摘要
第一部分MiR-590-5P和S100A10的表达及关系分析
目的探讨miR-590-5P和S100A10基因在人肝癌细胞及正常肝细胞中的表达差异。
方法培养人正常的肝细胞及多种肝癌细胞株，取对数生长期细胞，Trizol法提取Total RNA，使用特异性反转录引物反转录制备cDNA，设计针对成熟体miR-590-5P的PCR检测引物，荧光定量法检测成熟体miR-590-5P含量。取对数生长期细胞，抽提细胞总蛋白，BCA蛋白定量，Western blot检测S100A10蛋白含量。分析miR-590-5P及S100A10在肝癌细胞及正常肝细胞中的表达差异。
结果荧光定量检测结果显示，miR-590-5P在人肝癌细胞中低表达(与正常肝细胞比较，p＜0.01)；S100A10蛋白含量则在肝癌细胞中高表达。
结论miR-590-5P在人肝癌细胞中低表达，S100A10基因在人肝癌细胞中高表达，二者之间存在负相关趋势。
第二部分MiR-590-5P与S100A10之间调控关系分析
目的分析miR-590-5P与S100A10表达之间可能存在的调控关系。
方法提取人基因组DNA， PCR扩增miR-590-5P前体序列，构建pcDNA-miR-590-5P重组质粒。提取人Total RNA，反转录制备cDNA，PCR扩增S100A10基因的3’UTR区，将S100A10的3’UTR区克隆到PGL3-promoter荧光素酶表达载体，构建pS100A10-3’UTR-PGL3重组质粒。使用脂质体转染的方法将报告基因重组载体及miRNA重组载体共转染293细胞，使用双荧光素酶检测系统，以海肾荧光素酶作为参照，检测细胞干预前后荧光素酶的变化趋势。若miR-590-5P能够抑制pS100A10-3’UTR-PGL3报告基因的萤光素酶表达，则采用点突变的方法使S100A10-3’UTR中位于miR-590-5P上的结合位点即种子序列发生碱基突变，然后通过检测，观察其抑制作用是否还继续存在或消失。将慢病毒包装质粒混合物及重组表达载体pcDNA-miR-590-5P共转染慢病毒包装细胞系（293T），采用Lv-miR-590-5P重组慢病毒进行包装和生产，同时采用比例稀释法来检测其病毒滴度，通过预实验来确定研究中慢病毒感染HepG2细胞的最佳MOI值。以最佳MOI值使用慢病毒颗粒对HepG2细胞进行感染，在感染72h后，检测HepG2细胞的感染效率，然后收集HepG2细胞，再分别提取其总RNA和总蛋白，采用RT-PCR和Western blot的方法检测miR-590-5P和S100A10之间的关系。
结果成功构建了pcDNA-miR-590-5P重组表达载体及pS100A10-3’UTR-PGL3报告基因重组质粒。在293细胞中转染pcDNA-miR-590-5P，可以抑制pS100A10-3’UTR-PGL3载体表达荧光素酶，而当3’-UTR进行突变修饰后，pmir-590-5P不能够抑制其表达。通过慢病毒系统在HepG2细胞中高效表达mir-590-5P，细胞中mir-590-5P的过表达抑制了S100A10基因表达。
结论S100A10为miR-590-5P的靶基因。在肝癌细胞中过表达miR-590-5P，可以抑制S100A10基因表达。
第三部分MiR-590-5P对于肝癌细胞HepG2的影响
目的通过慢病毒途径在HepG2细胞中过表达miR-590-5P，观察基因干预对于细胞增殖活性，细胞周期，钙离子浓度，细胞侵袭能力及相关蛋白含量的影响。
方法使用重组慢病毒Lv-miR-590-5P和对照病毒Lv-GFP，以最佳MOI值感染HepG2细胞，感染后72h，荧光显微镜下观察细胞，通过绿色荧光蛋白（GFP）表达判断细胞的感染效率。取病毒感染后的细胞，胰酶消化的方法制备细胞悬液，接种细胞到96孔板中，正常条件培养24、48、72h后，CCK-8法检测细胞增殖活性。收集病毒感染后72h细胞，台酚蓝细胞染色后进行活细胞计数，接种细胞到6孔细胞培养板，正常条件培养24h，收集对数期细胞，然后使用75%乙醇固定细胞24h，Rnase处理细胞，PI染色，通过流式细胞仪检测细胞周期。感染后72h，胰酶消化的方法制备细胞悬液，Transwell检测细胞侵袭能力变化。取病毒感染后的细胞，台酚蓝细胞染色后进行活细胞计数，接种细胞到6孔细胞培养板，正常条件培养24h，收集对数期细胞，进行钙离子浓度检测。取病毒感染后的细胞，采用胰酶消化的方法将其制备成一定浓度的细胞悬液，再将接种细胞置于6孔细胞培养板上，在正常条件下培养接种细胞24h，收集对数期细胞，进行相关蛋白含量检测。使用细胞蛋白提取试剂盒提取细胞总蛋白，BCA法进行蛋白定量。使用Western blot检测WNT通路相关蛋白（WNT5a、E-cadherin、Caspase3、cMyc、CyclinD1、MMP7）含量，及β-catenin蛋白磷酸化程度。并使用光密度分析软件对目的条带进行灰度值分析。
结果通过慢病毒途径，可以在肝癌细胞HepG2中获得100%的基因转导效率。在肝癌细胞HepG2中过表达的miR-590-5P，不仅可以明显抑制患者肿瘤细胞的增殖活性，还会引起其细胞周期发生停滞，进而加剧对肿瘤细胞侵袭能力的抑制作用。在肝癌细胞中过表达miR-590-5P，可明显抑制WNT5a、cMyc、CyclinD1、MMP7蛋白表达，而促进E-cadherin、Caspase3蛋白表达，同时，miR-590-5P的过表达可引起β-catenin蛋白的磷酸化，加速其降解。
结论在人肝癌细胞中过表达miR-590-5P，可以抑制WNT通路的激活，可以抑制肿瘤细胞增殖活性，使肿瘤细胞停滞与G1期，降低细胞钙离子含量，抑制肿瘤细胞侵袭能力。miR-590-5P在HepG2细胞中过表达，可有效抑制细胞的肿瘤活性。
Part I Analysis on the Expression Profiles of miR-590-5P and S100A10
Objectives To analyze the expression differences of miR-590-5P and S100A10gene in hepatocarcinoma cells and normal hepatocytes.
Methods Normal human hepatocytes and several hepatoma cell lines were cultured,and total RNA was extracted from the cells at logarithmic growth phase using Trizol.Specific primers for reverse transcription were used to prepare cDNA, PCR primers for thedetection of mature miR-590-5P were designed, and mature miR-590-5P content wasdetected by the quantitative fluorescence method. The total cellular protein was extractedfrom the cells at logarithmic growth phase and quantified by the BCA assay, and S100A10protein content was detected by western blot. The expression differences of miR-590-5Pand S100A10in hepatocarcinoma cells and normal liver cells were analyzed.
Results Quantitative fluorescence detection results showed that the expression ofmiR-590-5P in human hepatocarcinoma cells was lower than that in normal liver cells (p<0.01) and S100A10protein content in hepatocarcinoma cells was higher than that innormal liver cells.
Conclusions The results suggest that miR-590-5P expression is down-regulated inhuman hepatocarcinoma cells and S100A10is highly expressed in hepatocarcinoma cells,with a negative correlation between the both. There is a negative correlation between thetwo.
Part II Analysis on the Regulatory Relation between miR-590-5P andS100A10
Objectives To analyze the possible regulatory relation between miR-590-5P andS100A10expression.
Methods Human genomic DNA was isolated, and the precursor sequence of miR-590-5P was amplified by PCR to construct a recombinant plasmid,pcDNA-miR-590-5P. Total human RNA was extracted, cDNA was prepared by reversetranscription, and3’UTR region of S100A10gene was amplified by PCR and cloned intopGL3-promoter vector to construct a recombinant plasmid, pS100A10A-3’UTR-PGL3.pcDNA-miR-590-5P and pS100A10A-3’UTR-PGL3were cotransfected into293cells bythe liposome method, and the dual-luciferase reporter assay system was used to detectedthe change of luciferase before and after the introduction of miRNA, with the Renillaluciferase as a reference. If miR-590-5P can inhibit the luciferase expression ofpS100A10-3’UTR-pGL3, the point mutation should be used to mutate the binding site(seed sequence) of miR-590-5P in S100A10-3’UTR, and the effect on the inhibitory roleshould be detected further.The lentiviral packaging mix and recombinantpcDNA-miR-590-5P vector were cotransfected into the lentiviral packaging cell line (293T)to produce the recombinant lentivirus, Lv-miR-590-5P. The titer was detected byproportional dilution and the optimal MOI for the infection of HepG2cells with thelentivirus was determined by preliminary experiments. HepG2cells were infected with thelentivirus at the optimal MOI and the infection efficiency was observed72hours afterinfection. Total RNA and total protein was extracted, and subjected to RT-PCR andwestern blot separately, to detect the relationship between miR-590-5P and S100A10.
Results The recombinant expression vector pcDNA-miR-590-5P and therecombinant reporter vector pS100A10-3’UTR-PGL3were constructed successfully. Thetransfection of pcDNA-miR-590-5P of293cells inhibited the luciferase expression ofpS100A10-3’UTR-PGL3. In addition, pmir-590-5P did not inhibit the luciferaseexpression after the mutation of3’-UTR. The efficient expression of mir-590-5P in HepG2mediated by the lentiviral system inhibited S100A10expression.
Conclusions S100A10is a target gene of miR-590-5P. Overexpression ofmiR-590-5P in HepG2cells inhibits the expression of S100A10gene.
Part III Effects of MiR-590-5P on HepG2cells
Objectives To study the effects of miR-590-5P overexpression in HepG2cellsmediated by the lentiviral system on cellular proliferative activity, cell cycle, Ca2+concentration, cell invasion ability and relevant proteins.
Methods HepG2cells were infected with the recombinant lentivirus Lv-miR-590-5P and the control lentivirus Lv-GFP at the best MOI, and the infectionefficiencies of the cells were determined by the fluorescence microcopy of greenfluorescent protein (GFP)72hours after infection.The cells infected with virus wereprepared into cell suspension by trypsinization, seeded into96-well plates and culturedunder normal conditions for24,48or72hours.10μl CCK-8reagent was added into eachwell, and the cells were incubated at37℃for another4hours. The cell proliferative abilitywas determined by cell counting, which was measured by the absorbance at450nm (A450)in a microplate reader.72hours after the infection, the viable cells were counted by trypanblue staining. The cells were seeded into6-well plates and cultured under normalconditions for24hours. The cells at exponential phase were collected and fixed in75%ethanol for24hours, treated with RNase, and stained with PI. The cell cycle was detectedwith flow cytometry.72hours after infection, the cell suspension was prepared bytrypsinization and subjected to Transwell assay to detect the change of cell invasion ability.The cells suspension was prepared with chemokine-free medium (containing5%BSA toadjust the osmotic pressure) at a concentration of1.0×106/ml.250μl of cell suspensionwas added into each insert, and500μl complete medium with10%FBS was added intoeach receiver well. The culture was covered, and incubated in an incubator at37°C and5%CO2for48hours. The cell suspension on the insert was aspirated, and the membranewas cut off with a scalpel and treated for fluorescence photography; at the same time, thefluorescence staining analysis was performed to count the cells on membrane.Afterinfection, the viable cells were counted by trypan blue staining. The cells were seeded to6-well plates and cultured under normal conditions for24hours Ca2+concentrationdetection was conducted on the cells at exponential phase. Fluo3-AM was solved inDMSO to prepare the stock solution of Fluo3-AM, which was diluted with dPBS to makethe working solution. The medium was removed and the cells were washed with cellbuffers three times, and added with Fluo-3-AM working solution and incubated at37°Cfor30min. The Fluo3-AM working solution was removed and the cells were washed withcell buffers three times to remove residual Fluo3-AM working solution thoroughly. Thecells were then incubated at37°C for20-30min, and then detected at an excitationwavelength of480-500nm and an emission wavelength of525-530nm.The cells infectedwith virus were prepared into the cell suspension by trypsinization, seeded into6-wellplates and cultured under normal conditions for24hours. The cells at exponential phase were collected for the detection of relevant protein levels. Total cellular protein wasextracted with the cellular protein extraction kit and quantified with the BCA method.Western blot was used to detect the levels of relevant proteins in Wnt pathway (WNT5a,E-cadherin, Caspase3, cMyc, CyclinD1and MMP7) and the phosphorylation level ofβ-catenin. The proteins were isolated by SDS-PAGE, and transferred to PVDF membranesat a constant current of400mA. The membranes were blocked with TBST containing5%nonfat milk for2hours at normal temperature, and incubated with the primary antibody at4°C overnight, and then reacted with the secondary antibody for1hour. The bands weredetected by chemiluminescence and imaged with X-ray films. The optical density analysissoftware was used to analyze the gray values of target bands on the scanned films.
Results The lentiviral system used in HepG2cells resulted in a transductionefficiency of100%. The overexpression of miR-590-5P in HepG2inhibited the cellularproliferative activity, caused cell cycle arrest, and suppressed the invasion ability of tumorcells effectively. Moreover, the overexpression of miR-590-5P reduced the expression ofWNT5a, cMyc, CyclinD1, and MMP7, and increased the expression of E-cadherin andCaspase3. Meanwhile, miR-590-5P expression induced the phosphorylation of β-catenin,so as to speed up its degradation.
Conclusions The overexpression of miR-590-5P in human hepatocarcinoma cellscan inhibit the activation of Wnt pathway and proliferative activity of tumor cells, causecell-cycle G1arrest, reduce intracellular Ca2+content, and inhibit the invasion ability oftumor cells. The overexpression of miR-590-5P in HepG2cell line effectively inhibits itstumor activity.
引文
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