【作者】 王立平；

【导师】 全成实；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 目的：探讨SB431542(SB)通过DNMT1调控CLDN6基因DNA甲基化及其表达的作用机制，揭示CLDN6通过EMT对乳腺癌细胞转移的调控作用。方法：采用RT-PCR和Western blot技术检测乳腺上皮细胞HBL-100、乳腺癌细胞MCF-7和SKBR-3中SMAD2和CLDN6的表达；采用Western blot法筛选SMADs抑制剂SB调控p-SMAD2和CLDN6表达的最适作用浓度与时间；用SB处理乳腺癌细胞，采用Western blot技术检测SMAD3和p-SMAD3的表达；采用甲基化特异性PCR(Methylation-Specific PCR，MSP)技术检测CLDN6基因DNA甲基化PCR产物的表达；采用亚硫酸氢盐测序（Bisulfite Sequencing PCR，BSP）法检测SKBR-3细胞中CLDN6启动子区甲基化位点；采用RT-PCR和Western blot技术检测DNA甲基转移酶1（DNA methyltransferase1，DNMT1）的表达，用DNMT1活性检测试剂盒检测DNMT1的活性；采用染色质免疫沉淀（ChromatinImmunoprecipitation assay，ChIP）技术检测DNMT1与CLDN6启动子区的结合；检测单层细胞跨上皮电阻；采用划痕实验和Transwell法检测细胞迁移和侵袭能力；采用RT-PCR和Western blot技术检测上皮标志物E-cadherin和间质标志物N-cadherin、Vimentin和SNAIL的表达；采用细胞免疫荧光法检测E-cadherin、N-cadherin及CLDN6的表达。利用RNAi技术沉默SB处理组的CLDN6基因后，检测单层细胞跨上皮电阻；采用划痕实验和Transwell法检测细胞迁移和侵袭能力；采用RT-PCR和Western blot检测E-cadherin、N-cadherin、Vimentin及SNAIL的表达；采用细胞免疫荧光法检测E-cadherin、N-cadherin及CLDN6的表达。结果：乳腺癌细胞MCF-7和SKBR-3细胞中CLDN6表达明显低于正常乳腺上皮细胞HBL-100，SMAD2表达明显高于HBL-100细胞；SB处理乳腺癌细胞对p-SMAD2和CLDN6表达影响的最适作用浓度与时间为10μM和24h，且具有时间和浓度依赖性；用10μM SB处理乳腺癌细胞24h，SMAD3总蛋白的表达不变，p-SMAD3的表达明显降低；CLDN6启动子区甲基化PCR产物减少，CpG位点甲基化概率减少；DNMT1表达下调，DNMT1活性降低，DNMT1与CLDN6启动子区的结合减少；细胞跨上皮电阻增加，迁移和侵袭能力降低，E-cadherin表达增高，N-cadherin、Vimentin及SNAIL的表达降低；沉默SB处理组CLDN6，细胞跨上皮电阻减小，迁移和侵袭能力增强，E-cadherin表达降低，N-cadherin、Vimentin及SNAIL的表达增高。结论：1．乳腺癌细胞MCF-7和SKBR-3中，SB下调DNMT1的表达，抑制DNMT1性并抑制DNMT1与CLDN6启动子的结合，降低CLDN6的甲基化水平，上CLDN6的表达。2．CLDN6通过逆转EMT抑制乳腺癌细胞的转移。更多还原

【Abstract】 Backgrounds:Breast cancer is a kind of malignant tumor derived from mammary glandepithelial. Recurrence and metastasis are the main causes of death of patients withbreast cancer and epithelial mesenchymal transition occur in metastasis of mostmalignant tumor derived from epithelial tissues. Currently considering that tightjunctions (TJs) proteins may play an important role in the epithelial-mesenchymaltransformation.TJs is composed of structural membrane proteins including atresia protein(Occludin), Claudins (CLDNs), tight junction adhesion molecules (JAMs) etc andperipheral cytoplasmic proteins such as ZO-l, ZO-2and ZO-3etc. TJs locate in thetop of junctional complexes and it is an important component of the formation andmaintenance of epithelial permeability barrier and cell polarity. As the cytoskeletalproteins, CLDNs include27family members and maintain the structure and functionof TJs, abnormal expression of CLDNs may affect tumor cells metastasis throughregulating EMT. In the previous studies, we confirmed that DNA methylation isassociated with low-expression of CLDN6in breast cancer cells and the bingding ofDNMT1with the DNA in the promoter of CLDN6is one of the key mechanisms ofthe down-regulation of CLDN6. Some signaling pathways and factors regulateDNMT1have been reported, such as TGFβ/SMAD, APC pathway, signal transducerand activator of transcription3(STAT3), IL-6, MicroRNA-342and MicroRNA-152etc. The expression level, activity and the binding of DNMT1with the promoter ofCLDN6would affect the regulation of its target genes. Recently it is reported thatSMADs regulated the expression of CLDN7through regulating DNMT1. We inferredthat SMADs might regulate the expression of CLDN6via regulating DNMT1.Our experiment aims to explore the mechanism of inhibitor of SMADs regulatingDNA methylation and the expression of CLDN6through affecting the expression andactivity of DNMT1and the binding of DNMT1with the promoter of CLDN6, SBregulating DNMT1and to reveal the CLDN6regulating metastasis of breast cancercells via EMT.Methods:1. The expression of CLDN6and SMAD2in breast cancer cells: the expression of CLDN6and SMAD2were detected by RT-PCR and Western blot in breast cancercells MCF-7, SKBR-3and mammary epithelial cells HBL-100.2. The effects of SB on the expression of SMADs and CLDN6: dose-andtime-dependent experiments were applied to examine the effects of SB on p-SMAD2and CLDN6in breast cancer cells and Western blot was conducted to determine theoptimum concentration and time of SB. The effects of SB on p-SMAD3weredetected by Western blot to confirm the inhibition of SB in SMAD signaling pathway.3. The mechanism of SB regulating CLDN6expression: Methylation-SpecificPCR (MSP) was applied to detect CLDN6methylation status in breast cancer cellstreated with SB. Bisulfite Sequencing PCR (BSP) was used to test the DNAmethylation sites of CLDN6promoter and DNMT1expression was detected byRT-PCR and Western blot. DNMT1activity was detected by DNMT1activity kit andthe binding of DNMT1and the promoter of CLDN6were detected by ChIP assay.4. The effect of CLDN6on the cells transepithelial electrical resistance andmetastasis: cells were treated with SB, the effects of SB on TEER was detected byTEER assay; the effects of SB on migration and invasion of breast cancer cells weredetected by Wound healing assay and transwell assay; CLDN6in breast cancer cellstreated with SB was silenced by RNAi and the silence efficiency was verified byRT-PCR and Western blot and then TEER assay was applied to detect the TEER,Wound healing assay and transwell assay were conducted to test migration andinvasion in breast cancer cells with CLDN6silenced.5． Mechanism of CLDN6regulating breast cancer metastasis via EMT: theeffects of SB on the cell morphology was observed under an inverted microscope;RT-PCR, Western blot and immunofluorescence assay were used to detect theexpression of epithelial and mesenchymal markers; and the markers were detected bythe same methods in breast cancer cells with CLDN6silenced after the treatment ofSB.Result:1. Expression of CLDN6and SMAD2in breast cancer cells: SMAD2isexpressed significantly higher and CLDN6expressed significantly lower in breastcancer cells than normal breast epithelial cells HBL-100.2. The effects of SB on the expression of SMADs and CLDN6: Western blotresults revealed that different concentrations from0.1μM of SB treated breast cancer cells, p-SMAD2was significantly down-regulated and CLDN6was significantlyup-regulated while10μM was the optimum concentration. Whereas total protein ofSMAD2remain unchanged with the treatment of SB. Different time points waschosen to treat cells with10μM SB, expression of p-SMAD2down-regulated and theexpression of CLDN6up-regulated significantly in8hours, the change was the mostsignificant when treated for24h, and with concentration and time dependence, whilethe total protein of SMAD2kept the same level. When10μM SB was used to treatcells for24h, p-SMAD3expression decreased while the total protein of SMAD3remained unchanged.3. Mechanism of SB regulating CLDN6expression:10μM SB treated cells for24h, MSP assay revealed that PCR products of CLDN6methylation decreased andBSP assay demonstrated that methylation sites in the promoter region of CLDN6decreased; the level of DNMT1was significantly down-regulated in breast cancercells treated with SB was confirmed by RT-PCR and Western blot. DNMT1activitykit confirmed that DNMT1activity was inhibited; ChIP assay demonstrated areduction in combination of DNMT1and CLDN6promoter.4. The effects of CLDN6on the cells transepithelial electrical resistance andmetastasis:10μM SB treated breast cancer cells for24h, TEER assay revealedincreasing TEER, Wound healing assay and transwell assay confirmed decreasedinvasiveness and migration of breast cancer cells; After CLDN6silenced in breastcancer cells treated with SB, TEER decreased and cell migration and invasion wereenhanced.5. Mechanism of CLDN6regulating metastasis:10μM SB treated breast cancercells for24h, morphology of MCF-7cells changed into short spindle from a longspindle and SKBR-3cells changed from polygon into slabs of stone like; RT-PCR andWestern blot revealed that expression of epithelial marker E-cadherin increased,expression of mesenchymal markers Vimentin, N-cadherin and SNAIL decreased;immunofluorescence results showed increased expression of CLDN6and E-cadherinand decreased N-cadherin. When silenced CLDN6expression in cells treated with SB,RT-PCR, Western blot and cell immunofluorescence verified that epithelial markermentioned above were all decreased and mesenchymal markers all increased.Conclusions:1. SMADs inhibitor SB up-regulates CLDN6through decreasing the CLDN6methylation by down-regulating the expression of DNMT1, inhibiting the activity of DNMT1and inhibiting the binding of DNMT1with CLDN6promoter in Breastcancer cells MCF-7and SKBR-3.2. CLDN6inhibits the metastasis of breast cancer cells by reversing EMT.更多还原