Glipr-2Promotes Ⅱ, Ⅲ Type EMT And Its Mechanism

Epithelial-mesenchymal transition (EMT) is considered as a process that epithelialcells lose epithelium characteristics and change to interstitial cells under somewhatphysiological and pathological conditions. During EMT, the differentiated epithelial cellslose their apical-basal polarity and epithelial adhesion (E-cadherin positive) and acquire amyofibroblast phenotype (α-smooth muscle actin and vimentin positive), which isaccompanied by enhanced cell migration and invasion. EMT is a process well known asthree types: In an early embryo, epithelial cells transform into mesenchymal cells and thistype of EMT has been classified as typeⅠ, which has no effect on organ fibrosis ormigration; EMT has been described to occur in a parenchymal organ (liver, lung, or kidney)when specific cells are lost in the course of a disease and are replaced by fibrotic tissue andthis type of EMT has been classified as typeⅡ, it was caused by chronic inflammation andleads to organ fibrosis; Another type of pathological EMT which has been described tooccur in carcinogenesis that the dedifferentiation of cells with loss of epithelial andacquisition of mesenchymal features and has been recently classified as type Ⅲ EMT.EMTis regulated by diverse genes and growth factors, such as HGF, EGF, TGF-β, which couldpromote EMT via their receptors.Thus, it is very import to further investigate themechanism of EMT for developing new strategy to treat correlative disease.Glioma pathogenesis related-2(GLIPR-2), whose aliases include GAPR-1andC9orf19, is a conserved mammalian protein belonging to the pathogenesis related-1(PR-1)family. GLIPR-2is mainly expressed in peripheral leukocytes and the lung in humanswhose molecular weight is17KD. It has been reported that GLIPR-2is correlated withEMT. Our studies showed that GLIPR-2was highly expressed in renal proximal tubularcells (PTCs) in the diabetic nephropathy (DN) and hepatocellular carcinoma (HCC) cellsspecifically. However, the mechanism of GLIPR-2-induced EMT remains to be clarified.ObjectiveTo detect the expression of GLIPR-2in DN and HCC tissues and investigate themechanism of GLIPR-2-induced EMT and relative signal pathway. Provide new insights into therapy of organ fibrosis and neoplasm metastasis.Methods1. GLIPR-2expression was detected by immunohistochemisty in DN and HCC tissues.DMEM low glucose medium with5,10,20,30mM glucose was applied to raise HK-2cellsfor7days, HepG2cells were raised in hypoxia conditions for72h. Western Blotting andreal-time RT-PCR were applied to detect the expression of GLIPR-2.2. HK-2cells with GLIPR-2stable transfection were constructed. SABiosciences RT2Profiler PCR Array of Human Epithelial to Mesenchymal Transition (PAHS-090A) wasapplied to detect the target genes which were identified by Western Blotting and qRT-PCR.3. HepG2cells with GLIPR-2transient transfection were constructed following bydetection of EMT markers via immunofluorescence, real-time RT-PCR and WesternBlotting. The signal pathway was detected by Western Blotting.4. Transwell assay was applied to examine the migration and invasion ability.5. GLIPR-2expression was suppressed by gene silencing under hypoxia conditions,the EMT markers and migration and invasion ability were detected as mentioned above.Results1. GLIPR-2was abundantly and specifically expressed in the renal tubular epithelialcells of DN kidney and HCC tissue samples, which contain the typical progressive renaltubulointerstitial fibrosis characteristic of this disease. Furthermore, we also found that theEMT marker E-cadherin decreased, whereas the vimentin and α-SMA EMT markers wereincreased in the same regions. High glucose could promote GLIPR-2expression in HK-2cells; hypoxia could promote GLIPR-2expression in HepG2cells.2. After EMT array analysis, EMT markers and target genes were screen which wasidentified by qRT-PCR and Western Blotting. The results showed that GLIPR-2overexpression in HK-2cells promotes cell EMT.3. The migration and invasion ability were increased after GLIPR-2transfection.4. Based on the EMT PCR Array analysis, we found that EGFR expression wasup-regulated after GLIPR-2overexpression. Our study indicates that p-ERK-1/2expressionis enhanced in PTCs from patients with DN consistent with the GLIPR-2transfection. Wefound that PD98059, a specific ERK1/2inhibitor, inhibited the GLIPR-2-initiatedactivation of ERK1/2signaling, EMT markers and cell migration in a dose-dependent manner.5. The suppression of GLIPR-2expression attenuates the hypoxia-induced EMT-likeprocess and cell migration and invasion.Conelusion1. GLIPR-2expression was increased in the renal tubular epithelial cells of DN kidneytissues and high glucose could promote GLIPR-2expression in HK-2cells.2. EMT array and Western Blotting analysis showed that GLIPR-2overexpression inHK-2cells promotes cell EMT and migration through ERK1/2activation.3. GLIPR-2expression was locally increased in HCC tissues and hypoxia couldpromote GLIPR-2expression in HepG2cells.4. GLIPR-2overexpression in HepG2cells promotes cell EMT and migration throughERK1/2activation also.5. Inhibition of GLIPR-2expression in hypoxia condition could suppress EMT processand following cell migration and invasion. GLIPR-2could be a new target to preventneoplasm metastasis.In a word, we are the first to find that GLIPR-2could promote cell EMT and migrationthrough ERK1/2activation in HK-2and HepG2cells. The suppression of GLIPR-2expression attenuates EMT process and GLIPR-2may be a new target to therapy of organfibrosis and neoplasm metastasis.