| BackgroundLung cancer is the most lethal cancer so far with its significant clinical features of obscure pathogenesis, easy recurrence and metastasis. It is reported that stem-cell-like cells, a small minority of malignant tumor, are universally called cancer stem cells(CSCs) or known as tumor initiating cells(TICs) and play an important role in the genesis, progression, recurrence and metastasis. There are also cancer stem cells in lung cancer, namely lung cancer stem cells(LCSCs) and play the same important role in the genesis, progression, recurrence and metastasis of lung adenocarcinoma. In other words, lung cancer is also a kind of stem cell disease, which makes it a new treatment strategy to study on lung adenocarcinoma stem-cell-like cells.Apart from chemotherapy resistance, stronger invasion and metastasis ability, the essential difference between cancer stem cells and common cancer cells is its stem cell feature, namely self-renewal. The genesis, maintenance, and invasion of tumor are closely related to inthe self-renewal capacity of cancer stem cells. Therefore, it is essential to study the mechanism of self-renewal ability in cancer stem cells.The self-renewal capacity of cancer stem cells is regulated by not only the inner signals but also mi RNAs, the small non-coding single-strain RNA molecules discovered in recent years that are 19-22 nucleotides long and regulate the target protein expression of target DNA post-transcriptionally. Some studies indicate the regulatory function of mi RNAs in cancerous genesis and development cannot be ignored. miRNAs are very important in proliferation, differentiation, apoptosis, drug-resistance and recurrence of cancer cells like oncogene or tumor suppress gene. mi RNA regulates various target genes and forms sequently enlarged gene regulation pathways. Moreover, miRNA can also be regulated by various genes and forms a large complex gene regulatory network. So far, studies have indicated that mi RNAs participate in the regulation of self-renewal and multi-directional differentiation potential of stem cells and cancer stem cells, which is the hot spot in miRNA research. For example, mi R-290, mi R-296 regulate the self-renewal and multi-directional differentiation of the embryonic stem cells through the self-renewal of related target transcription factor Oct-4, Tcf3, Sox-2 and Nanog. Let-7 regulates the self-renewal and malignant proliferation of breast cancer stem cells through H-ras and HMGA2. So how is the self-renewal of lung cancer stem cells regulated? Is there any mi RNA that regulates the self-renewal of lung cancer stem cells? How do they work? And what do they mean to the guidance of clinical work? This research will focus on all these problems.Objectives1. To test the expression of mi R-214 in LAC CSLCs.2. To find out the function of mi R-214 in LAC CSLCs self-renewal.3. To elucidate the mechanism which mi R-214 regulates LAC CSLCs self-renewal.MethodsWe adopted the following research methods according to the objectives above:1. Detect the expression of miR-214 in lung cancer stem-like cells(CSLCs).(1)Overexpressed miR-214 and compared the stem cell markers between non-CSLCs and CSLCs derived from A549, NCI-H1650 and lung cancer cell line by real-time PCR.(2) Overexpressed miR-214, tested the self-renewal ability by sphere formation assay and the percentage of CD133+/CD326+ by flow cytometry in A549, NCI-H1650 lung cancer cell lines.(3) To test the miR-214 expression in non-CSLCs and CSLCs derived from A549, NCI-H1650 lung cancer cell line by real-time PCR. To test the miR-214 expression during differentiation by re-differentiation test of CSLCs derived from A549, NCI-H1650.2.Functional assessment of miR-214 in the regulation of self-renewal of CSLCs.(1)Constructed sh-mi R-214 lentivirus, studied the effect of mi R-214 on self-renewal of CSLCs derived from A549, NCI-H1650 lung cancer cell line and primary lung cancer cell line in vitro by sphere formation assay.(2)Constructed the sh-miR-214 lentivirus, tested the expression of stem cell related markers(Nanog, Oct-4, Sox-2, CD326) by real-time PCR, immunofluorescence technique and western-blot.(3)Constructed the sh-miR-214 lentivirus, studied the effect of mi R-214 on self-renewal-related signal pathways using PCR array.(4)Constructed the sh-miR-214 lentivirus, studied the effect of mi R-214 on tumorigenic ability of CSLCs derived from NCI-H1650 lung cancer cell line by xenograft experiment in nude mice.3.Investigate the target of mi R-214 on LAC CSLCs.(1)Selected the target gene of miR-214 by bioinformatics methods and self-renewal-related genes and validated the gene by dual-luciferase report gene, real-time PCR, western-blot and immunofluorescence technique.(2)Transfected the sh-mi R-214& sh-CTNNBIP1 dually, validated the effect of mi R-214 on LAC CSLC through target gene CTNNBIP1 by sphere formation assay, real-time PCR, western-blot and immunofluorescence.(3)Studied the clinical significance of CTNNBIP1 on stage IIIa lung adenocarcinoma patients by immunohistochemical methods, etc.Results1. miR-214 over-expression enhances CSLCs properties of LAC cellsWe tested the effect of miR-214 on sphere-forming ability of A549, NCI-H1650 lung cancer cells and validated the expression of stem cell markers by mi R-214-overexpressed lentivirus technique; compared the expression difference of mi R-214 between non-CSLCs and LAC CSLCs derived from A549, NCI-H1650 lung cancer cell line and primary lung cancer; results of sphere formation assay and real-time PCR indicated sphere-like growth in A549, NCI-H1650 lung cancer cells with its stem cell percentage and marker expression higher than those in control group; results of real-time PCR also showed a higher expression of mi R-214 in CSLCs derived from A549, NCI-H1650 and primary lung cancer than that in non-CSLCs; it showed a decreasing tendency of miR-214 in re-differentiation. These results indicated important function of mi R-214 in self-renewal of LAC CSLCs. Therefore we chose to study on the effect of mi R-214 on LAC CSLCs self-renewal further.2. miR-214 regulates the self-renewal capacity and stemness of LAC CSLCs in vitro and in vivo.In order to further elucidate the function of miR-214 in LAC CSLCs, we transfected and made expression-stable LAC CSLCs by constructing the sh-mi R-214 lentivirus. We proved that mi R-214 promoted the self-renewal of LAC CSLCs by comparing the number and size of spheres between sh-mi R-214 transfection group and control group. Real-time PCR suggested that CSLCs derived from A549, NCI-H1650 showed a significant decrease of mRNA expression of stem cell markers(Nanog, Oct-4, CD326) by contrast with control group. The results of western-blot and immunofluorescence technique further proved evident protein expression decrease of Nanog, Sox-2 and Oct-4 in sh-mi R-214 transfection group. Results of PCR array indicated that the self-renewal-related signaling pathways were somewhat affected after mi R-214 down-regulation. Results from above suggested that mi R-214 promoted the self-renewal of LAC CSLCs. Taken together, xenograft experiment in nude mice proved the ability of mi R-214 to promote tumorigenesis of LAC CSLCs in vivo.3. CTNNBIP1 is a direct target of mi R-214 and the role of mi R-214 in regulating self-renewal of LAC CSLCs depends on the target gene CTNNBIP1.We predicted the potential target gene of mi R-214 through bioinformatics and discovered wnt pathway was the most severely affected one with mi R-214 down-regulation. We further screened out CTNNBIP1 to be the direct target gene of miR-214 by dual-luciferase report gene. Real-time PCR also showed the evident negative correlation between mRNA expression of CTNNBIP1 and miR-214 in lung cancer samples. Western-blot and immunofluorescence showed the significantly increased CTNNBIP1 protein expression after mi R-214 down-regulation. In order to clarify whether mi R-214 participated in self-renewal of cancer stem cells through target gene CTNNBIP1, we transfected sh-mi R-214&sh-CTNNBIP1 dually and examined the changes of the number and size of spheres in LAC CSLCs. We found that CSLCs with dually transfected were significantly higher than those in sh-miR-214 transfection group. Results of western-blot indicated that, compared with the sh-miR-214 transfection group, there showed significantly lower CTNNBIP1 expression and higher β-catenin, Cyclin D1, Oct-4, Sox-2 expression in sh-mi R-214&sh-CTNNBIP1 transfection group. Therefore, these results suggested that miR-214 participated in self-renewal of LAC CSLC through CTNNBIP1.To validate whether the expression of CTNNBIP1 in lung adenocarcinoma will affect the survival prognosis of the patients, we detected the CTNNBIP1 expression in pathological lung adenocarcinoma tissues of stage IIIa patients and discovered higher CTNNBIP1 expression in high-differentiation lung cancer tissues than low- differentiation lung cancer tissues. Statistical analysis revealed that CTNNBIP1 expression was positively correlated with the differentiation degree of lung adenocarcinoma while negatively related with Ki67 expression. Further study on Kaplan-Meier survival curve found the group with high CTNNBIP1 expression lived longer than lower-expression group, indicating a close correlation between CTNNBIP1 and prognosis of lung adenocarcinoma.ConclusionThe expression of miR-214 is significantly higher in LAC CSLCs and promotes the self-renewal in vivo and tumorigenesis in vivo through CTNNBIP1. Clinically, CTNNBIP1 expression was correlated with longer overall survival time in LAC patients. |
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