The Study Of MicroRNA-218about Its Role And Mechanism In Gastrointestinal Stormal Tumor Cells

Part One The Expression of microRNA-218and its Effects on Biological Featuresin Gastrointestinal Stromal Tumors CellsBackgroud: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymaltumors of the GI tract, arising from the interstitial cells of Cajal, primarily in the stomachand small intestine. Gastrointestinal stromal tumors (GISTs) occur primarily in olderpatients of either sex, with annual incidences between11and19.6per million peoleworldwide. This corresponds to between3300and6000new cases per year in the UnitedStates. Following surgical resection, GISTs often recur locally, spread diffusely throughoutthe serosal surfaces of the abdomen, and/or metastasize to the liver. Advanced disease isassociated with metastases to distant sites, including the lung and bone. GISTs manifest awide range of morphologies, from spindle cell to epithelioid, but are immunopositive forKIT (CD117) and/or DOG1in essentially all. Before the advent of targeted therapies, theprognosis for advanced GISTs was poor owing to their inherent resistance to bothchemotherapy and radiation therapy. The objectives of this study were to detect theexpressions of microRNA-218(miR-218) in human GISTs tissues and cells, explore itseffects on the biological features of GIST-T1cells, so as to provide new insights for GISTtreatment. Methods: Using the quantitative real-time polymerase chain reaction(qRT-PCR) to detecte the expressions of miR-218in the tissues and adjacent tissues ofGIST and in the GIST cell lines including GIST882, GIST430, GIST48, and GIST-T1.Forty-eight hours after the miR-218mimic was transfected into the GIST-T1cells, theexpression of miR-218in the GIST-T1cells was detected by qRT-PCR. The effect of miR-218on the GIST-T1cell viability was detected using MTT. The effect of miR-218onthe proliferation and apoptosis of GIST-T1cell was analyzed using flow cytometry.Transwell invasion chamber was applied to detect the effect of miR-218on the invasion ofGIST-T1cells. Results: As shown by qRT-PCR, compared with that in the GIST adjacenttissue, the expressions of miR-218in the tumor tissue and GIST cell lines weresignificantly decreased (P<0.0001). Compared with the control group, the expression ofmiR-218increased significantly in GIST-T1cells transfected with miR-218mimic for48hours (P<0.01). MTT showed that the cell viability decreased significantly after theover-expression of miR-218in the GIST-T1cells (P<0.01). Flow cytometry showed thatthe cell proliferation index significantly declined after the over-expression of miR-218(P<0.01), and meanwhile the apoptosis of cells also significantly increased (P<0.01).Detection using the Transwell invasion chamber showed that the number of cells passingthrough the Transwell chamber significantly dropped after the enhanced expression ofmiR-218(P<0.01). Conclusion: the expression of miR-218decreases in human GISTtissue and cell lines. miR-218can inhibit the proliferation and invasion of GIST cells.Treatment based on the enhanced expression of miR-218may be a promising strategy forGIST.Part Two The Study About Target Gene by microRNA-218and Their RegulatoryMechanismsBackgroud: MicroRNAs (miRNAs) are endogenous small noncoding RNAs with about22nucleotides length, which regulate the expression of target genes by interfering withtranscription and/or translation. miRNAs play important roles in physiologic developmentas well as in pathologic states, including cancer. Many cancer cells show aberrantexpression of miRNAs, and this aberrant expression has been suggested to play importantroles in the development and progression of cancers, including gastrointestinal stromaltumor. It has been predicted that more than one third of the human genes are conserved miRNA targets. The roles of miRNA in human disease, especially tumors, have beenattracting increasing attention. The first part studies have explored the expression ofmiR-218, and effects of miR-218on proliferation, apoptosis and invasion ofgastrointestinal stromal tumor cell. This part of the study was to explore the miR-218byregulating target genes to effect on the biological behavior of gastrointestinal stromaltumor cells. Methods: KIT was predicted one of the regulated target genes by miR-218using online software TargetScan. Luciferase reporter plasmid was constructed. Thefragments of the3’UTR of the target gene KIT, which complement miR-218, and those ofthe mutated KIT were chemically synthesized. The fragments of the3’UTR of the KIT thatpredicts the miR-218-binding sites and those of the mutated KIT were cloned into the XbaⅠ site of pGL3-Lueiferase vector. Luciferase plasmid containing the3’UTR of the KITwas cotransfected, together with miR-218mimic or non-specific control. The normalcontrol group was also established. Forty-eight hours after the transfection, the cells werecollected, detected using the luciferase assay kit, and read with microplate reader. Theeffect of miR-218on the expression of KIT protein was determined using Westernblotting. Results: Luciferase reporter gene assay showed that, compared with the controlgroup, the relative luciferase activity significantly declined in the miR-218mimictransfection group (P<0.01). Compared with the control group, the expression of KITprotein in the GIST-T1cells transfected with miR-218mimic for48hours significantlydecreased (P<0.01). Conclusion: KIT was one of the regulated target genes by miR-218.miR-218can negatively regulate the expression of KIT protein.Part Three microRNA-218Increase Chemo-sensitivity to Imitinib ThroughPI3K/Akt Pathway in Gastrointestinal Stromal TumorBackgroud：Gastrointestinal stromal tumors (GIST) are sarcomas arising in the musclewall of the gastrointestinal tract. Tyrosine kinase inhibitors (TKIs), such as imatinib andsunitinib, block the aberrant activation of KIT, leading to major clinical benefits of objective response and durable disease control. Imatinib represents the standard first-linetherapy for this disease when it is surgically incurable. The inhibition of KIT by imatinibis an example of posttranslational inhibition through competitively binding theATP-binding pocket of KIT. However, KIT inhibition by imatinib is imperfect becauseresistance can occur through putative mutations that can develop in the specific aminoacid sequences during treatment. The resultant imatinib resistance occurs through theinhibition of imatinib binding to the ATP-binding pocket of KIT. However, imatinib doesnot cure advanced GIST. Moreover, while the large majority of patients treated withimatinib do not show signs of primary resistance (disease progression within the first sixmonths of therapy), secondary resistance to imatinib emerges in at least half the patientsafter two years of therapy and in more than80%of patients after seven years. The purposeof study is to detect the expressions of microRNA-218(miR-218) in an imatinibmesylate-sensitive human gastrointestinal stromal tumor (GIST) cell line (GIST882) andan imatinib mesylate-resistant cell line (GIST430) and explore the roles of miR-218andGIST cells in the sensitivity of gastrointestinal stromal tumor to imatinib mesylate and itspotential signaling pathways, with an attempt to provide new insights for the treatment ofGIST. Methods: The GIST cell lines (GIST882and GIST430) were cultured in vitro.Quantitative real-time PCR (qRT-PCR) was utilized to determine the expression profilesof miR-218in both GIST cell lines. Forty-eight hours after the transfection of the miR-218mimic or miR-218inhibitor in the GIST cells, the changes in the expression of miR-218inthe GIST cells were detected with qRT-PCR. The effects of the ectopic expression ofmiR-218in GIST882or GIST430cells on the imatinib mesylate-induced GIST cellviability were determined by MTT. The effects of miR-218ectopic expression on theapoptosis of imatinib mesylate-induce GIST cells were determined by Annexin V/PIdouble staining method and flow cytometry. The effects of miR-218ectopic expression onthe AKT and p-AKT expressions of imatinib mesylate-induce GIST cells were determinedby Western blot and flow cytometry with the PI3K pathway inhibitor Wortmannin. Results: As shown by qRT-PCR, compared with that in the imatinib mesylate-sensitiveGIST882, the expression of miR-218in imatinib mesylate-resistant GIST430wassignificantly decreased (P<0.01). Compared with the control group, the expression ofmiR-218significantly increased in GIST882and GIST43048hours after the transfectionof miR-218mimic (P<0.01) and significantly declined after the transfection of miR-218inhibitor (P<0.01). As shown by MTT and flow cytometry, after the expression ofmiR-218was inhibited in GIST882under the effect of imatinib mesylate, the cell viabilitysignificantly increased (P<0.01) and the number of apoptotic cells significantly decreased(P<0.05); on the contrary, the over-expression of miR-218in GIST430under the effect ofimatinib mesylate resulted in the significantly decreased cell viability (P<0.01) and thesignificantly increased number of apoptotic cells (P<0.05). Western blot and flowcytometry showed that, in comparison to the control group, miR-218over-expressioncould significantly inhibit the expression of p-AKT in GIST430cells (p<0.01) andstimulated apoptosis (P<0.01). Conclusion: The expression of miR-218is down-regulatedin an Imatinib Mesylate-resistant GIST cell line (GIST430), whereas miR-218over-expression can improve the sensitivity of GIST cells to imtinib mesylate, withPI3K/akt signaling pathway possibly involved in the mechanism.