Effects Of Interaction Between Rorβ And Nrip2on The Expression Of EMT-related Transcription Factors In Gastric Cancer

BackgroundGastric cancer is a common malignant tumor with morbidity ranking second and mortality ranking third among all malignant tumors in China. At present, the pathogenesis of gastric cancer has not been fully elucidated. It is generally accepted that gastric cancer originates from gastric epithelial stem cells and is a kind of stem cell disease. The convertion of normal stem cells into cancer stem cells which results from the accumulation of genetic abnormalities or loss of heterozygosity can promote tumor development. A small subset of cancer stem cells exists in gastric cancer, which shares the ability of self-renewal and multi-differentiation, and can promote invasion and metastasis. It is one of the main reasons for the poor prognosis. The mechanisms of invasion and metastasis involved by gastric cancer stem cells need to be further elucidated. To clarify the mechanism is significant for early warning and blocking gastric cancer cells invasion and metastasis. Epithelial mesenchymal transition (EMT) is a process that epithelial cells lose cell polarity and convert into mesenchymal-like cells under the influences of various factors, which enables epithelial cells to obtain the ability of invasion and migration. EMT is one of the most important reasons for the malignant biological behaviors because it can promote tumor cell invasion and migration, and leads to metastasis. It plays an important role in primary invasion and secondary metastasis in a variety of cancers including gastric, colon, liver, breast, ovarian and lung cancer. To explore the regulatory machanisms of EMT is significant for anti-metastatic targeting therapy. In the previous study, we have discovered a new molecule Nrip2by constructing retroviral cDNA libraries of colorectal cancer stem cells. Nrip2can promote the self-renewal of colorectal cancer stem cells. Rorβ was the target protein of Nrip2. Rorp was identified as a member of orphan nuclear receptor family and its expression was restricted to the central nervous system, in particular in regions involved in modulation of circadian rhythms such as suprachiasmatic nucleus, pineal gland, and retina. Recently, with the increased sensitivity of detection, Rorβ has been found outside the nervous system such as normal bone tissue, pancreatic and endometrial cancer, and uterine leiomyosarcoma. Previously we have found the expression of Rorp in normal intestinal epithelial cells and intestinal tumors using the methods of mRNA FISH and qPCR. Further, we have found that the interaction between Nrip2, Rorβ and HBP1could negatively regulate the self-renewal of colorectal cancer stem cells by attenuating the activity of the Wnt pathway. However the effects of Rorβ on gastric cancer have not been clarified.ObjectiveIn this study, we aim to explore the effects of Rorp on the malignant biological behaviors in gastric cancer and clarify the way how Rorβ and Nrip2regulate the expression of EMT-related transcriptional factors and to detect the correlation between Nrip2and Rorβ based on our previous achievements. Methods1. Detection of Rorp expression:We detected Rorβ expression in gastric cancer tissues by immunohistochemical staining and Taqman real-time qPCR and subsequently analyzed the correlation between Rorβ and the prognosis of gastric cancer.2. Gene set enrichment analysis of Rorβ:mRNA profiles from200gastric cancer tissues were divided into two groups, the Rorβ high expression group and low expression group. Differential expressed genes were found by Gene Set Enrichment Analysis.3. The impact of Rorβ on the Wnt pathway:Wnt pathway activity was determined by dual-luciferase reporter assays and western blot after transfection of plasmid Rorβ-pReceiver in SGC7901gastric cancer cell lines.4. Detection of EMT-related transcriptional factors expression:We observed the morphology of SGC7901cells which were infected lentiviral Nrip2and Rorβ by confocal microscopy, then we constructed Rorβ-pEGFP-C1recombinant plasmid and detected the expression of EMT-related transcriptional factors after transiently transfection of Rorβ-pEGFP-C1, Nrip2-pEGFP-C1and Nrip2T152A recombinant plasmids via western blot.5. Cell localization of Nrip2and Rorβ:SGC7901cells were transiently transfected with the Nrip2-pEGFP-C1and Rorp-pEGFP-C1plasmids and then localization of Nrip2and Rorβ were observed under confocal microscopy and their expression levels were detected by western blot.6. Interaction between Nrip2and Rorp:SGC7901cells were transiently transfected with RorP-2A-Peptide-Nrip2/pCMV plasmid and then the isolated nuclear and cytoplasmic components were detected by western blot. Results1. Lower expression of Rorp in gastric cancer tissues:Rorβ was downregulated in gastric cancer tissues compared with normal pyloric glandular tissues showed by immunohistochemistry and real-time PCR and Rorβ mRNA was also decreased in gastric cancer tissues compared with cancer adjacent tissues. However, we didn’t find the correlation between the expression of Rorβ and the gastric cancer TNM staging.2. Rorβ promoted cell differentiation:The gene set enrichment analysis of200gastric cancer tissues showed that CD44, ALDH1, ALCAM/CD166were all decreased in Rorβ high expression group compared with low expressiongroup.3. Rorβ inhibited Wnt pathway activity.4. Rorβ downregulated the expression of EMT-related transcriptional factors:The morphology of SGC7901cells have changed after the lentiviral infection of Nrip2. Overexpression of Rorβ downregulated the expression of EMT-related transcriptional factors Snail/Slug, while overexpression of wild-type Nrip2showed the opposite effect that Snail/Slug and Zeb1were upregulated. The effect of Nrip2on the EMT-related transcription factors was changed after its phosphorylation site (152) was mutated.5. Cellular localization:Cellular localization experiments showed that Nrip2mainly distributed in the cytoplasm while Rorp located in the nucleus.6. The interation between Nrip2and Rorβ:Rorβ was significantly "arrested" in the cytoplasm in cells co-overexpressed with Nrip2and Rorp.ConclusionRorp is downregulated in gastric cancer. Overexpressed Rorβ in gastric cancer cells can downregulate the Wnt pathway activity and inhibit the expression of EMT-related proteins. The interaction between Nrip2and Rorβ weakens the inhibitory effects of Rorβ on the expression of EMT-related proteins.