Mechanism of Abnormal Chondrocyte Proliferation Induced by Piezo1-siRNA Exposed to Mechanical Stretch

Objective. To investigate the effect of small interfering RNA targeting mechanosensitive ion channel protein Piezo1 (Piezo1-siRNA) on abnormal chondrocyte proliferation exposed to mechanical stretch. Methods. Construct and screen effective Piezo1-siRNA sequences and explore an appropriate method to transfect lentiviral vector into chondrocytes exposed to mechanical stretch. Western blot and RT-PCR were used to detect the mRNA and protein expression of Piezo1, Kif18A, and β-tubulin, respectively. Flow cytometry was used to measure the changes in the chondrocyte cycle. The proliferation of chondrocyte was evaluated by cell counting kit-8. Results. According to the mRNA and protein expression of Piezo1, the effective siRNA sequence was successfully screened. Compared with the 0 h group, mechanical stretch upregulated the expression of Piezo1, Kif18A, and β-tubulin, resulting in chondrocyte cycle arrest and eventually inhibiting chondrocyte proliferation. Moreover, Piezo1-siRNA transfection effectively blocks this process and promotes the proliferation of chondrocyte. Conclusion. Piezo1-siRNA can reduce the inhibition of chondrocyte proliferation induced by mechanical stretch via downregulating the expression of Kif18A and inhibiting the depolymerization of microtubules. Piezo1-siRNA plays a protective role in chondrocytes, which provides a potential method for the treatment of OA under abnormal mechanical stimulation.

Inhibition of STAT3 in tubular epithelial cells prevents kidney fibrosis and nephropathy in STZ-induced diabetic mice

Recent evidences indicate that signal transducer and activator of transcription 3 (STAT3) is one of the crucial signaling pathways in the progression of diabetic nephropathy (DN). Here, we investigated the hypothesis that pharmacological blockade of STAT3 limits the progression of DN. Treatment with selective STAT3 inhibitor, S3I-201 for 16 weeks significantly attenuated kidney injuries in streptozotocin (STZ) induced diabetic mice, associated with downregulated expression of TGF-β1, ACE/AT1, and VEGF in diabetic mouse kidneys. Similar results were confirmed using genetic knockdown of STAT3 in mouse kidneys by injections of AAV2 expressing STAT3 shRNA in diabetic mouse. Further, STAT3 localization in kidney tissue was evaluated using immunofluorescent double-staining analysis, which indicated that STAT3 expression was mainly in the tubular epithelial cells. As expected, in renal tubular epithelial NRK-52E cells, high glucose (HG)-induced overexpression of TGF-β1, ACE/AT1, and VEGF were abrogated by S3I-201 pretreatment, as well as by genetic knockdown of STAT3 using specific siRNA sequence. This study found that renal tubular epithelial cells contributed to STAT3-mediated progression of DN and provided the first evidence that pharmacological inhibition of STAT3 attenuates DN.

Adenovirus-mediated small interfering RNA targeting ezrin induces apoptosis and inhibits metastasis of human osteosarcoma MG-63 cells

Osteosarcoma is a disease prone to recurrence and metastasis, and adenovirus expression vector is frequently studied as a therapeutic target of osteosarcoma in recent years. The present study attempts to explore the effect of adenovirus-mediated siRNA targetting ezrin on the proliferation, migration, invasion, and apoptosis of human osteosarcoma MG-63 cells. Human osteosarcoma MG-63 cell line was selected for construction of recombinant adenovirus vector. The mRNA and protein levels of ezrin, Bcl2-associated X protein (Bax), B cell lymphoma-2 (Bcl-2), p21, p53, Caspase-3, matrix metalloproteinase (MMP) 2 (MMP-2) and MMP-9, Cyclin D1, and cyclin-dependent kinase 4a (CDK4a) were determined. Through ELISA, the levels of Caspase-3, MMP-2 and MMP-9 were examined. Finally, human osteosarcoma MG-63 cell viability, growth, invasion, migration, and apoptosis were detected. Initially, adenovirus expression vector of ezrin was constructed by ezrin 2 siRNA sequence. Adenovirus-mediated siRNA targetting ezrin reduced expression of ezrin in MG-63 cells. The results revealed that adenovirus-mediated siRNA targetting ezrin elevated expression levels of Bax, p21, p53, and Caspase-3, Cyclin D1, and CDK4a and reduced expression levels of Bcl-2, MMP-2 and MMP-9. Furthermore, adenovirus-mediated siRNA targetting ezrin inhibited human osteosarcoma MG-63 cell viability, growth, invasion, and migration, and promoted apoptosis. Our study demonstrates that adenovirus-mediated siRNA targetting ezrin can induce apoptosis and inhibit the proliferation, migration, and invasion of human osteosarcoma MG-63 cells.

The DNA-Binding Protein High Mobility Group Box 1 (HMGB1) Interacts with RelB to Maintain Non-Canonical NF-κb Signaling, Cell Survival and Immune Evasion in Infant Acute Lymphoblastic Leukemia

Disordered NF-κB signaling is a hallmark of many hematologic malignancies but has not been well-studied in MLL-rearranged infant lymphoblastic leukemia (iMLL-ALL), a subtype of pediatric ALL with an extremely poor prognosis in need of new therapy. Given the stable genome and short latency of iMLL-ALL, targeting of oncoproteins that play a coordinated role in regulation of the leukemic transcriptome represent ideal targets. Here, we present one such potential target: a complex formed between the nucleosome-stabilizing protein high mobility group box 1 (HMGB1) and RelB, a transcriptionally active subunit of the noncanonical NF-κB pathway. Disruptions in the non-canonical NF-κB pathway, which normally functions to support lymphogenesis, are known to drive lymphoid malignancies. Specifically, changes in proteasomal processing of the p100 subunit modulate nuclear p52/RelB regulation of the NF-κB2 gene. In stable short-term culture, we observed that primary infant MLL-ALL cells (derived from the diagnostic pheresis specimen of a 9 month old with t(4;11)) express an endogenous NF-κB subunit profile consistent with constitutive non-canonical activation, including significantly increased levels of IKKa, RelB, and NF-κB2 by Western Blot and qPCR compared to healthy human B cells and cytogenetically normal ALL. First, we asked whether exogenous "drivers" of the canonical (p65/RelA) pathway could alter this expression profile by treating iMLL-ALL with TNFalpha and bacterial LPS. Exogenous cytokine treatment did not alter expression levels of IKKa, RelB or NF-κB2, nor did it increase p65RelA, likely due to the lack of pattern recognition receptors on precursor lymphoblastic cells. We have previously demonstrated that infant MLL-ALL express high intracellular levels of the nucleosome-stabilizing protein high mobility group box-1 (HMGB1). During infection, HMGB1 forms a transcription regulatory complex with RelB that, by epigenetic means, silences inflammatory cytokine promoters as a negative feedback. In this study, we hypothesized that HMGB1-RelB interactions stabilize and maintain a pro-leukemic NF-κB program. Because the function of HMGB1 is determined by post-translational modifications, we first verified the DNA-binding disulfide form of the protein in the nuclear fraction of iMLL-ALL cells by immunoprecipitation. Next, we proved a physiologically relevant interaction between HMGB1 and RelB by co-immunoprecipitation; both HMGB1 and RelB could be identified by immunoblot from iMLL-ALL extracts probed for either for RelB or HMGB1. Detection of the complex required minimal stabilization, suggesting a durable interaction. Preliminary studies suggest that the complex associates with the acetylated form of histone 3 (H3), rather than histone 1 as in sepsis models. We then determined the effect of HMGB1 siRNA knockdown on the NF-κB subunit expression profile, proliferation and survival of iMLL-ALL, nonleukemic control PBMC and 293T cells. HMGB1 siRNA produced 30-50% spontaneous apoptosis in MLL-ALL, but not nonleukemic control cells, measured by Annexin/PI. iMLL-ALL transfected with a nonsense siRNA sequence did not apoptose. HMGB1 siRNA also silenced expression of RelB and NF-κB 2, measured by Western Blot and qPCR. Unexpectedly, HMGB1 siRNA restored expression of critical canonical NF-κB subunits: p65/RelA, the TNF-associated factor TRAF3, and the proteasome regulator NEMO. Further, HMGB1 siRNA knockdown resulted in a 51.3% (+/- 3.47, p<0.0001) increase in engulfment by activated bone marrow macrophages in a dual-label phagocytosis assay, compared to nonsense siRNA and control iMLL-ALL- suggesting that HMGB1 expression plays a role in evasion of iMLL-ALL by the innate immune system. Taken together, these data indicate that HMGB1-RelB interactions represent an important regulatory component of the NF-κB signaling program in human infant MLL-ALL cells that support survival and leukemogenesis. Current studies are comparing p100 processing and proteasome activity in iMLL-ALL with competent and disrupted HMGB1-RelB interactions, as well as comparing the effectiveness of proteasome inhibition with bortezomib. Near-future studies will screen other high-risk ALL's for HMGB1-Rel activity and elaborate the downstream effects of complex binding on the MLL-ALL transcriptome to establish it as a rational target for drug development. Disclosures No relevant conflicts of interest to declare.

Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence

Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities.

Prediction of siRNA Efficacy Using BP Neural Network and Support Vector Machine

RNA interference (RNAi) is a mechanism for sequence-specific, post-transcriptional down-regulation of gene expression. The success of RNAi gene silencing depends on siRNA feature design. The shortcoming of previously reported methods which design siRNA sequences based on limited rules is that they are difficult to accurately predict the efficacy that a candidate siRNA sequence will silence the target gene. With validated siRNA databases have been developed in recent years, machine learning methods can be applied to predict siRNA accuracy and optimize design. This paper proposed a combined prediction method of BP neural network and support vector machine (SVM) for selecting effective siRNA sequences. With SVM, siRNA sequences were classified into effective or ineffective siRNAs. Subsequently, BP neural network model with great learning ability selected highly effective candidate sequences from effective siRNAs. We applied this method to published siRNAs datasets, and the experimental results confirmed good prediction capability.

RETRACTED ARTICLE: RNAi targeting CXCR4 inhibits proliferation and invasion of esophageal carcinoma cells

CXC chemokine receptor 4 was found to be expressed by many different types of human cancers and its expression has been correlated with tumor aggressiveness, poor prognosis and resistance to chemotherapy. However the effect of CXCR4 on the esophageal carcinoma cells remains unclear, the present study explored the effects of CXCR4 siRNA on proliferation and invasion of esophageal carcinoma KYSE-150 and TE-13 cells. Two siRNA sequence targeting CXCR4 gene were constructed and then were transfected into KYSE-150 and TE-13 cells by Lipofectamine™2000. Changes of CXCR4 mRNA and protein were analyzed by qRT-PCR and Western blot. Effect of CXCR4 siRNA on KYSE-150 and TE-13 cells proliferation was determined by MTT. Transwell invasion assay was used to evaluate the invasion and metastasis of KYSE-150 and TE-13 cells. Tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. qRT-PCR and Western blot demonstrate that the expression level of CXCR4 gene were obviously decreased in KYSE-150 and TE-13 cells transfected with CXCR4 targeting siRNA expression vectors. The average amount of cells transfected with CXCR4 siRNA penetrating Matrigel was significantly decreased (p<0.05). Injection of CXCR4 siRNA transfected cells inhibited tumor growth in a xenograft model compared with blank and negative control groups (p <0.05). CXCR4 silenced by siRNA could suppress the proliferation, invasion and metastasis of esophageal carcinoma cell lines KYSE-150 and TE-13 in vitro and in vivo. The results provide a theoretical and experimental basis for the gene therapy of ESCC using RNAi technology based on CXCR4 target site. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3502376691001138

Gene silencing of myofibrillogenesis regulator-1 by adenovirus-delivered small interfering RNA suppresses cardiac hypertrophy induced by angiotensin II in mice

Our previous studies proved that myofibrillogenesis regulator (MR)-1 has a close relationship with cardiac hypertrophy induced by ANG II. In the present study, we developed a recombinant adenoviral vector (AdSiR-MR-1) driving small interfering (si)RNA against MR-1 to evaluate its effect on cardiac hypertrophy in vivo. Cardiac hypertrophy was induced by chronic ANG II infusion in mice; AdSiR-MR-1 was administered via the jugular vein through one bolus injection. Thirteen days after the injection, viral DNA was still detectable in the heart, validating the efficiency of gene transfer. Expression levels of MR-1 mRNA and protein were increased by 2.5-fold in the heart after ANG II infusion; AdSiR-control, which contained a scrambled siRNA sequence, had no effect on them. AdSiR-MR-1 treatment abolished the upregulation of MR-1 induced by ANG II. The silencing effect of AdSiR-MR-1 was observed in many other tissues, such as the liver, lung, and kidney, except skeletal muscle. ANG II-induced cardiac hypertrophy was suppressed in mice treated with AdSiR-MR-1, as determined by echocardiography. Morphological and immnohistochemical examinations revealed that interstitial cardiac fibrosis as well as infiltrating inflammatory cells were increased after ANG II infusion; AdSiR-MR-1 greatly ameliorated these disorders. In ANG II-infused mice, MR-1 silencing also blocked the upregulation of other genes related to cardiac hypertrophy or metabolism of the extracellular matrix. In summary, our results demonstrate the feasibility of MR-1 silencing in vivo and suggest that MR-1 could be a potential new target to treat cardiac hypertrophy induced by ANG II.