| BackgroundNon-small cell lung caner (NSCLC) is a highly leading cause of tumor mortality. Inrecent decades, the survival of patients with has changed better due to the modifications indiagnosis and combined modality therapy, including neo-chemotherapy, high-precisionradiotherapy, and molecular targeted therapy, however, the overall5years survival haschanged little.Increasing evidence reveal that tumor mass contain a rare subpopulation, termedcancer stem cells(CSCs) or tumor initiating cells(TICs), possessing stronger capacity ofself-renewal, invasion and therapy-resistance, resulting in the collapse and metastasis. It isreported that TICs also reside in lung adenocarcinoma but its molecular markers remainsome controversial. Existing researches considered that CD133, a commonly molecularmarker, could represent TICs in primary lung cancer samples but its alone could not be aTICs marker in lung cancer cell lines including A549. Based on the feature ofdrug-resistance, the survival cells can represent TICs.Emerging evidence demonstrates that miRNAs are important regulators of TICs andcan serve as oncogenes or tumor suppressors involved in tumorigenesis and progression.Thus, a better recognition of abberant miRNAs in lung TICs might help to explain thepotential mechanism of lung cancer bio-behavior.ObjectiveBeing little research has been performed on the miRNAs in lung TICs, this study isaim to obtain the lung adenocarcinoma initiating cells from A549cell line based onpaclitaxel treatment combination with serum-free cultivation and to validate the spared cellscan represent the TICs. Next, we will investigate the abberant miRNAs expression in TICscompared to the normal A549cells with microarray and perform quantitative RT-PCR tovalidate the array data. We want to establish a systemic recognition of miRNAs in lungTICs and partly reveal the mechanism between lung TICs and these miRNAs. Methods1. In order to avoid the controversial on markers of lung tumor initiating cells, wecombined paclitaxel with serum-free cultivation to obtain TICs from A549. At the secondpassage after serum-free cultivation, paclitaxel was added at a dilution of100nmol/L for48h and then placed fresh medium once or twice per week until the new spheres appeared.2. To identify the survival cells possess the stemness, we carried out some experiments invitro and in vivo including flow cytometry analysis, immunofluores cence,immunohistochemistry on differentiation cells, xenografts on nude mice and detection ofCSCs-associated genes expression to validate this subpopulation can be regarded as tumorinitiating cells.3. MicroRNA array was performed to screening the abberant miRNAs expressionbetween this spared subpopulation and normal A549cells. As a further validation,10miRNAs were chosen for quantitative RT-PCR.4. Immunofluorescence was used to confirm if CD133+/CD326+subpopulation residesin the primary lung adenocarcinoma samples. Followed by magnetic activated cell sorting, weobtained the TICs and narrowed the array data to the future researches.Results1. Normal A549cells were resuspended in serum-free medium for expanding. At thesecond passage, paclitaxel was added into the culture for48h. After replaced with freshmedium, most spheroids were clearaged and the survival cells could gradually regrow intospheres approximately during a period of two weeks.2. The survival cells could expand continuously in vitro. At the4thpassage,immunofluorescence showed most of the sphere cells positive for CD133/CD326andcytometric showed about86%cells positive for CD133/CD326. Under condition ofdifferentiation, the spheroids-derived cells had same morphology with the normal A549cellsat the sixth day and almost all the cells expressed the differentiation antigens CK8or CK18.Xenografts on nude mice showed that1×104/ml CD133+/CD326+cells could generatexenografts while1×105/ml normal A549cells failed to do so. The CSCs-associated genesexpression including CD133, CD326, Nanog, Nestin, and OCT-4were tested by quantitativeRT-PCR and showed all higher expression in CD133+/CD326+subpopulation compared to thenormal A549cells. 3. MicroRNA array was performed to screening the abberant miRNAs expression inimmunofluorescence compared to the normal A549cells. We found36miRNAsdown-regulation and14miRNAs up-regulation. Considering tumorigenesis, invasion,therapy-resistant and epithelial-mesenchymal transition (EMT), we chose10miRNAs forquantitative RT-PCR including miR-29ab, miR-155, miR-127-3p, miR-17, miR-663, miR-183,miR-125, miR-520h, miR-18b.4. We performed immunofluorescence to detect whether the CD133+/CD326+subpopulation reside in the primary lung cancer samples and found the CD133+/CD326+cellsco-located in the same area in fresh sections. Next, we successfully isolated the doublepositive cells with magnetic activated cell sorting. The selected10miRNAs were alsovalidated on primary samples to narrow the future research range and enhance the cliniccorrelation and found the expression trend of miR-183, miR-17-5p, miR-127-3p.wereconsistent with the array data on A549cell line and primary samples level.Conclusions1. Paclitaxel treatment combination with serum-free cultivation could effectively enrichlung adenocarcinoma initiating cells from A549.2. The survival cells, marked by CD133/CD326, defined the features of stemnessincluding self-renew, multi-differentiation, stronger tumorigenesis, higher expression ofCSCs-associated genes and could be regarded as lung adenocarcinoma initiating cells.3. We found50abberant miRNAs expression in CD133+/CD326+subpopulation andchose10miRNAs to perform quantitative RT-PCR and6miRNAs expression trend wereconsist with array data.4. The CD133+/CD326+subpopulation reside in the fresh sections. Narrowed byvalidation on primary samples, miR-183, miR-17, miR-127expression trend were consistwith the result of cell line. We found most the potential target genes of above miRNAs wereinvolved in the cell cycle, invasion, transcription, signal transduction, and play an importantrole in regulating tumorigenesis and progression.
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