Effect Of RbAp48 Knockdown On Repression Of Proliferation And Metastasis In Human Cervical Cancer And Its Mechanism

Partl.Effect of RbAp48 knockdown on repression of proliferation in human cervical cancerObjective Although the cancer treatment has made great progress,according to the report of American Cancer Society in 2015,global cervical cancer has not been effectively controlled, still ranking fourth of women cancer incidence and mortality, seriously threatening women’s health. To find new ways to treat cervical cancer is urgent. In recent years, the study has showed that Retinoblastoma Associated Protein 48(RbAp48, also known as RBBP4) in lung cancer, liver cancer and leukemia cells have been abnormal elevated. It means that RbAp48 might be a close correlation with the tumor and a new potential anti-tumor target. This study was to explore the expression of RbAp48 in cervical cancer and to investigate the effect of RbAp48 knockdown on repression of proliferation in human cervical cancer cell line.Methods 1.Tissue microarray assay detected the expression of RbAp48 for 224 cases of cervical cancer tissues and adjacent cervical mucosa tissues.2. SiRNA was applied to knock down the expression of RbAp48.3. Western blot analysis was performed for siRbAp48 to the expression of RbAp48 in human cervical cancer cell lines.4. MTT analysis and cloning experiment detected the effect of RbAp48 knockdown on the proliferation in Hela, MS751 and SiHa cell lines.5. Beta-Galactose glucoside enzyme staining analysis was performed for siRbAp48 on the cell senescence in Hela and MS751 cell lines.6. Flow cytometry analysis was performed for siRbAp48 on the cell cycle and apoptosis in Hela and MS751 cell lines.7. Nude mice was performed to detect the effect of RbAp48 knockdown on the proliferation in vitro. Results 1.Expression of RbAp48 in cervical cancer was significantly higher than adjacent cervical mucosa tissues. In cervical cancer cells the RbAp48 staining was scored in high in 67.86%(152 cases) and the adjacent cervical tissues was scored high in in 19.15%(9 cases),**P<0.01.2. siRbAp48 can effectively reduce expression of RbAp48 in Hela and MS751 cell lines.3.The cell proliferation was inhibited by 20nM of siRbAp48 in Hela cell lines. Best inhibition effect was produced by 50nM of siRbAp48 with 36.11±4.47% survival rate after four days.4.The cell proliferation was inhibited by 10nM of siRbAp48 in MS751 cell lines. Best inhibition effect was produced by 50nM of siRbAp48 with 47.80±5.08% survival rate after seven days. The cell proliferation was inhibited by 30nM of siRbAp48 in Hela cell lines. Best inhibition effect was produced by 50nM of siRbAp48 with 79.35±8.50% survival rate after five days.5.The cell proliferation was inhibited by 30nM of siRbAp48 in SiHa cell lines. Best inhibition effect was produced by 100nM of siRbAp48 with 56.98±3.97% survival rate after five days.6. The clone numbers of siRNA NC group and siRbAp48 were 87.17±8.63 and 36.67±2.89(**p<0.01) in Hela cell lines,130±8.89 and 87.33±1.16 (**p<0.01) in MS751 cell lines,36.67±2.31 and 31.67±2.09 (p>0.05) in SiHa cell lines.7.In Hela cell lines,siRbAp48 group displayed obvious G2/M cell-cycle arrest with the rate of 32.33±7.14% after 48 hours(**P<0.01). In MS751 cell lines, siRbAp48 group displayed obvious G2/M cell-cycle arrest with the rate of 35.61±3.79% after 96 hours(**P<0.01).8.In Hela and MS751 cell lines,cells displayed obvious senescence with bigger, flatted and bluer intracellular with Senescence p-Galactosidase Staining Kit.8.In Hela cell lines,siRbAp48 group displayed obvious apoptosis with the rate of 54.65±9.06% after 72 hours(**P<0.01). In MS751 cell lines, siRbAp48 group displayed obvious apoptosis with the rate of 32.20±4.47% after 120 hours(*P<0.05).9. In vivo experiment,The effect of RbAp48 knockdown significantly inhibit the growth of tumors in vivo.In Hela cell lines, the turner volume of siRNA NC group was 654.30±191.90 mm3 and the turner weight of siRNA NC group was 353.83±84.11 mg. The turner volume of siRbAp48 group was 158.23±75.82 mm3 (**P<0.01) and the turner weight of siRbAp48 group was 107.92±68.51 mg(**P<0.01). In vivo experiment of MS751 cell lines, the turner volume of siRNA NC group was 182.20±76.09 mm3 and the turner weight of siRNA NC group was 163.78±76.86 mg. The turner volume of siRbAp48 group was 14.85±18.60 mm3 (**P<0.01) and the turner weight of siRbAp48 group was 20.28±22.59 mg(**P<0.01). 10.The IC50 fell from 2.5μg/ml to 1.25μg/ml after using DDP and siRbAp48 on 24 hours. Conclusion 1.Expression of RbAp48 in cervical cancer was significantly higher than para-cancer tissue.2.SiRbAp48 can effectively reduce expression of RbAp48 in human cervical cancer cells.3.Knockdown RbAp48 can effectively induce cell-cycle arrest,cell senescence, apoptosis and inhibit cell proliferation in Hela and MS751 cell lines.4.Knockdown RbAp48 can increase the sensitivity of cervical cancer cells to cisplatin.Part2.The mechanism of RbAp48 knockdown on repression of proliferation in human cervical cancerObjective We previously applied the second generation sequencing study to find that knocking down RbAp48 can inhibit expression of Cyclin protein family and cause cell cycle arrest in human lung carcinoma A549 cell line.So we infered that RbAp48 knockdown on repression of proliferation in human cervical cancer cells may also associate with Cyclin protein pathway.We investigated the mechanism of RbAp48 knockdown on repression of proliferation in human cervical cancer. Methods 1.Western blot analysis was performed for siRbAp48 to detect the expression of Cyclin protein family in human cervical cancer cells.2.RNA interference technique was applied to verify the effection of Cyclin protein family of RbAp48 knockdown on repression of proliferation in human cervical cancer.3.Western blot analysis was performed for siRbAp48 to detect the expression and activication of CDK2, CDK4,CDK6,Rb,E2F1 and so on which were related to Cyclin protein family.4.Combined total of transfection method was performed to survery the proteins which were related to RbAp48 on cervical cancer cells growth.5. MTT analysis detected the siRbAp48 on the proliferation of human cervical cancer in vitro.Results 1. The expression of Cyclin D1, Cyclin E1, Cyclin H,CDK2,CDK4,CDK6,PRb and E2F1 decreased after knockdown RbAp48 in Hela and MS751 cell lines,only the Rb protein expression increased.2. The expression of Cyclin E1,CDK2,CDK4,CDK6, PRb and E2F1 decreased after knockdown RbAp48 in SiHa cell lines,only the Rb, Cyclin D1 and Cyclin H protein expression didn’change.3. After knockdown the expression of Rb,the repression of siRbAp48 antagonize.4. The survival rate of siRb-siRbAp48 group increased from 34.94±2.71% to 56.12±2.72%(**P<0.01) in Hela cell lines, from 44.24±5.83% to 86.04±9.28% (**P<0.01) in MS751 cell lines, from 80.03±7.65% to 92.36±4.89% (**P<0.01) in SiHa cell lines.5.SiCyclin D1, siCyclin E1 and siCyclin H can effectively reduce expression of Cyclin D1,Cyclin E1 and Cyclin H.After knockdown the expression of Cyclin Dl or Cyclin H,the proliferation of cervical cancer cells was repressed,but Cyclin E1 didn’t. In Hela cell lines,the survival rate of siCyclin D1,siCyclin El and siCyclin H groups were 39.19±7.77%,82.83±5.94% and 40.10±4.02% respectively. In MS751 cell lines,were 51.55±11.70%,88.88± 12.80% and 49.61±7.51% respectively. In SiHa cell lines,were 64.34±4.45%,88.25±8.43% and 80.74±11.57% respectively.6. After knockdown the expression of Rb,the repression of siCyclin Dl antagonize. Compared with siCyclin D1-siRNA NC group,the survival rate of siRb-siCyclin D1 group increased from 34.00±3.43% to 46.54±2.96%(**P<0.01) in Hela cell lines, from 38.09±4.10% to 42.68±1.99%(**P<0.01) in MS751 cell lines, from 66.05±6.56% to 98.33±5.61%(**P<0.01) in SiHa cell lines.7.After knockdown the expression of Rb,the repression of siCyclin H antagonize.Compared with siCyclin H-siRNA NC group,the survival rate of siRb-siCyclin H group increased from 43.68±4.13% to 77.54±5.47%(**P<0.01) in Hela cell lines,from 56.29±5.00% to 86.84±6.95%(**P<0.01) in MS751 cell lines,from 85.78±2.47% to 97.54±3.57% (**P<0.01) in SiHa cell lines.Conclusion 1.The expression of Cyclin D1, Cyclin E1, Cyclin H significantly decrease after knockdown RbAp48 in cervical cancer cell lines.2.After knockdown the expression of Cyclin D1 or Cyclin H which is same with RbAp48,the proliferation of cervical cancer cells are repressed.3.SiRb can antagonize the repression of siRbAp48,siCyclin D1 and siCyclin H.4.The mechanism of siRbAp48 on repression of proliferation in human cervical cancer is knockdown the expression of Cyclin D1 and Cyclin H and cell cycle arrest.5. Reduced expression of Rb can reduce the effect of siRbAp48 on proliferation of cervical cancer The mechanism of siRbAp48 on repression of proliferation in human cervical cancer is achieved by adjusting the Rb-E2F pathway.Part3.Effect of RbAp48 knockdown on the migration and invasion of human cervical cancer cell line MS751Objective Metastasis is an important cause of cervical cancer incurable and migration resistance attacks is an important way to treat cervical cancer. So we explored the effect of RbAp48 knockdown on the migration and invasion in human cervical cancer cells and its mechanism. Methods Cell migration and invasion analysis was performed using the wound healing tests and the transwell tests. The expressions of RbAp48, Vimentin, N-cadherin, E-cadherin,Snail, Twist, MMP-2 and TIMP-2 were determined with Western blotting. Results 1.Scratches and Transwell exprements showed, showed the number of migrated and invaded cells significantly decreased than the siRNA NC after siRNA-mediated RbAp48 knockdown (**P<0.01).2.Knockdown of RbAp48 obviously induced the interstitial cells phenotype proteins Vimentin、N-cadherin、MMP-2 down-regulated and the epithelial cells phenotype proteins E-cadherin、TIMP-2 with up-regulated expressions. It showed that cells of Epithelial-mesenchymal transition was inhibited. Further study has found significantly down-regulated the expression of Snail and Twist which were EMT upstream regulatory protein. Conclusion Knockdown of RbAp48 can significantly inhibited the Epithelial-mesenchymal transition in cervical cancer cell line MS751 and markedly suppressed the cell migration and invasion. Its mechanisms is related to reduce the expression of Snail and Twist.