of KRAS expression in breast cancer and its relationship with immune infiltration. Firstly, the expression level and methylation of KRAS were analyzed. Then survival analysis was used to verify the prognostic capability of KRAS expression. After that, gene functional enrichment analysis was performed. The relationship between KRAS gene expression and immune infiltration was researched later. The expression level of KRAS in breast cancer was increased (P = 2.2e-16). Tumor KRAS expression in the subtypes of basal-like, HER2-enriched, Luminal A and Luminal B were 1.64, 1.67, 1.51 and 1.42 times of normal, respectively. 13 methylation sites were different between tumor and normal tissues and associated with KRAS expression. Subsequently, Kaplan-Meier analysis suggested that the high KRAS expression group had a poor prognosis (P = 0.0028). In multivariate Cox regression analysis, KRAS expression was an independent prognostic indicator (HR = 1.353, 95% CI 1.009-1.814, P = 0.044). Gene Ontology (GO) analysis showed enrichment of epidermal growth associated pathways. Additionally, different KRAS expression levels represented different tumor immune infiltration status, which may be caused by the influence of the RAS/MAPK and RAS/PI3K pathways on the level of PD-L1. This study suggests that KRAS expression can be used as a prognostic indicator of breast cancer, and it is closely related to tumor immune infiltration.

MiR-16 inhibits proliferation of cervical cancer cells by regulating KRAS.

The aim of this study was to explore the effects of micro ribonucleic acid (miR)-16 on the proliferation and apoptosis of cervical cancer (CC) cells and its related regulatory mechanism. The downstream regulatory targets of miR-16 were analyzed based on the miRNA online database. HCC94 cells were selected as experimental objects. Subsequently, the cells were transfected with miR-16 mimic (miR-16 mimic group), miR-16 small interfering RNA (siRNA) (miR-16 siRNA group) and only Lipofectamine 2000 transfection reagent [blank control group and miR-16 normal control (NC) group]. The expression level of miR-16 in HCC94 cells was measured via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) assay, 5-Ethynyl-2'-deoxyuridine (EdU) staining assay and flow cytometry were then conducted to detect the effects of miR-16 on the viability, proliferation and apoptosis of HCC94 cells, respectively. Additionally, the effect of miR-16 on the protein expression level of Kirsten rat sarcoma viral oncogene homolog (KRAS) in HCC94 cells was determined via Western blotting. MiRNA online database analysis showed that KRAS was the downstream target of miR-16. Compared with miR-16 NC group, the viability and proliferation ability of HCC94 cells increased significantly in miR-16 siRNA group but decreased significantly in miR-16 mimic group (p<0.05). However, the apoptosis rate evidently declined in miR-16 siRNA group while increased remarkably in miR-16 mimic group (p<0.05). In addition, the protein expression level of KRAS in HCC94 cells was significantly higher in miR-16 siRNA group but significantly lower in miR-16 mimic group when compared with miR-16 NC group (p<0.05). MiR-16 is lowly expressed in HCC94 cells. Moreover, highly expressed miR-16 represses the viability and proliferation of HCC94 cells and promotes their apoptosis by targeted regulation on KRAS.

Cytotoxicity study of the interleukin-12-expressing recombinant Newcastle disease virus strain, rAF-IL12, towards CT26 colon cancer cells in vitro and in vivo

BackgroundOncolytic viruses have emerged as an alternative therapeutic modality for cancer as they can replicate specifically in tumour cells and induce toxic effects leading to apoptosis. Despite the great potentials and promising results shown in multiple studies, it appears that their efficacy is still moderate and deemed as not sufficient in clinical studies. In addressing this issue, genetic/molecular engineering approach has paved its way to improve the therapeutic efficacy as observed in the case of herpes simplex virus (HSV) expressing granulocyte–macrophage colony-stimulating factor (GM-CSF). This study aimed to explore the cytotoxicity effects of recombinant NDV strain AF2240-i expressing interleukin-12 (rAF-IL12) against CT26 colon cancer cells.MethodsThe cytotoxicity effect of rAF-IL12 against CT26 colon cancer cell line was determined by MTT assay. Based on the IC50 value from the anti-proliferative assay, further downward assays such as Annexin V FITC and cell cycle progression were carried out and measured by flow cytometry. Then, the in vivo study was conducted where the rAF-IL12 viral injections were given at the intra-tumoral site of the CT26 tumour-burden mice. At the end of the experiment, serum biochemical, T cell immunophenotyping, serum cytokine, histopathology of tumour and organ section, TUNEL assay, and Nanostring gene expression analysis were performed.ResultsThe rAF-IL12 induced apoptosis of CT26 colon cancer cells in vitro as revealed in the Annexin V FITC analysis and also arrested the cancer cells progression at G1 phase of the cell cycle analysis. On the other hand, the rAF-IL12 significantly (p < 0.05) inhibited the growth of CT26 tumour in Balb/c mice and had regulated the immune system by increasing the level of CD4 + , CD8 + , IL-2, IL-12, and IFN-γ. Furthermore, the expression level of apoptosis-related genes (bax and p53) was up-regulated as a result of the rAF-IL12 treatment. Additionally, the rAF-IL12 had also down-regulated the expression level of KRAS, BRAF, MAPK1, Notch1, CCL2, and VEGF oncogenes. Besides, rAF-IL12 intra-tumoral delivery was considered safe and not hazardous to the host as evidenced in pathophysiology of the normal tissues and organs of the mice as well as from the serum biochemistry profile of liver and kidney.ConclusionsThese results indicated that rAF-IL12 had better anti-tumoral and cytotoxicity effects compared to its parental wild-type, AF2240-i in combatting the CT26 colon cancer model.

Effect of Hypoxia on RAGE Signaling in Pancreatic Cancer

Pancreatic cancer (PC) is one of the deadliest cancers owing to its late diagnostic and poor response to chemotherapeutic agents. PC tumors are characterized by large hypoxic regions that contribute to tumor growth, formation of metastasis and resistance to chemotherapeutic agents. Studies have shown that the Receptor for Advanced Glycation End products (RAGE) is upregulated in hypoxic conditions. The goal of this study was to determine the effect of hypoxia on RAGE signaling in two pancreatic cancer cell lines (Panc‐1 and CFPAC). In particular we investigated if hypoxia dependent RAGE upregulation could aftect RAGE signaling and lead to NF‐κB activation. To interrogate RAGE signaling, we first investigated the effect of hypoxia on KRAS upregulation, as well as on ERK and NF‐KB activity levels by Western blot analysis. We use cobalt chloride to create hypoxic‐like conditions by stabilizing HIF‐1α in cells. We confirmed up‐regulation of HIF‐1α and RAGE in cells incubated with cobalt chloride. We also observed increased expression levels of KRAS in hypoxic‐like conditions. Additionally, higher levels of NF‐κB (p65) and of phosphorylated ERK were observed in cells treated with cobalt chloride as compared to untreated cells. In many cell types, RAGE signaling leads to NF‐κB activation resulting in further RAGE upregulation through a feedforward mechanism. Based on these observations, we hypothesize that in hypoxic conditions, stabilization of HIF‐1α leads to RAGE upregulation and signaling, resulting in NF‐κB activation through the activation of KRAS and ERK. We will use a small molecule RAGE inhibitor (FPS‐ZM1) to interrogate the role of RAGE in the proposed KRAS/ERK/NF‐κB pathway.Support or Funding InformationDepartment of Pharmaceutical Sciences, College of Health Professions, NDSU, NIH‐COBRE: P20GM109024

A Triple-Regulated Oncolytic Adenovirus Carrying MicroRNA-143 Exhibits Potent Antitumor Efficacy in Colorectal Cancer

The cancer-targeting gene virotherapy might be a useful strategy for the treatment of cancer, because it could combine the advantages of both gene therapy and virotherapy. This study aimed to construct a triple-regulated oncolytic adenovirus, Ad-RGD-Survivin-ZD55-miR-143, carrying the therapeutic gene miR-143 and evaluate its possible antitumor effect in colorectal cancer. We observed that miR-143 was lowly expressed in patients with colorectal cancer. The upregulation of miR-143 could inhibit cell proliferation and induce cell apoptosis by targeting KRAS in colorectal cancer cells. Then, Ad-RGD-Survivin-ZD55-miR-143 was successfully constructed in this study. Cells infected with Ad-RGD-Survivin-ZD55-miR-143 could inhibit cell proliferation, suppress cell migration and invasion, arrest cells at the G1 phase, and induce cellular apoptosis. At the same time, Ad-RGD-Survivin-ZD55-miR-143 decreased the expression of PARP-1 and KRAS protein in vitro. In a HCT116 xenograft model, intratumoral injection of Ad-RGD-Survivin-ZD55-miR-143 resulted in reduced tumor growth. Furthermore, Ad-RGD-Survivin-ZD55-miR-143 induced apoptosis and decreased the expression level of KRAS in HCT116 xenograft cells. Our results suggested that Ad-RGD-Survivin-ZD55-miR-143 produced a strong antitumor effect by targeting KRAS and that this strategy could broaden the therapeutic options for treating colorectal cancer.

MiR-199a Inhibits Tumor Growth and Attenuates Chemoresistance by Targeting K-RAS via AKT and ERK Signalings.

Glioma is the most malignant brain tumors in the world, the function and molecular mechanism of microRNA-199a (miR-199a) in glioma is not fully understood. Our research aims to investigate miR-199a/K-RAS axis in regulation of glioma tumor growth and chemoresistance. The function of miR-199a in glioma was investigated through in vitro and in vivo assays. We found that miR-199a in tumor tissues of glioma patients was significantly downregulated in this study. Kinase suppressor of ras 1 (K-RAS), was indicated as a direct target of miR-199a, as well as expression levels of K-RAS were inversely correlated with expression levels of miR-199a in human glioma specimens. Forced expression of miR-199a suppressed AKT and ERK activation, decreased HIF-1α and VEGF expression, inhibited cell proliferation and cell migration, forced expression of K-RAS restored the inhibitory effect of miR-199a on cell proliferation and cell migration. Moreover, miR-199a renders tumor cells more sensitive to temozolomide (TMZ) via targeting K-RAS. In vivo experiment validated that miR-199a functioned as a tumor suppressor, inhibited tumor growth by targeting K-RAS and suppressed activation of AKT, ERK and HIF-1α expression. Taken together, these findings indicated that miR-199a inhibits tumor growth and chemoresistance by regulating K-RAS, and the miR-199a/K-RAS axis is a potential therapeutic target for clinical intervention in glioma.

Inhibition effects of Yuxiao San combined with cisplatin on transplanted tumor growths via upregulation of nm-23 and downregulation of K-ras in Lewis lung cancer mice.

Traditional Chinese medicine applied to cancer therapy has a long history. This study aimed to investigate the effect and underlying mechanism of the traditional Chinese medicine, Yuxiao San, in combination with cisplatin, on Lewis lung cancer mice. Lewis lung cancer mouse models were were established in mice, and were randomly divided into model (normal saline + egg white), cisplatin (cisplatin injection + egg white), traditional Chinese medicine (Yuxiao San + egg white) and combined medication (Yuxiao San + cisplatin injection + egg white) groups. The weight and tumor size of mice was measured every 3 days, and the number of lung surface metastasis nodules were calculated after 14 days. Nucleoside diphosphate kinase (nm-23) and K-ras expression levels in tumor tissues were determined by immunohistochemical methods and reverse transcription- quantitative polymerase chain reaction. The results suggested that compared with the model group, cisplatin, traditional Chinese medicine and combined medication groups significantly inhibited growth of transplanted tumors, reduced the number of lung metastasis nodules, increased the inhibition rate of lung metastasis (P<0.05 or P<0.01), significantly increased tumor suppressor gene nm-23 expression and significantly downregulated oncogene K-ras expression. Among all groups, the effect of the combined medication group for inhibiting tumor growth was most favorable (P<0.01). Compared with the cisplatin group, nm-23 expression in the traditional Chinese medicine group and the combined medication group was significantly increased, and K-ras expression in the traditional Chinese medicine and combined medication groups was significantly reduced (P<0.01). To conclude, Yuxiao San and cisplatin injection effectively inhibited growth of the transplanted tumor. The underlying mechanism may be associate with nm-23 upregulation and K-ras downregulation. Therefore, the Yuxiao San and cisplatin injection may become a potential therapeutic strategy for tumor treatment.