The Preliminary Research TM4SF1 Gene In Human Umbilical Vein Endothelial Cell Biology Behavior

Malignant solid tumor growth and metastasis depend on tumor angiogenesis, tumor treatment by inhibiting the growth of tumor angiogenesis has been applied in clinic.TM4SF1 is a family of TM4SF (Tetraspanins) of a distant relative, is highly expressed in many epithelial cancer and cultured human umbilical vein endothelial cells (HUVEC), meanwhile the highly expression of TM4SF1 is correlated with tumor cell migration, invasion, metastasis and poor prognosis. Preliminary studies of our group selected antigen TM4SF1 from the cDNA construction useing patients with epithelial ovarian cancer cells, and found that the serum TM4SF1 antibody can be used as a serological marker for ovarian cancer diagnosis,and down regulate the expression of TM4SF1,and inhibit the proliferation, migration and metastasis in ovarian cancer cell line HO8910PM. This research using lentivirus vector to decrease TM4SF1 expression in HUVEC, in order to find out the effects of TM4SF1 on HUVEC proliferation, migration and angiogenesis, then co-inject human ovarian cancer cell line HO8910PM and HUVEC-TM4SF1 cell line subcutaneously in nude mice, using an in vivo animal imaging technology, the growth of tumor transplanted subcutaneously in nude mice were observe and record, have any effects on metastasis and tumor angiogenesis.Part1. Lentivirus mediated TM4SF1 gene silencing of HUVEC and HO8910PM constructionObjective Using RNA interference technology to establish stable transmem brane four superfamily member 1 (Transmembrane 4 super family 1,TM4SF1) gene silencing in human umbilical vein endothelial cells (HUVEC) and human ovarian cancer cell line (HO8910PM) mediated by lentivirus.Methods To design and synthesis of targeting TM4SF1 and expression of two kinds of lentiviral vector of GFP or luciferase gene, named LV-TM4SF1-RNAi-GFP and LV-TM4SF1-RNAi-Luc, LV-TM4SF1-RNAi-GFP infection and the high expression of TM4SF1and HUVEC, were screened by flow cytometry with stable expression of GFP cell line LV-TM4SF1-RNAi-GFP/HUVEC. The infection of LV-TM4SF1-RNAi-Luc and the high expression of TM4SF1 HO8910PM, with puromycin stable and established the stable expression of luciferase geneLV-TM4SF1-RNAi-Luc/HO8910PM. In the negative control and blank control as the control, the gene and protein of two TM4SF1 were detected RT-PCR and Western blotting expression.Result The silencing of TM4SF1 and the expression of two GFP or luciferase lentiviral vector was constructed successfully, infection and screened stable expression cell strain LV-TM4SF1-RNAi-GFP/HUVEC and LV-TM4SF1-RNAi-Luc/HO8910PM, LV-CON-RNAi-GFP/HUVEC and negative control group (LV-CON-RNAi-Luc/HO8910PM) and HUVEC (HO8910PM) compared to the normal group, LV-TM4SF1-RNAi-GFP/HUVEC group and LV-TM4SF1-RNAi-Luc/HO8910PM TM4SF1 mRNA expression decreased significantly, "(0.06+0.02) vs (0.95+0.13)/(1.02+0.13), P<0.05] ([(0.05 +0.02) vs (0.91+0.13)/(1.04+0.13),P<0.05]);protein expression was also significantly reduced[(0.13+0.01)vs(0.75+0.03)/(0.75+0.04), P<0.05] ([(0.11+0.01) vs (0.58+0.02)/(0.65+0.03), P<0.05], LV-CON-RNAi-GFP/ HUVEC) and negative control (LV-CON-RNAi-Luc/HO8910PM) group and HUVEC(HO8910PM) blank control group had no significant difference, P was greater than 0.05.Conclusion Using RNA interference technology to construct TM4SF1 stable silencing of LV-TM4SF1-RNAi-GFP/HUVEC and LV-TM4SF1-RNAi-Luc/ HO8910PM cell lines.Part2. The effect of TM4SF1 gene silencing on the biological function of epithelial ovarian cancer cells in vitroObjective To investigate the effect of TM4SF1 gene silencing on HUVEC proliferation, migration and angiogenesis.Methods The experiment was divided into 3 groups:HUVEC group, LV-CON-RNAi-Luc/HO8910PM group and LV-TM4SF1-RNAi-GFP/HUVEC group. CCK-8 method was used to detect the changes of the ability of cell proliferation, FCM to detect changes in cell cycle, Transwell assay was performed to detect the changes of cell migration, tube formation in Matrigel angiogenesis assay the ability to generate change.Results After TM4SF1 gene silencing, the proliferation of HUVEC was inhi bited. [72h:(0.59+0.06) vs (0.98+0.04)/(1.05+0.05), P<0.05] cells were arrested at G0/G1 phase;[(69.30+5.48) vs (54.44+4.09)/(51.69+4.1 6), P<0.05] migration was; reduce [(14.20+4.59) vs (70.60+17.75)/(74.40 +16.06), P<0.05]; lumen like structure significantly reduced [(3.33+1.52)vs (19.66+1.52)/(22.66+2.08), P<0.05]. But there was no significant difference between HUVEC group and LV-CON-RNAi-Luc/HO8910PM group.Conclusion TM4SF1 gene silencing,the proliferation migration and angiogenesis wereinhibited by HUVEC.Conclusion TM4SF1 gene silencing,the proliferation migration and angiogenesis wereinhibited by HUVEC.Part3. The effect of TM4SF1 gene silencing on the biological function of ovarian cancer tumor angiogenesis in vivoObjective To construct the nude mice model suitable for small animal imaging system observation, to explore the effect of TM4SF1 gene silencing on the growth, metastasis and angiogenesis of ovarian cancer.Methods HO8910PM and HUVEC cells were co cultured with Transwell cells, two cells in vitro to determine the most suitable for growth of proportion. According to the determined the proportion of cells in the TM4SF1 HUVEC and HO8910PM stable knockdown cell lines wereinoculated subcutaneously in nude mice, nude mice transplantation tumor model was constructed to observe the small animal in vivo imaging system.36 female nude mice were randomly divided into 6 groups, respectively A:LV-CON-RNAi-Luc/HO8910PM; B:LV-TM4SF1-RNAi-Luc/HO8910PM; C:LV-CON-RNAi-Luc/HO8910PM+LV-CON1-RNAi-GFP/HUVEC; D:LV-CON-RNAi-Luc/HO8910PM+LV-TM4SF1-RNAi-GFP/HUVEC, E:LV-TM4SF1-RNAi-Luc/HO8910PM+LV-CON1-RNAi-GFP/HUVEC; F:LV-TM4SF1-RNAi-Luc/HO8910PM+LV-TM4SF1-RNAi-GFP/HUVEC。By in vivo imaging system in detecting tumor growth,metastasis, by immunohistochemical staining observed the xenograft tumor angiogenesis.Results The transwell cell co culture HO8910PM and HUVEC cells, two cells in vitro to determine the most suitable for the growth ofthe proportion is HO8910PM/HUVEC:2/1. Sensitivity test of animal imaging system,capable of cell number in the minimum detection biotin luminous in vitro forl02cells.36 mice the tumor formation rate was 100%, total 4 rats died in 6 weeks. On 6 week, The tumor volume of which were inoculated HUVEC group (C, D, E, F) was significantly larger than single vaccination HO8910PM group (A, B), Group B was significantly less than the negative control group A [(0.11±0.05) vs (0.87 ±0.05), P<0.05]; D, E, F group less than the negative control group C to varying degrees [(0.26±0.06)/(0.23±0.07)/(0.04±0.03) vs (0.72±0.08), P <0.05], completely silent group F were the smallest on tumor volume of inoculated groups, and part of silenting group D and E were fairly. The growth of xenografts was observed dynamiclly in vivo imaging system, with time, bioluminescence fluorescence signal is gradually increased, but metastases was not detected in six groups. Similarly, no metastases in the anatomyed nude. The xenograft tumor angiogenesis was observed by Immunohistochemical staining, The MVD of inoculated HO8910PM and HUVEC group (C, D, E, F) were significantly higher than single vaccination HO8910PM group (A, B) (P<0.05), but only the inoculate groups had humanized microvessels. In Single inoculation groups, the MVD of control group (6.67±1.53) and Silence group (6.33±0.57) was no significant difference (P> 0.05). The humanized MVD was more than the total number of 90% microvessels. From the total MVD, the group C (60.67±5.77) and E group (60.33±9.07) was higher than D group (36.35±6.42) and F group (31.56±.11) (P<0.05), and between group C and E, D and F were no significantly different (P> 0.05).Conclusion TM4SF1 gene silence have inhibit effect to tumor growth and angiogenesis of ovarian cancer, but it can not determine whether inhibition of metastasis of ovarian cancer.