Résumé
The water-soluble polypeptide drug oxytocin was encapsulated in liposomes by reverse-phase evaporation vesicle method to obtain oxytocin loaded liposomes (OT@LPs) which was further modified with cationic cell penetrating peptide—arginine octamer (R8) to get R8 modified oxytocin loaded liposomes (OT@LPs-R8) which showed enhanced mucoadhesive. The brain targeting efficiency was evaluated preliminarily after nasal administration. OT@LPs-R8 showed a round shape with a particle size distribution of 110.2 ± 7.3 nm, a surface potential as high as +18 mV, a drug loading (62.17 ± 1.88) %, an encapsulation rate (5.85 ± 0.72) %, and stood stable in nasal mucus. After nasal administration, it could significantly prolong the retention and enhance the distribution in the brain with no irritation to the nasal mucosa. The animal experiment in line with the regulations of the Department of Laboratory Animal Science of Fudan University on the ethics of animal experiments had been carried out after passing the review of the Animal Ethics Committee of Fudan University. The results showed nasal administration of OT@LPs-R8 could promote oxytocin directly into the brain from the nose which expected to become a new carrier for delivery of oxytocin to the brain.
Résumé
Inhibitor of DNA binding 1 (ID1) has an aberrantly high expression in multiple cancer tissues, including colon cancer, lung cancer, breast cancer, and so on, which is closely related to cancer aggressiveness and poor clinical outcomes in cancer patients. It has been reported that ID1 maintains colorectal cancer cells (CRCs) stemness traits and contributes to the CRC drug resistance. While, the biological molecular mechanisms have not been fully elucidated. In this research, we found that ID1 upregulates octamer binding transcription factor (OCT4) protein level as well as OCT4 signaling pathway via Western blot, gene set enrichment analysis (GSEA), dual-luciferase reporter assay, and real-time PCR. Through the in vitro sphere formation assay, we found that overexpression of OCT4 reverses the inhibitory effect of knocking down ID1 on CRC sphere formation ability. With the help of JASPAR and GEPIA database, we predicted a novel transcriptional repressor—forkhead box D3 (FOXD3) of OCT4. Finally, by using co-immunoprecipitation (Co-IP), confocal and real-time PCR, we demonstrated that ID1 interacts with FOXD3 to inhibit its transcriptional repression activity and therefore to upregulate OCT4 transcription and OCT4 signaling pathway. In conclusion, this study provides a new theoretical basis for the regulation mechanism of colon cancer stem cells, and the newly found protein-protein interaction of ID1-FOXD3 provides a potential drug target for the therapy of CRC.
Résumé
Background: The promising improvement in the clinical outcome of lung cancer can be possibly achieved by identification of the molecular events that underlie its pathogenesis. Cancer stem cell (CSC) being one of the subsets of tumor majorly participates in drug resistance and treatment failure because of the moderate cell cycle, lower proliferation, and increased expression of DNA repair and anti-apoptosis genes. Although many putative CSC markers exist, a precise characterization for non-small cell lung cancer is of utmost importance due to increased mortality rate and lack of targeted therapies. Hence, the article focuses on the expression of stemness-associated markers, namely octamer-binding transcription factor 4 (OCT4), NANOG, and sex-determining region Y-box 2 (SOX2) in non-small cell lung cancer (NSCLC) patients. Methods: The expression of OCT4, NANOG, and SOX2 were evaluated in 32 histopathologically confirmed NSCLC tissues using real-time polymerase chain reaction. The obtained expression was correlated with clinical and pathological manifestations using the statistical test such as Student's t-test and Pearson correlation in varied statistical software. Results: Results showed a significantly higher expression of OCT4 and NANOG compared to SOX2 in the tumor tissues. When the expression of these markers was correlated with the clinical parameters, higher expression was seen in males, patients with age above 60 years, and in adenocarcinoma subtype. In correlation with the habit, higher expression of OCT4 and SOX2 was observed in habituated patients. Expression of NANOG and OCT4 was higher even in patients with poor differentiation. Conclusion: The expression and prognostic significance of CSC markers obviously vary depending on histological NSCLC subtype. Importantly, our findings suggest that OCT4, SOX2, and NANOG network together may be promising for ongoing targeted therapies in specific NSCLC subgroups
Résumé
Objective To investigate the expressions of octamer-binding transcription factor 4 (Oct4) and phosphorylated-protein kinase B (p-Akt) proteins in colorectal cancer and their clinical significances, in order to explore the roles of Oct4 and p-Akt in the staging and grading of colorectal cancer. Methods Immunohistochemical technique was used to examine the expression of Oct4 and p-Akt proteins in 78 cases of colorectal cancer in Wuxi Second Hospital of Traditional Chinese Medicine and Wuxi First People's Hospital from January 2011 to December 2016. Relationship between expressions of Oct4 and p-Akt proteins and clinicopathological parameters were analyzed. Results The positive rate of Oct4 in tumors was 74.36 % (58/78), which was obviously higher than that in normal tissues [35.90 % (20/78), χ2= 23.32, P < 0.01]; and the positive rate of p-Akt in tumors was 67.95 % (53/78), which was obviously higher than that in normal tissues[28.21 %(25/78),χ2=24.68,P <0.01].The double positive and negative expression rate of these two proteins accounted for 80.8 %(63/78), with a linear positive correlation (r= 0.455, P < 0.000 1). In 78 cases of colorectal cancer, the expression of Oct4 protein was correlated with histological grade, lymph node metastasis, and Duke staging (all P < 0.05), and the expression of p-Akt protein was correlated with histological grade and lymph node metastasis (both P < 0.05). The multivariate logistic regression analysis showed that the expression of Oct4 protein was related to histological grade and Duke staging(both P<0.05),and the expression of p-Akt protein was only related to lymph node metastasis (P<0.05). Conclusion The combined detection of Oct4 protein and p-Akt protein has reliable and important clinical significance for judging the histological grade,lymph node metastasis and Duke staging of colorectal cancer.
Résumé
Background and purpose:Differentiation of tumor tissue is an important factor on determining the prognosis of gastric cancer. This study aimed to investigate the expression levels and clinical signiifcance of gender determining region Y-box 2 (SOX2) gene and octamer binding factor 4 (OCT4) gene in gastric cancer tissues varying different differentiation degrees. Methods: Sixty cases with gastric cancer were recruited in this study. The gastric cancer tissues and corresponding normal mucosa of the 60 cases were obtained. The mRNA and protein level of SOX2, OCT4 gene are evaluated by the quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry, respectively. The relationship between the expression levels of SOX2, OCT4 gene and clinical pathological parameters were also analyzed in this study. Results:The expression of SOX2 in both mRNA and protein levels had no signiifcant difference between the well-differentiated gastric cancer tissues and normal gastric mucosa (mRNA levels:t=0.1033, P>0.05;protein levels:t=0.116, P>0.05). However, both the mRNA and protein expression of SOX2 in patients with well-differentiated gastric cancer tissues were signiifcant higher than not only in patients with moderately differentiated gastric carcinoma (mRNA levels: t=12.48, P0.05;protein levels:t=1.064, P>0.05). Immunohistochemical study demonstrated that the positive rate of SOX2 in patients with well-differentiated gastric cancer tissues (10/21) were higher than in patients with not only moderately differentiated gastric carcinoma (7/20) but also poorly differentiated gastric carcinoma (2/19, P0.05). Nevertheless, the expression of SOX2, OCT4 were positive or negative correlated with the pathological staging, the degree of inifltration and lymph node metastasis (P<0.05). Conclusion:Decreased SOX2 expression and increased expression level of OCT4 can promote the formation, development and invasion of gastric cancer and they may become biomarkers or the diagnosis, treatment and prognosis evaluation in gastric carcinoma.
Résumé
Objective: To investigate the expression and the clinical significance of Oct4 in rectal carcinoma and canceradjacent tissues.
Résumé
Tumor marker research is a hot field of oncology.Previous studies of tumor markers such as placental alkaline phosphatase,alpha-fetoprotein,c-kit and CD30 and so on,lack sufficient sensitivity and specificity for the early diagnosis of germ cell tumor.Recent research demonstrated that octamer-binding transcription factor 4 (OCT4),sal-like gene 4 (SALL4) can be used as new germ cell tumor markers.This paper reviewed the research progress on the OCT4 and SALL4 in recent years,to provide the reference about the early diagnosis,tumor typing,condition estimation,prognosis and targeted therapy of germ cell tumors.
Résumé
Objective The aim of this study was to understand the role of specific markers of stem cells Oct-4 expression in the development of human epidermal non-melanoma cutaneous tumors.Methods The paraffin-embedded samples were retrieved from files of pathology department at our hospital,including 20 cases of skin squamous cell carcinomas (SCC),20 cases of basal cell carcinomas (BCC),20 cases of seborrhoeic keratosis (SK) and 20 cases of normal skin (from head,face,trunk,extremities).The expression of Oct-4 and PCNA were observed by immunohistochemical staining technique.Results Oct-4 protein was abnormally increased in SCC and BCC comparel to normal skin and SK ( P ＜0.05 ).However there were no significant difference of Oct-4 protein expression between SCC and BCC ( P ＞0.05).There were also no significantly different Oct-4 protein expression between Sk and the normal skin ( P ＞ 0.05 ),and no significantly different Oct-4 protein expression between SK and BCC( P ＞0.05 ).PCNA protein was abnormally increased in SCC and BCC compared to normal skin and SK ( P ＜0.01 ).There were significantly different PCNA protein expression between SCC and BCC( P ＜0.05).There were also significantly different PCNA protein expression between SK and the normal skin ( P ＜ 0.05 ).However there were no significant difference of PCNA protein expression between SK and BCC ( P ＞ 0.05 ).There were positive correlation between the expression intensity of Oct-4 and PCNA in SCC and BCC.Conclusions The abnormal expression of Oct-4 may have an important role in the development of BCC and SCC.Positive Oct-4 expression cells may be the tumor stem cell in SCC and BCC.There were positive correlation between the expression intensity of Oct-4 and PCNA in SCC and BCC.The over expression Oct-4 in BCC and SCC may play an important role in proliferation of tumor.
Résumé
Objective To investigate the expression of Oct4 protein and analyze its correlation with the clinic pathological features and prognosis of non-muscle-invasive bladder cancer.Methods The oct4 protein expression was assessed by immunohistochemical analysis in 87 specimens of bladder transitional cell carcinoma and 15 specimens of adjacent normal tissues.A correlation between Oct4 and clinic pathological features was analyzed.Results The positive rate of Oct4 protein was significantly higher in bladder cancer than that in normal bladder tissue (P＜0.01).The positive rate of Oct4 protein was 40.7％ in G1 bladder cancer,69.4％ in G2 bladder cancer and 91.7％ in G3 bladder cancer,and the differences was significant (P＜0.01).All patients were followed up for 3-78 months,and 63 of them relapsed.The expression of Oct4 protein was significantly higher in patients of recurrence than in non-recurrence (77.8％ ∶ 37.5％,P ＜ 0.01 ).21 patients of recurrence were in progression,and the expression of Oct4 protein had no significant differences between patients of progression and non-progression (71.4％ ∶65.2％,P ＞0.05).The positive rate of Oct4 protein was not related with gender,age,tumor number and size (P ＞0.05).Conclusion The detection of Oct4 protein is in favor of early detection of bladder tunor,estimation the degree of differentiation and surveillance for recurrence of superficial bladder cancer.
Résumé
Objective: To express the fusion protein of hOct4 and cell penetrating peptides using prokaryotic expression systems, and to optimize its expression methods and observe the membrane penetrating ability of the fusion proteins. Methods: The pET-based prokaryotic expression system was constructed by genetic engineering, and the fusion plasmid was transferred into E. coli BL21(DE3) and Rosetta2(DE3). The protein was purified by Ni affinity chromatography and identified by Western blotting analysis. The penetrating ability of the Rhodamine-labelled fusion protein was investigated using BJ cells. Results: We successfully constructed pET21a(+)-hOct4-11R-His and pET21a(+)-EGFP-11R-His vectors. Fusion proteins hOct4-11-His and EGFP-11R-His were generated by transfering the plasmids into E. coli. The fusion protein was verified by Western blotting analysis and was detected in BJ cells. Conclusion: We have successfully generated EGFP-11R-His and hOct4-11R-His fusion proteins, and the proteins can effectively enter the BJ cells and locate around the nuclei.
Résumé
The expression of octamer binding factor 4 (Oct4) gene in bladder cancer cell line T24 and its effects on the biological characteristics of the cells were investigated.RT-PCR and Western blot were employed to detect the expression of Oct4 in T24 cells.The changes of biological charac-teristics in T24 cells were analyzed before and after gene-silencing by Boyden chamber and MTT.The results showed that the expression of Oct4 gene was detectable in T24 cells by RT-PCR and Western blot.The expression of Oct4 gene and protein was down-regulated by siRNA,and average number of transwell cells in interference group,negative control group and blank control group was 101.40±4.56,104.20±10.03 and 111.00±11.90,respectively.There was significant difference in the proliferation ability of the cells from 48 h,72 h to 96 h after the interference by siRNA between in-terference group and negative group or blank control group (P<0.05).It was suggested that Oct4 gene was related with proliferation ability ofT24 cells,but not with invasive capability.
Résumé
PURPOSE: Nocodazole, a microtubule disrupting reagent, is known to arrest cells in the M phase, To gain insight on the regulatory mechanism of H2B histone gene expression by nocodazole in HL-60 cell, the binding pattern of nuclear proteins to cis element in the human H2B histone gene promoter has been investigated with DNase I footprinting and DNA mobility shift assay. MATERIALS AND METHODS: Northern blot hybridization was performed by the method of Virca et al. A Hinc II-Sac I fragment of pSPH28 was used as probe for Northern blot analysis of H2B histone mRNA. DNase I footprinting and DNA mobility shift assay were performed by the method of Lim et al. End labeled DNA oligomer (upper strand, 5'-CTTCACCTTATTTGCATAA GCGATTC-3') for octamer binding activity was mixed with nuclear extracts in a 20 ul reaction volume containing 60 mM KC1, 12 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 12% glycerol, and 2 ug of poly [dI-dC]. RESULTS: The level of H2B histone mRNA rapidly was reduced at 24 hours in nocodazole-treated HL-60 cells and the mRNA was repressed in proportion to the concentration of nocodazole. Nocodazole-dependent repression of H2B histone gene was restored by replacement with nocodazole-free media. In DNase I footprinting analysis, one nuclear factor bound at 42 bp site (octamer motif) in the absence of nocodazole. In the presence of nocodazole, the binding of nuclear factor on octamer motif partially vanished. In DNA mobility shift assay, one DNA-protein complex (Octl) was formed when octamer motif was incubated with nuclear extract of HL-60 cell. After nocodazole treatment, Octl binding activity was reduced by time dependent manner. CONCLUSION: These results suggest that nocodazole-dependent repression of H2B histone gene is correlated with reduction of Octl binding activity in HL-60 cell.