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@#CIP2A is an oncoprotein involved in the progression of several human malignancies. It has recently been described as a prognostic marker in many cancers. The present study aimed to investigate the immunohistochemical expression of CIP2A in benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN) and prostate cancer (PC), and to analyse the association with the clinicopathological parameters in PC cases to define its role in the development and progression of PC. Materials and Methods: Immunohistochemical staining for CIP2A was performed on the tissue microarray sections of 105 PC, 27 HGPIN and 27 BPH tissues. The CIP2A expression scores were compared with several clinicopathological parameters. Results: CIP2A was expressed in 96,2% of PC, 55,6% of HGPIN and 40,7% of BPH tissues. The expression of CIP2A in PC was significantly higher than in HGPIN (p<0.0001) and BPH (p<0.0001) cases. CIP2A expression score was significantly associated with Gleason score (p=0.032) and lymphovascular invasion (p=0.039). Nevertheless, there was no statistically significant association between the expression of CIP2A and perineural invasion, pT stage, metastasis and recurrence (p>0.05). Multivariate analysis indicated that GS, lymphovascular invasion, distant metastasis were independent prognostic factors for PC patients but, CIP2A expression score was not found to be a prognostic factor. Additionally, there was no significant difference between the survival times of patients according to CIP2A expression (p=0.174). Conclusion: According to our results, the expression of CIP2A protein is increased in PC and its expression may be involved in the development, differentiation, and aggressiveness of PC. However, further studies are needed to confirm our findings and to clarify the role of CIP2A in the development of PC.
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Objective: To investigate the effect of inhibiting CIP2A expression on the biological characteristics of HepG2 cells. Methods: The recombinant adeno-associated viral vector rAAV2-EGFP-CIP2A shRNA was successfully transfected into HepG2 cell lines by using previous experiments. Cell proliferation was detected by MTT assay, cell apoptosis was measured by flow cytometry, and cell invasion ability was detected by Transwell assay. A model of subcutaneous implant tumor in nude mice was established to observe the growth of xenograft tumor in nude mice. The volume and quality of tumor growth were detected in nude mice after 30 days, and the expression of CIP2A protein in the transplanted tumor tissues was detected by immunohistochemistry. Results: The results of MTT and flow cytometry showed that the proliferation and apoptosis of HepG2 cells were inhibited after downregulation of CIP2A expression. The Transwell invasion experiment indicated a decrease in cell invasion ability. The result of subcutaneously growing tumor in the nude mice found in the interference experiment group of nude mice, the subcutaneous tumor growth rate, the final volume and weight were much smaller than those in blank control group and no-load virus group, CIP2A protein expression in transplanted tumor tissues was significantly lower. Conclusion: CIP2A promotes the proliferation, invasion of HepG2 cells and malignant growth in vivo.
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Objective To construct the recombinant adeno-associated virus vector carrying cancerous inhibitor of PP2A (CIP2A)short hairpin RNA (shRNA)for preparation of high-titer viruses.Methods The small hairpin RNA of CIP2A (CIP2A shRNA)was designed,synthesized and cloned into pDC31 6-EGFP-U6 plasmid which was double digested by Bam HⅠ and Hin dⅢ.The resultant plasmid pDC31 6-EGFP-shRNA was confirmed and served as template to appraise primers.EGFP-CIP2A shRNA sequence was amplified by PCR,double digested with Eco RⅠand Sal Ⅰ and ligated to pSNAV2.0 plasmid digested with the same enzyme pair.pSNAV2.0-EGFP-CIP2A shRNA plasmid DNA was prepared,purified,identified and transfected into BHK-21 cells.BHK-21 cells expressing CIP2A shRNA (BHK-21/CIP2A-shRNA ) were obtained and subsequently infected with VGTC’s proprietary AAV packaging system to package the rAAV2-CIP2A shRNA.After purification,the functional and infectious virus was obtained and the titer of virus was detected.Real-time PCR and Western blot methods were used to detect the expression of CIP2A after infection with HepG2 cells,and the empty viral vector rAAV2-EGFP was used as control. Results A recombinant adeno-associated virus-2 vector carrying CIP2A shRNA was constructed successfully.The presence of the target sequence in the vector was confirmed by double enzyme digestion and sequencing.By transfecting the pSNAV2.0-EGFP-CIP2A shRNA plasmid into BHK-21 cells,BHK-21/CIP2A shRNA cells were infected with helper virus HSV1-rc/ΔUL2 to package the rAAV2-CIP2A shRNA to obtain a functional and infectious virus.The titer of the recombinant virus was 0.25×10 1 2 v.g./mL.The expression of CIP2A mRNA and screening value of 1×10 5 MOI effected HepG2 cells.Conclusion A high-titer recombinant adeno-associated virus-2 vector carrying CIP2A shRNA has been constructed successfully.
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Objective To construct eukaryotic vectors expressing short hairpin RNAs ( shRNAs ) targeting at the CIP2 A gene and to explore its effects on gastric cell line BGC-823 .Methods Four oligonucleotides targeting the CIP2A gene were synthesized and cloned into the eukaryotic expression plasmid pGPU 6.The recombinant plas-mids, pGPU6/GFP/Neo-CIP2A-shRNA-1, 2, 3 and 4, were introduced into BGC-823 cells by lipofectamine-me-diated transfection and the infection rate was observed by fluorescence microscope .The gene silencing efficiency was measured by real-time PCR and Western blot .The effects on proliferation of BGC-823 cells were detected by CCK-8 .Results DNA sequencing and enzyme digestion analysis confirmed the identity of the four recombinant shRNA expression vectors .Immunofluorescsence demonstrated that transfection efficiency was above 70%.Trans-fection of shRNA-1, 2, 3 and 4, significantly knocked down the expression of CIP 2A mRNA and protein at 24, 48 and 72 h after transfection .Compared with the 2 , 3 and 4 , shRNA-1 had the more strong inhibitory effect on the expression of CIP2A mRNA and protein.The CCK-8 assay showed that the anti-proliferation effect on BGC-823 cells was significant ( P<0.05 ) .Conclusions The recombinant vector may effectively inhibit the expression of CIP2A in BGC-823 cells and depress the proliferation of BGC-823 cells.
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Objective To investigate the expression of CIP2A and c‐Myc and their correlation in cervical carcinoma .Methods We detected CIP2A and c‐Myc expression using immunohistochemistry in 72 samples of cervical carcinoma and 12 samples of normal cervix tissues .Correlation between proteins and clinicopathologic features and relation between CIP2A and c‐Myc expression were analyzed .Results The positive expression of CIP2A and c‐Myc in cervical carcinoma tissues were 52 .8% and 56 .9% ,respec‐tively .While their positive expression in normal cervix tissues were 8 .3% and 25 .0% ,respectively .The differences had statistical significance(χ2 =8 .169 ,P=0 .004 ;χ2 =4 .208 ,P=0 .040 ,respectively ) .Clinicopathological analysis suggested that CIP2A and c‐Myc protein expression were associated with histopathological differentiation and clinical stage(P<0 .05) in cervical carcinoma ,but the protein expression was not related to age ,lymph node metastasis and pathological type .CIP2A was significantly positive correla‐ted with c‐Myc protein in cervical carcinoma(r=0 .673 ,P=0 .001) .Conclusion The high‐expression of CIP2A is correlated with malignant clinicopathologic characteristics ,and CIP2A is positively associated with c‐Myc ,suggesting that CIP2A may promote tumor initiation and development through maintaining c‐Myc protein in cervical carcinoma .
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Objective To explore the expression and correlation of CIP 2A and HPV16-E7 protein in cervical cancer.Methods The expressions of CIP2A and HPV16-E7 in 50 cases of cervical cancer ,34 cases of CINⅠ,40 cases of CINⅡ,45 cases of CINⅢand 12 cases of normal tissues were assessed by immunohistochemi -cal method.R esults The rate of expressions of CIP2A protein in cancer tissues and CINⅢ were 68%(34/50) and 13.3%(6/45)respectively;The rate of expressions of CIP2A protein in CINⅡ、CINⅠtissues and normal tis-sues were negative.The expressions of CIP2A protein in cancer tissues were stronger than in CIN tissues and nor-mal tissues(P0.05).The expression of CIP2A was positively associated with the expression of HPV16-E7 in cervical cancer(r=0.514,P<0.05).Conclu-sion The expressions of CIP2A and HPV16-E7 are very strong in cervical cancer;CIP2A and HPV16 may play important roles in the carcinogenesis and the development of cervical cancer .
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Objective This study is to explore the expression of CIP2A mRNA in hepatocellular carcinoma (HCC), and its correlations with clinicopathologic features and prognosis of HCC patients.Methods CIP2A mRNA expression was analyzed in four liver cancer cell lines (Hep-G2, MHCC97,SMMC-7721 and BEI-7402), one immortalized liver cell line L-O2, neoplastic tissues and adjacent matched non-neoplastic liver tissues in 120 HCC patients and normal liver tissues of 20 cases using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The correlations between CIP2A mRNA and clinicopathologic features and prognosis of HCC were analyzed. Results CIP2A mRNA was detected in Hep-G2, MHCC-97H, SMMC-7721 and BEL-7402, but not in L-O2.The positive rate of CIP2A mRNA expression was significantly increased in HCC tissues (78.3%)than in adjacent matched non-neoplastic liver tissues (28.3%) and normal liver tissues (5.0%,P＜0. 01). CIP2A mRNA expression was correlated with tumor size, differentiation and TNM stage (P＜0.05). Patients with positive expression of CIP2A mRNA had lower overall survival and diseasefree survival rates. Conclusions CIP2A mRNA, which is highly expressed in liver cancer cell lines and HCC tissues, may be involved in hepatocarcinogenesis. CIP2A mRNA may be a valuable biomarker for assessing the prognosis of HCC.