Knockdown Of RbAp48 Expression Leads To The Inhibition Of Human Digestive Tract Tumor Cells Proliferation

Background:Digestive tract tumor is one of the most common malignant tumors.Esophageal cancer, colorectal cancer, gastric cancer and other digestive tract cancer’s morbidity and mortality has been in the forefront of the various forms of cancer. The current treatment of digestive tract tumors mainly were surgery, radiotherapy and chemotherapy. But the effects were not satisfactory, what’s worse the side effects of radiotherapy and chemotherapy were serious. Therefore, it is particularly important to find new targets for drug treatment.RbAp48 (Retinoblastoma-associated protein 48), a member of WD-40 protein family, was originally identified as the Rb protein binding protein, whose molecular weight is 48kDa. RbAp48 is involved in a variety of compounds function, such as ATP-dependent recombinant complexes, histone deacetylase complex Sin3, Chromosome assembly factor and DNA sequence pairs complexes CAF-1, nuclear chromatin remodeling and deacetylase NuRD. Recent study found that RbAp48 is the core subunit of PcG (Polycomb group protein, PcG). PcG can specifically methylate nucleosomal histone H3 at lysine 27, thus silences target genes and affects the development of tumors. Study found that RbAp48 is abnormal high expression in many malignancies including lung cancer, cervical cancer, thyroid carcinoma, acute myelogenous leukemia, primary liver cancer, and so on, which suggests that RbAp48 has close relationship with the occurrence and development of tumor.Our previous study found that down-regulating the expression of RbAp48 cound inhibit the growth of lung cancer cells and reverse the cisplatin resistance. We envisaged that down-regulating the expression of RbAp48 may inhibit the growth of digestive tract tumors, and it may be a potential target for the treatment of digestive tract tumors, we has carried on the related research.Part 1 To explore the effects of down-regulation of RbAp48 expression on human esophageal cancer cells proliferationObjective:Study on the effects of down-regulation of RbAp48 expression on human esophageal cancer cells proliferation, explore the possibilities of RbAp48 to be an esophageal cancer treatment target. Method:1. Immunohistochemical detected RbAp48 expression of esophageal cancer tissues and adjacent esophageal mucosa tissues.2. Western blot detected the RbAp48 expression inhibitory effects of siRNA interfered cells.3. After RbAp48-siRNA transfected cells:①MTT assay detected the growth effect on human esophageal cancer cells EC 109 and human normal epithelial cells T132. ②Tablet clone forming experiment detected clone forming ability of EC109.(3)MTT assay detected the change of sensitivity to cisplatin in EC109, EC109 /DDP cells.4. In vivo experiment:EC 109 cells were implanted subcutaneously in BABL/C nude mice to build mouse tumor models, then RbAp48-siRNA were injected into the tumors. Compare the difference of tumors.5. ① β-galactose anhydride enzyme staining method to detect cell senesecence. ②Flow cytometry detected cell apoptosis and cell cycle. Results:1. The expression of RbAp48 in esophageal tissue is significantly higher than the adjacent esophageal mucosa tissue.2. RbAp48-siRNA can effectively inhibit the RbAp48 expression in EC 109.3. After RbAp48-siRNA transfected cells: ① The negative control group and the RbAp48-siRNA group inhibition rates were:(18.54 ± 2.80)%, (45.24 ± 2.74)%(p<0.01), RbAp48-siRNA group inhibition rate was significantly increased, while the L132 cell growth inhibition rate was no significant difference (p> 0.05). ② Clone number of the blank group, the negative control group, RbAp48-siRNA group were 165.00 ± 13.00,161.00 ± 11.53 and 101.00 ± 3.61, clone number of RbAp48-siRNA group was obviously reduced(p<0.01). ③In EC109 cells,the IC50 of cisplatin on the blank group, the negative control group and RbAp48-siRNA group were 4.76 ± 0.09μmol/L,2.87 ± 0.14μmol/L,0.89 ± 0.02μmol/L (p< 0.05). In EC 109/DDP cells, the IC50 of cisplatin on the blank group, negative control group and RbAp48-siRNA group were 25.69 ± 0.69μmol/L,15.58 ± 0.36μmol/L,8.26 ± 0.96μmol/L(p<0.01). It showed that the susceptibility to cisplatin were significantly increased on these two cells after down-regulation of RbAp48 expression.4. In vivo experiment:compared with the blank group, the weight of tumor on experiment group fell from 997.10 ± 141.50 mg to 202.50 ± 81.34 mg (p<0.01), and the volume fell from 1627.00 ± 261.90 mm3 to 371.40 ± 155.90 mm3, (p<0.01).5.± Compared with blank group, negative control group, RbAp48-siRNA group appear obvious aging phenomenon. ② It showed that RbAp48-siRNA transfection could induce G2/M phase arrest:the ratio of G2/M phase cells from (13.35 ± 2.84)%, (16.97 ± 2.64)% rise to (31.48 ± 11.39)%(p<0.01) on the third day after down-regulation of RbAp48 expression.③Compared with blank group, negative control group, RbAp48-siRNA group cells apoptosis rate increased: apoptotic rate from (5.44±1.36)%、(7.25 ±0.44)% rise to (12.18 ±0.50)%(p<0.01) on the fourth day after transfection. Conclusion:1. The expression of RbAp48 in esophageal cancer tissue was significantly higher than which in adjacent tissue.2. Down-regulation of RbAp48 expression in esophageal cancer could block the cell cycle, induce cell senesecence and apoptosis so that inhibi of cell proliferation. But there was no obvious effect on human normal cells.3. Down-regulation of RbAp48 expression can significantly improve the sensitivity to cisplatin of esophageal cancer cells and its cisplatin resistance cells.4. RbAp48 is expected to be a new target for the treatment of esophageal cancer.Part 2 To explore the effects of down-regulation of RbAp48 expression on human colorectal cancer cells proliferationObjective:Study on the effects of down-regulation of RbAp48 expression on human colorectal cancer cells proliferation, explore the possibilities of RbAp48 to be a colorectal cancer treatment target. Method:1.Immunohistochemical detected RbAp48 expression of colorectal cancer tissues and adjacent colorectal mucosa tissues.2. Western blot detected the RbAp48 expression inhibitory effects of siRNA interfered cells.3. After RbAp48-siRNA transfected cells: ①MTT assay detected the growth effect on human colorectal cancer cells HT-29 and human normal epithelial cells L132. ②Tablet clone forming experiment detected clone forming ability of HT-29.③MTT assay detected the change of sensitivity to 5-FU in HT-29 cells.4. In vivo experiment:HT-29 cells were implanted subcutaneously in BABL/C nude mice to build mouse tumor models, then RbAp48-siRNA were injected into the tumors. Compare the difference of tumors.5.① β-galactose anhydride enzyme staining method detected cell senesecence.②Flow cytometry detected cell apoptosis and cell cycle. Results:1. The expression of RbAp48 in colorectal tissue is significantly higher than the adjacent colorectal mucosa tissue.2. RbAp48-siRNA can effectively inhibit RbAp48 expression in HT-29.3.After RbAp48-siRNA transfected cells:①The negative control group and the RbAp48-siRNA group inhibition rates were:(5.08 ± 1.24)%, (33.69 ± 6.30)%,p<0.01, RbApP48-siRNA group inhibition rate was significantly increased, while the L132 cells growth inhibition rate was no significant change (p> 0.05). ② Clone number of the blank group, the negative control group, RbApP48-siRNA group were 108.70±4.67,103.00±2.08and68.00 ± 4.62, clone number of RbApP48-siRNA group was obviously reduced(p<0.01). ■ In HT-29 cells, the IC50 of 5-FU on the blank group,the blank group, the negative control group and RbApP48-siRNA group were 94.89 ±6.97μmol/L,57.42±2.74μmol/L and 17.80±0.42μmol/L (p<0.01). It showed that the susceptibility to 5-FU was significantly increased on HT-29 cells after down-regulation of RbAp48 expression.4. In vivo experiment:compared with the blank group, the weight of tumor on experiment group fell from 210.2±93.04 mg mg to 90.44±59.87 mg (p<0.05), and the volume fell from 377.20±193.40mm3 to 141.80±79.35 mm3, (p<0.05).5. ① Compared with blank group, negative control group, RbApP48-siRNA group appear obvious aging phenomenon. ② RbApP48-siRNA transfected HT-29 cells did not occur significantly apoptotic and cell cycle arrest during four days. Conclusion:1, The expression of RbAp48 in colorectal cancer tissue was significantly higher than which in adjacent tissue.2. Down-regulation of RbAp48 expression in colorectal cancer could induce cell senesecence and inhibition of cell proliferation. But there was no obvious effect on human normal cells.3. Down-regulation of RbAp48 expression can significantly improve the sensitivity to 5-FU of colorectal cancer cells.4. RbAp48 is expected to be a new target for the treatment of colorectal cancer.Part 3 To explore the effects of down-regulation of RbAp48 expression on human gastric cancer cells proliferationObjective:Study on the effects of down-regulation of RbAp48 expression on human gastric cancer cells proliferation, explore the possibilities of RbAp48 to be a gastric cancer treatment target. Method:1. Immunohistochemical detected RbAp48 expression of gastric cancer tissues and adjacent gastric mucosa tissues.2. Western blot detected the RbAp48 expression inhibitory effects of siRNA interfered cells.3. After RbAp48-siRNA transfected cells:①MTT assay detected the growth effect on gastric cancer cells HGC-27 and human normal epithelial cells L132. ②Tablet clone forming experiment detected clone forming ability of HGC-27. ③ MTT assay detected the change of sensitivity to 5-FU in HGC-27 cells.4. ①β-galactose anhydride enzyme staining method to detect cell senesecence. ② Flow cytometry detected cell apoptosis and cell cycle. Results:1. The expression of RbAp48 in gastric tissue is significantly higher than the adjacent gastric mucosa tissue.2. RbAp48-siRNA can effectively inhibit RbAp48 expression in HGC-27.3.After RbAp48-siRNA transfected cells: ① The negative control group and the RbAp48-siRNA group inhibition rates were:(35.00±2.04)%, (70.39±1.21)%,p< 0.01, RbAp48-siRNA group inhibition rate was significantly increased, while the L132 cell growth inhibition rate was no significant difference (p> 0.05). ②Clone number of the blank group, the negative control group, RbAp48-siRNA group on the number of clones were 111.30 ±8.62、31.33±2.19 和 4.00±1.73, clone number of RbAp48-siRNA group was obviously reduced(p<0.01). ③ In HGC-27 cells, the IC50 of 5-FU on the blank group,the blank group, the negative control group and RbApP48-siRNA group were 3.42±0.39μmol/L、1.84±0.04μmol/L and 0.07± 0.02μmol/L (p<0.01). It showed that the susceptibility to 5-FU was significantly increased on HGC-27 cells after down-regulation of RbAp48 expression..4.① Compared with blank group, negative control group, RbAp48-siRNA group appear obvious aging phenomenon. ② It showed that RbAp48-siRNA transfection could induce G2/M phase arrest:the ratio of G2/M phase cells from (8.42±0.28) %、 (13.33± 2.94)% rise to (30.75 ±1.65)%, (p<0.01) on the third day after down-regulation of RbAp48 expression. ③ Compared with blank group, negative control group, RbAp48-siRNA group cells apoptosis rate increased:apoptotic rate from (14.94+ 5.90)%、(16.02±2.12)% rise to (61.30± 8.07)%(p<0.01) on the fifth day after transfection. Conclusion:1. The expression of RbAp48 in gastric cancer tissue was significantly higher than which in adjacent tissue.2. Down-regulation of RbAp48 expression in gastric cancer could block the cell cycle, induce cell senesecenceand apoptosis so that inhibit of cell proliferation. But there was no obvious effect on human normal cells.3. Down-regulation of RbAp48 expression can significantly improve the sensitivity to 5-FU of gastric cancer cells.4. RbAp48 is expected to be a new target for the treatment of gastric cancer.