| Background:Thyroid cancer is a common endocrine malignant tumor,in recent decades,its incidence increasing significantly and tends to be younger.However,Papillary thyroid carcinoma（PTC）is the most frequent pathological type of thyroid carcinoma,and the incidence of PTC is more than 80%.PTC is an indolent endocrine tumor,but,some PTC patients may have cervical lymph node metastasis when diagnosed at an early stage,and the metastasis rate is high,up to about 30%,which seriously affects the quality of life and prognosis of patients.Therefore,to further reveal the potential mechanism involved in the progression of PTC,and to find intervention targets for anti-PTC diagnosis and treatment,it is of great significance to improve the early diagnosis rate and prognosis of PTC.The abnormal proliferation of cells is a crucial factor leading to the development of tumor,however,abnormal regulation of cell cycle is the initiating factor of abnormal cell proliferation,abnormal expression of cell division cycle-related proteins,it can directly cause abnormal regulation of biological processes such as cell division,proliferation and apoptosis.Cell division cycle-associated protein 2（CDCA2）is an important member of the cell division cycle-associated protein family,its bidirectional regulated by CDKl and Aurora B protein kinases with protein phosphatase 1 and protein phosphatase 2A during the cell division cycle,and CDCA2 is closely involved in mitosis,chromatin remodeling,nuclear membrane recombination,and repair of DNA damage responses.The reversible interaction of CDCA2 with chromatin in the cell cycle,it plays a vital role in maintaining normal cell division.At present,studies have suggested that the expression of CDCA2 is relative elevate in malignant tumor specimens and is associated with poor prognosis of patients,at the same time,the functional studies of tumor cells also suggested that CDCA2 could accelerate the proliferation of tumor cells,inhibit cell apoptosis,and then induce the invasion and metastasis of tumor cells and other malignant behaviors.Therefore,CDCA2 may be a new potential tumor therapeutic target,at present,the biological role and prognosis evaluation of CDCA2 in the development and progression of PTC are not clear,and the mechanism of CDCA2 involvement in the regulation of PTC malignant biological behavior is also unclear.Therefore,the role of CDCA2 in the progression of PTC and its potential regulatory mechanism are further studied in this study.Methods:1.Oncomine database was used to analyze and compare the expression of CDCA2 in human PTC tissues and paracancer tissues.Then,a total of 80 pairs of PTC tissues and adjacent normal tissues were collected by surgically resected（no cancer cells were found by pathological section during operation）.Immunohistochemistry（IHC）was used to detect the positive expression rate of CDCA2 in PTC tissues and adjacent normal tissues,Westen Blot and Quantitative real-time polymerase chain reaction（q-PCR）were used to detect the expression of CDCA2 protein and m RNA in PTC tissues and paired adjacent normal tissues,and analysis the relationship between the expression of CDCA2 and clinicopathological features in PTC patients.2.PTC cell lines（K1,TPC-1）and normal thyroid follicular epithelial cell line（Nthy-ori-3-1）were cultivated.The expression levels of CDCA2 protein and m RNA in PTC cells was detected by Western Blot and q RT-PCR.K1 and TPC-1 cells were transfected with plasmids or lentiviral tools,respectively,the transfection efficiency of CDCA2 was evaluated.The K1 and TPC-1 cell lines with stable knockdown and overexpression of CDCA2 gene were constructed and screened.3.The effect of CDCA2 on the activity and proliferation of PTC cells were detected by MTS,Ed U and plate clonogenesis experiments;the effect of CDCA2 on the migration and invasion ability of PTC cells were investigated by transwell invasion assay and Wound healing assay;Annexin V-FITC/PI double staining flow cytometry was used to detected the effect of CDCA2 on the apoptosis of PTC cells;the effect of CDCA2 on PTC cell cycle was detected by flow cytometry with Propidium Iodide（PI）staining.4.The samples were detected by Westen Blot,the protein expression levels of Cyclin Dl,Cyclin E,anti-apoptotic factor（Bcl-2）and apoptotic promoter（Bax）after changing the expression of CDCA2 gene in PTC cells,the expression levels of p-PI3K,PI3K,p-Akt and AKT protein in PI3K/AKT signaling pathway were detected,meanwhile,epithelial-to-mesenchymal transition was also detected,the expression of EMT markers E-cadherin,N-cadherin,vimentin and matrix metalloproteinase 2/9（MMP-2/9）.5.PTC cells overexpression group was used for recovery experiment,they were divided into non-transfected group,CDCA2 overexpression group and CDCA2 overexpression+PI3K inhibitor group,and the effects on biological function of PTC cells were detected,and the expression of related proteins in the above experiments.6.The xenograft tumor model of nude mice was constructed by TPC-1 cells of stably transfection CDCA2 gene,nude mice were randomly divided into knockout group,knockout no-load group,normal cell group,overexpression no-load group and overexpression group（n=5/group）,the growth of transplanted tumors was observed,the mice were sacrificed at 28 days after tumor formation,and the volume and weight of tumor were measured.Westen Blot were used to detect the expression levels of CDCA2 protein,EMT markers and key proteins in PI3K/AKT signaling pathway of transplanted tumor tissue.Results:1.Searching Oncomine database showed that CDCA2 was significantly overexpressed in human PTC tissues（P<0.01）.IHC results showed that the positive expression rate of CDCA2 staining in adjacent normal tissues was11.25%（9/80）,and in PTC tissues was 76.25%（61/80）（P<0.05）.Western Blot and q RT-PCR results showed that the relative expression levels of CDCA2 protein and m RNA in PTC tissues were significantly higher than in adjacent normal tissues（P<0.05）,the high level of CDCA2in PTC tissues was closely correlated with lymph node metastasis and multifocality tumor（P<0.05）,there were no significant differences in age,gender,tumor size and capsule invasion between patients（P>0.05）.2.The results of Western Blot and q RT-PCR showed that compared with thyroid follicular epithelial cell Nthy-ori-3-1,the expression levels of CDCA2 protein and m RNA in K1 and TPC-1 cells were significantly increased（P<0.01）.Compared with non-transfected group（con）and knockdown no-load group（neg-sh RNA）,the relative expression levels of CDCA2 protein and m RNA in knockdown group（CDCA2-sh RNA）were significantly decreased（P<0.05）,there was no significant difference between con group and neg-sh RNA group（P>0.05）.Compared with con group and pc DNA3.3overexpression no-load group,the expression of CDCA2 protein and m RNA in PCDNA3.3-CDCA2 overexpression group was significantly increased（P<0.05）,while there was no significant difference between con group and pc DNA3.3 group（P>0.05）.These results indicated that PTC cell lines with stable CDCA2 knockdown and overexpression were successfully constructed.3.The results of MTS,Ed U and plate clone formation experiments suggested that CDCA2 promoted the proliferation of PTC.MTS showed that compared with con group and neg-sh RNA group,the proliferation ability of PTC cells in CDCA2-sh RNA group was significantly decreased（P<0.05）;Ed U showed that the average cell counts of PTC cells in CDCA2-sh RNA group were significantly lower than in con group and neg-sh RNA group（P<0.05）;plate clone formation experiment,the average number of cell colonies in PTC CDCA2-sh RNA group was significantly lower than that in con group and neg-sh RNA group（P<0.05）.There was no significant difference between con group and neg-sh RNA group in the above three methods（P>0.05）.On the contrary,use the same experimental,the effect of CDCA2 gene overexpression on the proliferation ability of PTC cells was detected.The results showed that compared with con and pc DNA3.3 groups,the proliferation ability of PTC cells in PCDNA3.3-CDCA2 group was significantly increased（P<0.05）,while there was no significant difference between con and pc DNA3.3 groups（P>0.05）.4.The results of Transwell invasion and scratch assay showed that CDCA2 could enhance the migration and invasion ability of PTC cells.In PTC cells,the mean cell counts of Transwell invasion assay in CDCA2-sh RNA group was significantly decreased compared with that in con group and neg-sh RNA group（P<0.05）;the migration rate of PTC cell was measured by scratch test,the percentage of cell healing area in CDCA2-sh RNA group was significantly decreased（P<0.05）,while there was no significant difference between con group and neg-sh RNA group（P>0.05）.In contrast,the same method was used to detect the effect of CDCA2 gene overexpression on the migration and invasion ability of PTC cells,the results showed that compared with con and pc DNA3.3 groups,the migration and invasion ability of PTC cells in PCDNA3.3-CDCA2 group were significantly enhanced（P<0.05）,while there was no significant difference between con and pc DNA3.3 groups（P>0.05）.5.Annexin V-FITC/PI double staining flow cytometry showed that CDCA2 could inhibit the apoptosis rate of PTC cells.In PTC cells,the apoptosis of CDCA2-sh RNA group was significantly higher than that of con group and neg-sh RNA group（P<0.05）,but there was no significant difference between con and neg-sh RNA group（P>0.05）.On the contrary,the apoptosis rate of PTC cells was significantly decreased after overexpression of CDCA2 gene（P<0.05）,while there was no significant difference between con and pc DNA3.3 groups（P>0.05）.PI staining results indicated that in PTC cells,compared with the con group and the neg-sh RNA group,the proportion of G0-G1 phase in CDCA2-sh RNA group was increased,and the proportion of S phase was significantly decreased（P>0.05）,but there was no significant difference in G2-M phase（P<0.05）.There was no significant difference between con group and neg-sh RNA group at each stage（P>0.05）.On the contrary,overexpression of CDCA2 gene promoted the transition of PTC cells from G0-G1 phase to S phase.in pc DNA3.3-CDCA2 group,the proportion of G0-G1 phase was less and the proportion of S phase was significantly increased（P<0.05）,while the proportion of G2-M phase was not significantly changed（P<0.05）.However,there was no significant difference between con group and pc DNA3.3 group at each stage（P>0.05）.6.Western blot was used to detect the protein expression levels of Cyclin Dl,Cyclin E,Bcl-2,Bax and MMP2/9 after changing CDCA2 gene expression.The results showed that in PTC cells,the relative expression levels of Cyclin Dl,Bcl-2 and MMP2 proteins in CDCA2-sh RNA group were significantly lower than in con group and neg-sh RNA group（P<0.05）.The relative expression levels of Bax protein in CDCA2-sh RNA group was significantly higher than that in con group and neg-sh RNA group（P<0.05）.On the contrary,PTC cells overexpression of CDCA2 gene,the relative expressions levels of Cyclin Dl,Bcl-2 and MMP2 in pc DNA3.3-CDCA2 group were significantly higher than in con group and pc DNA3.3 group,the relative expression levels of Bax protein in pc DNA3.3-CDCA2 group was significantly lower than that in con group and pc DNA3.3group（P<0.05）,and there was no significant difference in the expression of the above proteins among the neg-sh RNA group,con group and pc DNA3.3 group（P>0.05）.Regulate the expression of CDCA2 gene in PTC cells,the protein levels of Cyclin E and MMP-9 were not significantly affected.Western blot was used to detect the effect of after CDCA2 gene expression on EMT and PI3K/AKT signaling pathway.The results showed that in PTC cells,the relative expressions of Vimentin,P-PI3K and p-Akt proteins in CDCA2-sh RNA group were significantly lower than in con group and neg-sh RNA group,while E-cadherin was significantly increased in CDCA2-sh RNA group（P<0.05）.On the contrary,after CDCA2 gene overexpression of PTC cells,the relative expressions of Vimentin,P-PI3K and p-Akt in pc DNA3.3-CDCA2 group were significantly higher than those in con group and pc DNA3.3 group,E-cadherin was significantly decreased in pc DNA3.3-CDCA2 group（P<0.05）.However,there was no significant difference in the expression levels of these proteins among the con group,neg-sh RNA group and pc DNA3.3 group（P>0.05）.The change of CDCA2 gene expression in PTC cells had no significant effect on the protein levels of N-cadherin,PI3K and AKT.7.The CDCA2 overexpression group was added with PI3K inhibitor for recovery experiment.In PTC cells,compared with the PCDNA3.3-CDCA2+PI3K inhibitor group,the protein levels of P-PI3K,p-Akt and PI3K in the PCDNA3.3-CDCA2+dd H2O group were significantly increased,and the differences between the groups were statistically significant（P<0.05）,these results suggested that PI3K inhibitor effectively inhibited the activity of PI3K/AKT signaling pathway（P<0.05）.The functional test results indicated that the proliferation,migration and invasion ability of pc DNA3.3-CDCA2 group was higher than that of the CDCA2 overexpression+PI3K inhibitor group,while the CDCA2overexpression+PI3K inhibitor group was higher than that the con group（P<0.05）;Annexin V-FITC and PI double staining flow cytometry showed that the apoptosis rate of PTC cells in pc DNA3.3-CDCA2 group was lower than that in CDCA2 overexpression+PI3K inhibitor group,and the apoptosis rate of PTC cells in CDCA2 overexpression+PI3K inhibitor group was lower than that in con group;the proportion of G0-G1 phase in PTC cells in pc DNA3.3-CDCA2 group was lower than that in CDCA2 overexpression+PI3K inhibitor group,and that in CDCA2 overexpression+PI3K inhibitor group was lower than that in con group,The proportion of S phase in pc DNA3.3-CDCA2 group was higher than that in CDCA2 overexpression+PI3K inhibitor group,and that in CDCA2overexpression+PI3K inhibitor group was higher than that in con group,the differences among the above groups were statistically significant（P<0.05）.Western blot analysis showed that the relative expressions levels of Cyclin Dl,Bcl-2,MMP2,Vimentin,p-PI3K and p-Ak T in PTC cells of pc DNA3.3-CDCA2 group were significantly higher than in CDCA2 overexpression+PI3K inhibitor group,while CDCA2 overexpression+PI3K inhibitor group were significantly higher than con group;the relative expression levels of Bax and E-cadherin proteins in pc DNA3.3-CDCA2 group were significantly lower than in CDCA2 overexpression+PI3K inhibitor group,and those in CDCA2 overexpression+PI3K inhibitor group were lower than those in con group,and the differences among the three groups were statistically significant（P<0.05）,however,the protein expression levels of Cyclin E,MMP-9,N-cadherin and AKT were not significantly changed between groups（P>0.05）.8.The test of xenograft in nude mice,tumor growth curve showed that the growth rate of CDCA2-sh RNA group was the slowest,while the pc DNA3.3-CDCA2 group was the fastest.The average volume and weight of the tumors in each group were measured,the CDCA2-sh RNA group was the lowest,and the pc DNA3.3-CDCA2 group was the highest,and the differences were statistically significant（P<0.05）,however,there was no significant difference among the con group,neg-sh RNA group and pc DNA3.3 group（P>0.05）.CDCA2 can promote the growth of TPC-1 cell xenografts in nude mice.The results of Western Blot showed that compared with con group,neg-sh RNA group and pc DNA3.3 group,the expression of CDCA2 protein in CDCA2-sh RNA group was significantly decreased（P<0.05）.pc DNA3.3-CDCA2 group was significantly increased（P<0.05）.The changes of PI3K/AKT and EMT related proteins in xenograft tumor tissues of nude mice showed that compared with the con group,the neg-sh RNA group and the pc DNA3.3 group,the expressions of Vimentin,p-PI3K and p-Akt proteins were significantly decreased in the CDCA2-sh RNA group.pc DNA3.3-CDCA2 group was significantly increased（P<0.05）;the expression of E-cadherin protein in CDCA2-sh RNA group was significantly increased,while the expression of pc DNA3.3-CDCA2 group was significantly decreased,and the differences were statistically significant（P<0.05）,however,there was no significant difference in the above proteins among con group,neg-sh RNA group and pc DNA3.3 group（P>0.05）.Conclusion:1.In the PTC patients,the expression of CDCA2 protein and m RNA in cancer tissues was significantly higher than that in adjacent normal tissues,and the difference was statistically significant;and the expression level of CDCA2 is closely related to lymph node metastasis and multifocal tumor in patients of PTC,which indicates that the high expression of CDCA2 may play a certain role in the progression of PTC.2.CDCA2 can significantly promote the proliferation,migration and invasion of PTC cells,and inhibit the apoptosis of PTC cells.3.CDCA2 regulates the proliferation,migration,invasion and EMT of PTC cells through PI3K/AKT signaling pathway.CDCA2 promotes the growth of subcutaneous xenografts of TPC-1 cells in nude mice through PI3K/AKT signaling pathway. |
