Pig copper zinc superoxide dismutase (CuZnSOD) is an important antioxidant enzyme. Some studies focused on the function of CuZnSOD gene, but the transcriptional regulation of the CuZnSOD gene is not yet fully elucidated. Therefore, the aims of the study were to determine the core promoter region and to explore its mechanism of transcriptional regulation. The 853 bp DNA sequence of 5'-flanking promoter was amplified by performing PCR. A series of CuZnSOD promoter fragments with gradually truncated 5'-end were produced by nested PCR and inserted into pGL3-Basic vector. The activities of the promoters were measured by the dual-luciferase assay system after transient transfection into the NIH/3T3 cells. The results demonstrated that there were 2 potential transcription start sites in the regions from initiation codon to -87 bp and -266 bp, respectively. The region from -383 bp to +67 bp in CuZnSOD gene promoter showed higher activity than other regions, and further deletion analysis demonstrated that the region from -75 bp to -32 bp contained an essential promoter sequence for pig CuZnSOD gene transcription. In addition, several potential transcription factor binding sites were predicted with bioinformatics method. These results suggest that these transcription factor binding sites may be involved in the transcriptional regulation of CuZnSOD gene.
Animals ; Cloning, Molecular ; Gene Expression Regulation ; Genetic Vectors ; Mice ; NIH 3T3 Cells ; Promoter Regions, Genetic ; Superoxide Dismutase ; genetics ; Swine ; Transfection

Objective The aim of this study was to investigate the relationship between phosphotyrosine interac-tion domain containing 1 ( PID1 ) gene and variation in intramuscular fat ( IMF ) content and the possibility to generate transgenic animals by testicular injection .Methods Expression vector pIRES2-acGFP-PID1 carrying pig PID1 gene was incubated with transfection reagents and injected into the testes of male New Zealand rabbits .We examined the F1 genera-tion by fluorescence detection , PCR, Western blotting and measuring the IMF content .The F1 generation gave birth to the F2 generation.Then we examined the F2 generation through detecting the positive rate and the IMF content .Results The exogenous PID1 gene and fluorescent protein gene were expressed at different levels both in the F 1 generation and the F2 generation, and the positive rates were 35.88%and 34.33%, respectively.The IMF content was significantly elevated (P<0.05) in the transgenic positive individuals compared with the negative ones and the control group , and the PID1 protein expression was similarly higher .Conclusions The results of this study demonstrate that PID 1 gene affects intramuscular fat content significantly .Moreover, the results of our analysis provide further evidence that transgenic animals can be gener -ated by testicular injection , and the exogenous gene can be inherited steadily .

OBJECTIVE: To analyze the clinicopathological characteristics and risk factors of 4L lymph node metastasis in left non-small cell lung cancer. METHODS: We retrospectively analyzed the data of 134 patients undergoing surgical resection of left non-small cell lung cancer and 4L lymph node dissection, including 60 patients with squamous cell carcinoma (SCC) and 74 with lung adenocarcinoma (ADC). The clinicopathological characteristics of the patients were analyzed, and logistic regression analysis was used to identify the predictors of station 4L metastasis. RESULTS: Of these patients, 16.4% (22/134) presented with station 4L metastasis. The patients with SCC and ADC showed significant differences in age, gender, smoking history, neoadjuvant chemotherapy, tumor size, tumor location and type, visceral pleural invasion, Ki-67 index, 4L metastasis and pathological TNM stage (stage Ⅱ). The rate of station 4L metastasis was significantly lower in SCC group than in ADC group. Univariate analysis revealed that pathological types (SCC or ADC), visceral pleural invasion, lymphovascular invasion, tumor markerabnormality, and station 5 to 10 metastasis were all high-risk factors for station 4L metastasis. Multivariate analysis suggested that the pathological type (OR=0.120, CONCLUSIONS In patients with left non-small cell lung cancer, station 4L metastasis is not rare and is more likely to occur in patients with lung adenocarcinoma. Dissection of the 4L lymph nodes should be performed in cases with low risk of damages of the adjacent tissues and high risk of station 4L metastasis.
Carcinoma, Non-Small-Cell Lung/pathology* ; Humans ; Lung Neoplasms/pathology* ; Lymph Node Excision ; Lymph Nodes/pathology* ; Lymphatic Metastasis ; Neoplasm Staging ; Prognosis ; Retrospective Studies ; Risk Factors

[Abstract] Objective: To explore the influence of miR-339-5p on the radio-sensitivity of lung cancer A549 cells by regulating the expression of Nudix hydrolase 5 (NUDT5). Methods: X-ray-resistant lung cancer A549 cells (RA549) were induced by treatment with low concentration gradient increment combined with large dose intermittent shock in vitro. The expression level of miR-339-5p in hu‐ man normal lung epithelial cells (BEAS-2B) and lung cancer cell lines (A549, L78, H1299, H460 and RA549 cells) was detected by qP‐ CR. According to the treatment, RA549 cells were divided into NC group, 5Gy group (treatment with 5Gy X-ray), 5Gy+miR-339-5p mimic group, 5Gy+si-NUDT5 group and 5Gy+si-NUDT5+miR-339-5p inhibitor group. CCK-8 assay, Annexin V-FITC/PI double stain‐ ing flow cytometry and WB were used to detect the proliferation, apoptosis and the protein expressions of NUDT5, γ-H2AX and H2AX in each group. The targeting relationship between mir-339-5p and NUDT5 was detected by Dual-luciferase reporter gene system. Results: The expression of miR-339-5p in lung cancer cell lines was significantly lower than that in BEAS-2B cells, with the lowest ex‐pression level in RA549 cells (all P<0.05). NUDT5 was the target gene of miR-339-5p. Compared with the NC group, the prolifera‐ tion activity and NUDT5 expression of RA549 cells in the 5 Gy group were significantly reduced (all P<0.01), and the apoptosis rate was significantly increased (P<0.01). Compared with the 5 Gy group, the proliferation activity of RA549 cells in the 5 Gy+miR-339-5p mimic group was significantly reduced (P<0.05), the apoptosis rate ([12.97±1.48]% vs [5.21±0.62]%, P<0.01) and the expression level of γ-H2AX (P<0.05) were significantly increased; the expression of NUDT5 (t=7.58, P<0.01) and cell proliferation activity (t=6.58, P<0.01) of RA549 cells in the 5 Gy+si-NUDT5 group were significantly reduced, while the apoptosis rate ([11.21±1.06]% vs [5.54±0.44%, P<0.01) and the expression of γ-H2AX (P<0.01) were significantly increased; and the above indicators in 5 Gy+si-NUDT5+miR-339-5p inhibitor group showed insignificant difference from the 5 Gy group. Conclusion: Overexpression of miR-339-5p enhances the radio-sensitivity of X-ray-resistant lung cancer A549 cells by targetedly down-regulating NUDT5 expression.