Value of serum galectin-3 and midkine level determination for assessing tumor severity in patients with thyroid cancer
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1 148 Journal of Hainan Medical University 2017; 23(3): Journal of Hainan Medical University Value of serum galectin-3 and midkine level determination for assessing tumor severity in patients with thyroid cancer Yan-Qing Liu Department of Otolaryngology Head and Neck Surgery, the First People s Hospital of Jingzhou Hubei Province, Jingzhou , China ARTICLE INFO Article history: Received 7 Jul 2016 Received in revised form 17 Jul 2016 Accepted 12 Jul 2016 Available online 24 Jul 2016 Keywords: Thyroid cancer Galectin-3 Midkine Proliferation gene Invasion gene ABSTRACT Objective: To investigate the value of serum galectin-3 and midkine level determination for assessing tumor severity in patients with thyroid cancer. Methods: 108 patients with thyroid cancer treated in our hospital between January 2013 and February 2016 were collected and divided into I II stage thyroid cancer group (n=39), III stage thyroid cancer group (n=55) and IV stage thyroid cancer group (n=14) according to TNM stage; 56 patients diagnosed with thyroid adenoma after physical examination in our hospital during the same period were selected as control group. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum galectin-3 and midkine levels of all groups; fluorescence quantitative PCR was used to determine proliferation gene and invasion gene mrna expression in tumor tissue; Pearson test was used to analyze the correlation of serum galectin-3 and midkine levels with proliferation and invasion gene mrna expression in patients with thyroid cancer. Results: Serum galectin-3 and midkine levels of patients with thyroid cancer were higher than those of control group (P<0.05), and the higher the tumor stage, the higher the serum galectin-3 and midkine levels; proliferation genes CyclinD1, BCL-2 and survivin mrna expression in tumor tissue of patients with thyroid cancer were higher than those of control group (P<0.05) while p27 mrna expression was lower than that of control group (P<0.05), invasion gene E-cadherin mrna expression was lower than that of control group (P<0.05) while S100A4 and SATB1 mrna expression were higher than those of control group (P<0.05), and the higher the tumor stage, the more significant the change of the above gene mrna expression; Pearson test showed that serum galectin-3 and midkine levels in patients with thyroid cancer were correlated with proliferation and invasion gene expression in tumor tissue. Conclusions: Serum galectin-3 and midkine levels can directly reflect the malignant degree of thyroid cancer, and is expected to become the new means for early disease diagnosis, curative effect evaluation and prognosis judgment. 1. Introduction Thyroid cancer is the most common malignant thyroid tumor, its incidence accounts for about 1% of the malignant tumors of the whole body, papillary carcinoma is seldom with lymphatic metastasis and is with good prognosis, but the follicular carcinoma and undifferentiated carcinoma are with high malignant degree, and Crresponding author: Yan-Qing Liu, Department of Otolaryngology Head and Neck Surgery, the First People s Hospital of Jingzhou Hubei Province, Jingzhou , China. Tel: ; Fund project: Scientific Research Fund Projects of Hubei Provincial Department of Education in 2015 (No: Q ). the prognosis is poor if they are not treated in time[1,2]. The main screening means B ultrasound for thyroid cancer has a certain error, and should be combined with other forms of indexes to increase the screening sensitivity and specificity. Galectin-3 and midkine are currently reported molecules that are with extremely high positive protein expression rate in thyroid carcinoma tissues, many scholars recommend to add them to the serum indexes for thyroid cancer screening, but there is still not much research at present about the directivity of their serum levels to the malignant degree of thyroid cancer[3,4]. In the following study, the value of serum galectin-3 and midkine level determination for assessing tumor severity in patients with thyroid cancer was analyzed.
2 Yan-qing Liu/ Journal of Hainan Medical University 2017; 23(3): Co., LTD., model eppendorf5702) to separate serum, and ELISA was used to determine galectin-3 and midkine levels in it. Elisa kits 2. Materials and methods 2.1. Research subjects 108 patients with thyroid cancer treated in our hospital between January 2013 and February 2016 were selected and divided into I II stage thyroid cancer group (n=39), III stage thyroid cancer group (n=55) and IV stage thyroid cancer group (n=14) according to TNM stage; 56 patients diagnosed with thyroid adenoma after physical examination in our hospital during the same period were selected as control group, all patients signed the informed consent themselves, and the study was approved by the hospital ethics committee. I II stage thyroid cancer group included 20 male cases and 19 female cases, they were years old, the body weight was kg and (62.17±8.09) kg in average; III stage thyroid cancer group included 30 male cases and 25 female cases, they were years old, the body weight was kg and (64.09±8.54) kg in average; IV stage thyroid cancer group included 8 male cases and 6 female cases, they were years old, and the body weight was kg and (64.88±9.53) kg in average. Control group included 30 male cases and 26 female cases, they were years old, and the body weight was kg and (63.28±8.15) kg in average. Differences in baseline information such as gender, age and body weight were not statistically significant among groups (P>0.05) Inclusion and exclusion criteria were purchased from Sigma Company in the United States, and the article number was RAB1015 and RAB1092 respectively Determination methods of malignant behavior gene expression in tumor tissue All groups of patients underwent surgical treatment, and tumor tissue samples were obtained during operation for target gene amplification by fluorescence quantitative PCR. Tissue samples were added in Trizol fluid (Sigma Company in the United States, the article number T9424), the RNA extraction kits (Sigma Company in the United States, the article number RNB400) were used to extract total RNA, the cdna synthesis kits (Roche Company in the United States, the article number ) were used to synthesize cdna, and the reaction system is as follows: buffer 2 μl, upstream and downstream primers 0.2 μl each, dntp 0.1 μl, reverse transcriptase 0.5 μl, template 2 μl, DEPC water 5 μl, total volume 10 μl. Amplified target gene mrna was as follows: proliferation genes Cyclin D1, p27, BCL-2 and survivin; invasion genes: E-cadherin, S100A4 and SATB Statistical analysis SPSS23.0 was used to input and analyze the data obtained in the study, measurement data was in terms of (x 依 s), comparison between groups was by t test and P<0.05 was set as the standard of statistical significance in differences. Inclusion criteria are as follows: (1) clearly diagnosed with thyroid tumors by B ultrasonography, and the tumor properties and staging were confirmed by biopsy; (2) 80 years old; (3) not associated with hyperthyroidism, hypothyroidism, Hashimoto's thyroiditis and other basic thyroid diseases; (4) without previous history of thyroid surgery. Exclusion criteria are as follows: (1) associated with systemic infectious diseases; (2) with serious heart, liver and kidney dysfunction; (3) with other primary malignant tumors; (4) with metastatic thyroid cancer; (5) pregnant or breastfeeding women Observation indexes Determination methods of serum galectin-3 and midkine levels 2 ml of fasting peripheral venous blood was extracted from all groups of patients immediately after admission, and centrifuged in low-speed centrifuge (Shanghai Ai Research Biological Technology 3. Results 3.1. Serum galectin-3 and midkine levels Variance analysis showed that differences in serum galectin-3 and midkine levels were statistically significant among the four groups of patients (P<0.05). Pair-wise comparison between groups showed that serum galectin-3 and midkine levels of patients with different stages of thyroid cancer were significantly higher than those of control group (P<0.05); the higher the tumor stage in patients with thyroid cancer, the higher the serum galectin-3 and midkine levels, and the differences between groups were statistically significant (P<0.05), shown in Table Proliferation gene mrna expression
3 150 Yan-qing Liu/ Journal of Hainan Medical University 2017; 23(3): Variance analysis showed that differences in proliferation genes CyclinD1, p27, BCL-2 and survivin mrna expression in tumor tissue were statistically significant among the four groups of patients (P<0.05). Pair-wise comparison between groups showed that CyclinD1, BCL-2 and survivin mrna expression in tumor tissue of patients with different stages of thyroid cancer were significantly higher than those of control group (P<0.05) while p27 mrna expression was significantly lower than that of control group (P<0.05); the higher the tumor stage in patients with thyroid cancer, the higher the CyclinD1, BCL-2 and survivin mrna expression in tumor tissue while the lower the p27 mrna expression, and the differences between groups were statistically significant (P<0.05), shown in Table Invasion gene mrna expression Variance analysis showed that differences in invasion genes E-cadherin, S100A4 and SATB1 mrna expression in tumor tissue were statistically significant among the four groups of patients (P<0.05). Pair-wise comparison between groups showed that E-cadherin mrna expression in tumor tissue of patients with different stages of thyroid cancer was significantly lower than that of control group (P<0.05) while S100A4 and SATB1 mrna expression were significantly higher than those of control group (P<0.05); the higher the tumor stage in patients with thyroid cancer, the lower the E-cadherin mrna expression in tumor tissue while the higher the S100A4 and SATB1 mrna expression, and the differences between groups were statistically significant (P<0.05), shown in Table Correlation analysis Pearson correlation analysis showed that serum galectin-3 and midkine levels in patients with thyroid cancer were directly correlated with proliferation gene and invasion gene expression in tumor tissue, specifically as follows: serum galectin-3 and midkine levels in patients with thyroid cancer were positively correlated with proliferation genes CyclinD1, BCL-2 and survivin mrna expression and negatively correlated with p27 mrna expression; they were positively correlated with invasion genes S100A4 and Table 1 Comparison of serum galectin-3 and midkine levels among groups ( x 依 s). Groups n Glactin-3 (ng/ml) Midkine (pg/ml) IV stage thyroid cancer group ±4.09 abc 57.63±8.23 abc III stage thyroid cancer group ±7.12 ab 34.71±4.63 ab I II stage thyroid cancer group ±2.31 a 9.51±1.09 a Control group ± ±0.68 F P <0.05 <0.05 Table 2 Comparison of proliferation gene mrna expression in tumor tissue among groups (x 依 s). Groups n Cyclin D1 p27 BCL-2 Survivin IV stage thyroid cancer group ±30.85 abc 23.74±2.83 abc ±23.56 abc ±30.12 abc III stage thyroid cancer group ±24.66 ab 40.68±4.52 ab ±19.72 ab ±23.14 ab I II stage thyroid cancer group ±15.09 a 78.63±8.94 a ±15.48 a ±18.66 a Control group ± ± ± ±11.75 F P <0.05 <0.05 <0.05 <0.05 Table 3 Comparison of invasion gene mrna expression in tumor tissue among groups (x 依 s). Groups n E-cadherin S100A4 SATB1 IV stage thyroid cancer group ±3.54 abc ±28.95 abc ±25.83 abc III stage thyroid cancer group ±7.43 ab ±23.59 ab ±19.62 ab I II stage thyroid cancer group ±9.83 a ±16.07 a ±15.98 a Control group ± ± ±10.73 F P <0.05 <0.05 <0.05
4 Yan-qing Liu/ Journal of Hainan Medical University 2017; 23(3): SATB1 mrna expression and negatively correlated with E-cadherin mrna expression (P<0.05). 4. Discussion Galectin-3 is the protein that can identify the specific oligosaccharide structure in glycolipids, study has shown that galectin-3 is highly expressed in ovarian cancer, lung cancer, gastric cancer and other malignant tumors, and it is considered as one of the key proteins directly involved in tumor progression[5]. At present, there is not much research about the correlation between galectin-3 and the malignant degree of thyroid carcinoma, but studies have confirmed that the positive expression rate of galectin-3 in thyroid papillary carcinoma reaches 90% 100%. midkine belongs to heparin-binding factor, and is related to cell growth and differentiation in embryonic development period. Recent studies have found that midkine expression increases in a wide variety of tumors, and it is closely related to the occurrence, progression and prognosis of tumors[6,7]. In order to define the characteristics of galectin-3 and midkine expression in thyroid cancer, serum galectin-3 and midkine levels in patients with different stages of thyroid cancer and benign thyroid adenoma were detected in the study at first, and it was found that compared with the control group, patients with different stages of thyroid cancer were with higher serum galectin-3 and midkine levels; compared with patients with I II stage thyroid cancer, patients with higher stages were with even higher serum galectin-3 and midkine levels. It indicates that galectin-3 and midkine are highly expressed in the patients with thyroid cancer, and with the increase of tumor malignancy, galectin-3 and midkine are in continuously over-expressed state. It can be confirmed that highly expressed galectin-3 and midkine participate in the occurrence and development of thyroid cancer, but it still cannot explain their internal relation with the malignant behaviors of tumor. Proliferation and invasion are the most typical characteristics of tumor cells and also the basis of their continuous progression, and the proliferation and invasion gene expression in patients with different severity of thyroid carcinoma and patients with thyroid adenoma were further compared in the study to back up the action of galectin-3 and midkine. CyclinD1, p27, BCL-2 and survivin are all thyroid carcinoma proliferation-related genes that have been reported in literatures, CyclinD1 is the cell cycle-related oncogene, it activates CDK4 to release a large number of growthregulating factors, and its label index increases with the increased malignant degree of thyroid[8,9]. p27 is negative regulatory gene of cell cycle, and it can inhibit the activity of Cyclin E-CDK2 and other kinase compounds, and terminate the cell cycle at G1 phase[10]. BCL-2 is a major gene involved in regulation of cell apoptosis, and it can inhibit the cell apoptosis and promote cell survival[11]. Survivin belongs to inhibitor of apoptosis proteins, it is not expressed in normal mature tissues, and it has become a new target for the diagnosis and treatment of many malignant tumors[12]. The study results showed that compared with the control group, patients with thyroid cancer are with higher CyclinD1, BCL-2 and survivin mrna expression in tumor tissue, and lower p27 mrna expression, and with the increase of malignant degree of thyroid carcinoma, the changes in above proliferation gene expression increase and are basically consistent with the changes in disease. The abnormal expression of invasion genes can directly affect the invasion and distant metastasis ability of tumor cells, and it is the core element of the malignant tumor progression[13]. E-cadherin is an important factor that regulates the epithelial cells and intercellular adhesion, and many studies have confirmed that the E-cadherin expression deletion is the key step that leads to the increased invasion and metastasis potential of epithelial tumor cells[14]. S100A4 is directly involved in thyroid cancer cell movement and intratumoral angiogenesis, and the study of Hou et al shows that S100A4 overexpression can increase the MMP-13 expression and prompt thyroid cancer metastasis[15]. SATB1 is an important signal molecule of tumors that can activate PI3K/Akt signal pathway and increase the expression of MMP-2, MMP-9 and inflammatory factors. The study of Wan and Zheng also shows that inhibiting SATB1 expression can decrease thyroid cancer invasion and metastasis ability[16]. The above gene mrna expression in tumor tissue was tested in the study, and it was found that compared with the control group, patients with thyroid cancer are with lower E-cadherin mrna expression in tumor tissue and higher S100A4 and SATB1 mrna expression, and with the increase of tumor stage, the changes in invasion gene expression increase, which confirms that invasion gene expression directly decides the malignant degree of thyroid carcinoma. Given that proliferation and invasive genes decide the malignant degree of thyroid cancer, the correlation of serum galectin-3 and midkine levels with proliferation and invasion gene mrna expression was analyzed at last in the study, and it was found that serum galectin-3 and midkine levels in patients with thyroid cancer were directly correlated with proliferation genes CyclinD1, p27, BCL-2 and survivin mrna expression as well as invasion genes E-cadherin, S100A4 and SATB1 mrna expression. So it can be concluded that serum galectin-3 and midkine levels are directly correlated with the malignant degree of thyroid carcinoma, and can be used as the effective supplementary means for tumor screening, early diagnosis, treatment guidance, etc.
5 152 Yan-qing Liu/ Journal of Hainan Medical University 2017; 23(3): [8] Martins-Costa MC, Cunha LL, Lindsey SC, et al. M918V RET mutation References [1] Lin YS, Li J. The interpretation of 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Carcinoma: New progress in radioactive iodine therapy of differentiated thyroid carcinoma. China Oncol 2016; 26(1): 1-3. [2] Sheng JG, Wang B, Diao ZP, et al. BRMS1 and Cx43 expression in fine needle aspiration thyroid cancer tissue and their correlation with tumor malignancy. J Hainan Med Univ 2016; 22(17): [3] Choi YW, Kim YH, Lee J, et al. Strong immunoexpression of midkine is associated with multiple lymph node metastases in BRAFV600E papillary thyroid carcinoma. Hum Pathol 2015; 46(10): [4] Mu WN, Qiang Y, Liu YT. Research progression of the clinical application of thyroid cancer molecular markers. Shandong Med J 2016; 56(14): [5] Xue G, Liu JC, Huang J, et al. Detection of glactin-3 in both serum and tissue for early diagnosis of thyroid carcinoma. J Southern Med Univ 2013; 33(7): [6] Sun CM, Wang LP, Liu M, et al. Effects of silencing lung cancer A549 midkine gene expression on cell apoptosis. Chongqing Med 2015; 44(5): [7] Wei WJ, Wang YR, Tao LY, et al. Relationship between midkine expression in differentiated thyroid cancer tissue and microvascular generation and the significance. Guangdong Med J 2014; 35(4): causes familial medullary thyroid carcinoma: study of 8 affected kindreds. Endocr Relat Cancer 2016; 23(12): [9] Smith J, Read ML, Hoffman J, et al. Germline ESR2 mutation predisposes to medullary thyroid carcinoma and causes up-regulation of RET expression. Hum Mol Genet 2016; 25(9): [10] Wu JL, Lu DC, Luo ZJ, et al. Effect of edible tulip on the BCL-2 and C-myc expression in thyroid cancer SW579 cells. Guangdong Med J 2014; 35(3): [11] Ting S, Synoracki S, Schmid KW. Thyroid C cells and their pathology: Part 1: normal C cells, - C cell hyperplasia, - precursor of familial medullary thyroid carcinoma. Pathologe 2015; 36(3): [12] Fang CH, Xu YJ, Sheng HM. Effect of HIC1 and SIRT1 expression levels in thyroid nodule on assessing benign or malignant nodules. J Hainan Med Univ 2016; 22(18): [13] Zhang LT, Wu WY, Lin XG, et al. Hypermethylation of HIC1 and aberrant expression of HIC1/SIRT1 contribute to papillary thyroid carcinoma. Chin J Endocrinol Metabol 2016; 32(5): [14] Qiu W, Yang Z, Fan Y, et al. MicroRNA-613 inhibits cell growth, migration and invasion of papillary thyroid carcinoma by regulating SphK2. Oncotarget 2016; 7(26): [15] Hou JY, Liu XA, Shan GY, et al. Effect of S100A4 on invasion of human thyroid carcinoma cells by regulation of MMP-13. Chin J Cancer Prevent Treat 2015; 22(7): [16] Wan L, Zheng XQ. Expression pattern and function of SATB1 in the invasiveness of thyroid carcinoma. Chin J Biochem Pharm 2014; 34(6):
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