The Establishment Of Choroidal Melanoma Cells Overexpressing KCTD12 And Investigation Of The Gene Biological Function

Human potassium channel tetramerization domain containing 12(KCTD12,also known as Pfetin)is a member of the KCTD family which consists of 26 members.It has been reported that KCTD12 regulates agonist potency and kinetics of GABAB receptor signaling.Proteomic analysis indicates that KCTD12 may be a potential biomarker for the diagnosis and prognosis of gastrointestinal stromal tumors.However,little has been reported concerning the role of KCTD12 in the other tumor types.So-called uveal melanoma usually refers to melanomas of the choroid,ciliary body and iris of the eye.In fact,choroidal and ocular melanomas are the alternative terms of this type of cancer.Uveal melanoma is the most common primary intraocular malignancy in adults.In the US,the incident rate of these cancers is ~5%,85% of which are uveal in origin.Patients with uveal melanoma experience painless loss or distortion of vision in the early stage,which even leads to retinal detachment at advanced stage.In addition,uveal melanoma is highly metastatic;patients with uveal melanoma are at risk for metastatic disease to the liver,lung and skin.Therefore,uveal melanoma is associated with high mortality in up to half of uveal-affected patients.Although the tumorigenesis and the metastasis of uveal melanoma are complex processes.However,to date,there have been no studies on the effects of KCTD12 on uveal melanomasIn the present study,we designed and subcloned full length and partial of KCTD12 genes.(OCM-1),and the preliminary results of overexpression of KCTD12 cells were obtained.It was found that overexpression of KCTD12 could upregulate the expression of tumor stability marker protein E-cadherin,meanwhile the Epithelial Mesenchymal Transition(EMT)marker Vimitin was downregulated.we designed and subcloned N-terminally Flag-tagged human KCTD12 into the p LVX Puro vector.We then generated a human uveal melanoma cell line(OCM-1)stably expressingKCTD12.We send the RNA sample from the new cell line to do the RNA sequencing detection and get the data for bioinformatics analysis.The changes of gene expression level were mainly focus on proliferation,cell adhesion,apoptosis,angiogenesis and so on.According to bioinformatics results.Using this stable cell line,we performed a series of experiments.Through the MTS assay,plate cloning and soft agar assay prove,the results showed that KCTD12 could inhibit the proliferation of OCM-1 cells.The scratch test,transwell invasion and migration and the expression of related proteins were tested.The expression of E-cadherin and ?-Catenin were up-regulated.The expression of Vimentin was decreased,and Snail and slug as a regulator of E-cadherin,the expression level were decreased.The results showed that overexpression of KCTD12 can limit the invasion and migration of OCM-1 cells.The cell cycle and apoptosis were detected by flow cytometry,and the cell cycle related proteins were detected.Protein expression showed a significant decrease in cyclin B expression.Indicate the prolongation of the progression of G2/M to G1 phase in the KCTD12-overexpressing OCM-1 cells.In addition,inhibition of KCTD12-overexpressing OCM-1 cell xenograft growth in nude mice was observed.The full length and part of the KCTD12 gene were ligated into the prokaryotic expression plasmid PGEX-4T vector.We obtained and purified the protein of KCTD12 full length and partial fragment with GST and HIS tag.The biological effects to OCM-1 cells were verified by full length protein.And mouse-derived antibodies were prepared by immunizing mice with partial fragment proteins.Taken together,KCTD12 may serve as a novel therapeutic target for patients with uveal melanoma.