Effect Of Small Interfering RNA Research Articles

Abstract 5860: Understanding the biological role of MMP16 in cisplatin resistance in ovarian cancer

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and lethal of Ovarian Cancer (OC) types, which accounts for eighty percent of deaths. Treatment for OC includes surgery and platinum-based therapy. However, around 70% of women with HGSOC become resistant to chemotherapy. Therefore, new therapeutic agents with higher specificity and efficacy are needed for women with advanced and drug-resistant OC. Matrix metalloproteases (MMPs) are a group of enzymes involved in the degradation of the extracellular matrix (ECM). Deregulation of MMPs contributes to tumor growth and metastasis in several cancer types. Preliminary studies of our research team found that MMP16 is higher in cisplatin-resistant as compared with cisplatin-sensitive OC cells. The aim of this project is to understand the role of MMP16 in the cisplatin resistance of OC cells. We hypothesize that MMP16 increased expression may contribute to cancer progression and drug-resistance. We performed experiments in A2780/A2780CP20 and OVCAR3/OVCAR3CIS human OC cells. To assess the MMP16 protein levels we used western blot analysis. To measure the mRNA levels, we used qRT-PCR. Kaplan-Meier survival curves were generated by interrogation of the KM Plotter patient database. The MMP16 protein and qRT-PCR experiments confirmed that protein and mRNA levels are more abundant in cisplatin-resistant as compared with cisplatin-sensitive cells. Interrogation of the KM plotter database showed that OC patients with higher MMP16 mRNA levels live less as compared with OC patients with lower MMP16 levels. Ongoing experiments are testing the biological effect of small interference RNA (siRNA) targeting MMP16 in cisplatin-resistant OC cells. Citation Format: Jesennia Bonilla-Liriano, Mariela Rivera-Serrano, Victor Reyes-Burgos, Jose Rolon-Perez, Fatima Valiyeva, Pablo Vivas-Mejia. Understanding the biological role of MMP16 in cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5860.

Effect of small-interfering RNA (As1974) and HFq-binding proteins on resistance gene and host microRNA (miR-30C) expression in Pseudomonas aeruginosa-infected patients from Iraq

Abstract Background: MicroRNAs (miRNAs) are a class of small RNAs encoded by the genome that regulate the production of cellular mRNAs that include either incomplete or complete miRNA-binding sites. Objectives: To evaluate the impact of sRNA (As1974) and HFq-binding proteins on the expression of resistance gene and host miRNA (miR-30C) in Iraqi urinary tract infections (UTIs) patients infected with Pseudomonas aeruginosa. Materials and Methods: Patients with UTIs from Baghdad, Iraq’s Baghdad Teaching Hospital, Ghazi Hariri Hospital, Central Laboratories in Medical City, and Al-Yarmouk Hospital were recruited during June 2022 and October 2022 to provide 200 clinical samples. Results: Out of 200 patients with UTIs, only 56 (38.14%) were diagnosed as P. aeruginosa from positive urine samples. Urine samples were analyzed for HFQ gene expression, and the results showed that HFQ is overexpressed in P. aeruginosa-resistant samples compared to sensitive clinical samples, as measured by fold change after normalization with housekeeping gene 16sRNA by folding (21.4971.241 vs. 1.92142 0.04598). Furthermore, normalization of As1974 gene expression in urine samples using 16sRNA revealed a downregulation of As1974 in resistance, with a fold change of 0.66220.0465 versus 2.0121.0243. The miR-30 gene was shown to be downregulated in urine and blood samples (1.360.34, 0.478210.03678) as compared to those of healthy subjects. Conclusions: All ages were susceptible to the UTIs, also females suffered from UTIs more than males. A significant over expression of HFQ-binding protein in P. aeruginosa compared to sensitive clinical samples. Downregulation of As1974 in resistance.

Inhibitory effect of specific small interference RNA targeted against transforming growth factor β1 in mice with hepatic fibrosis infected with Schistosoma japonicum

Objective To investigate the inhibitory effect of small interference RNA (siRNA) targeted against transforming growth factor β1 (TGFβ1) in mice with hepatic fibrosis infected with Schistosoma japonicum. Methods Three short hairpin RNAs (shRNA) targeting different positions of TGFβ1 and one unrelated control sequence (HK) were designed and cloned to a plasmid pGenesil-1 respectively to obtain four recombinant expression vectors. Thirty male BALB/c mice were randomly divided into six groups, including normal group, model group, control group (pGenesil-HK) and three treatment groups (pGenesil-TGFβ1-m1, pGenesil-TGFβ1-m2 and pGenesil-TGFβ1-m3) and each group had five mice. The hepatic fibrosis animal models infected with Schistosoma japonicum were constructed. The levels of hydroxyproline (HYP) in liver tissue were examined by biochemistry. Liver histopathology was examined by hematoxylin-eosin and Masson staining. The mRNA expression and protein expression levels of TGFβ1, mothers against decapentaplegic homolog (Smad) 3, Smad 7 and α-smooth muscle actin (α-SMA) in the livers were detected by quantitative real time polymerase chain reaction (RT-qPCR) and Western blot. Two independent samples t test was used to compare the measurement data between groups. Results The liver fibrogenesis was obviously improved in all treatment groups compared with model group.The levels of HYP of liver tissue in all treatment groups were significantly lower than that in model group (t=14.870, 7.097 and 10.741, respectively, all P<0.01). The mRNA expression levels of TGFβ1, Smad 3 and α-SMA(model group vs pGenesil-TGFβ1-m1 group, t=3.235, 5.141 and 10.026, respectively; model group vs pGenesil-TGFβ1-m2 group, t=3.396, 5.145 and 4.951, respectively; model group vs pGenesil-TGFβ1-m3 group, t=3.511, 5.429 and 6.485, respectively)and protein (model group vs pGenesil-TGFβ1-m1 group, t=8.847, 8.044 and 10.746, respectively; model group vs pGenesil-TGFβ1-m2 group, t=9.709, 7.484 and 10.847, respectively; model group vs pGenesil-TGFβ1-m3 group, t=9.672, 8.766 and 11.508, respectively) were significantly decreased in all treatment groups compared with model group (all P< 0.01), while the levels of Smad 7 mRNA and protein were significantly increased in all treatment groups compared with model group(t=11.742 and 11.211, respectively in pGenesil-TGFβ1-m1 group; t=14.446 and 13.736, respectively in pGenesil-TGFβ1-m2 group; t=10.892 and 10.908, respectively in pGenesil-TGFβ1-m3 group, all P< 0.01). Conclusions Specific siRNA targeting TGFβ1 could significantly inhibit the liver fibrogenesis in mice infected with Schistosoma japonicum. The anti-fibrosis mechanisms of the siRNA maybe associated with the down-regulation of TGFβ1, Smad 3 and α-SMA expressions and up-regulation of Smad 7 expression in liver tissue, which results in suppressing the activation of hepatic stellate cells. Key words: Transforming growth factor beta 1; RNA, small interfering; Schistosoma japonicum; Hepatic fibrosis

Small interfering RNA-mediated monocarboxylate transporter 1 silencing enhances sensitivity of nasopharyngeal carcinoma HNE1/DDP cells to cisplatin-induced apoptosis

To investigate the effect of small interfering RNA (siRNA)-mediated silencing of monocarboxylate transporter 1 (MCT1) on the sensitivity of drug-resistant nasopharyngeal carcinoma HNE1/DDP cells to cisplatin (DDP)-induced apoptosis and explore the possible mechanism. The expression of MCT1 was analyzed in HNE1 and HNE1/DDP cells and in HNE1/DDP cells transfected with siRNA using Western blot. MTT assay was used to assess the inhibitory effect of different concentrations of DDP alone or in combination with MCT1 siRNA on the proliferation of HNE1/DDP cells. The apoptosis of cells treated with MCT1 siRNA or/and DDP (8 µmol/L) was assessed using flow cytometry with PI staining, and the mitochondrial membrane potential was detected using JC-1 staining assay; the expressions of Mcl-1, Bak, Bcl-2, and Bax were analyzed using Western blotting. HNE1/DDP cells showed a high expression of MCT1, and MCT1 silencing using siRNA significantly increased the sensitivity of HNE1/DDP cells to DDP (P<0.05) and partly reversed DDP resistance of the cells. MCT1 silencing enhanced the sensitivity of HNE1/DDP cells to DDP-induced apoptosis. Treatment of HNE1/DDP cells with MCT1 siRNA combined with 8 µmol/L DDP for 24 h resulted in an apoptotic rate of (51.23∓2.86)%, significantly higher than that in cells treated with MCT1 siRNA or DDP alone (P<0.05). The combined treatment also reduced the mitochondrial membrane potential, down-regulated the expression of Mcl-1 and Bcl-2, and up-regulated the expression of Bax in the DDP-resistant cells. MCT1 siRNA can enhance the sensitivity of HNE1/DDP cells to DDP-induced apoptosis, the mechanism of which may involve the down-regulation of Mcl-1 and Bcl-2 and up-regulation of Bax expression.

SiRNAs with decreased off-target effect facilitate the identification of essential genes in cancer cells.

Since the essential genes are crucial to the proliferation and survival of cancer cells, the interference of these genes is promising to be an option for cancer therapy to overcome heterogeneity. However, the essential genes are highly overestimated by RNA interference (RNAi) screenings, which is mainly caused by the pervasive off-target effect of small interference RNA (siRNA) and short hairpin RNA (shRNA). In the present study, we designed Match-Mismatch paired siRNAs to discriminate the on-target effect from off-target effect of siRNAs on cell viability. Only one of the 7 potential essential genes was validated as essential to cell viability, which demonstrates the high false positive rate in RNAi screenings. We modified the siRNA by introducing random nucleotides (N) into the guide strand to mitigate the off-target effect, without significantly compromising the on-target effect. The whole transcriptome profile analysis of cells transfected with siRNAs with or without Nindicates that siRNA-dN (with Ns on both the 2nd and the 18th bases of the guide strand) weakens the off-target effect by decreasing the unintended targets. The optimized siRNAs can be applied in the characterization of essential genes in cancer cells.

肝細胞癌細胞株MHCC97Hのサバイビン発現,増殖およびアポトーシスに及ぼす低分子干渉RNAの効果【Powered by NICT】

Objective To observe the effect of small interfering RNA(siRNA)targeting Survivin gene on urviving expression, proliferation and apoptosis of hepatocellular carcinoma cell line MHCC-97H. Methods Survivin sequence specific siRNA was designed and synthesized.siRNA/liposome complex was transfected into hepatocellular carcinoma cell line MHCC- 97H.The MHCC-97H cells were divided into Survivin siRNA group(Si-survivin), negative control siRNA group(NC group)and blank group(normal control group). Survivin mRNA and protein expressions were detected by reverse transcription-PCR and Western blot, respectively.The proliferation of MHCC-97H was measured by methythiazolydiphenyl-tetrazolium bromide assay.The Annexin V/PI double labeled flow cytometry was employed to measure the apoptosis at 24 h after transfection in different concentrations of Survivin siRNA(12.5, 25.0 and 50.0 nmol/L, respectively). Results After 48 h of transfection, the Survivin mRNA levels were 0.55±0.16(Si-survivin group), 0.85±0.28(NC group)and 0.93±0.40(normal control group), respectively, which were significantly different among 3 groups(F=414, P<0.01). The level of Survivin mRNA was the lowest in Si-survivin group, which was statistically different with NC group and normal control group(t=-20.56, -28.37, all P<0.001). The levels of Survivin protein expression in 3 groups were 0.602±0.005 (Si-survivin group), 0.835±0.007(NC group)and 0.993±0.003(normal control group)at 48 h after transfection, which were statistically different among 3 groups(F=238, P<0.01). The lowest level of protein expression was in Si-survivin group, which was statistically different with NC group and normal control group(t=-40.17, -66.03, all P<0.001). After 72 h and 96 h of transfection, the inhibitory rate of cell growth was significantly higher in Si-survivin group[(19.5±3.6)%, (12.0±0.9)%] compared with that in NC group[(3.6±0.9)%, (-1.3±6.1)%](t=36.18, 42.53, all P<0.05). The apoptosis rates in 12.5, 25.0, 50.0 nmol/L Survivin siRNA were (22.64±2.54)%, (35.37±3.28)% and (53.28±4.35)%, respectively.However, in NC group and normal control group, the apoptosis rates were (8.77±1.25)% and (9.72±1.37)%.The rates were statistically different among those 5 groups(F=35.93, P<0.01). And in the apoptosis rates of siRNA groups in different concentrations were statistically different when compared between each two groups(t=-29.73, -38.57, all P<0.001). Conclusion Survivin specific siRNA can inhibit the proliferation and induce the apoptosis by blocking Survivin gene expression in hepatocellular carcinoma cell line MHCC-97H. Key words: Hepatocellular carcinoma; Survivin; Small interfering RNA

Si-RNA-Mediated Silencing of ADRBK1 Gene Attenuates Breast Cancer Cell Proliferation.

Abstract Breast cancer is the most prominent cause of cancer-related deaths among women worldwide. It has been found that genetic mutations play distinct roles in the onset and progression of breast cancer. Androgenic, beta, receptor kinase 1 (ADRBK1) has been reported to possess oncogenic characteristics vital for cancer cell viability. This study was designed to investigate the effects of small interference RNA (si-RNA)-mediated ADRBK1 knockdown on breast cancer cell growth in vitro. High-expression levels of ADRBK1 were observed in all tested breast cancer cell lines (MDA-MB-231, MCF-7, T-47D, and BT-474). ADRBK1 si-RNA was delivered to breast cancer cells using lentivirus delivery system. Depletion of ADRBK1 significantly attenuated the cell viability and colony-formation ability. Flow cytometry analysis further demonstrated that ADRBK1 silencing led to MDA-MB-231 cell arrest in the G0/G1 phase. Collectively, these results indicate that knockdown of ADRBK1 gene has detrimental effects on breast cancer cell growth, which may be a potential therapeutic approach for the treatment of breast cancer.

Silencing SRY-related high-mobility-group box-6 gene regulating proliferation of glioma cells in vitro

Objective To investigate the effects of small interfering RNA (siRNA)-induced specific silence of SRY-related high-mobility-group box-6 (SOX-6) gene on the proliferation of glioblastoma cells in vitro.Methods The LN229 cells in good conditions were selected to be transfected by SOX-6 silenced by small interfering RNA with fluorescent marker,and observed under the fluorescent microscope to check the transfection efficiency.The cells were divided into 3 groups:transfection of control siRNA-A as control group; transfection of SOX-6 silenced by small interfering RNA as experimental group,and normal culture as blank control group.The expression of SOX-6 as well as its transcriptional activity was examined using reverse transcription-polymerase chain reaction (RT-PCR).The proliferation of cells was analyzed by using counting kit-8 (CCK-8) assay.Results After transfection,RT-PCR results showed that SOX-6 gene expression in LN229 cells was inhibited specifically in experimental group as compared with control group and blank group (0.427 ± 0.032 vs.0.612 ± 0.055 vs.0.609 ± 0.049).CCK-8 assay results showed that cell proliferation in experimental group was decreased significantly as compared with the control group and blank group at 24,48 and 72 h (P ＜ 0.01).Conclusion SOX-6 gene plays an important role in regulating the proliferation of LN229 cells in vitro. Key words: Glioblastoma ; Small interfering RNA ; SRY-related high-mobility-group box-6 gene

Effect of tumor necrosis factor receptor associated factor 6 gene silencing by small interfering RNA on proliferation of odontoblast cells

To investigate the effect of small interfering RNA (siRNA)-mediated inhibition of tumor necrosis factor receptor associated factor 6(TRAF6) gene on murine odontoblast-like cell line, MDPC-23 cell and the effect of TRAF6 on MDPC-23 cell proliferation. The vectors expressing siRNA against TRAF6 were constructed and introduced into MDPC-23 cell with lipofectin , and the cell line with stable expression of siRNA of TRAF6 was obtained by G418 screening and colony culture. Reverse transcription-PCR (RT-PCR) and Western blotting were performed to detect the expression of TRAF6. The proliferation of transfected MDPC-23 cell was investigated through methabenzthiazuron (MTT) and flow cytometry (FCM) assay. The positive single colony was screened out, and was found to express siRNA against TRAF6 effectively because both TRAF6 mRNA and protein relative expression were significantly decreased in the experimental group (pSUPER-TRAF6siRNA: mRNA 0.163 ± 0.008, protein 0.215 ± 0.006) compared with controls (pSUPER: mRNA 0.778 ± 0.017, protein 0.964 ± 0.007 (P < 0.001). The A value of treated pSUPER-TRAF6siRNA cells (3 d: 0.46 ± 0.03, 5 d: 1.35 ± 0.06) was increased compared with controls (P<0.01). The result of proliferation index (PrI) was also increased compared with controls [pSUPER- TRAF6siRNA: (24.1 ± 2.2)%; pSUPER (11.2 ± 1.0)%; control (10.5 ± 0.7)%, P < 0.01]. The transcription and expression of TRAF6 gene were inhibited. The proliferation ability was increased in MDPC-23 cells by the constructed pSUPER-TRAF6siRNA vector. It may further influence the formation and repair of dentin, and may be involved in the regulation of normal tooth eruption and process of dentin repair after injury.

BCR/ABL mRNA targeting small interfering RNA effects on proliferation and apoptosis in chronic myeloid leukemia.

To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5 nmol/L~50 nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations (IC50) of siRNA1384, siRNA1276 and siRNA1786 for 24 hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50 nmol/L siRNA transfection. Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

Effect of down-regulation of astrocyte elevated gene-1 expression by small interfering RNA inhibits cell proliferation and induces apoptosis in human hepatocellular carcinoma HepG2 cells

Objective To investigate the effect of small interfering RNA (siRNA)-induced astro-cyte elevated gene-1 (AEG-1) down-regulation on the proliferation,apoptosis and cell cycle of human hep-atocellular carcinoma cells and its mechanism.Methods siRNA and AEG-1 siRNA were transfected to Human hepatocellular carcinoma HepG2 cells,respectively.The expression of AEG-1 mRNA was detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-PCR).The cell proliferation was detected by methyl thiazol tetrazolium (MTT) method.The apoptosis and cell cycle were analyzed by flow cytometry.Results Expression of AEG-1 mRNA in AEG-1 siRNA group (0.43 ±0.04)was significantly lower than that in control siRNA group (0.94 ± 0.08) and control group (0.96 ± 0.09)(P ＜0.01),respectively.After treated with AEG-1 siRNA for 72 h,the absorbance values of AEG-1 siRNA group (1.25 ± 0.08) was significantly lower than that in control siRNA group (2.49 ± 0.14) and control group (2.64 ± 0.14) (P ＜ 0.01),respectively.The cell rate in G0/G1 phase (61.38 ± 2.31) ％ was significantly higher than that in control siRNA group (42.15 ± 1.64)％ and control group (41.24 ± 1.97)％(P ＜ 0.01),and the cell rates in S phase (25.36 ± 1.21) ％,G2/M phase (13.26 ± 0.97) ％ were lower than those in control siRNA group (32.52 ± 1.23) ％,(25.33 ± 0.86) ％ and control group (33.60 ±1.24) ％,(25.16 ± 0.79) ％ (P ＜ 0.01),respectively.Conclusion The mRNA expression of AEG-1 is down-regulated by AEG-1 siRNA in human hepatocellular carcinoma cells.Knockdown of AEG-1 expres-sion in human hepatocellular carcinoma cells significantly inhibits cell proliferation and apoptosis,and induces cell arrest in G0/G1 phase of the cell cycle. Key words: Astrocyte elevated gene-1 ; Cancer, hepatocellular; Small interfering RNA ; Apoptotic

Silencing of CXCR4 Inhibits Tumor Cell Proliferation and Neural Invasion in Human Hilar Cholangiocarcinoma

Background/AimsTo evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing.MethodsAn immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay.ResultsThe expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation.ConclusionsCXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.

Inhibitory effect of small interfering RNA mediated by tumor target cell penetrating peptides on in hepatoma cell line SMMC-7721

Objective To observe the inhibitory effect of small interfering RNA (siRNA) mediated by tumor target cell penetrating peptides (CPPS) targeting metalloproteinase on hepatoma cell line SMMC-7721.Methods SMMC-7721 cells were cultured,and divided into experimental group and control group.In experimental group,siRNA/CCPS complex was added,and in control group,silencer negative control/CCPS was added.The cells were harvested after culture for 48 h.Flow cytometry was use to examine the cell cycle.The human telomerase reverse transcriptase (hTERT) gene expression level was detected by using quantitative polymerase chain reaction (PCR).Methyl thiazol tetrazolium (MTT) assay was used to meassure the inhibitory effect of siRNA/CCPS complex on SMMC-7721 cells.Results As compared with control group,the proportion of cells in G1 phase in experimental group was increased (75.14％ vs.62.55％),while that in G2 phase in experimental group decreased (11.39％ vs.12.14％),and that in the S phase also reduced (13.47％ vs.25.31％).Treatment of CPPS plasmid complexes could arrest the cells in the G1 phase,and the expression quantity of hTERT mRNA in experimental group was 74％ of that in control group.After treatment with CPPS plasmid complexes for 48 h,the proliferation inhibition rate of SMMC-7721 cells was 34.82％.Conclusion The CCPS can be a transmitter to transfect siRNA into hepatoma cells and suppress hepatoma cells to some extent. Key words: Carcinoma, hepatocellular; Cell penetrating peptides; Tumor targeting; Small interfering RNA

Involvement of microRNA-181b in the gemcitabine resistance of pancreatic cancer cells

Background/objectivesMicroRNAs (miRs) have been shown to regulate the sensitivity to several chemotherapeutic agents in various types of cancers. MiR-181b is one of such regulators, yet its importance in pancreatic cancer is not determined so far. The aim of this study was to investigate the relationship between microRNA (miR)-181b expression and gemcitabine resistance in pancreatic cancer cells. MethodsThe effects of overexpression or knockdown of miR-181b on four pancreatic cancer cell lines exposed to gemcitabine were examined. The induction of apoptosis and the changes of the cylindromatosis (CYLD) protein were examined. Furthermore, the effect of small interference RNA for CYLD (siCYLD) on cell viability and the relationship between CYLD and nuclear factor kappa B (NF-κB) were investigated. ResultsThe expression of miR-181b was higher in BxPC3, Panc1 and PSN1 cells compared with MiaPaCa2 cells. Pre-miR-181b transfection into MiaPaCa2 cells increased their gemcitabine resistance, whereas anti-miR-181b transfection into the other pancreatic cancer cell lines reduced their resistance to gemcitabine and led to the induction of apoptosis. The protein levels of CYLD were increased by anti-miR-181b in Panc1 and PSN1 cells. Inhibition of CYLD increased the NF-κB activity and gemcitabine resistance in Panc1 and PSN1 cells. ConclusionsThe present study demonstrated that miR-181b was associated with the resistance of pancreatic cancer cells to gemcitabine, and verified that miR-181b enhances the activity of NF-κB by inhibiting CYLD, leading to the resistance to gemcitabine. Our results suggest that miR-181b is a potential target for decreasing gemcitabine resistance.

Effects of small interfering RNA against protein kinase Cε gene on proliferation, migration and invasion of human renal clear cell carcinoma cell line 769P

Objective To evaluate the effects of small interfering RNA (siRNA) against protein kinase Ce (PKCe) gene on the proliferation,migration and invasion of human clear cell RCC cell line 769P cells.Methods PKCe-siRNA was transfected into the 769P cells.The mRNA and protein expression of PKCe was detected by using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting,respectively.The proliferation of 769P cells was measured by methyl thiazol tetrazolium (MTT) assay and colony formation assay.Cell migration and invasion was assessed by using wound-healing assay and CHEMICON cell invasion assay,respectively.Results PKCe-siRNA efficiently down-regulated the PKCe expression,which resulted in inhibition of the proliferation,migration,and invasion of clear cell RCC cell line 769P.Conclusion PKCeis may be related with proliferation,migration and invasion of 769P cells,and may be a potential therapeutic target for clear cell RCC. Key words: Protein kinase Ce; Renal clear cell carcinoma; Small interfering RNA

Small interfering RNA in silencing cox-2 expression enhances radiosensitivity of esophageal cancer EC9706 cell

To explore the effects of small interfering RNA (siRNA) specific to cox-2 gene on the radiosensitivity of esophageal cancer cell EC9706. The siRNA vector was established for cox-2 gene and then induced into esophageal cancer cell EC9706 by lipofectamine. G418 screening yielded stably transfected cells. After the irradiation of 0, 2 and 4 Gy, the cellular expression levels of cox-2, matrix metalloproteinase-2 (MMP2), Bax and Bcl-2 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and those of cox-2 protein, AKT protein and phosphorylation AKT protein (pAKT) by Western blot. Cell apoptosis was examined by flow cytometer. Invasion of cells was detected by invasive assay in vitro. The invasive and metastatic capacities of cancer cells were assessed by invasion assay in vitro. Proliferative potential was quantified by clone-forming assay. The sequencing result confirmed that siRNA vector pRNA-U6 for cox-2 gene was established. The results of 1-sinCox214, RT-PCR and Western blot showed that cox-2 gene expression of transfected EC9706 cell was silenced efficiently. After the irradiation of 0, 2 and 4 Gy, the expressions of MMP2, Bcl-2 mRNA, AKT protein and pAKT in silencing cox-2 gene expression significantly decreased. There was an inverse correlation with irradiation dose. The Bax mRNA expression evidently increased directly with irradiation dose; the apoptotic rate in cox-2 silencing groups was evidently higher than the control groups. And the difference was significant (P < 0.01); invasion cells in vitro in Cox-2 silencing groups evidently decreased with significant difference (P < 0.01). The colony formation rate of cells decreased obviously in cox-2 silencing groups after the irradiation of 0, 1, 2, 4, 6, 8 and 10 Gy (P < 0.01). Small interference RNA in silencing cox-2 gene expression can enhance significantly the radiosensitivity of esophageal cancer EC9706 cells. And the mechanism may be related with MMP2, Bax, Bcl-2, AKT protein and pAKT protein.

Silencing of a novel tumor metastasis suppressor gene LASS2/TMSG1 promotes invasion of prostate cancer cell in vitro through increase of vacuolar ATPase activity

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), is a newly found tumor metastasis suppressor gene in 1999. Preliminary studies showed that it not only suppressed tumor growth but also closely related to tumor metastasis, however, its molecular mechanisms is still unclear. There have been reported that protein encoded by LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase. Thus, in this study, we explored the effect of small interference RNA (siRNA) targeting LASS2/TMSG1 on the invasion of human prostate carcinoma cell line PC-3M-2B4 and its molecular mechanisms associated with the V-ATPase. Real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot revealed dramatic reduction of 84.5% and 60% in the levels of LASS2/TMSG1 mRNA and protein after transfection of siRNA in PC-3M-2B4 cells. The V-ATPase activity and extracellular hydrogen ion concentration were significantly increased in 2B4 cells transfected with the LASS2/TMSG1-siRNA compared with the controls. The activity of secreted MMP-2 was up-regulated in LASS2/TMSG1-siRNA treated cells compared with the controls; and the capacity for migration and invasion in LASS2/TMSG1-siRNA treated cells was significantly higher than the controls. Thus, we concluded that silencing of LASS2/TMSG1 may promote invasion of prostate cancer cell in vitro through increase of V-ATPase activity and extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2.

The effects of siRNA against RPL22 on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells

The aim of this study was to investigate the effects of small interference RNA (siRNA) against ribosomal protein L22 (RPL22) on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells (HPASMCs). HPASMCs were transfected with siRNA against RPL22, incubated in smooth muscle cell culture medium (SMCM) and ET-1. RPL22 gene expression and protein levels of HPASMCs were measured by real-time PCR and western blotting, respectively. PCNA, CCK-8 immunohistochemistry and flow cytometry analysis were used to evaluate HPASMC proliferation. Cyclin D1 (CCND1) expression was also assayed. Following transfection with siRNA against RPL22, RPL22 expression was significantly inhibited in the control and ET-1 groups. HPASMC proliferation was significantly suppressed by transfection with siRNA against RPL22. Moreover, CCND1 was also downregulated by inhibiting RPL22 expression. In conclusion, these data suggest that inhibition of RPL22 expression can suppress HPASMC proliferation and CCND1 expression. The effects of siRNA against RPL22 on HPASMC proliferation are considered to be mediated through inhibition of CCND1 expression.

SiRNA against presenilin 1 (PS1) down regulates amyloid β42 production in IMR-32 cells

BackgroundOne of the pathological hallmarks of Alzheimer's disease (AD) is the deposition of the ~4 kDa amyloid β protein (Aβ) within lesions known as senile plaques. Aβ is also deposited in the walls of cerebral blood vessels in many cases of AD. A substantial proportion of the Aβ that accumulates in the AD brain is deposited as Amyloid, which is highly insoluble, proteinaceous material with a β-pleated-sheet conformation and deposited extracellularly in the form of 5-10 nm wide straight fibrils. As γ-secretase catalyzes the final cleavage that releases the Aβ42 or 40 from amyloid β -protein precursor (APP), therefore, it is a potential therapeutic target for the treatment of AD. γ-Secretase cleavage is performed by a high molecular weight protein complex containing presenilins (PSs), nicastrin, Aph-1 and Pen-2. Previous studies have demonstrated that the presenilins (PS1 and PS2) are critical components of a large enzyme complex that performs γ-secretase cleavage.MethodsIn this study we used RNA interference (RNAi) technology to examine the effects of small-interfering RNA (siRNA) against PS1 on expression levels of PS1 and Aβ42 in IMR-32 Cells using RTPCR, western blotting and immunofluorescence techniques.ResultsThe results of the present study showed down regulation of PS1 and Aβ42 in IMR32 cells transfected with siRNA against PS1.ConclusionOur results substantiate the concept that PS1 is involved in γ-secretase activity and provides the rationale for therapeutic strategies aimed at influencing Aβ42 production.

SiRNA targeting of Cdx2 inhibits growth of human gastric cancer MGC-803 cells

To investigate the effects of small interference RNA (siRNA) targeting of Cdx2 on human gastric cancer MGC-803 cells in vitro and in vivo. The recombinant pSilencer 4.1-Cdx2 siRNA plasmids were constructed and transfected into gastric cancer MGC-803 cells in vitro. The stable transfectants were selected. The effects of Cdx2 siRNA on growth, proliferation, cell cycle, apoptosis, migration and invasiveness of human gastric cancer MGC-803 cells were evaluated and the expression of phosphatase and tensin homolog (PTEN), caspase-9 and caspase-3 was observed in vitro by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting analysis. We also investigated the effect of Cdx2 siRNA on growth of MGC-803 cells in nude mice in vivo. Cdx2 siRNA led to inhibition of endogenous Cdx2 mRNA and protein expression as determined by RT-PCR and Western blotting analysis. Cdx2 siRNA significantly inhibited cell growth and proliferation, blocked entry into the S-phase of the cell cycle, induced cell apoptosis, and reduced the motility and invasion of MGC-803 cells. Cdx2 siRNA also increased PTEN expression, and activated caspase-9 and caspase-3 in MGC-803 cells in vitro . In addition, siRNA targeting of Cdx2 inhibited the growth of MGC-803 cells and promoted tumor cell apoptosis in vivo in nude mice tumor models. Cdx2 was involved in regulating pro-gression of human gastric cancer cells MGC-803. Manipulation of Cdx2 expression may be a potential therapeutic strategy for gastric cancer.