PMC:6249243 / 28362-32781 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/6249243","sourcedb":"PMC","sourceid":"6249243","source_url":"https://www.ncbi.nlm.nih.gov/pmc/6249243","text":"Materials and methods\n\nHCC patients, samples, and data\nOne hundred and eighty randomly group selected patients (60 SHCC, 60 SLHCC, and 60 NHCC) who underwent HR for HCC and had detailed clinicopathological and prognostic data at Department of Surgery, Xiangya Hospital of Central South University (Changsha, China) from January 2007 to December 2010 were included in this study. Only patients with initially diagnosed with HCC were enrolled, and none of them accepted chemotherapy, transcatheter arterial chemoembolization or radiotherapy before the operation. Diagnosis of HCC was confirmed by two independent histopathologists blind to the information of patients. The surgical indication was HCC patient with enough residual liver volume, and lack of distant metastasis, decompensated cirrhosis, and organic dysfunction. The snap-frozen and paraffin embedded HCCs and ANLTs were collected and reserved. Tumor recurrence and metastasis were detected by serum AFP, ultrasonography and chest x-ray every 3 months in the first 2 years, and twice a year in the next 5 years. Contrast-enhanced Computed Tomography scan or Magnetic Resonance Imaging was performed if relapse or metastasis was suspected clinically. The OS defined as the time from HR to death for HCC or to the date of the last follow-up; DFS defined as the time from the date of HR to the date when relapse or metastasis was detected. The complete clinical, pathological and prognosis data of these patients were collected and stored in our database by a researcher fellow. The present prognostic study strictly follows the consensus of the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guideline41. All the human materials and matching information (all anonymous) were obtained with a written informed consent form, and approved by the ethics committee of Xiangya Hospital of Central South University. All the experiments and procedures were in accordance with the Helsinki Declaration.\n\nIn vivo assay\nFirst, luciferase-labeled HepG2 and MHCC97-L cell lines are built via transfecting by recombinant plasmid pSin-hyg-GFP/luc2 (Berthold, Bad Wildbad, Germany). Then, the HCC subcutaneous and orthotopic transplantation model in nude mice were constructed as described before12. Briefly, 5 × 106 treated cells were subcutaneously injected into the left buttock regions of 4 nude mouse (randomly selected, 3–4 weeks of age, male, BALB/c), and the untreated cells were injected into the right buttock of the same mouse as a control. After 4 weeks of implantation, the mice were sacrificed, and the subcutaneous tumor size was calculated and recorded used vernier caliper as follows: tumor volume (mm3) = (L × W2)/2, where L = long axis and W = short axis42. All the measurements were repeated three times. Then the subcutaneous tumor was cut into pieces of the size as 1 mm3 and implanted into the liver of each group (also random selected, 4 in each group). The activity of luciferase was detected by IVIS Lumina II (Xenogen, Hopkinton, MA) every week according to the protocol of IVIS. After 8 weeks of implantation, the mice were sacrificed, and the size of tumors was calculated and compared as mentioned above. Serial sections were subjected to IHC staining. The animal management and experimental procedures were carried out in accordance with the Guidelines of National Institutes of Health for the Care and Use of Laboratory Animals, and approved by the Medical Experimental Animal Care Commission of Central South University.\n\nStatistical analysis\nStatistical analyses were performed using SPSS 18.0 for Windows (SPSS Inc., Chicago, IL) and GraphPad Prism6 (GraphPad Software Inc., La Jolla, CA). Data were expressed as the mean ± standard error of the mean from at least three independent experiments. Quantitative data between groups were compared using the Student t test or analysis of variance. Dichotomous variables were analyzed by the χ2 test or Fisher exact test. Correlations between different protein expressions level were analyzed using Spearman’s rank analysis. Overall survival and disease-free survival curves were obtained by the Kaplan-Meier method, and differences were compared by the log-rank test. Univariate analysis and multivariate analysis were analyzed with Cox proportional hazard regression model to verify the independent risk factors. A P-value \u003c 0.05 was considered statistically significant.\n","divisions":[{"label":"Title","span":{"begin":0,"end":21}},{"label":"Section","span":{"begin":23,"end":1975}},{"label":"Title","span":{"begin":23,"end":54}},{"label":"Section","span":{"begin":1977,"end":3519}},{"label":"Title","span":{"begin":1977,"end":1990}},{"label":"Section","span":{"begin":3521,"end":4418}},{"label":"Title","span":{"begin":3521,"end":3541}}],"tracks":[{"project":"2_test","denotations":[{"id":"30464168-22642691-79085100","span":{"begin":1684,"end":1686},"obj":"22642691"},{"id":"30464168-23401231-79085101","span":{"begin":2262,"end":2264},"obj":"23401231"},{"id":"30464168-22911555-79085102","span":{"begin":2739,"end":2741},"obj":"22911555"}],"attributes":[{"subj":"30464168-22642691-79085100","pred":"source","obj":"2_test"},{"subj":"30464168-23401231-79085101","pred":"source","obj":"2_test"},{"subj":"30464168-22911555-79085102","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93deec","default":true}]}]}}