Study Of Mitotic Protein BubR1Expression In Esophageal Squamous Cancer And Its Influence On Paclitaxel Sensitivity

Mitosis is a very important phase in cell cycle progression. Spindleassembly checkpoint (SAC) is the system which monitors chromosomalproper arrangement and separation in mitosis and further guaranteegenomic integrity in posterity cells. The intact SAC function is prerequisitecondition for diploid cells’ division and growth. Abnormal SAC isfrequently observed in human tumors. Weaken cell cycle checkpoint in theearly stage of tumorigenesis is the crucial step to promote tumor growth.SAC is a signal pathway composed by a set of coordinated proteinsand BubR1is the most important sensor and executor of this system.Disordered BubR1expression has been found in several kinds of clinicaltumor reports, including overexpression of BubR1in breast, bladder,gastric and ovarian cancer, and down-regulation in colorectal cancer.Abnormal BubR1expression represents impaired SAC function andanti-mitosis drugs always depend on normal SAC for their drug-inducedcytotoxic effects. This study is aimed to investigate whether BubR1isup-regulated in esophageal squamous cancer and its’ relationship withanti-microtubule drug paclitaxel effect, and further explore the underlying mechanism of BubR1regulation. Real-time quantitative PCR assay wasemployed to compare BubR1mRNA expression in fifty collected clinicalesophageal squamous cancer tissues and adjacent non-cancerous tissues.Seventy two percentages of those samples (36of50) were found BubR1up-regulation. BubR1expression was high in three esophageal squamouscancer cell lines and relatively high BubR1expression cells showed lowresponse to anti-microtubule drug paclitaxel. To determine the role ofBubR1in esophageal squamous cancer, BubR1expression adenovirus andBubR1interference adenovirus were constructed using adenovirus basedtechnique. After BubR1knockdown in esophageal cancer cells by usingBubR1interference adenovirus, increased cells sensitivity to paclitaxel wasfound, represented by decreased drug IC50and augmentedpaclitaxel-induced cell death. Although down-regulated expression ofBubR1in ECA-109cells did not impact cell growth curve in vitro for shortterm, it largely limited tumor growth of ECA-109cells in nude mice.Conversely, overexpression of BubR1in three esophageal cancer cell linesfurther reduce mitotic index under anti-microtubule drug treatment. HighBubR1and oncogene C-Myc expression were found simultaneously high inesophageal squamous cancer tissues. To investigate whether C-Mycpotentially regulates BubR1, BUB1b gene promoter reporter vector wasconstructed. The result showed that C-Myc expression could activateBUB1b gene promoter and promote BubR1expression. Otherwise, C-Myc inhibition by10058-F4can suppress BubR1expression and recover cells’response to paclitaxel-induced cell growth inhibition, and further boostmitotic arrest. The study of BubR1expression in esophageal squamouscancer, its’ impact on drug effects and related regulation mechanism canhelp us understand why abnormal BubR1exists in tumors, and can providetheoretical foundation for effective esophageal squamous cancer therapy.PART ITHE EXPRESSION OF BUBR1IN ESOPHAGEAL SQUAMOUSCANCER AND CELL LINES AND ANALYSIS OF CLINICALMEANINGSObjective:1. Detect BubR1expression in clinical esophageal squamous cancer tissuesand adjacent no-tumor tissues;2.Analysis the relationship between BubR1expression and patients’clinical characteristics；3. Detect BubR1expression in three esophageal squamous cancer celllines.Methods:1. BubR1relative mRNA expression was detected in fifty pairedesophageal squamous cancer tissues and adjacent no-tumor tissues byreal-time quantitative PCR; 2. Statistically analysis the correlation of BubR1expression and clinicaltraits.3. Real-time PCR and Western Blot assays were used to detect BubR1expression in esophageal squamous cancer cell lines.Results:1. Thirty six of fifty esophageal squamous cancer samples showed BubR1up-regulation, twenty three of which BubR1expression exceed two foldcompared with adjacent no-tumor tissues.2. There is no significantly correlation between BubR1expression andclinical pathological traits.3. High BubR1expression can be observed in esophageal squamous cancercells, in which BubR1expression in ECA-109was lower than that inKYSE150and KYSE180cells.Conclusion:1. Abnormal BubR1expression was confirmed in esophageal squamouscancer tissues but it’s up-regulation was not related with patients’ clinicaltraits.2. BubR1expression level in esophageal squamous cell lines wasconsistent with the result of clinical study. PART ⅡTHE CONSTRUCTION, VERIFICATION AND AMPLIFICATIONOF BUBR1EXPRESSION AND INTERFERENCE ADENOVIRUSObjective:1. Construct BubR1expression adenovirus;2. Construct BubR1interference adenovirus;3. Verify the effect of BubR1expression adenovirus;4. Verify the effect of BubR1interference adenovirus.Methods:1. Total RNA was extracted from293cells and reversed transcript intocDNA and BubR1CDS district fragment was cloned by PCR andinserted into shuttle vector pAdTrack-Tox;2. Four siRNA fragments target to BubR1mRNA were designed andinserted into shuttle vector pSES-HUS.3. DNA sequencing technique was used to verify the accuracy of BubR1expression and interference fragments.4. Homologous recombination happened in BJ5183bacteria betweenadenoviral shuttle vector and backbone vector. Correct recombinedadenoviral vector was transfected into293cells for adenovirus packingand amplification;5. PCR and Western Blot assays were employed to verify the effect ofBubR1expression and interference adenovirus. Results:1. BubR1CDS district fragment was inserted into pAdTrack-Tox from293cDNA and the correct vector was named pAdTrack-Tox-BubR1;2. After homogeneous recombination between pAdTrack-Tox-BubR1andbackbone vector pAdEasy-1, Pac I enzyme cut recombined vector toconfirm right recombination.30kb large fragment and4.5kb smallfragment can be seen from enzyme cut result;3. BubR1expression adenovirus packing and amplification wereprogressed in293cells. GFP expression can be observed. Thisadenovirus was named Ad-BubR1;4. PCR and Western Blot detected elevated BubR1expression afterAd-BubR1infection;5. Four siRNA fragments were inserted into pSES-HUS vector. Aftercorrect sequencing, four BubR1interference vectors were transfectedinto ECA-109cells and BubR1expression was detected by PCR andWestern Blot. Result showed that pSES-HUS-siBubR1-4had significantffect. pSES-HUS-siBubR1-4vector and scramble vector were used forfurther experiment;6. After homogeneous recombination between pSES-HUS-siBubR1-4andbackbone vector pAdEasy-1, Pac I enzyme cut recombined vector toconfirm right recombination.30kb large fragment and3kb smallfragment can be seen from enzyme cut result; 7. BubR1interference adenovirus packing and amplification wereprogressed in293cells. RFP expression can be observed. Thisadenovirus was named Ad-siBubR1-4.Conclusion: BubR1expression and interference adenovirus weresuccessfully constructed and their effects were verified in293cells andECA-109cells, respectively, by PCR and Western Blot assays. PART IIITHE EFFECTS OF BUBR1EXPRESSION IN ESOPHAGEALSQUAMOUS CANCER ON PACLITAXEL SENSITIVITYObjective:1. Three esophageal cancers’ response to paclitaxel before and after BubR1knockdown;2. The difference of paclitaxel-induced cell death was compared before andafter BubR1knockdown;3. Mitotic index under paclitaxel treatment change was observed afterBubR1knockdown;4. Short-term growth of ECA-109cells with BubR1suppression in vitroand long-term tumorigenesis in nude mice was observed. Methods:1. Three cell lines with or without BubR1suppression were treated bygradient concentration of paclitaxel. MTT assay was employed to detectcell viability and drug IC50was calculated.2. FACS assay was carried out to detect cell cycle distribution of ECA-109cells with BubR1suppression under paclitaxel treatment;3. Trypan blue staining was used to count the number of dead cellpercentage of ECA-109cells with BubR1expression under paclitaxeltreatment;4. Mitotic cell percentages of three cell lines with BubR1overexrepssionby Ad-BubR1infection of different time points were observed afterNocodazole treatment;5. MTT assay was used to detect cell growth curve after BubR1knockdown in ECA-109;6. ECA-109cells with BubR1suppression were injected into nude mice fortumorigenesis observation. Tumor’s weight and volume was detected.Results:1. Esophageal squamous cancer cell lines KYSE150and KYSE180withrelatively high BubR1expression showed lower sensitivity towardpaclitaxel treatment. After BubR1knockdown, paclitaxel drug IC50isdecreased in all three cell lines;2. After BubR1was down-regulated in ECA-109, sub-G1phase of cell cycle increased and cell death correspondingly increased underpaclitaxel treatment;3. Overexpression of BubR1in three cell lines can further suppress mitoticcell percentage induced by Nocodazole, which represented as decreasedmitotic index;4. ECA-109cell growth in vitro was not affected by BubR1knockdown,but tumorigenesis in vivo was largely inhibited. Tumor weight andvolume was significantly smaller than control group.Conclusion:1. Relatively high BubR1expression cells showed low response topaclitaxel and mitotic index was also low in those cells;2. BubR1interference can promote esophageal squamous cancer cellsresponse to paclitaxel, represented as cell death induced by paclitaxelelevated;3. BubR1interference can inhibit tumor formation of esophageal squamouscancer cells in vivo. Part ⅣTHE REGULATION OF C-MYC ON BUBR1IN ESOPHAGEALSQUAMOUS CANCER INDIRECTLY AFFECT PACLITAXELSENSITIVITYObjective:1. Confirm the correlation of BubR1and C-Myc expression in esophagealcancer tissues；2. Verify C-Myc expression in three esophageal squamous cancer cell linesand its influence on BubR1expression;3. Determine the effect of C-Myc on paclitaxel sensitivity.Methods:1. Immunohistochemistry was employed to detected BubR1and C-Mycprotein expression in esophageal squamous cancer tissues;2. PCR and Western Blot assays were carried out to detect C-Mycexpression in three esophageal squamous cancer cell lines;3. The activity of alkaline phosphatase was detected in three esophagealsquamous cancer cell lines after pSEAP2BubR1-P2000transfection.pSEAP2-BubR1-P2000activation was confirmed in293cells infectedby Ad-C-Myc; 4. PCR and Western Blot assays were used to detect BubR1mRNA androtein expression under the condition of C-Myc overexpression andC-Myc suppression by10058-F4, respectively;5. MTT assay was used to detect cell viability under paclitaxel treatmentafter C-Myc suppression;6. DAPI staining was carried out to demonstrate cell mitotic index changesunder combination of C-Myc inhibitor and low concentration ofpaclitaxel.Results:1. BubR1and C-Myc expression showed close correlation in esophagealsquamous cancer tissues.2. C-Myc expression in three cell lines showed similar results with BubR1expression;3. C-Myc can activate BubR1promoter vector and up-regulate BubR1expression;4. C-Myc suppression can promote paclitaxel-induced cell growthinhibition and help increase mitotic index.Conclusion:1. C-Myc could be upstream regulator of BubR1in esophageal squamouscancer;2. C-Myc expression in esophageal squamous cancer indirectly affects cellsresponse to paclitaxel.