| Part 1 Expression of CARLo-5 in esophageal carcinoma tissues and cells and its effect on its biological characteristicsObjective:To investigate the expression of cancer-associated region long non-coding RNA(CARLo-5)in esophageal carcinoma and cells,and the effection on the proliferation,migration,invasion,apoptosis and cells cycle of esophageal cancer cells.Methods:1 Expression levels of Lnc RNA CARLo-5 in esophageal carcinoma tissues,adjacent tissue and cells were detected by Realtime-PCR.2 Proliferation,migration and invasion abilities of esophageal carcinoma cells with CARLo-5 over-expression or low-expression were detected by MTS,transwell,matrigel chamber and wound scrape assays,respectively.3 Flow cytometry assay detected the effect of low-expression of CARLo-5 on cell cycle and apoptosis of KYSE30 and KYSE180 cells.4 MTS and flow cytometry assays detected the effect of CARLo-5 gene on the drug sensitivity of 5 fluorouracil in KYSE30 and KYSE180 cells.Results:1 Realtime-PCR demonstrated that expression levels of CARLo-5 in esophageal carcinoma tissues were higher than those in the adjacent normal esophageal tissues(P<0.05).The expression levels of CARLo-5 in esophageal cancer cells were highter than those in normal esophageal epithelial cells(P <0.05).2 MTS assay demonstrated that low-expression of CARLo-5 inhibited proliferation abilities of KYSE30 and KYSE180(P<0.05),while over-expression of CARLo-5 promoted their proliferation abilities(P<0.05).Transwell migration assays demonstrated that low-expression of CARLo-5 inhibited migration and invasion abilities of KYSE30 and KYSE180(P<0.05),while over-expression of CARLo-5 promoted their migration and invasion abilities(P <0.05).3 Flow cytometry analysis revealed that compared with the control group,the apoptosis of low-expression CARLo-5 were significantly increased in KYSE30 and KYSE180 cells(P<0.05).In addition,compared with the control group,there was an increased relative proportion of low-expression CARLo-5 KYSE30 and KYSE180 cells in G0 phase(P<0.05),and a reduced relative proportion in S phase(P<0.05).4 MTS assay demonstrated that low-expression of CARLo-5 could decrease the sensitivity of 5 fluorouracil in KYSE30 and KYSE180 cells(P<0.05).Flow cytometry analysis revealed that over-expression of CARLo-5 could reduce the rate of apoptosis after 5 fluorouracil treatment(P<0.05).Part 2 Study on the mechanism of CARLo-5 in esophageal cancer cellsObjective: To study the mechanism of interaction between CARLo-5 and proteins or micro RNAs in order to investigate the possible role of CARLo-5 in promoting cancer.Methods:1 Expression levels of cell cycle-related proteins,invasion-related proteins,apoptosis-related proteins,were detected by Western blot assay in KYSE30 and KYSE180 cells after knockdown of CARLo-5 gene.2 RNA-pulldown combining with RIP and westem blot assay were performed to detect CARLo-5 binding PTBP1 protein.3 Proliferation and migration abilities of KYSE30 and KYSE180 cells were detected by MTS and transwell migration chamber assays,with PTBP1 low-expression or over-expression CARLo-5 or combination with low-expression PTBP1,respectively.4 Effects of CARLo-5 over-expression on expression of PTBP1 in KYSE30 and KYSE180 cells after treated with cycloheximide or with MG132 and cycloheximide were detected by Western blot.5 Expression levels of apoptosis-related proteins and metabolic-related protein expression were detected by Western blot in KYSE30 and KYSE180 cells.6 Expression levels of different micro RNA in esophageal carcinoma cells with CARLo-5 over-expression or low-expression were detected by Realtime-PCR,and screened out the significant difference of mi R-218-5p for future research.7 Proliferation,migration and invasion abilities of esophageal cancer cells with mi R-218-5p over-expression or low-expression were detected by MTS,transwell migration,matrigel invasion chamber and wound scrape assays,respectively.8 Proliferation and migration abilities of KYSE30 and KYSE180 cells with over-expression CARLo-5 and combination with low-expression mi R-218-5p were detected by MTS and transwell migration chamber assays,respectively.9 Expression levels of different target gene in esophageal carcinoma cells with mi R-218-5p over-expression or low-expression were detected by Realtime-PCR,and screened out the significant difference of target gene for future research.10 Expression levels of ROBO1,BMI1,RELN proteins with mi R-218-5p over-expression or low-expression were detected by Western blot in KYSE30 and KYSE180 cells.Results:1 Western blot assay demonstrated that low-expression of CARLo-5 could decrease cell cycle related proteins,such as cyclin B1,cyclin D1,CDK2,CDK4 and increase the protein of P21(P<0.05);also could decrease invasion-related proteins,such as MMP9 and MMP2(P<0.05);and could increase apoptosis-related proteins,such as Caspase-3 and decrease the protein of MCL-1(P<0.05).2 RNA—pulldown combining with RIP and Western blot assay demonstrated that CARLo-5 co?Ld binding PTBP1 proteins.3 MTS assay demonstrated that low-expression of PTBP1 inhibited proliferationabilities of KYSE30 and KYSE180 cells,compare with control group(P<0.05).Transwell migration assays demonstrated that low-expression of PTBP1 inhibited migration abilities of KYSE30 and KYSE180 cells,compare with control group(P<0.05).4 MTS assay demonstrated that low-expression of PTBP1 can reduce the enhancement of CARLo-5 over-expression on the proliferation of KYSE30 and KYSE180 cells(P<0.05).Transwell migration assays demonstrated that low-expression of PTBP1 can reduce the enhancement of CARLo-5 over-expression on the migration of KYSE30 and KYSE180 cells(P<0.05).5 Western blot demonstrated that over-expression of CARLo-5 in KYSE30 and KYSE180 cells treated with cycloheximide reduced the d egradation of PTBP1 compared with the control group(P<0.05).6 Western blot demonstrated that over-expression of CARLo-5 in KYSE30 cells treated with MG132 and cycloheximide have no difference in expression.7 Western blot assay demonstrated that low-expression of PTBP1 co?Ld decrease Mcl-1 and PKM2 proteins,increase the protein of PKM1(P<0.05).8 The results of Realtime-PCR showed that mi R-218-5p expression was consistent and most significant in knockdown of CARLo-5 and overexpression of CARLo-5(P<0.05).9 MTS assay demonstrated that low-expression of mi R-218-5p promoted proliferation abilities of KYSE30 and KYSE180(P<0.05),while over-expression of mi R-218-5p inhibited their proliferation abilities(P<0.05).Transwell migration assays demonstrated that low-expression of mi R-218-5p promoted migration and invasion abilities of KYSE30 and KYSE180(P<0.05),while over-expression of mi R-218-5p inhibited their migration and invasion abilities(P<0.05).10 MTS assay demonstrated that low-expression of mi R-218-5p can reduce the enhancement of CARLo-5 over-expression on the proliferation of KYSE30 and KYSE180 cells(P<0.05).Transwell migration assays demonstrated that low-expression of mi R-218-5p can reduce the enhancement of CARLo-5 over-expression on the migration of KYSE30 and KYSE180 cells(P<0.05).11 The res?Lts of Realtime-PCR showed that ROBO1,BMI1 and RELN expression was consistent in knockdown of mi R-218-5p and overexpression of mi R-218-5p(P<0.05).12 Western blot assay demonstrated that mi R-218-5p over-expression or low-expression co?Ld increase and decrease the level of ROBO1 respectively(P<0.05),the level of BMI1 and RELN have no difference in expression.Part 3 Study on the effect of knockdown of CARLo-5 on the growth of esophageal cancer cells in nude miceObjective: To investigate the effect of CARLo-5 on the proliferation of esophageal cancer in vivo.Methods:1 Stably transfected KYSE30 cells with a lentivirus construct containing CARLo-5 low-expression,The cells were then injected subcutaneously into nude mice and observe tumors proliferation rate.2 The expression levels of PTBP1 in nude mice transplanted tumors were detected by immunohistochemical.Results:1 The growth rate of nude mice transplanted tumors in sh-CARLo-5 group was significantly lower than that in control group(P<0.05).2 The immunohistochemical assay showed that the expression levels of PTBP1 in nude mice transplanted tumors with CARLo-5 low-expression were lower than that in control group.Conclusion:The level of CARLo-5 in esophageal cancer tissue and cell were up-regulation.And in vitro and in vivo can promote the proliferation of esophageal cancer cells.In vivo can also promote the migration and invasion of esophageal cancer cells.In addition,CARLo-5 can bind to PTBP1 and mi R-218-5p to induce the development of esophageal cancer. |
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