| Part? High mobility group box 1 mediated the sternness traits by regulating the Janus Kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway.ObjectiveTo investigate whether high mobility group box 1 involves in mediating the stemness traits by regulating the Janus Kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway.Method(1) We established the HMGB1 and STAT3 recombinant plasmid followed by restriction enzyme digestion and sequencing. HMGB1and STAT3 were transfected into SiHa cells, the efficiency of overexpression were determined by qRT-PCR and Western Blot Analysis, simultaneous construct siRNA-HMGB 1 empty vector served as control, the efficiency of overexpression were determined for qRT-PCR and by Western Blot analysis, Knock-down efficiency were determined by qRT-PCR and Western Blot Analysis, Selection of highest interference rate were carried out in subsequent experients.(2) The expression of cancer stem cell markers (Nanog), and p-JAK, JAK, p-STAT3, STAT3 protein were measured by Western Blot. Immunofluorescence technique were employed to measure the variations of p-JAK, JAK, p-STAT3 and STAT3 protein in the nucleus and cytoplasm; Flow cytometry were utilized to analyze the ratios of stem cell markers (OCT4, Sox2 and Nanog); Tumor spheres formation test were utilized to investigate the formation level of tumor spheres on the up or down of HMGB1 expressed genes.(3) The expression of cancer stem cell markers Nanog were measured by Western Blot. Flow cytometry were utilized to analyze the ratios of stem cell markers (OCT4, Sox2 and Nanog); Tumor spheres formation test were utilized to investigate the formation level of tumor spheres on the up or down of STAT3 expressed genes.Result(1) HMGB1 CDS was chemically synthesized and recombinanted to overexpressing plasmid of GV230. STAT3 CDS was extracted by PCR and recombinanted to vector of GV316. This evaluation of vector construction was carried out by duplicate.Enzyme cleavage, PCR Expression, Western Blot and qRT-PCR. demonstrating that current carrier show successful formation that exceeded experimental requirements. Knock-down efficiency are seen in over 80 percent of siRNA-HMGB1.(2) The expression levels of Nanog, pJAK, JAK, pSTAT3, STAT3, OCT4 and Sox2 protein were significantly higher in overexpressing HMGB1 cells than that in siRNA-HMGB1 cells and empty vector (P<0.05). Immunofluorescence technique showed that compared with siRNA-HMGB1 cells and empty vector, phosphorylation of STAT3 and JAK increased and mainly concentrated in overexpressing HMGB1 cells; it showed that HMGB1 can regulate the STAT3 and JAK into the nucleus. Flow cytometry showed that compared with siRNA-HMGB1 cells and empty vector, the stem cell markers (OCT4, Sox2 and Nanog) ratio increased significantly in overexpressing HMGB1 cells (P<0.01); Tumor sphere formation experiment showed that the efficiency of tumor spheres were the highest in the overexpression of and HMGB1 group (P<0.01), while it exhibited only loose cell aggregates in the siRNA-HMGB1+ STAT3 inhibitor in cervical cells (p<0.01). The experiment confirmed that overexpressing HMGB1 can provoke the formation of tumor sphere.(3) The expression levels of Nanog protein were significantly higher in overexpressing STAT3 than that in STAT3 inhibitor cells and empty vector (P<0.05). Flow cytometry showed that compared with STAT3 inhibitor cells and empty vector, the stem cell markers (OCT4, Sox2 and Nanog) ratio increased significantly in overexpressing STAT3 cells (P<0.01); Tumor sphere formation experiment showed that the efficiency of tumor spheres were the highest in the overexpression of and STAT3 group (P<0.01), while it exhibited only loose cell aggregates in the STAT3 inhibitor (P<0.01). The experiment confirmed that overexpressing STAT3 can provoke the formation of tumor sphere.ConclusionHMGB1 is capable of mediating sternness traits by regulating the signal pathway of JAK/STAT3, thereby regulating the sternness traits of cervical cancer; JAK/STAT3 act as the linkage between HMGB1and Nanog in this cascade reaction.Partll The influnce of HMGB1/STAT3/Nanog signal pathway on the DDP of chemoresistanceObjectiveTo examine whether HMGB1/STAT3/Nanog pathway can mediate the cervical cancer cells sensitive to the DDP.Methods(1) We cultured Siha/DDP in vitro with different concentration and duration of cisplatin. (2) we used colony formation method to measure levels of cell proliferation, expression of pH2AX, p53BP1, pSTAT3, and Nanog were analyzed by Westernblot. In addition, we used AnnexinV/PI double staining method to measure apotosis, PI simple staining method to detect cell cycle and flow cytometry method to detect biomarkers of stem cell including OCT4, Sox2 and Nanog.Results(1) SiHa/DDP cell cisplatin resistance index was 8.58, suggesting that SiHa/DDP cells were significantly resistant.(2) Colony formation assay showed that compared with empty vector group with Siha /DDP, lowexpressing HMGB1 and lowexpressing HMGB1+ overexpressing STAT3 with Siha/DDP could result in poor grow of cervical cancer cells (both p<0.05). Experiments have shown that HMGB1 knockdown significantly enhances ddp-induced to inhibit cancer cells proliferation in SiHa/DDP. Western blot assay results showed that compared with empty vector group with Siha/DDP, lowexpressing HMGB1 and lowexpressing HMGB1+ overexpressing STAT3 with Siha/DDP could could increase the levels of p53BPl and pH2AX(both p<0.05), and decrease pSTAT3 and Nanog levels (p<0.05 or p<0.001); we further found that down-regulation of HMGB1 expression at the protein levels was also observed in association with degradation of STAT3 and Nanog and decreased ddp-induced to repair DNA damage in SiHa and SiHa/DDP. AnnexinV/PI double staining and PI single staining showed that compared with empty vector group with Siha/DDP, lowexpressing HMGB1 and lowexpressing HMGB1+ overexpressing STAT3 with Siha/DDP could increase apoptosis and G1 phase proportion (both p<0.05). We further found out that HMGB1 knockdown can inhibit cancer cells proliferation via arresting cells in the G1 phase in SiHa and SiHa/DDP. Flow cytometry showed that compared with empty vector group with Siha/DDP, lowexpressing HMGB1 and lowexpressing HMGB1+ overexpressing STAT3 could decrease significantly stem cell markers (OCT4, Sox2 and Nanog) ratio (both p<0.05). This experiment demonstrated that HMGB1 knockdown can decrease the conversion of tumor stem cell in SiHa and SiHa/DDP.ConclusionHMGB1/STAT3/Nanog pathway mediates the cervical cancer cells sensitivity to the DDP. Knockdown of HMGB1 is useful to increase the DDP sensitivity of the cervical cancer cells.Part? Correlation of stem cell-related transcription factors with a high mobility group Box-1 protein and stem cell-related transcription factors in cervical cancerObjectiveTo detect the correlation of stem cell-related transcription factors with a high mobility group Box-1 protein and stem cell-related transcription factors Nanog in cervical cancer and the effect on prognosis.MethodsImmunohistochemistry was applied to detect HMGB1, STAT3 and Nanog expressions in cervical carcinoma in 78 cases. t test,x2 test, Spearman rank correlation analysis and Cox regression were performed to analyze the expression levels, correlation,and prognosis.ResultsHMGB1, STAT3, and Nanog were highly expressed in CC. As HMGB1 and STAT3 expressions increased, Nanog increased. As HMGB1 and STAT3 expressions decreased, Nanog decreased. Positive correlations were also found between the HMGB1, STAT3, and Nanog. In the group with low HMGB1 and STAT3 expressions, the survival time of the group with a high Nanog expression was significantly lower than that of the group with a low Nanog expression. A multivariate Cox regression model showed that HMGB1, differential degree, lymph node metastasis, invasive depth and surgical margin were independent prognostic factors. STAT3 and Nanog were not independent prognostic factors.ConclusionHMGB1 protein, STAT3 and Nanog were highly expressed in CC. Patients who exhibit increased HMGB1 expression or increased STAT3 consequently show enhanced Nanog expressions, increased poor prognosis. |
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