Objective To construct vectors that express phosphatidylinositol-3-kinase, catalytic, beta polypeptide （PIK3cb） shRNA in eukaryon plasmid catalyzed by PI3K in rat, then test their effects on intimal hyperplasia in transplanted vein graft. Methods One hundred and fifty SD rats were randomly divided into six groups （n=25, in each group）: blank (25% Pluronic F-127), shRNA-1, shRNA-2, 1/2 （shRNA-1+shRNA-2）, negative control (pGenesil-1 scramble shRNA) and positive control （wortmannin） group. The jugular vein in rats were interpositioned autologously into the common carotid artery. shRNA and 25% Pluronic F-127 were mixed and coated around the transplanted vein in three PIK3cb shRNA groups. Every 5 samples were removed according to the time point (1, 3, 7, 14 and 28 days after operation), respectively. The thickness of intima and neointima area were calculated and analyzed by computer system. The PCNA expression was detected by Western blot and SP immunohistochemistry. Results The intimal thickness of three PIK3cb shRNA groups were lower than those in the blank group and negative control group on day 3, 7, 14, 28 after operation (P＜0.05); The neointima area in three PIK3cb shRNA groups (except shRNA-2 group on day 3, 7) began to decrease significantly from day one (P＜0.05). The protein expression of PCNA in three PIK3cb shRNA groups on day 3 after operation were decreased compared with blank group and negative group (P＜0.05). The percentage of the PCNA positive cells area in three PIK3cb shRNA groups were significantly lower than those in blank group and negative control group in each time point (Plt;0.05). There were no significant differences between blank and negative control group in different time points (Pgt;0.05). Conclusion The PIK3cb shRNA can effectively inhibit the proliferation of vascular smooth muscle cell, which may provide a new gene therapy for the prevention of vein graft restenosis after bypass grafting.
ObjectiveTo establish MCF-7 cell lines with different ERα/ERβ expression level and observe their biological behaviors. MethodsERα or ERβ gene were silenced by RNA interference, and the cell lines with different ERα or ERβ expression level were obtained in MCF cell lines. MTT assay, flow cytometry, RT-PCR, double layer softagar clony formation test, and Matrigel adhesion assay were used to detect the abilities of cell proliferation, apoptosis, tumorigenesis, and adhesion. ResultsThe stable expression cell lines with ERαlow/ERβhigh or ERαhigh/ERβlow were established successfully. After ERα gene knockdown, MCF-7 grew slowly and was arrested at phase G0-G1. Apoptosis of MCF-7 was induced and the capacity of tumorigenesis and adhesion in vitro were weakened. However, the characteristics mentioned above except for adhesion changed to the opposite sides after ERβ gene knockdown. ConclusionsThe ERα gene silence can inhibit the formation of tumor, however the ERβ gene silence can promote tumor growth, invasion, and metastasis. Therefore, may be a useful approach on the breast cancer therapy.
【 Abstract 】 Objective To observe the effect of disruption of hypoxia inducible factor-1 α (HIF-1 α ) pathway by small hairpin RNA (shRNA) on chemosensitivity of human hepatocellular carcinoma (HCC) cells and to reveal the correlative mechanisms. Methods Plasmid of pshRNA-HIF-1α was transfected into HepG2 cells by lipofectamine. HepG2/pshRNA-HIF-1α (HepG2/pshRNA) cell lines were obtained by selection of HepG2 cells in G418. Meanwhile, plasmid of empty vector (pHK) was transfected as a control (HepG2/pHK). The mRNA and protein expression levels of HIF-1α and mdr1 were investigated by RT-PCR and Western blot respectively. Using CoCl2 to simulate the hypoxia condition, growth inhibition and apoptosis rates of HepG2 cells under different dosages of chemotherapeutic agents (adriamycin) were measured by MTT assay and flow cytometry (FCM) ． ResultsCompared with HepG2/pHK cells, the mRNA and protein expression levels of HIF-1αand mdr1 were obviously down-regulated in HepG2/pshRNA cells. At the same time, the proliferation inhibition and apoptosis rates were evidently increased after transfection with pshRNA-HIF-1α(P＜0.05)，which decreased the expression of HIF-1αto 82.18% at mRNA level and 75.51% at protein level. There was no significant effect of transfection pHK (Pgt;0.05). Conclusion These data demonstrates that HIF-1α interference by shRNA increased the sensitivity of HCC chemotherapy and the reversal of multidrug resistance, which may be done by down-regulating the transcription of mdr1 and the translation of P-gp. Blocking HIF-1αin HCC cells may offer an new avenue for gene therapy．
ObjectiveTo observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells. MethodsCultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000±500) Lux for 6 hours to establish the light injured model. Light injured cells were divided into HtrA1 siRNA group, negative control group and blank control group. HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively. The proliferation of cells was assayed by CCK-8 method. Transwell test was used to detect the invasion ability of these three groups. Flow cytometry was used to detect the cell cycle and apoptosis. The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively. ResultsThe mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62, 15.09; P<0.05). Compared with negative control group and blank control group, the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37, 4.52), migration (t=9.56, 12.13), apoptosis (t=23.37, 29.08) and decreased mRNA (t=17.36, 11.32, 7.29, 4.05) and protein levels (t=12.02, 15.28, 4.98, 6.24) of HtrA1 and VEGF-A. Cells of HtrA1 siRNA group mainly remained in G0/G1 phase, the difference was statistically significant (t=6.24, 4.93; P<0.05). ConclusionKnockdown of HtrA1 gene may reduce the proliferation, migration capability and apoptosis of light-injured RPE cells, and decrease the expression of VEGF-A.
Objective To investigate the influence of RNA interference targeting c-Jun gene on the proliferation of rat vascular smooth muscle cells (VSMCs). Methods The experiment was performed with c-Jun siRNA （c-Jun siRNA group）, control reverse sequence siRNA (control siRNA group) or no siRNA (control group). VSMCs were transfected with siRNA targeting c-Jun gene by liposome. Effects of c-Jun siRNA on mRNA and protein expressions of c-Jun were examined by RT-PCR analysis and Western blot respectively. MTT test and 3H-TdR incorporation were used to detect VSMCs proliferation. Cell cycle analysis of VSMCs in vitro was determined by flow cytometer. Results The expression levels of mRNA and protein of c-Jun in c-Jun siRNA group were significantly lower than those in control group （P＜0.05， P＜0.01). There was no significant difference between control group and control siRNA group （Pgt;0.05）. Proliferation activity of VSMCs decreased significantly in c-Jun siRNA group compared with that in control group （P＜0.05） and VSMCs was blocked in the G0/G1 phase of cell cycle significantly （P＜0.05）. There was no significant difference between control group and control siRNA group （Pgt;0.05）. Conclusion c-Jun gene silenced by RNA interference can inhibit VSMCs proliferation effectively in vitro.
Objective To study the inhibitory effect of RNA interference (RNAi) on bcl-2 expression of vascular smooth muscle cells （VSMCs) in rabbit. Methods The expression vector of bcl-2 gene-targeting small interference RNA (pshRNA-bcl-2) was constructed and was transfected into VSMCs by lipofectamine, and the unloaded vector was used as control. The expression of bcl-2 mRNA was identified by RT-PCR and Western blot, respectively. The growth of the transfected VSMCs was examined by MTT. Results The pshRNA-bcl-2 may inhibit the expression of bcl-2 gene at the levels of transcription and translation. There were significant differences (P＜0.01) of the expressions of bcl-2 mRNA between the VSMCs that were transfected with pshRNA-bcl-2 and the ones in plasmid transfected group and control group, respectively. There was a significant difference (P＜0.01) in the growth of VSMCs between the plasmid transfected and the control groups. Conclusion The plasmid containing the small interference RNA of bcl-2 may have an inhibitory effect on the cell growth and endogenous expression of bcl-2 gene at the levels of transcription and translation in VSMCs.
Objective To observe the effect of RNA interference (RNAi) on HepG2 hepatic cancer cell by small interfering RNA (siRNA). Methods siRNA targeting vascular endothelial growth factor (VEGF) gene was transfected into HepG2 cells by LipofectimineTM 2000. The VEGF mRNA and protein were respectively detected by real-time quantitive PCR and Western blot, and the concentration of VEGF protein in the cell culture supertant was determined by ELISA at 48 h after culture. Results The average efficiency of siRNA transfection was (90.4±2.9)% after 6 h cell culture. The expressions of VEGF mRNA and protein in HepG2 cells could be effectively suppressed by siRNA, and the concentration of VEGF protein in the cell culture supertant was also decreased. Conclusion siRNA can knock down the expression of VEGF gene and decrease the concentration of VEGF protein in HepG2 cells.
Objective To study the interferencing and anti-tumor effects of lentiviral vector of siRNA targeting IGF1R and EGFR gene of the liver cancer cell. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and connected to the pLVTHM vector, named pLVTHM-IGF1R, into whom the EGFR-siRNA expression frame containing H1 promotor synthesized by RT-PCR was cloned to generate pLVTHM-IGF1R-EGFR-siRNA. The 293T cells were cotransfected by 3 plasmids of pLVTHM-IGF1R-EGFR-siRNA, psPAX2 and pMD2G to enclose LVTHM-IGF1R-EGFR-siRNA, which was amplified in large amount and purified by caesium chloride density gradient centrifugation for measurement of virus titer. SMMC7721 cells infected by LVTHM-IGF1R-EGFR-siRNA were infection group, the untreated SMMC7721 cells and blank vector plasmid LVTHM were two control groups (SMMC7721 cell group and blank vector group). The effect of LVTHM-IGF1R-EGFR-siRNA on IGF1R and EGFR expressions of SMMC7721 cells were detected by RT-PCR and Western blot. The antitumor potential of LVTHM-IGF1R-EGFR-siRNA to SMMC7721 cells was evaluated by Cell Counting Kit-8 assay for cell growth and TUNEL for apoptosis respectively. Results LVTHM-IGF1R-EGFR-siRNA was constructed successfully. Functional pfu titers of LVTHM-IGF1R-EGFR-siRNA was 4.58×109 pfu/ml. Protein and mRNA expression of IGF1R and EGFR of infection group were less than those of blank vector group and SMMC7721 cell group （P＜0.05）, LVTHM-IGF1R-EGFR-siRNA was more effective to inhibit the proliferation and promote apoptosis of SMMC7721 cells （P＜0.05）. Conclusion LVTHM-IGF1R-EGFR-siRNA expressing IGF1R-EGFR-siRNA can inhibit the expression of IGF1R and EGFR, and may be used for further investigation of gene therapy of liver cancer.
【Abstract】Objective To explore the application of RNA interference (RNAi) in colorectal cancer gene therapy. Methods The related literatures in recent years were reviewed. Results RNAi causes a high effective and distinctive degradation of mRNA homologous in sequence to the dsRNA. This new technology has been successfully applied to research the genesis and the growth of colorectal cancer.Conclusion RNAi has been a new focus in gene therapy for colorectal cancer.
Objective To investigate the radiation-sensitizing effects of Ku80 silencing by siRNA interference for A549 lung cancer cells. Methods The sequences of Ku80 siRNA and negative siRNA were chemically synthesized and transfected into A549 lung cancer cells by lipofectamine. RT-PCR and Western bolt analysis were used to determine Ku80 gene expression. The transfected cells in culture dishes were irradiated with X ray at doses of 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy, respectively. Once all treatments were completed, the cells were processed with the colony formation assay. Results RT-PCR detection showed that Ku80 mRNA levels in A549 lung cancer cells were reduced after transfected with Ku80 siRNA at 24 h, 48 h and 72 h time points. Western blot analysis showed that Ku80 protein content decreased at 48 h and 72 h time points compared with the control group ( P lt; 0. 05 ) . Cloning formation assay indicated that radiosensitivity of A549 lung cancer cells was enhanced after transfected with Ku80 siRNA. Conclusion Ku80 siRNA can effectively inhibit Ku80 gene expression of A549 lung cancer cells, and therefore enhance its radiosensitivity.