| Part I The construction of GCSC cell line,and detection of the CD 133 expression in GCSC cell line.Background:Gastric cancer is the fourth most common cancer and the second leading cause of malignancy-related death for both sexes in the world.Cancer stem cells(CSC)are a subgroup of tumor cells that have the capacity of self-renewal and multilineage differentiation.The cancer stem cells not only initiate tumor development,metastasis and recurrence,but also make routine treatment of cancer unsuccessful.To identify the gastric cancer cells,several studies have been conducted on some cell surface markers.CD 133(prominin-1 gene)on chromosome 4p15 encodes a transmembrane glycoprotein with a molecular weight of 120 kDa involved in stem cells attachement to their niche,maintenance of cell polarity and migration through interactions of cells with each other and the matrix.This marker was originally identified as a marker of hematopoietic stem cells,but then its association with brain cancer,kidney,ovary,liver,colon and gastric cancers has been reported.MKN-45 cells have demonstrated tumorigenicity in vivo and are greater resistant to radiotherapy compared to the other cell lines,which seems to closely resemble gastric cancer stem cells(GCSCs)that are prone to metastasis,refractory,and resistance to conventional treatment.CD44 has been shown to be a potential marker of GCSCs.In addition,CD 133-expressing GCSCs have been identified as stem-like cells in molecular imaging and are also implicated as target to overcome therapeutic resistance in GCSCs.Aim:To construct the GCSC cell line,and detect the expression of CD133 in GCSC cell line.Methods and results:Through FACS assay,CD133+ and CD133-MKN-45 cells were clearly separated.CD133+ or CD133-cells were collected,and the protein level of CD 133 was determined after several passages by immunofluorescence staining.Furthermore,CD44v8-10 is a cancer-specific marker for GCSCs;we found that CD133 and CD44v8-10 merged perfectly,which indicated the GCSCs could be enriched with CD 133+ as the specific marker.Moreover,consistent with CD 133 level,several key factors for stemness,including OCT4,SOX2,and NANOG,were significantly increased at transcriptional level and protein levels in CD 133+ cell subset,compared with those in CD 133-cell subset.Taken together,these data validated the stem cell characteristics of CD133+ subset isolated from MKN-45 gastric cancer cell line.Next,we proceeded to evaluate the expression of IncRNA ROR in GCSCs.Both CD133+ and CD133-subsets showed detectable levels of IncRNA ROR,whereas CD133+ subset presented a strikingly higher level than that in CD 133-subset.Consistently,we analyzed the mRNA levels of CD133 and the lncRNA ROR in cancer tissues obtained from 33 gastric cancer patients and found that the expression of lncRNA ROR was positively correlated with CD 133 level.Conclusion:Hence,our results suggested that ROR was closely related to CD 133 and might be involved in the development of GCSCs.Part Ⅱ The influence of IncRNA ROR in the proliferation,invasion and apoptosis in GCSCs.Background:The proliferation and differentiation of CSC are tightly regulated by a variety of epigenetic mechanisms.One such regulatory mechanism involves noncoding RNAs(ncRNAs),including microRNAs(miRNAs)and long noncoding RNAs(lncRNAs).LncRNAs are recently identified regulators of the transcriptional and epigenetic networks.A number of recent studies have revealed that IncRNAs are important and powerful cis and trans regulators of gene activity.They can be used as enhancers and mediators of long-range chromatin interactions,and they also can be used by chromatin-modifying complexes and nuclear bodies as scaffolds.Recently,ROR,a human lncRNA that is only 2.6 kb in length,has been shown to reprogram differentiated cells to induced pluripotent stem cells.Importantly,ROR directly targets major transcription factors that are essential for pluripotent stem cell phenotype,including OCT4,SOX2,and NANOG,through colocalization of these three factors in close proximity to ROR promoter region.In addition,lncRNA ROR is also involved in DNA damage and stem cell selfrenewal.Aim:To explore the biological function of IncRNA ROR in GCSCs.Methods and results:MTT assay and EdU assay were performed to investigate whether the IncRNA ROR regulates GCSCs growth.Interestingly,we found that lncRNA ROR overexpression in CD 133-cells significantly increased cell proliferation compared to the vector control.On the contrary,knockdown of the IncRNA ROR could remarkably attenuate the cell growth rate of CD133+ cells(Fig.3B).Similarly,EdU analysis revealed that CD133-cells with ROR overexpression contained a higher percentage of S phase cells than the vector control(Fig.3C),while knockdown of ROR in CD 133+ cells displayed a significantly lower potential in promoting cell proliferation than scramble control.We exploited transwell invasion assay to investigate the regulatory effect of the IncRNA ROR in GCSCs invasion.The percentage of viable cancer cells migrated into the lower chamber was significantly higher in the IncRNA ROR OE CD 133-cells than vector control(Vector)CD 133-cells,while knockdown of the IncRNA ROR dramatically decreased the invasion capability of CD 133+ cells.In addition,we found that the apoptotic rates between IncRNA ROR OE and vector-treansfected(Vector)CD133-cells were almost similar.However,a significantly higher percentage of apoptotic cells was seen in the CD133+ cells with the IncRNA ROR knockdown(ROR-si)compared to the scramble control(Con-si).Conclusion:LncRNA ROR promoted the proliferation,stimulated invasion and inhibited apoptosis in GCSCs.Part III The influence of IncRNA ROR on sternness of GCSCs.Background:Tumors are comprised of heterogeneous cell subpopulations,and these subsets respond distinctly and differently to various therapeutics.One subset of these cells consists of CSCs,which are largely similar to normal stem cells with respect to both their behavior and their regulation.CSCs are pluripotent,capable of self-renewal,highly resistant to cytotoxic therapies,and drive tumorigenesis.The octamer-binding transcription factor 4(OCT4),the sex determining region Y-box 2(SOX2)and Nanog homeobox(NANOG)genes are three genes encoding transcription factors that have been reported to be involved in the regulation of GCSCs.These proteins make up the core transcriptional network responsible for the regulation of stem cell self-renewal and pluripotency.Aim:To determine whether key transcriptional factors for sternness were under the regulation of the IncRNA ROR.Methods and results:Since a strikingly higher level of the IncRNA ROR was detected in the CD 133+ subset cells and the IncRNA ROR demonstrated an important role in GCSCs proliferation and invasion,we proceeded to investigate whether ROR mediates the sternness of GCSCs.Indeed,the expression of ROR was closely related to the levels of several key transcriptional factors for sternness.Specifically,we found that the IncRNA ROR OE in CD133-cells led to moderate upregulation of OCT4,SOX2,NANOG,and CD 133 at both the transcriptional level and translational level.Importantly,compared to scramble siRNA control,CD133+ cells treated with ROR siRNA dramatically suppressed the mRNA or protein expression of OCT4,SOX2,NANOG,and CD133.Conclusion:LncRNA ROR regulated the expression of key sternness transcriptional factors. |
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