Study On MiR-9Enhances The Radiosensitivity By Targeting NRP1in A549Cells

Lung cancer is one of the most common malignancies and the leading cause ofcancer related mortality in the world. Lung cancer is broadly divided into non-smallcell lung cancers (NSCLC) which approximately account for80%of lung cancer, andsmall cell lung cancers (SCLC) which approximately account for20%of lung cancer. Radiation therapy is commonly used to treat lung cancer in the clinical, but could notcompletely kill cancer cells. NSCLC is more resistant to radiation than SCLC.Whether radiation therapy combined with gene therapy is required for improvingradiotherapy for NSCLC needs to be solved urgently. With the progress of cancerresearch in molecular biology, considerable attention has been devoted to moleculartargeted therapy which is widely used in clinical practice. Recent studies have foundthat microRNA opens up a new filed for the early diagnosis and treatment of NSCLC.Research background:MicroRNAs (miRNAs) are~22-nucleotide non-coding RNAs that pair to3’UTRof messages of protein-coding genes to guard the cleavage of target mRNA direct thepost-transcriptional repression of these mRNAs, and widely are found in diverseorganisms, including animals and plants. Accumulating evidence demonstrated thataberrant miR-9expression has been reported in many types of cancer, suggesting thatmiR-9is involved in tumor formation or progression. MiR-9is deceased in tissues ofgastric, colon, ovarian, esophageal cancer and renal cell carcinoma and inhibits theirgrowth and metastasis. In contrast, miR-9was upregulated in human breast, brain, andbiliary tract cancer, and enhance the growth of these cells. In addition, high-levelexpression of miR-9in breast cancer is associated with poor overall survival. Theseresults indicate that miR-9may play an important role in cancer development and play different roles in various cancers.However, the clinical significance of miR-9has not been fully understood inhuman non-small cell lung cancer. Volinia et al. first reported that miR-9wasup-regulated in lung cancer tissue using miRNA microarray analysis with lungcancers and adjacent normal lung tissues. Thereafter, Vosa et al. also reported similarresults using miRNA expression array analysis; however, Yanaihara et al. foundopposite pattern between tumor tissue and normal tissue. Therefore, furtherinvestigation is needed to understand the precise expression pattern of miR-9and itsprognostic implication in non-small cell lung cancer.Himanshu et al. demonstratedthat miR-9expression is reduced in H1299cells after irradiation. Meanwhile, theyalso found that miR-9could increase the radiosensitivity of cancer cells throughregulation of NF-κB1.Neuropilin1(NRP1) was expressed in lung, breast cancer andinvolved in the process of tumor angiogenesis.Bioinformatics analysis showed thatthere is a correlation between miR-9and NRP1. Until recently, little research has beendirected at miR-9enhancing radiosensitivity via regulation NRP1in domestic andinternational institutions. Therefore, the present study is to investigate the effects oftargeted regulation of NRP1by miR-9on lung cell radiosensitivity and clarify theregulation mechanism. The research will provide fundamental evidences and newideas for improving radiotherapy of NSCLC.Research objectives:1. To verify that NRP1is a target of miR-9.2. To investigate the effects of miR-9on IR induced A549cell proliferation,apoptosis and cycle, migration and invasion.3. To clarify the mechanism of miR-9regulation NRP1on enhancingradiosensitivity of A549cells.Research methods:1. Bioinformatics was used to analyze the potential binding sites inNRP1-3′UTR for hsa-miR-9. NRP1gene3′-UTR luciferase reporter plasmid (pEZX-MT05-NRP1-3′UTR) and mutant plasmid (pEZX-MT05-NRP1-3′UTRMUT)were constructed using conventional molecular biology methods and identified byrestriction enzyme digestion and sequencing. Luciferase activity assay was performedwith the dual luciferase reporter assay system to testify whether miR-9regulatesNRP1through binding to NRP13’UTR.2. Chemical synthetic miR-9mimics were used to transfect into A549cell. TheNRP1mRNA and protein expression were detected by real-time PCR western blotrespectively.3. A549cells were transfected with miR-9mimics with or without10Gy X-raytreatment. Cell proliferation, apoptosis, migration and invasion were measured using acolony-forming assay, Annexin V apoptosis kit and Trans Well assay.Research results:1. The relationship between miR-9and NRP1MiR-9significantly reduced the luciferase activity of the WT construct of theNRP13’UTR compared with the negative control, whereas such a suppressive effectwas not observed in the Mut construct of NRP13’UTR cells. The future relationshipbetween miR-9and NRP1was to prove from the negative, we used miR-9inhibitorand pEZX-MT05-NRP1-3’UTR cotransfected to test the luciferase activity and foundluciferase activity was significantly increased (p <0.05), whereas miR-9inhibitor andpEZX-MT05-NRP1-3’UTRMUT cotransfected did not significantly change theluciferase activity values. NRP1protein and mRNA expression in A549cells weredecreased after the cells transfected with miR-9mimic.2. The effect of miR-9targeted NRP1on radiosensitivity in A549cellCompared with the parent group, over-expression of miR-9significantlyinhibited cell proliferation. MiR-9combined with ionizing radiation furthersignificantly inhibited cell proliferation than that of miR-9treatment alone, indicatingthat miR-9could reduce proliferation of A549cells. Radiosensitivity is different atdifferent phases in the cell cycle. The experimental results showed various phases of the cell cycle have the mostobvious changes at48h after irradiation The results showed that number of G1phase,S phase cells were decreased and G2/M phase cells were increased. Therefore,over-expression of miR-9can cause A549cells G2/M phase arrest. Over-expressionof miR-9significantly enhanced IR-induced apoptosis in A549cell compared withcontrol transfected with miR-NC at48h after irradiation. Transwell assay showedover-expression of miR-9and miR-9combined with ionizing radiation could suppressA549cell migration and invasion.3. miR-9targeted NRP1enhanced radio-sensitivity of A549cellsColony formation assay showed that the survival fraction of cells in each groupwere deceased gradually with increasing irradiation dose; Compared to the parentcells, miR-9over-expression significantly reduced the survival fraction (survivingfraction, SF) at the same dose. We found that miR-9over-expression decreased D0and SF2values, indicating that increased expression of miR-9could enhance theradiosensitivity of A549cells.4. The signaling pathway mechanism of miR-9enhanced radio-sensitivity ofA549cellsCompared with control group, the level of NRP1expression was decreased inmiR-9over-expression group. Meanwhile, the expression pattern of PI3KⅠ, NF-κB,p-ERK, p-AKT, pP38was consistent with the change of NRP1. However, theexpression of ERK, AKT, and p38increased slightly. Compared with the controlgroup, the expression of NRP1, PI3KⅠ, NF-κB, pERK, pAKT, pP38, ERK, AKT,and P38were also reduced in miR-9combined with ionizing radiation group, but notstatistically significant, indicating that miR-9could regulate NRP1to induceactivation of multiple signaling pathways.Conclusions:1. NRP1is a target of miR-9and its translation and transcription was inhibited.2. Overexpression of miR-9decreased the cell proliferation of A549cells via induction of cell cycle arrest at the G2/M phase, inhibited cell migration and invasionand enhance cell apoptosis,.3. MiR-9could increase the radiosensitivity of A549cells, which is mainlyreflected the decrease in the value of D0, Dq and SF2.4. MiR-9could induce radiosensitivity of A549cell via NRP1activating PI3KⅠ-AKT, MARK-ERK, P38and NF-κB signaling pathway.