| Part one B7-H4 expression in esophageal tissue of mice during esophagealsquamous cell tumorigenesisObjective: Esophageal squamous cell carcinoma(ESCC) has been a common cancer with serious health threat. The prognosis of ESCC is very poor with the 5 year survival rate only about 20% because most patients were diagnosed in the advanced state, when there is no obviously effective treatment. Esophageal precancerous condition is a kind of pathological change from normal esophagus to ESCC. Fortunatly, the prognosis of esophageal precancerous condition is much better with 5 year survival rate more than 90% provided that the proper diagnosis and treatment are employed. Therefore, the prevention and treatment of precancerous condition is a good choice to reduce the incidence and mortality of ESCC. Costimulatory molecule B7-H4 is an important member of the B7 family, which is highly expressed in various tumor tissues and negatively correlated with prognosis. In this paper, we established an esophageal precancerous lesion model in mice with 4NQO drinking water, detected B7-H4 and relative molecules expression as well as lymphocytes infiltration in tumor microenvironment during the whole ESCC tumorigenesis procession in order to elucidate the pathogenesis of precancerous lesions and to evaluate the potiential prospect of B7-H4 as a new biomarker or treatment target of esophageal precancerous condition.Methods:1 4NQO drinking water was introduced to induce esophageal precancerous lesions in mice. During the procession, general conditions and body weights of mice were observed, and the pathological changes of esophagi were evaluated by HE staining.2 B7-H4 expression and CD4+T, CD8+T, CD11b+macrophages infiltration in esophageal tissues of mice were detected by immunohistochemistry staining technology during the whole procession of ESCC formation. The correlations between B7-H4 expression and lymphocytes infiltration were also analyzed.3 The expression of B7-H4, IL-6, IL-10, TGF-β, IFN-γ, STAT3, and p-STAT3 were measured by q PCR, Western blot and ELISA assays. Besides the correlations between B7-H4 expression and relative molecules were also analyzed.4 Western blot and ELISA assays were used to detect the expression of SCCA-2 and s B7-H4 in sera of mice with precancerous lesions, and the correlation between their expression and the grades of pathological changes were also analyzed.Results:1 Body weight changes of miceThe general condition and weight gain of control mice were normal during the whole ESCC tumorigenesis procession. However, compared with the control mice, the general condition of 4NQO mice were poor during the tumorigenesis. The amounts of the diet or drinking water were smaller than that of control mice. Besides, the body wights gain of 4NQO female and male mice were significantly slower. Especially after 17 th week, the weights of 4NQO male and female mice were gradually decreased.2 General observations of esophageal tissues lesionsFrom 16 to 28 weeks, all control esophageal tissues showed smooth surface and normal size. However, esophageal tissues of 4NQO mice exhibited pathological characteristics, such as surface roughness, uneven thickening, and many masses with different sizes. Therefore, it suggested that, with the inducing time going, the degree of the lesions increased gradually.3 Esophageal pathological changes evaluated by HE stainingDuring the whole process of ESCC tumorigenesis, the esophageal tissues of 4NQO mice underwent the pathological changes from normal esophagus, low grade intraepithelial neoplasia(LGIN) to high grade intraepithelial neoplasia(HGIN). Normal esophageal epithelial cells showed regular distribution, natural nuclear size and polarity. LGIN, however, exhibited that the mucosal epithelium became thicker, and the low half cells lost polarity as well as the cell nucleus enlarged and been hyperchromatic. Moreover, HGIN represented that the epithelial layer became thicker further, and more than half cells to the whole epithelial layer lost polarity with cell nucleus enlarging more seriously. Esophageal pathological grades were positively correlated with the inducing time, χ2 = 33.5476, P < 0.0001. In addition, Kruskal-Wallis analysis showed that there was no significant difference of esophageal pathological conditions between male and female mice, P > 0.05.4 B7-H4 expression and lymphocytes infiltration in esophageal tissues analyzed by immunohistochemistry stainingB7-H4 was expressed in the cytoplasm and membrane of esophageal epithelial cells. According to the expression score, B7-H4 was highly expressed in 2 of 22 cases of normal esophagi(9.1%), 11/27 cases of LGIN tissues(40.7%) and 17/21 cases in HGIN tissues(81%). Spearman correlation analysis showed that B7-H4 expression was positively correlated with the pathological grade(P < 0.0001). However, no significant correlation was observed between B7-H4 expression and gender of mice(P = 0.2959).CD4, CD8 and CD11 b are membrane molecules expressed on the CD4+T, CD8+T and macrophage cells surface. The three types of lymphocytes were mainly located in the submucosa layer of esophageal epithelial tissue, while less invasion in the mucosa. In addition, B7-H4 expression was positively correlated with the CD11 b macrophage infiltration(P = 0.0002). Whereas there was no significant correlation between B7-H4 expression and CD4+T and CD8+T cells infiltration(P = 0.8507, P = 0.3166, seperately).5 B7-H4 and relative genes expression detected by q PCR assayCompared with the control mice, B7-H4, IL-6, IL-10, and TGF-β expression were significantly increased with the inducing time going. Especially 26 and 28 weeks, these genes expression increased significantly with statistical difference. In addition, B7-H4 expression was positively correlated with IL-6(r = 0.5952, P < 0.0001), or IL-10(r = 0.6561, P < 0.0001), or TGF-β(r = 0.6586 and P < 0.0001 with Spearman correlation analysis. However, no significant difference of STAT3 gene expression was found during the whole tumorigenesis.6 B7-H4 and relative proteins expression detected by Western blot and ELISACompared with the control mice, B7-H4 and IL-6 expression were all increased in esophageal tissues of 4NQO mice with the inducing time going. Especially at 26 th and 28 th week, the two proteins increased significantly with statistical difference, P < 0.05, P < 0.01. In addition, p-STAT3 expression was significantly increased in the 28 th week in esophagi of 4NQO mice, P < 0.01. On the other side, the expression of B7-H4 was positively correlated with the expression of IL-6 and p-STAT3 in the whole process of ESCC tumorigenesis,(r = 0.6145, P < 0.0001; r = 0.6180, P < 0.0001). In conclusion, the results showed that B7-H4 expression was positively correlated with the activation of IL-6/STAT3 signaling pathway during the formation process of esophageal precancerous lesions in mice.7 s B7-H4 and SCCA-2 proteins expression in sera of mice detected byWestern blot and ELISA assaysCompared with the normal mice, the concentrations of s B7-H4 and SCCA-2 in the sera of HGIN mice were significantly increased,P < 0.05, P < 0.05. The results suggested that s B7-H4, similar to SCCA-2 protein, may be a potential tumor marker for esophageal precancerous condition. Part two B7-H4 promotes the proliferation of esophageal squamous cellcarcinoma cells and activates IL-6/STAT3 signaling pathwayObjective:In this part, the effects of B7-H4 on Eca109, TE1 and TE13 cells proliferation and IL-6/STAT3 signal pathway activation were investigated to study the role of B7-H4 in ESCC cells biological procession and esophageal precancerous lesions formation.Methods:1 MTS cell proliferation assayCells pretreated with control sh RNA and B7-H4 sh RNA were collected and planted in 96 well plates, after 0 h, 24 h, 48 h and 72 h incubation, 20μl MTS was added to wells. Then the absorbance values were detected2 Colony formation assayCells pretreated with control sh RNA and B7-H4 sh RNA were collected and planted in culture plate of 3 cm-diameter, after 12 days incubation, colonies were stained and counted.3 Cells cycle was detected by Flow cytometryCells pretreated with control sh RNA and B7-H4 sh RNA were collected and stained with PI, the DNA and cell cycle profiles were analyzed by Flow cytometry.4 Apoptosis was detected by Flow cytometryCells pretreated with control sh RNA and B7-H4 sh RNA were collected and stained with Annexin- Ⅴ and 7-AAD, then the apoptosis rates were analyzed by Flow cytometry.5 B7-H4 and relative molecules expression detected by q PCR, Western blot and ELISA assaysCells pretreated with control sh RNA and B7-H4 sh RNA were collected. Then the expression of B7-H4, IL-6, STAT3, p-STAT3, JAK2, p-JAK2, Bcl-2, BAX and Survivin were detected by q PCR, Western blot and ELISA.6 Location of total STAT3 and p-STAT3 detected by Immunofluorescence staining and Western blot assayOn the one hand, cells pretreated with control sh RNA and B7-H4 sh RNA were collected, then total STAT3 and p-STAT3 location in cytoplasm and nucleus were observed by Immunofluorescence staining. On the other hand, cells pretreated with control sh RNA and B7-H4 sh RNA were collected, then the cytoplastic and nuclear proteins were extracted to evaluate the total STAT3 and p-STAT3 expression in cytoplasm and nucleus of ESCC cells.7 The effect AG490 on the B7-H4 facilitating IL-6 secretion in ESCC cells.ESCC cells pretreated with 40 μM AG490 were transfected with control sh RNA or B7-H4 sh RNA. After 48 h incubation, supernatant of cells was collected and IL-6 concentration was detected by ELISA.8 The effect of Tocilizumab on the B7-H4 activating JAK2/STAT3 in ESCCcellsESCC cells pretreated with Tocilizumab(200 ng/ml) were transfected with control sh RNA or B7-H4 sh RNA. After 48 h incubation, cells were harvested and total JAK2, p-JAK2, toal STAT3 and p-STAT3 expression were detected by Western blot.Results:1 B7-H4 was highly expressed in ESCC cellsB7-H4 expression was seldom detected in normal esophageal tissue. Compared with normal esophageal tissue, B7-H4 was highly expressed in Eca109, TE1 and TE13 cells at a similar level. There was no significant difference of B7-H4 expression among the three cell lines, P > 0.05.2 B7-H4 silence inhibited ESCC cells proliferation and colony formationMTS result showed that, compared with control cells, the proliferation rates of cells pretreated with B7-H4 sh RNA were significantly reduced. Besides, colony formation result exhibited that, compared with control cells, the colony numbers of Eca109, TE1 and TE13 cells pretreated with B7-H4 sh RNA also decreased markedly, P < 0.05, P < 0.001, and P < 0.001 seperatley.3 Effect of B7-H4 on ESCC cells cycleCompared with control cells, the G1 phase ratios of Eca109, TE1 and TE13 cells pretreated with B7-H4 sh RNA were significantly enlarged(P all < 0.05), while the S and G2 ratios exhibited reduction to a certain degree, but with no statistical difference, P > 0.05.4 Effect of B7-H4 on ESCC cells apoptosisCompared with the control cells, the total apoptosis rate of Eca109, TE1 and TE13 cells pretreated with B7-H4 sh RNA were significantly increased, P < 0.001, P < 0.01, and P < 0.05, seperately. In addition, the early apoptosis rate and late apoptosis rate of B7-H4 silence cells were also upregulated to a certain degree, P < 0.05 or P < 0.01.5 Effect of B7-H4 on the expression of apoptosis relative molecules in ESCC cellsCompared with control cells, B7-H4 silence Eca109, TE1 and TE13 cells showed significant downregulation of apoptosis inhibitory molecules, Bcl-2 and Survivin expression as well as upregulation of apoptosis promoting molecule BAX expression, P < 0.01 or P < 0.001.6 Effect of B7-H4 on IL-6 secretionCompared with control cells, the concentration of IL-6 of supernatant of Eca109, TE1 and TE13 cells decreased significantly, P < 0.05, P < 0.01, or P < 0.001.6 Effect of B7-H4 on JAK2/STAT3 activationCompared with control cells, p-JAK2 expression was significantly inhibited in B7-H4 silence Eca109, TE1 and TE13 cells, P all < 0.01. In addition, B7-H4 silence Eca109, TE1 and TE13 cells also showed reduction of p-STAT3 expression, P < 0.01, P < 0.05, P < 0.01 seperately. However, the total JAK2 and total STAT3 expression was not significantly affected by B7-H4.7 Effect of B7-H4 on total STAT3 and p-STAT3 translocation from cytoplasm to nucleusThe location of p-STAT3 in Eca109, TE1 and TE13 nucleus was markedly suppressed by B7-H4 silence(P < 0.01, P < 0.05, P < 0.05). However, total STAT3 translocation from cytoplasm to nucleus was not effect by B7-H4, P > 0.05.8 AG490 blocked the inhibitory effect of B7-H4 silence on IL-6 secretion inhibitionCompared with the control cells, the IL-6 expression was significantly inhibited by B7-H4 sh RNA pretreated Eca109, TE1 and TE13 cells. However, compared with cells pretreated with control sh RNA and AG490, IL-6 secretion of Eca109, TE1 and TE13 cells pretreated with B7-H4 sh RNA and AG490 can was not significantly affected(P > 0.05), which suggested that B7-H4 silence dampened IL-6 secretion in Eca109, TE1 and TE13 cells through STAT3 inhibition.9 Tocilizumab didn’t block the inhibitory effect of B7-H4 silence on JAK2/STAT3 activationCompared with control cells, the p-JAK2 and p-STAT3 expression were significantly inhibited in TE13 cells pretreated with B7-H4 sh RNA(P < 0.05, P < 0.05). Besides, compared with cells pretreated with control sh RNA and Tocilizumab, p-JAK2 and p-STAT3 expression in TE13 cells pretreated with B7-H4 sh RNA and Tocilizumab were also significantly suppressed(P < 0.05, P < 0.05), which suggested that B7-H4 silence dampened JAK2/STAT3 activation directly. Part three IL-6 facilitates ESCC cells proliferation and regulates B7-H4expressionObjective:The effects of IL-6 and Tocilizumab on B7-H4 expression as well as ESCC cells proliferation were investigated in order to elucidate whether IL-6 can induce the expression of B7-H4 in ESCC cells and whether the IL-6 upregulation induced by B7-H4 plays the role in the effect of B7-H4 facilitating ESCC cells proliferation.Methods:1 Effect of IL-6 on ESCC cells proliferationEca109, TE1 and TE13 cells were cultured in medium with 0, 10, 20, 40 ng/ml human recombinant IL-6. Then cells proliferation rates were tested by MTS assay.2 Effect of Tocilizumab on ESCC cells proliferationEca109, TE1 and TE13 cells pretreated with control sh RNA or B7-H4 sh RNA were collected and cultured in medium with or without 200 ng/ml Tocilizumab. Then the proliferation rates and colony numbers were detected by MTS and colony formation assays.3 Effect of IL-6 on B7-H4 expressionEca109, TE1 and TE13 cells were cultured in medium with or without 40 ng/ml IL-6 for 48 h. Then cells were harvested and B7-H4 was measured by Western blot assay.4 Effects of Tocilizumab B7-H4 expressionEca109, TE1 and TE13 cells were cultured in medium with or without 200 ng/ml Tocilizumab for 48 h. Then cells were harvested and B7-H4 was detected by Western blot assay.Results:1 IL-6 didn’t promote the proliferation of ESCC cellsCompared with control cells, the proliferation rates of Eca109, TE1 and TE13 cells pretreated with 10, 20 and 40 ng/ml IL-6 were not significantly affected, P > 0.05.2 Tocilizumab inhibited ESCC cells proliferation and colony formationCompared with control cells, proliferation rates and colony numbers of Eca109, TE1 and TE13 cells pretreated with 200 ng/ml Tocilizumab were significantly inhibited.3 Tocilizumab didn’t inhibit the proliferation rates and colony numbers of B7-H4 silence ESCC cellsCompared with cells pretreated with B7-H4 sh RNA alone, proliferation rates and colony numbers of Eca109, TE1 and TE13 cells pretreated with 200 ng/ml Tocilizumab and B7-H4 sh RNA were not obviously affected, P all > 0.05.4 IL-6 didn’t induced B7-H4 expression in ESCC cells40 ng/ml IL-6 had no effect on the B7-H4 expression in Eca109, TE1 and TE13 cells, P > 0.05.5 Tocilizumab inhibited B7-H4 expression in ESCC cellsTocilizumab significantly inhibited B7-H4 expression in Eca109, TE1 and TE13 cells, P < 0.05, P < 0.001 and P <0.01, seperately.Conclusions:1 The esophageal precancerous condition was successfully induced by 4NQO drinking water in mice.2 B7-H4 plays an important role in the activation of IL-6/STAT3 positive feedback. Furthermore, the positive feedback of B7-H4/IL-6/STAT3 activation is the inner motivation of B7-H4 promoting ESCC cells proliferation esophageal precancerous lesion and ESCC formation. |
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