The Research Of The Role Of B7-H4 In Breast Cancer Progression And Its Downstream Molecules

B7-H4 belongs to the B7 protein family along with B7-H1 / PD-L1 and B7-H2/ICOS-L.As a co-inhibitory molecule,B7-H4 is primarily thought to promote tumor immune escape via suppressing T cell immunity through binding to its corresponding receptor on T cells at the beginning of its discovery.Nowadays,an accumulating body of studies have indicated that aberrant expression of B7-H4 similarly plays a vital role in the malignant biological behavior of cancer cells.PRDX3 is a member of the peroxidase family.PRDX3 mainly have a hand in regulating the mitochondrial hydrogen peroxide level induced by cytokine induction.Studies have displayed that overexpression of PRDX3 is found in breast,hepatocellular,colorectal,lung,and nasopharyngeal cancers and protects cancer cells from apoptosis and chemical destruction.Therefore,many scholars have started to take note of the role it plays in tumorigenesis.The E2F transcription factor family consists of 8 members（E2F1、E2F2、E2F3、E2F4、E2F5、E2F6、E2F7、E2F8）,all of which are the main transcriptional regulators of the expression of cell cycle-related genes and are very important for the regulation of cell cycle.Abnormal expression of E2 F family members in cancer cells is associated with poor prognosis of many kinds of cancers.E2F2 has been reported to be overexpressed in invasive or non-invasive ductal breast cancer and invasive lobular breast cancer.Because of its strong association with poor prognosis,it is important in breast cancer progression.Transcriptome sequencing results showed downregulation of PRDX3 and E2F2 at m RNA levels by interfering with B7-H4 in breast cancer cells.Nevertheless,the function of B7-H4 in breast cancer carcinogenesis and roles of its possible downstream molecules PRDX3 and E2F2 remain to be explored.Objective: To investigate effects of interfering with B7-H4 on cell proliferation,cell cycle distribution,total apoptosis,and intracellular ROS level in breast cancer cell lines,we interfered with the expression of B7-H4 in breast cancer cell lines by using siRNA specifically targeting B7-H4.Validation of the expression correlations between B7-H4 and PRDX3 or E2F2 were analyzed at the m RNA and protein levels.Then,we interfered with the expression of PRDX3 or E2F2 in breast cancer cell lines by using siRNAs specifically targeting PRDX3 or E2F2 to explore effects of interfering with PRDX3 or E2F2 on cell proliferation and cycle distribution of breast cancer cell lines.Meanwhile,the effect of interfering with PRDX3 on the intracellular ROS level of breast cancer cells was also be explored.PRDX3 and E2F2 overexpression vectors were constructed,and we overexpressed PRDX3 in breast cancer cell lines that interfered with B7-H4 to explore roles of PRDX3 on proliferation,cell cycle distribution and ROS level changes of breast cancer cells induced by interfering with B7-H4.Furthermore,we overexpressed E2F2 in breast cancer cell lines that interfered with B7-H4 to explore roles of E2F2 on proliferation and cell cycle distribution changes of breast cancer cells induced by interfering with B7-H4.Methods:Part 1: Exploring effects of interfering with B7-H4 on the malignant phenotype of breast cancer cells and its possible downstream molecules（1）Total RNA was extracted from breast cancer cell lines maintained in our laboratory.RNA expression levels of B7-H4 within breast cancer cell lines were determined using RT-PCR experiments.（2）The proteins of the above cells were extracted,and the protein expression levels of B7-H4 within each cell line were detected by WB experiments.（3）siRNA specifically targeting B7-H4（siB7-H4）was synthesized and then,we transfected it into breast cancer cell lines using Lipofectamine^TM 2000,interference efficiency was identified by RT-q PCR and WB experiments.（4）Flow cytometry was used to investigate effects of interfering with B7-H4 on ROS levels,apoptosis,and cell cycle distribution in breast cancer cells.（5）Cell proliferation assay was used to explore the effect of interfering with B7-H4 on the proliferation of breast cancer cells.（6）The RNA and protein expression changes of PRDX3 and E2F2 were detected by RT-q PCR and WB experiments after interfering with B7-H4 in breast cancer cells.Part 2: Exploring roles of PRDX3 on proliferation,cell cycle distribution and ROS level changes of breast cancer cells induced by interfering with B7-H4（1）The changes of cell proliferation were detected by cell proliferation assay after interfering with PRDX3 in breast cancer cell lines by using siRNA specifically targeting PRDX3（siPRDX3）.（2）The effects of interfering with PRDX3 on ROS level and cell cycle distribution of breast cancer cell lines were detected by flow cytometry.（3）Primers were designed to synthesize the full length of the PRDX3 coding sequence by PCR.The coding sequence was inserted into the pc DNA 3.1（+）vector to construct the PRDX3 overexpression vector named pc DNA 3.1-PRDX3,and the plasmid was used in subsequent experiments after sequencing without error.（4）A scrambled sequence（siNC）and pc DNA3.1（+）empty vector were co-transfected into breast cancer cell lines by using Lipofectamine^TM 2000,which were named as control group（MCF-7 or T47D/siNC+pc DNA 3.1 vector）,siB7-H4 and pc DNA3.1（+）empty vector were co-transfected into breast cancer cell lines,which named as interference group（MCF-7 or T47D/siB7-H4+pc DNA3.1 vector）,siB7-H4 and pc DNA3.1-PRDX3 were co-transfected into breast cancer cell lines,which named as the PRDX3 revertant group（MCF-7 or T47D/ siB7-H4+pc DNA3.1-PRDX3）.The RNA and protein from the cells of each group were extracted,and expression changes of B7-H4 and PRDX3 within the cells of each group were detected by RT-q PCR and WB experiments,respectively.（5）Changes of ROS levels and cell cycle distribution in each group were detected by flow cytometry.（6）The cell proliferation change of cells in each group were detected using cell proliferation assay.Part 3: Exploring roles of E2F2 on proliferation and cell cycle distribution changes of breast cancer cells induced by interfering with B7-H4（1）Changes of cell proliferation were detected by cell proliferation assay after interfering with E2F2 in breast cancer cell lines by using siRNA specifically targeting E2F2（siE2F2）.（2）The effect of interfering with E2F2 on cell cycle distribution of breast cancer cell lines was detected by flow cytometry.（3）According to the same method in Part 2,the E2F2 overexpression vector pc DNA3.1-E2F2 was constructed and was used in subsequent experiments after sequencing without error,control group（MCF-7 or T47D/siNC +pc DNA3.1vector）,interference group（MCF-7 or T47D/siB7-H4+pc DNA3.1vector）and E2F2 revertant group（MCF-7 or T47D/ siB7-H4+pc DNA3.1-E2F2）were constructed by using the same method according to the Part 2,expression changes of B7-H4 and E2F2 within the cells of each group were detected by RT-q PCR and WB experiments,respectively.（4）Cell proliferation assay was used to detect cell proliferation changes among the three groups.（5）Cell cycle distribution changes in each group were detected by flow cytometry.Results: Part 1:（1）RT-PCR and WB results showed that B7-H4 was highly expressed in MCF-7 and T47 D cells,which were chosen for subsequent experiments.RT-q PCR and WB experiments showed the downregulation of B7-H4 in cells transfected with siB7-H4 compared to cells transfected with siNC.（2）Interfering with B7-H4 significantly inhibited the proliferation of MCF-7 and T47 D cells and arrested cells in the G1-S phase accompanied by increased intracellular ROS levels,however it had no significant effect on overall apoptosis rates in both cell lines.Results of RT-q PCR and WB experiments showed downregulation of PRDX3 and E2F2 after interfering with B7-H4 in MCF-7 and T47 D cells.Part 2:（3）RT-q PCR and WB experiments showed the downregulation of PRDX3 in cells transfected with siPRDX3 compared to cells transfected with siNC.Interfering with PRDX3 significantly inhibited cell proliferation accompanied by increased intracellular ROS levels in MCF-7 and T47 D cells,meanwhile,MCF-7 cells were arrested in the G1-S phase,however,no significant change of cell cycle distribution was found in T47 D cell after interfering with PRDX3.（4）The coding sequence of PRDX3 amplified by PCR was inserted into pc DNA3.1（+）vector.,the PRDX3 overexpression vector pc DNA3.1-PRDX3 was successfully constructed according to the NCBI database after sequencing.RT-q PCR and WB experiments showed successful suppression of B7-H4 expression in interference group and PRDX3 revertant group cells.In contrast to the control group,not only B7-H4,but also the expression of PRDX3 decreased significantly in the interference group,however,the expression of PRDX3 in the PRDX3 revertant group increased significantly in contrast to the interference group.Results of cell proliferation experiments showed that in contrast to the control group,the cell proliferation of the interference group was significantly inhibited,while the cell proliferation of the PRDX3 revertant group was slightly inhibited.We observed a significant increase in intracellular ROS levels of the interference group in contrast to the control group,however,we found no significant change in the PRDX3 revertant group.At the same time,the cells of both interference group and PRDX3 revertant group were arrested in the G1-S phase compared to the control group.Part 3:（5）RT-q PCR and WB experiments showed the downregulation of E2F2 in cells transfected with siE2F2 compared to cells transfected with siNC.Interfering with E2F2 significantly inhibited cell proliferation of MCF-7 and T47 D cells and arrested them in the G1-S phase.（6）E2F2 was overexpressed according to the method described above while interfering with B7-H4,RT-q PCR and WB experiments showed successful suppression of B7-H4 in both interference group and E2F2 revertant group cells.In contrast to the control group,the expression of E2F2 decreased significantly in the interference group,however,the expression of E2F2 in the E2F2 revertant group increased significantly compared to the interference group.The results of cell proliferation experiments showed that the cell proliferation of the interference group was significantly inhibited compared to the control group,while the cell proliferation of the E2F2 revertant group was slightly inhibited.Flow cytometry analysis showed that both interference group and E2F2 revertant group cells were arrested in the G1-S phase compared to the control group.Conclusion:（1）Interfering with B7-H4 inhibited breast cancer cell proliferation and caused cell cycle retarding at G1-S phase accompanied by increased intracellular ROS levels and had no significant effect on cell apoptosis.（2）Interference with B7-H4 could affect the intracellular ROS level and inhibit cell proliferation partially by downregulating PRDX3 in breast cancer cells.（3）Interference with B7-H4 could inhibit cell proliferation partially by downregulating E2F2 in breast cancer cells.（4）Reverted expression of PRDX3 or E2F2 did not cause significant changes in interfering with the B7-H4-led cell cycle arrest of the breast cancer cells.In summary,this study explored function of B7-H4 in breast cancer cell tumorigenesis and found its downstream molecules PRDX3 and E2F2,which played important roles in the changes of malignant phenotype of breast cancer cells caused by interfering with B7-H4.It contributes new ideas to investigate the molecular mechanism of B7-H4 in breast cancer tumorigenesis.