| Hypopharyngeal Squamous Cell Carcinoma(HSCC)is relatively uncommon in head and neck malignancies.But recent years have seen a relatively fast upward trend in its incidence,which accounts for about 5%of that of head and neck cancer.The early clinical manifestations of HSCC include the blockage of foreign bodies in pharynx and the feeling of being choked when swallowing something.As the tumor becomes larger and especially when there is ulcer and erosion on the surface,people will find it uncomfortable to swallow things,accompanied by regional ache,and reflectively,the ear on the same side may also ache and there may be bloodshot in the phlegm.Sometimes,people can also find it difficult to swallow.Worse,when the laryngeal cavity is affected,a hoarse voice will be developed and it will become difficult even to make voices and breathe,let alone other relevant activities.As for its incidence areas,70%-80%of HSCC involve the pyriform sinuses,followed by the posterior pharyngeal wall,which accounts for 5%-22%,and it is rarely observed in the postcricoid area.As more than 95%of hypopharyngeal malignancies are squamous cell cancers and most of these cells are poor in their differentiation performance and are easy to spread beneath the mucosa,HSCC is now considered one of the most severe malignant tumors of all head and neck cancers.Besides,patients are usually asymptomatic and early presentations are unfortunately uncommon,so they are usually diagnosed as in the late stages of cancer(periods ?&?)when coming to see a doctor.And in this stage,tumor lesions have spread to the root of the tongue,throat and cervical esophageal and other body parts,often accompanied by multiple cervical lmph node metastasis,thus leading to poor prodnosis.At present,the goal of hypopharyngeal cancer management is to eradicate the tumor as far as possible,reduce the complication of operation,prolong the life of patients and improve the living quality.With the level of diagnosis and treatment of head and neck tumors improving,therapies like radiotherapy,chemotherapy and surgery or the three combined have been applied to the clinical treatment of HSCC.Among these,the therapy combining surgery and radiotherapy represents the major treatment method.Nowadays,all the three therapies have gained great progress and patients'survival rate have increased,but HSCC's recurrence rate remains high because it is often diagnosed as in the late stage when patients first come to a doctor and its malignancy degree is relatively high,its anatomical position is deep and it is vulnerable to regional spreading and distant metastasis.According to the clinical study,the 5-year survival rate for patients with clinical Phase III and IV of HSCC is only 25%-40%.Therefore,it has become an urgent topic for surgeons of head and neck tumors to study the biological characteristics of HSCC in a more profound manner and according to this,find what can signify cancers.And they also need to explore methods to diagnose HSCC at an early stage and find out an efficient therapy.The ETS family is the largest signaling-dependent transcription factor family ever known to all.It is known to have more than 30 members and is involved in numerous physiological and pathological processes by regulating cell proliferation and differentiation,regulating cell apoptosis and epithelial-mesenchymal interactions.The proliferation and migration of endothelial cells and the differentiation of lumen morphology will lead to the formation of tumor vessels.According to the research conductedrecently,ETS-1,a member of ETS family,can affect the formation of tumor vessels and plays a significant role in the formation and development process as well as the process of invasion and spreading.ETS family is encoded by v-ETS,a kind of proto-oncogene,and so far,altogether over 30 members have been found,including ETS-1,ETS-2,GABPa(GA-binding protein a),Spi-1(salmonella pathogenicity island 1)and so on.ETS-2,also known as E26 oncogene homolog 2,is a member of the ETS oncogene homolog family.It is the effective product of cell signaling pathway,and can regulate many downstream target genes,and further regulate the proliferation,differentiation,apoptosis and angiogenesis of the effector cells by regulating various signaling pathways or the interaction of various effective proteins.ETS2 is expressed in many tissues and cells,and research has shown that differences exist in terms of the amount and function of ETS2 expression when it is in different tissues.ETS2 gene expression abnormalities can be found in many tumors,such as liver cancer,esophageal cancer,thyroid cancer and malignant blood tumors.It is speculated that the abnormal expression or translocation of ETS-2 will lead to the formation of fusion protein,which may be closely related to the occurrence and progression of tumors,and that transcription factor ETS-2 can be a potential sign of tumors and it may be a new therapeutic target.Therefore,experiments designed to study the role of ETS-2 in the expression and development of HSCC can help develop theoretical basis and practical guidance for understanding the pathogenesis and biological therapy of HSCC,thus enjoying great theoretical and clinical significance.At present,no research report has been found about the expression of ETS-2 in HSCC tissues and cells as well as about the related mechanisms.In conducting this research,the expression of ETS-2 in HSCC tissues and adjacent mucosa tissues was examined from the perspective of mRNA and protein levels.Besides,it is guaranteed that patients received regular post-operative follow-up and related data were recorded in details and the relationship between ETS-2 expression and age,gender,location,tumor classification,cervical lymph node metastasis,histopathological grading,clinical staging and prognosis of patients suffering from HSCC was analyzed and discussed.In this research,small interfering RNA technique was also applied to exclude the influence of ETS-2 in human HSCC FaDu cell line and thus designed many experiments to study the effect of low expression of ETS-2 on cell cycle,proliferation,apoptosis,invasion and metastasis of FaDu cells.And the possible mechanisms were also explored and discussed accordingly.Part1 The expression and corresponding clinical significance of ETS2 in HSCCObjective:To study the expression of ETS-2 in HSCC,and analyze the relationship between the expression of ETS-2 and the clicicopathological features as well as prognosis of patients.Methods:1.All the histological specimens were from inpatients in Otorhinolaryngology Head and Neck Surgery of Qilu Hospital of Shandong University.Postoperative tumor tissue specimens from patients with HSCC and morphologically normal mucosa tissue samples which is 2 cm above the lumps were collected.Patients'clinical records were also recorded in details.2.Apply immunohistochemical staining methods to detect the expression of ETS-2 in cancer tissues and in non-cancer mucosa tissue specimens beside cancer cells.3.The Trizol method was applied to extract RNA from tumor tissues and non-tumor mucosa tissues.The Real-time PCR method was also used to compare differences of ETS-2 expression in cancer tissue and non-cancer mucosa tissue specimens beside cancer cells at the mRNA level.4.Extract total proteins from tissue specimens and apply protein immune-imprinting(Western blot)to examine and compare differences of ETS-2 expression in cancer tissue and non-cancer mucosa tissue specimens beside cancer cells at the protein level.5.Pearson Card-Square Test was applied to analyze the comparative study on the positive rate of immunohistochemical staining of ETS-2 in HSCC cancer tissue and non-tumor mucosa tissue specimens;Wilcoxon rank-sum test was used to analyze the mRNA expression of ETS-2 in cancer tissues and non-tumor mucosa tissues and analyze the comparison of quantitative results of protein imprinting analysis.Relative amount of ETS-2 expression was calculated by the way of average value and standard deviation and analysis of relationship between ETS-2 expression and HSCC clinicopathological features was analyzed through adopting the card-square test method.Kaplan-meier method was turned to so as to draw the patients' survival curve and the difference analysis was completed with the help of Log-rank test method.A single-factor Cox regression analysis was employed to analyze the influence on patients' prognosis of ETS-2 positive expression and clinical staging and multivariate Cox regression was used to analyze the independent risk factors that influence the prognosis of HSCC patients.All data were statistically analyzed with the help of GraphPad Prism 5.0 and SPSS 18.0 and all statistical analyses were bilateral and were deemed statistically significant when p<0.05.Results:1.Altogether 58 HSCC patients were involved in this research and 58 tumor specimens were collected,52 normal non-tumor mucosa tissue specimens beside cancer cells collected,among which,44 were pyriform sinus carcinoma and postcricoid area cancer and posterior hypopharyngeal wall carcinoma totaled 14.Patients who were in the periods T3 and T4 amounted to 40,accounting for 69%and those with lymph node metastasis totaled 45,accounting for 77.6%.Those who were clinically diagnosed as Highly Differentiated Squamous Cell Carcinoma amounted to 16,accounting for 27.6%,with Medium and lowly-differentiated ones totaling 42,accounting for 72.4%.Among those patients who were in the periods ? and ?,43 were close to 3/4(74.1%)of the selected cases.All selected patients were pathologically confirmed as squamous cell carcinoma,and all the specimens showed negative in their cutting margins.2.According to the results of immunohistochemical examination:the expression of ETS-2 was positive in the nucleus and cytoplasm but mainly occurred on the nucleus.ETS-2 presented positive expression in 81%(47/58)of cancer tissues and presented positive expression in 23.1%(12/52)of normal mucosa tissues beside cancer cells(p<0,001).According to the statistical results:the expression of ETS-2 in the tumor tissue of hypopharyngeal squamous cell carcinoma was obviously higher than that of normal mucosa tissue.3.Real-time PCR results showed that the relative expression degree of ETS-2 mRNA in the tumor tissues of hypopharyngeal carcinoma(2.497 ±0.163)was significantly higher than that of cancer-adjacent non-tumor mucosa(1.095 ± 0.058).Statistical analysis showed that the differences had a statistical significance(p<0.001).4.The results of Wester Blot showed that the relative protein expression of ETS-2 in the tumor tissue of hypopharyngeal carcinoma was 0.674±0.026,and the relative protein expression(0.211±0.035)was significantly higher than that of the tumor-adjacent mucosa.And statistical analysis showed that the differences had a statistical significance(p<0.001).5.The positive expression of ETS-2 had something to do with the primary tumor classification(p<0.001),and was also related to the clinical TNM staging of tumors(p<0.001)as well as histopathological grading(p<0.001)and regional lymph node metastasis(p=0.005)in the neck.In addition,the expression of ETS-2 had nothing to do with tumor primary sites,age and gender of patients.6.Of all patients that were involved in this research,survival rate of ETS-2 with a positive expression was far below that with a negative expression(p<0.01).According to the results from single-factor Cox regression analysis,the size and grading of primary tumor(p=0.0043),regional lymph node metastasis(p=0.0012),clinical TNM staging(p=0.0008),and ETS-2 positive expression(p<0.001)of tumor specimens were all related to the prognosis of the patients.But according to the multi-variate Cox regression analysis for proportional hazards,positive expression of ETS-2 in tumor specimens was a risk factor that independently influenced patients'prognosis(p=0.003,HR 6.343,95%Cl 2.211-15.786).Conclusion:ETS2 increased significantly in the expression in squamous cell carcinoma of the stomach tumor tissue,the ETS2 abnormal expression is related to the HSCC primary tumor classification,regional lymph node metastasis,histopathological grading and the clinical TNM staging of the tumor.ETS2 with positive expression is associated with poor prognosis in patients.In addition,the tumor specimen ETS2 positive expression is independent risk factors affecting prognosis of patients,these experimental results indicate that ETS2 gene may become a new target for the treatment of HSCC molecules.Part 2 The effects and mechanism of ETS2 knockdown on cell cycle,proliferation,apoptosis,invasion and metastasis of the FaDu cell line of hypopharyngeal squamous cell carcinomaObjective:ETS2-siRNA was transfected into the FaDu cell line of human hypopharyngeal squamous cell carcinoma,thereby studying the influence and mechanism of ETS2 knockout on cell cycle,proliferation,apoptosis,invasion and metastasis.Methods:1.To develop the FaDu cell lines of hypopharyngeal cancer cells,3 groups of siRNA,siRNA-1,siRNA-2 and siRNA-3 were designed and synthesized,and NC siRNA of the negative control group was set up,which was transfected with Lipofectamin 2000 liposome method.Experiments in three kinds of concentration,25 nm,respectively,50 nm.100 nm,in turn,after the success of the transfection,application of Real-time PCR and Western blot experimental method to detect the transfection efficiency of siRNA,and selected the most suitable siRNA into the next phase of the experiment2.The experiment was divided into three groups:Control group,NC group and siRNA-ETS2 group.CCK8 and Brdu were used to detect the proliferation of cells ineach group.The effects of ETS2 on proliferation of HSCC FaDu cell lines were studied.3.The effects of ETS2 on the cell cycle of HSCC FaDu were detected by flowcytometry.4.The cell apoptosis of three cells was detected by flow cytometry and Hoechst staining,and the effects of ETS2 on apoptosis of HSCC FaDu cell line were studied.5.Western Blot was used to detect the expression of apoptosis-related proteins Bcl2 and Caspase3.The effect of ETS2 gene on apoptosis of HSCC FaDu cell lines was investigated.6.Transwell invasion experiment and cell scratch were used to detect the migration and invasion ability of three groups of cells.The changes in the invasion ability of FaDu cells after ETS2 were studied.7.Using Western Blot technique to detect epithelial markers in cells:e-cadherin,zq-1 and interstitial markers:Vimentin,?-SMA expression to detect the effect of ETS2 gene on the epithelial cell-mesenchymal transformation(EMT)of FaDu cell line.8.Western Blot was used to detect the expression of p38 and p-p38,ERK and p-ERK,JNK and p-jnk related proteins in MAPK signaling pathway.The influence of ETS2 on the MAPK signaling pathway of cancer cells and the pathway of cell apoptosis and migration and metastasis was explored.9.GraphPad Prism 5.O software and SPSS 18.0 statistical analysis software for statistical analysis were used.Each experiment was repeated at least three times independently,and all the experimental data were expressed in terms of mean or standard deviation.The differences between different groups were evaluated by t test or single factor variance analysis.All statistical data and analysis were P<0.05,which was considered statistically significant.Results:1.Three groups of siRNA,siRNA-1,siRNA-2,siRNA-3 were successfully designed and synthesized and the NC siRNA of the negative control group was set up.In order to find the best experimental concentration and improve the efficiency of transfection,the experiment was carried out in three concentrations:25nM,50nM and 100nM respectively.Lipofectamin 2000 liposome was used for transfection,and the transfection efficiency of siRNA was detected by real-time PCR.The experiment showed that the optimum transfection concentration was 100nM.At 100nM,the nc-siRNA group,siRNA-1 group,siRNA-2 group,and the relative expression levels of ETS2 in the siRNA-3 group were 1.000 ± 0.046,0.561 ± 0.034,0.334± 0.023 and 0.156 ± 0.016.The difference between the groups was statistically significant(p<0.001).Among them,siRNA-3 transient transfection efficiency can reach about 80%.The Western blot experimental result shows:After siRNA-3 transfected with FaDu cell lines of hypopharyngeal cancer,the expression of ETS2 protein was significantly inhibited.ETS2 proteins in the Control group,the NC group,siRNA3 relative expression of transfection group was 1.012 ± 0.031,0.992 ± 0.022 and 0.022 ± 0.015.The difference was statistically significant(p<0.001).2.The proliferation of cells was detected by Brdu staining.The results showed that compared with the control group,the number of staining cells in the siRNA-ETS2 group decreased significantly,indicating that the proliferation activity of the cells was significantly inhibited.CCK8 was applied to detect cell proliferation in each group,and the results showed that:compared with the Control group and the NC group,there was no significant difference in the proliferation of FaDu cells in 12h after the knockout of ETS2,but the proliferation of FaDu cells was significantly inhibited after 48h and 72h(p<0.05).The experiment showed that the low expression of ETS2 was caused by ETS2-siRNA transfection with FaDu cell line,and the proliferation activity of FaDu cells was obviously inhibited.3.The effect of ETS2 on the FaDu cell cycle was detected by flow cytometry,and the results showed that:The proportion of cell cycle in G0/G1 phase was 59.4%,57.2%and 69.3%in Controlgroup.NC group ? siRNA group.The proportion of cell cycle in S phase was 31.4%,33.4%and 21%.The proportion of cell cycle in G2/M period was 9.2%,9.4%and 9.7%,respectively.Therefore,compared with the control group,the siRNA-ETS2 group of the experimental group with ETS2 gene was able to induce FaDu cells to accumulate in G0/G1 phase,thus reducing the cell rate of S phase(p<0.01).4.Hoechst was used to detect the apoptosis of three groups of cells,and the nuclei were found to be blue clumps under the microscope.After apoptosis,it can be found that the blue clumps and the nuclei are broken,and the chromatin is highlighted by shrinkage.The apoptosis rate of the three groups was detected by flow cytometry:4.65%± 0.53%,4.52%± 0.48%and 29.12%± 1.02%.The apoptosis rate of cells was significantly higher than that in the siRNA-ETS2group(p<0.01).5.Western Blot was used to detect the expression of apoptosis-associated protein Bcl2/Caspase3.The results showed that the relative expression of Caspase3 in the Control group,NC group and siRNA-ETS2 group was 1.012 ± 0.033,1.098 ±0.064 and 2.859 ± 0.102.Compared with the three,the relative expression of Caspase3 in the siRNA-ETS2 group was significantly increased,and the difference was statistically significant(p<0.001).The relative expression of Bcl2 in Control group,NC group and siRNA-ETS2 group is 1.119±0.029,1.090±0.018 ?0.549±0.013.Compared with the three,the relative expression of Bc12 in the siRNA-ETS2 group decreased significantly,and the difference was statistically significant(p<0.05).6.After the Transwell invasion test was used to detect the changes in the invasion ability of the FaDu cells after the deletion of ETS2 gene,the cell migration and invasion in the lower chamber of Transwell were observed and measured.Statistical analysis shows that:Compared with the Control group and NC group,the invasion and migration of siRNA-ETS2 cells decreased significantly(p<0.01).Cell scratch test shows:the migration distance of the FaDu cells after being knocked off the ETS2 gene was smaller than that of Control group and NC group after 48h,and the difference between them was statistically significant(p<0.01).The experiment showed that when the FaDu cells were knocked off the ETS2 gene,the invasion and metastasis of the cells were significantly reduced.7.Detection of epithelial markers in cells using Western Blot technique.The results showed that the relative expression of the epithelial marker e-cadherin in the Control group,the NC group and the siRNA-ETS2 group was respectively 1.097±0.043,0.986±0.023? 3.879±0.124.The relative expression of ZQ-1 in the three groups of epithelial markers was respectively 1.055±0.075,0.093±0.056 and 4.198±0.176.SiRNA-ETS2 group compared with control group,E cadherin and ZQ-1 the relative expression quantity increase obviously,there are significant difference statistically significant(p<0.001).The relative expression of Vimentin between the three groups was 1.118±0.033,1.123±0.081 and 0.335±0.022.The relative expression quantity of a-SMA was 1.012±0.021,1.114±0.062 and 0.298±0.014.SiRNA-ETS2 group compared with control group,the expression of Vimentin and a-SMA significantly lowered.The difference was statistically significant(p<0.01).The analysis showed that after inhibiting the expression of ETS2,tumor cells inhibited the process of intercellular filling transformation by increasing the expression of e-cadherin,zq-1 and decreasing the expression of Vimentin and a-SMA.8.Western Blot was used to detect the expression of p38 and p-p38,ERK and p-ERK,JNK and p-JNK related proteins in MAPK signaling pathway.The results showed that the relative expression of p-p38,p-ERK and p-ERK protein in cells was significantly higher than that in the control group after TGF-3 induced cell activation,and the difference was statistically significant(p<0.01).However,p-p38,p-ERK and p-ERK protein and TGF-? group of TGF-? +siRNA group,TGF-? +NC group,were significantly less elevated and significantly different(p<0.01).Research has proved that after ETS2 is knockout,will cause the MAPK/p38/ERK/JNK signaling pathway is restrained,show the TGF-? induced tumor ectomesenchymal transformation of epithelial cells-(EMT)in the process of ETS2 through MAPK signal pathway plays an important role.Conclusion:Through the ETS2-siRNA transfection Hypopharyngeal squamous cell carcinoma FaDu cell line to knock down ETS2,compared with the control group to induce FaDu cells in GO/G1 phase,significant inhibition of cell proliferation,and promote the FaDu cells apoptosis.By inhibiting the signaling pathway of MAPK/p38/ERK/JNK,the process of cell mesenchymal transformation was inhibited and the invasion and metastasis ability of the cells was weakened. |
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