| Purpose:The incidence of breast cancer has been the highest currently among the female cancer and it is also growing rapidly. Triple Negative Breast Cancer(TNBC) is a particular type of breast cancer, of which estrogen receptor(ER), progesterone receptor(PR) and human epidermal growth factor receptors-2(Her-2) are all negative. TNBC accounts for 10.0% ~ 20.8% of all breast cancer and is a subtype of the worst prognosis. It characterized by early onset, poor prognosis, easy early recurrence, high mortality and easy invasive and metastasis. B7 homolog 4(B7-H4) expresses in a variety of malignant tumors with inhibiting T cell proliferation and cytokine secretion, blocking cell cycle, and thereby promoting tumor development. Expression of B7-H4 has a close correlation with the clinical pathological features and the poor prognosis of cancer patients.Otherwise, it can inhibit the proliferation of tumor-associated lymphocytes. In this study, by lowing the influence of B7-H4, the biological characteristics of TNBC cell, referring to proliferation, invasion, metastasis and apoptosis and the correlation of B7-H4 and the clinical pathological features, were involved, and the mechanisms of B7-H4 molecules during the development in TNBC was investigated.Methods:1 Immunohistochemistry was used to detect the expression of B7-H4 molecule protein levels in Luminal A, Luminal B, Her-2 and TNBC, and analyzed the correlation between the expressions and clinic pathological features and prognosis.2 Control-sh RNA and B7-H4 sh RNA were transfected into TNBC cells(MDA-MB-231, MDA-MB-453, MDA-MB-468).3 Fluorescence microscopy and Western-blot were used to detect control-sh RNA and B7-H4-sh RNA’s transfection efficiency in MDA-MB-231, MDA-MB-453, MDA-MB-468 cells and one proper cell line was chose for the following tests.4 MTT was used to detect whether B7-H4 expression was relative with the MDA-MB-453 cells growth pattern.5 We used the Transwell to detect the influence of cell invasion after B7-H4 expression interference in reduced MDA-MB-453 cells.6 Flow cytometry was used to detect apoptosis after B7-H4 expression interference in reduced MDA-MB-453 cells.7 Real-time quantitative PCR and Western-blot method were used to detect the m RNA and protein expression of p65 after down regulation of B7-H4 expression interference in MDA-MB-453 cells.Results:1 The results of Immunohistochemical analysisIn 111 cases of breast cancer, the positive rate of B7-H4 was 73.9%(82/111), B7-H4 expresses in cancer cell cytoplasm and membrane. According to the molecular phenotype of breast cancer classified as: Luminal A, Luminal B, Her-2 and TNBC, according to the results, B7-H4 was highly expressed in this four tissue type. B7-H4 positive rate in TNBC tissues was 90.2%(37/41), significantly higher than the Luminal A type: 66.7%(14/21), Luminal B type: 68.0%(17/25) and Her-2 type: 58.3%(14/24), the difference was statistically significant(χ2=9.708, P<0.05). Compared the positive rate of B7-H4 in Luminal A, Luminal B and Her-2 with TNBC respectively, the difference was statistically significant(P ≤ 0.0167). Results indicated that the molecule B7-H4 highly expressed in breast cancer, TNBC group was even higher.In 41 cases of TNBC, the results were statistically significant(χ2=7.315, P<0.05); The positive expression rate of the TNBC patients with lymph node metastasis was significantly higher than those without lymph node metastasis(χ2=6.667, P<0.05); the expression of B7-H4 was correlated with the expression of androgen receptor(AR)(χ2 = 5.857, P<0.05). Study did not find the correlation between B7-H4 expression and other clinicopathological features of TNBC patients, such as age, TNM stage, histological grade, presence of Ki-67 expression(P>0.05).For TNBC group survival analysis showed that B7-H4 positive expression group’s 5-year disease-free survival rate was 59.5%, was significantly lower than the negative expression group 75.0%, and the difference was statistically significant(P<0.05), indicating that poor prognosis was associated with high expression of B7-H4 molecules.In summary, among four types of breast cancer, the B7-H4 expression in TNBC was the highest. In TNBC, B7-H4 expression was positive correlation with pathological type, lymph node metastasis, negative expression of AR, and was an independent poor prognostic factors of TNBC.2 Transfection efficiency test resultsFluorescence microscopy and Western-blot were used to detect the transfection efficiency, the result is: MDA①-MB-231 cells: The relative Gray value of control-sh RNA and B7-H4-sh RNA of B7-H4 were 0.236 ± 0.180 and 0.180 ± 0.018, B7-H4-sh RNA transfection efficiency was 23.6 ± 0.03%. ② MDA-MB-453 cells: The relative Gray value of control-sh RNA and B7-H4-sh RNA of B7-H4 were 0.871 ± 0.190 and 0.217 ± 0.006, B7-H4-sh RNA transfection efficiency was 75.1 ± 0.8%. ③MDA-MB-468 cells: The relative Gray value of control-sh RNA and B7-H4-sh RNA of B7-H4 were 0.691 ± 0.018 and 0.358 ± 0.061, B7-H4-sh RNA transfection efficiency was 48.2 ± 8.0%. In summary, B7-H4 protein was highly expressed in MDA-MB-453 cells and the transfection efficiency of B7-H4-sh RNA to MDA-MB-453 cells was significantly higher than the other two cells(P<0.01), therefore MDA-MB-453 was suitable for TNBC biological function test.3 MTT resultsThe control-sh RNA group and B7-H4-sh RNA group of MDA-MB-453 cells were seeded in 96-well plates, after 24 h, 48 h, 72 h, the absorbance value of control-sh RNA cells are detected as 0.326 ± 0.093, 0.612 ± 0.123, 1.115 ± 0.111; B7-H4-sh RNA cells are detected as 0.282 ± 0.072, 0.372 ± 0.100, 0.693 ± 0.099. T-test analysis that 24 h after inoculation, no significant difference in cell proliferation, but 48 h and 72 h after inoculation, the absorbance value of the B7-H4-sh RNA was significantly lower than the control-sh RNA cell proliferation(P<0.01). The growth inhibition rates of B7-H4-sh RNA in 24 h, 48 h, 72 h were 12.8 ± 4.1%, 39.9 ± 4.9% and 38.0 ± 2.8%, which may be related to high transfection efficiency of B7-H4-sh RNA group at 48 h and 72 h. These results suggested that reducing the expression of B7-H4 could inhibit vitro proliferation of MDA-MB-453 cells in vitro.4 Results of cell invasion and migrationThe amount of MDA-MB-453 cells invasion through the transwell membrane to the lower chamber by an inverted microscope: control-sh RNA group and B7-H4-sh RNA group were 200.3 ± 8.0 and 93.7 ± 6.0/high power field(×100), B7-H4-sh RNA group was significantly less than the control-sh RNA group(P<0.01); The amount of MDA-MB-453 cells invasion through the transwell membrane to the lower chamber by an inverted microscope: control-sh RNA-transfected group and B7-H4-sh RNA group were 295.0 ± 8.7 and 147.3 ± 3.5/high power field(×100), transfected with B7-H4-sh RNA group was significantly less than the control-sh RNA group(P<0.01). Results described that reducing B7-H4 expression could inhibit the invasion and migration MDA-MB-453 cells.5 Flow cytometry resultsFlow cytometry results showed that after being transfected with B7-H4-sh RNA, the apoptosis of the MDA-MB-453 cells was significantly increased compared with the control-sh RNA group, and the cell cycle arrested in G0/G1 phase(P<0.01). It suggested that B7-H4 low expression can promote apoptosis and regulate the cell cycle G0/G1 phase of TNBC cells6 Real-time PCR and Western-blot test resultsReal-time PCR was used to detect the expression of p65 factor in MDA-MB-453 cells at m RNA levels, the results showed that B7-H4-sh RNA group p65 expression was significantly lower than control-sh RNA group, and the difference was statistically significant(P<0.05), and Spearman correlation analysis showed that p65 and B7-H4 m RNA expression was positively correlated(P<0.01).Western-blot was used to detect the expression of p65 factor in the MDA-MB-453 cells at protein levels, the results showed that B7-H4-sh RNA group p65 expression was significantly lower than that of control-sh RNA group, and the difference was statistically significant(P<0.05), and B7-H4 and p65 expression were positively correlated.In summary, in TNBC MDA-MB-453 cells, the expression of p65 and B7-H4 were positive correlation.Conclusions:B7-H4 molecules highly express in different subtypes of breast cancer,which is highest in TNBC. In TNBC, the expression of B7-H4 is correlated with pathological type, lymph node metastasis, the negative expression of AR and the poor prognosis. After downregulation of molecule B7-H4, the proliferation, invasion and migration of TNBC cells are weakened, while the apoptosis is induced, and the expression of p65, which is the major transcriptional regulatory subunit of the NF-κB pathway,was inhibited. Therefore B7-H4 is expected to become a new index of TNBC targeted therapy. |
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