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.
Non-coding RNA (ncRNA) is a newly discovered functional RNA different from messenger RNA, which can participate in the regulation of tumor occurrence and development. Studies have shown that ncRNA can participate in the regulation of radiotherapy response to gastric cancer, and its mechanism may be related to its influence on DNA damage repair, gastric cancer cell stemness, apoptosis, and activation of epidermal growth factor receptor signal pathway. This article summarizes the mechanism of ncRNA regulating the response of gastric cancer to radiotherapy, and looks forward to the potential clinical application of ncRNA in the resistance of gastric cancer to radiotherapy.
Objective To investigate the molecular mechanisms by which the long non-coding RNA (lncRNA) MIR223HG affects the proliferation, migration and apoptosis of lung adenocarcinoma cells. MethodsDNA damaging agent Zeocin was used to treat human embryo lung cell (MRC-5) and lung cancer cell (A549 and H1299), and the expression of MIR223HG was tested by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Moreover, the ataxia-telangiectasia mutated (ATM) protein and ATM pathway downstream factor Cell cycle checkpoint kinase 2 (Chk2), p53 tumor suppressor protein (p53) in the lung cancer cell (A549 and H1299) with Zeocin were also tested by qRT-PCR. Cell transfection and Transwell migration assay, colony formation assays, apoptosis assays were performed to verify the role of ATM in the expression of MIR223HG in lung adenocarcinoma. ResultsThe expression of MIR223HG was reduced markedly in the lung cancer cells (A549 and H1299) compared with human embryo lung cell (MRC-5) after treated with Zeocin. ATM protein and its downstream factors Chk2, p53 involved in the process, and ATM regulated the expression of MIR223HG in the lung cancer cells with Zeocin. Futhermore, ATM joined in the processes that MIR223HG regulated the lung cancer cells proliferation, migration and apoptosis. Conclusions The expression of MIR223HG is related to the DNA damage response in the lung cancer, and MIR223HG regulates lung cancer cells proliferation, migration and apoptosis by ATM/Chk2/p53 pathway. MIR223HG may be a potential therapeutic target for lung adenocarcinoma treatment.
Hematopoietic stem cells (HSCs) are tissue specific stem cells that replenish all mature blood lineages during the lifetime of an individual. Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Recently, people have learned a lot about the embryonic HSCs on their development and homing. During their differentiation, HSCs are regulated by the transcription factors, such as Runx1 and Notch signaling pathway, etc. MicroRNAs also regulate the self-renewal and differentiation of hematopoietic stem/progenitor cells on the post-transcriptional levels. Since the onset of circulation, the formation of HSCs and their differentiation into blood cells, especially red blood cells, are regulated by the hemodynamic forces. It would be of great significance if we could treat hematologic diseases with induced HSCs in vitro on the basis of fully understanding of hemotopoietic stem cell development. This review is focused on the advances in the research of HSCs' development and regulation.
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 detect expressions of transient receptor potential channel C5 (TRPC5) and microRNA-320a (miR-320a) in thyroid cancer and explore clinical significances of them in thyroid cancer.MethodsThe expressions of TRPC5 and miR-320a mRNA in the thyroid cancer were investigated by searching the Ualcan database. While the expressions of TRPC5 and miR-320a mRNA in 80 cases of thyroid cancer, 35 cases of thyroid adenoma and 32 cases of normal thyroid tissues adjacent to thyroid adenoma tissues in the Zhengzhou Seventh People’s Hospital from March 2014 to March 2015 were tested. Real time PCR was used to detect the expressions of TRPC5 mRNA and miR-320a mRNA in the various tissues and Western blot was used to detect the TRPC5 protein in the thyroid cancer tissues. Therelationships between the expressions of TRPC5 and miR-320a mRNAs and clinicopathologic features of thyroid cancer were analyzed. The correlation between expressions of TRPC5 and miR-320a mRNA was analyzed by Pearson method. The risk factors influencing the prognosis were analyzed by univariate and multivariate Cox proportional hazards regression model.ResultsThe results of Ualcan database showed that the expression level of TRPC5 mRNA in the thyroid cancer was higher than that in the normal thyroid tissue (P<0.001), while the expression level of miR-320a mRNA was lower than that in the normal thyroid tissue (P<0.001). The results of clinical cases showed that the expression level of TRPC5 mRNA was significantly higher, while the expression of miR-320a mRNA was significantly lower in the thyroid cancer tissues as compared with the normal thyroid tissues (P<0.05). There was a negative correlation between the expression level of TRPC5 and miR-320a mRNA in the thyroid cancer (r=−0.653, P<0.001). The expressions of TRPC5 and miR-320a mRNA were correlated with the degree of differentiation, lymph node metastasis, and TNM stage (P<0.05). Kaplan-Meier survival curve analysis found that the patients with higher expression level of TRPC5 and lower expression level of miR-320a showed the poor prognosis, and multivariate analysis found that the lower tumor differentiation, later TNM stage, with lymph node metastasis, higher expression level of TRPC5 mRNA, and lower expression level of miR-320a mRNA were the risk factors affecting prognostic survival (P<0.05).ConclusionsFrom the database and clinical case data, it is concluded that TRPC5 mRNA is highly expressed, while miR-320a mRNA is lowly expressed in thyroid cancer tissues, and expressions of TRPC5 and miR-320a mRNA are related to degree of tumor differentiation, lymph node metastasis, TNM staging, and prognosis in patients with thyroid cancer. TRPC5 and miR-320a mRNA might be used as potential indicators for clinical and prognostic monitoring.
Objective To investigate the effects of expression of TNFα mRNA on glucose uptake in both the liver and skeletal muscle after endotoxemia. Methods In the mice with intraperitoneal injection of lipopolysaccharide (LPS), the changes of TNFα level of plasma and uptake of 2-deoxyglucose (2-DG) in the isolated soleus muscle and hepatic tissues were determined, then the reinstatement of glucose uptake by injecting TNF-McAb for 3 days was also observed. In addition, changes of TNFα mRNA expression of liver were evaluated. Results The expression of TNFα mRNA in the liver showed markedly increased in the first 3 hours post endotoxemia and remaind high for 3 days, and the plasma TNFα level paralleled with TNFα mRNA expression of liver also was elevated. The basal uptake of 2-DG both in muscle and liver were markedly increased, but the stimulated 2-DG uptake with insulin was greatly reduced as compared with the control. In addition, these abnormalities of 2-DG uptake can be partially corrected by neutralization of the circulatory TNFα by administration of TNF-McAb. Conclusion The disorders of glucose uptake of the liver and the muscle due to the overexpression of TNFα mRNA and elevated circulatory TNFα level may be the mechanism of insulin resistance after endotoxemia.
ObjectiveTo explore the effect of DDX46 silencing on growth and apoptosis in esophageal squamous cell carcinoma cell TE-1 by the shRNA. MethodsThe relative expression of DDX46 mRNA in TE-1 cells was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and compared with immortalized human esophageal squamous cell Het-1A. DDX46 shRNA-expressing lentivirus was applied to silence DDX46 (experimental group), and non-silencing control lentivirus was added (control group) with a multiplicity of infection of 5 in TE-1 cells. In both groups, cell growth was monitored using high content screening, cell colony-forming capacity was measured by colony formation assay, cell apoptosis were determined by flow cytometry. Further, the Stress and Apoptosis Signaling Antibody Array Kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown. ResultsCompared with the control group, cell counting after DDX46 silencing showed that TE-1 cell growth was significantly inhibited (P<0.001). Colony formation assay showed that cell colony-forming capacity was significantly inhibited (P<0.01). Annexin V-APC flow cytometry showed a significant increase in apoptosis (P<0.001). In PathScan® Antibody Array, the expression levels of Akt (Ser473, phosphorylation) and IκBα (Total, N/A) significantly decreased (P<0.01), and the expression of Caspase-3 (Asp175, cleaved) increased (P<0.05). ConclusionDDX46 is overexpressed in TE-1 cells. Targeted gene silencing of DDX46 inhibits cell growth, and induces cell apoptosis. DDX46 silencing probably by negative regulation of Akt/NF-κB signaling pathway, to play a role in inhibiting TE-1 cells growth and inducing apoptosis.
Objective To observe whether Nogo-66 can inhibit the neurite outgrowth during the neuronal differentiation of the neural stem cells (NSCs) and remove such an inhibitory effect by the small interfering RNA (siRNA) mediated knockdown of the Nogo66 receptor (NgR). Methods NSCs derived from the rat spinal cord were collected, and were cultured by the suspension culture in vitro. NSCs were transfected by siRNA to knock downtheexpression of NgR. Immunofluorescence and Western blot were used to assess the knockdown efficiency. NSCs were divided into four groups and differentiated in the medium containing 10% FBS. In the control group, no intervention was applied to NSCs; in the Nogo-P4 group, NSCs were differentiated in the presence of Nogo-P4 (active segment of Nogo-66); in the siRNA group, NSCs were transfected by siRNA to knock down NgR before they were differentiated; in the siRNA and Nogo-P4 group, NSCs were transfected by siRNA to knock down NgR before they were differentiated in presence of Nogo-P4. The differentiated neurons were labeled by immunofluorescence, and the neurite length was measured by the ImagePro Plus 5.0 software. The differentiation of the neurite length was compared in each group. Results The suspension-cultured cells became the nerve bulb, which could positively expresses Nestin by immunofluorescence. At 1 week of the differentiation in the medium containing 10% FBS, the positively-labeled neuron specific enolase, the glial fibrillary acidic protein, and the myelin basic protein were observed. Both immunofluorescence and Western blot approved that the expression of NgR was knocked down by transfection of siRNA at 24 hours after the transfection. The knockdown efficiency was 90.35%±3.10%. The neurite length was 97.80±6.97 μm, 80.54±6.75 μm,92.14±7.27 μm, and 94.01±8.37 μm in the control group, the Nogo-P4 group, the siRNA group, and the siRNA and Nogo-P4 group, respectively. The Nogo-P4 group had a significant difference when compared with the otherthree groups (Plt;0.01), and the other three groups had no significant difference when compared with each other（Pgt;0.05）. ConclusionNogo-66 can inhibit the neuronal neurite outgrowth during the differentiation ofNSCs. Such an inhibitory effect can be removed by the siRNA mediated knockdown of NgR.
Objective To systematically evaluate the correlation between the expression of microRNA (miRNA)-21 and the prognosis of esophageal cancer. Methods PubMed, Cochrane Library, Embase, Wanfang Data, China National Knowledge Infrastructure and VIP Databases were searched by for the literature on the correlation between miRNA-21 and the prognosis of esophageal cancer till July 10, 2022. Two researchers independently performed literature screening, quality evaluation, and data extraction. Statistical analysis was conducted with Stata 14.0. Results A total of 13 articles were included, including 1 204 patients. The results of meta-analysis showed that: the overall survival (OS) of patients with high expression of miRNA-21 was lower than that of patients with low expression of miRNA-21 [hazard ratio (HR)=2.11, 95% confidence interval (CI) (1.56, 2.84), P<0.001]. miRNA-21 expression was not associated with disease free survival [HR=2.53, 95%CI (0.67, 8.22), P=0.182]. The OS of Asian patients with high expression of miRNA-21 was significantly lower [HR=2.44, 95%CI (1.71, 3.49), P=0.005], while the OS of non-Asian patients was not related to miRNA-21 expression [HR=1.34, 95%CI (0.94, 1.91), P=0.363]. The high expression of miRNA-21 was correlated with the decreased OS in patients with esophageal squamous cell carcinoma [HR=2.22, 95%CI (1.52, 3.26), P=0.001], while the OS in patients with esophageal adenocarcinoma was not correlated with the expression of miRNA-21 [HR=1.39, 95%CI (0.63, 3.06), P=0.409]. Conclusion The overexpression of miRNA-21 is associated with poor prognosis and might be regarded as a potential prognostic biomarker for patients with esophageal cancer.