Lung cancer cell-derived Sema4D mediates osteolytic bone destruction in lung cancer by inhibiting osteoblast differentiation / 肿瘤

Objective: To determine the role of semaphorin 4D (Sema4D) in osteolytic bone destruction of bone metastasis in lung cancer, and to explore its mechanism. Methods: Immunohistochemistry was used to detect the expression of Sema4D in bone metastases of lung cancer and normal bone tissues. The expression levels of Sema4D protein and mRNA in four kinds of lung cancer cell lines were detected by Western blotting and real-time fluorescent quantitative PCR, respectively. The conditioned medium of lung cancer cells was collected, and ELISA was used to detect the secretion level of Sema4D in lung cancer conditioned medium. The conditioned medium from lung cancer cells with Sema4D high expression (PC9 cells) or low expression (A549 cells) was collected and co-cultured with osteoblastic precursor MC3T3-E1 cells, then the osteoblast differentiation ability was detected by alkaline phosphatase staining, the mineralization ability of osteoblasts was detected by alizarin red staining, the expression levels of osteoblast differentiation genes alkaline phosphatase, liver/bone/kidney (ALPL), Oxterix and collagen type alpha 1 (Col1α1) were detected by real-time fluorescent quantitative PCR. Sema4D shRNA was transfected into lung cancer PC9 cells, then the expression and secretion levels of Sema4D in lung cancer cells were detected by Western blotting, real-time fluorescent quantitative PCR and ELISA, respectively. The conditioned medium of lung cancer PC9 cells transfected with Sema4D shRNA was collected and co-cultured with preosteoblasts, and the effect of the conditioned medium from lung cancer cells with Sema4D interference on the osteoblast differentiation was determined by alkaline phosphatase staining, alizarin red staining and real-time fluorescent quantitative PCR, respectively. Results: Compared with the normal bone tissues, Sema4D was highly expressed in osteolytic bone metastases of lung cancer. Sema4D was expressed and secreted in different lung cancer cell lines in different degrees. The expression and secretion levels of Sema4D were the highest in lung cancer PC9 cells, but lowest in lung cancer A549 cells (P < 0.05). The conditioned medium of lung cancer cells significantly inhibited the osteogenic differentiation of osteoblasts induced by osteogenic inducer, which was characterized by the reduction of relative staining area of alkaline phosphatase staining and alizarin red staining (both P < 0.05), and the decreased expression levels of osteogenic differentiation genes ALPL, Oxterix and Col1α1 (all P < 0.05). The conditioned medium of PC9 cells with Sema4D high expression exhibited more aggressive inhibitory effect on osteoblast differentiation than the conditioned medium of A549 cells with Sema4D low expression (P < 0.05). shRNA transfection significantly reduced the expression and secretion of Sema4D in lung cancer cells (both P < 0.05), and significantly attenuated the inhibitory effect of lung cancer conditioned medium on osteoblast differentiation (all P < 0.05). Conclusion: Lung cancer-derived Sema4D plays an important role in osteolytic bone destruction by inhibiting osteoblast differentiation, suggesting that it is expected to become a new therapeutic target for bone metastasis of lung cancer.

Regulation of semaphorin 4D expression and cell proliferation of ovarian cancer by ERalpha and ERbeta

Ovarian cancer is one of the most common malignancies in women. Semaphorin 4D (sema 4D) is involved in the progress of multiple cancers. In the presence of estrogen-like ligands, estrogen receptors (ERα and ERβ) participate in the progress of breast and ovarian cancers by transcriptional regulation. The aim of the study was to investigate the role of sema 4D and elucidate the regulatory pattern of ERα and ERβ on sema 4D expression in ovarian cancers. Sema 4D levels were up-regulated in ovarian cancer SKOV-3 cells. Patients with malignant ovarian cancers had significantly higher sema 4D levels than controls, suggesting an oncogene role of sema 4D in ovarian cancer. ERα expressions were up-regulated in SKOV-3 cells compared with normal ovarian IOSE80 epithelial cells. Conversely, down-regulation of ERβ was observed in SKOV-3 cells. Forced over-expression of ERα and ERβ in SKOV-3 cells was manipulated to establish ERα+ and ERβ+ SKOV-3 cell lines. Incubation of ERα+ SKOV-3 cells with ERs agonist 17β-estradiol (E2) significantly enhanced sema 4D expression and rate of cell proliferation. Incubated with E2, ERβ+ SKOV-3 cells showed lower sema 4D expression and cell proliferation. Blocking ERα and ERβ activities with ICI182-780 inhibitor, sema 4D expressions and cell proliferation of ERα+ and ERβ+ SKOV-3 cells were recovered to control levels. Taken together, the data showed that sema 4D expression was positively correlated with the progress of ovarian cancer. ERα positively regulated sema 4D expression and accelerated cell proliferation. ERβ negatively regulated sema 4D expression and inhibited cell multiplication.

Effect of semaphorin 4D on the proliferation, migration and angiogenic of human pancreatic carcinoma cells / 新乡医学院学报

Objective To investigate the effects of semaphorin 4D(Sema4D) on the proliferation,migration and angiogenic of human pancreatic carcinoma cells.Methods Sema4D-siRNA was designed and synthesized and transfected into human pancreatic carcinoma cells.After 48 hours of transient infection,the changes of expression of Sema4D mRNA before and after transfection were detected by reverse transcription-polymeruse chain reaction method.And after 72 hours of transient infection,the changes of expression of Sema4D protein before and after transfection were detected by Western blot method.The changes of growth of the transfected cells were observed by methyl thiazolyl terazolium assay.Using transwell migration test and scratch repair test to detect the changes of migration ability of human pancreatic carcinoma cells after transfection.Using tubule formation assay to observe the effect of supernatant of pancreatic carcinoma cell cultures on angiogenesis after transfection.Results Compared with the negative control group and blank control group,the expression of Sema4D mRNA and Sema4D protein and the growth rate of pancreatic carcinoma cells decreased significantly (P ＜ 0.05).In transwell migration test and scratch repair test,it was observed that Pancreatic cancer cells penetrating cell number and scratch repair rate were significantly lower than that in negative control group and blank control group (P ＜ 0.05).Tubule formation assay showed that there were significant differences in angiogenesis numbers among siRNA transfection group(0.5 ± 0.02),negative control group(1.45 ± 0.60) and blank control group (1.37 ± 0.52) (P ＜ 0.05).Conclusion Sema4D-siRNA can induce RNA interference in pancreatic carcinoma cells and down-regulate the expression of Sema4D gene,which can inhibit the proliferation of pancreatic carcinoma cells,significantly reduce the migration ability of pancreatic carcinoma ceils and inhibit angiogenesis.

Role of inhibition of osteogenesis function by Sema4D/Plexin-B1 signaling pathway in skeletal fluorosis in vitro / 华中科技大学学报（医学）（英德文版）

Skeletal fluorosis is a chronically metabolic bone disease with extensive hyperostosis osteosclerosis caused by long time exposure to fluoride. Skeletal fluorosis brings about a series of abnormal changes of the extremity, such as joint pain, joint stiffness, bone deformity, etc. Differentiation and maturation of osteoblasts were regulated by osteoclasts via Sema4D/Plexin-B1 signaling pathway. Furthermore, the differentiation and maturation of osteoclasts are conducted by osteoblasts via RANKL/RANK/OPG pathway. Both of these processes form a feedback circuit which is a key link in skeletal fluorosis. In this study, an osteoblast-osteoclast co-culture model in vitro was developed to illustrate the mechanism of skeletal fluorosis. With the increase of fluoride concentration, the expression level of Sema4D was decreased and TGF-β1 was increased continuously. OPG/RANKL mRNA level, however, increased gradually. On the basis of that, the inhibition of Sema4D/Plexin-B1/RhoA/ROCK signaling pathway caused by fluoride promoted the level of TGF-β1 and activated the proliferation of osteoblasts. In addition, osteroprotegerin (OPG) secreted by osteoblasts was up-regulated by fluoride. The competitive combination of OPG and RANKL was strengthened and the combination of RANKL and RANK was hindered. And then the differentiation and maturation of osteoclasts were inhibited, and bone absorption was weakened, leading to skeletal fluorosis.

Sema4D deficiency reduces colorectal carcinoma xenograft growth and vascularity in nude mice / 中国肿瘤临床

Objective:Semaphorin 4D (Sema4D) acts as a regulator for axon guidance in central nervous system development. However, new evidence indicates that Sema4D has a previously unrecognized function, namely, compensatory angiogenic factor. This study aimed to investigate the effect of Sema4D on tumor growth and vascularity of colorectal carcinoma (CRC) in nude mice. Meth-ods:Sema4D was knocked down in CRC cells by infecting the cells with lentiviruses coding for Sema4D shRNA. Two groups of cells, namely, those infected with control viruses and those infected with Sema4D shRNA viruses, were subjected to migration assay to test their ability induce endothelial cell migration. The two cell groups were subcutaneously injected into nude mice. Tumor growth was documented, and the tumors harvested from the mice were subjected to immunohistochemistry or immuno fl uorescence analyses. Re-sults:In vitro migration assay results indicated that media conditioned by HCT-116 cells infected with Sema4D shRNA lentiviruses in-duced low endothelial cell migration. The two groups of subcutaneously inoculated cells showed 100%tumorigenicity. However, tumor growth rates were significantly different between the two groups. Xenografts in which Sema4D was downregulated showed marked re-duction in tumor size and vascularity. Conclusion:Cancer cells may highly express Sema4D to trigger net neo-angiogenesis and gener-ate a tumor blood supply system. Thus, Sema4D could potentially be a target in anti-angiogenic therapy of CRC patients.