The Effects Of Nucleostemin RNAi On The Expression Of CDX2 In HT29 Cells

Background and ObjectiveBarrett's esophagus (BE) is a metaplastic process in which the normal squamous epithelium of the distal esophagus is replaced by columnar lined epithelium and is the result of chronic gastroesophageal reflux. The majority of esophageal adenocarcinoma (EC) arises in BE with specialized intestinal-type glandular epithelium (SIM) with an estimated 0.5–1.0% annual risk of developing EC. Accumulating clinical and experimental studies indicate that BE might arise through multipotential stem cells. Caudal-related homeobox 2 (CDX2) is a homeobox transcription factor that plays an important role in the early differentiation and maintenance of intestinal epithelium.It is a useful marker of intestinal metaplasia in the diagnosis of Barrett esophagus. Nucleostemin is p53-binding protein mainly existing in the nucleoli of embryonic stem cells and various cancer cell lines,but does not express in committed and terminally differentiated cells. It is essential for stem and cancer cell proliferation and implicated in oncogenesis , and keeps cells in non-differentiated state. However, its role in the pathogenesis of BE and subsequently esophageal adenocarcinoma is yet to be elucidated.In the present study, We hypothesized that nucleostemin down-regulates CDX2 expression, and the loss of nucleostemin expression in the esophageal"stem cells"may result in activation of CDX2 expression, leading to the intestinal differentiation and subsequent formation of intestinal metaplasia. It has been demonstrated that HT29 cells can be used to serve as an in vitro model for the study of the effects of different components of gastroduodenal refluxate on cellular and molecular events in the development of BE. To test our hypothesis, we further determined the effects of silencing nucleostemin expression on the expression of CDX2 in HT29 cells with the RNAi technique to see whether siRNAs that targets nucleostemin would enhance CDX2 expression in vitro. In addition, we observed the expression of nucleostemin and CDX2 in HT29 cells after chenodeoxycholic acid (CDC) exposure.Methods 1. Three siRNAs targeted nucleostemin were designed by a program available online(www.genscript.com). siRNA-expressing plasmids were constructed by cloning the siRNA sequences into pRNAT-U6.1/Neo via BamHI and HindIII digestion. The plasmids were extracted following the manufacturer's instruction and then sequenced to confirm the correct insertion, The new plasmids were named pRNAi-1, pRNAi-2 and pRNAi-3. HT29 cells were seeded on 6-well culture plates and grown to 80%~90% confluence before the transfection. The recombinant pRNAi-1, pRNAi-2 and pRNAi-3 were used for the transfection in the corresponding experimental groups. LipofectamineTM 2000 was used for the transfection in the blank control group whereas the empty plasmid pRNAT-U6.1/Neo was used in the negative control group.The transfected cells were selected by G418. Until positive clones were discovered after 14 days,the cells were cultured and finally selected by G418 for a further 10 days. Single clones were selected to build a stable transfected cell line.2. Changes of biological behaviour of HT29,including cell proliferation, cell cycle distribution, clone formation after NS RNA interference were investigated.3. HT29 cells were placed in serum-free RPMI 1640 for 24 h and then exposed to 100, 500, and 1,000μM CDC in serum-free medium for 2, 12, and 24 h, respectively. Cells were harvested at the end of each time point with 0.25% trypsin solution. 4. Detection of expression of nucleostemin and CDX2 in HT29 cells by Reverse Transcription Polymerase Chain Reaction and Western blot analysisResults1. The recombinant plasmids were identified to have correct sequences by DNA sequencing analysis, and the result confirmed that the DNA chains had ligated to the vectors. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. Green fluorescene of the stable transfected HT29 cell lines could be observed under inverted fluorescence microscope.2. MTT result showed that cell proliferation ability of pRNAi-1 cells was significantly lower than that of empty plasmid group and untreated controls. Plate clone formation experiment demonstrated that single cell clone formation capacity of pRNAi-1 cells was obviously lower than that of empty plasmid group and untreated controls. Flow cytometry demonstrated that specific NS siRNA caused accumulation in the G0/G1 phase, and decreased the number of cells of G2/M phase in comparison of empty plasmid group and untreated controls.3. Nucleostemin expression were down-regulated significantly in HT29 cells after transfection with pRNAi-1, pRNAi-2 and pRNAi-3 (all P<0.05). Since the pRNAi-1 was the most effective vector, it was selected for further experiment on the effect of RNAi on the expression of CDX2. It was shown that CDX2 expression in HT29 cells transfected with pRNAi-1 was significantly increased, compared with that in HT29 cells transfected with pRNAT or untransfected HT29 cells4. A low level of CDX2 expression was detected in HT29 cells without CDC exposure. CDX2 protein expression was highly up-regulated by CDC treatment in a dose- and time-dependent fashion. Although CDC exerted no significant effect of on CDX2 expression in HT29 cells at 100μM for up to 24 h and at 500μM for up to 12 h, CDX2 expression was significantly increased after treatment with 500μM CDC at 24 h, or 1,000μM CDC at 2 h, with the maximal effect being achieved with 1,000μM CDC at 24 h. Furthermore, nucleostemin expression was decreased in a dose- and time-dependent fashion in HT29 cells treated with CDC.ConclusionIn vitro, inhibition of nucleostemin results in not only a decreased cellular proliferation,but an increased expression of CDX2. In addition, CDC dose-dependently increases CDX2 production and decreases nucleostemin production in HT-29 cells. These findings suggest that the inhibition of nucleostemin expression in"esophageal stem cells"may be involved in the pathogenesis of BE through up-regulating CDX2 expression. Further studies are needed to investigate whether the inhibition of nucleostemin results in the activation of the CDX2 promoter via a transcription factor binding site (e.g. NFκB).