Effect Of MiR-96Down-regulation On The Proliferation And Apoptosis Of Lung Adenocarcinoma Cells By Targeting FOXO3

BackgroundLung cancer is the leading cause of cancer-related deaths all over the world. One ofthe main pathology types is small cell lung cancer (SCLC), which has a rapiddevelopment and is13%-15%of the total. The other85%of lung cancers arenon-small cell lung cancer (NSCLC), including adenocarcinoma, squamous cellcarcinoma, large cell carcinoma and neuroendocrine carcinoma, of which lungadenocarcinoma has the highest incidence. Lung cancer with poor prognosis hasmany reasons. Most of the patients have reached an advanced stage when to bediagnosed. Therefore, early detection and early operation are still the best treatment.As to lung cancer patients in malignant or advanced stage, the optional treatments arechemiotherapy, radiotherapy, biological target therapy and so on. These treatments,especially chemiotherapy and radiotherapy, have some effect, but result in poorertolerance, higher side effects and repeated recurrence. Invasion and metastasis of lungcancer also affect patients’ effects and survival time. The clear pathogenesis andmetastasis mechanism is still unclear, and the clinical work lacks effective therapy. Itis necessary to search the new tumor marker for the early diagnosis and the besttreatment of lung cancer.MicroRNAs (miRNAs) are small, non-coding RNAs approximately22nucleotides inlength that negatively regulate gene expression at the post-transcriptional and/ortranslational level by binding to complimentary sequences in the3’UTR-untranslated regions (UTRs) of target mRNAs. MiRNAs affect the stability and translationalefficiency of their target mRNAs. They have been implicated in an increasing numberof biological processes, including carcinogenesis. The up-or down-regulation ofspecific miRNAs during carcinogenesis has been documented widely, and themolecular pathways that mediate the effects of miRNAs on different aspects of cancerdevelopment, including proliferation, angiogenesis and metastasis, have beenidentified. Dysregulation of miRNAs plays roles in the pathogenesis of humandiseases, including malignancy. miRNAs may function as both oncogenes and tumorsuppressors. MiR-96is a member of the miR-183family （miR-96, miR-182andmiR-183）. It maps to the chromosomal region17. Recent studies showed that miR-96plays a role in a variety of tumors, and not only the abnormal expression of thismiRNA in tumor tissue, but also its involvement in the biological behavior of certaintumors Some research also validated that miR-96was significantly increased in bothhuman NSCLC tissues and cell lines. Forkhead transcription factors of the O class(FoxOs) consist of four members, FoxO1, FoxO3, FoxO4and FoxO6. Thesetranscription factors have been identified as important regulators involved in cellulardifferentiation, apoptosis, oxidative stress, glucose metabolism and other cellularfunctions. MiR-96and FOXO3are interrelated and interact on each other bybiological information analysis.The present study examined the effect and mechanism of miR-96on the proliferationand apoptosis of lung adenocarcinoma cell line A549.Methods:1. Cell culture and Synthetic miR-96inhibitor and miRNA scramble2. Human lung adenocarcinoma cell line A549was cultured in DMEM culturemedium with10%fetal bovine serum. miR-96inhibitor and miRNA scramblewere synthesized by Shanghai GenePharma Co.,Ltd.3. Cell transfection miR-96inhibitor and miRNA scramble were transfected intoA549cells by electrotransfection. The experiment was designed three groups:miRNA-96inhibitor group, miRNA scramble and blank group. After transfectionfor24h-48h, we checked the relative exprements.4. The expression of FOXO3mRNA was analyzed by RT-PCR and the protein level was detected by western blotting. CCK-8assay was used to assess the effect ofmiR-96on A549cell proliferation. Wound healing assay was used to investigatethe effect of miR-96on invasion and migration of A549. FACS was used to assessthe effect of miR-96on A549apoptosis.5. Statistical analysis: All the dates were analyzed by SPSS17.0statistical package.The mean among more groups uses the ANVOA. P＜0.05is considered assignificance.Results:1. The mRNA and protein expression level of FOXO3was significantly increased inmiR-96inhibitor group (P <0.05).2. The migration ability of A549cell. Both Blank and miRNA scramble groups hadreached a higher cell density at24h post-wounding compared to the miR-96inhibitor group. These data indicate that inhibiting miR-96restricts the migrationand invasion capacity of A549cell.3. In the CCK8assay, we observed that, compared with the Blank and miRNAscramble groups, the viability of the miR-96inhibitor group decreased (P <0.05).In contrast we found no significant difference between the Blank and the miRNAscramble groups (P>0.05).4. Cells apoptosis results showed that apoptosis cells were significantly increasedafter transfection miR-96inhibitor （P <0.05）. There was no statisticallysignificant difference between the Blank and the miRNA scramble groups (P>0.05).Conclusion:1. Down-regulation of miR-96can suppress FOXO3expression. Therefore, miR-96may be a potential therapeutic in NSCLC treatment in the future.2. Up-regulated expression of FOXO3induced apoptosis and suppressed cellproliferation, invasion and metastasis in the lung adenocarcinoma cell line A549.3. MiR-96has a correlation with FOXO3and regulates cell proliferation, invasion,metastasis and apoptosis by regulating the expression of FOXO3.