The Experimental Study Of The Regulation Of HEXIM1 Protein On The Dormancy And Activation Of Stem Cell In Head And Neck Squamous Cell

Objective:At present,the proliferation of cancer cells as the target for the proliferation of cancer cells,G0 phase(dormant)cancer cells are not obvious,and under certain conditions caused by recurrence.Cancer cells import G0 mechanism is not clear,if the mechanism of cancer cells in and out of the cell cycle can be stayed in G0 phase through the regulation of cancer cells(dormant)long time without proliferation,so as to achieve the purpose of cancer cell and organism coexist.Therefore,it is of great significance to further explore the mechanism of cell cycle regulation of cancer stem cells in order to improve the prognosis of head and neck squamous cell carcinoma.It is possible to clarify the mechanism of cancer stem cells in and out of the G0 phase and applicate anticancer agent to kill residual cancer stem cells,so as to prevent the recurrence of the objective.In recent years,studies have found that Sox2 plays an important role in tumor formation,its abnormal expression has been linked to lung cancer,breast cancer,esophageal cancer and nasopharyngeal carcinoma,are closely related.SOX2 involves epigenetic modification and stem cell characterization during tumor development.Japanese scholar induced mouse fibroblasts into exogenous stem cells with embryonic stem cell characteristics,iPS cells,by exogenous introduction of Oct4,Sox2,c-myc and Klf4.The following year they used the same method to reconstruct human skin fibroblasts and human neonatal fibroblasts into iPS cells.Therefore,SOX2 is also regarded as a marker of cancer stem cells.5 fluorouracil(5-fluorouracil,5-FU)is widely used in clinical medicine cell cycle specificity,its main function is to S,is the basic treatment of malignant tumor chemotherapy,chemotherapy has many containing 5-fluorouracil.Vera and the research group found that 5-FU enrichment of cancer cells resistant to chemotherapy has many of the characteristics of stem cells,such as colony forming ability,rich in the phenotype of SP cells,the high expression of stem cell markers,low expression of differentiation markers.Serum free culture: it has been reported that some mammalian adult stem cells in serum-free medium(SFM).In the SFM,most of the differentiated tumor cells can not adhere to the apoptosis,leaving some of the suspension was spherical growth,proliferation ability,similar to stem cells of tumor cells.Serum-free suspension culture can be used as an effective method for enrichment of tumor stem cells.P-TEFb complex phosphorylation CTD-Ser2 triggers transcriptional activity extension,so CTD-Ser2 phosphorylation and transcriptional activity extension is a sign of eukaryotic cell gene expression.In 2008,Zhou found that at the end of the cell mitosis,when blocking P-TEFb from being raised to the chromosome,the cells were blocked in the G0 phase and could not enter the S phase.Therefore,CTD-Ser2 phosphorylation can be seen as the cell from the dormancy period into the proliferative phase(activation period)of the sign,and vice versa for dormancy.In recent years,Pol II-regulated gene transcription mechanism has become a hotspot,and hexamethylene diacetamide-induced protein HEXIM1 has been studied as a potential tumor suppressor gene in the pathogenesis of breast cancer proliferation,metastasis and acute leukemia.And HEXIM1 or P-TEFb in cancer cells and cancer stem cell dormancy and activation of the regulatory mechanism of the study is rarely reportedIn this study,human pharyngeal squamous cell carcinoma cell line Fadu cells as the experimental object,using 5 fluorouracil and effect on Fadu cell stem cell enrichment and purification effect of serum culture,observe the dynamic changes of stem cell transcription factor SOX2 expression,HEXIM1 expression,positive transcription elongation factor P-TEFb and phosphorylation of CTD-Ser2 amino acids and stem cell enrichment and in the process of differentiation.The regulation of P-TEFb and CTD-Ser2 p was detected by transfection of HEXIM1.To investigate the regulatory mechanism of HEXIM1 protein on the Fadu cell stem cells from G0 phase to S phase,namely,the dormancy and activation of Fadu stem cells.Materials and Methods:1.Immunohistochemical staining was used to detect the expression of HEXIM1 in laryngeal carcinoma.2.10%FBS and 100U/ml of penicillin and streptomycin MEM medium 37 C and 5% CO2 of Fadu cell culture.3.The morphology of human pharyngeal squamous cell carcinoma Fadu cells was observed by inverted microscope.4.MTT assay was used to determine the concentration of 5-Fu inhibition(IC50)of the proliferation of Fadu cells.5.According to 5-Fu(IC50)Fadu cells,5-Fu cell model of Fadu cells was established by 0h and 48 h before and after treatment.6.Indirect immunofluorescence staining was used to detect the expression of SOX2 and CTD-Ser2 p in Fadu cell model of 5-Fu cells.7.Fadu cells were cultured in serum-free medium(SFM)to collect suspended Fadu stem cells.8.SiRNA was transiently transfected into HEXIM1 by Lipofectamine 2000 reagent.9.The expression of SOX2,HEXIM1,CDK9,Cyclin-T1 and CTD-Ser2 p protein was detected by Western blot.10.Real-time PCR technology was used to detect the expression level of SOX2,HEXIM1,CDK9,Cyclin-T1 and CTD-Ser2 p in mRNA.11.The cell cycle was detected by flow cytometry.12.,all data using the results of 3 independent experiments,the mean + standard deviation(+ s)to express.SPSS 17 statistical analysis software was used for statistical analysis.T test was used for comparison between groups,P< 0.05 was statistically significant.Results:1?HEXIM1 positive expression in head and neck squamous cell carcinoma and expression trend and cell differentiationHEXIM1 was found to be expressed in normal laryngeal squamous cell carcinoma and laryngeal squamous cell carcinoma by HEXIM1 staining by immunohistochemical staining.HEXIM1 was expressed in normal laryngeal squamous epithelium and negative in basal layer Expression,in laryngeal squamous cell carcinoma,HEXIM1 is highly expressed in the cancer nest.2?5 fluorouracil inhibited proliferation of human nasopharyngeal carcinoma cell line FaduThe 5-fluorouracil(5-fluorouracil)was used to observe the proliferation inhibition rate of Fadu cells at 24 h,48h and 72 h after 0-1000?g / ml drug concentration gradient.Compared with the control group,5-Fu Fadu cell proliferation was inhibited to varying degrees,and this inhibition was positively correlated with drug concentration and duration of action.According to MTT test results,this experiment selected 1000?g / ml,the role of 48 hours as 5-FU Fadu cells on the half inhibitory dose(IC50).3?5-Fu can enrich the stem cells of Fadu cellsSOX2 immunofluorescence staining of Fadu cells was observed by 5-Fu(IC50)before and after treatment with 5-Fu(IC50),and SOX2 was almost expressed after 5-Fu,and the expression of SOX2 was significantly increased after 5-Fu effect.The expression of SOX2 in Fadu cells was detected by Western blot.The expression of SOX2 was significantly increased after 5-Fu treatment,and the expression of SOX2 was significantly decreased after 48 hours of normal culture.The results of Realtime PCR were consistent with those of Western blot.At the same time,we used stem cells to extract Fadu cells with serum-free culture method with clear stem cell purification,and the expression of SOX2 protein was detected by Western blot.The expression of SOX2 in serum-free culture group was significantly higher.The results showed that 5-Fu Fadu cells had high expression of stem cell marker SOX2,and 5-Fu could enrich the stem cells of Fadu cells.4? HEXIM1,CTD-Ser2 p,P-TEFb and Fadu cell cycle regulation relatedThe expression of HEXIM1,CTD-Ser2 p,CDK9 and Cyclin-T1 in Fadu cells before and after 5-Fu treatment was detected by Western blot and Realtime PCR.The expression of HEXIM1 was significantly increased after 5-Fu recovery,CTD-Ser2 p,CDK9 and Cyclin-T1 expression decreased accordingly.The expression of CTD-Ser2 p,CDK9 and Cyclin-T1 was up-regulated,and the expression of HEXIM1 was completely opposite to that of CTD-Ser2 p,CDK9 and Cyclin-T1,and the expression of HEXIM1 was down-regulated by 5-Fu.The results suggest that the effect of 5-Fu on the expression of HEXIM1,CTD-Ser2 p,CDK9 and Cyclin-T1.The results of cell cycle test showed that the proportion of cells in G0 phase was significantly increased and the proportion of S phase cells was significantly decreased after recovery of 0-h after 5-Fu effect,and the proportion of S phase cells in recovery group was significantly higher than that in recovery group The proportion of cells decreased significantly.These results suggest that the stem cells of Fadu cells are associated with HEXIM1,CTD-Ser2 p,CDK9 and CyclinT1 from G0 phase to S phase.5?HEXIM1 inhibits the transcription elongation activity by inhibiting P-TEFb activity in Fadu cellsFadu/Si and its blank vector control cell line Fadu/N,Fadu/NC were established by using the interfering sequence to transiently silence HEXIM1 gene.The expression of HEXIM1,CTD-Ser2 p,CDK9 and Cyclin-T1 protein and RNA level in HadIM1 gene were detected by Western blot and Realtime PCR.The expression of HEXIM1 was down-regulated and the expression of CTD-Ser2 p,CDK9 and CyclinIndicating that HEXIM1 can negatively regulate the expression of CTD-Ser2 p,CDK9 and Cyclin-T1.The results suggest that HEXIM1 inhibits transcriptional elongation by inhibiting P-TEFb activity.6?HEXIM1 inhibits cell cycle progression of Fadu cells by inhibiting P-TEFb activityThe cell cycle of Fadu cells with transient silencing HEXIM1 gene was detected by flow cytometry.Compared with the control group,the proportion of S phase cells in Fadu cells was significantly increased and the proportion of cells in G0 phase was significantly decreased,suggesting that DNA synthesis increased,Fadu Cells are transformed from G0 to S phase.The expression of HEXIM1,CTD-Ser2 p,CDK9 and Cyclin-T1 in HEXIM1 gene silencing group was observed.These results suggest that HEXIM1 in Fadu cells inhibits transcriptional elongation by inhibiting P-TEFb-regulated signaling pathway and prevents Fadu cells from transforming to proliferative phase during dormancy.Conclusion:1.The expression of HEXIM1 was positively correlated with the degree of cell differentiation.2.HEXIM1 by inhibiting P-TEFb,down-regulation of CTD-Ser2 p expression,and thus impede the Fadu cell stem cells from dormancy to proliferative phase transformation,that HEXIM1 stable cancer stem cells in dormant role.3.Inhibition of HEXIM1 can promote stem cells from dormancy into the proliferative phase,and stem cells are driven into the proliferative phase is easy to be anti-cancer drugs to kill,this study to reduce and eliminate cancer stem cells provide a new way.