High Glucose Or Hypoxia Promotes EMTs Of Renal Tubular Epithelial Cells Or Human Hepatocellular Cell Via Inducing GLIPR-2Expression

Objectives:EMT is a process that Epithelial cells transform into mesenchymal phenotype cellsunder certain conditions. Research has confirmed that the EMTs play an important roles inrenal fibrosis and the process of tumor metastasis. Previous studys have shown that EMTand the high expression of GLIPR gene simultaneously discovered in mice renal fibrosistissue. Follow-up study has also found that the high expression of GLIPR-2in humandiabetic kidney renal fibrosis and primary hepatocellular carcinoma tissue samples, but itsmechanism is unclear. To explore the the role of GLIPR-2in the process of EMT of humanrenal tubular epithelial and hepatocellular cells, we detect the expression of GLIPR-2anddiscuss the relationship between the gene and EMT renal tubular epithelial cells under thehigh glucose condition, and detect the change of the migration and invasive ability viainducing GLIPR-2expression under the hypoxia conditions in hepatocellular cells.So as tofind out the mechanism of GLIPR-2in the occurrence of diabetic nephropathy andmetastasis of primary hepatocellular carcinoma. Further enrich the understanding for theoccurrence of diabetic nephropathy and metastasis of primary hepatocellular carcinoma.Methods:1. Acording to siRNA targets for GLIPR-2gene and negative control siRNA targets, todesign and synthesis double-stranded oligonucleotide, then connect to lentiviral vectorpMAGic7.1. To identify the correctness of pMAGic7.1-shRNA-GLIPR-2plasmid by PCRamplification, gel electrophoresis and DNA sequencing.2. Lentiviral recombinant plasmid, packaging plasmid and membrane proteinexpression plasmid transfect293T cells to get lentiviral coarse particle, then to obtain highdegree of purification of lentiviral particles by collection and concentration.3. To establish a stable expression of shRNA-GLIPR-2experimental group and control group in HK-2cells and HepG2cells by infection of lentivira. Then using flow separationtechnology to screen out tag fluorescence GFP positive cells.4. Under the condition of high glucose or hypoxia, using Western Blotting techniqueto detect the expression level of GLIPR-2, EMT related markers, and the correspondingsignal pathway in HK-2cells and HepG2cells. Using Tanswell migration and invasionexperiment testing to detect the change of cell migration and invasion ability.5. Under the induction condition, to detect the expression level of GLIPR-2, EMTrelated markers, the corresponding signal pathway and cell migration and invasion ability inexperimental group with stable expression of shRNA-GLIPR-2experimental group cellsand the control group cells.Results:1. The result of Gel electrophoresis and DNA sequencing show that double-strandedoligonucleotide targeting GLIPR-2siRNA and the negative control siRNA targets haveconnected to lentiviral vector pMAGic7.1successfully. And it is able to stable expression incells.2. When cultivate HK-2cells with the condition of high glucose, GLIPR-2expressionlevel will increase, EMT related biomarkers show that the epithelial markers E-caherinexpression decreased significantly, mesenchymal cells markers of alpha SMA and Vimentinexpression increased significantly, p-ERK1/2expression also increased significantly andcell number increased in the migration experiment. After the application of ERK1/2inhibitor PD98059, the expression of α-SMA and of Vim can be obviously inhibited andthe expression of E-ca can increase. At the same time, cell number decreased in themigration experiment. Indicate that high glucose can promote the expression of GLIPR-2,at the same time activate ERK1/2signaling pathways to promote the occurrence of EMT,enhance the capacity of cell migration.3. Using RNA interference, even though the HK-2cells in the condition of highglucose, the expression GLIPR-2is inhibited significantly. When the expression ofGLIPR-2is suppressed, EMT markers have obvious reverse changes, including E-caincreased expression, α-SMA and Vim reduced expression. The expression level ofp-ERK1/2also decrease significantly. cell number decreased in the migration and invasionexperiment. Show that under the condition of high glucose, interference the expressionGLIPR-2can inhibit ERK1/2signaling pathways to inhibit the occurrence of EMT, thus todecreased cell migration ability. 4. When cultivate HepG2cells under hypoxia condition, GLIPR-2expression levelwill increase, E-ca expression decreased significantly, α-SMA and Vim expressionincreased significantly, p-ERK1/2expression also increased significantly and cell numberincreased in the migration and invasion experiment. After the application of ERK1/2inhibitor PD98059, the expression of α-SMA and of Vim can be obviously inhibited andthe expression of E-ca can increase. At the same time, cell number decreased in themigration and invasion experiment. Indicate that hypoxia can promote the expression ofGLIPR-2, at the same time activate ERK1/2signaling pathways to promote the occurrenceof EMT, enhance the capacity of cell migration and invasion.5. Using RNA interference, even though the HepG2cells in the condition of hypoxia,the expression GLIPR-2is inhibited significantly. When the expression of GLIPR-2issuppressed, EMT markers have obvious reverse changes, including E-ca increasedexpression, α-SMA and Vim reduced expression. The expression level of p-ERK1/2alsodecrease significantly. cell number decreased in the migration and invasion experiment.Show that under the condition of hypoxia, interference the expression GLIPR-2can inhibitERK1/2signaling pathways to inhibit the occurrence of EMT, thus to decreased cellmigration and invasion ability.Conclusions:1. Successfully construct RNAi lentiviral vectors targeting human GLIPR-2gene andestablish a stable expression of pMAGic7.1-shRNA-GLIPR-2and pMAGic7.1-NCrecombinant plasmid in HK-2cells and HepG2cells.2. The results show thatGLIPR-2can regulate the process of EMT induced by highglucose in HK-2cells by activating p-ERK1/2signaling pathways, thus promote cellmigration ability.3. GLIPR-2can regulate the process of EMT induced by hypoxia in HepG2cellsthrough the activation of p-ERK1/2signaling pathways, thus promote cell migration andinvasion ability.In conclusion, through independently construct RNAi lentiviral vectorstargeting human GLIPR-2gene, we reveal the relation between the expression of GLIPR-2and the process of EMT under the condition of high glucose or hypoxia, To provide a newtrain of thought for the treatment of fibrosis and cancer metastasis related diseases.