MiR-421Induces Cell Proliferation And Apoptosis Resistance In Nasopharyngeal Carcinoma Via Downregulation Of FOXO4

Nasopharyngeal carcinoma (NPC)is one of the most common malignanttumors in the Southern ofChina,and it ranks the first in head-neck Cancer. The place where NPC originates from is covert and it is difficult to be discovered. The nasopharynx is neighbor to nose cavity,nasal sinuses and encephalic structure. So clinic symptoms appear late,and early signs are multiple. There is the highest incidence of distant metastasis for NPC among head and neck cancer. For the reasons stated above, NPC is easy to be misdiagnosed or fail to be diagnosed, and it’s curative effect is not satisfactory. The maximum age incidence is the fouth to fifth decade. It brings great suffering to patient’s family and society. It is very important to research early diagnosis and therapy of NPC. Nowadays, the major therapeutic modality of NPC is radiotherapy. The chemotherapy only is used in the patients who aren’t sensitive to radiotherapy or the patients who have recurrence tumors after previous radiotherapy,or patients who are found lately. As an adjunctive therapeutic modality,operation only is limited to the special conditions. In recent years, some authors attempt to use photodynamic therapy and microwave hyperthermia therapy to treat NPC, but those methods also are regarded as adjunctive therapy.The purpose of this study is to construct recombination plasmids which express precursor of miRNA by simulating natural and to attempt to regulate the expression of cancer-related genes-FOXO4.The experiment is divided into two sections:Section one:To construct the plasmid which regulates target genes, and to screen effective plasmid,and to confirm efficient positions in which miRNA direct against FOXO4gene, and to observe regulative effect of candidate plasmid. These studies will provide the foundation for follow-up experiments in vitro and in vivo. Section two:To identify miR-421inhibits the FOXO4signaling pathway. negative control plasmid, are used to transferred to CNE-2cell line separately,and to generate stable cell lines that constitutively express miRNA by antibiotics selection. The effects of stable transfeetion of the miRNA plasmids are observed by qRT-PCR、Western blotting、soft agar colonies formation assay. Result find that four cell lines express stably miRNA,and miRNA regulate expression of target gene in stable cells.Conclusions:(1)We construct successfully the recombination plasmid vectors which target FOXO4; the plasmid vectors which express miRNA can down-regulate the expression of FOXO4gene efficiently,(2)miR-421induces cell proliferation and apoptosis resistance in nasopharyngeal carcinoma.MicroRNA as a recently discovered small RNA in post-transcriptional level of regulation of protein expression has become the focus of our attention. MicroRNAs are small RNA molecules present in the genome of plants and animals, about20-24nt (a small number of less than20nt), a hairpin loop structure consists of a section, a length of70-80nt single-stranded RNA precursors (pre-miRNA formation) after shearing. Its target mRNA molecules in the3’untranslated region (3’ from a untranslated region3’UTR) is completely non-complementary match to participate in the regulation of gene transcription levels in physiological and pathological conditions and plays a very important role.miRNA as non-coding RNA was first discovered in the embryonic development of the nematodes (Caenothabditis, elegans) in1993by Lee et al. First of all, the primary transcripts of miRNA genes (the Pri-miRNAs) cutting the precursor miRNA (pre-miRNAs) by the RNase m Drosha in the nucleus. Transporter protein exPortin-5plays a role in the initial shear Pre-miRNA from the nucleus to the cytoplasm, and then further cut by another RNase Ⅲ Dicer to generate mature miRNA. The mature miRNA with other proteins form RISC (RNA-induced sileneing complex) complex and they lead to the target mRNA degradation or translational repression.MicroRNAs have important tissue-specific functions and play a general regulatory role in the regulation of gene expression. miR-421is a specific expression in tumors, and most rich in microRNA.Part Ⅰ1. Upregulation of miR-421promotes cell proliferation and renders apoptosis resistance in human nasopharyngeal carcinomaObjective:To identify the ability of miR-421promoting the proliferation of detection of miR-421for nasopharyngeal carcinoma cells.Methods:The use of viral transfection techniques, miR-421transfection of CNE2observed in vitro miR-421on CNE2proliferation and use of Annexin V method and TUNEL assay cell apoptosis.Results:A colony formation assay showed that proliferation rate of miR-421-overexpressing cells were about3fold of control cells, indicating that miR-421upregulation significantly promoted nasopharyngeal carcinoma cell growth. Moreover, overexpressing miR-421also augmented the apoptosis resistance of nasopharyngeal carcinoma cells to treatment of chemotherapeutic agent cisplatin as indicated by the Annexin V and TUNEL staining assays.Conclusion:miR-421Induces Cell Proliferation and Apoptosis Resistance in Nasopharyngeal Carcinoma2. Inhibition of miR-421reduces cell proliferation and induces apoptosis in human nasopharyngeal carcinoma We further examined the effect of miR-421inhibition on nasopharyngeal carcinoma progression. Consistent with abovementioned results, colony formation assays showed that miR-421suppression dramatically inhibited the growth rate of CNE2cells as compared with that of control cells. Moreover, miR-421inhibition could also decrease the resistance to apoptosis in nasopharyngeal carcinoma cells treated with cisplatin as indicated by Annexin V and TUNEL staining analysis.Part II1. miR-421inhibits the FOXO4signaling pathwayWe further investigated the underlying molecular mechanism through which miR-421promotes cell proliferation and render apoptosis resistance. Since FOXO4signaling pathway could reduce cell proliferation and induce cell apoptosis through transcriptionally upregulation of its targets, such as p21, p27, Bim and FASL. Moreover, FOXO4is found frequently inactivated in cancers, and considered key tumor suppressors. Hence, we then examined whether miR-421regulated the FOXO4activity. As shown in our research, the luciferase activity of FOXO4reporter significantly decreased in the miR-421-overexpressing cells, but increased in the miR-421-inhibited cells. Consistently, we found that both mRNA and protein expression levels of4classical FOXO4target genes were downregulated in miR-421-transduced cells but upregulated in miR-421-inhibited cells. Thus, our results indicated that miR-421inhibits the FOXO4signaling pathway.2. miR-421directly targets FOXO4Interestingly, analysis using TargetScan program tool for miR-421target prediction showed that FOXO4is the potential target of miR-421. Importantly, western blotting analysis showed that ectopic expression of miR-421dramatically decreased, but inhibition of miR-421increased FOXO4protein expression in CNE2cells. To examine whether miR-421-induced FOXO4downregulation was mediated by the3’-untranslated region (3’UTR) of FOXO4, we subcloned the FOXO43’UTR fragment, containing the miR-FOXO4binding site into pGL3dual luciferase reporter vectors. As shown in our research, overexpression of miR-421decreased and inhibition of miR-421increased the luciferase activity of the FOXO4-3’UTR. However, neither miR-421overexpression nor inhibition altered the luciferase activity of the FOXO4-3UTR-mutant, containing point mutations in the miR-421-binding seed region of FOXO4-3-UTR, suggesting that miR-421specifically targets the3’-UTR of FOXO4. Collectively, our results demonstrate that FOXO4is a bonafide target of miR-421.3. FOXO4expression is critical for miR-421-induced cell growth and anti-apoptosisWe further investigated the role of FOXO4expression in miR-421-induced cell growth and anti-apoptosis. As shown in our research, we found that the miR-421inhibition induced cell growth arrest and apoptosis promotion could be abolished by downregulation of FOXO4, indicating that FOXO4plays an important role in miR-421-mediated biological functions.4. Clinical correlation between miR-421and FOXO4in nasopharyngeal carcinoma tissuesFinally, we examined whether the miR-421-induced FOXO4repression identified in our study is clinically relevant. As shown in our research, we found that miR-421levels in7freshly collected nasopharyngeal carcinoma tissue samples inversely correlated with the FOXO4expression levels (r=-0.798, P=0.032).Taken together, our results suggest that miR-421upregulation inactivates FOXO4signaling, and consequently leads to cell proliferation and apoptosis resistance in nasopharyngeal carcinoma.