The Impacts Of Silencing Indoleamine2,3-dioxygenase-1 Using RNAi On The Angiogenesis In Lewis Lung Cancer

Objective:This study was designed to investigate the effect of indoleamine 2,3-dioxygenase-1(IDO1)on the biological characteristics of Lewis lung cancer cell(LLC)line,and observe the effect of IDO1-shRNA on tumor growth and angiogenesis in LLC-bearing mice.We aimed to clarify the relationship between tumor angiogenesis and IDO1 expression,and to explore the potential of IDO1-targeting molecular therapy for lung cancer.Methods:1.The IDO1 gene in LLC cell line was silenced using IDO1-siRNA.The expressions of IDO1 mRNA and protein levels were detected by Real-time quantitative PCR(qRT-PCR)and Western blot.2.After IDO1 gene silencing,the invasion,migration and vasculogenic mimicry(VM)formation ability of LLC cells were detected by transwell invasion assay,transwell migration assay and Matrigel three-dimensional culture assay respectively.3.A lung cancer model was established by inoculating 1x106 LLC cells subcutaneously into the upper hind leg of C57BL/6 mice.Then the cancer-bearing mice were treated with 50μg of IDO1-shRNA by hydrodynamic injection through the tail vein.The tumor growth was observed and recorded daily.4.The expression of IDO1 and microvessel density(MVD)labeled by CD34 and CD146 were detected by immunohistochemical staining,and analyzed the relationship between IDO1 and MVD.Results:1.The results of qRT-PCR and Western blot showed that the expression of IDO1 was markedly decreased by more than 80% at both of the mRNA and protein levels after transfecting with IDO1-siRNA in LLC cell line.2.Transwell cell invasion assay showed that the invasion ability of LLC cells was significantly inhibited after silencing IDO1 gene.The results of transwell migration assay also showed that the migration ability of LLC cells was significantly decreased after IDO1 gene silencing.In addition,the Matrigel three-dimensional culture experiments showed that the numbers of vasculogenic mimicry(VM)tubers formed by the IDO1 gene silenced LLC cells was significantly reduced.3.After treatment with IDO1-shRNA in Lweis lung cancer-bearing mice,the tumor growth was significantly slower as comparing with scrambled shRNA-treated mice and sham-treated mice.Both tumor size and weight were less in IDO1 shRNA-treated mice than in scramble shRNA-treated mice and sham-treated mice.4.Immunohistochemical staining results showed that the expression of IDO1 and microvessel density(MVD)labeled by CD34 or CD146 both decreased significantly after IDO1 shRNA treatment.And IDO1 expression was positively correlated to both CD34+MVD(r2=0.8632)and CD146+MVD(r2=0.761).Conclusions:1.Using siRNA to knock down the IDO1 gene alters the biological characteristics of the LLC cell line including inhibit the abilities of tumor cells invasion,migration,and vasculogenic mimicry formation.2.IDO1-shRNA effectively inhibits tumor growth and angiogenesis in a murine lung cancer model.3.IDO1 expression is positively correlated with MVD marked by CD34 or CD146.IDO1 has the potential to participate in the formation of new capillaries.