| Esophagus cancer is one of the most malignant cancers in the world, and its incidence is region-related. The incidence of esophagus cancer in China is the highest, and over a half of newly diagnosed esophagus cancers occurred in China. The highest esophagus cancer incidence area locates in Wsetern Anyang, which lies in the north part of Henan province, China, with an incidence around478per100thousands inhabitants. Majority of the esophagus cancers in China are squamous. The survival of the esophagus cancer has been increased greatly by using surgery, chemotherapy and radiotherapy, but relatively high recurrence rate and metastasis hinder the removal of cancer cells completely, causing poor prognosis of the patients. Therefore it is important to continuously explore molecular factors that determine prognosis of squamous esophagus cancers and then develop novel therapeutic modalities, in order to improve the survival of esophagus cancer patients after surgery.C-kit, or called CD117or mast/stem cell growth factor receptor (SCFR), is a known proto-oncogene. It is a tyrosine kinase type â…¢ receptor. Upon binding to its ligand stem cell factor (SCF), c-kit will phosphorylate and activate its downstream signal pathway, playing an important role in the proliferation, apoptosis and differentiation of cells. Recent research shows that coexpression of c-kit and SCF plays a central role in the occurrence and development of ovarian, small cell lung, and intrahepatic cholangiocarcinomas, and higher level expression of c-kit has close relation with the poor prognosis of many cancers. However, to our best knowledge no report on coexpression of c-kit and SCF and the prognostic correlation of esophagus cancer is available in literature.In view of the above reasons, we designed our study in two parts:The first part is a clinicopathological study on the expression of c-kit and SCF in a series of esophageal squamous cell carcinomas and their prognostic correlations; The second part is s functional study by c-kit gene knockdown using RNA interference technology, and then to study the role of the cell and biological consequences in esophageal squamous cell cancer cell line in vitro.Part â… :The Expression of c-kit and SCF in Esophageal Squamous Cell Carcinoma and Its Relationship with Clinical PrognosisAim:To explore the expression of c-kit and its ligand SCF in human esophageal squamous cell carcinomas and their relationships with the clinicopathological and clinical consequences/prognosis.Methods:1. C-kit and SCF protein expression was immunohistochemically examined in a series of157patients with esophageal squamous cell carcinoma and10normal esophageal epithelia.2. Immunohistochemistry scoring:Immunostaining of each slide was semiquantitatively scored for both intensity (1, absent/weak;2, moderate;3, strong) and extent of staining (percentage of the positive tumor cells:1,<10%;2,10-50%;3,>50%). The scoring results of intensity and extent were multiplied to give composite scores ranging from0to9for each section. Then we divide the results into4groups:negative (0), weak (1and2), mild (3and4) and strong (6and9) positivity.3. Statistical analyses:Statistical analyses were performed using SPSS17.0software. Association analysis between the ordinal variables was run using Spearman. Correlation coefficient is used to determine the correlation between the intensity of the variable factors. Comparison between the categorical variables was tested using Pearson’s χ2function. The survival curves were plotted using the Kaplan-Meier method. The difference between each group was analyzed using log-rank test. The Cox risk model was employed to assess their independent effect on the prognosis of the cancers. It was considered to be statistically significant when P≤0.05.Results:1. No c-kit and SCF expression was detected in the10normal esophageal mucosa, while38of the157esophageal squamous cell carcinoma samples coexpressed both c-kit and SCF, accounting for24.1%. Among the157esophageal squamous cell carcinoma samples47were positive for c-Kit, accounting for29.9%positivity. For these47c-Kit positive tumors15were weakly positive (9.5%), another15were moderately positive (9.5%) and17were strongly positive (10.8). Membrane positivity for SCF was observed in52tumors (33.1%) where19were weakly positive (12.1%),17were moderately positive (10.8) and16were strongly positive (10.2). By using Spearman’s correlation test model, it was discovered that c-kit and SCF expression were positively corrected, and the correlation coefficient was0.553(P<0.001).2. The expression of c-kit was closely correlated with T stage, namely the depth of invasion (P<0.001), the metastasis of lymph node (P=0.019), distant metastasis (P=0.015) and the clinical stage (P=0.021). The Spearman correlation coefficients were0.435,0.194,0.194and0.190, respectively. The recurrence and metastasis rate of the cancer was34.8%for the patient whose c-kit express was positive, while the recurrence and metastasis rate was18.9%for the patient whose c-kit express was negative with P=0.034.3. The expression of SCF was also closely correlated with the depth of invasion (P=0.000), tumor size (P=0.014), metastasis of the lymph node (P=0.018), distance metastasis (P=0.002) and clinical stage (P=0.003). The Spearman’s correlation coefficients for these factors were0.373,0.227,0.186,0.240and0.239, respectively. The recurrence and metastasis rate was32.7%for the SCF-positive patients, while it was18.1%for the SCF-negative patients with P=0.042.4. The median DFS was80.284(95%CI63.760-96.808) months for positive c-kit expression group patients, while the value for negative c-kit expression group patients was126.875(95%CI115.375-138.385) months, P=0.010. The median DFS was96.038(95%CI75.786-116.290) months for positive SCF expression group patient, while the value was126.862(95%CI115.347-138.377) months for negative SCF expression group patient, P=0030.5. The median overall survival (OS) of positive c-kit expression group was much shorter than that of the negative c-kit expression group (P<0.001),55(95%CI33.142-76.858) months and127(95%CI89.546-164.454) months, respectively. The median OS gradually shortened for the weak expression group, moderate group and strong groups. The median OS for the positive SCF expression group was60(95%CI30.155-89.845) months, while the OS for the negative SCF expression group was127(95%CI94.922-159.078) months, P=0.001.6. The median OS time was39months (95%CI9.397-77.603) for the c-kit and SCF coexpression group patients, while the OS was119(95%CI88.260-149.740) months for the patients without coexpression of c-kit and SCF. The overall survival of the patients with coexpression of c-kit and SCF was significantly shorter than that in those tumors without coexpression (P=0.000).7. The c-kit expression (P=0.002), SCF protein expression (P=0.025), distant metastasis (P=0.023) and the lymph node metastasis (P=0.045) were all discovered as independent factors that determined the prognosis of the patients with esophageal squamous cell carcinoma.Conclusion:1. Normal esophageal sqaumous epithelial cells are negative for c-kit and SCF expression, while both c-kit and SCF are expressed in human esophageal squamous cell carcinoma, suggesting that SCF/c-Kit signaling may be involved in the occurrence or development of esophageal squamous cell carcinoma.2. The findings that the expression of c-kit and its receptor SCF is closely correlated with the invasion depth, lymph node metastasis, distant metastasis and clinical stage of the tumors suggest that SCF/c-kit signaling may be involved in the invasion and metastasis process of the esophageal squamous cell carcinoma.3. Significantly shorter DFS and OS for the patients with c-kit and SCF positive tumors strongly indicate their prognostic roles in esophageal squamous cell carcinoma.Part â…¡:The Effect of C-kit Gene Knock-down on the Biological Behaviors of KYSE70Cells in VitroAim:To explore the expression of c-kit and its ligand SCF in human esophageal squamous cell carcinoma cell KYSE70and the effect of the c-kit gene knockdown on the biological behaviors.Methods:1. Expression status of c-kit, SCF and ABCG2in KYSE70cells cultured in hypoxic and in normal culture conditions were examined using RT-PCR and Western-blotting.2. To silence the expression of c-kit in KYSE70cells RNA interference technology was applied, and interference efficiency was explored using RT-PCR and Western blotting.3. Expression level of SCF and ABCG2mRNA in KYSE70cells before and after the interference of RNA was checked using RT-PCR and Western blotting.4. Cell and biological effect of c-kit knock-down on KYSE70cells was examined using colony formation assay, MTT, flow cytometry and chemosensitivity test.5. SPSS17.0software was used to for statistical analyses. Gray values of Western blots and RT-PCR results were performed with Gene tools. The comparison between two samples was carried out using t-test, and the comparison among multiple samples was performed using one-way ANOVA analysis. It was considered to be statistically significant when P<0.05.Results:1. Levels of mRNA and protein expressions of c-kit, SCF and ABCG2in esophageal squamous cell carcinoma cell KYSE70were detected. The expression of c-kit, SCF and ABCG2was significantly increased after hypoxia culture.2. Compared with the normal control group and the non-specific interference group, the expression of c-kit and ABCG2mRNA and protein in the specific c-Kit gene interferenced KYSE70cells was decreased (P<0.05), while there was no SCF mRNA and protein expression difference in these cells.3. The clone formation assay revealed that the colony formation rate was53.63±4.68%for normal control group, while it was55.32±6.78%for non-specific siRNA interference group. No statistically significant difference can be found in these two groups,(P>0.01). While the colony fomration rate was17.01±4.83%for the c-kit siRNA group, with a statistically significant difference compared with the above two groups (p<0.01).4. MTT assay results showed that there was no significant difference for the growth rate of KYSE70cells in normal control group and the non-specific siRNA group (P>0.05). But the growth rate of the cells in the c-kit siRNA group was significantly inhibited, and the difference was statistically significant (P<0.05). No obvious difference could be detected for the inhibition rate of normal KYSE70cells and non-specific small RNA processed KYSE70cells at the same5-Fu concentration (P>0.05). Nevertheless, in the c-kit siRNA group, the survival rate of the cells decreased greatly, while the gowth inhabitation rate of the cells was significantly increased (P<0.05).5. The results obtained from the flow cytometry showed that the G0/G1detection rate for the KYSE70cells under normal control group and non-specific siRNA interference group were46.8±1.34%and49.2±1.37%, respectively, with no significant difference in these two groups. While the G0/G1detection rate for the c-kit siRNA group KYSE70cells was greater, about77.8±1.45%. Compared with the above two groups, the difference was statistically significant (P<0.05). There was no early apoptotic rate for KYSE70cells in normal control group and the non-specific siRNA interference group (P>0.05). The early apoptosis rate was27.36%±1.09%for the c-kit siRNA cells, significantly decreased compared with the above two groups.Conclusions:1. KYSE70esophageal squamous cancer cells express c-Kit, SCF and ABCG2, and their expression is upregulated under hypoxia conditions. 2. The proliferation of the esophageal squamous cell carcinoma cells is positively correlated with the c-kit gene, and the proliferation of the KYSE70cells can be inhibited by knocking-down c-kit expression. The molecular mechanism may be due to that fact that it blocks the KYSE70cells at G0/G1state, and induces their apoptosis.3. C-kit gene knock-down using siRNA decreases the drug-resistance of KYSE70cells in esophageal squamous cell carcinoma cells, and the possible mechanism may be due to the down-regulation of ABCG2, which functions mainly by pumping drugs out of cells.4. C-kit may play an important role in esophageal cancer cell stemness maintenance. |