| ObjectiveTo observe the expression of motilin (MTL) in paraventricular hypothalamic nucleus (PVN), pituitary and thyroid gland, and to compare the sequences of MTL precursor mRNA in thyroid and intestine. To study the expression differences of MTL peptide or precursor mRNA in thyroid tumors and its clinical significance.To research the modulating function of PVN-pituitary-thyroid axis on the secretion of thyroidal MTL and gastric motility, and further to study the feedback of the thyroidal MTL to the expression of MTL in PVN and pituitary.To explore the expression, secretion and regulation of MTL in human medullary thyroid carcinoma (TT) cells, and try to explain the role and clinical significance of MTL in the proliferation, migration and invasion of TT cells.Methods1. The MTL expression in PVN, pituitary and thyroid gland was detected by immunofluorescence histochemistry. The real-time polymerase chain reaction (Real-time PCR), Southern Blot, molecular cloning and double-staining immunofluorescence histochemistry were used to show the differences of MTL precursor mRNA sequences between thyroid and intestines, and to compare the expression of MTL peptide and precursor mRNA in normal thyroid, benign thyroid tumor and malignant thyroid cancer.2. After PVN excitation/lesion, hypophysectomy or thyroidectomy, manometric recording was employed to observe the phaseâ…¢-like migrating myoelectric complex (MMC) contraction of stomach, radioimmunoassay (RIA) was used to detect the changes in the concentrations of plasma T3, T4 and MTL. The effects of PVN excitation/lesion on the expression of MTL precursor mRNA in PVN-pituitary-thyroid were detected by real-time PCR. The expression of MTL in the thyroid after the PVN excitation and c-Fos in the PVN after the thyroidectomy were observed by immunohistochemical staining. 3. The expression of MTL mRNA and peptide in TT cells were checked with Southern Blot, Western Blot and immnuflurescence cytochemistry. After decreasing the MTL expression in TT cells with small interfering RNA technique, the proliferation, invasion and migration of tumor cells were analysed with MTT test, Transwell assay, and scratch experiments.Results1. MTL immunoreactive cells could be observed in the hypothalamus PVN, pituitary, thyroid of rats and the pituitary, thyroid of human. There was co-expression of thyroid MTL and calcitonin (CT) in thyroid C cells.2. RT-PCR and Southern blot showed that MTL mRNA expressed in human thyroid was identical to that expressed in duodenum from Genbank (BC112314, NCBI, USA).3. The precursor mRNA and protein of MTL were shown in normal and tumor tissues of human thyroid. The expression of MTL precursor mRNA and protein in thyroid medullar carcinoma were significantly higher than those of normal thyroid and other thyroid tumors (thyroid medullar, acidophilic adenoma, follicular, papillary and nodular goiter) (P<0.01). The expression of MTL precursor mRNA and protein in thyroid follicular and papillary were significantly lower than those of normal thyroid tissue, nodular goiter and acidophilic adenoma (P<0.05). There is no difference among the nodular goiter, acidophilic adenoma and normal thyroid tissue (P>0.05).4. The electric excitation of PVN promoted the expression of MTL mRNA and protein in pituitary and thyroid (P<0.05), increased the concentration of MTL in plasma (P<0.05), and further improve the corresponding gastric motility of rats (P<0.05), which was partially inhibited by the administration of the MTL receptor antagonist, GM-109 (P<0.05). In thyroidectomized group, the PVN excitation increased the phaseâ…¢-like contraction and plasma MTL concentration (P<0.05), which were weaker than those of the sham operation control group (P<0.05). After the electric lesion of PVN, the concentration of plasma MTL, the expression of MTL mRNA and peptide in pituitary and thyroid decreased (P<0.05), and the MMC phaseâ…¢-like contraction was attenuated significantly (P<0.05).5. Hypophysectomy reduced the frequency of MMC (P<0.05). The frequency of MMC was higher in rats with hypophysectomy and MTL injection in PVN than that of rats with hypophysectomy and saline injection in PVN (P<0.01), but was lower than that of rats only with sham operation and MTL injection in PVN (P<0.05).6. Thyroidectomy decreased the plasma MTL concentration (2d, P<0.01; 4d, P<0.05), and reduced the MMC phaseâ…¢-like contraction (2d, P<0.01; 4d, P<0.05). After thyroidectomy, the expression of MTL mRNA in hypothalamus and pituitary increased (2h, P<0.05), and the c-Fos immunoreactive cells in PVN rised (2h, P<0.05).7. There were MTL precusor mRNA and immunoreactive substances in TT cells. MTL and CT co-expressed in the same cells. The TT cells secreted MTL, which was promoted by growth hormone (GH) and thyrotrophin (TSH) (P<0.05). MTL administration increased the proliferation, invasion and migration of TT cells.Conclusions1. MTL procusor mRNA and peptide were expressed in the thyroid and TT cells, and there was co-expression of MTL and CT in the C cells.2. The sequence of MTL precursor mRNA from human thyroid was identical to that expressed in duodenum.3. The expressions of MTL precursor mRNA and protein in thyroid medullar carcinoma were high, while in thyroid follcular carcinoma and thyroid papillary carcinoma, their expressions were significantly lower than those of normal thyroid tissue.4. MTL was involved in the modulation of gastric motility by PVN-pituitary-thyroid axis, and thyroid affect the MTL expression in hypothalamus and pituitary by feedback pathway.5. GH and TSH promoted the MTL secretion of TT cells, and MTL improved the proliferation, invasion and migration of TT cells.
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