Mechanisms Of Down-regulation The Expression Of RbAp48 In Inhibiting Proliferation And Increasing Cisplatin Sensitivity Of Tumor Cells

Part1 Mechanisms of down-regulation the expression of RbAp48 in inhibiting of tumor cell proliferationObjective RbAp48 belongs to the WD family and is a highly conserved protein found in almost eukaryotes.Our study found that down-regulation the expression of RbAp48 could induce cycle arrest and inhibit cell proliferation in lung,cervical,gastric and colorectal cancer cells.However,the mechanism remains unknown.Therefore,we mainly studied the mechanism of down-regulation the expression of RbAp48 in inhibiting tumor cell proliferation.Methods The expression of protein was down-regulated by RNA interference.Next Generation Sequencing was used to detect the expression of genes after down-regulating the expression of RbAp48 and differential expressed genes were analyzed by GO and KEGG analyse.Western blot assay was used to detect the expression level of protein after down-regulating the expression of RbAp48.MTT assay was used to detect the effect of siRbAp48 in cell proliferation after down-regulation or over-expression(lentiviral transfection technique)of protein.Result 1.Next Generation Sequencing was used to detect the expression of genes in A549 cell lines after down-regulating the expression of RbAp48.A total of 468 gene expressions were altered,of which 164 genes were down-regulated and 304 genes were up-regulated.GO analysis showed that differential genes mainly regulated cell proliferation(P=4.07E-05,corrected P=0.035);KEGG analysis showed that there were 56 genes associated with non-small cell lung cancer pathway,only CCND1 was closely associated with down-regulation the expression of RbAp48.2.Western blot confirmed that the expression of CyclinD1 in lung cancer A549 and cervical cancer Hela cells was significantly decreased after down-regulating RbAp48 expression.The MTT results showed that over-expression of CCND1 with lentiviral vectors could significantly antagonize the inhibitory effect of siRbAp48 on the proliferation of A549.3.After down-regulation of RbAp48 in gastric cancer HGC-27 and colon cancer HT-29 cells by siRbAP48,the expression of CyclinDl was not down-regulated,indicating that its inhibition of cell proliferation was not achieved by inhibiting CyclinD1.However,down-regulation of RbAp48 inhibited the expression of Cdc25C in HGC-27 and HT-29 cells.Over-expression of Cdc25C with a lentivirus vector significantly antagonized the inhibitory effect of siRbAp48 on the proliferation of HGC-27 and HT-29 cells.Conclusion:1.Down-regulation of CyclinD1 expression is the main mechanism of siRbAp48 inhibiting the proliferation of lung cancer and cervical cancer cells.2.Down-regulation of Cdc25C expression is the main mechanism of siRbAp48 inhibiting the proliferation of gastric cancer and colorectal cancer cells.Part2 Mechanisms of down-regulation the expression of RbAp48 in increasing cisplatin sensitivity of tumor cellsObjective Our previous study found that down-regulation of RbAp48 expression could significantly increase the sensitivity of A549 cisplatin resistant cell lines to cisplatin.Based on the results of Next Generation Sequencing,we investigated the mechanism of down-regulation the expression of RbAp48 in sensitizing tumor cells to cisplatin..Methods The sensitivity of cells to cisplatin was determined by MTT assay.Atomic absorption spectrophotometry was used to determine the platinum content in cells.MTT analyse was used to detect the effect of down-regulation the expression of protein on cisplatin sensitivity,the lentiviral vector was constructed to over-express the target gene.Results 1.MTT results showed that down-regulating the expression of RbAp48 increased the sensitivity of lung cancer,cervical cancer and its drug-resistant cells to cisplatin,but over-expression of RbAp48 decreased the sensitivity.2.After down-regulating the expression of RbAp48,the platinum content and most of the genes that associated with cisplatin resistance did not change significantly,but the expression of CCND1 was down-regulated.3.Down-regulation of CyclinD1 expression increased the sensitivity of cells to cisplatin.Over-expression of CyclinDl reversed the increase in cisplatin sensitivity of cells induced by siRbAp48.Conclusion Down-regulation of RbAp48 expression significantly increased the sensitivity of tumor cells to cisplatin by inhibiting the expression of CyclinD1.