Expression Of Ndrg2in Human Medullary Thyroid Carcinoma And Preliminary Study On Its Function

Thyroid carcinoma is the most frequent primary malignancy in theneuroendocrine system. Of these, approximately5%are medullary thyroidcarcinoma which derives from the parafollicular C-cells of the thyroid gland andthere has been a dramatic increase in the incidence of it in the past few decadesin our country, which suggests that new pathogenesis and effective treatmentoptions should be explored. Despite widespread use of total thyroidectomy andcentral neck compartment dissection, survival rates have not improved much inthe past few decades. Radioiodine therapy should prolong survival and improvequality of life by elimination of microscopic residual or metastatic disease as anadjuvant treatment. However, radioiodine therapy for dedifferentiated thyroidcarcinoma and medullary thyroid carcinoma are often of limited benefit becauseof their respond poorly to the specific uptake of iodine into thyroid cells. So it isan urgent task to explore the road for improves the effectiveness of the radioiodine therapy.The Sodium/Iodide Symporter (NIS) is an integral plasma membranelycoprotein that mediates active I-transport into the thyroid follicular cells, thefirst step in thyroid hormone biosynthesis. Data indicate that the ability of thethyroid to accumulate I-via NIS is a close correlation with Na+/K+-ATPase.Na+/K+-ATPase locate on the basolateral side of thyroid cells and can pump Na+outside by expending energy, and this can form the Na+concentration difference.NIS-mediated I-accumulation in the thyroid is an active transport process thataccompany with2-fold Na+. Previous observations demonstrated thathNIS-transfected medullary thyroid carcinoma TT cells showed perchlorate-sensitive iodide uptake, accumulating125I about12-fold in vitro withorganification of4%of accumulated iodide resulting in a significant decrease iniodide efflux. NIS provides the basis for the radioiodine therapeuticmanagement of medullary thyroid carcinoma.Human NDRG2was discovered and cloned firstly as a novel gene by thedepartment of biochemistry and molecular chemistry of our university. We havedemonstrated that NDRG2was a candidate tumor suppressor and is possiblyrelated to cell proliferation and differentiation and stress reaction. Previousobservations indicated that NDRG2is the diffenentiated gene between normalthyroid tissues and thyroid carcinoma and might participate in the pathogenesisof medullary thyroid carcinoma. Yeast two-hybrid approach was used to screenthe proteins which can interact with NDRG2,3fragments encodedNa+/K+-ATPaseβ1, and found that NDRG2and Na+/K+-ATPaseβ1couldco-localized in the perinuclear region of the cytoplasm. And the activity ofNa+/K+-ATPaseβ1is directly proportional to NDRG2protein level. NDRG2could increase the Na+/K+-ATPaseβ1stability and extend Na+/K+-ATPaseβ1 protein half-life.Previous observations suggest strongly that NDRG2may play an importantrole in the pathogenesis of medullary thyroid carcinoma, especially closelyrelated to the expression and function of NIS and iodide uptake of active I-. somore investigation and verification will be done in the present research.Objectives1．To clear the expression pattern of NDRG2and NIS in the thyroid tissueand thyroid carcinoma;2．To investigate the function of NDRG2in medullarythyroid carcinoma cells and the correlation with NIS;3．To explore the effect ofaccumulate I-and radionuclide imaging on medullary thyroid carcinomathroughout NDRG2.Methods1．Immunostainning analysis was employed to clear the expression patternof NDRG2and NIS in the thyroid carcinoma using the tissue microarray;2．Amplified of NDRG2over-expression lentivirus and transfected respectivelyinto human medullary thyroid carcinoma TT cells, stably transfectedTT-NDRG2and TT-CHERRY cells were established through selecting withBlasticidin, RT-PCR and Western-blot were employed to validate the effect ofover-expression lentivirus of NDRG2in stably transfected cells and theexpression level of NIS.3．MTT assay、Colony formation assay、Monolayerwound healing assay、Transwell invasion assay and Cell cycle and apoptosisanalysis were employed to detect the contribution of NDRG2on TT cells,respectively. Then iodide uptake studies were employed to detect the ability of TT cells to accumulate I-via NIS.4．Tumorigenesis assay were employed todetect to the influence of NDRG2over-expression on the tumor development ofMTC TT cells in vivo and99mTcO4-radionuclide imaging of xenografted tumors.Results1．Immunostainning analysis revealed that significant staining of NDRG2and NIS were observed in normal thyroid tissues while the expression ofNDRG2and NIS were almost undetectable or at a significantly low level inthyroid carcinoma, and there was a positive correlation between NDRG2andNIS. NDRG2immunoreactivity could be seen mainly in cell cytoplasm and NISwas located to cell membrane.2．RT-PCR and Western-blot assay showed thatthe levels of NDRG2and NIS protein and mRNA transcripts in TT-NDRG2cells increased significantly，and infection with control lentivirus did not affectthe expression of NDRG2as the levels of protein and mRNA transcripts inTT-CHERRY cells were similar to that in parent TT cells.3．MTT and Colonyformation assay showed that the growth of TT-NDRG2cells was much slower,as compared with that in control groups on day9(p<0.05). A similar pattern ofinhibitory effect of NDRG2over-expression in TT cells was achieved in colonyformation assay. The result indicated that up-regulating NDRG2expressioninhibited the ability of proliferation significantly. Following incubation ofphysically wounded cells for48h, the mobile distance of TT-NDRG2wassignificantly shorter than that of controls (P <0.05). Transwell invasion assayrevealed that up-regulation of NDRG2expression reduced the invasiveness ofMTC TT cells. The results of FACS analysis indicated that the frequency ofTT-NDRG2cells at the G0/G1phase statistically increased while lowerfrequency of cells progressed at the S-phase, as compared with that in control groups (P <0.01), and the apoptotic cells in TT-NDRG2cells were much morethan that in the control cells. Iodide uptake was measured in stably transfectedTT cells and showed that TT-NDRG2cells concentrated125I about17-fold, ascompared with TT-cherry.4．Tumorigenesis assay showed that the developmentof TT-NDRG2cell-related solid tumors grew slowly and the mean weight oftumors was significantly lighter, as compared with that in control groups (P <0.05), and99mTcO4-radionuclide imaging of the solid tumors in TT-NDRG2groups indicated significantly that medullary thyroid carcinoma acquired theability of99mTcO4-accumulation after tumor-specific NDRG2gene expression.ConclusionsWe identified that NDRG2is a critical molecule in the regulation of cellproliferation and invasion in medullary thyroid carcinoma and provided thepreliminary evidence that NDRG2can up-regulate the expression level of NISand enhance the ability of the medullary thyroid carcinoma to accumulate I-viaNIS. Our data lay the foundation for the research of radioiodine therapy formedullary thyroid carcinoma.