Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1 (Rac1-siRNA) on rat retinal neovascularization in retinae. Methods Retinal vein occlusion was induced by retinal photodynamic medthod in 25 Sprague-Dawley rats. Rac1-siRNA vector DNA was injected into the vitrous of one eye of those rats (gene intervention group), and empty vector DNA was injected into the fellow eye (blank control group). Rac1-siRNA vector was injected in other 25 SD rats without retinal vein occlusion (blank intervention group). Two weeks after injection, fluorescein isothiocyanate (FITC)-dextran was perfused into the hearts of all the rats, and the retinal wholemount was made to observe the neovascularization. The numbers of endothelial cells which break through the internal limiting membrane were counted after hematoxylin-eosin staining. Results A massive of neovascularization and FITC leakage were found in blank control group. Small part of neovascularization and a little FITC leakage were observed in the gene intervention group. Retinal vessels were normal in blank intervention group. Compared with blank contrast group and blank intervention group, the difference of the mean numbers of endothelial cells which broke through the internal limiting membrane in the gene intervention group was significant（t=? P=0.000??lt;0.05）. Conclusion Rac1-siRNA can inhibit retinal neovascularization induced by retinal vein occlusion in rats.
ObjectiveTo analyze the expression of cold-induced RNA-binding protein (CIRBP) in lung adenocarcinoma and its clinical significance based on bioinformatics, in order to provide a new direction for the study of therapeutic targets for lung adenocarcinoma.MethodsThe CIRBP gene expression data and patient clinical information data in lung adenocarcinoma tissues and adjacent tissues were downloaded from The Cancer Genome Atlas and Gene Expression Omnibus databases. The expression of CIRBP in lung adenocarcinoma was analyzed. Furthermore, its relationship with clinicopathological features and prognosis in patients with lung adenocarcinoma was analyzed. GO and KEGG enrichment analysis were carried out for the screened genes. The CIRBP protein interaction network was constructed by STRING, and the correlation analysis was carried out using the GEPIA online website.ResultsThe expression level of CIRBP gene in lung adenocarcinoma tissues was significantly lower than that in adjacent tissues (P<0.01), and its expression level was correlated with T stage and N stage in clinicopathological features. The prognosis of patients with high CIRBP expression in lung adenocarcinoma was significantly better than that with low CIRBP expression. Univariate and multivariate Cox regression analysis showed that CIRBP was an independent prognostic factor in patients with lung adenocarcinoma. GO functional annotation showed its enrichment in organelle fission, nuclear fission, chromosome separation, and DNA replication, etc. KEGG analysis showed that it was mainly involved in cell cycle and DNA replication. Protein interaction network and GEPIA online analysis showed that the expression level of CIRBP was negatively correlated with the expression level of cyclin B2.ConclusionCIRBP gene is down-regulated in lung adenocarcinoma tissues, and its expression level is closely related to patient prognosis. CIRBP gene may be a potential therapeutic target and prognostic marker for lung adenocarcinoma.
Objective To investigate the inhibitory effects of RNA interference (RNAi) expression vector on the expression of survivin in pancreatic cancer cell PANC-1. Methods The protein and mRNA expressions of survivin were examined with immunofluorescence and RT-PCR. The survivin gene was cloned into the T-vector and sequenced. The RNAi expression vectors targeting survivin, named si-svv-1 and si-svv-2 respectively according to whether they harbored a mutation or no mutation, were constructed and transfected into PANC-1 cells with liposome. The expression of survivin mRNA was detected with RT-PCR. Apoptosis of PANC-1 cells was analyzed with DNA ladder and FACS. Results There was a high degree expression of survivin in PANC-1 cells. The expression of survivin was not inhibited by RNAi expression vectors si-svv-1, but inhibited about (72.43±8.04)% by si-svv-2 and the apoptosis rate of PANC-1 cells increased to （12.36±1.44）% after 72 h. Conclusion The RNAi expression vector can effectively inhibit the expression of survivin in pancreatic cancer cell PANC-1 cells and induce the apoptosis in PANC-1 cells.
Objective To investigate the association of the expression of CD15 mRNA with the invasion and prognosis of hepatocellular carcinoma (HCC) and the expression of nm23H1 mRNA. Methods In situ hybridization and immunohistochemistry methods were used to detect the expression of CD15 mRNA and protein nm23H1 mRNA in HCC.Results In 99 cases of HCC, the positive rate of CD15 mRNA,its protein and nm23H1 mRNA were 38.4%, 36.4% and 76.8%, respectively. The expression of CD15 mRNA was consistent with its protein and negatively correlated with the expression of nm23H1 mRNA. The expression of CD15 mRNA and its protein, nm23H1 mRNA were associated with the invasiveness and metastasis of HCC and the prognosis of HCC patients. Conclusion The detection of CD15 expression could be a new pathological biology index to judge the metastasis and prognosis of HCC.
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.
Objective To explore the relationship between nasopharyngeal microecology and diseases in children with bronchial asthma. Methods A total of 41 children with asthma who were treated in Hainan Provincial Hospital of Traditional Chinese Medicine between November 2020 and March 2023 were retrospectively included in the study, and 26 healthy children undergoing adenoid examination in the same period were selected as the control group. Samples of nasal mucosa were collected from the anterior and medial side of inferior turbinate, and the expression of DEFB2, IL17A, TSLP, IL13, IL5 and T1R3 genes was analyzed by polymerase chain reaction. Nasal swabs were collected from the children, and the bacterial composition was analyzed by 16S ribosomal RNA gene sequencing. Results Compared with the control group, the rate of atopy cases in the asthma group increased significantly (53.7% vs. 19.2%, P<0.05). At the phylum level, compared with the control group, the phylum Chloroflexi, the phylum Patescibacteria, the phylum Tenericutes and the phylum Nitrospirae in the asthma group increased significantly (P<0.05), and the phylum Elusimicrobia decreased significantly (P<0.05). At the genus level, compared with the control group, the members of Bacillus (Fimnicutes), Ruminococcus (Fimnicutes), Rhodococcus (Actinobacteria), Acinetobacter (Proteobacteria), Moraxella (Proteobacteria) and Asaia (Proteobacteria) in the asthma group increased significantly (P<0.05), and the members of Enterococcus (Fimnicutes), Alkanindiges (Proteobacteria), Rickettsia (Proteobacteria), and Rhizobium (Proteobacteria) in the asthma group decreased significantly (P<0.05). Compared with the control group, the Shannon index of the asthma group decreased significantly (2.63±1.45 vs. 3.90±1.44; t=2.708, P=0.010). According to receiver operating characteristic curve analysis, the optimal cut-off point of Shannon index was 3.10. In all study populations, compared with children whose Shannon index was higher than the cut-off point, children whose Shannon index was lower than the cut-off point were characterized by increased expression of IL17A and T1R3 (P<0.05) and decreased expression of TSLP (P<0.05). Conclusion The composition and abundance of nasopharyngeal microbiota are significantly different between children with asthma and healthy control children.
ObjectiveTo investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats.MethodsOne hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group (n=35), the rats in SCI group were given SCI according to Allen’s method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above (n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins.ResultsWestern blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury (P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury (P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment (P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B (P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B (P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B (P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B (P<0.05).ConclusionLentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.
ObjectiveThis study is aimed to determine the expression of ubiquitin-specific peptidase 39 (USP39) protein in the colorectal cancer (CRC) tissues, and the effect of silencing USP39 gene on the cell growth and cell cycle distribution of CRC cells.Methods① The expressions of USP39 protein in CRC tissues and its paracancerous tissues were determined by immunohistochemical staining method. ② By lentiviral infection, Lv-shUSP39 (KD-1 and KD-2 group) and Lv-shCon (shCon group) were transferred into SW1116 and HCT116 cells, and cells of blank control group did not received any treatment (Con group). To determine the role of USP39 gene in cell growth, MTT assay was performed to draw growth curve, and cell cycle distribution of CRC cells in the 4 groups were determined by flow cytometer.Results① The expression of USP39 protein was higher in CRC tissues compared to adjacent tissues (P=0.007). ② For SW1116 and HCT116 cells, the cell proliferation ability of KD-1 and KD-2 groups were remarkably decreased than those in corresponding shCon and Con groups on 3, 4, and 5-day (P<0.05). ③ Flow cytometry assay showed that, the percentage of G0/G1 phase cells were decreased obviously (P<0.05), while increased significantly in percentage of G2/M phase and number of sub-G1 phase cells in KD-1 group compared with that in the Con group and shCon group of SW1116 and HCT116 cells (P<0.05).ConclusionsThe expression of USP39 protein is highly expressed in CRC tissues. Knockdowning of USP39 gene can inhibit cell proliferation and promote cell apoptosis.
【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 construct small hairpin RNA (shRNA) expression plasmid targeting rat opticin gene.Methods Four pairs of opticin oligonucleotides were synthesized and inserted into the plasmid vector, resulting into four plasmids: shRNA-1, shRNA-2, shRNA-3 and shRNA-4. Then the four constructed shRNA expression vectors and empty vector were transfected into rat ciliary non-pigment epithelium (NPE) cells by lipofectmaine 2000. Nontransfected NPE cells were set as control group.The expression of opticin mRNA and protein were measured by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot respectively.Results The opticin mRNA expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were decreased compared with the control group (F=10.239，P＝0.000)；the inhibitory rate were 85.7%，62.87%，54.87% and 48.77% respectively.The opticin protein expression of the shRNA-1,shRNA-2,shRNA-3,shRNA-4 group were also decreased compared with the control group (F=17.870，P=0.000)；the inhibitory rate were 78.7%，34.6%，31.1% and 16.8% respectively.Conclusions The shRNA-1 expression plasmid has most potent inhibitory effect on opticin expression in rat ciliary NPE cells.