The effects of casein kinase 2 interacting protein-1 on the growth and development of craniomaxillofacial soft and hard tissues in mice / 口腔疾病防治

Objective@# To investigate the effect of casein kinase 2 interacting protein-1 (CKIP-1) on craniofacial soft tissues and hard tissues, to provide the basis for the study and treatment of craniomaxillofacial related diseases.@*Methods@#6-month- old male CKIP-1 knockout (KO) mice were selected as the experimental group, and wild-type (WT) mice were selected as the control group. The craniomaxillofacial hard tissues (parietal bone, nasal bone, incisors and molars) were analyzed through micro- CT, and the morphological changes of maxillofacial soft tissues (nasal cartilage, lip mucosa and tongue) were analyzed through HE staining and toluidine blue staining.@* Results@#CKIP-1 negatively regulated bone mass of cancellous bone of cranial and maxillofacial bones and dentin mineralization. Compared with the WT mice, the thickness of the parietal baffle layer increased by 93% in KO mice, while cortical bone showed no significant difference between the two groups. The nasal cancellous bone thickness increased by 160% in KO-mice, while cortical bone showed no significant difference between the two groups; the enamel thickness was normal, but the pulp cavity became smaller and the dentin thickness increased by 48%. Compared with the WT mice, the HE staining and toluidine blue staining analyses of the soft tissues revealed that the thickness of the alar cartilage plate of KO mice increased by 57%, and local ossification was found within the cartilage plate. The thickness of the keratinized layer of the labial mucosa increased by 170% in KO mice and the muscle fiber diameter of the lingual muscle increased by 45%. @*Conclusion@#CKIP-1 genes have different effects on the growth and development of various soft and hard tissues in the maxillofacial region of mice.

Effects of casein kinase 2 interacting protein-1 on the osteogenic differentiation ability of human periodontal ligament stem cells / 口腔疾病防治

Objective @#To investigate the effects of casein kinase 2 interacting protein-1 (CKIP-1) on the osteogenic differentiation ability of human periodontal ligament stem cells (hPDLSCs).@*Methods @#The hPDLSCs were obtained by primary culture with periodontal ligament tissues that were collected from normal humans. Then, a lentiviral vector containing a CKIP-1-specific siRNA sequence was constructed, and the transcriptional level of CKIP-1 in hPDLSCs was downregulated after vector infection. The P4 cells were divided into four groups: the control group, negative control group (infected with a control vector), CKIP-siRNA group (infected by a CKIP-1 siRNA lentivirus) and CKIP-1 group (infected by a CKIP-1 overexpression virus). All of the cells were cultured under osteogenic induction for 21 days. Then, alizarin red staining and quantitative determination were performed to detect the osteogenic differentiation ability of the hPDLSCs. In addition, qPCR was used to detect the transcriptional level of osteogenesis-related regulatory factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), and receptor activator of nuclear factor kappa-B ligand (RANKL), and the osteogenesis-related regulatory factors of the bone morphogenetic protein (BMP) signaling pathway.@*Results@#There were no differences in the indexes between the negative control group and the control group (P > 0.05). Compared with the negative control group, the CKIP-siRNA group demonstrated more mineralized nodules (P < 0.05), significantly increased calcium salt deposition (P < 0.05), and increased mRNA levels of osteogenesis-related regulatory factors, such as Runx2 , ALP, OCN, and RANKL, and the osteogenesis-related regulatory factors of BMP signaling pathway (P < 0.05). @*Conclusion@#Downregulation of CKIP-1 could promote the osteogenic differentiation of hPDLSCs, which is related to the transcription level of osteogenic-related regulatory factors.

CK2 inhibitor CX4945 reverses cisplatin-resistance of lung cancer A549/DDP cells / 肿瘤

Objective: To investigate the effect of CK2 (casein kinase 2) inhibitor CX4945 on the cisplatin (DDP)-resistance of lung cancer A549/DDP cells and the underlying molecular mechanism. Methods: The CCK-8 assay was used to detect the half maximal inhibitory concentration (IC50) of DDP in lung cancer A549 and A549/DDP cells, and to compare the DDP-resistance of two cell lines. The effect of CX4945 on DDP-resistance of A549/DDP cells was tested by CCK-8 method. Western blotting was used to detect the expressions of Wnt signaling pathway-related proteins (CK2α, β-catenin and cyclin D1), drug resistance-related proteins [multidrug resistance-associated protein 1 (MRP1) and lung resistance-related protein (LRP)] and apoptosis-related protein [cleaved caspase-3 (c-caspase-3)] in A549 and A549/DDP cells treated with DDP or not. The A549/DDP cells were treated with no drug (as the control group), CX4945, DDP and their combination (as CX4945+DDP group), then the expressions of Wnt signaling pathway-, drug resistance- and apoptosis-related proteins were detected by Western blotting, and the apoptosis of A549/DDP cells was detected by FCM method. Results: The IC50 value of DDP in A549/DDP cells was 4.59 times higher than that in A549 cells, and the DDP-resistance of A549/DDP cells was decreased by CX4945 pretreatment (P 0.05). Compared with the control group and DDP group, the expression levels of β-catenin, cyclin D1, MRP1 and LRP proteins in A549/DDP cells of CX4945 group and CX4945+DDP group were significantly declined (all P < 0.01). In addition, the apoptosis rate of A549/DDP cells and the expression level of c-caspase-3 in CX4945+DDP group were significantly higher than those in the control group and DDP group (all P < 0.001). Conclusion: CK2 inhibitor CX4945 can reverse the DDP resistance of lung cancer A549/ DDP cells through blocking Wnt signal pathway and decreasing the expressions of drug resistance-relatied proteins.

Effect of CKIP-1 siRNA lentivirus transfection on proliferation of glioma U87-MG cells / 中华行为医学与脑科学杂志

Objective@#To investigate the effects of Casein kinase 2-interacting protein 1 (CKIP-1) gene silencing on the proliferation of glioma cells U87-MG.@*Methods@#The recombinant lentiviral vectors targeting CKIP-1 gene or negative control were constructed and then used to infect glioma U87-MG cell line.The effects of knock-down on the mRNA or protein expression of CKIP-1 were evaluated by real-time qPCR and western blotting.Cell cycle was detected by the flow cytometry assay, and cell proliferation changes were evaluated by cell counting, MTT, and BrdU assay, respectively.Lastly, the colony formation was used to investigate the effect of CKIP-1 knock-down on the clone formation.@*Results@#Compared with the group of Ctrl, CKIP-1 siRNA was observed to significantly inhibit CKIP-1 expression at the mRNA levels (Ctrl (1.01±0.13) vs CKIP-1 siRNA (0.23±0.02), P<0.01) and protein levels in the U87-MG cells.Also, CKIP-1 suppression mediated by RNAi decreased the ratio of G0/G1 phase (Ctrl (69.64±0.79) vs CKIP-1 siRNA(62.64±0.66), P<0.01), increased that of G2/M phase (Ctrl (8.36±0.52) vs CKIP-1 siRNA(13.87±2.90), P<0.05), and significantly inhibited the cell proliferation and clone formation (Colony number: Ctrl (25±2) vs CKIP-1 siRNA(2±1), P<0.05) of transfected U87-MG cells.@*Conclusion@#Knocking down the expression of CKIP-1 significantly inhibit cell proliferation in human U87-MG glioma cells, indicating that CKIP-1 is involved in the development of gliomas and could promote the cell proliferation.

Effect of E1A gene on radiosensitivity of human nasopharyngeal carcinoma cells and its possible mechanism / 中华放射肿瘤学杂志

Objective To investigate the effect of E1A gene on the radiosensitivity of human nasopharyngeal carcinoma cells and its possible mechanism. Methods The E1A gene was transfected into nasopharyngeal carcinoma CNE-2R cells by adenovirus vector. The expression of E1A gene was detected by RT-PCR. Untransfected CNE-2R cells(PBS group)and CNE-2R cells transfected with empty vector Ad-β-gal(Ad-β-gal group)and E1A(Ad-E1A group)were given 0 Gy,2 Gy,4 Gy,6 Gy,8 Gy 6 MV X-ray irradiation. The changes in radiosensitivity of CNE-2R cells were determined by colony-forming assay. Flow cytometry was used to analyze cell apoptosis in each group. The expression of NF-κB, CK2α, Bcl-2, and cleaved caspase-3 was measured by Western blot. Results RT-PCR confirmed that the E1A gene was transfected into CNE-2R cells and stably expressed. The Ad-E1A group had a significantly lower plating efficiency than the PBS group and the Ad-β-gal group(P<0.05). The Ad-E1A group had significantly lower cell survival rate at 2 Gy irradiation than the PBS group and the Ad-β-gal group(0.217 vs. 0.602, P<0.05;0.217 vs. 0.585, P<0.05). The Ad-E1A group had a significantly higher α/β value than the PBS group and the Ad-β-gal group(24.680 vs. 5.268, P<0.05;24.680 vs. 5.132, P<0.05). Flow cytometry results showed that irradiation alone could promote the apoptosis of CNE-2R cells,when combined with E1A gene,the apoptosis rate was significantly increased(P<0.05). Western blot results showed that E1A gene down-regulated the expression of NF-κB/p65,CK2α,and Bcl-2 and up-regulated the expression of cleaved caspase-3. Conclusions E1A gene can enhance the radiosensitivity of nasopharyngeal carcinoma cells by inhibiting the expression of CK2 to block the NF-κB signaling pathway and promoting cell apoptosis.

Inhibitory effect of siRNA targeting CK2αgene on growth of HCT1 16 cells and its mechanism / 吉林大学学报(医学版)

Objective To investigate the inhibitory effect of siRNA targeting casein kinase 2 (CK2α)gene on the growth of HCT1 1 6 cells and to clarify its mechanism.Methods CK2α-siRNA sequence was designed according to mRNA sequence of CK2α. The in vitro cultured HCT1 1 6 cells were divided into normal control group (without transfection),negative control group (transfected with siRNA)and CK2α-siRNA group (transfected with CK2α-siRNA ),and the HCT116 cells were transfected with Lipofectamine 2000.The expression levels of CK2α,cyclin H,P53,and P21 proteins in the HCT116 cells were detected by Western blottting method,and the proliferation activities of the HCT116 cells were detected by MTT method,and the cell cycle was measured by flow cytometry. Results Compared with negative control group,the expression levels of CK2α,and cyclin H proteins in CK2α-siRNA group were decreased(P0.05), and the expression level of P21 protein was increased significantly(P<0.01).Compared with negative control group,the survival rate in CK2α-siRNA group was decreased markedly 48 and 72 h after transfection detected by MTT method(P<0.01).Flow cytometry analysis showed the percent of the cells at G1 phase in CK2α-siRNA group was significantly higher than that in negative control group and the percent of the cells at S phase in CK2α-siRNA group was lower than that in negative control group(P<0.01),and the cell cycle was arrested at G1 phase. Conclusion siRNA targeting CK2αcan inhibit the proliferation of HCT116 cells and induce the arrest of G1 phase, which may be associated with inhibiting the expression of cyclin H and recovering the P53 activity after silencing CK2α.

Detection of expressions of Ck2α,β-catenin and survivin in ovarian cancer tissue and its clinical application value / 吉林大学学报(医学版)

Objective To study the expressions of casein kinase 2α(Ck2α),β-catenin, survivin in ovarian cancer tissue and analyze the relationships between them, and to explore the clinical application value. Methods Immunohistochemistry staining was used to detect the expressions of Ck2α,β-catenin and survivin in 8 cases of simple ovarian cysts tissue,8 cases of ovarian benign tumor tissue and 29 cases of ovarian cancer tissue;the correlations of the expressions of Ck2α,β-catenin,survivin in ovarian cancer tissue and their associations with the clinicopathologic characteristics were analyzed.Results The expression levels of Ck2α,β-catenin and survivin in ovarian cancer tissue were significantly higher than those in simple ovarian cysts tissue and ovarian benign tumor tissue(P<0.05);the Ck2αexpression level in ovarian cancer tissue was positively corelated with the expression levels ofβ-catenin and survivin(r=0.438,r=0.479,P<0.05);the expression levels of Ck2α,β-catenin and survivin in low-medium differentiation group were significantly higher than those in high differentiation group (P<0.05);the expression levels of Ck2α,β-catenin and survivin in stagesⅡandⅢ group were significantly higher than those in stage Ⅰ group(P<0.05).Conclusion The protein expression levels of Ck2α,β-catenin and survivin are increased in ovarian cancer tissue, and three are positive corelations between them;Ck2α,β-catenin and survivin may play an important role in the occurrence and development of ovarian cancer;the combined detection of them has important clinical value.

A preliminary study on recombinant expression and function in vitro of proteasome activator REGγ / 军事医学科学院院刊

Objective To study the expression of the fused proteasome activator REGγ(11S regulator complex gamma subunit) using gene recombination technology and to further study the interaction between REGγ and casein kinase-2 interacting protein-1(CKIP-1)in vitro.Methods Firstly, the full length cDNA fragment of REGγ was amplified through PCR using the plasmid pCMV-Myc-REGγ as template and subcloned into the prokaryotic expression vector pGEX-4T-2 before being transformed into E.coli BL21 cells. The protein expression was induced by isopropyl-β-D-thiogalactoside(IPTG) .Secondly, the protein expression was monitored by SDS-PAGE and Western blotting after ultrasonication. Finally, the GST Pull-down assay was performed to investigate the interaction between REGγ and CKIP-1 in vitro.Results The prokaryotic expression construct pGEX-4T-2-REGγ was generated successfully and confirmed by DNA sequencing. Expression analysis showed that the GST-REGγ protein was easily expressed and isolated mainly in the lysate supernatant after sonication and centrifugation. The GST Pull-down assay revealed the strong mutual interaction between REGγ and CKIP-1 in vitro.Conclusion The proteasome activator REGγ could interact with the negative regulator of osteoblastogenesis CKIP-1 in vitro and the current study has shed light on further investigations of their physiological relevance.