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08 February 2019, Volume 46 Issue 2 Previous Issue    Next Issue

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Effect of targeted silencing Notch1 on proliferation and apoptosis of human nonsmall cell lung cancer stem cells  Liu Hengyao1,2, Mu Yanling2, Wang Yan2, Wang Fuwen2, Zhao Guoli1,3, Wang Zhaopeng3, Zhou Shuping3, Cai Haibo4, Zhang Yueying3 2019, 46 (2):  65-71.  doi: 10.3760/cma.j.issn.1673422X.2019.02.001 Abstract ( 536 )   PDF (2381KB) ( 504 )   Save Objective  To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells. Methods  Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group, Nc-shRNA group and Notch1-shRNA group. The Nc-shRNA group was a negative control RNAi lentivirus group, and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group. The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1. The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay. Apoptosis was detected by Annexin V/7-AAD double staining. Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1. Results  The results of qRT-PCR showed that the relative expression levels of Notch1 in control group, Nc-shRNA group and Notch1shRNA group in A549 cells and SPC-A-1 cells were 1.000±0.000, 0.937±0.025, 0.490±0.036 and 1.000±0.000, 1.077±0.070, 0.373±0.038, with statistically significant differences (F=359.707, P<0.001; F=210.455, P<0.001), further paired comparison, the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P<0.05). Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results. MTT assay showed that the 24 h A values of A549 cells in control group, Nc-shRNA group and Notch1shRNA group were 0.209±0.005, 0.219±0.009, 0.159±0.006, 48 h A values were 0.293±0.004, 0.302±0.004, 0.205±0.005, 72 h A values were 0.450±0.003, 0.430±0.012, 0.348±0.017, with statistically significant differences (F=79.487, P<0.001; F=508.664, P<0.001; F=57.156, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of NcshRNA group at 24, 48, 72 h (all P<0.05). The 48 h A values of SPC-A-1 cells in control group, Nc-shRNA group and Notch1shRNA group were 0.438±0.022, 0.412±0.015, 0.364±0.010, 72 h A values were 0.540±0.016, 0.519±0.009, 0.438±0.019, with statistically significant differences (F=15.667, P=0.004; F=37.299, P<0.001), further paired comparison, the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P<0.05). The sphere sizes of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667±6.506)μm, (136.667±7.095)μm, (86.676±7.638)μm, with statistically significant difference (F=65.940, P<0.001). The sphere sizes of the three groups in SPC-A-1 cells were (118.667±6.658)μm, (128.000±7.000)μm, (60.675±4.509)μm, with statistically significant difference (F=105.372, P<0.001). Further paired comparison, the sphere size of Notch1-shRNA group was significantly smaller than that of Nc-shRNA group in the two kinds of cells (all P<0.05). The apoptosis rates of control group, Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were (0.489±0.014)%, (0.633±0.021)%, (1.683±0.221)% and (1.323±0.194)%, (1.690±0.188)%, (3.017±0.356)%, with statistically significant differences (F=77.660, P<0.001; F=32.200, P=0.001), further paired comparison, the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P<0.05). Western blotting showed that the expressions of PCNA, Bcl-2 and Hes-1 in control group, Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F=155.343, P<0.001; F=22.576, P=0.002; F=70.108, P<0.001), and the expressions of PCNA, Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F=49.419, P<0.001; F=28.090, P=0.001; F=12.040, P=0.007). Further paired comparison, the expressions of PCNA, Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells, and the differences were statistically significant (all P<0.05). Conclusion  Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis, which may be related to the down-regulation of its downstream gene Hes1 Related Articles | Metrics

miR-182-5p enhances proliferation and inhibits apoptosis of A549 lung cancer cells by targeting forkhead box O3a Gong Qian1, Chen Yun1, Liao Dehua1, Fu Yilan1, Cao Lizhi1, Yao Dunwu1, Yang Xiaohong2 2019, 46 (2):  72-76.  doi: 10.3760/cma.j.issn.1673422X.2019.02.002 Abstract ( 482 )   PDF (1120KB) ( 454 )   Save Objective  To evaluate the effect of microRNA-182-5p (miR-182-5p) on proliferation and apoptosis of non-small cell lung cancer (NSCLC) A549 cells by targeting forkhead box O3a (FOXO3a). Methods  The difference of miR-182-5p expression between human normal lung epithelial cells BEAS-2B and NSCLC cells A549 was compared. The A549 cells were chosen, and miR-182-5p mimic (miR-182-5p mimic group), miR-182-5p inhibitor (miR-182-5p inhibitor group), negative control mimic (NC mimic group) and negative control inhibitor (NC inhibitor group) were transfected respectively. The expression of miR-182-5p was detected by reverse transcriptionpolymerase chain reaction (RT-PCR). The protein expression of FOXO3a was detected by Western blotting. The cell proliferation activity was detected by methyl thiazolyl tetrazolium (MTT) method. The cell apoptosis was detected by flow cytometry. The targeted relationship between miR-182-5p and FOXO3a was detected by dualluciferase experiment. Results  The miR-182-5p expression of A549 cells and BEAS-2B cells respectively was 3.21±0.24 and 1.01±0.11, and the difference was statistically significant (t=14.209, P＜0.001). The miR182-5p expression of NC mimic group, miR-182-5p mimic group, NC inhibitor group and miR-182-5p inhibitor group respectively was 1.09±0.20, 12.80±1.10, 1.03±0.11 and 0.47±0.08, and the difference was statistically significant (F=87.872, P＜0.001). The FOXO3a expression of the above four groups respectively was 118.34±16.71, 50.89±11.58, 125.33±20.87 and 289.26±34.51, and the difference was statistically significant (F=62.125, P＜0.001). The 72 h proliferation activity of the four groups respectively was 1.12±0.13, 1.70±0.14, 1.07±0.13 and 0.71±0.11, and the difference was statistically significant (F=31.336, P＜0.001). The proliferation activity of miR-182-5p mimic group was significantly higher than that of NC mimic group (P＜0.05), and the proliferation activity of miR-182-5p inhibitor group was significantly lower than that of NC inhibitor group (P＜0.05). The apoptosis rate of the four groups respectively was (5.51±1.80)%, (1.41±0.50)%, (6.24±1.71)% and (47.93±5.12)%, and the difference was statistically significant (F=211.081, P＜0.001). The apoptosis rate of miR1825p mimic group was significantly lower than that of NC mimic group (P＜0.05), and the apoptosis rate of miR-182-5p inhibitor group was significantly higher than that of NC inhibitor group (P＜0.001). The miRNA target genes prediction software test results showed that miR-182-5p could act on FOXO3a 3′-untranslated region (UTR). Compared with transfection NC mimic, co-transfection miR-182-5p mimic and FOXO3aWt could make luciferase activity of A549 significantly decreased (1.20±0.14 vs. 0.62±0.10; t=5.839, P=0.004）. Conclusion  miR-182-5p can enhance proliferation and inhibit apoptosis of A549 cell by targeting FOXO3a. Related Articles | Metrics

Effect of up-regulation of miR-193b on HBV positive hepatocellular carcinoma cells Huang Rui1, Wu Gang1, Xu Jian1, Zheng Bo1, Huang Lingyuan2, Zhong Zhendong1 2019, 46 (2):  77-81.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.003 Abstract ( 414 )   PDF (2557KB) ( 459 )   Save Objective  To investigate the effect of miRNA-193b (miR-193b) up-regulation on the apoptosis, invasion and metastasis of hepatitis B virus (HBV)positive hepatocellular cells. Methods  HepG2.2.15 cells were cultured until logarithmic growth phase and transfected with miR-193b mimic at a concentration of 25, 50, 100 pmol. The control group was transfected with blank mimic (0 pmol). After 48 hours, the expression of miR-193b in each group was detected by realtime quantitative polymerase chain reaction (qRT-PCR), the apoptosis rate in each group was detected by flow cytometry, the cell migration was detected by cell scratch assay, the cell invasion was detected by Transwell assay, and the expressions of Bax, Bcl-2, matrix metalloprotease (MMP)-9 and MMP-2 protein were detected by Western blotting. Results  qRT-PCR results showed that the miR-193b expressions of miR-193b mimic (25, 50 and 100 pmol) groups and control group in HepG2.2.15 cells were 1.05±0.09, 1.53±0.12, 2.08±0.17 and 0.49±0.12, and the difference was statistically significant (F=261.35, P<0.001). Compared with the control group, miR-193b expression significantly increased in each transfection group (P=0.036; P=0.029; P=0.022). Flow cytometry results showed the apoptosis rates of miR193b mimic (25, 50 and 100 pmol) groups and control group were (30.28±5.22)%, (53.41±6.18)%, (79.89±7.13)% and (1.02±0.13)%, and the difference was statistically significant (F=357.19, P<0.001). Compared with the control group, the apoptosis rate significantly increased in each transfection group (P=0.025; P=0.010; P=0.007). Cell scratch assay results showed that the migration distances of miR-193b mimic (25, 50 and 100 pmol) groups and control group were (27.53±1.54)mm, (19.24±2.12)mm, (13.42±1.53)mm and (34.95±1.92)mm, and the difference was statistically significant (F=408.62, P<0.001). Compared with the control group, the migration distance significantly decreased in each transfection group (P=0.032; P=0.007; P=0.006). Transwell experimental results showed that the absorbance values of miR-193b mimic (25, 50 and 100 pmol) groups and control group were 1.02±0.12, 0.59±0.13, 0.42±0.10 and 1.68±0.16, and the difference was statistically significant (F=511.68, P<0.001). Compared with the control group, the cell invasion ability significantly weakened in each transfection group (P=0.028; P=0.005; P=0.002). Western blotting results showed that the expressions of Bax, Bcl-2, MMP-9 and MMP-2 protein of miR-193b mimic (25, 50 and 100 pmol) groups and control group had statistically significant difference (F=264.38, P<0.001; F=437.19, P<0.001; F=377.46, P<0.001; F=208.79, P<0.001). Further paired comparison showed that the expressions of Bax, Bcl-2, MMP-9 and MMP-2 protein between mimics groups and control group were statistically significant (all P<0.05). Conclusion  miR-193b can induce the apoptosis and inhibit the invasion, metastasis of HBVpositive hepatocellular cells, and the mechanism may be related to the regulation of Bax, Bcl-2, MMP-9 and MMP-2 protein expression. Related Articles | Metrics

Clinical significance of preoperative platelet count in laryngeal squamous cell carcinoma prognosis Hu Yanhong, Zhao Guofeng, Wang Donghai 2019, 46 (2):  82-86.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.004 Abstract ( 566 )   PDF (911KB) ( 533 )   Save Objective  To explore the effect of preoperative platelet (PLT) count on the prognosis of patients with laryngeal squamous cell carcinoma. Methods  The clinical data of 286 patients with laryngeal squamous cell carcinoma were retrospectively analyzed to determine the optimal critical value of PLT count for end point of recurrence and death. The effects of preoperative PLT count on the recurrence and 5-year survival rates of patients with laryngeal squamous cell carcinoma after surgery were analyzed. Results  The optimal critical value of PLT count for end point of recurrence was 242.5×109/L. The patients were divided into PLT≥242.5×109/L group (n=115) and PLT<242.5×109/L group (n=171). Single factor analysis indicated that the recurrence was not related to age (χ2=0.005, P=0.942), gender (χ2=0.309, P=0.579) and pathological differentiation (Z=2.858, P=0.240), and was related to T staging (χ2=10.509, P=0.001), lymph node metastasis (χ2=7.297, P=0.007), primary tumor site (χ2=16.797, P<0.001) and preoperative PLT count (χ2=12.081, P=0.001). Multivariate analysis indicated that T staging (OR=0.518, 95%CI: 0.2810.954, P=0.035), primary tumor site (OR=2.371, 95%CI: 1.2834.382, P=0.006), and PLT count (OR=2.885, 95%CI: 1.6075.179, P<0.001) were the independent factors affecting the recurrence of laryngeal squamous cell carcinoma. The optimal critical value of PLT count for end point of death was 251.5×109/L. The patients were divided into PLT≥251.5×109/L group (n=94) and PLT<251.5×109/L group (n=192). Single factor analysis indicated that the 5year survival rate was not related to age (χ2=0.030, P=0.863), gender (χ2=0.000, P=0.945) and pathological differentiation (χ2=4.050, P=0.133), and was related to T staging (χ2=41.630， P<0.001), lymph node metastasis (χ2=58.110， P<0.001), primary tumor site (χ2=36.250, P<0.001) and preoperative PLT count (χ2=4.790， P=0.029). Multivariate analysis indicated that T staging (HR=0.353, 95%CI: 0.1930.645, P=0.001), primary tumor site (HR=2.151, 95%CI: 1.3123.526, P=0.002), lymph node metastasis (HR=2.819, 95%CI: 1.6334.867, P<0.001), and PLT count (HR=1.853, 95%CI: 1.1602.960, P=0.010) were the independent factors affecting 5year survival rates of laryngeal squamous cell carcinoma. KaplanMeier survival analysis indicated that the 5year survival rate of PLT≥251.5×109/L group and PLT<251.5×109/L group were 58.23%, 67.87%, with significant difference (χ2=4.79, P=0.029). Conclusion  Preoperative PLT count is the influence factor of recurrence and 5-year survival rate of laryngeal squamous cell carcinoma patients, which has important significance to the prognosis of laryngeal squamous cell carcinoma patients. Related Articles | Metrics

Mechanism study and immunotherapy of immune checkpoint PD-1/PD-L1 Hu Gengwei1, Zhang Ying2, Wu Zhihao3 2019, 46 (2):  87-90.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.005 Abstract ( 844 )   PDF (675KB) ( 966 )   Save Programmed death ligand-1 (PD-L1) is highly expressed on most tumor cells, and it interacts with programmed death-1 (PD-1) on the surface of immune cells, which mainly inhibits T cell proliferation and plays an important role in tumor immune escape. The studies find that PD1/PDL1 pathway can promote tumor cell glycolysis and epithelialmesenchymal transition, and can induce PDL1 expression on macrophages and enhance immunosuppression in tumor microenvironment. Therefore, PD-1/PD-L1 is considered to be an important immunoassay point, and a series of antiPD-1 and PD-L1 antibodies, such as pembrolizumab, nivolumab, atezolizumab, durvalumab and avelumab, have clinically shown good effects. Further understanding of its mechanism may provide new ideas for the treatment of malignant tumors such as lung tumors. Related Articles | Metrics

Effect of tumor metabolism on tumor immunity Yang Qianlu, Huang Yunchao 2019, 46 (2):  91-93.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.006 Abstract ( 1022 )   PDF (666KB) ( 932 )   Save Tumor metabolism is closely related to tumor occurrence, development and the behavior of tumor immune escape. The local nutrient consumption (such as glucose metabolism, amino acid metabolism and oxygen metabolism) of tumor metabolism and the effect of metabolites on immune cells are the hotspots in the field of tumor immunology and it can also guide the research of new anticancer drugs related to tumor immunity. Related Articles | Metrics

Medical treatment in head and neck squamous cell carcinoma Chai Yue, Dong Mei 2019, 46 (2):  94-97.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.007 Abstract ( 893 )   PDF (676KB) ( 617 )   Save Medical treatment is an important strategy for patients with head and neck squamous cell carcinoma (HNSCC). Studies have shown that threeweekly cisplatin regimen is a promising treatment for patients with locally advanced HNSCC and cetuximab plus platinum and 5fluorouracil can be the firstline therapy for patients with recurrent or metastatic HNSCC. Current results from immunotherapy trials have shown an improved response rate and overall survival in patients with recurrent or metastatic HNSCC while maintaining a safe and tolerable toxicity profile. Immunotherapy is becoming the fourth treatment strategy towards cancer. There is still insufficient evidence that patients with HNSCC can benefit from induction chemotherapy and further study is warranted. Related Articles | Metrics

Advancement of targeted agents in the treatment of anaplastic thyroid carcinoma Wu Yuanyuan, Wang Jun 2019, 46 (2):  98-101.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.008 Abstract ( 1797 )   PDF (676KB) ( 846 )   Save Anaplastic thyroid carcinoma (ATC) is very rare and aggressive in humans, and its survival time is most often expressed in weeks or months. For now, there is no standard treatment for longterm survival, even if the multidisciplinary approach could prolong survival in some patients. The development of oncomolecular biology promotes the understanding about the pathogenesis of ATC from genetic level, and then providing a theoretical basis for targeted therapy. Some targeted drugs have been approved for the treatment of refractory advanced differentiated thyroid cancer, in the meantime, the clinical trials of targeted therapy bring new hope for ATC patients. Related Articles | Metrics

Expression and clinical significance of p53 gene of non-small cell lung cancer Wen Jie, Chen Shaoshui 2019, 46 (2):  102-104.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.009 Abstract ( 946 )   PDF (664KB) ( 613 )   Save The development of nonsmall cell lung cancer (NSCLC) is a complex process which referring to multifactor interaction. In this process, p53 gene regulates the normal growth of cells, but p53 gene after mutation can induce the occurrence of NSCLC, promote tumor distant metastasis, induce chemotherapy resistance, and cause poor prognosis of patients. Therefore, the restoration of normal expression of p53 gene is very significant for the treatment of NSCLC. In recent years, the rapid progress of p53 gene therapy has opened up a new way for the treatment of NSCLC. Related Articles | Metrics

Treatment of non-small cell lung cancer with EGFR-TKI Long Lili1, Liang Yanling2, Zhang Xingmei2, Shi Yusheng3 2019, 46 (2):  105-108.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.010 Abstract ( 907 )   PDF (674KB) ( 1058 )   Save Epidermal growth factor receptortyrosine kinase inhibitors (EGFR-TKIs) are the key to the treatment of advanced nonsmall cell lung cancer (NSCLC), but there are various drugresistant mutations in the latter stage. The first and second generation of EGFRTKIs significantly prolong the survival of patients with EGFR-activated mutant tumors. The third generation of EGFR-TKIs effectively inhibit the progress of T790M mutant tumors. The fourth generation of EGFR-TKIs inhibit both L858R, T790M and L858R, T790M, C797S mutant tumors. Using circulating tumor DNA and exogenous RNA can effectively detect the mutation types of EGFR, and choose EGFR-TKIs therapy or combined chemotherapy according to the mutation types. Related Articles | Metrics

Analysis of current status of neoadjuvant chemotherapy for non-small cell lung cancer Li Qin, Fu Zhenming 2019, 46 (2):  109-112.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.011 Abstract ( 611 )   PDF (674KB) ( 663 )   Save Lung cancer has been the most dangerous malignant tumor in the worldwide. Nowadays, for patients with resectable early or local advanced nonsmall cell lung cancer (NSCLC), both domestic and foreign guidelines recommended multidisciplinary treatments like surgery, chemotherapy and radiotherapy. Recent studies have shown that neoadjuvant chemotherapy can significantly improve the prognosis of patients with resectable NSCLC and has better treatment compliance and tolerance. Related Articles | Metrics

Radiotherapy for elderly esophageal cancer Liao Ye, Zhao Lina, Shi Mei 2019, 46 (2):  113-116.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.012 Abstract ( 535 )   PDF (675KB) ( 876 )   Save For resectable elderly esophageal cancer, neoadjuvant chemoradiotherapy combined with surgery is still the preferred treatment under the condition of fully measuring the patient′s physical function. Definitive chemoradiation is the first choice for patients with inoperable esophageal cancer, and may be a good alternative for elderly operable patients with good response to tumor regression. For older patients (>80 years old), radiotherapy alone may be mild and conservative enough to be the preferred option. At the same time, compared with conventional radiation dose (50.4 Gy), selective regional lymph node irradiation, standard PF regimen (cisplatin + 5fluorouracil), radiotherapy of high dose (≥60 Gy), involving field irradiation combined with singledrug chemotherapy (such as tegio) can bring better prognosis. Related Articles | Metrics

Application of shear wave elastography in cervical cancer Zeng Manting1, Liu Jihua1, Zhou Ningbo2, Wang Jian1, Li Xuanxuan1, Zhu Hong1 2019, 46 (2):  117-120.  doi: 10.3760/cma.j.issn.1673-422X.2019.02.013 Abstract ( 601 )   PDF (673KB) ( 505 )   Save Shear wave elastography (SWE) is used to quantitatively analyze the hardness of the tissue by Young′s modulus. The hardness of the tissue is visualized in the form of color coding to distinguish the benign and malignant tissue detected. SWE has higher sensitivity, accuracy and specificity compared with traditional color doppler, which is more objective than elastography, safer, cheaper and simpler than MRI. SWE has a good application prospect in the diagnosis, clinical staging and curative effect monitoring of cervical cancer. Related Articles | Metrics