RNAi Targeting Layilin Inhibits Proliferation, Invasion And Metastasis Via Lymphatic Vessels In A549 Lung Carcinoma Cell Line And Its Xenografts In Nude Mice

Background and objectiveLymphatic metastasis is an important early event occurred in human lung carcinoma. Hyaluronan(HA), a kind of polysaccharids with high molecular weight, was found to be involved in the process of proliferation, adhesion, migration and invasion of various tumor cells including human lung carcinoma cell, morever, as a member existed in mesenchymal of many kinds of tumors including human lung carcinoma, and degraded in lymphatic system, one of its specific receptors, Layilin, was found to express on human lung carcinoma A549 cell line in our previous research. Therefore, we doubt whether Layilin activated by HA might promote tumor metastasis via lymphatic vessels. In the present study, we further examined whether inhibition of Layilin by gene silencing using mammalian expression vectors expressing small interfering RNA (siRNA) might lead to inhibition of Layilin expression, primary tumor growth, experimental metastasis, and increase of host survival using a murine lung carcinoma model.MethodsRNAi vector that can express siRNA targeting Layilin(Layilin siRNA Vector)or siRNA that does not match any known human coding mRNA (Control siRNA Vector)was designed, constructed, and lipofected into A549 cells line. A549 cells carrying layilin siRNA Vector(A549-siLayilin) or Control siRNA Vector (A549-siControl) were selected with 500μg/mL G418-sulfate. Stably transfected A549-siLayilin and A549-siControl cells were obtained and routinely maintained in selection media containing 250μg/mL of G418-sulfate to avoid overgrowth of nontransfected cells. Expressions of Layilin mRNA and protein were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR), Real time RT-PCR, Western blot, and immunofluorescence stain in A549-nontransfection, A549-siLayilin and A549-siControl cells respectively. With or without exogenous HA existence, ability of proliferation, adhesion, migration and invasion...