Hypoxia And TGF-?1 Induced PLOD2 Expression Improve The Metastasis Of Cervical Cancer Cells By Promoting Epithelial-to-Mesenchymal Transition(EMT)and Focal Adhesion Formation

Background Intra-tumoral hypoxia and increases in extracellular level of Transforming Growth Factor ?1(TGF-?1),which are common findings in cancer,are associated with an increased risk of metastasis and mortality.Moreover,metastasis is the leading cause of death of patients with cervical cancer.PLOD2 is an intracellular enzyme required for the biogenesis of collagen and its expression can be induced by hypoxia and TGF-?1.Accordingly,PLOD2 is up-regulated in several types of cancer,including cervical cancer,and has been associated with cancer metastasis.Thus,we aimed to investigate the role of PLOD2 in the motility of cervical cancer cell lines and to show the molecular mechanism underlying this effect.Objective and meaning This research aimed at investigating the effect of PLOD2 on the migration and invasive ability of cervical cancer cells and the involved molecular mechanism in vitro and demonstrating that it mediates the promotion of metastasis in vivo,and then providing new insights into the mechanism of cervical cancer metastasis and a new potential prognosis indicator and treatment target.Methods siRNA was used to knockdown genes of interest in the cervical cancer cell lines HeLa and SiHa to investigate the effect of PLOD2 in vitro.The ability of these cells to migrate and invade,their adhesion to type I collagen,and their capacity for Epithelial-to-Mesenchymal Transition(EMT)and focal adhesion formation were analyzed.Gene expression changes were validated by qRT-PCR,Western blotting and immunocytochemistry.The morphological status of cells was examined using Phalloidin staining.Differences in PLOD2 expression among patients with cervical cancer were identified by referring to public databases,including Oncomine and TCGA.sh-RNA expression vectors targeting PLOD2 packaged by Lentiviruses were used to stably knockdown the expression of PLOD2 in Hela cells.Tail vein cell injection assay was conducted in nude mice to observe the effect of PLOD2 on cervical cancer metastasis in vivo.Results Hypoxia and TGF-?1 enhanced the expression of PLOD2 in HeLa and SiHa cells,and knockdown of PLOD2 inhibited cell motility and EMT.Moreover,the depletion of PLOD2 attenuated hypoxia-mediated cell migration and invasion and inhibited TGF-?1-induced phenotypic EMT-like changes by preventing p-catenin from entering the nucleus.In addition,PLOD2 depletion decreased cell adhesion to extracellular collagen by inhibiting the formation of focal adhesions.Moreover,a database analysis showed that PLOD2 expression is associated with human cervical cancer progression.At last,animal experiments on nude mice demonstrated that the stable knockdown of PLOD2 by Lentiviruses packaged sh-RNA inhibites the metastasis of cervical cancer in vivo.Conclusions Overall,our results indicated that hypoxia-and TGF-?1-induced PLOD2 expression promotes the migratory,invasive and adhesive capacities of cervical cancer cells in vitro by participating in TGF-?1 induced EMT and the formation of focal adhesions.Moreover,the knockdown of PLOD2 inhibits the metastasis of cervical cancer cells in vivo.