| Background and ObjectiveCraniopharyngioma is formed by germ layer outside cranial pharynx tube of a common embryonic cell development residual incidence rate, tumor is the most common congenital children intracranial tumor,12-14% of intracranial tumor in children, accounting for over 50% of the children saddle area tumors. Craniopharyngioma was first put forward by professor Erdheim of Austria for the first time in 1904 its associated with embryonic eustachian tube, and is described as a kind of separate saddle area tumors.Cushing 1932 in its published his book cerebral tumor, in order to "craniopharyngioma" as the name on the detailed description, established the naming of the tumor, and has been in use today.For more than one hundred years according to the origin of craniopharyngioma, scholars both at home and abroad in areas such as the extensive and in-depth research, but limited to research technical limitations, the tumor pathology features of tumor biological characteristics of know there are still great controversy.Craniopharyngioma is belong to the WHO grade I histologically benign tumor, its pathological types can be divided into:type ameloblastic craniopharyngioma (adamantinomatous craniopharyngioma ACP) and squamous papillary type cranial pharynx tube (papillary craniopharyngioma PCP) two subtypes, the former occurs in children patients, there are many calcification, inflammation, cystic change, such as performance, the latter to adults see more, more solid tumors, rare calcification.Origin area of craniopharyngioma and growth way is diversiform, tumors in saddle area including the pituitary stalk, optic nerve and optic chiasma, hypothalamus, several important structure, such as in the surgical treatment if you want to total excision of craniopharyngioma, due to the important structure around tumor tissue and adhesion, lead to the total excision of the surgery is difficult, severe postoperative complications, postoperative morbidity and mortality is high, so also known as benign tumors, with malignant behavior.And if only the tumor resection, the recurrence rate is extremely high. So good to remove craniopharyngioma and try not to damage the surrounding the important structure has been the important problem of the neurosurgeon.In a lot of risk factors of craniopharyngioma treatment, calcification is among the more important one.Craniopharyngioma calcification rate is high, calcification in patients with children even more than 80%. A large number of clinical data, the results showed that the craniopharyngioma calcifications with pituitary stalk, optic chiasma and third ventricle bottom structure closely linked, in the operation of the complete detachment is difficult.Calcifications composition is mainly hydroxyapatite combined with a variety of fiber, the massive calcifications texture more tenacious, intraoperative are inseparable resection, overall is likely to cause damage to surrounding structures and cause serious consequences.Clinical data shows: occurrence calcification of craniopharyngioma is ameloblastic type more, calcification and squamous papillary type basic will not occur.Histologically, ameloblastic type of craniopharyngioma and tooth source sex epithelial tumors and organizations exist great similarities.Peculiar to enamel source sex tissue protein in ameloblastic type is also expressed in craniopharyngioma, and is not found in squamous papillary craniopharyngioma.Combined with the "pituitary" development path in the process of differentiation, although some scholars think that on the source, ameloblastic type craniopharyngioma and squamous papillary type craniopharyngioma were from embryonic "pituitary. Oropharyngeal tube" remnants of the organization, but its origin position is not the same, which originated in the mouth concave tooth source sex groups, which originated in the buccal mucosal tissue.Literature reported that bone morphogenetic proteins (bonemorphogeneticprotein BMP) and osteopontin osteopontin (OPN) in type ameloblastic craniopharyngioma especially calcification in high expression serious craniopharyngioma, and expressed in squamous papillary type craniopharyngioma is low, the formation of craniopharyngioma calcification.The forming mechanism of craniopharyngioma calcification is still not very clear, it needs to be studied further.Small RNA (miRNA, miRNA) is a kind of highly conservative on evolution, non-coding proteins of single small RNA molecules that consist of about 22 nucleotides.MiR-27 is one of many in the family of the miRNA function significantly, humans have miR-a and miR-27 b two subtypes, the present study found that miR-27 is closely related to the occurrence and development of human diseases miR-27 in breast cancer, colon cancer, pancreatic cancer and gastric cancer and other tumors in the expression of normal tissue significantly raised, miR-27 in odontoblast and is especially significant role in the process of osteoblast differentiation, miR-27 in human bone marrow mesenchymal stem cells (hMSC) humanmesenchymalstemcells, into the process of bone cells inhibits Wnt signaling pathways in the colonic tumor polyposis gene expression, prompting the accumulation of beta serial protein (beta-catenin), which affects the bone cell differentiation.By miR-27 expression can promote the degradation of (beta catenin), inducing osteoblast apoptosis, lead to osteoporosis.Schoolmeetters also found that miR-27 in hMSC can affect a chiral calcium binding protein expression of transcription factors, affect the formation of bone.Akhtar etc. The study found that miR-27 b can cut chondrocytes matrix metalloproteinases (MMPS) secretion, its abnormal expression is easy to lead to the occurrence of osteoarthritis.Furthermore, miR-27 can role in shaping the BMP receptor protein signaling pathways of bone-1 a gene and BMPR2 gene, promote the osteoblast differentiation.Classic Wnt signaling pathways are described when Wnt proteins and cell surface Frizzled (FZD) receptor family after a series of reactions, including beta catenin is the key factor in classic Wnt signaling pathways, beta catenin most abundant in the cytoplasm, can exist in the cell membrane and the nucleus.When the Wnt signaling pathway has not been activated in beta-catenin and APC in the cytoplasm, AXIN, GSK-3 beta these factors together, form a combination.After phosphorylation-catenin then start the beta TrCP mediated by ubiquitin/proteasome pathway degradation. When Wnt signaling pathways in activation, beta catenin molecules escape from the degradation and a large number of accumulation, into the nucleus, and in TCF/LEF1 combination, so as to activate the purpose gene expression.Build glaze type craniopharyngioma proved to exist beta-catenin CTNNB1 genes mutations can lead to the activation of Wnt signaling pathways, and in squamous papillary type does not exist the mutation of craniopharyngioma.This study by detecting miR-27 a and beta-catenin expression in craniopharyngioma, and USES the transfection raised miR-27 expressed in craniopharyngioma, a key protein beta testing Wnt signaling pathways in cells catenin protein involved in the changes and calcification of ALP, the expression of BMP2 situation, looking for miR-27 a role in the Wnt signaling pathway to promote evidence of craniopharyngioma calcification, and discussed the formation mechanism of craniopharyngioma calcification.Content and Methods1. Beta-catenin and miR-27 a expression in craniopharyngioma.Collect southern medical university affiliated hospital neurosurgery in April 2013 to 2014,10 menstrual pathological diagnosis of 33 cases of craniopharyngioma specimen, which made 27 cases craniopharyngioma glaze type, squamous papillary type 6 cases craniopharyngioma, the above cases all have complete preoperative, postoperative radiographic and clinical data.Experimental methods:using immunohistochemical method to detect different pathological and calcification of the level of beta-catenin expression in specimens of craniopharyngioma, and analyze the beta-catenin expression and the pathological types and calcium levels of correlation, further using Western blot method of detection of craniopharyngioma in beta-catenin expression levels in the organization, to analyze its expression and pathological type and amount of calcium level correlation;By using the method of rt-pcr detection miR-27 a in different pathological type and level of calcification of craniopharyngioma expression quantity in the organization, and analyze the pathological types and craniopharyngioma and the relationship between the level of calcification.2. miR -27a by acting on Wnt signaling pathways promote craniopharyngioma calcification of the preliminary studyThrough the method of fresh specimens of craniopharyngioma, to craniopharyngioma the real part of the tumor tissue cells of primitive culture, cell model of craniopharyngioma and validation.By transient transfection raised miR-27 a craniopharyngioma cells as the experimental group (miR-27 a+), set up blank control group, through the qRT-PCR method to detect the expression of micrornas level to confirm transfection is successful, further after 72 hours under the condition of the same culture with Western blot test beta-catenin proteins in the cells in each group and calcification BMP2, protein involved in the expression of ALP levels, at last, by alizarin red staining method to detect each group of cell calcium salt deposition, to further verify the results of the test, take raising miR-27 a successful tumor cells, the experimental group to join Wnt signaling pathway blockers, the same conditions continue to develop 72 hours, again by Western blot method validation calcification ALP protein involved in experimental group and the blank control group, the expression of BMP2 level.3. The statistical methodsUsing SPSS 19.0 software for statistical processing.Measurement data with x+s, group comparison between the two independent samples t test, multiple comparison between different level of calcification SNK method (Student-Neuman-Keuls).Statistically difference at P< 0.05.Results1. Beta-catenin and miR-27a expression in craniopharyngioma and with different pathological types and craniopharyngioma calcification hierarchical relationship. Using immunohistochemical method to detect different craniopharyngioma beta-catenin expression levels in the organization, the results showed type ameloblastic craniopharyngioma tissue specimens of beta-catenin expression strength higher than that of squamous papillary type craniopharyngioma, ameloblastic type craniopharyngioma and tissue specimens seen in beta-catenin expression within the nucleus, which type ameloblastic craniopharyngioma is beta-catenin nucleus expression cells often appeared in the shape of vortex and calcifications area surrounding cells.And tissue samples of squamous papillary type craniopharyngioma beta catenin expression on cell membrane,not found its expression in the nucleus.Further using western blot method to all craniopharyngioma tissues in the beta testing, the expression of catenin made glaze type according to the results of craniopharyngioma tissues beta catenin protein expression level is higher than squamous papillary type craniopharyngioma (P=0.013,<0.05);And ameloblastic type craniopharyngioma tissues in beta-catenin protein expression level have higher consistency with calcification (rs=0.791, P<0.001), high calcium level organization of craniopharyngioma beta-catenin protein expression level is higher, but not seen calcification type ameloblastic craniopharyngioma beta-catenin expression level and the expression of craniopharyngioma squamous papillary type has no statistical significance (P=0.237).Using qRT-PCR technique to detect craniopharyngioma miR-27 a expression levels in the organization, and the results showed type ameloblastic craniopharyngioma tissue samples of miR-27a expression level than squamous papillary type craniopharyngioma (P=0.024,<0.05), and in type ameloblastic craniopharyngioma tissues miR-27 a expression level in the different level of calcification group expression differences statistically significant (P<0.05), with the increase of calcium level, the expression levels of miR-27 a is raised (rs= 0.680, P<0.001).While without calcification type ameloblastic craniopharyngioma miR-27 a expression in squamous papillary type and craniopharyngioma expression differences of no statistical significance (P=0.31).2. miR-27 a by acting on Wnt signaling pathway to promote research of craniopharyngioma calcificationCraniopharyngioma cells using qRT-PCR miR-27 a expression detection, transfection group (miR-27a+) expression level higher than the blank control group, validation transfection success.Using Western blot test beta-catenin and calcification in the experimental group and control group in cell related proteins BMP2, ALP expression level, the result shows that the experimental group BMP2 protein involved in beta-catenin and calcification in the cells, the expression of ALP levels were higher than the control group (P<0.05);Alizarin red staining results showed that the experimental group of calcium salt deposit more significantly than control group. In order to further verify the above test results, using Western blot test Wnt signal blocking group (Wnt-) and control group in beta-catenin and calcification related proteins in the cell BMP2, ALP expression level, the result shows that the experimental group BMP2 protein involved in intracellular calcium, the expression of ALP levels were lower than the control group (P< 0.05).ConclusionMiR-27 a and the activation of Wnt signaling pathways in craniopharyngioma plays an important role in the process of calcification.In the type ameloblastic craniopharyngioma miR-27a expression can promote the calcification of craniopharyngioma cells, the promoting effect is produced by activation of Wnt signaling pathways.Blocking the activation of Wnt signaling pathways can inhibit the promoting effect.miR-27 a activation of Wnt signaling pathways of craniopharyngioma cells calcification is craniopharyngioma form one of the possible mechanisms of calcification, after the activation of Wnt signaling pathways to improve the calcification of craniopharyngioma cells gene specific mechanisms need further study. |
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