Effect Of Short Hairpin RNA Research Articles

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A.

BACKGROUNDColorectal cancer (CRC) is one of the most common malignant tumors worldwide. The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative. Immunohistochemical analysis has revealed high expression of centromere protein K (CENPK) in CRC. However, the role of CENPK in the progression of CRC is not well characterized.AIMTo evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A (CUL4A) in RKO and HCT116 cells.METHODSHuman colon cancer samples were collected and tested using a human gene expression chip. We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis. In vitro experiments verified the function of this gene. We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction (qPCR), western blot, and flow cytometry. The effect of short hairpin RNA (shRNA) virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging. To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells, we performed a series of in vitro experiments, using qPCR, western blot, MTT assay, and flow cytometry.RESULTSWe demonstrated overexpression of CENPK in human colon cancer samples. CENPK was an independent risk factor in patients with CRC. The downstream genes FBX32, CUL4A, and Yes-associated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells. Significantly delayed xenograft tumor emergence, slower growth rate, and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group. The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo. The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells, with overexpression of the CUL4A.CONCLUSIONWe indicated a potential role of CENPK in promoting tumor proliferation, and it may be a novel diagnostic and prognostic biomarker for CRC.

Knockdown of long non-coding RNA KCNQ1OT1 suppresses the progression of osteoarthritis by mediating the miR-211-5p/TCF4 axis in vitro.

Numerous studies have reported the critical roles of long non-coding RNAs (lncRNAs) in the regulation of osteoarthritis (OA) development. The present study aimed to assess the function and regulatory mechanism of a lncRNA, KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1), in OA in vitro. C28/I2 cells were treated with lipopolysaccharide (LPS) to generate an in vitro OA model. The relative expression levels of KCNQ1OT1, microRNA (miR)-211-5p and transcription factor 4 (TCF4) were determined via reverse transcription-quantitative polymerase chain reaction. The associations between KCNQ1OT1, miR-211-5p and TCF4 were confirmed using a dual-luciferase reporter assay. Furthermore, cell viability was assessed using the MTT assay. Inflammatory cytokine levels were measured using ELISA. The protein expression levels of matrix metalloproteinase-3/13, collagen II/X and TCF4 were detected by western blotting. KCNQ1OT1 and TCF4 were highly expressed in the cartilage tissues of patients with OA and C28/I2 cells treated with LPS (OA cells), whereas miR-211-5p was downregulated concomitantly in OA tissues and cells. Knockdown of KCNQ1OT1 stimulated cell viability, and suppressed the inflammation and degradation of the extracellular matrix (ECM) in OA cells. In addition, overexpression of miR-211-5p stimulated cell viability, and inhibited inflammation and degradation of the ECM in OA cells. Notably, miR-211-5p was revealed to be the target of, and was negatively regulated by, KCNQ1OT1. TCF4 was targeted and negatively modulated by miR-211-5p. Transfection of cells with the miR-211-5p inhibitor or pcDNA-TCF4 reversed the suppressive effects of short hairpin RNA (sh)-KCNQ1OT1 on inflammation and ECM degradation, as well as the promotive effect of sh-KCNQ1OT1 on viability in OA in vitro. Therefore, KCNQ1OT1 may regulate the miR-211-5p/TCF4 axis to ameliorate OA in vitro.

NAD-dependent methylenetetrahydrofolate dehydrogenase inhibits oral squamous cell carcinoma cell proliferation and promotes apoptosis.

BackgroundNAD-dependent methylenetetrahydrofolate dehydrogenase catalyzes the conversion of 10-formyltetrahydrofolate to formate in embryonic and adult mammalian mitochondria. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) is a folate cycle enzyme that is involved in the development of various diseases including cancer. However, the specific mechanisms in oral squamous cell carcinoma (OSCC) are unclear. We analyzed the functional routes of MTHFD1L in OSCC cells.MethodsMTHFD1L expression in OSCC was analyzed using data from The Cancer Genome Atlas (TCGA) database. Then, the levels of mRNA were measured in OSCC and para-tumor oral tissues using Affymetrix microarrays. Additionally, the effects of short hairpin RNA (shRNA)-induced MTHFD1L silencing on the biological behavior of OSCC were assessed in vitro and in vivo, and the potential molecular mechanisms underlying MTHFD1L activity were also investigated.ResultsA TCGA database analysis of RNA sequencing revealed that MTHFD1L levels were higher in tumor tissue than in adjacent tissues. Immunohistochemical staining and Kaplan-Meier survival analysis also indicated that MTHFD1L upregulation is associated with a poor prognosis in OSCC. The knockdown of MTHFD1L suppressed cell proliferation, colony formation, and tumorigenesis, while it induced apoptosis in OSCC. Mechanistically, a microarray analysis showed that MTHFD1L suppressed c-MYC and activated p53 signaling by regulating the protein expression of TP53, GADD45A, FAS and JUN.ConclusionsMTHFD1L may be involved in OSCC progression via the c-MYC gene and p53 signaling and may serve as a novel target and orientation for tumor therapy.

Effect of Phosphoribosyltransferase Down-regulation on Malignant Glioma Cell Characteristics.

Nicotinamide phosphoribosyl-transferase (NAMPT) is a rate-limiting enzyme in the pathway synthesizing nicotinamide adenine dinucleotide (NAD (+)) from nicotinamide (NAM). Glioma tissues exhibit up-regulated NAMPT expression associated with a poor prognosis of patients. To determine if NAMPT can be a molecular therapeutic target, we investigated the effects of short hairpin RNA (shRNA)-mediated NAMPT down-regulation. We designed shRNA to NAMPT and transfected to T98G cells. The characteristics of these cells were analyzed. The NAMPT shRNA-transfected cells exhibited delayed cell growth. However, there was no difference in the increase of sensitivity to temozolomide (TMZ) or X-ray irradiation between the NAMPT and scramble shRNA-transfected cells. The expression of NAMPT in the NAMPT shRNA-transfected cells increased with cell passage. Additionally, the shRNA-mediated transfection was associated with enhanced expression of quinolinic acid phosphoribo-syltransferase (QPRT). shRNA-mediated NAMPT down-regulation may not decrease the NADt to a sufficient level to increase TMZ/radiation sensitivity.

Effects of shRNA on Cisplatin-resistant Non-small Cell Lung Cancer Cell A549 via Silencing CCAT2

To investigate the effects of short hairpin RNA (shRNA) on the proliferation, invasion, apoptosis and tumor formation of non-small cell lung cancer cisplatin-resistant cell line (A549/DDP) via silencing of colon cancer associated transcript 2 ( CCAT2). TA549/DDP cells were transfected with shRNA- CCAT2 (sh- CCAT2) or shRNA-negative control (shRNA-NC), and untransfected A549/DDP cells were used as the control group. CCAT2 mRNA expression in three groups of A549/DDP cells was detected by quantitative real-time PCR (qRT-PCR). The proliferation of three groups of A549/DDP cells treated with different mass concentrations of DDP (0-8 mg/L) was detected by MTT. According to the proliferation experiment results, 2 mg/L was selected as DDP concentration for subsequent experiments. The effects of 2 mg/L DDP treatment on the proliferation, apoptosis, and invasion ability of each group of cells (with untreated A549/DDP cells as the control group) were tested by clone formation experiments, flow cytometry analysis and Transwell experiments. The expression levels of cell proliferation marker proteins (Ki67, PCNA), apoptosis marker proteins (Caspase-3, Caspase-9) and invasion marker proteins (VEGF, MMP-14) were detected by Western blot. Nude mice were injected subcutaneously with A549/DDP cells, A549/DDP cells transfected with shRNA-NC or A549/DDP cells transfected with sh- CCAT2. DDP was intraperitoneally injected at the concentration of 2 mg per kilogram of mice body weight totally for 7 times with an interval of 3 d. A control group was injected subcutaneously with A549/DDP cells, and an equal volume of normal saline instead of DDP was injected intraperitoneally. The tumor volume was detected every 5 d for a total of 30 d. Mice were sacrificed and tumor tissues were taken out 30 d later. CCAT2 mRNA expression level in tumor tissues was detected by RT-PCR, and tumor cell apoptosis was detected by TUNEL staining. Compared with the control group and the shRNA-NC transfection group, the expression level of CCAT2 mRNA was decreased in sh- CCAT2 transfected A549/DDP cells ( P<0.01). The decrease degree of cell proliferation was more pronounced after treating with 2 to 8 mg/L of DDP ( P<0.01). Compared with the control group, in the three groups that treated with DDP, the formation of clones and the expression of proliferation marker proteins Ki67 and PCNA were reduce ( P<0.01), while the rate of apoptosis and the expression of apoptosis marker proteins Caspase-3 and Caspase-9 were increased ( P<0.01). Also, the number of invasion cell and the expression of invasion marker proteins VEGF and MMP-14 were reduced in the three groups that treated with DDP ( P<0.01). Among the three groups of DDP-treated cells, the changes in sh- CCAT2 transfected cells was the most obvious ( P<0.01). Compared with the control group, the tumor volume of the three DDP treatment groups was smaller and the differences were statistically significant at 30 d ( P<0.01). The expression of CCAT2 mRNA was decreased in tumor tissues ( P<0.01), while apoptosis increased ( P<0.01). Among the three DDP treatment groups, the A549/DDP cell group transfected with sh- CCAT2 showed the most notable changes ( P<0.01). sh- CCAT2 can inhibit the proliferation of A549/DDP cells, induce apoptosis and reduce the cell invasion ability, thereby inhibiting the growth of A549/DDP cells.

Enhanced Expression of TGFBI Promotes the Proliferation and Migration of Glioma Cells

Background/Aims: Transforming growth factor beta-induced protein (TGFBI) is an extracellular matrix protein induced by TGF-β. Previous studies have reported that the abnormal expression of TGFBI is related to the occurrence and development of some types of cancers, while the role of TGFBI in glioma is uncertain. Methods: The association between TGFBI expression and the prognosis of patients with glioma was analyzed based on data obtained from The Cancer Genome Atlas database. TGFBI expression was analyzed in 3 normal human brains and 57 cases of human gliomas by immunohistochemistry followed by an evaluation of the relationships between TGFBI expression and clinic-pathological features. Furthermore, the RNA interference plasmid pSUPER-shTGFBI was constructed and transfected into U87 and U251 cells to explore the effect of short hairpin RNA against TGFBI (shTGFBI) on cell proliferation, migration, invasion and apoptosis. Western blot analysis was performed to examine the expression of proteins related to apoptosis and proteins in the PI3K/Akt signaling pathway. Results: High TGFBI expression was found to be associated with poor prognosis in patients with glioblastoma multiforme. Immunohistochemistry showed that TGFBI expression was significantly higher in glioma tissue than in normal human brain tissues. The expression level of TGFBI showed no significant correlation with age, sex, lymph-node metastasis, or pathological grade. sh-TGFBI could inhibit proliferation, invasion and migration and induce apoptosis in U87 and U251 cells in vitro. Furthermore, the phosphorylation levels of AKT and mTOR declined significantly in sh-TGFBI transfected U81 and U251 cells when compared with control. Conclusion: TGFBI was up-regulated in glioma cells and played a promoting role in the growth and motility of U87 and U251 cells. These results suggested that TGFBI has the potential to be a diagnostic marker and to serve as a target for the treatment of gliomas.

Inhibition of the Wnt/β-catenin signaling pathway improves the anti-tumor effects of sorafenib against hepatocellular carcinoma

Sorafenib, a multikinase inhibitor, is currently the only approved drug for advanced hepatocellular carcinoma (HCC). The current study tested the hypothesis whether inhibition of the Wnt/β-catenin signaling pathway could improve the anti-tumor effects of sorafenib in HCC. ICG-001, a small molecule which blocks the interaction of β-catenin with its transcriptional coactivator CBP, dose-dependently enhanced the growth-suppressive and apoptosis-induction effects of sorafenib in multiple HCC cell lines. Downregulation of β-catenin by RNA interference increased sorafenib sensitivity, whereas overexpression of β-catenin reduced sorafenib sensitivity in Huh7 cells. The sorafenib-sensitization effect of short hairpin RNA (shRNA)-mediated β-catenin downregulation in Huh7 cells was attenuated by β-catenin overexpression. Mechanistically, sorafenib combined with ICG-001 or shRNA-mediated β-catenin downregulation augmented the induction of apoptosis, and resulted in a significant downregulation of Mcl-1 in HCC cells. In Huh7 cell mouse xenograft model, the combination of ICG-001 and sorafenib showed a more significant growth-retarding effect than single agent treatment of sorafenib or ICG-001. Our data indicate that inhibition of the Wnt/β-catenin signaling pathway improves the antitumor effects of sorafenib against HCC in vitro and in vivo.

Antiproliferative Effect of Specific Inhibitor XAV939 for β-catenin on MCL Jeko-1 Cells

To investigate the effect of short hairpin RNA (shRNA) and XAV939, a specific inhibitor for β-catenin, on growth and apoptosis of mantle cell lymphoma(MCL) Jeko-1 cell line. β-catenin shRNA eukaryotic expression vector was transfected into Jeko-1 cells, the antiproliferative effect of shRNA on Jeko-1 cells was detected by RT-PCR and Western blot. The proliferation inhibitory rate of Jeko-1 cells treated by different doses of XAV939 was assayed by MTT method; the level of apoptosis of Jeko-1 cells was detected by flow cytometry; the expression levels of apoptosis-related protein BCL-2, BAX, CyclinD1, C-MYC and Caspase-3 in Jeko-1 cells were determined by Western blot. The expression of β-catenin mRNA and growth of Jeko-1 cell line were inhibited by shRNA; after Jeko-1 cells treated with 0,2 and 8 µmol/L XAV939 for 48 hours, the cell proliferation rate decreased, while the cell apoptosis rate increased, the expressions of apoptosis-related protein BCL-2, CyclinD1 and C-MYC were down-regulated, on the contrary, the expression of BAX and caspase-3 were up-regulated. The specific inhibition of β-catenin can inhibit Jeko-1 cell proliferation and promote the cell apoptosis.

G-protein coupled receptor 34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro.

Background:Overexpression of G-protein coupled receptor 34 (GPR34) affects the progression and prognosis of human gastric adenocarcinoma, however, the role of GPR34 in gastric cancer development and progression has not been well-determined. The current study aimed to investigate the effect of GPR34 knockdown on the proliferation, migration, and apoptosis of HGC-27 gastric cancer cells and the underlying mechanisms.Methods:The expression of GPR34 in gastric cancer cell line HGC-27 was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. HGC-27 cells were employed to construct the stable GPR34 knockdown cell model in this study. Real-time RT-PCR and Western blotting were applied to validate the effect of short hairpin RNA (ShRNA) on the expression of GPR34 in HGC-27 gastric cells. The proliferation, migration of these cells were examined by Cell Counting Kit-8 and transwell. We also measured expression profile of PI3K/PDK1/AKT and ERK using Western blotting.Results:The ShRNA directed against GPR34 effectively inhibited both endogenous mRNA and protein expression levels of GPR34, and significantly down-regulated the expression of PIK3CB (P < 0.01), PIK3CD (P < 0.01), PDK1 (P < 0.01), phosphorylation of PDK1 (P < 0.01), Akt (P < 0.01), and ERK (P < 0.01). Furthermore, GPR34 knockdown resulted in an obvious reduction in HGC-27 cancer cell proliferation and migration activity (P < 0.01).Conclusions:GPR34 knockdown impairs the proliferation and migration of HGC-27 gastric cancer cells in vitro and provides a potential implication for therapy of gastric cancer.

RNA interference targeting human integrin α6 suppresses the metastasis potential of hepatocellular carcinoma cells

BackgroundIncreased metastasis has been proved to be associated with a poor prognosis for hepatocellular carcinoma (HCC). There are higher-level expressions of integrin α6 in the tissues of HCC patients with a higher fatality rate. The aim of this study is to investigate the effect of short hairpin RNA (shRNA) silencing integrin α6 expression on the proliferation and metastasis in HCC cell lines.MethodsTwo human HCC cell lines, HepG2 and Bel-7402 were transfected with shRNA targeting human integrin α6. Protein and mRNA expression level were determined by western blot and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to detect the transfected efficacy. The metastasis potential of HCC cells was evaluated by their proliferation, adhesion and invasion abilities. Cell proliferation was measured by MTT assay. Adhesion ability was measured by adhesion and spreading assays. The expression of matrix metalloproteinases (MMPs) was measured by qRT-PCR. The potential of invasion was measured by qRT-PCR and Transwell chamber assay. PI3K inhibitor LY294002 was used to explore the signal pathways of integrin α6 in HCC cells.ResultsWestern blot and qRT-PCR detection showed that over 75% of integrin α6 expression in HCC cells was through knockdown by shRNA. Proliferation, adhesion, spreading and invasion of HepG2 and Bel-7402 cells were dramatically decreased in cells transfected with shRNA compared to the control cells. P-ERK and p-AKT were reduced by shRNA targeting integrin α6 and PI3K inhibitor LY294002.ConclusionKnockdown integrin α6 can inhibit the proliferation and metastasis of HCC cells through PI3K/ARK and MAPK/ERK signal pathways by shRNA in vitro. Integrin α6 can mediate the metastasis potential, and can be used as a candidate target for therapy in HCC resulting in improved patients’ survival.

Abstract 391: Short hairpin RNA targeting autotaxin reduces the human gastric carcinoma AGS cell proliferation, migratory and invasive capability in vitro and causes tumor regression in vivo.

Abstract To investigate the effect of short hairpin RNA(shRNA) targeting autotaxin on the migratory and invasive capability of human gastric cancer cell AGS and the growth xenografts in nude mice. The pSUPER-ATX and pSUPER-mock (non-specific), which were constructed in corresponding to the ATX-shRNA and negative control mock-shRNA synthesized on basis of gene sequence, were transfected with blank plasmid pSUPER-control into human gastric cancer cell AGS by Lipofectamine. At 24 h, 48 h and 72 h post-transfection, the cells were harvested and analyzed. The endogenous ATX mRNA and protein of different group AGS cells were detected by RT-PCR and western blot assays. The cell proliferation was measured by MTT assay. In vitro Transwell and Matrigel assay were used to detect the cell migratory and invasive capability. Tumor xenograft model was generated by subcutaneous injection of AGS cells into the dorsum of nude mice. The growth of xenograft tumor was monitored and measured; change of the tumor morphology and mice organs were observed by HE staining. The expressions of autotaxin, MMP-2 and MMP-9 protein in xenograft tumor tissue were detected by immunohistochemistry and western blot. in vitro, both the mRNA and protein level of ATX in pSUPER-ATX group were significantly down-regulated (P&amp;lt;0.01), and the cell proliferation, migration and invasive capability were significantly decreased as well. In vivo, no obvious damage of the organs was found. The pSUPER-ATX significantly suppressed the tumor volume and weight since 7th week after cell transplantation, when compared to the pSUPER-mock, pSUPER-control and WT groups, the inhibition rates were about 50%(P&amp;lt;0.05). However, no significant differences of these parameters were found among the WT, pSUPER-mock and pSUPER-control group. Furthermore, autotaxin, MMP-2/MMP-9 were expressed at significantly lower level in pSUPER-ATX group than in other three groups (P&amp;lt;0.05), and a significant correlation between ATX and MMP-2 expression was displayed (r=0.869, P&amp;lt;0.01). The specific shRNA targeting ATX down-regulated the endogenous ATX of human gastric cancer AGS cells, and could inhibite the AGS cell proliferation, migratory and invasive capability. Moreover, the shRNA targeting autotaxin inhibited the growth of human gastric cancer xenograft in nude mice. Citation Format: Zhouxun Chen, Yingpeng Huang, Xian Shen, Henning Dralle, Cuong Hoang-Vu, Oliver Gimm. Short hairpin RNA targeting autotaxin reduces the human gastric carcinoma AGS cell proliferation, migratory and invasive capability in vitro and causes tumor regression in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 391. doi:10.1158/1538-7445.AM2013-391 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

P38 MAPK regulates the expression of ether à go-go potassium channel in human osteosarcoma cells

BackgroundThe ether à go-go (Eag) channel has been shown to be overexpressed in a variety of cancers. However, the expression and function of Eag in osteosarcoma are poorly understood. In addition, the molecular mechanisms responsible for Eag overexpression in cancer cells remain unclear.MethodsThe expression of Eag in human osteosarcoma cell line MG-63 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of Eag inhibition on MG-63 cell proliferation was assessed in vitro. The effect of short hairpin RNA (shRNA) mediated knockdown of Eag on osteosarcoma growth was evaluated in xenograft model in vivo. The activation of mitogen-activated protein kinase (MAPK) pathway and p53 in MG-63 cells was detected by Western blot analysis.ResultsEag was overexpressed in MG-63 cells. Imipramine or Eag shRNA significantly suppressed the proliferation of MG-63 cells in vitro and in vivo. MG-63 cell proliferation was specifically inhibited by p38 MAPK inhibitor SB203580 or small interference RNA (siRNA). The inhibition of p38 MAPK activation by SB203580 or siRNA reduced Eag protein level but increased p53 protein level. Moreover, the activation of p53 by nutlin-3 induced cell growth arrest in MG-63 cells and reduced Eag protein level, while the inactivation of p53 by pifithrin-alpha (PFT-α) promoted MG-63 cell growth and increased Eag protein expression.ConclusionsEag channel functions as an oncogene to promote the proliferation of human osteosarocma cells. Furthermore, the high expression of Eag in osteosarcoma cells is regulated by p38 MAPK/p53 pathway.

Short hairpin RNA targeting autotaxin reduces human gastric carcinoma AGS cell proliferative, migratory and invasive capabilities in vitro and causes tumor regression in vivo

The aim of this study was to investigate the effect of short hairpin RNA (shRNA) targeting autotaxin (ATX) on the migratory and invasive capability of AGS human gastric cancer cells and the growth of xenografts in nude mice. pSUPER-ATX and pSUPER-mock (non-specific), which were constructed corresponding to the ATX-shRNA and negative control mock-shRNA synthesized based on gene sequence, were transfected with blank plasmid pSUPER-control into AGS human gastric cancer cells using Lipofectamine. At 24, 48 and 72h post-transfection, the cells were harvested and analyzed. The endogenous ATX mRNA and protein of the different groups of AGS cells were detected by RT-PCR and western blot assays. Cell proliferation was measured by MTT assay. Invitro Transwell and Matrigel assays were used to detect the cell migratory and invasive capabilities. A tumor xenograft model was generated by subcutaneous injection of AGS cells into the dorsum of nude mice. The growth of xenograft tumors was monitored and measured; changes in tumor morphology and the organs of mice were observed by H&E staining. The expression of ATX, MMP-2 and MMP-9 protein in xenograft tumor tissues was detected by immunohistochemistry and western blotting. Invitro, both the mRNA and protein levels of ATX in the pSUPER-ATX group were significantly downregulated (P<0.01), and the cell proliferative, migratory and invasive capabilities were also significantly decreased. Invivo, no obvious damage to the organs was found. pSUPER-ATX significantly suppressed the tumor volume and weight from the 7th week after cell transplantation, compared to the pSUPER-mock, pSUPER-control and WT groups; the inhibition rates were approximately 50% (P<0.05). However, no significant differences in these parameters were found among the WT, pSUPER-mock and pSUPER-control groups. Furthermore, ATX, MMP-2 and MMP-9 were expressed at significantly lower levels in the pSUPER-ATX group compared to the levels in the other three groups (P<0.05), and a significant correlation between ATX and MMP-2 expression was found (r=0.869, P<0.01). The specific shRNA targeting ATX downregulated the endogenous ATX of AGS human gastric cancer cells, and inhibited AGS cell proliferative, migratory and invasive potentials. Moreover, shRNA targeting ATX inhibited the growth of human gastric cancer xenografts in nude mice.

GOLGA2/GM130, cis-Golgi Matrix Protein, is a Novel Target of Anticancer Gene Therapy

Achievement of long-term survival of patients with lung cancer treated with conventional chemotherapy is still difficult for treatment of metastatic and advanced tumors. Despite recent progress in investigational therapies, survival rates are still disappointingly low and novel adjuvant and systemic therapies are urgently needed. A recently elucidated secretory pathway is attracting considerable interest as a promising anticancer target. The cis-Golgi matrix protein, GOLGA2/GM130, plays an important role in glycosylation and transport of protein in the secretory pathway. In this study, the effects of short hairpin RNA (shRNA) constructs targeting GOLGA2/GM130 (shGOLGA2) on autophagy and lung cancer growth were evaluated in vitro and in vivo. Downregulation of GOLGA2/GM130 led to induction of autophagy and inhibition of glycosylation in A549 cells and in the lungs of K-ras(LA1) mice. Furthermore, downregulation of GOLGA2/GM130 decreased angiogenesis and cancer cell invasion in vitro and suppressed tumorigenesis in lung cancer mice model. The tumor specificity of sequence targeting GOLGA2/GM130 was also demonstrated. Taken together, these results suggest that induction of autophagy by shGOLGA2 may induce cell death rather than cell survival. Therefore, downregulation of GOLGA2/GM130 may be a potential therapeutic option for lung cancer.

Effect of shRNA-mediated silencing of CTGF and TIMP-1 on mRNA expression of CTGF, TIMP-1, and PC I and secretion of extracellular matrix in rat hepatic stellate cells

To investigate the effect of short hairpin RNA (shRNA)-mediated silencing of CTGF and TIMP-1 in hepatic stellate cells (HSCs) on mRNA expression of TIMP-1, CTGF, and procollagen type-I (PC I), as well as secretion of extracellular matrix (ECM) proteins. Two recombinant expression plasmids harboring shRNAs against CTGF and TIMP-1 (psiRNA-GFP-CTGF and psiRNA-GFP-TIMP-1) were transfected alone or together into TGFb1-activated HSC-T6 cells. The mRNA expression levels of CTGF, TIMP-1, and PC I were detected by fluorescence quantitative PCR (FQ-PCR). The concentrations of secreted PC type-III, hyaluronate (HA), and laminin (LN) were measured by radioimmunoassay (RIA) of culture supernatants. FQ-PCR analysis showed that CTGFshRNA and TIMP-1shRNA specifically inhibited the expression of CTGF, TIMP-1, and PC I mRNA in activated HSC-T6 cells. The concentrations of secreted PC III, HA, and LN were decreased significantly in HSC-T6 cells with shRNA-silenced CTGF or TIMP-1 (P less than 0.01 or P less than 0.05). Moreover, HSC-T6 cells with shRNA-silenced CTGF and TIMP-1 showed a more robust decrease in synthesis of PC III, HA and LN (all, P less than 0.01), as well as in mRNA expression of PC I (P less than 0.05). CTGFshRNA and TIMP-1shRNA effectively inhibit expression of the respective target genes, as well as of PC I, and decrease secretion of ECM components from HSC-T6 cells. Silencing of both CTGF and TIMP-1 produces more robust effects than either in isolation. These data suggest that CTGF and TIMP-1 may be effective targets of shRNA-based gene therapy to treat liver fibrosis.

PKCδ signalling regulates SR-A and CD36 expression and foam cell formation

The formation of foam cells is crucial in the initiation and progression of atherosclerosis. One of the critical steps in foam cell formation is the uptake of low-density lipoprotein (LDL) by macrophages via scavenger receptors (SRs). This study examined the role of protein kinase C (PKC) isoforms on foam cell formation. The effects of short-hairpin RNA (shRNA) and small interfering RNA (siRNA) against classical PKC and novel PKC isoforms were investigated in THP-1-derived macrophages and primary macrophages. The knockdown of PKCδ inhibited oxidized LDL (OxLDL) uptake and intracellular cholesterol accumulation in both cell models. The reduction of PKCδ resulted in decreased expression of SR-A and CD36. Similar conclusions were obtained in examining the effects of a PKCδ inhibitor, rottlerin. Molecular investigation revealed that a decrease in PKCδ inhibited protein kinase B (PKB/Akt) expression and extracellular-signal-regulated kinase (ERK) phosphorylation. Surprisingly, PKCδ-knockdown selectively decreased protein but not the mRNA level of PKCβI and PKCβII. We showed that the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt upstream of ERK decreased SR-A and CD36 expression; however, the inhibition of ERK or PKCβ downstream of ERK attenuated SR-A but not CD36 expression. We further demonstrated that PKCδ could be induced by pro-atherogenic mediators, OxLDL and interferon-γ. Notably, PKCδ, phosphorylated ERK, Akt, and SR-A were highly expressed in human atherosclerotic arteries and CD68-positive macrophages as visualized by immunohistochemical staining. Through regulating PI3K/Akt and ERK activity, PKCδ affects SR-A and CD36 expression and foam cell formation. The results suggest PKCδ as a potential target for atherosclerosis therapeutics.

Effect of short hairpin RNA of Bcl-xL gene on biological behaviors of HepG-2 cells

To investigate the inhibitory effect of short hairpin RNA (shRNA) targeting the Bcl-xL gene on the apoptosis and proliferation of hepatocellular carcinoma HepG-2 cells. We constructed Bcl-xL shRNA, and transfected it into HepG-2 cells, then detected Bcl-xL gene expression on mRNA and protein levels by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Cell proliferation was detected by MTT assay. Cell apoptosis and Bcl-2 and Bax expression were detected by flow cytometry. The expression of Bcl-xL was obviously inhibited by RNA interference. The inhibition rates of Bcl-xL mRNA and protein were 86.6 and 70.2%, respectively. Bcl-xL shRNA inhibited cell proliferation (P < 0.05), whereas promoted apoptosis (P < 0.01). After transfection of Bcl-xL shRNA in HepG-2 cells, the expression of Bcl-2 did not change, but Bax increased significantly. Bcl-xL shRNA effectively inhibits Bcl-xL gene expression and proliferation of HepG-2 cells and promotes apoptosis.

Targeting transforming growth factor βRII expression inhibits the activation of hepatic stellate cells and reduces collagen synthesis

Abnormal production of extracellular matrix (ECM) components significantly contributes to the development of liver fibrosis. This study aimed at examining the effects of short-hairpin RNA (shRNA)-mediated transient knockdown of transforming growth factor βRII (TGFβRII) expression on the proliferation and activation of hepatic stellate cells (HSCs) and synthesis of fibrogenic ECM components in HSC cells. Three different shRNA-expressing plasmids were constructed for the expression of shRNA-(a, b, c) targeting to the rat TGFβRII mRNA beginning at nucleotide position 339, 444 and 528 and they were transfected into a rat stellate cell line, HSC-T6 cells, respectively. The levels of TGFβRII, α-smooth muscle actin (α-SMA), and type I and III collagen expressions were characterized by reverse transcription polymerase chain reaction and Western blot assays. The concentrations of hyaluronic acid (HA) and type IV collagen in the supernatants of cultured cells were measured by enzyme-linked immunosorbent assay. Transfection with the TGFβRII-specific shRNAs resulted in varying levels of inhibition in the expression of TGFβRII in HSC-T6 cells, and transfection with the potent shRNA-c inhibited the expression of TGFβRII in a dose-dependent manner. Knockdown of TGFβRII expression significantly reduced the levels of α-SMA, type I, III and IV collagen, and HA expression in HSC-T6 cells (P < 0.01). In conclusion, our data indicated that knockdown of TGFβRII expression inhibited the activation of HSCs and the production of fibrogenic ECM components in HSC-T6 cells. Therefore, our findings support the notion that TGFβRII is an important factor of the pathogenic process of liver fibrosis.

Effection of short hairpin RNA targeting extracellular signal-regulated kinase 2 on proliferation of Eca109 cells

Objective To study the effect of short hairpin RNA (shRNA) targeting extracellular signal-regulated kinase 2 (ERK2) on proliferation of human esophageal cancer cell line Eca109. Methods The recombinant plasmid (pGeneClipTM-shRNA-ERK2) was constructed and transfected into the human esophageal cancer cell line Eca109 by liposome, and the transfection efficiency was observed by reverse fluorescence microscope. The proliferation ability of esophageal cancer Eca109 cells after transfection was analyzed by methyl thiazolyl tetrazolium (MTF). The expression of ERK2 and inhibitor apoptosis gene Survivin following transfection was detected by Western blotting. Results Sequencing proved that recombinant plasmid was successfully constructed, and the transfection efficiency was about 50%-70% after 72 h following transfection. The Eca109 cell growth at 96 h after transfection was significantly inhibited by 10.45% as compared with negative group and blank group,P ＜0. 05. The protein expression of ERK2 and Survivin was decreased in transfection group as compared with the U0126 group and negative group at 72 h and 96 h ( P ＜ 0. 05 ). Conclusion The recombinant plasmid ( pGeneClipTM-shRNA-ERK2) has been proved not only to be effective for down-regulation of ERK2, but also can affect the expression of Survivin and significantly inhibit the Eca109 cells proliferation. Key words: Esophageal carcinoma; Small interference; Extracellular signal-regulated kinase; Cell proliferation

Mpz gene suppression by shRNA increases Schwann cell apoptosis in vitro

We investigated the effects of short hairpin RNA (shRNA) on myelin protein zero (MPZ) gene expression in Schwann cells (SCs) in vitro and determined the effects of the MPZ gene suppression on the survival of SCs. The MPZ-specific shRNA was introduced into a lentiviral vector for expression under the U6 promoter, and the viral vector-based shRNAs were used to infect cultured SCs. The efficiency of MPZ knockdown was analyzed by real time-PCR (RT-PCR) and western blotting. Flow cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to determine the cell cycle and the amount of apoptosis of SCs. We found that MPZ shRNAs significantly inhibited the expression of the MPZ gene and induced SC apoptosis in vitro. These results provided interesting experimental evidence for understanding the mechanism of demyelinating neuropathies caused by MPZ gene malfunction.