| Background Kaposi’s sarcoma (KS) is a multicentric angioproliferative tumour of mesenchymal origin. The disease was first described in 1872 by Moritz Kaposi, and it mostly affects elderly men of Italian, Jewish, or Mediterranean origin. In China, more than 90% of KS cases occur in Xinjiang, which is a multiethnic gathering place. The pathogenesis of Kaposi’s sarcoma is not clear at present, many studies have clarified human herpes virus 8(HHV-8) is the most important pathogens Kaposi’s sarcoma. It has been reported that there was a high seroprevalence of HHV-8 in Xinjiang, but HHV-8 is not the only cause of KS. Owing to the lack of genomics research, the pathogenesis of KS in this population is unclear. The particular geography, environment and ethnic composition may have led to the specific characteristics of KS in Xinjiang. MicroRNAs (miRNAs) are small (19-25 nucleotides; NT), single stranded non-protein-coding RNAs that play a role in many biological processes. MiRNAs regulate target mRNA expression by binding to complementary sequences of their target mRNA genes in the 3’-untranslated regions. The functions of miRNAs include signal transduction, cell differentiation, cell proliferation and apoptosis. The role of miRNAs in cancer is important, and they include oncogenic/tumor suppressor miRNAs and those involved in regulation of proliferation, angiogenesis, etc. MiRNAs generally have specific expression profiles in cancer, and therefore miRNAs can serve as markers for diagnosis ind the response to therapy.Objective (1) We performed a miRNA microarray analysis by detecting six paired KS and matched adjacent healthy tissues using the 7th generation of miRCURYTM LNA Array (v.18.0) (Exiqon).(2) Through a series of experiment of cell biology (cell proliferation, cell migration, invasion, apoptosis and cell cycle) in vitro, we observed the biological traits of SLK cell lines after transfection miR-126-3p mimic or inhibitor.(3) Combined with three miRNA prediction software, we found that miR-126-3p can be combined with PIK3R2 3’UTR. It assumed that PIK3R2 was likely target genes of miR-126-3p.The part validated that PIK3R2 was target gene regulated by miR-126-3p.MethodsA The expression profiles of microRNAs in Kaposi’s sarcoma(1) Diseased tissue samples from patients with KS and matched adjacent healthy tissues were obtained from surgical specimens immediately after resection, from January 2012 to September 2013 in the Department of Dermatology. Total RNA (including miRNAs) was isolated using TRIzol. After isolation of RNA from the samples, the miRCURYTM Hy3TM/Hy5TM Power labeling kit was used according to the manufacturer’s guidelines for miRNA labelling.After washing, scanning, and data analysis, we analysis the expression profiles of microRNAs. We selected part significant differentially expressed miRNAs, which were confirmed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in 18 paired KS and matched adjacent healthy tissue specimens. We also investigated the associations between clinical features (the stage, HIV, HHV-8 and the lesions area) and miRNA expression.B The function research of miR-126-3p in SLK cell lines(1) Optimization of transfection conditions:the optimal transfection concentration and time were determined by transfection with different concentrations of positive control followed by MTT assay.(2) Cell proliferation activitys determined by MTT:We compared each cell proliferation determined by optical density value (OD value) on the enzyme-linked immune detector atter 48 h transfection miR-126-3p mimic, inhibitor, negative control, inhibitor negative control of SLK.(3) After 24h transtection miR-126-3p minic or inhibitor, cell migration and invasion was determined by transwell assay followed by hematoxylin staining.(4) We detected the cell apoptosis and cell cycle after 48 h transfection miR-126-3p mimic, inhibitor of SLK by flow cytometry.C The mechanism research of PIK3R2 regulated by miR-126-3p(1) The target genes of miR-126-3p was predicted by biology software.(2) We detected the expession level of PIK3R2 after 48h transfection miR-126-3p mimic, inhibitor, negative control using the qRT-PCR detection(2-ΔΔCT).(3) Using Western Blot method to detect PIK3R2 protein expression changes in SLK after transfection the miR-126-3p mimic and inhibitor.(4) We synthetic containing target 3’end in miR-126-3p loci, and then cloning the segment in the luciferase report gene carrier pmiRGIO. We detected the luciferase activity of 293T cells with together transfection of PIK3R2 mutant and wild type carrier and miR-126-3p mimic and inhibitor by double luciferase report gene.ResultsA The expression profiles of microRNAs in Kaposi’s sarcoma(1)A11 patients were Uyghur nationality. The male-to-female ratio was 17:1 (17/1). The mean age was 60.1±14.1 years. The duration of disease was 24.2±33.8 months. In the 11 cases of CKS, the viral (HHV-8) prevalence was 81.82%(9/11). Among the seven cases of AIDS-KS, six patients were infected with HHV-8 (85.71%).(2) Among the 3100 human miRNA probes in the microarrays, we identified 170 differentially expressed miRNAs (69 upregulated and 101 downregulated miRNAs) in KS versus normal skin adjacent to the tumors. The most significantly upregulated miRNAs were miR-126-3p, miR-199a-3p, miR-16-5p, and 13 upregulated KSHV-related miRNAs. Among the most significantly downregulated miRNAs were miR-125b-1-3p and miR-1183. To validate the results of the microarray data, we first examined the expression of ten disregulated miRNAs by qRT-PCR in the 18 paired KS tissues used for the microarray. It also showed the consistency of the results between qRT-PCR and microarray.(3) There was no statistically significant difference in expression of miR-125b-1-3p and miR-16-5p in association with the clinical data, except for HHV-8 and HIV infections.B The function research of miR-126-3p in SLK cell lines(1)MTT results:Compared with NC group, miR-126-3p mimic can inhibit cell proliferation, which inhibition rate was 19.62%+1.68 (P<0.05). Inhibition expression of miR-126-3p in SLK cells, it promoted cell proliferation (12.42%+1.79) (P<0.05). It showed that over expression of miR-126-3p significantly inhibited the proliferation of SLK cells (P<0.01).(2) We detected the cell migration and invasion ability of SLK after transfection miR-126-3p mimic, inhibitor, negative control group, negative control by using transwell test.The results showed that overexpression of miR-126-3p reduced migration and invasion of SLK cell and low expression of miR-126-3p can increase migration and invasion of SLK cell (P<0.05). It suggested that miR-126-3p may have inhibited migration and invasion ability of SLK cell.(3) We detected the cell cycle of miR-126-3p on SLK by flow cytometry. The proportion of G2/M phase[(30.75±0.64)%] in miR-126-3p mimic transfection group was significantly higher than that of the negative control group of[(4.70±0.99)%] (P=0.00).The proportion of S phase[(10.8±1.49)%] in miR-126-3p mimic transfection group significantly lower than the negative control group[(40.80±0.99)%] (P=0.00). the proportion of S phase[(52.50±2.69)%] in miR-126-3p inhibitor transfection group was significantly higher than that of negative and inhibitor negative control group [(40.10±1.27)%](P<0.05). It indicated that miR-126-3p can be arrested at G2/M phase in SLK cells and hindered the cells into the secretory phase.(4) We studied the cell apoptosis of miR-126-3p on SLK by flow cytometry. The early apoptosis rate of SLK cells after transfection miR-126-3p mimic(20.53%±1.70) increased than the NC group (9.67%±0.31). The early apoptosis rate of SLK cells after transfection miR-126-3p mimic(11.10%±0.95) had no obvious change compared with the NC group (9.67%±0.31).It showed that miR-126-3p maybe promote the cells apoptosis of SLK.C The mechanism research of PIK3R2 regulated by miR-126-3p(1) Intersection datas were taken through three online miRNA target gene prediction software Mirbase, Miranda and Targetscan to predict the miR-126-3p downstream target genes. It found that PIK3R2 may be the target genes of miR-126-3p.(2)Compared with the negative control group, the level expression of PIK3R2 was decreased after overexpression of miR-126-3p. The level expression of PIK3R2 was upregulated after transfection of miR-126-3p inhibitor. We speculated that miR-126-3p can generate the silencing effect on PIK3R2 mRNA.(3) Compared with the negative control group, the expression of PIK3R2 protein was decreased after transfection of miR-126-3p mimic, whereas the expression of PIK3R2 protein was upregulated after transfection of miR-126-3p inhibitor.It showed that miR-126-3p may be a negative regulation to the expression of PIK3R2 protein.(4) Compared with the negative control group, miR-126-3p was cotransfected with the wild type plasmid decreased the firefly luciferase activity and the ratio of luciferase relative (P<0.05), whereas the PIK3R2-3’UTR-MUT cotransfected with miR-126-3p, the fluorescence intensity is not obviously changed (P> 0.05).Conclusion(1) We identified 170 differentially expressed miRNAs (69 upregulated and 101 downregulated miRNAs) in KS versus normal skin adjacent to the tumors. The results of the microarray data were reliable through verified by qRT-PCR it reminded that the miRNAs were closely related with the pathogenesis of KS and played an important role in the disease process.(2) MiR-126-3p can inhibit cell proliferation, invasion and migration, promote cell apoptosis, induce SLK cells to G2/M arrest, and shorten the cell ratio of S phase. It suggested that miR-126-3p may have the similar role of tumor suppressor genes in Kaposi’s sarcoma and negative regulate the biological behaviors of Kaposi’s sarcoma.(3) Bioinformatics analysis showed that the binding site of PIK3R2 3’UTR and miR-126-3p was highly conserved. MiR-126-3p can downregulate the gene expression of PIK3R2, negative regulate the expression of PIK3R2 protein. Dual luciferase report gene activity assay confirmed that PIK3R2 was a direct target gene of miR-126-3p. |
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