Bioinformatics Analysis Of The Regulatory Mechanism Of Metastatic Melanoma And The Effect Of CLDN4 On The Biological Behavior Of Melanoma

Objective To find the core gene of metastatic malignant melanoma and predict its transcriptional regulatory factors by integrating and analyzing the sequence data of human malignant melanoma chip in TCGA database with bio-informatics methods.At the same time,we constructed a ce RNA regulatory network based on lnc RNA-micro RNA-RNA.Methods The chip data of 471 melanoma tissues in TCGA database were downloaded and analyzed by R Language Pack 3.4 software.The core genes of malignant melanoma were screened by weighted gene co-expression network analysis,and the protein-protein interaction network was constructed.Furthermore,we use bio-informatics website to predict the transcription factors regulating core genes,and construct a ce RNA regulatory network based on lnc RNA-micro RNA-RNA using multiple Bio-informatics databases.Results A total of 24 important core genes were found to be involved in the process of immune response and tumorigenesis of metastatic malignant melanoma(including RASGRP2,IKZF1,CXCR5,LTB,BLK,LINGO 3,CCR6,P2RY10,RHOH,JUP,KRT14,PLA2G3,SPRR1 A,KRT78,SFN,CLDN4,IL1 RN,PKP3,CBLC,KRT16,EM79,KLKK8,LYPD5).It is predicted that GATA1,STAT1,SP1 and PSG1 may be involved in transcription factors regulation of core genes.The ce RNA regulatory network was constructed for 25 mi RNAs,18 lnc RNAs and 18 m RNAs screened.Non-coding RNA and RNA survival analysis showed that there was a significant correlation between the abnormal expression of 5 mi RNAs,6 lnc RNAs and 7 m RNAs and the survival time of melanoma patients.Conclusion The core genes involved in the pathogenesis of metastatic melanoma were identified and predicted by bio-informatics,and the transcription factors and ce RNA regulatory network regulating the core genes were predicted.These findings provide useful research ideas for us to further understand the metastasis mechanism and pathogenesis of malignant melanoma.Objective To investigate the expression difference of CLDN4 gene and its gene expression products in metastatic melanoma and in situ melanoma,and to analyze the effect of overexpression and knockout of CLDN4 gene on the biological behavior of human malignant melanoma cells.Methods The expression of CLDN4 gene in clinical melanoma samples was semi-quantitatively determined by RT-q PCR,Western blotting and immunofluorescence.The effects of CLDN4 gene expression on cell cycle,apoptosis,invasiveness and proliferation of human melanoma cell lines A375-S2 and SK-MEL-2 were observed by using lentivirus packaging overexpression and CRISPR gene knockout techniques.To explore whether CLDN4 knockout and overexpression are specific through PI3K/Akt signaling pathway.Results The expression of CLDN4 gene and its protein in melanoma metastasis tissue was significantly lower than that in situ tissue.Immunofluorescence and Western blotting showed that CLDN4 overexpression and knockout stable cell lines were successfully constructed.Experiments confirmed that overexpression or knockout of CLDN4 gene had no significant effect on the proliferation,cell cycle and apoptotic process of melanoma cells.However,after CLDN4 knockout,the expression of EMT-related markers was significantly up-regulated,which could significantly enhance its EMT ability.On the contrary,over-expression of CLDN4 significantly reduced the expression of EMT-related markers,which could significantly reduce its EMT ability.At the same time,PI3K/Akt signaling pathway related marker protein was significantly up-regulated in CLDN4 knockout cell lines.In CLDN4 overexpression cell lines,it was significantly down-regulated.Blocker3-Methyladenine can reverse the EMT process.Conclusion CLDN4 gene is low expressed in metastatic melanoma.CLDN4 gene may participate in the mesenchymal process of malignant melanoma cells and play a role through PI3K/Akt signaling pathway.