The Expression Of Id1/Id3in Endometrial Carcinoma And Role Of Id1/Id3in Biological Function Of Endometrial Carcinoma Cell

[Background]The Id (inhibitor of DNA differentiation) proteins can react with alkaline HLH transcription factor, and form the inactive heterodimer, which can block the integration of bHLH and E promoter on the target gene DNA. This would suppress the expression of differentiation-related genes indirectly and affect the growth of tumor cell, the formation of vein and the ability of invasion and metastasis. In addition, the Idl/Id3have many overlapping effects, which possess a type of synergistic effect in promoting the tumor cells’ proliferation, invasion and metastasis. Therefore, the Idl/Id3is considered as the upstream regulatory gene that associated with tumor’s aggressiveness.In recent years, Gene and targeted therapy are given more and more attention, because of their better effects in the treatment of cancer. However, until now, there are no particularly effective specific-markers in the treatment endometrial cancer. Besides, the molecular mechanisms of growth and metastasis have not yet been fully understood. And the relationship of Idl/Id3gene and the occurrence, development, invasion and metastasis of endometrial cancer have not been studied. We hope that we could find a new treatment strategy by studying the mechanism of endometrial cancer’s occurrence, development, invasion and metastasis on biological behavior.[Purpose]We aim to detection the expression of Idl/Id3in endometrial carcinoma.we constructe Id1/Id3double-knockdown adenovirus by RNA interference technology, and then infected endometrial cancer cell lines HEC-1-B and RL-952. By observing the influence of MMP2, CXCR4and P21on the level of gene and protein after Idl/Id3double-knockdown, we speculated the relationship between dl/Id3and P21,MMP2,CXCR4,and investigate that Idl/Id3whether participate in their signaling pathways. The mechanism of infiltration, proliferation and metastasis of endometrial cancer cells was studied. We especially discussed the influence of double-knockdown Idl/Id3genes on the ability of proliferation, invasion, migration of endometrial cancer cells. We also inspect whether targeting Idl/Id3treatment can be used as an effective new strategy for the treatment of endometrial cancer, to provide a theoretical basis for clinical diagnosis and treatment.[Materials and methods]1.Expression of Idl and Id3in endometrial cancer tissues were detected by Western blot:Total protein was extracted from4patients’ fresh endometrial cancer tissues and its para-cacinoma tissues, which were selected randomly from the patients who accepted the operation in department of obstetrics and gynecology of Nanfang Hospital of Southern Medical University during June-October in2011. According to the BCA quantitative kit, A562was determined. The average absorbance of each protein with different concentration level was calculated, then standard curve was draw and the regression equation was calculated.2×SDS protein sample buffer (1:1) was added to the total extracted protein, then the protein was heated to100℃for5min,and preserved in-20℃. Idl and Id3expression in4frensh endometrial cancer tissues and its para-cacinoma tissues was detected by Western blot. After Western blot, we observe the gel images and by using the automatic image analyze to quantitative analysis of the relative content of Id1/Id3(gray).2. To detect the relative expression of Idl, Id3, MMP2, CXCR4, P21mRNA in endometrial cancer cell lines HEC-1-B and RL-952after different recombinant adenovirus vector infection by real-time PCR:Each cell was divided into four groups, include untreated group (abbreviations control), blank virus group(vector), the promoter group (promoter) and Id1/Id3double-knockdown group (RNAi). Then the RNA extraction, genome elimination, purity detection and gel electrophoresis detection, reverse transcription and quantitative PCR operation of the four groups of cells were conducted. ΔΔ CT method was used to the quantitative analysis of the results of real-time PCR, then to calculate the expression inhibition rate of MMP2, CXCR4, P21after Idl/Id3double-knockdown.3. To detect the relative expression of Idl, Id3, MMP2, CXCR4, P21protein in endometrial cancer cell lines HEC-1-B and RL-952after different recombinant adenovirus vector infection by Western blot:Total protein of four groups of each cell line was extracted, the specific steps ibid.4. To detect the effect of different recombinant adenoviral vectors on the proliferation of endometrial cancer cell lines HEC-1-B and RL-952by MTT assay:MTT was added to the four groups of each cell line, and refer to the blank control orifice, the average optical density (OD) and inhibition rate were counted by Universal Microplate Spectrophotometer. Cell growth curve was draw with time as abscissa, the absorbance as a vertical coordinate. The experiment was repeated3times.5. Transwell test was used to evaluate the invasion of endometrial cancer cell lines HEC-1-B and RL-952after transfection, generally the number of cells that across the membrane in6high-power fields was selected randomly.6. To detect the effect of different recombinant adenoviral vectors on the migration of endometrial cancer cell lines HEC-1-B and RL-952by Wound-healing assay, and usually A sample was taken from Oh,6h,12h,24h,48h, untreated cells were took as comparison. The wound area of pixels was measured by phoshop after photographed.7. Using cell adhesion experiments to observe the influence on the ability of cell adhesion of endometrial carcinoma cell lines RL-952and HEC-1-B after infected by different recombinant adenovirus vector. Cells need been treated by MTS assay after camera processing and six composite holes were done in each group.8. Statistical processingThe statistical software SPSS14.0was used for data analysis. Data were expressed in mean±tandard deviation format, two independent samples t test was used for the comparison of expression of Id1/Id3in4endometrial cancer tissues and its para-cacinoma tissues. The single factor variance analysis named ANOVA was used for the comparison of the rest experimental groups, Tamhane’s T2test was used for comparison of each two groups. The test level a=0.05. P <0.05was thought to be statistically significant.[Result]1. Expression of Idl in endometrial carcinoma tissues was higher than its para-cacinoma tissues, and compared two groups have statistical significance (F=9.656t=0.000); Id3expression in endometrial carcinoma also raised. Compared with the expression of its para-cacinoma tissues there are significant differences (F=14.600t=0.000).2. After Id1/Td3double-knockdown of HEC-1-B cells, Idl mRNA expression significantly inhibited (F=283.920P=0.000), Id3mRNA expression significantly inhibited (F=318.073P=0.000), CXCR4mRNA expression significantly inhibited (F=185.749P=0.000), and MMP2mRNA expression levels was reduced (F=57.383, P=0.000), and P21mRNA expression significantly increased (F=37.590P=0.000). For RL-952cells, Idl mRNA expression significantly inhibited (F=211.715P=0.000), Id3mRNA expression significantly inhibited (F=228.218P=0.000), CXCR4mRNA expression levels reduce (F=99.462P=0.000), and MMP2mRNA expression of a lower level (F=19.060P=0.001), higher P21mRNA expression levels (F=104.071P=0.000).3. After Idl/Id3double-knockdown of HEC-1-B cells, Idl protein expression inhibition (F=454.836P=0.000), Id3protein expression inhibition (F=34.112P=0.000), CXCR4protein expression inhibition (F=510.178P=0.000), MMP2protein expression inhibition (F=286.765P=0.000), and P21protein expression elevated (F=1757.754P=0.000). For RL-952cells, Idl protein expression inhibition (F=882.161P=0.000), Id3protein expression inhibition (F=1190.847P=0.000), CXCR4protein expression inhibition (F=963.998P=0.000), MMP2protein expression inhibition (F=145.342P=0.000), and P21protein expression elevated (F=4799.080P=0.000). The addition to P21Idl/Id3double knockdown group showed no significant difference (P=0.998) with the promoter group, the other two groups was observed by Tamhane’s T2test were statistically significant.4. Thiazolyl blue (MTT) colorimetric assay confirmed after double knockdown Idl/Id3expression to inhibit the proliferation of HEC-1-B and RL-952cells:for HEC-1-B cells with12h after transfection, compared with untreated cells group, blank virus group, the promoter group and Id1/Id3double knock low group mean,there are the significant difference between the four groups (F=19.358P=0.001);24h after transfection mean compared to a significant difference (F=26.536P=0.000);48h after transfection compared with a significant difference (F=30.078P=0.000); RL-952cells transfected with viral12h, there are the significant difference between the four groups (F=4.557P=0.038); transfected24h, there are the significant difference between the four groups (F=14.059P=0.001);48h after transfection, there are the significant difference between the four groups (F=28.356P=0.000); growth curve indicate that interference the expression of Id1/d3, cell growth activity is significantly reduced, and the lowest proliferative capacity in48h time point.5. In the transwell test, HEC-1-B and RL-952cells transfected with Id1/Id3double-knockdown obviously reduced invasion:comparison and analysis the number of cells that reach the next room between the untreated group, blank virus group, the promoter group and Idl/Id3double-knockdown group of HEC-1-B cell by statistical analysis, and there are the significant difference between the four groups (F=79.116, P=0.000). RNAi group with the other three groups have significant differences (P<0.001); For RL-952cells, there are the significant difference between the four groups (F=85.899P=0.000), RNAi group with the other three groups have significant differences (P<0.001).6. The ability of endometrial carcinoma cells to migration was obviously weakened after Id1/Id3double-knockdown by wound-healing assay:after scratches6h of RL-952cells, the mobility between the four groups showed significant differences (F=124.307P=0.000), after scratches12h, mobility between the four groups there is a significant difference (F=106.879P=0.000), after scratches24h, mobility between the four groups showed significant differences (F=25.808P=0.000), scratches after48h mobility between the four groups showed significant differences (F=49.600P=0.000), and scratches after48h, compared the migration speed between the RNAi group and the other three groups, the RNAi was slowest, migration minimum distance (P<0.001). after scratches6h of HEC-1-B cells, the mobility between the four groups showed significant differences (F=51.267, P=0.000), after scratches12h, mobility between the four groups there is a significant difference (F=15.869P=0.001), after scratches24h, mobility between the four groups showed significant differences (F=15.179P=0.001), scratches after48h mobility between the four groups showed significant differences (F=51.066, P=0.000), and scratches after48h, compared the migration speed between the RNAi group and the other three groups, the RNAi was slowest, migration minimum distance, there are statistically significant (P<0.05).7. The ability of endometrial carcinoma cells to adhesion was obviously weakened after Idl/Id3double-knockdown by Adhesion assay:there are the significant difference between the untreated group, blank virus group, the promoter group and Idl/Id3double-knockdown group of RL-952cells (F=40.314P=0.000), and RNAi group with the other three groups have significant differences; there are the significant difference between the untreated group, blank virus group, the promoter group and Idl/Id3double-knockdown group of HEC-1-B cells (F=48.945P=0.000), and Idl/Id3double knockdown group with untreated cells group, blank virus group and the promoter group, there were significant differences.[Conclusion]1. Idl and Id3were obviously up-regulated in endometrial carcinoma tissue than para-carcinoma tissues with a significant difference. This can provide a proof whether inhibition Idl/Id3can effective treatment of endometrial cancer. 2. Idl/Id3double-knockdown decreased the expression of MMP2and CXCR4and increased the expression of P21in both mRNA and protein level. Groups were compared between each other have significant difference, and Idl/Id3double-knockdown group compared with the other three groups have significant difference. We speculate that, Idl/Id3can up-regulation MMP2and CXCR4, and down-regulation P21, which can raised the CDK2, Cyclin E, CDK1, Cyclin B expression, and further up the non-phosphorylated Rb protein expression. Visible, Idl/Id3upstream regulatory gene for P21, MMP2and CXCR4can participate in them-mediated cell survival, cell proliferation or chemotactic signals to the path, inhibits endometrial cancer cell infiltration, proliferation and metastasis.3. The endometrial carcinoma cells proliferation, invasion, migration and adhesion ability were inhibited after Idl/Id3double-knockdown.In short, Idl/Id3play an important role in the development of endometrial cancer, targeting Idl/Id3would be a novel strategy for the prevention and treatment of endometrial carcinoma, but the detailed mechanism Idl/Id3endometrial cancer still needs further research.