shRNA Plasmid Research Articles

Role and mechanism of FOXG1 in invasion and metastasis of colorectal cancer

This study was aimed to investigate the effect of Forkhead Box G1 (FOXG1) on the epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells and the underlying mechanism. For this purpose, FOXG1 lentiviral interference (shRNA) plasmid and expression plasmid were constructed. Western blotting was used to analyze the expression of FOXG1 protein in five CRC cells, namely RKO, SW480, SW620, LoVo and DLD-1. The shRNA fragment of FOXG1 (shFOXG1) was designed and synthesized. Recombinant plasmids were obtained with the aid of DNA recombination technique. Double digestion and sequencing were used to identify the recombinant plasmids, and then lentivirus packaging, purification and stable transfection were carried out. Additionally, stable CRC cell lines were screened out. The changes of FOXG1 knockdown and overexpression efficiency, E-cadherin, Vimentin, Fibronectin, Snail, Twist mRNA and protein were investigated respectively by Western blotting and qRT-PCR analysis. Furthermore, the changes of cell morphology after knockdown and cell migration ability were evaluated respectively with optical microscopy, scratch test and Transwell assay. FOXG1 had the highest protein expression in RKO and the lowest in DLD-1 among the five CRC cells. Compared with those of the control group, the cell morphology in FOXG1 knockdown RKO group was changed from spindle into round or polygonal shape, cell polarization was enhanced and tight junction assembly was acclerated while cell migration distance was noticeably decreased. Moreover, the number of cells invaded and migrated through chambers was significantly reduced. Among these key factors of EMT, the expression of E-cadherin was increased while the expressions of Vimentin, Fibronectin, Snail and Twist were decreased. The opposite was the case in the overexpressed FOXG1 group. The overexpression of FOXG1 in CRC promoted the invasion and metastasis of CRC cells and played a crucial role in regulating the EMT. Thus, FOXG1 might be a novel therapeutic target in CRC treatment.

Pro-apoptotic effects of micro-ribonucleic acid-365 on retinal neurons by targeting insulin-like growth factor-1 in diabetic rats: An in vivo and in vitro study.

Aims/ObjectiveThe present study aimed to explore the effects of micro‐ribonucleic acid‐365 (miR‐365) on apoptosis of retinal neurons by targeting insulin‐like growth factor‐1 (IGF‐1) in diabetes mellitus rats.Materials and MethodsHigh glucose‐induced retinal neurons were assigned into the blank (with no plasmid transfection), negative control (with plasmid transfection), anti‐miR‐365 (transfected miR‐365 antagomir), transfected IGF‐1 short hairpin RNA plasmid (sh‐IGF‐1) and transfected miR‐365 antagomir and IGF‐1 shRNA plasmid (anti‐miR‐365 + sh‐IGF‐1) groups. Proliferation and apoptosis of retinal neurons were detected by 5‐ethynyl‐2′‐deoxyuridine assay and Hoechst 33342 staining, respectively. Expressions of miR‐365, IGF‐1, Bcl‐2‐associated X protein (Bax) and Bcl‐2 were determined by reverse transcription quantitative polymerase chain reaction and western blotting. A control group contained 10 healthy rats. Terminal deoxynucleotidyl transferase dUTP nick‐end labeling staining was used to evaluate apoptosis of retinal neurons in rats.ResultsIn the anti‐miR‐365 group, the apoptosis rate and Bax expression were reduced in comparison with the negative control and blank groups, whereas the sh‐IGF‐1 and anti‐miR‐365 + sh‐IGF‐1 groups presented an opposite trend. Compared with the normal group, expressions of miR‐365 and Bax were increased, and expressions of IGF‐1 and Bcl‐2 were decreased, with more apoptotic cells in diabetes mellitus rat models. The sh‐IGF‐1 group had lower Bax expression, and higher expressions of IGF‐1 and Bcl‐2 with fewer apoptotic cells. Additionally, Bax expression was upregulated, expressions of IGF‐1 and Bcl‐2 were downregulated, and apoptotic cells were higher in the anti‐miR‐365 + sh‐IGF‐1 groups than the anti‐miR‐365 group.ConclusionThe results of the present study suggest that suppressed miR‐365 increases the IGF‐1 expression, leading to anti‐apoptotic effects on retinal neurons in diabetic rats.

Silencing TRPC6 Gene Attenuates Hypertension‐induced Renal Injury in 5/6 Ablation/Infarction Model

The transient receptor potential canonical 6 (TRPC6) channel is a member of the TRPC family. A “gain of function” mutation of TRPC6 causes familial focal segmental glomerulosclerosis (FSGS) and that overexpression of TRPC6 in podocytes causes a kidney disease consistent with FSGS in animals. Moreover, TRPC6 participates in the mechanical or pressure sensation in various cell types, including in the podocyte slit diaphragm. Therefore, TRPC6 may be the upstream mediator that transmits the pressure stress into downstream molecular pathways to cause hypertension‐induced renal injury. The present study tested the hypothesis that up‐regulated TRPC6 in the kidney mediates the hypertension‐induced renal injury in rats with 5/6 renal ablation/infarction (A/I), a model in which the renal injury is closely associated with blood pressure. Adults male Sprague Dawley rats were divided into shame group, 5/6 A/I control group and 5/6 A/I shRNA group. The left kidneys of shame and 5/6 A/I control groups were treated with intra‐renal transfection of control plasmids and the 5/6 A/I shRNA group TRPC6 shRNA plasmids. The results showed that the levels of renal TRPC6 was increased in 5/6 A/I control group compared with shame group and that the increase of TRPC6 was blocked in 5/6 A/I shRNA groups, the relative protein levels of TRPC6 were 1.0±0.26, 3.9±0.16 and 1.4±0.17 in shame, 5/6 A/I control and 5/6 A/I shRNA groups, respectively. TRPC6 shRNA had no effect on hypertension in 5/6 A/I rats (MAP: 153.9 ± 8.1 mmHg in control groups vs. 154.7 ± 8.9 mmHg in shRNA groups). However, the albuminuria was attenuated in 5/6 A/I shRNA group compared with 5/6 A/I control group. The levels of urinary albumin were 0.8±0.34 mg/Kg/24h in shame, 20.82± 3.7 mg/Kg/24h in 5/6 A/I control and 11.9±1.5 mg/Kg/24h in 5/6 A/I shRNA group, respectively (P=0.037 for control vs. shRNA groups). The regression slop between the blood pressure and the % of glomerulosclerosis was significantly steeper in 5/6 A/I control group than in 5/6 A/I shRNA group (P=0.028 by ANCOVA), indicating a significant attenuation of hypertension‐induced glomerular injuries. These data suggested that hypertension‐induced activation of TRPC6 may be the molecular sensor of pressure stress to produce kidney damage associated with hypertension. (Supported by NIH grants DK107991 to NL and DK54927 to PL).Support or Funding InformationNIH grants DK107991 to NL and DK54927 to PLThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decreased PECAM1-mediated TGF-β1 expression in the mid-secretory endometrium in women with recurrent implantation failure.

Is recurrent implantation failure (RIF) associated with decreased expression of platelet and endothelial cell adhesion molecule 1 (PECAM1) and transforming growth factor β1 (TGF-β1) in the endometrium during the implantation window? The present study demonstrates that the expression of PECAM1 and TGF-β1 is significantly decreased in the mid-secretory endometrium in women with RIF, which may account for embryo implantation failure. RIF has become a bottleneck issue that hampers the improvement of pregnancy rates in IVF-embryo transfer (IVF-ET). The causes of RIF are complex and may involve the dysregulation of various growth factors, metabolites, and inflammatory cytokines. At present, the precise pathogenesis of RIF has not been elucidated. This was a prospective case-control study. Endometrial tissue samples were obtained from January 2014 to December 2016 from two groups of women who had undergone IVF (RIF group, 22 women who underwent ≥3 ETs including a total of ≥4 good-quality embryos without pregnancy, control group, 18 women who conceived in their first treatment cycle). At the same time, samples were obtained from 18 women with infertility secondary to tubal factor in the early proliferative, late proliferative and mid-secretory phases of the menstrual cycle (n = 6 per group). Samples used for isolation of primary human endometrial epithelial cells and stromal cells (HEECs and HESCs) were collected in December 2017 from six women with infertility secondary to tubal factor. We investigated gene expression using integrative whole genome expression microarray analysis, including differentially expressed gene screening, principal component analysis, and functional enrichment analysis. RT-qPCR, western blotting, immunohistochemistry, immunofluorescence co-localization analysis and short hairpin RNA (shRNA) plasmid transfection in Ishikawa cell line, HEECs and HESCs were used to investigate the expression of PECAM1 and TGF-β1. Integrative data mining of whole-genome expression profiles identified cell adhesion as a key regulator in RIF. Database retrieval and literature review screened several novel cell adhesion-related genes that might participate in embryo implantation, which include PECAM1, intercellular adhesion molecule 2 (ICAM2), integrin subunit β2 (ITGB2), selectin P (SELP) and TEK receptor tyrosine kinase (TEK). Among these targets, the mRNA and protein levels of PECAM1 were significantly lower in the RIF group than those in the control group. During the menstrual cycles of women with secondary infertility, the protein expression level of PECAM1 was the lowest in early proliferative phase, slightly increased in late proliferative phase and was the highest in mid-secretory phase. While the expression level of HOXA10, an endometrial receptivity marker, kept at a low level in early proliferative phase and increased in late proliferative phase, then maintained at a high level in the mid-secretory phase. Furthermore, TGF-β1, mediated by PECAM1, was also decreased significantly in the RIF group. Using shRNA-based approach, we demonstrated that the depletion of PECAM1 significantly decreased the expression of TGF-β1 in Ishikawa cells, as well as in primary HEECs and HESCs. These results indicated that PECAM1 and TGF-β1 might play a pivotal role in modulating endometrial receptivity. Although we have shown that PECAM1 and TGF-β1 were down-regulated in the women with RIF, the molecular mechanism of the effect of the factors on the endometrial receptivity remain unclear. Our findings provide insight into the contribution of PECAM1 and TGF-β1 in regulating implantation, which could be used to develop potential therapeutic methods for RIF. This work was supported by grants from the National Natural Science Foundation of China (Nos. 81771656 and 81370763), Special fund for clinical research of the Chinese Medical Association (No. 16020480664), and the Merck Serono China Research Fund for Fertility Agreement. The authors have no competing interests.

The effects of targeted inhibition of hedgehog signaling pathway on proliferation, apoptosis and metastasis of hepatocellular carcinoma

Objective To observe Hedgehog signaling pathways of liver cancer cell growth and the influence of the metastatic potential targeted inhibit Hedgehog. Methods Construction of Smo shRNA plasmid, The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened after lipofection. The stable and low-expressed Smo-expressing HCC QGY-7701 cell line was screened, The cell cycle, apoptosis, invasion and metastasis of QGY-7701 cells were detected by Western blot, flow cytometry, CCK8 and transwell assay. Subcutaneous implantation of hepatocarcinoma cells in nude mice. Study on the growth and metastasis of hepatocarcinoma cells with low expression of Smo in. The ultrastructural changes of hepatoma cells with low expression of Smo were observed under electron microscope. Results RT-PCR and Western blot showed stable shR-Smo cell line was successfully constructed. Cell cycle test showed that compared with the control group, G0/G1 cells increased in shR-Smo, cells in S phase decreased; apoptosis, CCK8 and Transwell tests showed that Smo-gene silencing could significantly increase the apoptosis percentage of the hepatic cancer cells to (5.46%±1.46%), proliferation activity decreasedand and the migration rates reduced to (7.82%±2.14)%; nude mice model showed that Smo-gene silencing could inhibit the growth of hepatocellular carcinoma cells in vivo, electron microscopy revealed that lysosomes increased significantly in Smo-gene silence cells. Conclusions Blocking Hh signaling pathways , liver cancer cells in vitro malignant degree of decline. Hedgehog in treating liver cancer have hidden meaning. Key words: Hepatocelluar Carcinoma; Signal transduction; Hedgehog signaling pathway

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Loss of PIM1 correlates with progression and prognosis of salivary adenoid cystic carcinoma (SACC)

BackgroundIncreasing evidence indicates that PIM1 is a potential prognostic marker and target for cancer treatment but its precise mechanisms of action remain to be determined in salivary adenoid cystic carcinoma (SACC). This study aims to decipher the prognostic and mechanistic role of PIM1 in progression of SACC cells and tumor tissues.MethodsA SACC cell line (ACC-M) was transfected with shRNA plasmids targeting the PIM1 gene. The expression levels of PIM1, RUNX3 and p21 were measured by quantitative real-time PCR and western blot. Subcellular translocalization of RUNX3 and p21 proteins was assessed using immunofluorescence, and cell cycle phase was quantified using flow cytometry. A total of 97 SACC patients were retrospectively analyzed by clinicopathologic characteristics and survival outcomes.ResultsAfter down-regulation of PIM1 in ACC-M cells, RUNX3 and p21 proteins were translocated from cytoplasm to nucleus, with a decrease of p21 expression and increase of G0/G1 phase cells. PIM1 and RUNX3 levels show a distinct covariance. PIM1 is associated with T-status, lymph node involvement, nerve invasion, and distant metastasis in SACC tissues. Patients with low PIM1 level had a better outcome than those with higher PIM1 level.ConclusionsPIM1 is multifunctional in ACC-M cells and it serves as a neoteric therapeutic target and potential prognostic marker for SACC patients.

Self-sensibilized polymeric prodrug co-delivering MMP-9 shRNA plasmid for combined treatment of tumors

Polymeric prodrugs are of immense interest as anticancer drug-delivery system owing to their superior drug stability during circulation and satisfactory drug loading capacity. However, they are usually less effective than free drugs due to imperfect degradable characteristics or active sites blockage. A polymeric prodrug (HPAA-MTX) with chemotherapeutic self-sensibilization effect consisting of glutathione (GSH)-triggered hyperbranched poly(amido amine) (HPAA) and methotrexate (MTX) was designed and synthesized in this work. This prodrug not only showed better inhibition effect on the tumor cells proliferation compared with free MTX, but also displayed selective sensibilization to tumor cells rather than normal cells. Meanwhile, HPAA-MTX was also explored as a MMP-9 shRNA plasmid delivery vector due to their rich amino group of HPAA, accompanying with MTX for simultaneous inhibiting tumor cells proliferation and migration. As expected, HPAA-MTX possessed excellent gene delivery capacity with significant down-regulation expression of MMP-9 protein and further inhibition of MCF-7 cells migration. Benefiting from the self-sensibilization effect and MTX/MMP-9 co-delivery strategy, this HPAA-MTX/MMP-9 co-delivery system exhibited significantly improved therapeutic efficacy to breast cancer in a combined manner which was confirmed through in vitro and in vivo assays. The strategy established in this study provided a facile “all-in-one” platform to integrate the drug/gene co-delivery strategy and self-sensibilization effect into one single nanocomposite for potential cancer treatment. Statement of significanceA cationic polymeric prodrug with chemotherapeutic self-sensibilization effect was designed and showed better inhibition effect on tumor cells proliferation compared with its free drug, as well displayed the selective sensibilization effect to tumor cells rather than normal cells. Moreover, the prodrug could also deliver MMP-9 shRNA plasmid for a combined therapy. As expected, the prodrug possessed excellent gene delivery capacity with significant down-regulation expression of MMP-9 protein and further inhibition of MCF-7 cells migration. Benefiting from the self-sensibilization effect and the drug/gene co-delivery strategy, this prodrug exhibited significantly improved therapeutic efficacy to breast cancer in a combined manner.

P62 as a therapeutic target for inhibition of autophagy in prostate cancer.

To test the hypothesis that p62 is an optimal target for autophagy inhibition and Verteporfin, a clinically available drug approved by FDA to treat macular degeneration that inhibits autophagy by targeting p62 protein, can be developed clinically to improve therapy for advanced prostate cancer. Forced expression of p62 in PC-3 cells and normal prostate epithelial cells, RWPE-1 and PZ-HPV7, were carried out by transfection of these cells with pcDNA3.1/p62 or p62 shRNA plasmid. Autophagosomes and autophagic flux were measured by transfection of tandem fluorescence protein mCherry-GFP-LC3 construct. Apoptosis was measured by Annexin V/PI staining. Tumorigenesis was measured by a xenograft tumor growth model. Verteporfin inhibited cell growth and colony formation in PC-3 cells. Verteporfin generated crosslinked p62 oligomers, resulting in inhibition of autophagy and constitutive activation of Nrf2 as well as its target genes, Bcl-2 and TNF-α. In normal prostate epithelial cells, forced expression of p62 caused constitutive Nrf2 activation, development of apoptosis resistance, and Verteporfin treatment exhibited inhibitory effects. Verteporfin treatment also inhibited starvation-induced autophagic flux of these cells. Verteporfin inhibited tumorigenesis of both normal prostate epithelial cells with p62 expression and prostate cancer cells and decreased p62, constitutive Nrf2, and Bcl-xL in xenograft tumor tissues, indicating that p62 can be developed as a drug target against prostate cancer. p62 has a high potential to be developed as a therapeutic target. Verteporfin represents a prototypical agent with therapeutic potential against prostate cancer through inhibition of autophagy by a novel mechanism of p62 inhibition.

Inhibiting trophoblast PAR-1 overexpression suppresses sFlt-1-induced anti-angiogenesis and abnormal vascular remodeling: a possible therapeutic approach for preeclampsia.

Is it possible to improve vascular remodeling by inhibiting the excessive expression of protease-activated receptor 1 (PAR-1) in trophoblast of abnormal placenta? Inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, offering an alternative therapeutic approach for preeclampsia. PAR-1 is high-affinity receptor of thrombin. Thrombin increases sFlt-1 secretion in trophoblast via the activation of PAR-1. It is reported that the expression of both thrombin and PAR-1 expression are increased in placentas of preeclampsia patients compared with normal placentas. Trophoblast cells were transfected with PAR-1 short hairpin RNA (shRNA) or PAR-1 overexpression plasmids in vitro. Tube formation assays and a villus-decidua co-culture system were used to study the effect of PAR-1 inhibition on placental angiogenesis and vascular remodeling, respectively. Placentas from rats with preeclampsia were transfected with PAR-1 shRNA to confirm the effect of inhibiting PAR-1 overexpression in placenta. The trophoblast cell line HTR-8/SVneo was transfected with PAR-1 shRNA or PAR-1 overexpression plasmids. After 48 h, supernatant was collected and the level of sFlt-1 secretion was measured by ELISA. Human umbilical cord epithelial cells and a villus-decidua co-culture system were treated with conditioned media to study the effect of PAR-1 inhibition on tube formation and villi vascular remodeling. A preeclampsia rat model was established by intraperitoneal injection of L-NAME. Plasmids were injected into the placenta of the preeclampsia rats and systolic blood pressure was measured on Days 15 and 19. The effect of different treatments was evaluated by proteinuria, placental weights, fetal weights and fetal numbers in study and control groups. The level of serum sFlt-1 in rats with preeclampsia was also measured. Changes in the placenta microvessels were studied by histopathological staining. PAR-1 shRNA inhibited PAR-1 expression and significantly suppressed sFlt-1 expression in trophoblasts. Soluble Flt-1 level in the supernatant was suppressed by PAR-1 inhibition plasmid transfection and increased by PAR-1 overexpression plasmids (46.93 ± 5.22 vs. 25.21 ± 4.18 vs. 67.84 ± 3.58 ng/ml, P < 0.01). Tube formation assays showed that conditioned media from shPAR-1 transfected cells resulted in an increase in the total number of branching points compared with that of blank controls (P < 0.05). The villus-decidua co-culture system confirmed down-regulation of PAR-1 was conducive to angiogenesis and vascular remodeling. Transfecting placenta with PAR-1 shRNA plasmids improved placental vascular development and ameliorated the symptoms of preeclampsia in rats. After treatment with shRNA, blood pressure was controlled (140.83 ± 1.08 vs. 123.6 ± 1.47 mmHg, P < 0.001) and proteinuria levels were decreased (4.48 ± 0.36 vs. 2.64 ± 0.25 μg/μl, P < 0.01). sFlt-1 protein levels were significantly higher in preeclampsia group than in the control group (1.44 ± 0.33 vs. 2.92 ± 0.85 ng/ml, P < 0.001), but was reduced (0.92 ± 0.06 ng/ml, vs. PE, P < 0.001) in the treatment group. The histopathological changes of the placental microvessels showed that in the preeclampsia group, the number of blood vessels was reduced, while in treatment group, the placental microvasculature was improved (P < 0.001). N/A. Despite our promising results, the evaluation of kidney damage was studied only by proteinuria measurement. Histochemistry of kidney damage will be supplemented in a further study. The data showed that inhibition of trophoblast PAR-1 overexpression may promote placental angiogenesis and vascular remodeling, potentially offering an alternative therapeutic approach for preeclampsia. This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81100442 and 81771605 for Y.Z. and 81179584 for L.Z.) and the Hubei Province Health and Family Planning Scientific Research Project (Grant No. WJ2017 M093 for Y.Z.). The authors declare that there is no conflict of interest.

Transferrin-functionalized chitosan-graft-poly(l-lysine) dendrons as a high-efficiency gene delivery carrier for nasopharyngeal carcinoma therapy

Transferrin-functionalized chitosan-graft-poly(l-lysine) dendrons (CS-PLLD-Tf) were synthesized in this work and then used to deliver the MMP-9 shRNA plasmid to HNE-1 cells for gene therapy. The obtained CS-PLLD-Tf displayed excellent gene transfection in vitro, verifying the strategy of constructing a high-efficiency gene carrier by conjugating the multiple low generation PLLDs to one molecule of CS to form a copolymer. Particularly, CS-PLLD-Tf showed much higher gene transfection efficiency than PEI-25k and CS-PLLD in the presence of serum, and more than 40% HNE-1 cells were transfected. This result led to an obvious decrease in MMP-9 protein expression and the apoptosis of HNE-1 cells as well as inhibition of the growth and invasion of HNE-1 tumors. The in vivo anti-tumor assay showed that CS-PLLD-Tf delivered the MMP-9 plasmid effectively and the tumor volume decreased more than 77% compared with the PBS control group. Moreover, CS-PLLD-Tf displayed higher bioavailability and good biocompatibility such as lower cytotoxicity, excellent blood compatibility and non-toxicity to organs, suggesting a potential application in gene therapy of tumors.

Transcriptional control of the MUC16 promoter facilitates follicle-stimulating hormone peptide-conjugated shRNA nanoparticle-mediated inhibition of ovarian carcinoma in vivo

Ovarian cancer is the leading cause of cancer death among gynecological malignancies. The high mortality rate has not been significantly reduced despite advances in surgery and chemotherapy. Gene therapy shows therapeutic potential, but several key issues must be resolved before clinical application. To minimize toxicity in noncancerous tissues, tumor-specific ligands are conjugated to vectors to increase the selectivity of drug delivery. The expression pattern of follicle-stimulating hormone (FSH) receptor in normal and cancer tissues provides an opportunity for highly selective drug delivery in ovarian cancer. Furthermore, tumor-specific promoters can conditionally regulate therapeutic gene expression in tumor or normal tissues. The mucin 16 (MUC16) promoter might be a potential tool to drive ovarian cancer-localized gene expression since MUC16/CA125 is overexpressed in most ovarian carcinomas. Here, we screened the possible MUC16 promoter sequences and constructed MUC16 promoter-driven gro-α shRNA plasmid vectors. The vectors were specifically delivered into ovarian cancer cells via FSH peptide-conjugated nanoparticles. The predicted promoter sequence with TAAA repeats showed high transcriptional activity. The nanoparticle complex containing MUC16 promoter-driven gro-α shRNA and FSH peptides had the ability to decrease gro-α protein secretion in ovarian cancer cells and block tumor growth without obvious toxic effects in a nude mouse model bearing ovarian cancer. Our study provides a novel gene delivery system using a MUC16 promoter trigger and FSH peptide-mediated active targeting in ovarian cancer, and this system may be a promising strategy for specific genetic therapeutic delivery.

NADPH oxidase 4 promotes cisplatin-induced acute kidney injury via ROS-mediated programmed cell death and inflammation

The goal of this study was to elucidate the functional role of Nox4 during acute kidney injury (AKI). NADPH oxidases are a major source of reactive oxygen species (ROS) in the kidney in normal and pathological conditions. Among NADPH oxidase isoforms, NADPH oxidase4 (Nox4) is highly expressed in the kidney and has an important role in kidney diseases, such as diabetic nephropathy and renal carcinoma. We previously found that Nox4 expression significantly increased in the toxic AKI model. However, its functional role and mechanism of action in AKI are still unknown. We scavenged ROS with apocynin in vitro and in vivo and found it attenuated cisplatin-triggered renal function decline. It also alleviated programmed cell death and renal inflammation, indicating a critical role for ROS in mediating AKI. Nox4 protein and mRNA levels were substantially upregulated by cisplatin in vivo and in vitro. Nox4 knockdown alleviated cisplatin-induced cell death and inflammatory response, while Nox4 overexpression aggravated them. Moreover, N-acetyl-L-cysteine (NAC)-mediated inhibition of ROS suppressed cell injury led by Nox4 overexpression, indicating Nox4-mediated ROS generation may be the key mediator in cisplatin-induced nephrotoxicity. Mechanistically, excessive expression of Nox4 induced programmed cell death, especially RIP-mediated necroptosis. Finally, we tested whether Nox4 is a potential therapeutic target using an AKI mouse model by injecting a lentivirus-packaged Nox4 shRNA plasmid through tail vein. Disruption of Nox4 led to renal function recovery, kidney damage relief and reduced inflammation. We conclude that Nox4 aggravates cisplatin-induced nephrotoxicity by promoting ROS-mediated programmed cell death and inflammation. Thus Nox4 may serve as a potential therapeutic target in the treatment of AKI.

Prognostic significance of long non-coding RNA MALAT1 for predicting the recurrence and metastasis of gallbladder cancer and its effect on cell proliferation, migration, invasion, and apoptosis.

The objective of this study is to explore the role of MALAT1 as a molecular indicator in predicting the recurrence, metastasis, and prognosis of gallbladder cancer (GBC) and its effect on the proliferation, invasion, migration, and apoptosis of GBC cells in vitro. GBC tissues and adjacent normal tissues were collected from 102 patients. MALAT1 short hairpin RNA (shRNA) plasmids were first constructed to transfect the GBC-SD cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was applied to detect MALAT1 expression. CCK-8 assay, flow cytometry, and Transwell assay were applied to testify the cell proliferation, cell cycle, apoptosis, invasion, and migration. A receiver operating characteristic (ROC) curve was used to evaluate the values of MALAT1 in GBC recurrence, metastasis, and prognosis. COX regression analysis was applied to analyze the independent influencing factors of GBC patients' survival status. ROC curve results showed that the MALAT1 expression could be a predictor of the GBC recurrence, metastasis, and prognosis. According to the COX regression analysis, MALAT1 expression, tumor size, and TNM stage were independent influencing factors of GBC patients' survival condition. Compared with the GBC-SD cells transfected with empty plasmids, those transfected with MALAT1 shRNA plasmids showed higher apoptosis rates, weakened proliferation, migration, and invasion. In conclusion, our findings demonstrate that lncRNA MALAT1 can be considered as an indicator for evaluating the recurrence, metastasis, and prognosis of GBC patients. We also demonstrate how the overexpression of MALAT1 confers an oncogenic function in GBC.

CD40 Knocked Down Tolerogenic Dendritic Cells Decrease Diabetic Injury.

Type-1 diabetes (T1D) is an autoimmune disease in which T lymphocytes destroy insulin-producing β-cells. Control of self-reactive T lymphocytes and recovery of diabetic injury is the end point of T1D. To investigate generation of tolerogenic dendritic cells (tolDCs) as an innovative method of diabetes therapy. Lentivirus vector production was achieved by GIPZ mouse CD40 shRNA, psPAX2 and pMD2G plasmids DNA. Purified bone marrow derived DCs were treated with CD40 shRNA, and expression of CD40 and mRNA level were evaluated by flow cytometry and Real-Time PCR, respectively. CD40 knock-down DCs were injected into STZ-induced diabetic mice. Blood glucose; glucose tolerance test and weight were analyzed in different groups. Mice treated with CD40 shRNA transfected DCs showed considerable differences in blood glucose, glucose tolerance, and weight compared to other groups. Also cytokine assays indicated an increase in IL-13 production in the CD40 shRNA group. In streptozotocin-induced diabetic mice model, administration of tolerogenic dendritic cells could improve diabetic parameters.

Heat shock protein 47 effects on hepatic stellate cell-associated receptors in hepatic fibrosis of Schistosoma japonicum-infected mice.

The study aimed to explore the regulation of heat shock protein 47 (HSP47) on expressions of receptors associated with hepatic stellate cell (HSC) in liver fibrosis mouse models induced by Schistosoma japonicum (S. japonicum). Mouse fibroblasts (NIH/3T3) were transfected with HSP47 shRNA plasmid by lipofectamine transfection, and experimental fibrosis in HSCs was studied in S. japonicum mouse models treated with HSP47 shRNA in vivo. HSP47 expression was assessed using Western blot and real-time PCR. Flow cytometry was adopted to determine the expression of cell membrane receptors. HSP47-shRNA could markedly down-regulate the expression of collagen (Col1a1 and Col3a1). The expressions of HSP47, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) significantly increased in the liver tissue of infected mice. However, the expressions of ETAR and HSP47 and ETBR remarkably decreased after the administration of HSP47 shRNA in vitro and in vivo. ETAR and ETBR levels were found to be positively correlated with HSP47 expression. HSP47 might exert influence on liver fibrosis via the regulation of ETAR and ETBR.

Wnt4 overexpression promotes thymoma development through a JNK-mediated planar cell polarity-like pathway.

Thymoma is the most common neoplasm of the anterosuperior mediastinum. Activation of the Wnt signaling pathway has a role in a variety of human cancers. The present objective was to examine c-Jun N-terminal kinase (JNK) mRNA and protein expression in thymoma cells undergoing apoptosis subsequent to downregulation of Wnt4. Wnt4 and JNK mRNA and protein expression was analyzed by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry, respectively, in 15 thymoma tissues and 6 thymus cyst tissues. Thymoma cells were cultured and transfected with shRNA plasmids targeting the Wnt4 gene. Wnt4 and JNK protein expression was detected by western blot analysis. Apoptosis was analyzed using Wright-Giemsa staining, Hoechst-33342/propidium iodine double staining and flow cytometry. The results showed that Wnt4 and JNK mRNA and protein expression were significantly increased in thymoma compared with normal thymus tissue. Subsequent to transfection, thymoma Wnt4 and JNK mRNA and protein expression were significantly decreased in shRNA-treated groups, with the strongest inhibition being 52.37%. Characteristic apoptotic morphological changes were observed and apoptosis increased. Overall, the present concluded that Wnt4 has an important role in thymoma development, which appears to be activated through a JNK mediated planar cell polarity-like pathway.

Aptamer-targeted delivery of Bcl-xL shRNA using alkyl modified PAMAM dendrimers into lung cancer cells.

RNAi-based gene therapy has been recently considered as a promising approach against cancer. Targeted delivery of drug, gene or therapeutic RNAi-based systems to tumor cells is one of the important issues in order to reduce side effects on normal cells. Several strategies have been developed to improve the safety and selectivity of cancer treatments including antibodies, peptides and recently aptamers with various attractive characteristics including higher target specificity, affinity and reduced toxicity. Here we described a novel targeted delivery platform comprising modified PAMAM with 10-bromodecanoic acid (10C) and 10C-PEG for improvement of transfection efficiency, AS1411 aptamer for targeting nucleolin ligand on target cancer cells and shRNA plasmid for specific knockdown of Bcl-xL protein. Modified vector could significantly improve the transfection efficiency even after covalent or non-covalent aptamer binding compared to the non-targeted vector in A549 cells. The results of gene silencing and apoptosis assay indicated that our targeted shRNA delivery system could efficiently down-regulate the Bcl-xL expression up to 25% and induce 14% late apoptosis in target cancer cells with strong cell selectivity. This study proposed a novel targeted non-viral system for shRNA-mediated gene-silencing in cancer cells.

In vitro gene silencing effect of chitosan/shRNA PDGF-D nanoparticles in breast cancer

Breast cancer is the most common cancer worldwide in women and it is highly malignant and fatal. PDGF-D plays role in regulation of many cellular processes such as angiogenesis. PDGF-D is overexpressed in many types of cancers and promote tumor growth and metastasis. Silencing of PDGF-D gene by using shRNA with an appropriate carrier system may decrease tumor growth and metastasis. In our study, we prepared chitosan nanoparticles loaded with five different shRNA plasmids targeting different exons of PDGF-D gene. Then, nanoparticles were characterized in vitro and transfection efficiency of these nanoparticles were investigated in breast cancer cell lines (MCF- 7, MDA-MB-231 and MDA-MB-435). The effects of single and multiple shRNA sequences, molecular weight of chitosan (150 kDa and 400 kDa) and the amount of shRNA (100 and 500 μg) on the characterization and transfection efficiencies of nanoparticles have been studied. Size of nanoparticles changed between 200-400 nm and approximately 95-100% encapsulation efficiency were obtained. Release of shRNA changed with the molecular weight of chitosan. It was obtained that formulation containing shRNA plasmid targeting PDGF-D exon 6 (NP1) has the highest silencing efficiency in MDA-MB-231 cell line. It was also evaluated that chitosan can be a suitable gene delivery system for shRNA targeting PDGF-D.

Involvement of Bmi-1 gene in the development of gastrointestinal stromal tumor by regulating p16Ink4A/p14ARF gene expressions: An in vivo and in vitro study

ObjectiveThis study was conducted in order to explore the role that Bmi-1 plays during the development of a gastrointestinal stromal tumor (GIST) by regulation of the p16Ink4A and p14ARF expressions. MethodsEighty-six patients diagnosed with GIST were selected to take part in this experiment. The Bmi-1 protein expressions in GIST and adjacent normal tissues were detected using immunohistochemistry and further analyzed by using photodensitometry. To monitor and track the progression of the GIST, a 3-year follow-up was conducted for all affected patients. After cell transfection, the GIST cells were assigned into the control group (without transfection), the negative control (NC) group (transfected with Bmi-1-Scramble plasmid), and the Bmi-1 shRNA group (transfected with the pcDNA3.1-Bmi-1 shRNA plasmid). Protein and mRNA expressions collected from Bmi-1, p16lnk4A, P14ARF, cyclin D1, and CDK4 were measured using both the RT-qPCR and western blotting methods Cell senescence was assessed and obtained by using the β-Galactosidase (β-Gal) activity assay. The use of a Soft agar colony formation assay and CCK-8 assay were performed in order to detect the cell growth and subsequent proliferation. Cell invasion and migration were analyzed using the Transwell assay and scratch test. ResultsBmi-1 in the GIST tissues was found to be significantly higher and the p16lnk4A and P14ARF expressions were lower than those in the adjacent normal tissues. Bmi-1 was negatively correlated with p16lnk4A and P14ARF expressions according to the correlation analysis. Bmi-1 expression was associated with the TNM stage, postoperative recurrence, metastasis, tumor size, and the 5-year survival rate. Area under ROC curve was calculated at 0.884, and sensitivity, specificity, and accuracy of Bmi-1 predicting the GIST were 67.44%, 97.67%, and 65.12%, respectively. Patients exhibiting a high Bmi-1 expression in the GIST tissues had lower survival rates than those with low Bmi-1 expression. In comparison with the control group, P14ARF, and p16lnk4A were up-regulated, while cyclinD 1 and CDK4 were down-regulated, cell senescence was promoted, and cell proliferation, invasion, and migration also showed some regression in the Bmi-1 shRNA group. ConclusionsThese collection of data indicated that the down-regulated Bmi-1 might inhibit the proliferation, invasion, and migration of GIST cells and can be subsequently linked to the incidence and developing a prognosis of GIST.