| Objective To clarify the expression level and role of miR-155 in Kaposi’s sarcoma(KS),and to analyze and verify the molecular mechanism of miR-155 affecting KS by targeting GATA3,so as to provide possible theoretical basis for targeted therapy and prognosis evaluation of tumors.Methods First,Real time PCR and Western blot were used to detect the expression of GATA3 m RNA and protein in KSHV infected and uninfected cells;secondly,the effects of GATA3 on the migration and invasion of islk-219 and islk-bac cells were detected by wound healing assay and Transwell assay;in addition,Western blot was used to detect the expression of GATA3 Effects of GATA3 on the expression of E-cadherin,ZO-1 and N-cadherin and slug in EMT.Through bioinformatics methods(micro RNA,targetscan,mirwalk database),we predicted and verified a number of candidate miRNAs that may target GATA3,and selected miR-155 as the research object.The expression of miR-155 in KSHV infected cell lines and uninfected control cell lines was detected by real-time fluorescent quantitative PCR;the serum of KS patients with pathological diagnosis in Xinjiang was collected,and the real-time fluorescent quantitative PCR experiment was used to verify whether miR-155 was differentially expressed in KS patients and non KS patients.In KSHV infected cells,miR-155 mimic and miR-155 inhibitor were transfected respectively,and the changes of GATA3 m RNA and protein expression levels after miR-155 expression was enhanced or decreased were verified by realtime quantitative PCR;CCK-8 experiment and clone experiment were used to detect the changes of cell proliferation ability;wound healing and Transwell assay were used to detect the changes of GATA3 m RNA and protein expression The effects of miR-155 on the migration and invasion of KSHV infected cells were detected,and the expressions of ZO-1,E-cadherin,slug and N-cadherin were detected by real-time PCR and Western blot.GATA3 3’UTR with miR-155 binding site was cloned into reporter gene vector to construct GATA3 wild-type and mutated luciferase reporter gene plasmids.Control plasmid,wild-type plasmid,mutated plasmid and miR-155 mimic were cotransfected into 293 T cells of human embryonic kidney respectively,and GATA3 luciferase activity was detected after cleavage.The above experiment was repeated after preparing KSHV virus and infecting 293 T cells.it was verified that miR-155 could still bind specifically to GATA3 3’UTR region after KSHV infection.The rescue experiment proves that miR-155 can inhibit its transcription activity by targeted regulation of GATA3,thus promoting cell proliferation,migration and invasion.The reporter gene plasmid of miR-155 promoter region was constructed,doxycycline was used to induce i SLK-219 cells to express the cleavage stage switch protein RTA encoded by KSHV,and RTA interference plasmid was transfected after inducing RTA expression.double luciferase reporter gene experiment verified the effect of RTA on transcription activity of miR-155 promoter region.Results 1.The results of real-time PCR and Western blot showed that the expression level of GATA3 in KSHV infected cells was lower than that in KSHV uninfected cells(P < 0.05).2.The results of cell scratch and Transwell test showed that overexpression of GATA3 could inhibit the migration and invasion of KSHV infected cells.The results of Western Blot showed that overexpression of GATA3 could increase the protein expression of epithelial markers Ecadherin and ZO-1,and inhibit the expression of N-cadherin and Slug(P < 0.05).3.Real time PCR results showed that the expression level of miR-155 in KSHV infected cells was significantly higher than that in KSHV uninfected cells,and the expression level of miR-155 in KSHV positive serum was also significantly higher than that in KSHV negative serum(P < 0.05).4.The results of CCK-8 assay,plate cloning assay,scratch assay and Transwell assay showed that miR-155 mimics could promote the proliferation,migration and invasion of KSHV infected cells,while miR-155 inhibitor could weaken the proliferation,migration and invasion of KSHV infected cells,suggesting that miR-155 can promote the proliferation,migration and invasion of KSHV infected cells Proliferation,migration and invasion of infected cells.5.The results of real-time PCR and Western blot showed that the expression of GATA3 was down regulated after transfection of miR-155 mimic in KSHV infected cell lines,while the expression of GATA3 was up-regulated after transfection of miR-155 inhibitor,indicating that miR-155 can negatively regulate the expression of GATA3.6.The results of double luciferase reporter gene experiment showed that after constructing the plasmid containing the 3’UTR region of GATA3 gene,the luciferase activity of GATA3 was obviously decreased by co-transfection of the plasmid with miR-155 mimic,which suggested that miR-155 could inhibit the transcription activity of GATA3.The experiment was repeated after infection with KSHV virus in 293 T cells,and the transcriptional regulation of miR-155 on GATA3 3’UTR region was verified again.7.Real time PCR and Western blot results showed that overexpression of miR-155 could inhibit the expression of E-cadherin and ZO-1,and promote the expression of N-cadherin and slug,while inhibition of miR-155 had the opposite effect(P < 0.05).8.After doxycycline induced successful expression of RTA in i SLK-219 cells,the results of double luciferase reporter gene experiment showed that RTA could enhance the activity of miR-155 promoter region,while the activity of miR-155 promoter region decreased after interfering with the expression of RTA,which indicated that RTA could promote the transcription activity of miR-155 promoter region.Conclusion 1.GATA3 was low expressed in KSHV infected cells and inhibited cell migration,invasion and EMT.2.miR-155 is highly expressed in serum and cells of Kaposi’s sarcoma and KSHV infection,and can promote the proliferation,migration and invasion of KSHV infected cells.3.miR-155 can promote the proliferation,migration and invasion of KSHV infected cells by targeting GATA3,and the cleavage stage protein RTA encoded by KSHV can promote the transcription activity of miR-155 promoter region. |
