The Function And Regulation Network Analysis Of SPLUNC1 Related Differentially Expressed MiRNA

Nasopharyngeal carcinoma(NPC) is a polygenetic inherited malignant tumor with a high incidence in South China and Southeast Asia. SPLUNC1 gene,considered as the NPC candidate tumor suppressor gene, is a tissue-specific gene of human nasopharyngeal epithelium with low expression in both NPC cell lines and tissue.Previous studies in our lab found that SPLUNC1 is a secreted protein which covered on the surface of epithelium and can bind to the Gram negative nanobacteria,indicating that it might function as an innate immune defensive molecule. SPLUNC1 protein can bind to bacterial lipopolysaccharide,inhibit not only P.aeruginosa,but also the EB virus.Meanwhile,our lab also have cloned some other NPC candidate tumor suppressor gene,such as BRD7, NGX6,UBAP1,LPLUNC1,NAG7,but the mechanism how those genes interact with each others which cause the tumorigenesis of NPC is far from full known.Since miRNAs are short RNAs that post transcriptionally regulate messenger RNAs,and the formation of miR-Gene network depended on the hundreds of miRNAs and their numerous target genes,which might be an answer to the puzzle of NPC candidate tumor suppressor genes above mentioned.To uncover the molecular mechanisms between SPLUNC1 and miRNA in NPC,we did the research below,and also acquired some interesting results [Hsa-mir-141 and hsa-mir-205 were downregulated in SPLUNC1 overexpressed NPC ceLll lines]We transfected the full length of SPLUNC1 into the highly invasive and matastatic NPC cell line 5-8F,and constructed a stable cell line.Via microRNAarray analysis from CapitalBio Corporation(Beijing,China), we found that 29 miRNAs were upregulated and 25 miRNAs were downregulated in SPLUNC1 overexpressed cell line.(Fold change＞3) Two of the downregulated miRNA.(Fold change＞10),Hsa-mir-141 and hsa-mir-205 were confirmed by northern blot and qRT-PCR in the same cell lines.We further confirmed that expression of hsa-mir-141 was upregulated in 10 laser capture microdessection NPC tissues compared to the normal nasopharyngeal epithelium tissues,while has-mir-205 showed no difference between them.[Hsa-mir-141 and hsa-mir-205 play a role as oncogenes in NPC cell Iine5-8f.]Since SPLUNC1 gene was a tumor suppressor gene that could inhibit the cell growth of NPC cell lines-HNE1,we then want to know whether the change of hsa-mir-141 level could affect cell growth.Via MTT assay, Although there were no difference among these groups during the first three days after transfection,by day 5 and 6 we detected inhibiting hsa-mir-141 group grew more slowly than other groups with about 30% inhibition,and upregulated hsa-mir-205 group grew more quickly than other groups with about 40%promotion.Since 5-8f is a highly invasive and matastatic cancer cell line,we accomplished cell wound healing assays and transwell assays to identify whether hsa-mir-141 and hsa-mir-205 could influence cell migration and invasive ability.The result showed a significant reduction in both cell migration and invasion in the hsa-mir-141 and hsa-mir-205 inhibitor groups.Via flow cytometry analysis,we further found inhibition of hsa-mir-141 and hsa-mir-205 could slightly increase the cells apoptosis rate,and arrested cells in G0-G1 with a concurrent reduction in S phase cells compared to other control groups.These data suggested the inhibition of hsa-mir-141 and hsa-mir-205 may promote the NPC cell apoptosis and delay cell cycle progression from G1 to S phase.To explore the possible mechanism involved in a delay of G1 to S phase progression by 141 and 205 inhibitor groups in 5-8F NPC cells,cell cycle-related effectors were examined by western blot.And we found an obvious change in the expression of caspase 3,caspase8,NF-κB,I-κα,JNK2,P-ERK in 141 and 205 inhibitor groups.This suggests that inhibition of 141 and 205 could inhibit cell growth and cell cycle progression in 5-8F cells by regulating MAPK/ERK and TNFR pathway. [The Whole Human Genome Oligo Microarray Shows the Differences in genes expression from the transfection of SPLUNC1 also involve in the same signaling pathway as hsa-mir-141 and has-mir-205's prediction target genes do]Firstly,we used both pictar and targetscan to search the possible targets of hsa-mir-141 and has-mir-205.Secondly,we used DAVID 2008 Functional Annotation Bioinformatics Microassay Analysis Tools to classify the function of the target gene which were predicted both from TargetScanS 4.2 and pictar.The result revealed that mostly(＞50%) target genes of hsa-mir-141 and has-mir-205 involved in the signaling pathways as MAPK,Wnt,JAK-STAT,Tight junction,Cell cycle,Adherens junction,which are important for tumorigenesis and tumor development Since the celluar function showed mir-141 and has-mir-205 may play a role as oncogene in NPC cell lines,we were interested to know whether tumor suppressor genes were potential regulated by hsa-mir-141.From Tumor Suppressor Gene Database,we found some hsa-mir-141 targeted genes,such as MAP2K4,MAP3K3,DLC1,TP53BP2 were considered as classical tumor suppressor genes.More interestingly,we found UBAP1 gene,one of the NPC candidate tumor suppressor gene previously cloned from our lab,was predicted as a hsa-mir-141 targeted gene by both pictar and targetscan.We then used the same cell line of transfected SPLUNC1 for the use of the Whole Human Genome Oligo Microarray.We found 1698 upregulated genes and 2135 downregulated genes between the transgenic group and the blank group.There was almost no perfect one to one genes match between the differences in mRNA and miRNA prediction target genes,but the Differences in genes expression from the transfection of SPLUNC1 involve in the gnaling pathway such as MAPK,Wnt,JAK-STAT,Cell cycle,which was also the same pathway hsa-mir-141 and has-mir-205's prediction target genes involve in.We may draw a conclution that SPLUNC1 can influence these tumor related pathways not only in direct way,but also can regulate the pathways in indirect way by miRNA.[hsa-mir-141 regulated NPC tumor suppressor gene UBAP1 via directly targeting its 3'UTR]The UBAP1 gene was originally cloned in our lab,which has been found to be down-regulated in NPC tissues.The result showed that when co-transfected with hsa-mir-141 mimic in 5-8f cells,luciferase reporter of UBAP1 3'UTRs was dramatically inhibited(down over 50%),but no obvious changes were found when co-transfected with hsa-mir-141 inhibitor and mimic negative control;whereas mutation of the miR-141 binding sites(RL-UBAP1 M) could diminish the regulating function of miR-141 on UBAP1,which verified that the effect of the miR-141 is due to direct interaction with the binding sites in UBAP1 3'UTRs.The next experiment was to detect whether transfection with hsa-mir-141 inhibitor could affect the UBAP1 protein levels.The cell extracts from 5-8f,141 inhibitor group,inhibitor nagetive control group, 5-8F/pcDNA3.1/his-SPLUNC1 and 5-8F/pcDNA3.1/his were used for western blot.The result demonstrated that UBAP1 were upregulated in both SPLUNC1 overexpressed cells and miR-141 inhibitor group,while hardly or barely detectable in the other groups.The result not only indicated that mir-141 could influence the expression of UBAP1,but also showed that the transfection of SPLUNC1 could upregulate UBAP1 via do wnregulating hsa-mir-141.In our previous studies,SPLUNC1 may affect some key molecules such as JNK2,NF-icB,and also influence the expression of caspase 8 and caspase 3 in TNFR pathway,which led to the DNA fragmentation and finally cell apoptosis.In this study,we confirmed the inhibitor of hsa-mir-141 had the same affect to influence the ERK/MAPK and TNFR pathway by western blotting.With our present study,SPLUNC1 might downregulate hsa-mir-141,while the downregulation of hsa-mir-141 might upregulate its target gene UBAP1 which may inhibit tumor growth by degrading the key proteins of tumorigeness.Hsa-mir-141 played a role as a turning point in this system, the NPC tumor suppressor genes SPLUNC1 may upregulate UBAP1 via do wnregulating hsa-mir-141.Whether these genes in ERK/MAPK pathway also involve in the mechanism of tumor inhibition need to be further studied and verified.