Objective To investigate the inhibitory effect of survivin antisense oligonucleotides (ASODN) on proliferation of pancreatic cancer cells PANC-1. Methods The ASODN and sense oligodeoxynucleotides (SODN) were complementary to survivin sequences. FAM-marked ASODN was transfected into PANC-1 cells mediated by positive ion liposome as ASODN group. Blank control group (normal cells), negative control group (normal medium)， and SODN group were established for comparison. The transfection efficiency was detected by flow cytometry (FCM) after transfection; MTT assay was used to detect cytotoxicity; Cell morphological changes were examined by transmission electron microscopy; The cell cycle and apoptotic rate were analyzed by FCM; Immunohistochemical staining techniques were used, and the expressions of survivin were observed under light microscopy, examined and analysed by computer image. Results ①The transfection efficiency was 31.9%, 37.4%, 41.4%, 52.6%, 24.2%, 11.4%, 16.1%, and 15.5% when the transfecting concentration of ASODN was 50, 100, 150, 200, 250, 400, 600, and 800 nmol/L, respectively; The transfection efficiency was 12.0%, 50.8%, and 11.2% when the inoculated cells was 2×104/well, 2×105/well, and 2×106/well, respectively; The transfection efficiency was 58.8%, 34.0%, and 23.6% when 2 μl, 3 μl, and 4 μl liposome was used during transfection, respectively. ②Cell gap was oversize, morphous was round, adherent cells were less after transfection under fluorescence microscope. ③The inhibition rate in the ASODN group was higher than that in each control group (Plt;0.05) on 24, 36, 48 h after treating by survivin ASODN, which increased as time prolonged (Plt;0.05). ④The apoptosis showed a ladder-shaped line in the ASODN group. ⑤Apoptotic morphology was demonstrated in the ASODN group, such as apoptotic cells with nuclear chromatin highly concentrated, crescent nuclear staining aggregated by the side nuclear membrane, nucleolus disappeared by AO and EB stains. ⑥The apoptotic rate 〔（38.1±3.4）%〕 in the ASODN group was higher than that in the SODN group 〔（4.16±1.7）%〕, Plt;0.05. ⑦G2/M cell cycle arrested in the ASODN group. ⑧After transfection, the expression of survivin protein in the ASODN group was significantly lower than that of each control group (Plt;0.05). Conclusions The optimal transfection conditions are as following: the cell count of 2×105/well, concentration of ASODN 200 nmol/L, and cationic liposome oligofectamine 2 μl, respectively. Survivin ASODN can inhibit the proliferation of pancreatic cancer cells and induce their apoptosis.
The pathogenesis of diabetic retinopathy (DR) is complex. Antisense non-coding RNA (ANRIL) in the INK4 locus in long-chain non-coding RNA (lncRNA) is closely related to cell proliferation, differentiation, and individual development. It plays an important role in the dysplasia of retinal vascular endothelial cells and is a new field in the study of the pathogenesis of DR. According to the researches at present, ANRIL may plays its role in the occurrence and development of DR through the signal pathway of nuclear factor-κB and ROS/polyadenylation diphosphate ribose polymerase, and interact with p300, miR-200b, and EZH2 to regulating the expression and function of VEGF. Specific blocking ANRIL and its related pathways may become a new target in the treatment of DR.
【Abstract】ObjectiveTo study the effect of down-regulation of E-cadherin on the invasion ability of tumor cells. MethodsHuman pancreatic carcinoma cell line JHP-1 was treated with E-cadherin antisense oligodeoxynucleotied (ASODN). The immunocytochemistry, Western blot were used to detect the expression and the contents of E-cadherin in the tumor cells, and the invasive ability of tumor cells were evaluated by invasive-MTT assay. Results Treated with E-cadherin ASODN, the expression of E-cadherin on JHP-1 cells were reduced, and the protein contents were decreased as well compared with control groups and ODN group. The invasive ability of JHP-1 cells to the basement membrane was increased (P＜0.001) compared with ODN group and control group. ConclusionE-cadherin was related to the invasive ability of tumor cells.
Objective To investigate the effect s of T lymphoma invasion and metastasis inducing factor 1 ( Tiam 1) antisense oligonucleotides (ASODN) on morphological remodeling of gast ric cancer cells. Methods The high-invasive and metastastic subgroup (MH ) was separated f rom human gast ric cancer cell line MKN245 (M0 ) by laminin adhesion method in vi t ro. And they were divided into four group s according to different further t reatment s : no t ransfection group (cont rol group ) , liposome t ransfection group , sense oligonucleotides2liposome t ransfection group ( SODN t ransfection with liposome group ) and antisense oligonucleotides2liposome t ransfection group (ASODN t ransfection with liposome group) . Then the expressions of Tiam 1 mRNA and protein were detected by RT-PCR and flowcytomet ry , respectively. The morphology changes between Tima 1 ASODN t ransfected MH cells and no t ransfected cells were observed by using HE stain , cytoskeletal protein stain and scanning elect ronic microscope (SEM) . Results Compared with the other group s , the expressions of Tiam 1 mRNA and protein in MH cells were significantly decreased af ter the cells were t ransfected with 0. 43 μmol/ L Tiam 1 ASODN ( P lt; 0. 01) . Additionally , it was observed that the t ransfected MH cells had less membrane surface projections , fewer or shortener pseudopodia , less irregular cytoskeletal network and less spotted-like actin bodys than no t ransfected MH cells did. Conclusion ASODN t ransfection could effectively suppress the expression of Tiam 1 and the remodeling in gast ric cancer cells , which may play an important role in the invasion and metastasis of gast ric cancer cells.
Objective To evaluate the effect of vascular endothelial cell growth factor (VEGF) antisense oligodeoxynucleotides (ASODNs) on the expression of VEGF in rats with oxygen-induced retinopathy. Methods Thirty newborn Sprague-Dawley (SD) rats were randomly divided into 3 groups:normal control group, disposal group and non-disposed group, The animal models with oxygen-induced proliferative retinopathy were established by raising the rats in hyperoxic environment. Retrobulbar injection was performed with VEGF ASODNs or normal saline on the rats in 3 groups respectively. The intraocular tissues (all the tissues except the cornea, sclera, and lens) and serum were collected, and the expressions of VEGF were determined by using competitive enzyme immunoassay.Results The expressions of VEGF in intraocular tissues of rats in disposal group were significantly lower than those in non-disposed group (P＜0.05), and there was no statistical difference between the disposal and normal control group (P＞0.05). There was no significant difference of the expressions of VEGF in serum of rats between the disposal and non-disposed group (P＞0.05), which were both lower than those in the normal control group (P＜0.05). Conclusion VEGF ASODNs could significantly inhibit the expression of VEGF in intraocular tissues. (Chin J Ocul Fundus Dis，2003，19：172-174)
Objective To investigate the effect on expression of c-myc and proliferating cell nuclear antigen （PCNA） of vein grafts transferred by c-myc antisense oligodeoxynucleotides（ODN） of soluble stent. Methods A rabbit model of common carotid arteries grafted by external jugular veins was constructed in 50 New Zealand rabbits and were randomly divided into five groups, 10 rabbits each group. Control group： no stents ; group 1： soluble stent ; group 2： soluble stent with sense-ODN; group 3： soluble stent with antisense-ODN; group 4.. soluble stent with mismatch-ODN. At 7 d, 28 d and 90 d after surgery, vein grafts were harvested. The expression of c-myc and PCNA were identified by immunochemistry methods. Results At 7d, 28d, 90d after surgery, the expression of c-myc and PCNA of the intima and media of vein grafts in control group, group 1, group 2, group 4 were higher significantly than that in group 3 （P〈0. 01）. At 28d, 90d after surgery, the expression of c-myc in five groups were higher than that in the same group at 7d after surgery （P〈0. 01）. Conclusion Soluble stent can transfer ODN effectively. C- myc antisense-ODN transferred by soluble stent can inhibit significantly the expression of c-myc and PCNA in the intima and media of vein grafts.
ObjectiveTo construct the recombinant adenovirus vector carrying antisense multidrug resistanceassociated protein (MRP) and transfect the human drugresistant hepatocellular carcinoma cell line(SMMC7721/ADM). MethodsThe fragment of MRP gene encoding 5′region was cloned reversely into the shuttle plasmid pAdTrackCMV, with the resultant plasmid and the backbone plasmid pAdEasy1,the homologous recombination took place in the bacteria and the recombinant adenoviral plasmid was generated. The adenoviruses were packaged and amplified in 293 cells. Then the cell line of SMMC7721/ADM was transfected with the resultant adenoviruses.ResultsThe recombinant adenovirus vector carrying antisense MRP was constructed successfully. The viral titer was 2.5×109 efu/ml, and more than 90% SMMC7721/ADM cells could be transfected when the multiplicity of infection（MOI） was 100. ConclusionThe recombinant adenovirus vector constructed by us could introduce the antisense MRP into the human drugresistant hepatocellular cell line effectively, which would provide experimental basis for the mechanisms and reversal methods of the multidrug resistance in human hepatocellular carcinoma.
Objective To study the effects on MCF-7 breast cancer cells with combination of tamoxifen（TAM） and antisense oligonucleotide （ASODN） targeting survivin mRNA. Methods MCF-7 breast cancer cells were treated with a 20 mer ASODN targeting survivin mRNA and TAM, which were divided into three groups: TAM group (treated by TAM only), ASODN group (by ASODN only), and TAM+ASODN combined group (by TAM+ASODN combination). The growth inhibition of MCF-7 cells, the changes of cell cycles and apoptotic rate, the positive rate of survivin mRNA expression, and the activity of caspase-3 were tested by MTT, flow cytometry, hybridization in situ, and spectrophotometric method, respectively.Results The rate of growth inhibition of MCF-7 cells in the TAM+ASODN combined group was (62.26±3.92)%, which was significantly higher than that in the TAM group 〔(42.30±6.63)%〕 or ASODN group 〔(54.77±9.99)%〕, Plt;0.05. The apoptotic rate of MCF-7 cells was (28.08±4.32)% in the TAM+ASODN combined group, which was significantly higher than that in the TAM group 〔(18.94±4.01)%〕 or ASODN group 〔(21.12±3.95)%〕, Plt;0.01. The effect of arresting MCF-7 cells in G0/G1 phase in the TAM+ASODN combined group was ber than that in the TAM or ASODN group (Plt;0.05, Plt;0.01). The positive rate of survivin mRNA in the TAM+ASODN combined group was (13.38±3.45)%, which was significantly lower than that in the TAM group 〔(39.67±7.42)%〕 or ASODN group 〔(27.50±5.80)%〕, Plt;0.01. The activity of caspase-3 in the TAM+ASODN combined group （0.93±0.13） was significantly higher than that in the TAM group (0.50±0.09) or ASODN group (0.64±0.08), Plt;0.01. Conclusion The ASODN targeting survivin mRNA can promote the apoptosis of MCF-7 breast cancer cells, and make MCF-7 cells more sensitive to tamoxifen.
Objective To observe the inhibitory effects of local co-transfection of tissuetype plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNAASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNAASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcriptionPCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty.Results The mRNA expression of tPA gene in the transplanted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups(P＜0.01).The number of PCNA positive cells in the transplanted arteries in PCNAASODN, tPA and tPA+PCNAASODN groups were significantly lower than that of control group(P＜0.05，P＜0.01). The intimal areas and degrees of luminal stenosis of PCNAASODN, tPA and tPA+PCNAASODN groups were lower than those of control group(P＜0.05，P＜0.01), and those of tPA＋ PCNA-ASODN group were lower than those of PCNA-ASODN and tPA groups(P＜0.05). Scanning electron microscopy showed that there were a few thrombocytes lining the vessel wall of tPA group and tPA+PCNAASODN group and no thrombus, whereas there were abundant thrombocytes and thrombi lining the vessel wall of the control group. Conclusion Co-transfection of tPA gene and PCNA-ASODN can effectively inhibit the proliferation of VSMC, hyperplasia of intima and restenosis of transplanted artery.
Objective To observe the effects of vascular endothelial growth factor antisense oligonucleotide (VEGF-ASODN) on expression of vascular endothelial growth factor (VEGF) and growth in gastric cancer cells. Methods The VEGF-ASODN was synthesized artificially with phosphorothioic acid. After transfecting with VEGF-ASODN in gastric cancer cells SGC-7901, the initial copy number of mRNA was detected by real-time RT-PCR, and the quantity of VEGF protein in both cell and supernatant were detected by ELISA. The levels of expression of survivin protein in cells were measured by Western blot. FCM and MTT method were used to detect cellular apoptosis and the activity of cells, respectively. The effect of transfection on the growth of cells was evaluated by growth curve. Results The copy number of VEGR mRNA, protein levels of VEGF in the cells and in culture fluid all decreased when the concentration of transfected VEGF-ASODN increased, as well as the levels of survivin protein （P＜0.05）. The ratio of apoptosis increased, the activity of cells also decreased as the concentration of transfected VEGF-ASODN increased （P＜0.05）. Conclusion Transfection with VEGF-ASODN in gastric cancer cells SGC-7901 can inhibit the expressions of VEGF and survivin remarkably. It can enhance cellular apoptosis and suppress growth of cells.