| BackgroundGastrointestinal stromal tumors (GISTs) are most common primary gastrointestinal mesenchymal tumors, accounting for 2% of all gastrointestinal tumors. GIST patients had a very poor prognosis because of their poor response to conventional chemotherapy and radiotherapy or because of the limited surgical options. Until 1983, said Mazur determine named for gastrointestinal stromal tumor research, its features and named mainly based on the differentiation of the tumor.Gastrointestinal stromal tumor was seen most frequently in the elderly, patients under the age of 40 were rare, the onset of the median age was the age of 50 to 60 years old.Generally 60-70% more in the stomach,20% to 30% occur in the small intestine, only less than 10% occurred in the esophagus, colon and rectum.Gastrointestinal stromal tumor mass substantial part was low signal on T1, muscle signal and has high signal on T2, tumor necrosis of the central can be found that the gas cavity and no signal area of the tumor calcification, if appeared hemorrhage within the tumor central, there would be a time of different mixed signals.Gastric gastrointestinal stromal tumor accounted for 2% to 3% in stomach tumor, risk generally located in gastric body, gastric antrum incidence was low, form, size were differ, the head can also more than 30 cm, most of the lesions tumors can outside the stomach tumor growth, or to the spread of the peritoneal cavity.Gastrointestinal stromal tumor in small intestine growth accounted for 20% of the entire gastrointestinal stromal tumor, the segment of small intestine all can happen, 30% of the disease develops in the duodenum, jejunum lesions than the ileum, usually occurs in the small intestine of gastrointestinal stromal tumor, a larger tumor volume, high malignant degree and the lesion generally located in the gut, pathological changes is partial mind more, some of the small intestine is kind like aneurysm expansion, its main mechanism is the insufflate shrinkage caused by tumor and nerve plexus is damaged, oral contrast imaging examination can discover more intestinal fistula, the lumen endogenous elders, performance for the intestinal obstruction;Occurred in the anus and the incidence of gastrointestinal stromal tumor of workplace less than one over ten, for the most part in the event of for malignant, more than two-thirds of malignant degree is high, has the possibility of invasion, tumor tissue to grow within the lumen, clear boundary, central necrosis lesions and cysts.The incidence of gastrointestinal stromal tumor of the colon, the lowest is usually malignant stromal tumors of small intestine and stomach tumors metastasis to the colon, generally above the stromal tumors of the colon injury and intestinal wall, spread to the abdominal cavity.Gastrointestinal stromal tumor of the esophagus accounts for a quarter of the entire gastrointestinal stromal tumor, the median age was 63 years old, high malignant degree, usually gastrointestinal stromal tumor of the esophagus with other lesions esophageal identification, small esophageal neoplasm, more for esophageal leiomyoma, if the tumor volume is larger, with advanced esophageal cancer and lymphoma or mediastinum tumor invasion of esophagus.The most common gastrointestinal stromal tumor metastasis to the liver metastasis, metastases CT scans, can show low density, also can show such as density, metastases of necrotic lesions, but seldom hemorrhage after necrosis, if appeared gastrointestinal stromal tumor metastasis, more performance for all multiple abdominal mass, usually small, state clearly, generally do not appear mesenteric invasion and pull.Gastrointestinal stromal tumor lymph node metastasis is rare, so if in tumor tissues surrounding lymph node enlargement, gastrointestinal stromal tumor is unlikely, gastrointestinal stromal tumor metastasis is rare, therefore, gastrointestinal stromal tumor is usually along veins, this feature is also a gastrointestinal stromal tumor and leiomyosarcoma, the main difference between the tumor cell is long and narrow, assumes the wavy, slightly eosinophilic cytoplasm and cell line is not clear, the nuclear spindle, fine on both ends.Gastrointestinal stromal tumor is the most characteristic markers CD117 and main application of immunohistochemical method to detect, and part of the gastrointestinal stromal tumor cells can also express CD34, CAJAL nuclei mast cells in the normal gastrointestinal muscle layer CD117 positive, and smooth muscle cells and vascular smooth muscle cell nucleus nerve fibers not CD117 expression.Gastrointestinal stromal tumor cells originated in the gastric wall muscle layer, Hal mesenchymal cells, its role is largely gastrointestinal nerve plexus rhythm pacemaker cells.Under the electron microscope, the tumor cell surface have more dendritic protrusions, cytoplasm contains a small amount of filaments and neurosecretory granules, similar to CAJAL cells.Usually gastrointestinal stromal tumor to the stomach is greater than 5.5 cm in diameter, in the intestine is greater than 4 cm, fission phenomenon in the stomach is greater than 5/50 HPE, HPE is greater than 1/50, the intestines, tumor characterized by necrosis of appearance, nuclear pleomorphism, tumor cells is rich, active growing, epithelioid cells in cell nests or gland bubble general disgusting degree is high, but now there is no effective indicators can predict the gastrointestinal stromal tumor metastasis and degradation behavior, some experts recommend the size of the tumor and evaluation index of the dangers of nuclear fission as a transfer, but the method is not reliable.Gastrointestinal stromal tumor has long been a diagnosis of leiomyosarcoma and malignant schwannoma, with immunohistochemistry, electron microscope, and the development of molecular biology techniques, find the tumor is C-kit gene mutation and protein expression, only has the new breakthrough progress, many histologically or undifferentiated pluripotent of spindle cells and epithelioid cells.Stromal tumor in nature is not the same with other tumor, on its biological properties can be divided into benign, border and malignant tumor.The performance of the malignant gastrointestinal stromal tumor mainly tumor infiltrating erosion resistance, often at the grassroots invasion or mucous membrane layer invasion, or tumor line distance organs and lymph node metastases.The following indicators can author as potentially malignant gastrointestinal stromal tumor index, tumor diameter greater than 5 cm, fission that is greater than 5/50 HPF, occurrence in tumor tissue necrosis, cancer cells have obvious nuclear atypia, judgement index of benign and malignant, usually with malignant index one or more than two potential malignant index diagnosed as malignant gastrointestinal stromal tumor, if there is only a potential malignant indicators for gastrointestinal stromal tumor border sex, if not the above indexes can be diagnosed as benign.The difference between the gastrointestinal leiomyoma and stromal tumor mainly include:gastrointestinal leiomyoma generally younger age, tumor cells are scarce, form A single, A-Mat (+), CD117 (-), CD34 (-), no C-KIT gene mutations, and gastrointestinal stromal tumor average onset age, tumor cell morphology and changeful, arrangement structure diversity, A-Mat (-), CD117 (+), CD34 (+), AC-KIT gene mutations.Major clinical treatment, gastrointestinal stromal tumor with surgical treatment is given priority to, complete excision is curative effect to improve the performance of the operation, disease-free operation in the operation, and to prevent the tumor has burst in the operation, postoperative recurrence or not surgery patients generally can choose the treatment of tumor molecular targeted drugs, the gastrointestinal stromal tumor the size of its diameter can be from 1 to 2 cm to 20 cm, usually a limited growth, most tumors without complete capsule, sometimes with psuedocapsule, malignant degree is high, sometimes accompanied by necrosis of focal hemorrhages and tumor tissue is broken, and sometimes can wear out mucous membrane layer, leading to the digestive tract ulcer and gastrointestinal stromal tumor generally seen in the submucosa, usually accounted for sixty percent of the whole stromal tumor, lowest proportion is limited to the muscular layer, only one over ten of the entire gastrointestinal stromal tumor, general multidirectional gastrointestinal lumen gastrointestinal stromal tumor growth, polyps in shape, often accompanied by ulcer polyp, to form a subserosal tumors from serous layer growth.Generally located in the interstitial cell tumor mass within the abdominal cavity volume is larger, nodules incision tumor or lobulated, cut red or gray, internal hard, mucosal surface generally have ulceration, hemorrhage, necrosis, mucus and cystic change between can and so on.Surgical resection is the only way to cure early gastrointestinal stromal tumor, general can do local excision and wedge resection, the scope of the general removal is greater than 2 cm tumor capsule, patients at high risk of gastrointestinal stromal tumor recurrence after surgery is high, according to reports, the recurrence rate can reach 80-91%, about 85% of the patients in 2-3 years can relapse after the operation, usually the recurrence process accompanied by the transfer of the liver, once a relapse, the possibility of its further treatment is very low, according to the related research, even if the surgery again, it is hard to improve the survival rate of patients, if found early gastrointestinal stromal tumor, coated no transfer complete, the 5-year survival rate after surgical removal of the half, if coated incomplete resection, the mucous membrane layer, or with incomplete resection,5-year survival rate is less than 35%, after the surgical removal of the tumor cannot be a total survival time of a year.Imatinib, a small molecule inhibitor of both KIT and PDGFRA, has improved the clinical outcome of these patients significantly, with a median overall survival (OS) of 4-5 years in the metastatic phase expected. Imatinib suppresses KIT by its binding to ATP-binding pocket to inhibit the KIT activation and blocks the activation of MAP kinase and PI3 kinase-AKT pathways. However, the frequently-occurred mutations in highly conserved positions on several kinases promote the Imatinib resistance and reduce its inhibitory effects. And the observed shift towards the active form of these kinases would allow ATP to outcompete the inhibitor. Therefore, alternative strategies are needed to inhibit the KIT activity, such as approaches via posttranscriptional and posttranslational mechanisms.microRNAs (miRNAs) are a class of endogenous small non-coding RNAs regulating gene expression in a wide range of cellular processes of various organisms. Recently, numerous miRNAs have been recognized to target the 3’untranslated regions (UTRs) of oncogenic or tumor suppressive genes, and thus regulate the cancer tumorigenesis. miRNA expression has been recently reported to exert a relevant role in the GIST biological processes such as tumorigenesis, progression, prognosis and drug resistance. Overexpressed levels of miR-125a-5p and miR-107 have been indicated to be associated with Imatinib resistance in GIST via regulating the expression of PTPN18. miRNA-218 negatively regulates KIT expression and inhibits the GIST cell proliferation and invasion, and thus regulates the Imatinib sensitivity of GIST cells through PI3K/AKT pathway.matinib for protein complex amino acid kinase small molecule inhibitors, and complex amino acid kinase can catalytic substrate phosphorylation, transmit signals inside the cell, stimulate cell proliferation, complex amino acid kinase active site are ATP in combination with pockets, complex amino acid kinase activity is through catalytic ATP transfer phosphate groups exactly the coning amino acid residues of content, known as the phosphorylation of kinases.Imatinib is the first indication of chronic myelogenous leukemia, indications and the second is a gastrointestinal stromal tumor.Imatinib to selectively make PDGERA receptor kinase inhibitory amino acid for the middle-late or surgery can’t treatment for gastrointestinal stromal tumor, preventive treatment in patients with malignant metastasis or dangerous, for improving the prognosis of patients and prolong survival time has the vital significance.Imatinib resistance is mainly due to gastrointestinal stromal tumor cells expressing wild-type KIT or exon 9 mutations, the main mechanism is mutations can lead to the stability of the KIT structure, to prevent the rest of the combination of imatinib.Imatinib as selective KITPDGFRA winding glycine receptor kinase inhibitor, is mainly used in not surgical resection, or transfer cases after resection, or recurrence after resection of patients, after complete resection or preventive treatment, in order to prevent the gastrointestinal stromal tumor recurrence.Usually, if the surgical treatment and found the following phenomenon is highly suspected recurrence of gravity, the first is the removal of part or the original site and discover new tumor tissue, or the original site of distant metastases occur around or the patient is in the process of implementation of imatinib therapy found that the tumor is gradually increasing in oven, imaging diagnosis, found that increased density of the original site., if can directly for surgical treatment of surgery as soon as possible, if not surgery, to advance imatinib therapy after surgery if possible.Research has shown that surgery combined imatinib and chemotherapy drug combination therapy can significantly improve the prognosis of gastrointestinal stromal tumor.But, in the process of imatinib therapy of gastrointestinal stromal tumor resistant rate is higher, to 70%, even some patients at the beginning of the performance for the treatment of drug resistance, general incidence was 20%, usually imatinib six months after a continuity of drug in the treatment of gastrointestinal stromal tumor found to tumor growth, and growth of the multiple lesions, general drug resistance in patients with its genotype of GIST wild-type KIT mutation of exon 9;Secondary main academic point of view is now resistant mutations can lead to the stability of the KIT, the stability of the KIT block the KIT structure domain and the combination of imatinib and other parts of mutations is exon 12,14 and 17. Micrornas-21 and cancer research in recent years, a large number of real-time fluorescence PCR or western blot experiments found that micrornas-21 in a variety of positive expression in cancer tissues or cells, mainly including the glial cell tumor, digestive system tumors (stomach cancer, esophageal cancer and bile duct cancer, liver cancer, oral cancer) reproductive system cancer (ovarian cancer, cervical cancer), and so on.As mentioned carcinogenic mechanism of micrornas-21 is not yet clear, mainly because of its targets is difficult to confirm, but in recent years, with the use of Northern blotting method, in-depth study of all the bioinformatics analysis technology, now the main findings of micrornas-21 main target genes are 33 loci, the main locus of PTEN, the main role is to inhibit cell migration.Proliferation, the main NFTB, play a role of transcriptional inhibitory factor, STAT3, regulation of transcription, cell movement, etc.APAF-1, regulation of cell apoptosis, etc.The above factors are confirmed by the miRNA-21 direct target genes, the dynamic changes of the above factors are directly regulated by miRNA-21.But there are not direct target genes of micrornas-21, but also by the regulation of micrornas-21, for example, the BCL-2 and TIMP3 etc, they are often mediated by other factors in order to control by micrornas-21.The above factors are associated with the miRNA-21, but also many process and associated micrornas-21, not only is the coordination of micrornas-21 other micrornas work together, as a result, the miRNA-21 regulation process is more complex network structure, some scholars research target genes that are sensitive to alcohol, found the JAG-1 is the miRNA-153, the miRNA-335 and the miRNA-21 common genetic targets, knock out one of these alone, will not affect the sensitivity of the nerve cells to alcohol.The expression of micrornas-21 process is usually in the nucleus by the RNA polymerase, transcription precursor formation by processing into the hairpin to the cytoplasm, the final shear mature. In the eighty s, the Japanese ten copies of the first from the huo gold lymphoma cells chromosome in the Bcl-2 gene was isolated, and the existence of the gene is associated with the Bcl-2 protein expression.The Bcl-2 protein size to 26 kd, which contains BH4 homologous structure, the structure and the structure of antiapoptotic proteins, BH3 structure is similar to other promoting apoptosis protein structure, so the role of Bcl-2 protein to the ratio of its two homologous structures, high proportion of antiapoptotic structure, the Bcl-2 protein appeared antiapoptotic effect, and vice versa.The Bcl-2 mainly exist in the mitochondrial outer membrane and nuclear membrane and tastes on the retina.The Bcl-2 protein family are divided into two kinds, one kind is to promote apoptosis proteins, such as the Bcl-XL, the Bcl-W, such as A1, promoting apoptosis proteins BAX, BAK, etc.Usually the Bcl-2 family is divided into three categories, antiapoptotic Bcl-2 protein contained in the main structure of BH1 BH2/BH3/BH4, directly or indirectly inhibit cell death, promoting apoptosis proteins BAX, BAK, its structure mainly contains BH1/BH2/ BH3, can promote cell apoptosis, inhibit the action of the cancer cells to generate.BH3-only, its main function for sensitization agent and inducers, can promote the apoptosis effect, can promote the role of inhibiting apoptosis.The Bcl-2 inhibits apoptosis mainly has five pathway, maintain the stability of cell calcium state, respectively, to prevent the mitochondrial membrane potential decreased;Inhibition effect on promoting apoptosis of BAX/BAK cell toxicity, maintain the stability of blocks the formation of the other polymers.Protect mitochondrial membrane function inhibition of the mitochondrial cytochrome C release, AIF, such as to promote the release of apoptosis protein, at the same time can reduce the formation of oxidation and oxidation and cells. Mutations of KIT or PDGFRA, both of which are tyrosine kinase receptor genes, occur in about 70-80% GIST cases, and are recognized to be the most known molecular event in GIST pathogenesis and development. The downstream molecular pathways upon to the KIT mutation include PI3 kinase-AKT, Src family kinase, Ras-ERK, and JAK-STAT. Activation of these molecular pathways, in response to KIT activation, results in GIST tumorigenesis through cell proliferation activation and apoptotic signal inhibition.The aim of the present study was to investigate the expression of miRNA-21 in GIST specimens, and explore the association of miRNA-21 with Imatinib response, clinic-pathological features of GIST patients. We then explored the functional and potential role of miRNA-21 in Imatinib sensitivity of GIST-T1 cells.ObjectivemiRNA-21 has recently been recognized to tumor suppressive in various types of cancers. However, the role of miRNA-21 in gastrointestinal stromal tumors (GISTs) is still ambiguous. In this study, we investigated the expression and the role of miRNA-21 on the development and Imatinib-sensitivity of GISTsMethodGIST tissue samples, GIST-T1 cell culture and treatmentThe 31 GISTs included in this study were identified in the Department of internal at The affiliated Hospital of Inner Mongolia medical university and Nanfang hospital of Southern medical university between June 2012 and 2014 August for molecular marker studies. Authorization to use these tissues for research purposes was obtained from the Institutional Review Board of The affiliated Hospital of Inner Mongolia medical university and Nanfang hospital of Southern medical university. All 31 specimens were permitted with written consent from each patient for scientific research. Clinic-pathologic characteristics including anatomic site, risk, and tumor size were indicated in Table 1.RNA preparation and TaqMan miRNA assayTotal mRNA samples from the GIST/control gastric specimens or from GIST-T1 cells were extracted with the TRIzol reagent (Life Technologies, Grand Island, NY, USA) and were added with 1μl SUPERase·InTM RNase Inhibitor (Thermo Scientific, Rockford, IL, USA). The quantitative analysis of Bcl-2 mRNA level was performed with Takara One Step RT-PCT kit (Takara, Tokyo, Japan). And the RT-qPCR was performed at 42℃ for 5 min, and then at 95℃ for 10 sec for the reverse transcription, at 95℃ for 5 sec and at 60℃ for 20 sec for the PCR reaction, with 40 cycles. The miRNA samples were isolated from GIST-T1 cells with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). And the expression of miRNA-21 were quantified by the mirVanaTM qRT-PCR miRNA Detection Kit (Thermo Scientific, Rockford, IL, USA) on the Applied Biosystems 7300 Real-Time PCR system. Fold changes of Bcl-2 or miRNA-21 were calculated, with β-actin or U6 as internal control, using the formula 2-(△△Ct), where △△Ct is △Ct(stimulus)-△Ct(solvent), △ct is Ct (target gene)-Ct(contol gene) and the Ct is the cycle, at which the threshold is crossed. Basal expression levels were calculated using the formula 2-(△Ct).Luciferase reporter assayFor the Luciferase reporting assay, the pGL3-luciferase vector (Promega, Madison, WI, USA) was inserted with three copies of miRNA-21-targeted sites in 3’ UTR of Bcl-2 (for Bcl-2 Reporter) or with three copies of mutant 3’UTR of Bcl-2 sequence (for Bcl-2mut Reporter) post the luciferase coding sequence. And the Bcl-2 Reporter or the Bcl-2mut Reporter was transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) into the 85%-confluent GIST-T1 cells, which were also transfected with 30 or 60 nM miRNA-21 or control mimics. Post an inoculation for 24 hours, the luciferase activity was assayed with the dual luciferase reporter assay kit (Promega, Madison, WI, USA).Cell proliferation assay, MTT assay and apoptosis assayThe GIST-T1 cells were seeded in a 12-well plate (300 cells per well),12 hours later, cells were treated with 0 or 5 μM Imatinib and were transfected with 60 nM nontargeting miRNA or miRNA-21. Then cells were incubated at 37 ℃ containing 5% CO2, for 96 hours. Then the GIST-T1 cells were stained with crystal violet for 30 minutes and the colony numbers were counted. MTT assay was performed to examine the cellular viability of GIST-T1 cells post treatment. In brief, GIST-T1 cells were seeded in 96-well plates to 85% confluence, and then were treated with 0 or 5 μM Imatinib and were transfected with 60 nM nontargeting miRNA or miRNA-21 for 0,24 or 48 hours. Then cells were updated with 1×MTT solution for 2-hour incubation at 37 ℃. The optical density was then measured at 450 nm using a spectrophotometer. The cell viability was expressed as relative viable cells (%) to control GIST-T1 cells. Apoptosis induction in GIST-Tl cells post Imatinib treatment or (and) miRNA transfection was examined by flow cytometry. GIST-T1 cells were firstly stained with the Annexin V-FITC Apoptosis Detection Kit (Abcam, Cambridge, UK) according to the product’s manual, and then was assayed with a flow cytometry.ResultsDownregulated miRNA-21, in association with an upregulated Bcl-2 mRNA levelA total of 31 human GIST specimens from patients were analyzed in this study. Clinical characteristics of these GIST patients were presented. We quantified the levels of miRNA-21 and Bcl-2, in these GIST specimens. The mean relative miRNA-21 level to U6 was 0.6729 ± 0.07632 in the GIST specimens, markedly lower than 1.0000 ± 0.1085 in the normal gastric tissues (p= 0.0129). However, the mRNA level of Bcl-2 was significantly upregulated in these GIST specimens to 1.500± 0.1484, compared to 1.0000±0.1193 in the control groups (p= 0.0103). In addition, we correlated miRNA-21 level with Bcl-2 mRNA level in the specimens, as shown in Figure 1C, there was a significant negative correlation between the Bcl-2 mRNA level and the miRNA-21 level (R2= 0.2450, p= 0.0046). Taken together, miRNA-21 was downregulated, in an association with upregulated Bcl-2, in GIST specimens.miRNA-21 downregulates Bcl-2 via targeting the 3’UTR of Bcl-2 in GIST-T1 cellsTo further explore the correlation between the miRNA-21 downregulation and the Bcl-2 upregulation in GISTs, we promoted the miRNA-21 level by transiently transfecting miRNA-21 mimics into GIST-T1 cells, and then examined the Bcl-2 expression. Figure 2A indicated that there was a marked upregulation of miRNA-21 level in the GIST-T1 cells, by the miRNA-21 mimics transfection (p<0.001 or p<0.0001 for 30 or 60 nM), compared with the control miRNA-transfected cells. In contrast, the Bcl-2 mRNA level was significantly downregulated by the miRNA-21 mimics transfection (p<0.05 or p<0.01 for the 30 or 60 nM). Moreover, the Bcl-2 downregulation was reconfirmed in protein level by the miRNA-21 mimics transfection (p<0.05 or p<0.01).To identify whether the Bcl-2 downreguation by miRNA-21 was the targeting inhibition 3’UTR of Bcl-2, we performed the luciferease reporter assay with the reporter plasmid with the 3’UTR of Bcl-2 or with the 3’UTR of Bcl-2 (Bcl-2mut reporter) in GIST-T1 cells, which were transfected with miRNA-21 mimics or control miRNA. The construction of the reporter plasmid or the Bcl-2mut reporter was indicated in Figure 3A. And the reporting assay demonstrated that the transfection with 30 or 60 nM miRNA-21 mimics significantly reduced the luciferease activity than the control miRNA (p<0.001 for 30 or 60 nM). However, there was no such luciferase reduction by miRNA-21 mimics with the Bcl-2mut reporter. Therefore, we confirmed that the miRNA-21 targeted the 31 UTR of Bcl-2 and inhibited Bcl-2 expression in GIST-T1 cells.miRNA-21 sensitizes GIST-T1 cells to ImatinibTo further investigate the regulatory role of miRNA-21 on the sensitivity of GIST-T1 cells to Imatinib, we performed the colony assay in GIST-Tl cells. As indicated, the treatment with 5 μM Imatinib markedly reduced the colonies which were formed by GIST-T1 cells (p<0.01, column 3 vs column 1). Moreover, such reduction was more significant in the GIST-T1 cells which were transfected with 60 nM miR-21 mimics, than with miR Control (p<0.001 p<0.05).We also examined the levels of viability and apoptosis in the Imatinib-treated GIST-T1 cells which were transfected with 60 nM miR-21 mimics or with miR Control. Figure 5A demonstrated that the transfection with 60 nM miR-21 mimics or with miR Control posed no regulation on the viability of GIST-T1 cells. However, the cellular viability significantly decreased in the GIST-T1 cells, post the treatment with 5 μM Imatinib at 24 or 48 hour post treatment (p<0.05 or p<0.01). Moreover, such viability reduction was more significant when cells were transfected with 60 nM miR-21 mimics than with miR Control (p<0.05 for 24 or 48 hours post treatment,). In addition, Imatinib induced apoptosis in GIST-T1 cells (p<0.01 or p<0.001); and the Imatinib-induced apoptosis was also markedly aggravated by the miR-21 mimics transfection (p<0.05 for 24 or 48 hours post treatment). Therefore, miRNA-21 sensitizes GIST-T1 cells to Imatinib.research. Clinic-pathologic characteristics including anatomic site, risk, and tumor size were indicated in Table 1.RNA preparation and TaqMan miRNA assayTotal mRNA samples from the GIST/control gastric specimens or from GIST-T1 cells were extracted with the TRIzol reagent (Life Technologies, Grand Island, NY, USA) and were added with 1μl SUPERase·InTM RNase Inhibitor (Thermo Scientific, Rockford, IL, USA). The quantitative analysis of Bcl-2 mRNA level was performed with Takara One Step RT-PCT kit (Takara, Tokyo, Japan). And the RT-qPCR was performed at 42℃ for 5 min, and then at 95℃ for 10 sec for the reverse transcription, at 95℃ for 5 sec and at 60℃ for 20 sec for the PCR reaction, with 40 cycles. The miRNA samples were isolated from GIST-T1 cells with the mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). And the expression of miRNA-21 were quantified by the mirVanaTM qRT-PCR miRNA Detection Kit (Thermo Scientific, Rockford, IL, USA) on the Applied Biosystems 7300 Real-Time PCR system. Fold changes of Bcl-2 or miRNA-21 were calculated, with β-actin or U6 as internal control, using the formula 2-(△△Ct), where △△Ct is △Ct(stimulus)-△Ct(solvent), △Ct is Ct (target gene)-Ct(contol gene) and the Ct is the cycle, at which the threshold is crossed. Basal expression levels were calculated using the formula 2-(△Ct).Luciferase reporter assayFor the Luciferase reporting assay, the pGL3-luciferase vector (Promega, Madison, WI, USA) was inserted with three copies of miRNA-21-targeted sites in 3’ UTR of Bcl-2 (for Bcl-2 Reporter) or with three copies of mutant 3’ UTR of Bcl-2 sequence (for Bcl-2mut Reporter) post the luciferase coding sequence. And the Bcl-2 Reporter or the Bcl-2mut Reporter was transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) into the 85%-confluent GIST-T1 cells, which were also transfected with 30 or 60 nM miRNA-21 or control mimics. Post an inoculation for 24 hours, the luciferase activity was assayed with the dual luciferase reporter assay kit (Promega, Madison, WI, USA).Cell proliferation assay, MTT assay and apoptosis assayThe GIST-T1 cells were seeded in a 12-well plate (300 cells per well),12 hours later, cells were treated with 0 or 5 μM Imatinib and were transfected with 60 nM nontargeting miRNA or miRNA-21. Then cells were incubated at 37 ℃ containing 5% CO2, for 96 hours. Then the GIST-T1 cells were stained with crystal violet for 30 minutes and the colony numbers were counted. MTT assay was performed to examine the cellular viability of GIST-T1 cells post treatment. In brief, GIST-T1 cells were seeded in 96-well plates to 85% confluence, and then were treated with 0 or 5 μM Imatinib and were transfected with 60 nM nontargeting miRNA or miRNA-21 for 0,24 or 48 hours. Then cells were updated with 1× MTT solution for 2-hour incubation at 37 ℃. The optical density was then measured at 450 nm using a spectrophotometer. The cell viability was expressed as relative viable cells (%) to control GIST-T1 cells. Apoptosis induction in GIST-T1 cells post Imatinib treatment or (and) miRNA transfection was examined by flow cytometry. GIST-T1 cells were firstly stained with the Annexin V-FITC Apoptosis Detection Kit (Abcam, Cambridge, UK) according to the product’s manual, and then was assayed with a flow cytometry.ResultsDownregulated miRNA-21, in association with an upregulated Bcl-2 mRNA levelA total of 31 human GIST specimens from patients were analyzed in this study. Clinical characteristics of these GIST patients were presented. We quantified the levels of miRNA-21 and Bcl-2, in these GIST specimens. The mean relative miRNA-21 level to U6 was 0.6729 ± 0.07632 in the GIST specimens, markedly lower than 1.0000 ± 0.1085 in the normal gastric tissues (p= 0.0129). However, the mRNA level of Bcl-2 was significantly upregulated in these GIST specimens to 1.500 ± 0.1484, compared to 1.0000±0.1193 in the control groups (p= 0.0103). In addition, we correlated miRNA-21 level with Bcl-2 mRNA level in the specimens, as shown in Figure 1, there was a significant negative correlation between the Bcl-2 mRNA level and the miRNA-21 level (R2= 0.2450, p= 0.0046). Taken together, miRNA-21 was downregulated, in an association with upregulated Bcl-2, in GIST specimens.miRNA-21 downregulates Bcl-2 via targeting the 3’UTR of Bcl-2 in GIST-T1 cellsTo further explore the correlation between the miRNA-21 downregulation and the Bcl-2 upregulation in GISTs, we promoted the miRNA-21 level by transiently transfecting miRNA-21 mimics into GIST-T1 cells, and then examined the Bcl-2 expression. Figure 2 indicated that there was a marked upregulation of miRNA-21 level in the GIST-T1 cells, by the miRNA-21 mimics transfection (p<0.001 or p<0.0001 for 30 or 60 nM), compared with the control miRNA-transfected cells. In contrast, the Bcl-2 mRNA level was significantly downregulated by the miRNA-21 mimics transfection (p<0.05 or p<0.01 for the 30 or 60 nM). Moreover, the Bcl-2 downregulation was reconfirmed in protein level by the miRNA-21 mimics transfection (p<0.05 or p<0.01).To identify whether the Bcl-2 downreguation by miRNA-21 was the targeting inhibition 3’UTR of Bcl-2,... |
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