Short Hairpin RNA Expression Research Articles

CD9 knockdown suppresses cell proliferation, adhesion, migration and invasion, while promoting apoptosis and the efficacy of chemotherapeutic drugs and imatinib in Ph+ ALL SUP‑B15 cells.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is regarded as a prognostically unfavorable subgroup, as this ALL subgroup has an increased risk of relapse/refractory disease. CD9, which belongs to the tetraspanin membrane proteins, is implicated in several pathological processes, including tumor progression. However, the role of CD9 in the pathogenesis of Ph+ ALL and the potential benefit of applying CD9-targeted RNA interference strategies for treatment of Ph+ ALL require further investigation. The aim of the present study was to determine the effects of CD9 on leukemic cell progression and the efficacy of therapeutic agents in Ph+ ALL cells, in addition to assessing the in vitro anti-leukemia activity of CD9-targeted RNA interference in Ph+ ALL cells. In the present study, a lentiviral short hairpin RNA (shRNA) expression vector targeting CD9 gene in Ph+ ALL SUP-B15 cells was constructed. The present results demonstrated that treatment of SUP-B15 cells with lentiviral-mediated shRNA against CD9 decreased CD9 mRNA and protein expression compared with the shControl cells transduced with a blank vector. In addition, CD9 knockdown could suppress cell proliferation, adhesion, migration and invasion, and promote apoptosis and the efficacy of chemotherapeutic drugs (such as vincristine, daunorubicin, cyclophosphamide and dexamethasone) and the tyrosine kinase inhibitor imatinib in SUP-B15 cells. Furthermore, CD9 knockdown suppressed cell proliferation and promoted apoptosis in SUP-B15 cells via a p53-dependent pathway. These findings suggested that gene silencing of CD9 using a shRNA-expressing lentivirus vector may provide a promising treatment for Ph+ ALL.

NCSTN Gene Silencing Inhibits the Retinoic Acid Signaling Pathway in Human Immortalized Keratinocytes

Abstract Objective: Acne inversa is a multifactorial chronic debilitating disease. Genetic factors are involved in 40% of patients, especially the nicastrin (NCSTN) gene. However, the role of the mutated NCSTN gene in the pathogenesis of acne inversa remains unclear. Retinoic acid is recommends to treat moderate to severe acne inversa, therefor we conduct this in vitro research to study the association between NCSTN gene mutation and the retinoic acid signaling pathway in human immortalized skin keratinocyte (HaCaT) cells. Methods: HaCaT cells were infected with a lentivirus-mediated short hairpin RNA (shRNA) expression plasmid specifically targeting the NCSTN gene. Real-time polymerase chain reaction (PCR) and Western blotting were used to detect the interference efficiency of NCSTN. RNA sequencing was used to detect differential genes in the NSCTN-deficient HaCaT cells. Based on bioinformatics analysis and clinical treatment data, the retinoic acid signal pathway was selected for screening. Quantitative PCR was used to verify the changes in the expressions of retinoic acid signaling pathway-related receptors and molecules in the HaCaT cell line after NCSTN silencing. The Student t test and one-way analysis of variance were used to evaluate intergroup differences. Results: Sequencing showed that the NCSTN-shRNA lentiviral recombinant expression plasmid was successfully constructed. After lentivirus infection of HaCaT cells, real-time PCR results showed significantly reduced NCSTN mRNA expression in the interference group compared with the negative control group, and the interference efficiency was 75.0%. Western blotting showed that the inhibition rate of NCSTN protein expression in the shRNA group was 71.7%. RNA sequencing revealed significant differential expression of some genes, and changes in signaling pathways. Compared with the control group, the group with the silenced NCSTN showed significantly decreased expression of retinoic acid receptors (RARα: F=23.482, RARβ: F=603.241, RXRα: F=69.689, and RARRES1: F=167.482, and all P < 0.001), and peroxisome proliferator-activated receptor γ (F=8.138, P < 0.01). Conclusion: Defective function of the NCSTN gene leads to an impaired retinoic acid signaling pathway in HaCaT cells, which suggests that the retinoic acid signaling pathway may play a role on the onset of acne inversa caused by NCSTN gene mutation.

Activation-Induced Cytidine Deaminase Expression Facilitates the Malignant Phenotype and Epithelial-to-Mesenchymal Transition in Clear Cell Renal Cell Carcinoma.

Although advances have been made in the development of antiangiogenesis targeted therapy and surgery, metastatic clear cell renal cell carcinoma (ccRCC) is still incurable. Activation-induced cytidine deaminase (AID) is mainly expressed in a variety of germ and somatic cells, and induces somatic hypermutation and class-switch recombination, playing a vital role in antibody diversification. We confirmed that AID was expressed at a higher level in ccRCC tissues than in the corresponding nontumor renal tissues. We explored the impact of AID on ccRCC proliferation, invasion, and migration. In 769-p and 786-0 cells, expression of an AID-specific short hairpin RNA significantly reduced AID expression, which markedly inhibited tumor cell invasion, proliferation, and migration. Previous studies showed that AID is associated with Wnt ligand secretion mediator (WLS/GPR177), cyclin-dependent kinase 4 (CDK4), and stromal cell-derived factor-1 (SDF-1/CXCL12) regulation, which was further confirmed in human ccRCC tissues. Therefore, we studied the relationship between AID and these three molecules, and the impact of AID on epithelial-to-mesenchymal transition in ccRCC. WLS/GPR177, SDF-1/CXCL12, and CDK4 were sensitive to 5-azacytidine (a DNA demethylation agent), which reverted the inhibition of carcinogenesis caused by AID repression. In summary, AID is an oncogene that might induce tumorigenesis through DNA demethylation. Targeting AID may represent a novel therapeutic approach to treat metastatic ccRCC.

ATF5 involved in radioresistance in nasopharyngeal carcinoma by promoting epithelial-to-mesenchymal phenotype transition.

This study aimed to investigate the effects of activating transcription factor-5 (ATF5) on nasopharyngeal carcinoma (NPC) cell radioresistance. HONE-1 nasopharyngeal carcinoma cells were irradiated by conventional fractionation to generate HONE-1R radiotherapy-resistant cells. Short hairpin RNA (shRNA) expression plasmids targeting the ATF5 gene were constructed and transfected into the HONE-1R cell line. The proliferation assay, colony formation analysis, Transwell Boyden chamber assay and other experimental methods were performed to verify changes in the radiosensitivity and other biological of NPC cells. X-ray irradiation significantly promoted the upregulation of ATF5 protein levels in HONE-1 cells, and the protein expression of ATF5 increased with the dose of X-ray irradiation (p < 0.05). The colony formation rate, cell survival rate and migration ability of HONE-1R cells were significantly higher than those of HONE-1 cells (p < 0.05). Simultaneously, X-ray could also promote the morphology of epithelial-mesenchymal transition (EMT) in HONE-1 cells, inducing a lower expression level of E-cadherin and a higher expression level of N-cadherin in a dose-dependent manner (p < 0.05). Inhibiting ATF5 significantly reduced the colony formation rate, cell survival rate, migration and invasiveness of HONE-1R cells (p < 0.05). Moreover, sensitizing HONE-1R cells to X-ray irradiation significantly upregulated the expression of E-cadherin and downregulated the expression of N-cadherin in these cells (p < 0.05). ATF5 may be a potential therapeutic target to improve radiosensitivity in NPC.

Identification and comparison of the porcine H1, U6, and 7SK RNA polymerase III promoters for short hairpin RNA expression.

RNA polymerase III is an essential enzyme in eukaryotes for synthesis of tRNA, 5S rRNA, and other small nuclear and cytoplasmic RNAs. Thus, RNA polymerase III promoters are often used in small hairpin RNA (shRNA) expression. In this study, the porcine H1, U6, and 7SK RNA polymerase III type promoters were cloned into a pcDNA3.1( +) expression vector containing a shRNA sequence targeting enhanced green fluorescent protein (EGFP). PK and DF-1 cells were cotransfected with the construction of recombinant interference expression vector and the EGFP expression vector, pEGFP-N1. The average fluorescence intensity of EGFP in transfected cells was measured by fluorescence microscopy and flow cytometry. Real-time PCR was used to detect expressed shRNAs and the relative expression of EGFP, to confirm the activity of the promoters. The results showed that the activity of porcine 7SK promoter is stronger than the U6 promoter, which is in turn stronger than porcine H1. While the high levels of expression of the U6 and 7SK promoters saturate the shRNAs level in the host cell, which can cause cytotoxicity and tissue damage. Therefore, porcine H1 promoter is effective for expression of shRNA, and may be an excellent tool to knockdown gene expression in pigs for functional genomics studies. The results also lay a foundation for the development of porcine RNAi technology and genetically modified porcine research.

Evaluation of combination gene therapy with SLC22A18 upregulation and sequence binding protein 1 downregulation for glioma U251 cells in vitro and in vivo

Background and Aim: Our previous study demonstrated that SLC22A18 downregulation through promoter methylation and Sequence binding protein 1 (SATB1) upregulation are associated with the development and progression of glioma. This study aimed to examine the effect of combined SLC22A18 and short hairpin RNA (shRNA) targeting SATB1 gene therapy on glioma growth and invasion. Materials and Methods: Here, a combined gene therapy to upregulate SLC22A18 and downregulate SATB1 in malignant glioma was evaluated both in vitro and in vivo. Glioma U251 cells overexpressing SLC22A18 and underexpressing SATB1 were generated to investigate the effects of these changes on cell survival, as measured by the methyl thiazol tetrazolium assay, and on cell invasion, as measured by cell invasion and migration assays. In addition, analysis of the cell cycle was performed using flow cytometry in vitro. The animal experiments were approved by the Ethics Committee of Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Zhongnan Hospital of Wuhan University Ethics Committees (approval No. ZNHWHU0389, NTPHSHJTUSM046). Results: The upregulation of SLC22A18 and downregulation of SATB1 significantly inhibited growth and invasion in vitro and reduced in vivo tumor growth of human glioma U251 cells. Furthermore, we revealed that U251 cells were arrested at the G0/G1 phase. Similar data for apoptotic glioma cell death were obtained in tumor cells from an in vivo glioma xenograft model after transfection. At the same total dose, the therapeutic effect was markedly better in the glioma xenografts transfected with both SLC22A18 gene and SATB1 short hairpin RNA (shRNA) expression vectors compared with tumors transfected with either agent alone. The levels of SLC22A18 and SATB1 protein expression were respectively increased and decreased in the glioma cells.Conclusion: These results demonstrate that combination treatment with SLC22A18 gene and SATB1 shRNA expression vectors effectively inhibits the growth of human malignant glioma cells both in vitro and in glioma xenografts in vivo, suggesting a promising novel strategy for glioma therapy that warrants further study.

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

Cutaneous T-Cell Lymphoma (CTCL) Cell Line-Derived Extracellular Vesicles Contain HERV-W-Encoded Fusogenic Syncytin-1

Inhibition of human lung adenocarcinoma growth and metastasis by JC polyomavirus-like particles packaged with an SP-B promoter-driven CD59-specific shRNA.

Lung cancer ranks first in both incidence and mortality and is a major health concern worldwide. Upon recognition of specific antigens on tumor cells, complement-dependent cytotoxicity (CDC) is activated, arresting cell growth or inducing apoptosis. However, by overexpressing CD59, a membrane complement regulatory protein (mCRP), lung cancer cells develop resistance to CDC. We previously showed that virus-like particles (VLPs) of human JC polyomavirus (JCPyV) could be used as a gene therapy vector to carry a suicide gene expression plasmid with a lung-specific promoter (SP-B (surfactant protein B)) for lung adenocarcinomas. Herein, we designed a CD59-specific short hairpin RNA (shRNA) expression plasmid driven by SP-B (pSPB-shCD59) to effectively and specifically inhibit CD59 overexpression in lung cancer cells. Treatment of lung cancer cells in vitro with JCPyV VLPs containing pSPB-shCD59 (pSPB-shCD59/VLPs) induces CDC and death of cancer cells. Mice that were subcutaneously injected with human lung cancer cells showed an 87% inhibition in tumor growth after tail vein injection of pSPB-shCD59/VLPs. Moreover, in a mouse model of lung cancer metastasis, a reduction in the lung weight by 39%, compared with the control group, was observed in mice treated with pSPB-shCD59/VLPs after tail vein injection of human lung cancer cells. Furthermore, tissue sectioning showed that the number and size of tumors produced was significantly reduced in the lungs of mice in the treatment group than those of the untreated group, indicating inhibition of metastasis by pSPB-shCD59/VLPs. Together, these results demonstrate the potential of pSPB-shCD59/VLPs as a therapeutic agent for CD59 overexpressed lung cancer.

P5725Identification of novel cardiomyogenic factors by transcriptome analysis of conditionally immortalized atrial myocytes

Abstract Background Cardiac development involves the properly timed expression of cardiomyogenic differentiation factors (CDFs). CDFs have mainly been discovered using animal models and, more recently, pluripotent stem cell-derived cardiomyocytes (PSC-CMCs). These models are, however, laborious, time-consuming and costly. Also, cardiomyogenic differentiation of CMCs is heterogeneous and yields phenotypically immature CMCs. Recently, our research group generated a monoclonal line of conditionally immortalized atrial myocytes, called iAM-1. After removal of the proliferation stimulus these cells spontaneously and synchronously differentiate into mature atrial myocytes, making them ideally suited for transcriptome analysis and the discovery of novels factors involved in cardiomyogenic differentiation. Methods Whole transcriptome analysis of iAM-1 cells was performed at 9 different time points during cardiomyogenic differentiation and subsequent dedifferentiation by RNA sequencing. Six genes upregulated during cardiomyogenic differentiation were selected for knockdown studies in differentiating iAM-1 cells. Each of these genes was targeted by a bicistronic lentiviral vector (LV) driving expression of a specific short hairpin RNA (shRNA) and of enhanced green fluorescent protein (eGFP). Knockdown effects during cardiomyogenic differentiation were studied by immunocytology. The LVs were also used in primary neonatal atrial and ventricular rat cardiomyocytes to study the role of the selected genes in cardiomyocyte homeostasis. Results Whole transcriptome analysis of differentiating iAM-1 cells identified the dynamic expression levels of ± 13.000 genes, including the expected profile for genes known to play a role in atrial myocyte differentiation, like Nkx2–5, Tbx3, Tbx5 and Nppa. Six genes with an unknown role in cardiomyocyte differentiation and homeostasis were selected based on significant upregulation during iAM-1 differentiation, substantial mRNA levels and selective expression in cardiac tissue. Inhibiting gene expression by lentiviral RNA interference resulted for Nkx2–5 as well as for 3 out of 6 target genes in disturbed iAM-1 differentiation, as evinced by loss of sarcomeric cross-striations. Similar effects were observed in shRNA-expressing (i.e. eGFP-positive) primary atrial and ventricular neonatal rat myocytes. Taken together, these results highlight the importance of these novel genes during cardiomyogenic differentiation and homeostasis in atrial as well as ventricular myocytes. Conclusions Transcriptome analysis of cardiomyogenic differentiation in conditionally immortalized atrial myocytes combined with genetic knockdown experiments led to the identification of several novel factors involved in the differentiation and homeostasis of atrial and ventricular myocytes. These results highlight the suitability of iAM-1 as model for fundamental research of cardiomyogenic differentiation. Acknowledgement/Funding ZonMW

Expression of short hairpin RNA (shRNA) targeting AC2 gene of Mungbean yellow mosaic India virus (MYMIV) reduces the viral titre in soybean

Mungbean yellow mosaic India virus (MYMIV) belonging to the family Geminiviridae and the genus Begomovirus is a severe pathogen of tropical legumes including soybean. The absence of genetically mapped loci conferring resistance together with the genetic diversity of begomoviruses infecting soybean warrants the utilization of RNA interference (RNAi) technology to develop virus resistance. However, viral suppressors of RNAi (VSRs) reduce the effectiveness of RNA silencing. Here, we report the effectiveness of Agrobacterium-mediated transient expression of shRNA, targeting a conserved region of AC2 ORF (a VSR) of MYMIV, in conferring virus resistance in soybean. Transient expression of shRNA showed progressive reduction of the viral titre estimated by the MYMIV-derived AC2 gene copy numbers from the initial inoculum by approximately 80-fold 20days post-application. In addition, the newly emerging leaves exhibited symptom recovery. Thus, this study proves that AC2 of MYMIV is a potent target gene for obtaining RNAi-mediated virus resistance in soybean. Agro-infiltration-based delivery of shRNA was an efficient means of gene silencing and could pave way for the development of transgenic virus-resistant soybean genotype.

Downregulation of lncRNA AWPPH inhibits colon cancer cell proliferation by downregulating GLUT-1.

Long non-coding RNA (lncRNA) associated with poor prognosis of hepatocellular carcinoma (AWPPH) serves pivotal roles in bladder cancer and liver cancer; however, to the best of our knowledge, its functionality in colon cancer has not been characterized. The present study aimed to investigate the involvement of lncRNA AWPPH in colon cancer. Serum levels of lncRNA AWPPH and glucose transporter 1 (GLUT-1) in patients with early stage colon cancer and healthy controls were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA. Correlation between lncRNA AWPPH and GLUT-1 expression was analyzed by Pearson's correlation coefficient. χ2 test was performed to investigate the associations between serum levels of lncRNA AWPPH and clinical data of patients with colon cancer. lncRNA AWPPH short hairpin RNA and GLUT-1 expression vectors were transfected into colon cancer cells, and the effects on lncRNA AWPPH, GLUT-1 and cell proliferation were detected by RT-qPCR, western blotting and Cell Counting Kit-8 assay. It was observed that serum levels of lncRNA AWPPH and GLUT-1 were significantly higher in patients with colon cancer patients compared with healthy controls. Serum levels of AWPPH and GLUT-1 were significantly positively correlated in patients with colon cancer. Serum levels of lncRNA AWPPH were associated with the tumor size. Furthermore, AWPPH-silencing significantly inhibited GLUT-1 expression and inhibited cancer cell proliferation. GLUT-1 overexpression promoted cancer cell proliferation and attenuated the inhibitory effects of AWPPH-silencing on cancer cell proliferation. However, GLUT-1 overexpression failed to significantly affect the expression of AWPPH. Therefore, it can be concluded that a downregulation of lncRNA AWPPH may inhibit colon cancer cell proliferation by downregulating GLUT-1.

Downregulation of miR-144 by triptolide enhanced p85α−PTEN complex formation causing S phase arrest of human nasopharyngeal carcinoma cells

Selective pharmacologic targeting of cell cycle regulators is a potent anti-cancer therapeutic strategy. Here, we show that caspase-3-mediated p21 cleavage involves p53 independent of triptolide (TPL)-induced S phase arrest in human type 1 nasopharyngeal carcinoma (NPC) cells. Coimmunoprecipitation studies demonstrated that TPL causes S phase cell cycle arrest by suppressing the formation of cyclin A–phosphor (p)-cyclin-dependent kinas 2 (CDK2) (Thr 39) complexes. Ectopic expression of constitutively active protein kinase B1 (Akt1) blocks the induction of S phase arrest and the suppression of cyclin A expression and CDK2 Thr 39 phosphorylation by TPL. Expression of the phosphomimetic mutant CDK2 (T39E) rescues the cells from TPL-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TPL-triggered S phase arrest. Treatment with TPL induces an increase in the formation of complexes between unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and p85α in the plasma membrane. Decreased microRNA (miR)-144 expression and increased PTEN expression after TPL treatment were demonstrated, and TPL-enhanced p85α–PTEN complexes and inhibitory effects on Akt (Ser 473) phosphorylation and S phase arrest were suppressed by ectopic PTEN short hairpin RNA or miR-144 expression. Knockdown of endogenous miR-144 by miR-144 Trap upregulated PTEN expression and accordingly enhanced p85α–PTEN complex formation and S phase arrest. Collectively, the effect of TPL on S phase arrest in human NPC cells is likely to enhance the p85α–PTEN interaction in the plasma membrane by suppressing miR-144 expression, resulting in the attenuation of cyclin A–p-CDK2 (Thr 39) complex formation via Akt inactivation.

HCN4 knockdown in dorsal hippocampus promotes anxiety-like behavior in mice.

Hyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channels mediate the I h current in the murine hippocampus. Disruption of the I h current by knockout of HCN1, HCN2 or tetratricopeptide repeat‐containing Rab8b‐interacting protein has been shown to affect physiological processes such as synaptic integration and maintenance of resting membrane potentials as well as several behaviors in mice, including depressive‐like and anxiety‐like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus‐based behaviors in mice. We show that HCN4 is expressed in various regions of the hippocampus, with distinct expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno‐associated viral (rAAV) vectors mediating expression of short hairpin RNA against hcn4 (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was evident in mice treated with shHcn4 compared to control littermates. Our findings suggest that HCN4 specifically contributes to anxiety‐like behaviors in mice.

Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

Cells to Surgery Quiz: December 2018

Cells to Surgery Quiz: December 2018

Long intergenic non‑protein coding RNA 152 promotes multiple myeloma progression by negatively regulating microRNA‑497

Long intergenic non‑protein coding RNA 152 (LINC00152, also known as cytoskeleton regulator RNA) is reportedly involved in the development and progression of various types of human malignancy. However, the functional role of LINC00152 in multiple myeloma (MM) and the underlying molecular mechanisms have remained elusive. The present study aimed to investigate the role of LINC00152 in the genesis of MM and the potential molecular mechanisms. It was identified that the expression of LINC00152 was significantly upregulated in plasma cells from patients with MM vs. healthy subjects, and that a high expression of LINC00152 was correlated with a shorter overall survival in patients with MM. Functional assays demonstrated that knockdown of LINC00152 by transfecting MM cells with LINC00152‑specific short hairpin RNA expression plasmids significantly inhibited cell proliferation by inducing cell cycle arrest at the G0/G1 phase. Furthermore, knockdown of LINC00152 promoted caspase‑3/9 activity and apoptosis in MM cells. In addition, knockdown of LINC00152 significantly attenuated tumor growth in mice bearing a myeloma xenograft. A luciferase reporter assay indicated that microRNA (miR)‑497 is a direct target of LINC00152, and its expression levels were inversely correlated with those of LINC00152 in MM tissues. Furthermore, repression of miR‑497 partly abrogated the inhibitory effects of LINC00152 knockdown on MM cells. Collectively, the results indicated that LINC00152 has an oncogenic effect in MM by targeting miR‑497, and may be a novel diagnostic marker and therapeutic target for MM.

Therapeutic advantage of genetically engineered Salmonella typhimurium carrying short hairpin RNA against inhibin alpha subunit in cancer treatment

BackgroundIn contrast to its well-known endocrine function, the role of inhibin in cancer development and therapeutic response is unclear. Salmonella, particularly less toxic attenuated Salmonella strains, are used to treat cancer in two ways. First, Salmonella accumulate around tumors, penetrate the cell barrier, and replicate inside the tumors. Second, Salmonella can act as a vehicle for delivering anticancer agents or proapoptotic genes to attack tumors. In this study, we aimed to develop a suitable cancer therapeutic strategy by genetically modifying attenuated Salmonella typhimurium to harbor short hairpin RNA (shRNA) expression plasmids targeting alpha subunit of inhibin (sh-INHA). MethodsWe analyzed the expression of human INHA in normal and cancer cells and tissues. We developed genetically engineered attenuated S. typhimurium harboring sh-INHA (S. typhimurium/sh-INHA) and assessed its cancer therapeutic effects by using cell culture models and syngeneic mouse tumor models. ResultsINHA expression levels were markedly higher in colon cancer and melanoma cells and tissues than in their normal counterparts. Suppression of INHA expression mildly reduced cancer cell survival and induced caspase activation and downregulation of anti-apoptotic Bcl-2 and Bcl-xL expressions. Although the genetically engineered S. typhimurium mildly interfered with the invasion of S. typhimurium into host colon cancer and melanoma cells, S. typhimurium/sh-INHA caused remarkable cytotoxicity in cancer compared with unmodified S. typhimurium or S. typhimurium expressing a control scrambled shRNA (S. typhimurium/sh-Cont). Salmonella typhimurium/sh-INHA-treated mice also showed a significantly inhibited growth of colon cancers and melanomas, with a survival advantage. ConclusionOur results suggest that tumor-targeted therapy using S. typhimurium/sh-INHA may provide a novel cancer treatment option.

The roles and mechanism of chemokine receptor type 5 in colorectal cancer proliferation

Objective To examine the expression of chemokine receptor type 5 (CXCR5) in colorectal cancer (CRC) and to explore its relationship with clinicopathological features and cell proliferation in CRC. Methods Immunohistochemical staining method was used to detect the protein expression of CXCR5 in 132 paired specimens of CRC, adjacent and 62 samples of adenoma. Analysing the relationship between CXCR5 with clinicopathological features in CRC. RNA interference (RNAi) technique was applied to construct four CXCR5 short hairpin RNA (shRNA) expression vectors. Lipofectamine 2000 was used to transfer plasmids into colon cancer cell lines LoVo and HCT116. Real-time PCR and Western blotting were used to observe the inhibitory effect of RNAi on CXCR5 expression. Lentivirus production and transduction were used to construct CXCR5 shRNA stably expressed CRC cell lines. CXCR5 overexpressed plasmid was used to construct CXCR5 stably expressed CRC cell lines. cell counting kit-8 (CCK-8) assay and colony formation assay were used to detect the influence of specific CXCR5 on CRC cell. Tumor formation in nude mouse was performed to detect the effects of CXCR5 on the proliferation ability of CRC cells in vivo. FCM were used to detect the cell cycle. Results CXCR5 protein was expressed in 46.97% (62/132) of CRC tissues and 24.19% (15/62) of adenoma tissues, respectively. The protein expression of CXCR5 was negative in adjacent tissues. The protein expression level of CXCR5 in CRC tissues was significantly higher than ademona tissues (P=0.020). The expression of CXCR5 was correlated with relapse, TNM stage, lymph node metastasis and distant metastasis (P=0.004, 0.025, 0.002, 0.008). CCK-8 and colony formation results showed that the CXCR5 shRNA inhibited the proliferation of CRC significantly, whereas overexpression of CXCR5 promoted CRC proliferation in vitro. Tumor formation results in nude mouse showed that CXCR5 silencing inhibited the proliferation and metastasis ability of CRC cells, whereas overexpression of CXCR5 promoted proliferation of CRC cells in vivo. CXCR5 may influence CRC cells proliferation through cell cycle in vitro. Conclusion CXCR5 may play a crucial role in carcinogenesis, development of CRC. CXCR5 could promote proliferation of colon cancer cell both in vitro and in vivo, and may influence CRC development through cell cycle. Key words: Colorectal cancer; Chemokine receptor type 5; Proliferation

Down-regulation of CD19 expression inhibits proliferation, adhesion, migration and invasion and promotes apoptosis and the efficacy of chemotherapeutic agents and imatinib in SUP-B15 cells

The survival rate of childhood acute lymphoblastic leukemia (ALL) has increased while that of Philadelphia-positive (Ph+) ALL remains low. CD19 is a B-cell specific molecule related to the survival and proliferation of normal B cells. However, there is little information available on the effects of CD19 on the biological behavior of Ph+ ALL cells. In this study, we explored a lentiviral vector-mediated short hairpin RNA (shRNA) expression vector to stably reduce CD19 expression in Ph+ ALL cell line SUP-B15 cells and investigated the effects of CD19 downregulation on cell proliferation, apoptosis, drug sensitivity, cell adhesion, cell migration and cell invasion in vitro. CD19 mRNA and protein expression levels were inhibited significantly by CD19 shRNA. Down-regulation of CD19 could inhibit cell proliferation, adhesion, migration and invasion, and increase cell apoptosis and the efficacy of chemotherapeutic agents and imatinib in SUP-B15 cells. Moreover, we found that down-regulation of CD19 expression inhibits cell proliferation and induces apoptosis in SUP-B15 cells in a p53-dependent manner. Taken together, our results suggest that lentiviral vector-mediated RNA interference of CD19 gene may be a promising strategy in the treatment of Ph+ ALL.

EIF4A2 is a host factor required for efficient HIV-1 replication

Host factors are required for efficient HIV-1 replication. To identify these factors, genome-wide RNA interference screening was performed using a human T cell line. In the present study, we assessed whether eukaryotic translation initiation factor 4A isoform 2 (eIF4A2), a DEAD-box protein identified in our screen, is necessary for efficient HIV-1 replication. Exploiting MT4C5 cells depleted of eIF4A2 by stable expression of eIF4A2-specific short-hairpin RNA (shRNA) using a lentiviral system, we found that depletion of eIF4A2 markedly inhibited the infection of a replication-competent reporter HIV-1. eIF4A2 depletion reduced the efficiency of viral cDNA synthesis with virion entry into target cells being unaffected. Depletion of eIF4A2 also inhibited HIV-1 spreading infection in a knockdown level-dependent manner. These results suggest that HIV-1 requires eIF4A2 for optimal replication in human T cells.