B7-H3Regulating Myeloid-derived Suppressor Cells Apoptosis In Mouse Prostate Cancer Progression

Objective: We regulated murine prostate cancer RM-1cells B7H3gene expressionusing gene transfection techniques. We observed mouse prostate cancer cell line of RM-1proliferation in the role of B7H3by in vivo and in vitro experiments, to explore its possiblemechanism.Methods: We retrieved mice B7H3the nucleotide sequence, synthesized murineB7H3gene fragment, and transferred to the destination vector, using Gateway Technology.B7H3gene high expression plasmid vector was constructed. Transfected genes wereconsistent with the purpose of gene detected by gene sequencing. RT-PCR was used todetect mRNA expression before and after the plasmid construction. The above twomethods identified that the plasmids were successfully constructed. Prostate cancer in miceRM-1cells were stable transfection, and then transfected with plasmid vector B7H3geneRM-1cells as a control. Application observed by fluorescence microscopy transfected withgreen fluorescent protein expression to determine transfection success. After transfectionthe B7H3gene expression level was used to detect changes in mRNA expression byRT-PCR. The B7H3ene expression at the protein level changes in the two groups of cellswas detected by flow cytometry. Detection further confirmed that the B7H3gene wasstable transfection. In the part of in-vitro experiments,the RM-1cells were divided intotwo groups: the experimental group (B7-H3expression group) and negative control group(B7-H3low expression group only transfected with GFP). Cultured in vitro under the sameconditions, the murine prostate cancer cell changes were observated by counting thenumber of the cells. RM-1cell growth rate of the two groups were detected for comparingB7H3gene expression with low B7H3gene expression in mouse prostate cancer cellgrowth in vitro. Mice spleen was taken out and cut into pieces. The tissue was washed,then centrifuged into single cell suspension after grinding to obtain cell pellet. Add to Gr-1~+CD11b~+antibody on sorting by flow cytometry, MDSC was separated. Gr-1~+CD11b~+ cells from the spleen separation and purification were mixed culture with prostate cancerRM-1cells of the experimental group and the control group in vitro. The cells werecollected and detected by flow cytometry after cultured.We found the group of B7H3highexpression inhibit myeloid derived suppressor cell apoptosis in vitro. When THP-1cellsB7H3gene expression were lowered by small interfering RNA， marrow-derived myeloidTHP-1cell apoptosis were observed by flow cytometry. In the part of in vivoexperiments,high B7H3expression RM-1cells and low B7H3gene expression RM-1cellswere implanted in mice inguinal subcutaneous. Tumor growth was continuously observedafter implantion. The time of tumor formation and tumor size changes were recorded. Invivo testing to22days, experimental mice were sacrificed, and specimens were taken fromthe tumor and the spleen of experimental mice.The specimens were cut into smallpieces.After grinding and PBS fully washing blood cells and impurities, the specimenscentrifuged to obtain cell pellet. The cells were added to different antibodies on flowcytometry. B7H3gene expression and apoptosis were detected in transplanted tumor cellsand spleen cells. Myeloid derived suppressor cells expression and apoptosis were detected.Finally, cyclophosphamide was role in the spleen of experimental mice.to suppress bonemarrow lymphocytes. The size of the tumor volume changed by the B7H3gene expressionof experimental mice was observed without the interference of the bone marrow immunecells.Results: The B7H3gene expression plasmid vector was successfully constructed.PCR amplification experiments confirmed that the the PCR identification bands around theplasmid construct theoretical size match the size of1200bp, with the purpose of Articlebelt. DNA sequencing showed that the nucleotide sequence of the insertion sites intoexperimental gene target gene fragment is entirely correct. The recombinant plasmidvectors by Gateway Technology B7H3gene were transducted into murine prostate cancerRM-1cells. The constructed plasmid B7H3support both at the mRNA level and at theprotein level could improve B7H3gene expression with the high transfection efficiency.After repeatedly subcultured the cells of the two experimental groups were extracted.Flowcytometry showed there are significant differences in the two groups of cells B7H3geneexpression. The experiment further confirmed that the B7H3gene was stable transfection.The in vitro cell growth rate results showed that the experimental group (B7-H3+group)and the negative control group RM-1cells were no significant difference. Purification Gr-1~+CD11b~+spleen cells were respectively mixed culture with RM-1prostate cancer cellsin mice of experimental group and control group. The cells were collected and detected byflow cytometry after cultured. B7-H3~+group, as compared to B7-H3-group, showedpreferentially inhibiting MDSC apoptosis. Marrow the endogenous THP-1cells apoptosisincidence rised after small interfering RNA reduced THP-1cells expression of humanB7H3. The two groups of mice prostate cancer cells were subcutaneously inoculated inmice groin. The tumorigenic time was recorded and tumor size were measured every twodays.There were significant differences in the two groups of tumor size. Tumors in miceappeared in B7H3gene high expression group earlier than the the B7H3genes lowexpression group.Tumor in experimental group grew faster than that in the controlgroup.The average tumor volume of experimental group increased significantly. Flowcytometry detected that the B7H3expression of mice transplanted tumor cells in B7H3gene regulation group was significantly increased. The in vivo experiments showed, theRM-1cells of B7H3gene regulation group in vivo tumorigenicity increased (P<0.01).After in Vivo cultured to22days, tumor-bearing mice were sacrificed by broken theneck. Specimens from tumor and spleen of experimental mice were extracted, recordingtumor volumes. Tumor cells and spleen cells were extracted from the two groups ofexperimental mice. The B7H3gene expression in tumor cells in mice inguinalsubcutaneous were detected by flow cytometry.That was also recorded including cellproliferation and apoptosis of myeloid derived suppressor cell proliferation and prostatecancer tumors in mice. The B7H3gene expression weakened after in vivo cultivation in thethe B7H3gene high expression group, but it was still higher than the B7H3gene lowexpression group.There was statistically significant difference between the two groups. Themyeloid derived suppressor cells of experimental group accelerated proliferation andapoptosis decreased while prostate cancer tumors in mice accelerated proliferation andapoptosis decreased. That was confirmed the B7H3gene in vivo promoted thedevelopment of tumors at the same time adjusted myeloid derived cells.The two groups ofexperimental mice were treated by Cyclophosphamide and then inoculated two groups ofprostate cancer cells RM-1in mice groin subcutaneous. There was no significant differencebetween the two groups of mice on transplanted tumor growth rate and size of tumor. Theexperiment proved B7H3promote tumor formation in vivo through the role of immunecells in the body. Conclusions: The plasmid vector was constructed successfully，and B7H3gene wasstably transfected in RM-1cell line. That the stable transfection of B7H3gene expressionby plasmid-mediated was no obvious promotion RM-1cell apoptosis in vitro. But itinhibited RM-1cell growth; promoted the RM-1tumors in vivo tumorigenicity andprogress. B7H3gene stably transfected regulated MDSC to promote RM-1tumorprogression.The B7H3site is expected to become auxiliary potential target on gene therapyfor prostate cancer.