Enhancement Effect Of MiR-18b On Chemosensitivity Of Human Breast Cancer MDA-MB-231Cells To DDP And Its Mechanism

Breast cancer is one of malignant tumor that endanger women’ life. Triplenegative breast cancer (TNBC) lacks of estrogen receptor, progesterone receptor andhuman epidermal growth factor receptor (HER2). There was no significanttherapeutic effects treated by endocrinotherapy. Compared with other kinds of breastcancers, TNBC showed poor prognosis and survival phase. At present, Chemotherapywas the main methods in clinical therapy. Several reports showed that cisplatin playeda significant role in the treatment of TNBC. The increasement of chemosensitivity incisplatin to TNBC could largely improve the prognosis with breast cancer.MicroRNAs (miRNAs), were short (20~22nucleotides) non-coding RNAs, andhighly conservatived in Genetics. MiRNAs has attracted broad attention in medicalwith sophisticated biological functions. MiRNAsbind by incomplimentary basepairing to the3’-untranslated region (3’-UTR) of their target mRNA and played animportant role in regulating target gene. miRNA was not only play a significant role inprogression, development and metastasis of tumor, but also is associated withradio-chemosensitivity of tumor. The new researches showed that the expression ofmiR-18b was largely increased and regulated invasive genes in breast cancer cells.There was uncertain about the mechanism in which miR-18b induced-biologicaleffects in the process of chemotherapy.Programmed cell death (PCD) is associated with morphogenesis, homeostasisand eliminating cancer cells. Apoptosis was not the only death way in organism.Several researches showed that cells often devour long-life protein and damagedorganelle inorder to acquire survival material and energy when cells were in the statesof hypoxia. Autophagy is other death way besides of apoptosis.miRNA regulatestumor cells’ apoptosis, autophagy and senescence and cell cycle, which playingdouble-edge sword effects of anti-cancer and promoting-cancer.miRNA wasdifferential expression in breast cancer, lung cancer, thyroid cancer and liver cancer,and biological functions were consider as oncogene and tumor suppressors.miRNAregulates tumor’s occurrence, development and distant metastasis through thesemechanisms. miRNA was not only used as diagnosis, treatment and prognosis, butalso was consider as oncogene and tumor suppressors. Autophagy as otherprogrammed cell death besides apoptosis, also has these similar doubleeffects.Besides of regulating mechanisms of miRNA, more researches needs schedule on miRNA.Many researches showed that the expression of miR-18b was largely increasedand regulated invasive genes in breast cancer cells. There was uncertain about themechanism in which miR-18b induced-biological effects in the process ofchemotherapy. This study was designed to investigate the mechanism of miR-18b inwhich regulate chemosensitivity..ObjectiveTo investigate the chemosensitivity effects of miR-18b on human breast cancercells MDA-MB-231and possible mechanism.MethodsHuman breast cancer MDA-MB-231cell was cultured and logarithmic phasecells were cryopreserved. The breast cancer cell divided into MDA-MB-231group,MDA-MB-231+DDP group, MDA-MB-231-NC group, MDA-MB-231-NC+DDP group, MDA-MB-231mimic group,and MDA-MB-231mimic+DDPgroup.Negative control (NC) was used as control, CCK8assays was used to detectcells’chemosensitivity of DDP. qRT-PCR was used to detectd endogenous expressionchanges of miR-18b;The target gene of miR-18b was predicted by bioinformaticsmethods;3’UTR fragments of target genes were acquired by PCR and dual luciferaseexpression vector was built by genetic engineering. Western blot was used to analyzethe expression of ATM after transfection.Results1. The prediction of miR-18b’ s target geneWe reported miR-18b’ s target gene by bioinformatics methods (PicTar、miRBaseandTaregestscan). miR-18b’ s target gene was ATM.2. The expression of miR-18b in breast cancer MDA-MB-231cells in variousgroup.Compared with NC, the expression of miR-18b increased by20folds aftertransfected with mimic. The expression of miR-18b in Mimic+DDP groupincreased by60folds (p<0.001).3. Luciferase and western blot showed that the ATM was miR-18b target gene.1）MiR-18b overexprssion inhibit3’UTR luciferase of ATM in breast cancerMDA-MB-231cellsluciferase of pMIR-ATM decreased to70%after MiR-18b overexprssion,which means MiR-18b overexprssion inhibit3’UTR luciferase of ATM. ATM was targetgene of MiR-18b and played regulatory effects through combining with ATM’s UTR.2）MiR-18b overexprssion inhibit the expression of ATM in breast cancerMDA-MB-231cellsIn order to confirm was target gene of MiR-18b, we transfected mimic intoMDA-MB-231cells aiming to miR-18b overexpression. Compared with miR-18b NCgroup, miR-18b mimic group can improve the expression of miR-18b inMDA-MB-231cells, mimic miR-18b+DDP group can further improve the miR-18bexpression in MDA-MB-231cells; miR-18b overexpression inhibit the expression ofATM in MDA-MB-231cells.4. The effect of miR-18b overexprssion on MDA-MB-231cells’ chemosensitivitycompared with negative control group, cck8method showed that miR18b mimicgroups of different concentrations of DDP inhibition on MDA-MB-231cells wereincreased, and indicates MDA-MB-231cells for DDP chemotherapy sensitivity.ConclusionmiR-18b enhance Human breast cancer cells’chemosensitivity to DDP throughtarget-regulating ATM.