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 summarize the research progress of microRNA （miRNA） as a tumor marker in peripheral blood. Methods The domestic and international published literatures about circulating miRNA as a tumor marker in recent years were reviewed. Results The miRNA expression has universality，stability and specificity，and it is related to the occurrence and development of viarous diseases. Conclusion Circulating miRNA shows a broad application prospect in clinical diagnosis， treatment， and prognosis of tumor and other diseases.
ObjectiveTo evaluate the inhibitory effect of small interfering RNA (siRNA) targeting peroxisome-proliferator-activated receptor-γcoactivator-1α(PGC-1α) on retinal neovascularization in the mouse. MethodsEighty seven-day-old C57BL/6J mice were divided into normal group, model blank group, model control group and PGC-1αsiRNA group, twenty mice in each group. Mice in the normal group were kept in normal room air. Mice in the model blank group, model control group and PGC-1αsiRNA group were induced for retinal neovascularization by hypoxia. Liposome with PGC-1αsiRNA (1 μl) and liposome with negative control siRNA (1 μl) were injected into the vitreous in the PGC-1αsiRNA group and model control group respectively when mice were moved out to room air from the cabin (Postnatal 12). No injection were performed in the model blank group. At postnatal 17, fluorescein angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. PGC-1αand vascular endothelial growth factor (VEGF) level in retina were measured by real-time polymerase chain reaction (real-time PCR) and Western blot. Inhibition efficiency of PGC-1αsiRNA on PGC-1αand VEGF was calculated. ResultsMice in the normal group showed reticular distribution of retinal blood vessels. Central nonperfused retina, neovascular tufts and fluorescein leakage were seen in the model blank group and model control group. Neovascular tuft and fluorescein leakage were decreased in the PGC-1αsiRNA group compared to the model blank group and model control group. The neovascular nuclei were increased in the model blank group and model control group compared to the normal group (P < 0.05). The neovascular nuclei were decreased in the PGC-1αsiRNA group compared to the model blank group and model control group (P < 0.05). The expression of PGC-1αmRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group, while decreased 54% and 53% respectively in the PGC-1αsiRNA group as compared with model blank group and model control group (P < 0.05). The expression of VEGF mRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group, while decreased significantly in the PGC-1αsiRNA group (decreased 48% and 40% respectively) as compared with model blank group and model control group (P < 0.05). ConclusionsIntravitreal injection of PGC-1αsiRNA mediated by liposome can inhibit retinal neovascularization in the mouse effectively.
Objective To study the effect of knockdown of signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA (shRNA) on proliferation, apoptosis and invasion of human gastric cancer cell line MKN-45 in vitro . Methods Specific shRNA plasmids to STAT3 were constructed, and then transfected into MKN-45 cells by lipofectamine methods. Cells were divided into three groups: control group, psiRNA-H1 transfected group as negative group and psiRNA-H1/STAT3 transfected group. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of STAT3 mRNA and protein, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. The invasion of the transfected MKN-45 cells was measured by Boyden chamber. Results Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed that the expressions of STAT3 mRNA and protein were down-regulated in the psiRNA-H1/STAT3 transfected group ( P ＜ 0.05) . The subcloned recombinant plasmid expressing shRNA effectively inhibited MKN-45 cell growth and proliferation while empty plasmid had no such specific effect. Cell apoptosis rate increased significantly in psiRNA-H1/STAT3 transfected group （ P ＜ 0.01）, and the invasion of MKN-45 cells was efficiently inhabited in psiRNA-H1/STAT3 transfected group as compared with control group and psiRNA-H1 transfected group( P ＜ 0.01).Conclusion Recombinant plasmid psiRNA-H1/STAT3 shRNA significantly inhibits the proliferation and invasion of MKN-45 cells and promotes their apoptosis.
ObjectiveTo summarize the role of circular RNA (circRNAs) in thyroid papillary carcinoma (PTC) and the emphasis of future research.MethodRelevant literatures in recent years about the biological function of circRNA and its role in PTC were reviewed.ResultscircRNAs had abnormal expression in PTC tissues. Besides, by working as miRNA sponges or interacting with RNA-binding proteins, circRNAs regulated the expression of proteins that were associated with cell proliferation, apoptosis, invasion, and metastasis, which could affect the biological characteristics of tumor cells.ConclusioncircRNAs are expected to be the biomarkers for early diagnosis of PTC or potential targets for PTC therapy and provid therapeutic bases to prevent PTC.
Objective To summarize the domestic and abroad articles related to the research on the relation between microRNA （miRNA） and pancreatic cancer，and explore the important effects of miRNA expression patterns in diagnosis of pancreatic cancer. Methods “microRNA and pancreatic cancer” were searched as key words by PubMed and CNKI series full-text database retrieval systems from 2000 to 2012. Totally 60 English papers and 15 Chinese papers were obtained. Choice criteria：the basic research of miRNA and pancreatic cancer，the clinical research of miRNA and pancreatic cancer， and the prospect of miRNA in pancreatic cancer diagnosis and treatment. According to the choice criteria，31 papers were finally analyzed. Results The miRNA expression spectrum and specific miRNA expression such as miR-21，miR-34，miR-217，miR-196a，miR-10a，miR-155，miR-221，miR-222，miR-181a，miR-181b，miR-181d， and the family members of miR-200 and let-7 might be used as tumor markers to differentiate pancreatic cancer from normal pancreas，chronic pancreatitis or pancreatic endocrine tumors，and might be used as prognostic factor to predict the outcome. Conclusions miRNA expression spectrum are not only related to diagnosis of pancreatic cancer， but also have provided a new research direction and method for gene therapy of pancreatic cancer．
Considering the low accuracy of prediction in the positive samples and poor overall classification effects caused by unbalanced sample data of MicroRNA (miRNA) target, we proposes a support vector machine (SVM)-integration of under-sampling and weight (IUSM) algorithm in this paper, an under-sampling based on the ensemble learning algorithm. The algorithm adopts SVM as learning algorithm and AdaBoost as integration framework, and embeds clustering-based under-sampling into the iterative process, aiming at reducing the degree of unbalanced distribution of positive and negative samples. Meanwhile, in the process of adaptive weight adjustment of the samples, the SVM-IUSM algorithm eliminates the abnormal ones in negative samples with robust sample weights smoothing mechanism so as to avoid over-learning. Finally, the prediction of miRNA target integrated classifier is achieved with the combination of multiple weak classifiers through the voting mechanism. The experiment revealed that the SVM-IUSW, compared with other algorithms on unbalanced dataset collection, could not only improve the accuracy of positive targets and the overall effect of classification, but also enhance the generalization ability of miRNA target classifier.
ObjectiveTo study value of long noncoding RNA H19 and HOTTIP in plasma in predicting efficacy of neoadjuvant chemotherapy (NAC) for resectable locally advanced gastric cancer. MethodsForty patients with T3–4aN+M0 gastric cancer and 40 patients with benign gastric diseases treated in the Yantai Yuhuangding Hospital Affiliated to Qingdao University from August 2020 to May 2021 were prospectively included. The expressions of H19 and HOTTIP in the plasma of gastric cancer and benign gastric diseases patients without any treatment after admission were detected before treatment (CAPEOX regimen was used in the patients with gastric cancer), then which were detected after 2 NAC courses for patients with gastric cancer. Meanwhile, some clinical items were detected and the efficacy of NAC was evaluated. The complete remission (CR) and partial remission (PR) were classified as objective remission, CR, PR, and disease stability were classified as disease control. The expressions of H19 and HOTTIP between the different patients were compared and the receiver operating characteristic (ROC) curve was used to evaluate their values in the diagnosis of resectable locally advanced gastric cancer. ResultsThere were 13 cases of T downstaging and 27 cases of T non-downstaging and 25 cases of objective remission and 35 disease control after NAC. The median relative expression levels of H19 and HOTTIP before NAC in the patients with gastric cancer were higher than those in the patients with benign gastric diseases (H19: 1.42 versus 0.98, Z=–3.835, P<0.001; HOTTIP: 2.15 versus 1.04, Z=–5.062, P<0.001), and which were in the patients with T downstaging and disease control were lower than those in the patients with T non-downstaging and 5 cases of disease progression (For T staging, H19: 1.12 versus 1.54, Z=–2.960, P=0.002; HOTTIP: 1.49 versus 2.30, Z=–2.310, P=0.019. For efficacy of NAC, H19: 1.39 versus 2.48, Z=–3.211, P<0.001; HOTTIP: 1.96 versus 3.25, Z=–2.393, P=0.014). The median relative expressions of H19 and HOTTIP after NAC were lower than those before NAC in the patients with gastric cancer (H19: 1.12 versus 1.42, Z=–3.965, P<0.001; HOTTIP: 1.30 versus 2.15, Z=–4.839, P<0.001). There were no significant differences in the changes of H19 and HOTTIP before and after NAC between the patients with T downstaging and T non-downstaging, and between disease control and disease progression (P>0.05). The areas of ROC curve of H19, HOTTIP, and combination of H19 and HOTTIP in diagnosis of resectable locally advanced gastric cancer were higher than 0.7. ConclusionsLncRNA H19 and HOTTIP might be potential tumor markers in gastric cancer, and their diagnostic values for resectable locally advanced gastric cancer are higher. Gastric cancer patients with low expressions of H19 and HOTTIP in plasma might be more sensitive to NAC.
Objective To study the molecular characteristics of RNA binding protein aldehyde dehydrogenase 18 family member A1 (ALDH18A1) in esophageal carcinoma cells (KYSE150 cells) and its effect on tumor growth. MethodsHuman esophageal squamous cell (KYSE150 cells) was cultured in vitro. At the same time, RNA co-immuno precipitation technology was used to study the binding of RNA and protein in the cell, and the corresponding RNA-protein complex was precipitated by the antibody of the target protein to separate and purify the captured RNA. The molecular characteristics of ALDH18A1 binding RNA were analyzed, and KyotoEncyclopedia of Genes and Genomes cluster analysis was performed for ALDH18A1 binding target genes. Results Protein immunoblotting experiments showed that the target protein was well enriched by antibodies. ALDH18A1 had extensive RNA binding activity, with significant enrichment in regions such as coding sequences, intron, and 5’untranslated region. ALDH18A1 mainly bound to the UGUAAUC motif of RNA. The cluster analysis showed that the RNA molecules bound to ALDH18A1 mainly participated in focal adhesion, central carbon metabolism in cancer, cell cycle, spliceosome, RNA transport, and ubiquitin mediated protein hydrolysis. Conclusion ALDH18A1 has the function of binding to RNA molecules and may play a role in the expression of esophageal cancer-related genes and related biological processes.
【Abstract】ObjectiveTo study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinoma cell line QBC-939. MethodsThe constructed antisense DNMT3b gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC-939 by using lipofectamine transfection reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfected into QBC-939 cells successfully was confirmed by amplifying the exogenous neoR gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively. ResultsFollowing the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0.956±0.053 to 0.209±0.023, and the protein level of DNMT3b gene also decreased from (75.38±3.22)% to (29.87±3.46)%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group (P＜0.01). ConclusionTransfection with antisense DNMT3b gene eukaryotic expression vector significantly reduces the expression level of DNMT3b gene in human cholangiocarcinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.