Objective:To evaluate the effect of regulation of zinc finger protein 281 (ZNF281) expression by miR-203 on the proliferation and migration of melanoma cell lines.Methods:The human vascular endothelial cell line ECV304, as well as human melanoma cell lines A375, M14, SK-MEL-28 and SK-MEL-2 were subjected to conventional culture. Real-time fluorescence-based quantitative PCR was performed to measure the miR-203 expression, and Western blot analysis to determine the ZNF281 protein expression in the above cell lines. Both A375 and M14 cells were divided into 5 groups: control group (normally cultured) , miR-203 mimic control group transfected with the miR-203 mimic negative control, miR-203 inhibitor control group transfected with the miR-203 inhibitor negative control, miR-203 mimic group transfected with a miR-203 mimic, miR-203 inhibitor group transfected with a miR-203 inhibitor. Real-time fluorescence-based quantitative PCR was conducted to measure intracellular miR-203 expression, cell counting kit (CCK) -8 assay to assess cellular proliferation activity, Transwell assay to determine the number of migratory cells, and Western blot analysis to measure the protein expression of ZNF281 in A375 and M14 cells in the above groups. Dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-203 and ZNF281. One-way analysis of variance was used for comparison of multiple groups, and the Student-Newman-Keuls- q (SNK- q) test for multiple comparisons. Results:Lower miR-203 expression and higher ZNF281 protein expression were observed in the melanoma cell lines A375, M14, SK-MEL-28, SK-MEL-2 compared with the vascular endothelial cell line ECV304 (all P < 0.05) . Compared with the control group, miR-203 mimic control group and miR-203 inhibitor control group, the miR-203 mimic group showed significantly increased miR-203 expression in A375 and M14 cells ( F = 487.632, 68.454, respectively, both P < 0.05) , significantly decreased number of migratory A375 and M14 cells (both P < 0.05) , and significantly decreased ZNF281 protein expression (both P < 0.05) , while the miR-203 inhibitor group showed significantly decreased miR-203 expression in A375 and M14 cells (both P < 0.05) , significantly increased number of migratory A375 and M14 cells (both P < 0.05) , significantly increased ZNF281 protein expression in A375 cells ( P < 0.05) , significantly increased proliferative activity of A375 cells at 36, 48, 60 and 72 hours (all P < 0.05) , and significantly increased proliferative activity of M14 cells at 24, 36, 48, 60 and 72 hours (all P < 0.05) . Dual-luciferase reporter assay showed interaction sites between miR-203 and ZNF281. Conclusion:Up-regulating the miR-203 expression can inhibit the proliferation and migration of melanoma cell lines, and vice versa, likely by regulating the expression of ZNF281.

Objective To explore the effect of glioma stem cells with high expression of protein tyrosin phosphatase receptor type Z1 (PTPRZ1 )on the phenotypic polarization and phagocytosis of tumor-associated macrophages and its regulatory mechanism.Methods GSCs and non-stem tumor cells (NSTCs) were screened out from human glioblastoma (GBM) specimens using flow cytometry,and the PTPRZ1 expression in paired GSCs and NSTCs were detected.Human peripheral blood mononuclear cells (PBMC)-derived CD14+monocytes were exposed to the conditioned medium from glioma cells or recombinant chemokine C-C motif ligand 20 (CCL20)for TAM polarization.Stable PTPRZ1 knockout GSCs (PTPRZ1-KO GSCs) were constructed using CRISPR/Cas9. TAM phagocytosis to GSCs,NSTCs,PTPRZ1-Control GSCs (PTPRZ1-Ctrl GSCs)and PTPRZ1-KO GSCs and the expression of immunosuppressive phenotype (M2) polarization marker CD163 were examined using flow cytometry.Differentially expressed genes (DEGs ) between paired GSCs and NSTCs were determined using a bulk RNA-sequencing dataset (GSE54791 )from Gene Expression Omnibus (GEO).A gene set informing worse outcome of patients with GBM was generated using The Cancer Genome Atlas (TCGA)-GBM cohort.By intersecting the aforementioned gene set with the gene set that encodes for human membrance proteins,the PTPRZ1 gene is obtained.Gene set enrichment analysis (GSEA)was used for pathway enrichment analysis to compare the differentially regulated pathways between GBMs with high or low PTPRZ1 expression.Bulk RNA sequencing,qRT-PCR and Western blotting were used to identify the DEGs between PTPRZ1-KO GSCs and PTPRZ1-Ctrl GSCs.Results GSCs were more capable of escaping from TAM phagocytosis than NSTCs (P<0.05 )and had specifically up-regulated PTPRZ1 expression.PTPRZ1-KO significantly suppressed GSCs escaping from TAM phagocytosis (P<0.01 ). GBMs with high PTPRZ1 expression showed significant inhibition of pathways mediating phagocytosis (P<0.05).The expression of CCL20 as a M2 TAM polarization chemokine was significantly down-regulated in PTPRZ1-KO GSCs (P<0.05 ).Treatment with recombinant CCL20 up-regulated the expression of CD163 as a M2 TAM marker in TAM.Conclusion PTPRZ1+GSCs mediate M2 TAM polarization and inhibit TAM phagocytosis,which may be related to the up-regulation of CCL20 in PTPRZ1+GSCs.