Epidermal Growth Factor Receptor siRNA Research Articles

Retraction for: Multiwalled carbon nanotubes co-delivering sorafenib and epidermal growth factor receptor siRNA enhanced tumor-suppressing effect on liver cancer

Retraction for: Multiwalled carbon nanotubes co-delivering sorafenib and epidermal growth factor receptor siRNA enhanced tumor-suppressing effect on liver cancer

Dexmedetomidine-Induced Aortic Contraction Involves Transactivation of the Epidermal Growth Factor Receptor in Rats.

In this study, we examined whether aortic contraction, induced by the alpha-2 adrenoceptor agonist dexmedetomidine, is involved in the transactivation of the epidermal growth factor receptor (EGFR) in isolated endothelium-denuded rat aortas. Additionally, we aimed to elucidate the associated underlying cellular mechanisms. The effects of the alpha-2 adrenoceptor inhibitor rauwolscine, EGFR tyrosine kinase inhibitor AG1478, Src kinase inhibitors PP1 and PP2, and matrix metalloproteinase inhibitor GM6001 on EGFR tyrosine phosphorylation and c-Jun NH2-terminal kinase (JNK) phosphorylation induced by dexmedetomidine in rat aortic smooth muscles were examined. In addition, the effects of these inhibitors on dexmedetomidine-induced contraction in isolated endothelium-denuded rat aorta were examined. Dexmedetomidine-induced contraction was inhibited by the alpha-1 adrenoceptor inhibitor prazosin, rauwolscine, AG1478, PP1, PP2, and GM6001 alone or by a combined treatment with prazosin and AG1478. AG1478 (3 × 10−6 M) inhibited dexmedetomidine-induced contraction in isolated endothelium-denuded rat aortas pretreated with rauwolscine. Dexmedetomidine-induced EGFR tyrosine and JNK phosphorylation were inhibited by rauwolscine, PP1, PP2, GM6001, and AG1478. Furthermore, dexmedetomidine-induced JNK phosphorylation reduced upon EGFR siRNA treatment. Therefore, these results suggested that the transactivation of EGFR associated with dexmedetomidine-induced contraction, mediated by the alpha-2 adrenoceptor, Src kinase, and matrix metalloproteinase, caused JNK phosphorylation and increased calcium levels.

Transforming Growth Factor-α Acts in Hypothalamic Paraventricular Nucleus to Upregulate ERK1/2 Signaling and Expression of Sympathoexcitatory Mediators in Heart Failure Rats

Activation of epidermal growth factor receptor (EGFR) tyrosine kinase is associated with increased extracellular signal-regulated kinase (ERK) 1/2 signaling in the hypothalamic paraventricular nucleus (PVN), which contributes to the sympathetic excitation in heart failure (HF). Transforming growth factor (TGF)-α is a major endogenous ligand for EGFR. The present study sought to determine whether TGF-α increases in the PVN in HF and promotes the activation of EGFR to increase ERK1/2 activity. Male rats received bilateral PVN microinjections of an EGFR siRNA or a scrambled siRNA followed by an intracerebroventricular (ICV) injection of TGF-α or vehicle one week later. In rats pretreated with the scrambled siRNA, ICV TGF-α increased phosphorylated (p-) EGFR and upregulated the expression of p-ERK1/2 and mRNA levels of proinflammatory cytokines (PICs) and renin-angiotensin system (RAS) components in the PVN, when compared with the untreated age-matched control rats. These responses to ICV TGF-α were significantly attenuated in rats pretreated with the EGFR siRNA. Furthermore, bilateral PVN microinjection of a TGF-α siRNA in HF rats significantly decreased the elevated levels of TGF-α, p-EGFR, p-ERK1/2 and the mRNA expression of PICs and RAS components in the PVN, compared with the HF rats treated with a scrambled siRNA. The TGF-α siRNA-treated HF rats also exhibited lower plasma norepinephrine levels and improved peripheral manifestations of HF. These data suggest that TGF-α expression is upregulated in the PVN in HF and induces the activation of EGFR-mediated ERK1/2 signaling to augment the inflammation and RAS activity that drives sympathetic excitation in HF.

Epidermal growth factor receptors siRNA-conjugated collagen modified gold nanoparticles for targeted imaging and therapy of lung cancer

Epidermal growth factor receptor (EGFR), a critical factor promotes lung cancer cell proliferation and survival. Knockdown of EGFR expression thus promise beating lung cancer clinically. Functionalized gold nanoparticles may serve an effective vehicle carrying theranostic bio-active materials. Herein, physically gold nanoparticles were fabricated with biocompatible collagen to improve siRNA loading capacity carrying EGFR siRNA to treat lung cancer. Physic-chemical properties were comprehensively characterized for the collagen gold nanoparticle (C–Au), and with EGFR siRNA conjugation, C–Au-EGFRsi namely. Issues of biocompatibility were addressed. Interestingly, C–Au appeared more biocompatible to normal airway epithelial cells (BEAS-2B) than to cancer cells (A549) in terms of ROS production, cell cycle behavior, and cell growth influence. The C–Au demonstrated comparable or even more efficient, compared with lipofetamine, in carrying siRNA to knockdown EGFR of A549 cells. Endocytosis mediated cell entry for the collagen gold nanoparticles, and endosome-lysosomal pathway involved transporting and metabolizing these nanoparticles. In xenograft mice model, substantial tumor suppression effects were observed treating with C–Au-EGFRsi, in which tumor weight reduced 30% for lipofetamine carrier, and down to 70% for C–Au carrier. Particularly, overall survival rate was improved for both treatment groups with lipofetamine and C–Au carrier, respectively.

Simultaneous Quantitative Analysis of Ginsenosides Isolated from the Fruit of Panax ginseng C.A. Meyer and Regulation of HO-1 Expression through EGFR Signaling Has Anti-Inflammatory and Osteogenic Induction Effects in HPDL Cells.

Periodontitis is a set of chronic inflammatory diseases caused by the accumulation of Gram-negative bacteria on teeth, resulting in gingivitis, pocket formation, alveolar bone loss, tissue destruction, and tooth loss. In this study, the contents of ginsenosides isolated from Panax ginseng fruit extract were quantitatively analyzed, and the anti-inflammatory effects were evaluated in human periodontal ligament cells. The major ginsenosides, Re, Ra8, and Rf, present in ginseng fruit were simultaneously analyzed by a validated method using high-performance liquid chromatography with a diode-array detector; Re, Ra8, and Rf content per 1 g of P. ginseng fruit extract was 1.01 ± 0.03, 0.33 ± 0.01, and 0.55 ± 0.04 mg, respectively. Ginsenosides-Re, -Ra8, and -Rf inhibited the production of pro-inflammatory factors and the expression of important cytokines in periodontitis by inducing the expression of heme oxygenase 1 (HO-1), promoting osteoblast differentiation of periodontal ligament cells, suppressing alveolar bone loss, and promoting the expression of osteoblast-specific genes, such as alp, opn, and runx2. An inhibitory effect of these ginsenosides on periodontitis and alveolar bone loss was observed via the regulation of HO-1 and subsequent epidermal growth factor receptor (EGFR) signaling. Silencing EGFR with EGFR siRNA confirmed that the effect of ginsenosides on HO-1 is mediated by EGFR. In conclusion, this study evaluated the contents of ginsenosides-Re, -Ra8, and -Rf isolated from P. ginseng fruit extract. Therefore, these results provide important basic data for future P. ginseng fruit component studies and suggest that ginsenosides Re, Ra8, and Rf have potential as future treatment options for periodontitis.

Multiwalled carbon nanotubes co-delivering sorafenib and epidermal growth factor receptor siRNA enhanced tumor-suppressing effect on liver cancer.

Objective: This study aimed to investigate the effects of multiwalled carbon nanotubes (MWNTs) co-delivering sorafenib (Sor) and epidermal growth factor receptor (EGFR) siRNA (MWNT/Sor/siRNA) on tumor growth in liver cancer (LC).Results: MWNT/Sor/siRNA was proved to possess increased Sor release, high siRNA stability, and enhanced cellular uptake. In addition, MWNT treatment has few effects on cell proliferation and apoptosis in HepG2 cells; however, MWNT/Sor/siRNA treatment significantly inhibited clone number and induced cell apoptosis, which shows a more favorable antitumor effect than MWNT/Sor and free Sor and free siRNA in HepG2 cells. Moreover MWNT/Sor/siRNA treatment has the most significant antitumor effect in vivo.Conclusions: MWNT/Sor/siRNA exhibited a superior antitumor effect in vitro and in vivo.Methods: The MWNT/Sor and MWNT/Sor/siRNA were prepared, and then the morphologies of MWNT/Sor/siRNA were analyzed. In vitro Sor release assay, siRNA stability and cellular uptake of MWNT/Sor/siRNA were performed as well. Next, the effects of MWNT, free Sor, free siRNA, MWNT/Sor and MWNT/Sor/siRNA were evaluated by colony-forming assay, and cell apoptosis assay in HepG2 cells. Meanwhile, the level of EGFR and proteins associated with apoptosis was tested. Furthermore, the anti-tumor effects of MWNT/Sor/siRNA on LC xenograft mice were also unraveled.

MicroRNA-126-5p suppresses cell proliferation, invasion and migration by targeting EGFR in liver cancer

The expression status and potential mechanistic involvement of microRNA (miR)-126-5p in hepatocellular carcinoma (HCC) are still elusive currently. Here we set out to address this issue both in cell lines and in patients' tissues. The relative expression levels of endogenous miR-126-5p and epidermal growth factor receptor (EGFR) were quantified by real-time polymerase chain reaction. Cell viability and proliferation were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays, respectively. Cell invasive and migrative capacities were determined by transwell and wound healing assays, respectively. The regulatory action of miR-126-5p on EGFR was interrogated by luciferase reporter assay. Translational level of EGFR was analyzed by Western blotting. MiR-126-5p was significantly down-regulated in both HCC patients' tissues and cell lines. Forced expression of miR-126-5p greatly compromised cell viability, proliferation, invasion and migration, while miR-126-5p-specific inhibitor promoted these oncogenic phenotypes. MiR-126-5p mimics inhibited endogenous expression of EGFR and suppressed EGFR 3'-untranslated region-fused luciferase activity. Co-expression of EGFR in miR-126-5p-proficient cells completely restored cell migrative and invasive capacities, while co-transfection with EGFR siRNA significantly inhibited miR-126-5p inhibitor-induced cell invasion and migration. MiR-126-5p was aberrantly decreased in HCC and subsequently relieved the suppression on EGFR expression, which consequently contributed to the tumor biology of HCC.

Transforming Growth Factor‐α Acts via Epidermal Growth Factor Receptor to Increase p44/42 Mitogen‐Activated Protein Kinase Signaling and Expression of Excitatory Mediators in the Hypothalamic Paraventricular Nucleus in Rats

Myocardial infarction (MI)‐induced heart failure (HF) in rats leads to activation of brain p44/42 mitogen‐activated protein kinase (MAPK) signaling, which may increase proinflammatory cytokine expression and upregulate renin‐angiotensin system (RAS) activity in the brain, contributing to sympathetic excitation. We recently reported (Hypertension, 2019; 74: AP2010) that activation of the epidermal growth factor receptor (EGFR) in the hypothalamic paraventricular nucleus (PVN), a critical cardiovascular and autonomic center, increases p44/42 MAPK signaling in rats with HF. Both transforming growth factor (TGF)‐α and TGF‐β1 can activate EGFR and p44/42 MAPK signaling in peripheral tissues. Both are expressed in the PVN. In the present study, we first examined whether HF alters TGF‐α and TGF‐β1 expression in the PVN. Male rats underwent coronary artery ligation (CL) to induce HF or sham surgery, and brains were collected 4 weeks after CL. Compared with sham rats, HF rats had significantly (*P<0.05) increased TGF‐α mRNA expression (1.93±0.83* vs 1.03±0.29, fold change) in the PVN, but TGF‐β1 mRNA expression in the PVN was comparable between HF and sham rats (1.42±0.63 vs 1.01±0.20). We next examined whether TGF‐α increases p44/42 MAPK activity in the PVN via EGFR. Male rats received bilateral PVN microinjections of a lentiviral vector encoding a small interfering RNA (siRNA) for EGFR or a scrambled siRNA. One week later, these rats received an intracerebroventricular (ICV) injection of TGF‐α (10 μl, 0.1 μg/μl over 5 min) and the brains were collected 4 hours after the TGF‐α injection. Normal rats without any treatments served as control. Compared with control rats, scrambled siRNA+TGF‐α rats had significantly increases in protein levels of phosphorylated p44/42 MAPK (0.70±0.15* vs 0.23±0.11, corrected by total p44/42 MAPK) and in mRNA expression of the proinflammatory cytokines tumor necrosis factor‐α (5.45±2.10* vs 1.24±0.77, fold change) and interleukin‐1β (2.58±0.33* vs 1.00±0.09, fold change), with a tendency toward an increase in mRNA expression of the RAS component angiotensin II type 1a receptor (1.40±0.29 vs 1.02±0.24, P = 0.08, fold change) in the PVN. All of these findings were ameliorated in the EGFR siRNA+TGF‐α rats (by 21–48%), in which EGFR mRNA expression was reduced by 57%. These results indicate that TGF‐α, an endogenous ligand for EGFR, increases in the PVN in HF and may increase p44/42 MAPK signaling, thereby contributing to the augmented inflammation and RAS activity that drives sympathetic excitation in HF.Support or Funding InformationSupported by NIH grant R01HL136149 and the Department of Veterans Affairs.

Cytoplasmic expression of EGFR shRNA using a modified T7 autogene-based hybrid mRNA/DNA system induces long-term EGFR silencing and prolongs antitumor effects

Unusual activation or overexpression of epidermal growth factor receptor (EGFR) has been found in various cancers, and therefore down-regulation of EGFR expression is recognized as a promising strategy for cancer treatment. For decades, RNAi has emerged as an effective solution to regulate gene overexpression, but transient effects of exogenous siRNA have limited the development of EGFR-targeting siRNA therapeutics. Recently, we developed T7 autogene-based hybrid mRNA/DNA system as a non-viral vector for shRNA production and reported the feasibility of long-term silencing for RFP expression as a concept of proof. To investigate its therapeutic availability in cancer therapy, we here modified the hybrid system to stably express EGFR shRNA and confirmed the antitumor effects. When autocatalytic production of T7pol protein in cytoplasm was combined with pT7-driven cytoplasmic transcription for EGFR shRNA, a single transfection lead to stable EGFR silencing in SKOV3 cells for more than 13 days. Also, liposomal systemic administration at two-week intervals resulted in significant inhibition of tumor growth in both SKOV3-bearing mice and PDX models, contrast to the conventional siRNA approach. Our results show an efficient strategy to overcome the temporary effects of synthetic EGFR siRNA in cancer treatment and ultimately provide a potential candidate as an anticancer drug.

Abstract P2010: Silencing Epidermal Growth Factor Receptor Reduces P44/42 Mitogen-Activated Protein Kinase Signaling in the Hypothalamic Paraventricular Nucleus and Ameliorates Sympathetic Excitation in Rats With Heart Failure After Myocardial Infarction

The p44/42 mitogen-activated protein kinase (MAPK) signaling pathway is activated in the brain in myocardial infarction (MI)-induced heart failure (HF). Activation of brain p44/42 MAPK signaling can induce endoplasmic reticulum (ER) stress, increase proinflammatory cytokine expression and upregulate renin-angiotensin system (RAS) activity in the brain, contributing to sympathetic excitation. Multiple excitatory agonists (e.g., angiotensin II, aldosterone and pro-inflammatory cytokines) activate brain MAPK signaling in HF, but the mechanisms are poorly understood. We tested the involvement of the epidermal growth factor receptor (EGFR), which has recently been shown to mediate MAPK signaling in peripheral cardiovascular tissues. Male rats received bilateral microinjections of a lentiviral vector encoding a small interfering RNA (siRNA) for EGFR, or a scrambled siRNA, into the hypothalamic paraventricular nucleus (PVN), a critical cardiovascular and autonomic center. One week later, some rats were euthanized to confirm the effectiveness of EGFR knockdown in PVN. mRNA for EGFR was significantly (*P<0.05) lower in rats that received the EGFR siRNA (0.54±0.17* vs 1.10±0.20, fold change). Other rats underwent coronary artery ligation (CL) to induce HF. Four weeks later, HF rats treated with siRNA for EGFR, compared with HF rats treated with scrambled siRNA, had lower protein levels of phosphorylated p44/42 MAPK (0.20±0.08* vs 0.34±0.06), lower mRNA levels of the ER stress markers BIP (0.77±0.10* vs 1.00±0.12) and ATF4 (0.71±0.14* vs 1.03±0.25), the inflammatory mediators TNF-α (0.73±0.11* vs 1.01±0.15), IL-1β (0.65±0.14* vs 1.02±0.21) and COX-2 (0.71±0.16* vs 1.00±0.14), and the RAS components ACE (0.68±0.26* vs 1.06±0.37) and AT 1a R (0.70±0.11* vs 1.03±0.28) in the PVN, lower urinary norepinephrine levels (28±9* vs 39±7 ng/ml), and improved peripheral manifestations of HF as indicated by reduced right ventricular/body weight and lung/body weight and left ventricular end-diastolic pressure. These results suggest that EGFR in the PVN activates p44/42 MAPK, as well as RAS, neuroinflammation and ER stress, in MI-induced HF. Brain EGFR may be a novel target for therapeutic intervention in the MI-induced HF syndrome.

Activation of GPR30 with G1 attenuates neuronal apoptosis via src/EGFR/stat3 signaling pathway after subarachnoid hemorrhage in male rats

BackgroundNeuron apoptosis plays a vital role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies showed that the activation of G protein-coupled receptor 30 (GPR30) with GPR30 agonist G1 was anti-apoptotic after experimental trauma brain injury and global cerebral ischemia in male rats or mice. However, the role of GPR30 activation with G1 has not been clarified in SAH. The aim of this study was to investigate the anti-apoptotic effect of GPR30 activation and the underlying mechanism of src/EGFR/stat3 signaling pathway in a male rat model of SAH. MethodsA total of 215 male rats and 18 female rats were used. SAH was induced by intravascular perforation. G1 was administrated intravenously 1 h after SAH. For mechanism study, the GPR30 antagonist G15 or epidermal growth factor receptor (EGFR) antagonist AG1478 was administrated intravenously 1 h before SAH, small interfering ribonucleic acid (siRNA) for GPR30 and EGFR were administered intracerebroventricularly 48 h before SAH. Post-SAH assessments included SAH Grade, neurological deficits, western blot, terminal deoxynucleotidyl transferase dUTP-biotin nick end labeling (TUNEL) staining, Fluoro-Jade C (FJC) staining, Nissl staining and immunofluorescence. ResultsThe expression of endogenous GPR30 in male rats was increased at 3 h and peaked at 24 h after SAH, which mainly co-localized with neurons, but there was no significant increase in intact female rats at 24 h after SAH. The G1 post-treatment significantly reduced the short-term and long-term neurological deficit as well as neuronal apoptosis in male rats, but it did not significantly improve the short-term outcome of intact female rats. Mechanistic studies indicated that G15 or GPR30 siRNA and AG1478 or EGFR siRNA reversed the anti-neuronal apoptosis effects of G1 and its effects on protein expressions of src/EGFR/stat3 signaling pathway. ConclusionG1 reduced EBI through attenuating neuronal apoptosis after SAH in male rats, partly via activating src/EGFR/stat3/signaling pathway. G1 may provide a promising therapeutic strategy for SAH patients.

Effect of Cetuximab and <i>EGFR</i> Small Interfering RNA Combination Treatment in NSCLC Cell Lines with Wild Type <i>EGFR</i> and Use of <i>KRAS</i> as a Possible Biomarker for Treatment Responsiveness

The epidermal growth factor receptor (EGFR) is a therapeutic target for patients with non-small cell lung cancer (NSCLC). Cetuximab is an anti-EGFR monoclonal antibody that inhibits EGFR signaling and proliferation of colorectal cancer and head and neck cancers. Since only few NSCLC patients benefit from cetuximab therapy, we evaluated a novel combination treatment using cetuximab and EGFR small interfering RNA (siRNA) to strongly suppress EGFR signaling and searched for a biomarker in NSCLC cell lines harboring wild-type EGFR. Alterations in EGFR and its downstream genes in five NSCLC cell lines (A549, Lu99, 86-2, Sq19 and Ma10) were assessed through sequencing. The protein expression levels of these molecules were assessed through western blotting. The effect of combination treatment was determined through cell proliferation assay, caspase-3/7 assay, invasion assay, and migration assay. All cell lines were harboring wild-type EGFR, whereas KRAS, PTEN, TP53 and TP53 were mutated in A549 and Lu99; Lu99 and Sq19; Lu99, 86-2, Sq19 and Ma10; and A549, 86-2, and Sq19 cell lines, respectively. PTEN was not expressed in Sq19, and LKB1 was not expressed in both A549 and Sq19. TP53 was not expressed in both A549 and Lu99. The combination of cetuximab and EGFR siRNA significantly suppressed cell proliferation in 86-2, Sq19 and Ma10, which express wild-type KRAS. It induced apoptosis in A549, 86-2 and Ma10 cells, which express wild type PTEN. The combination treatment had no effect either on cell invasion nor migration in all cell lines. EGFR targeted therapy using the combination of cetuximab and EGFR siRNA is effective in NSCLC cell lines harboring wild-type EGFR. Wild-type KRAS may act as a potential biomarker for response to combination treatment by the induction of apoptosis in cells with wild-type PTEN.

Resolvin D1, but not resolvin E1, transactivates the epidermal growth factor receptor to increase intracellular calcium and glycoconjugate secretion in rat and human conjunctival goblet cells

PurposeTo identify interactions of the epidermal growth factor receptor (EGFR) with the pro-resolving mediator receptors for RvD1 and RvE1 to stimulate an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion from cultured human and rat conjunctival goblet cells. MethodsGoblet cells from human and rat conjunctiva were grown in culture using RPMI media. Cultured goblet cells were pre-incubated with inhibitors, and then stimulated with RvD1, RvE1, EGF or the cholinergic agonist carbachol (Cch). Increase in [Ca2+]i was measured using fura-2/AM. Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-1. Western blot analysis was performed with antibodies against AKT and ERK 1/2. ResultsIn cultured human conjunctival goblet cells RvE1 -stimulated an increase in [Ca2+]i. RvD1-, but not the RvE1-, stimulated increase in [Ca2+]i and mucin secretion was blocked by the EGFR inhibitor AG1478 and siRNA for the EGFR. RvD1-, but not RvE1-stimulated an increase in [Ca2+]i that was also inhibited by TAPI-1, an inhibitor of the matrix metalloprotease ADAM 17. Inhibition of the EGFR also blocked RvD1-stimulated increase in AKT activity and both RvD1-and RvE1-stimulated increase in ERK 1/2 activity. Pretreatment with either RvD1 or RvE1 did not block the EGFR-stimulated increase in [Ca2+]i. ConclusionsWe conclude that in cultured rat and human conjunctival goblet cells, RvD1 activates the EGFR, increases [Ca2+]i, activates AKT and ERK1/2 to stimulate mucin secretion. RvE1 does not transactivate the EGFR to increase [Ca2+]I and stimulate mucin secretion, but does interact with the receptor to increase ERK 1/2 activity.

Migfilin promotes migration and invasion in glioma by driving EGFR and MMP-2 signalings: A positive feedback loop regulation

Glioma is the most common type of primary brain tumors in the central nervous system (CNS). Migfilin occurs in human glioma and enhances cellular motility via the epidermal growth factor receptor (EGFR) pathway. However, the underlying molecular mechanism is not fully understood. In this study, we found that Migfilin promoted matrix metalloproteinase-2 (MMP-2) activity, and restrained the expression of tissue inhibitor of metalloproteinase 2 (TIMP2), which is an MMP-2 inhibitor. Functional and structural studies showed that the LIM1 domain of Migfilin was required for Migfilin-mediated TIMP2 expression inhibition and MMP-2 activity, and was also necessary in promoting cell motility. Furthermore, Migfilin-induced EGFR phosphorylation was greatly reduced by MMP-2 inhibitor (GM6001) or siRNA, while Migfilin-induced MMP-2 activation was also blocked by the EGFR inhibitor (AG1478) or siRNA. MMP-2 and EGFR inhibitors and their siRNAs can block Migfilin-induced migration and invasion, respectively. These results demonstrated that EGFR and MMP-2 signalings may form a positive feedback loop to enhance Migfilin-induced migration and invasion. Finally, we detected that the expression of Migfilin, EGFR phosphorylation (Tyr1173) and MMP-2 activity had a positive correlation in the clinical glioma sample. Taken together, these results suggest that Migfilin is a critical regulator in cellular motility by driving the EGFR-MMP-2 feedback loop, and may be considered as a potential therapeutic target in glioma.

Effects of small interfering RNA silencing in epidermal growth factor receptor on proliferation and migration of vascular smooth muscle cells

Objective To investigate the effect of silencing epidermal growth factor receptor (EGFR) by small interfering RNA (siRNA) interference on the proliferation and migration of vascular smooth muscle cells (VSMCs), and the related mechanisms. Methods VSMCs of rats were isolated and cultured. VSMCs were infected with EGFR siRNA lentiviral vectors. The cells were divided into siRNA-EGFR group, siRNA-negative control group and blank control group. The cell proliferation abilities were detected by 5’-bromo-2’-deoxyuridine (5-BrdU) method. The cell migration abilities in different transfection groups were examined by Transwell chamber and scratch assays. The expression levels of EGFR, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 genes in different transfection groups were detected using real-time quantitative polymerase chain reaction (Real-time PCR) technology. The expression levels of EGFR, p-ERK, p-JNK and p-p38 proteins in different transfection groups were detected by Western blotting. Results The number of migration cells in the siRNA-EGFR group was 48.5±6.1, which were significantly lower than the siRNA-negative control group and blank control group, which were 86.2±7.2 and 88.3±7.6; the difference was statistically significant (F=37.261, P=0.000). Compared with 0 h, after 24 h, the migration rate of VSMC in the siRNA-EGFR group was (35.7±3.8)%, which was significantly lower than that of siRNA-EGFR group and blank group, which were (74.2±5.7)% and (75.5±6.1)%; the differences were statistically significant (F=40.315, P=0.000). The relative expression levels of ERK, JNK and p38 mRNA in the siRNA-EGFR group (0.53±0.09, 0.47±0.11, 0.41±0.07) were lower than the siRNA-negative control group (0.82±0.12, 0.64±0.10, 0.68±0.09) and blank control group (0.85±0.14, 0.66±0.13, 0.70±0.12), the differences were statistically significant (F=15.391, 12.817, 11.264, P=0.000, 0.000, 0.000). The relative expression levels of p-ERK, p-JNK and p-p38 proteins in the siRNA-EGFR group (0.46±0.09, 0.38±0.06, 0.33±0.06) were lower than the siRNA-negative control group (0.71±0.12, 0.57±0.08, 0.52±0.07) and blank control group (0.73±0.13, 0.56±0.07, 0.54±0.09), the differences were statistically significant (F=20.152, 14.375, 13.843, P=0.000, 0.000, 0.000). Conclusion Specific inhibition of EGFR could effectively reduce proliferation and migration capacities of VSMCs, which might be related to inhibition of mitogen-activated protein kinase signaling pathway. Key words: Vascular smooth muscle cells; Epidermal growth factor receptor; Small interference; Cell proliferation; Cell migration

Precision knockdown of EGFR gene expression using radio frequency electromagnetic energy.

Electromagnetic fields (EMF) in the radio frequency energy (RFE) range can affect cells at the molecular level. Here we report a technology that can record the specific RFE signal of a given molecule, in this case the siRNA of epidermal growth factor receptor (EGFR). We demonstrate that cells exposed to this EGFR siRNA RFE signal have a 30-70% reduction of EGFR mRNA expression and ~60% reduction in EGFR protein expression vs. control treated cells. Specificity for EGFR siRNA effect was confirmed via RNA microarray and antibody dot blot array. The EGFR siRNA RFE decreased cell viability, as measured by Calcein-AM measures, LDH release and Caspase 3 cleavage, and increased orthotopic xenograft survival. The outcomes of this study demonstrate that an RFE signal can induce a specific siRNA-like effect on cells. This technology opens vast possibilities of targeting a broader range of molecules with applications in medicine, agriculture and other areas.

A tumor-targeting cRGD-EGFR siRNA conjugate and its anti-tumor effect on glioblastoma in vitro and in vivo

The epidermal growth factor receptor (EGFR) is an important anti-tumor target. The development of novel molecular-targeted anti-tumor drugs that can target the interior of tumor cells and specifically silence EGFR expression is valuable and promising. In this work, a promising anti-tumor conjugate comprising methoxy-modified EGFR siRNA and cyclic arginine-glycine-aspartic acid (cRGD) peptides, which selectively bind to αvβ3 integrins, was synthesized and examined. To prepare cRGD-EGFR siRNA (cRGD-siEGFR), cRGD was covalently conjugated to the 5′-end of an siRNA sense strand using a thiol-maleimide linker. The cellular uptake and cytotoxicity of cRGD-siEGFR in vitro were tested using an αvβ3-positive U87MG cell line. In vivo bio-distribution, anti-tumor activity, immunogenicity and toxicity were investigated in a nude mouse tumor model through repeated i.v. administration of cRGD-siEGFR (7 times over a 48 h interval). Analyses of in vitro data showed that cRGD-siEGFR silenced EGFR expression effectively, with high tumor targeting ability. Administration of cRGD-siEGFR to tumor-bearing nude mice led to significant inhibition of tumor growth, obvious reduction of EGFR expression and down-regulation of EGFR mRNA and protein in tumor tissue. Furthermore, serum biochemistry and pathological section evaluation did not indicate any serious toxicity of cRGD-siEGFR in vivo. cRGD-siEGFR is likely a promising candidate with high targeting ability, substantial anti-tumor effects and low toxicity in vitro and in vivo.

Targeted in vivo delivery of EGFR siRNA inhibits ovarian cancer growth and enhances drug sensitivity.

A functionalized nanohydrogel siRNA delivery system and a mouse model of serous ovarian cancer were used to test predictions from previous cell line studies that knockdown of EGFR (epidermal growth factor receptor) may be of clinical significance in the treatment of epithelial tumors especially with respect to the enhancement of platinum based therapies. Our results support these predictions and suggest that targeted delivery of EGFR siRNA may be an effective strategy for the treatment of ovarian and other epithelial tumors associated with elevated levels of EGFR and especially those demonstrating resistance to platinum-based therapies.

Novel cell-penetrating peptide-loaded nanobubbles synergized with ultrasound irradiation enhance EGFR siRNA delivery for triple negative Breast cancer therapy

The lack of safe and effective gene delivery strategies remains a bottleneck for cancer gene therapy. Here, we describe the synthesis, characterization, and application of cell-penetrating peptide (CPP)-loaded nanobubbles (NBs), which are characterized by their safety, strong penetrating power and high gene loading capability for gene delivery. An epidermal growth factor receptor (EGFR)-targeted small interfering RNA (siEGFR) was transfected into triple negative breast cancer (TNBC) cells via prepared CPP-NBs synergized with ultrasound-targeted microbubble destruction (UTMD) technology. Fluorescence microscopy showed that siEGFR and CPP were loaded on the shells of the NBs. The transfection efficiency and cell proliferation levels were evaluated by FACS and MTT assays, respectively. In addition, in vivo experiments showed that the expression of EGFR mRNA and protein could be efficiently downregulated and that the growth of a xenograft tumor derived from TNBC cells could be inhibited. Our results indicate that CPP-NBs carrying siEGFR could potentially be used as a promising non-viral gene vector that can be synergized with UTMD technology for efficient TNBC therapy.

PA-MSHA in combination with EGFR tyrosine kinase inhibitor: A new strategy to overcome the drug resistance of non-small cell lung cancer cells

The inhibition of epidermal growth factor receptor (EGFR) signaling by Gefitinib provides a promising treatment strategy for non-small cell lung cancer (NSCLC); however, drug resistance to Gefitinib and other tyrosine kinase inhibitors presents a major issue. Using NSCLC cell lines with differential EGFR status, we examined the potency of PA-MSHA (Pseudomonas aeruginosa-mannose-sensitive hemagglutinin) in combination with Gefitinib on proliferation, apoptosis, cell cycle arrest, EGFR signaling and tumor growth. PC-9, A549, and NCI-H1975 cells were treated with PA-MSHA, Gefetinib, or PA-MSHA plus Gefetinib at different concentrations and times. The effects of the drugs on proliferation, cell cycle distribution and apoptosis were evaluated. The activation of EGFR and apoptotic signaling-related molecules was evaluated by Western blotting in the presence or absence of EGFR siRNA. Tumor growth and pathway signaling activation was assessed by xenografts in nude mice. A time-dependent and concentration-dependent cytotoxic effect of PA-MSHA was observed in all NSCLC cells tested. The combination of PA-MSHA plus Gefitinib enhanced the growth inhibition, sub-G1 content and apoptosis over that observed with either agent alone. Furthermore, the combination of PA-MSHA plus Gefitinib resulted in caspase-3/caspase-9 cleavage and increased inhibition of EGFR-dependent activation of AKT and ERK phosphorylation. Combination treatment was more effective in reducing tumor size and EGFR activation than either agent alone. These data suggest that PA-MSHA and Gefitinib function additively to suppress the proliferative effects of NSCLC cells of differential EGFR status. The combination of PA-MSHA and Gefitinib provides a potential new strategy to conquer drug resistance for anti-EGFR-targeted therapy of NSCLC.