The pGenesil-1-Beclin1 eukaryotic expression vectors were constructed to establish an SH-SY5Y cell line stably expressing shRNA-Beclin1. The shRNA was connected to pGenesil-1 to construct the recombinant plasmid pGenesil-1-Beclin1, which was transformed into JM109 E.coli. Positive clones were identified by digestion with restriction endonuclease and DNA sequencing. SH-SY5Y cells were cultured by the conventional method. The pGenesil-1-Beclin1 and pGenesil-1 plasmids were transfected into SH-SY5Ycells, and the cells were screened by G418 until the stable G418-resistant monoclonal cells were acquired. Beclin1 mRNA and Beclin1 protein were detected by RT-PCR and Western blot analysis respectively. The results of restriction endonuclease analysis and DNA sequencing confirmed the correct construction of the eukaryotic expression vector pGenesil-1-Beclin1. Two SH-SY5Y transfected cell lines were successfully selected. Compared with the control group, RT-PCR and Western blot showed that the expression of Beclin1 mRNA and protein were down regulated 71.28%±1.45%(P<0.05)and 75.50%±2.63%(P<0.05), respectively. The results indicated that the eukaryotic expression vector pGenesil-1-Beclin1 was successfully constructed and the SH-SY5Y cell lines with inhibited Beclin1 expression were established. It provides a useful cell model for studying the biological function of Beclin1.
ObjectiveTo systematically review the correlation between Beclin1 protein expression and cervical cancer as well as its different clinical pathologic features. MethodsWe electronically searched databases including The Cochrane Library (Issue 1, 2014), PubMed, EMbase, Ovid, CNKI, VIP, CBM and WanFang Data from inception to February 2014, to collect the correlation between Beclin1 protein expression and cervical cancer as well as its different clinical pathologic features. Two reviewers independently screened literature according to the inclusion and exclusion criteria, extracted data, and assessed the methodological quality of the included studies. Then meta-analysis was conducted using RevMan 5.2 software. ResultsA total of 5 case-control studies involving 637 patients were included, of which, 388 cases in the cervical cancer group, 130 cases in the cervical intraepithelial neoplasia (CIN) group, and 119 cases in the normal cervical tissue group. The results of meta-analysis showed that, a) as for Beclin1 expression, significant differences were found in cervical cancer vs. normal cervical tissues (OR=0.07, 95%CI 0.02 to 0.25, P < 0.000 1), cervical cancer vs. CIN (OR=0.37, 95%CI 0.23 to 0.59, P < 0.000 1), CIN vs. normal cervical tissues (OR=0.23, 95%CI 0.06 to 0.88, P=0.03), and cervical cancer tissues with vs. without lymph node metastasis (OR=0.29, 95%CI 0.17 to 0.49, P < 0.000 01). However, no significant difference was found in medium/low differentiation vs. well differentiation (OR=0.50, 95%CI 0.16 to 1.56, P=0.23), tumour diameter no less than vs. less than 4 cm (OR=0.72, 95%CI 0.44 to 1.18, P=0.20), myometrial invasion depth no less than vs. less than 1/2, and FIGO Ⅰ vs. Ⅱ (OR=0.70, 95%CI 0.44 to 1.10, P=0.12). ConclusionBeclin1 protein expression is notably associated to cervical cancer. Due to the limited quantity and quality of the included studies, the above conclusion still needs to be further verified by performing more high quality studies.
ObjectiveTo analyze the expression and significance of NF-κBp65 and autophagy-related proteins Beclin1 and p62 in patients with papillary thyroid carcinoma (PTC).MethodsOne hundred and sixty cases of PTC patients' tumor tissue specimens and paracancerous tissue specimens in our hospital from March 2013 to February 2015 were collected, and 90 cases of cervical lymph node metastasis tissue specimens of the above patients were collected. The expressions of NF-κBp65, Beclin1 and p62 in PTC tissues, metastatic lymph node tissues and paracancerous tissues were detected by immunohistochemical method, and the relationship between the above indexes and the clinicopathological characteristics and prognosis of PTC patients was analyzed.ResultsThe positive rates of expression of NF-kappa Bp65 and p62 in PTC tissues and metastatic lymph node tissues were higher than those in paracancerous tissues (P<0.05). The expression rate of Beclin1 in PTC tissues and metastatic lymph node tissues was lower than that in paracancerous tissues (P<0.05). The positive rate of NF-κBp65 expression in PTC tissues was not related to the clinicopathological characteristics of patients (P>0.05). The expression of p62 decreased with the increase of tumor differentiation (P<0.05). The expression of Beclin1 in patients with stage Ⅲ+Ⅳ and lymph node metastasis were lower than those in patients with stage Ⅰ+Ⅱ and without lymph node metastasis (P<0.05), while the expression of p62 was opposite. Spearman correlation analysis showed that the expression of Beclin1 and p62 in PTC tissues was negatively correlated (r=–0.656, P<0.01). In metastatic lymph node tissues, the expression of Beclin1 and p62 was also negatively correlated (r=–0.562, P<0.01). The 3-year survival rates of patients with positive expression of p62 and NF-κBp65 in PTC tissues were lower than that of patients with negative expression (P<0.05). The 3-year survival rate of patients with positive expression of Becrin1 was higher than that of negative expression (P<0.05). TNM stage, lymph node metastasis, NF-κBp65 and p62 were independent risk factors for PTC prognosis, and Beclin1 was protective factor.ConclusionsNF-κBp65 and p62 are highly expressed in PTC tissues and lymph node metastasis tissues, while Beclin1 is poorly expressed, which could be used as independent prognostic factors for PTC patients. In addition, Beclin1 and p62 are related to PTC biological behavior and may become potential indicators for PTC diagnosis.