Semaphorin 6D And Its Receptor Plexin-A1 Expression In Gastric Cancer And Their Relationship With Tumor Angiogenesis

The semaphorin and plexin family of ligand and receptor proteins were early found provideimportant axon growth and guidance cues required for development.In recent years,studies have expanded the role of them in the regulation of cardiac morphogenesis and tumorgenesis. However the mechanism of their rolein regulating cancer development and progression has not been recognized. The present study found Semaphorin6D(Sema6D) and its receptor plexin-A1 expressed at high levels in vascular epithelial cells within gastric cancer, and were positively correlated with vascular endothelial growth factor receptor 2(VEGFR2).These findings suggestthat Sema6 D and plexin-A1 may closely associatewith tumor angiogenesis.Combined with experimental observations in MGC803 gastric cancer cell line, we found that knock down plexin-A1 signaling led to a decreased expression of VEGFR2 at mRNA and protein levels. Sema6 D recognized and activatedplexin-A1, which subsequently activated its down-stream target VEGFR2. The activation of VEGFR2 functioned as a positive regulator of tumor angiogenesis.Our data indicated Sema6D/plexin-A1/VEGFR2 may be a complex signaling pathway involving in angiogenesis-related pathway in tumor cells.In light of our observations, pharmacological interventions targeting this signaling may affect the tumor angiogenesis, which potentially be expected to provide a novel thinking in the treatment of gastric cancer. Objective:To detect Sema6 D and plexin-A1 expression in gastric cancer and their effects on tumor angiogenesis, and further explore the molecular mechanism of tumor angiogenesis. Methods:1. We used PCR technology to detect the m RNA expression of Sema6 D and its receptor plexin-A1 in gastric cancer and normal gastric mucosa tissue.2. We used western blot technology to detect the protein expression of Sema6 D and its receptor plexin-A1 in gastric cancer and normal gastric mucosa tissue.3. We used RT-PCR technology to detect the mRNA expression of plexin-A1, plexin-A2 and plexin-A4 in three gastric cancer and one normal gastric mucosa cell lines.4.We used immunofluorescencetechnology to detect theexpression and location of Sema6 D and its receptor plexin-A1 in gastric cancerandnormal gastric mucosa tissue.5. We used immunofluorescence staining technology to detect the colocalization of Sema6D/plexin-A1 and VEGFR2 respectively in gastric cancer tissue and normal gastric mucosa tissue.6.We used0、5、10μM of ISO to incubate gastric cancer cells for 3 hours, and extract total RNA and proteins ofcells respectively; In addition, we used5μM of ISO to incubate gastric cancer cells for 0、1、2、3、6 hours, collect cells and extract total RNA and proteins ofcells.7. We used plasmid transfection technique to decrease the expression of plexin-A1 in MGC803 gastric cancer cell lines, and further detect theexpression of VEGFR2. Results:1. Real-time PCR analyses of the mRNA expression of Sema6 D increased remarkably in tumor when compared with normal gastric mucosa tissue. Western blot analyses of the protein expression of Sema6 Dincreased remarkablyin tumor when compared with normal gastric mucosa tissue.2. RT-PCR was performed to show the expression of Sema6 D receptors plexin-A1, plexin-A2 and plexin-A4 in MGC803, HGC27 and MNK45 three gastric cancer and GES-1 cell lines. The mRNA expression of plexin-A1 was obviously increased in all the three gastric cancer when compared with normal gastric mucosa cell line, but there was no significant difference of plexin-A2 and plexin-A4 between gastric cancer and normal gastric mucosa cell lines. The expression of plexin-A1 wasobviously increased when compared with plexin-A2 and plexin-A4 in all the three gastric cancer cell lines.3. Real-time PCR analyses of the mRNA expression of plexin-A1 increased remarkablyin tumorwhen compared with normal gastric mucosa. Western blot analyses of the protein expressionof plexin-A1 in allcases of tumor were enhanced.4. Immunofluorescence analysis showed that Sema6 D was highly expressed in thevascular endothelial cells within gastric cancer compared withnormal gastric tissue.The similar results showed the expression of plexin-A1 was highly expressed ingastric cancer vascular endothelial cells.5. Fluorescence images show the double-labellingof Sema6D/plexin-A1 and VEGFR2 respectivelyinperi-cancerous andcancerous tissue. The results show Sema6 D expression was strongly correlated with VEGFR2 in vascular epithelial cells in the cancerous tissue, but there was no significant phenomenon in normal gastric mucosa.Similarly, the double-labelling cells of plexin-A1 and VEGFR2 can also be detected invascular epithelial cellsin gastric cancer tissue.6. MGC-803 and HGC-27 cells were stimulated with increased concentrations ofISO. Real-time PCR analyses show ISO induced plexin-A1 expression at mRNA levels in gastric cancer cells. Western blot analyses showthe expression of plexin-A1 protein was up-regulated after ISO treatment. MGC-803 cells weretreated with increased timesof ISO. Real-time PCR and westernblot analysesshowISO stimulation also upregulated plexin-A1 expression in a time-dependent manner at mRNA and protein levels.7. MGC803 andHGC27 gastriccancer cell lines were treated with increased time of ISO, Real-time PCR and westernblot analysesshowISOstimulation also promotethe mRNA and protein expression levels of VEGFR2.8.Using shplexin-A1 transfection to knock down plexin-A1 expression in MGC803 cells, and the expression of VEGFR2 also decreased. Further, Real-time PCR and western blot analyses showISO treatment was unable to upregulate VEGFR2 expression at mRNA and protein levels when plexin-A1 was knocked down. Conclusion:1. Sema6 D and its receptor plexin-A1 are highly expressionin gastric cancer, and mainly located in endothelial cells of vascular2. Sema6 D and plexin-A1 may affect tumor angiogenesis through mediate VEGFR2 signals3.Isoprenaline promotes plexin-A1 and VEGFR2 expression in gastric cancer cells, and promotes VEGFR2 expression dependent on plexin-A1...