The Study On The Role Of IncRNA HOTAIR、MDA9and GRP78in Invasion And Metastasis Of Melanoma

Malignant melanoma is the most lethal skin cancer among all cutaneousdiseases. Major obstacles in treatment to improve patient survival arethe invasion and metastasis of the cancer. The mechanism that controlsmelanoma invasion and metastasis is not completely understood. It istherefore necessary to illustrate the regulators of melanoma invasion andmetastasis, in order to develop new methods for the diagnosis andtreatment of malignant melanoma.Purposes:1. Identify the lncRNA that correlates with malignancy of melanoma.Study the mechanism by which lncRNA affects the melanoma metastasis.2. Screen regulators of melanoma invasion and metastasis by comparingprimary and metastasis tissue of melanoma. Identify the mechanism by whichthese markers regulate the malignant progression of melanoma.Methods:1. Harvest total RNA from from primary and metastatic melanoma. Test theexpression levels of metastasis-related lncRNAs in these tissues.2. Harvest whole tissue lysate from primary and metastatic melanoma.Screen for metastasis-related targets by2-D gel electrophoresis. Verifythe results by Q-RT-PCR.3. Clone and verify the expression of the genes that are differentiallyexpressed in primary and metastatic melanoma.4. Design and test the efficacy of siRNAs against the lncRNA and identifiedgenes.5. Test the role of potential metastasis regulators by transfecting thecDNAs or siRNAs into A375cells, followed by wound-healing and matrixinvasion test.6. Down-regulate the expression of lncRNA and test the secretion of matrix metalloproteinases in A375cells by ELISA.7. Test the interaction between potential metastasis-regulators byco-immunoprecipitation.8. Predict the possible interaction mechanism between potentialmetastasis-regulators and test the important of such interaction formelanoma metastasis.Results:1. Expression levels of lncRNA HOTAIR correlates with metastaticprogression of melanoma. HOTAIR promotes the metastasis of melanoma bystimulating the migration and basement-invasion of melanoma cells.2. Expression levels of MDA9and GRP78correlate with metastasis ofmelanoma. As confirmed by wound-healing assay and Matrigel invasion test,MDA9and GRP78promote the invasion and metastasis of malignant melanoma.It is yet to be examined if MDA9and GRP78interact with each other. Themechanism by which MDA9and GRP78affect melanoma metastasis remains tobe elucidated.Discussion：GRP78and MDA9are up-regulated in metastatic melanoma and contribute toits invasion and metastasis. Further study is required to test if thesetwo proteins interact with each other, and to find out the mechanism bywhich these two proteins promote melanoma metastasis. Elucidating thefunction and mechanism of these two metastasis-related markers shallprovide more insight in designing new methods for the diagnosis,prevention and therapeutic intervention of melanoma in human patients.The role for lncRNA HOTAIR in promoting the invasion and metastasis ofmelanoma is also confirmed. HOTAIR promotes the migration andbasement-invasion of melanoma cells, and may represent a potential targetfor therapeutic intervention against malignant melanoma in humans.