MicroRNA-339-5p/3p在结直肠癌中作用的研究
摘要
研究背景和目的
结直肠癌是危害人类生命和健康的恶性肿瘤之一。在世界范围内,每年有近100万的新发病例,在西方国家的死亡率达33%。在我国,随着人们生活水平的提高,饮食习惯与结构的改变,结直肠癌发病率和死亡率均呈明显上升,且具有年轻化,家族聚集,遗传易感等特征。
结直肠癌与其他类型肿瘤一样,是复杂的基因表达异常性疾病,其中包括编码及非编码基因的结构和表达的异常。那么调控结直肠癌细胞特异的基因表达及其发展过程中基因表达变化的分子生物学机制究竟如何?
近年来,非编码RNA(non. coding RNAs),特别是微小RNA(microRNA, miRNA)调控基因表达研究的兴起为该问题的解决提供了新的视点。MicroRNA(miRNA)是一类大小约为19-22个核苷酸的小分子RNA。miRNA的加工成熟过程分为两步：pri-miRNA在核内被Drosha酶加工成前体：pre-miRNA。 Pre-miRNA转运到细胞质后在Dicer酶的作用下,通过剪辑加工后形成两种成熟序列,根据丰度多少,称作miRNA/miRNA*。而当前未通过实验确定丰度高低的,以诸如miR-5p(5‘臂)和miR-3p(3‘臂)这种命名形式来区分。最近越来越多的文献表明：位于同一发卡结构上的5p端和3p端可作用于相同或不同的靶基因,发挥相同或不同的功能。
人类的pre-miR-339可产生两种不同序列的剪接体：miR-339-5p和miR-339-3p。它们在结肠癌中的表达特点如何?它们又如何调控结肠癌细胞生物学过程?目前尚未见研究报道。为深入探讨这些问题,我们拟通过实时定量PCR技术的方法,检测miR-339-5p/3p在结直肠癌组织和细胞株中表达特性；利用RNA干扰技术及构建]miRNA过表达慢病毒载体,通过CCK-8、细胞周期、细胞迁移和侵袭、裸鼠皮下成瘤等实验检测miR-339-5p/3p对结肠癌细胞的增殖及转移能力的影响；根据生物信息学预测结果,通过实时定量PCR技术、蛋白质电泳、荧光素酶报告实验确定miR-339-5p的靶基因,而miR-339-3p靶基因的研究将在后续研究中进行。这些疑问的解答有助于明确miR-339-5p/3p在结直肠癌生长及转移过程中的作用,为探讨结直肠癌发生、转移机理提供重要实验依据,为探索新的治疗靶点提供线索和思路。
研究方法
1. miR-339-5p/3p在结直肠癌组织及细胞中的表达
采用荧光定量RT-PCR的方法检测了30例结直肠癌组织标本及其配对的正常组织中miR-339-5p/3p的表达。检测miR-339-5p/3p在SW480、SW620、HT29、 LOVO、HCT116、LS174T6种结直肠癌细胞的表达。
2. miR-339-5p/3p对结直肠癌细胞体内外生物学特性的影响
(1)利用阳离子脂质体法,将miR-339-5p/3p mimics瞬时转染至结直肠癌细胞株SW620中,利用CCK8法、细胞周期和Transwell迁移、侵袭实验,检测miR-339-5p/3p对体外癌细胞增殖、迁移和侵袭能力的影响。
(2)利用阳离子脂质体法,将miR-339-5p/3p inhibitor瞬时转染至结直肠癌细胞株HCT116中,利用CCK8法、细胞周期和Transwell迁移、侵袭实验,检测miR-339-5p/3p对体外癌细胞增殖、迁移和侵袭能力的影响。
(3)设计并构建慢病毒表达载体pLVTHM-pre-miR-339,病毒包装后感染SW620细胞,通过流式细胞仪分选鉴定稳定过表达niR-339-5p/3p的结直肠癌细胞株SW620-p1VTHM/pre-miR-339。通过CCK8、平板克隆形成实验和流式细胞术细胞周期检测pre-miR-339过表达对细胞体外增殖能力的影响,通过Transwell小室观察pre-miR-339过表达对结直肠癌细胞的运动、迁移能力的影响,利用整体可视化动物模型,进一步观察pre-miR-339对裸鼠皮下成瘤能力的影响。
3. miR-339-5p/3p靶基因的预测、验证及相关通路分子检测
(1)利用生物信息学在线网站TargetScan、Pictar和microRNA.ORG预测miR-339-5p/3p的靶基因,筛选miR-339-5p/3p的候选靶基因。
(2)利用荧光定量PCR检测miR-339-5p的候选靶基因PRL-1在6株不同结直肠癌细胞株中的表达；利用荧光定量PCR、Western Blot检测瞬时miR-339-5p/3p及稳定过表达miR-339-5p/3p的细胞SW620、干扰miR-339-5p/3p表达的HCT116中靶基因PRL-1的mRNA及蛋白表达变化。
(3)扩增包含PRL-13'UTR种子区域的基因片段并将其亚克隆至psiCHECK-2载体,并对此重组载体进行定点突变构建psiCHECK-2-PRL-1-Mut载体。利用荧光素酶报告系统检测PRL-1与miR-339-5p/3p的结合情况,明确PRL-1是否为miR-339-5p/3p直接作用的靶基因。
(4)结直肠癌中miR-339-5p参与结直肠癌信号通路分子的检测：利用WesternBlot检测过表达miR-339-5p的结直肠癌细胞株SW620-p1VTHM7pre-miR-339和干扰miR-339-5p表达的HCT116中ERK1/2、p-ERK1/2蛋白质的表达变化。
4.统计学分析
采用SPSS13.0统计软件进行数据分析。组织的荧光定量PCR结果比较2-△Ct采用非参数检验(nonparametric test Mann-Whitney-Wilcoxon),细胞荧光定量PCR结果比较2-ΔΔCt值,采用单因素方差分析(One-Way ANOVA),方差不齐时采用Welch近似方差分析；方差齐性的多重比较采用LSD法、SNK(Student-Newman-Keuls)法,方差不齐时采用Dunnett's T3法。亦采用两独立样本t检验(Independent-Samples T text),方差不齐时采用近似t检验。细胞周期检测、平板克隆形成实验、Transwell迁移实验,荧光素酶活性检测结果方差齐性时采用两独立样本t检验(Independent-Samples T text),方差不齐时采用近似t检验。荧光素酶活性检测结果方差齐性时采用两独立样本t检验(Independent-Samples T test),方差不齐时采用近似t检验。CCK8体外增殖实验采用析因设计的方差分析和单因素方差分析。P<0.05为差异有统计学意义。
研究结果
1. miR-339-5p/3p在结直肠癌组织及细胞中的表达
(1)miR-339-5p/3p在结直肠癌组织中的表达
在30对新鲜结直肠癌配对组织中,荧光定量RT-PCR检测结果显示：miR-339-5p/3p在结直肠癌组织中的表达低于正常粘膜组织(z=-5.671, P<0.001)(z=-5.597, P<0.001)。miR-339-5p/3p在伴有淋巴结转移的结直肠癌组和不伴有淋巴结转移的结直肠癌组之间的表达有统计学差异(t=2.063,P=0.041)；临床病理分析显示miR-339-5p/3p在30例结直肠癌配对组织中的表达与患者性别、年龄、以及肿瘤分化程度均无显著关系(P=0.853,P=0.179,P=0.397)。
(2) miR-339-5p/3p在不同转移潜能结直肠癌细胞系中的表达
荧光定量PCR结果显示,以正常组织作为对照,6种细胞之间miR-339-5p/3p的表达差异有统计学意义(F=66.554,P<0.001)(F=74.603,P<0.001)。经Dunnett'sT3多重比较发现,miR-339-5p在HCT116细胞中的表达高于其他6种细胞(P=0.03, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001)。而miR-339-3p在HT-29细胞中的表达高于其他6种细胞(P<0.001, P=0.112, P<0.001, P<0.001, P<0.001)。
2. miR-339-5p/3p对结直肠癌细胞体内外生物学特性的影响
a.转染miR-339-5p/3p mimics对结直肠癌细胞体外生物学特性的影响
(1)将商品化的miR-339-5p mimics分别转染结直肠癌SW620细胞,荧光定量PCR结果表明：和miR mimics control组相比,转染后的SW620细胞中miR-339-5p的表达水平上升,差异具有统计学意义(t=-5.780,P=0.029)。将商品化的niR-339-3p mimics转染结直肠癌SW620细胞,荧光定量PCR结果表明：和miR mimics control组相比,转染后的SW620细胞中miR-339-3p的表达水平上升,差异具有统计学意义(t=-7.020,P=0.002)说明干扰效果显著。
(2)CCK8法对细胞体外增殖能力的改变进行检测结果显示：与miR mimics control组相比,转染miR-339-5p mimics的SW620细胞增殖能力减弱(F=60.735,P<0.001)。与miR mimics control组相比,转染miR-339-3p mimics的SW620细胞增殖能力减弱(F=7.175,P<0.001)。
(3)采用流式细胞仪分析细胞周期分布,与miR mimics control组相比,过表达miR-339-5p后导致SW620细胞S期细胞数减少(t=8.516,P<0.001),而相应G1期则增多(t=-13.178,P<0.001)。过表达miR-339-3p对SW620细胞周期无明显影响,S期细胞数无明显变化(t=-5.220,P=0.629),相应G1期细胞数也无明显变化(t=0.598,P=0.582)。
(4) Transwell小室迁移实验发现与miR mimics control组相比,转染miR-339-5p mimics后的SW620细胞的运动迁移能力减弱,差异具有统计学意义(t=14.219, P<0.001)。Transwell小室迁移实验发现与miR mimics control组相比,转染miR-339-3p mimics后的SW620细胞的运动迁移能力减弱,差异具有统计学意义(t=10.862,P<0.001)。
(5)铺基层胶后Transwell小室侵袭实验发现与miR mimics control组相比,转染miR-339-5p mimics后的SW620细胞的侵袭能力减弱,差异具有统计学意义(t=8.817,P<0.001)。Transwell小室侵袭实验发现与miR mimics control组相比,转染miR-339-3p后的SW620细胞的侵袭能力减弱,差异具有统计学意义(t=9.014,P<0.001)。
b.转染miR-339-5p/3p inhibitor对结直肠癌细胞体外生物学特性的影响
(1)将商品化的niR-339-5p及miR-339-3p inhibitor分别转染结直肠癌HCT116细胞,荧光定量PCR结果表明：miR inhibitor control组相比,转染后HCT116细胞中miR-339-5p的表达水平下降,差异具有统计学意义(t=-36.847,P=0.001)。与miR inhibitor control组相比,转染后HCT116细胞中miR-339-3p的表达水平下降,差异具有统计学意义(t=-6.924,P=0.02),说明干扰效果明显。
(2)CCK8法对细胞体外增殖能力的改变进行检测结果显示：与miR inhibitorcontrol组相比,转染miR-339-5p inhibitor的HCT116增殖能力增强(F=33.604,P<0.001)。与miR inhibitor control组相比,转染miR-339-3p inhibitor的HCT116增殖能力增强(F=18.204,P<0.001)。
(3)采用流式细胞仪分析细胞周期分布,与miR inhibitor control组相比,抑制miR-339-5p表达后导致HCT116细胞S期细胞数增多(t=-8.335,P=0.001),而相应G1期则减少(t=7.393,P=0.002)。抑制miR-339-3p表达后对HCT116细胞细胞周期无明显影响,S期细胞数无明显变化(t=-0.518,P=0.632),G1期细胞数无明显变化(t=0.508,P=0.6)。
(4) Transwell小室迁移实验发现与niR inhibitor control组相比,转染miR-339-5p inhibitor后的HCT116细胞的运动迁移能力增强,差异具有统计学意义(t=-17.882, P<0.001)。Transwell小室迁移实验发现与miR inhibitor control组相比,转染miR-339-3p inhibitor后的HCT116细胞的运动迁移能力增强,差异具有统计学意义(t=-13.917,P<0.001)。
(5)铺基层胶后Transwell小室侵袭实验发现与miR inhibitor control组相比,转染miR-339-5p inhibitor后的HCT116细胞的侵袭能力增强,差异具有统计学意义(t=-7.178,P<0.001). Transwell小室侵袭实验发现与miR inhibitor control组相比,转染miR-339-3p inhibitor后的HCT116细胞的侵袭能力增强,差异具有统计学意义(t=-8.243,P<0.001)。
3.慢病毒表达载体pLVTHM/pre-miR-339对结直肠癌细胞体内外生物学特性的影响
(1)分子克隆技术构建慢病毒表达载体pLVTHM/pre-miR-339。
(2)流式细胞仪筛选稳定过表达miR-339-5p和miR-339-3p的细胞株SW620,命名为S W620/pLVTHM-pre-miR-339。
(3)荧光定量PCR鉴定miR-339-5p和miR-339-3p表达,与转染空载pLVTHM的SW620细胞(命名为SW620/pLVTHM-NC)相比,SW620/pLVTHM-pre-miR-339组中miR-339-5p和miR-339-3p的表达量高于SW620/pLVTHM-NC组(t=7.621, P=0.02)(t=-5.638, P=0.03);说明pre-miR-339转染成功。
(4)CCK8法对细胞体外增殖能力的改变进行检测结果显示：与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞增殖能力减弱(F=12.189,P=0.001)。
(5)采用流式细胞仪分析细胞周期分布,与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞细胞S期细胞数减少(t=5.056,P=0.007),而相应G1期则增多(t=-3.669,P=0.021)。
(6)克隆形成实验结果显示,与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞形成克隆的能力降低(t=11.841,P<0.001)。
(7) Transwell小室迁移实验发现与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞的运动迁移能力降低,差异具有统计学意义(t=7.606,P<0.001)。
(8)铺基层胶后Transwell小室侵袭实验发现与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞的侵袭能力降低,差异具有统计学意义(t=8.682,P<0.001)。
(9)裸鼠皮下成瘤实验显示,分别接种SW620/pLVTHM-NC组、SW620/pLVTHM-pre-miR-339细胞后,裸鼠皮下肿瘤生长具有明显组间差异(F=13.984, P<0.001); SW620/pLVTHM-pre-miR-339组与SW620/pLVTHM-NC组相比,皮下肿瘤生长速度明显减慢(F=5.024,P=0.008)。
4. miR-339-5p和miR-339-3p作用的靶基因的生物信息学分析,miR-339-5p作用靶基因的鉴定及下游信号分子检测
(1)生物信息学预测软件TargetScan、PicTar、MICRORNA.ORG对miR-339-5p/3p的靶基因进行预测,取交集获得与肿瘤的发生发展及转移过程相关的基因,通过查阅文献,初步候选PRL-1作为miR-339-5p的靶基因进行研究。miR-339-3p的靶基因只是进行了预测,最终的筛选结果将在后续的实验中得到认证。
(2)荧光定量PCR检测6种细胞株SW480、SW620、HT29、HCT116、LS174T、 LOVO中PRL-1的表达水平。在6种结直肠癌细胞株中,miR-339-5p和PRL-1的表达趋势相反。
(3)荧光定量PCR与Western blot结果显示：与miR mimics control组相比,转染miR-339-5p mimics的SW620细胞中,PRL-1的mRNA (t=26.858, P=0.001)及蛋白表达水平均明显下降；但是与miR mimics control组相比,转染miR-339-3p mimics的SW620细胞中,PRL-1的mRNA (t=0.452, P=0.675)及蛋白表达水平均未见变化。同样在稳定转染的细胞株检测实验中,与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞中PRL-1的mRNA及蛋白表达水平均下降。与miR inhibitor control组相比,转染miR-339-5p inhibitor的HCT116细胞中,PRL-1的mRNA (t=-9.036, P=0.012)及蛋白表达水平均上升;与miR inhibitor control组相比,转染miR-339-3p inhibitor的HCT116细胞中,PRL-1的：mRNA (t=-1.501, P=0.272)及蛋白表达水平均未见改变。
(4)构建psiCHECK-2/PRL-13'UTR载体及其突变载体。psiCHECK-2/WT-3'UTR和miR-339-5p mimics、miR mimics control共转染后,293FT细胞和HCT116细胞荧光素酶活性分别降低,经统计分析,差异均有统计学意义(P<0.001,P=0.002)。突变质粒psiCHECK-2/MUT-3'UTR与miR-339-5p mimics、miR mimics control共转染均不能改变荧光素酶活性(P=0.069,P=0.453),表明miR-339-5p与PRL-13'UTR可发生特异性结合。psiCHECK-2/WT-3'UTR和miR-339-3p mimics、miR mimics control共转染后,293FT细胞和HCT116均不能改变荧光素酶活性(P=0.466、P=0.243)。突变质粒psiCHECK-2/MUT-3'UTR与miR-339-3p mimics、miR mimics control共转染也均不改变荧光素酶活性(P=0.273,P=0.224),表明miR-339-3p与PRL-13'UTR未发生结合。
(5) Western blot结果显示：与miR inhibitor control组相比,转染miR-339-5p inhibitor后的HCT116细胞中的ERK1/2表达水平未见改变,而ERK1/2磷酸化水平升高,相反,与pLVTHM-NC组相比,转染pLVTHM-pre-miR-339的SW620细胞中的ERKl/2表达水平未见改变,而ERK1/2磷酸化水平降低。说明miR-339-5p能抑制p-ERK1/2的活性。
结论
1.MiR-339-5p通过靶基因PRL-1抑制结直肠癌细胞的增殖、侵袭和转移能力。
2. MiR-339-3p抑制结直肠癌细胞的增殖、侵袭和转移能力,其靶基因的鉴定将在后续实验中展开
3.初步验证MiR-339-5p影响ERK信号通路的活化,从而抑制结直肠癌细胞的增殖、侵袭和转移过程。
Backgroud and Objective
Colorectal cancer is one of the most prevalent carcinomas throughout the world. Every year, more than1million individuals will develop colorectal cancer, and the disease-specific mortality is nearly33%in the developed world. In our country, with the improvement of living standards and dietary changes, colorectal cancer has been increasing gradually year by year. With the change of dietary habits and structure, the morbidity and mortality were significantly increased. And younger, familial aggregation, genetic susceptibility are the characteristics of the disease in our country.
Colorectal cancer, like other tumor types, is a disease full of complex gene expression abnormalities, including abnormal and expression of coding and noncoding gene structure. Then the the molecular biological mechanism which controls node specific gene expression and gene expression in the process of development of the disease is need to study.
In recent years, non-coding RNA (non.coding RNAs), especially the microRNA (microRNA, miRNA) can regulate gene expression in the world, which offers a new perspective to solve this problem. MicroRNA (miRNA) is a class of small molecule RNA size of about19-22nucleotides. The processing of miRNA ripening process is divided into two steps:pri-miRNA Drosha enzyme processing into the precursor in the nucleus is called pre-miRNA. Pre-miRNA translocation to the cytosol in Dicer is under the action of the enzyme, the formation of two mature sequence by editing processing, which are called miRNA/miRNA*. And now the abundance of miRNAs does not be decided through experiments, are named miR-5p (5'arm) and miR-3p (3' arm). More recent literatures show that:5p and3p can be applied to the same or different target genes, play the same or different function.
Human pre-miR-339has two different sequences of the splicing:miR-339-5p and miR-339-3p. What is their expression in colon cancer? How do they control the biological cells process? There are no reports about it. To discuss these issues, we examined the expression level of miR-339-5p in human colon cancer cells and cancer tissues using qRT-PCR, and tested its effects on cell growth, cell-cycle distribution, and colony formation and invasion capacity in vitro using RNA interference technology and construction of miRNAs expression by lentiviral vector. We administered miR-339-5p precursor to a mouse colon cancer tumor xenograft model and further demonstrated that it could suppress colon tumor growth in vivo. We determined the target gene of miR-339-5p according to the prediction of biological information, real-time quantitative PCR, protein electrophoresis, luciferase reporter experiment. The solution of questions would indicate the function of miR-339-5p/3p in colorectal cancer growth and metastasis of colorectal cancer. These results indicate that miR-339-5p/3p can function as a tumor suppressor, regulating the maintenance and progression of cancers, providing the clue and thinking for exploring new therapeutic targets.
Methods
1. Mature miR-339-5p/3p expression in tissues and cells was detected using Real-time RT-PCR
Mature miR-339-5p/3p expression in tissues and cells was detected using Real-time RT-PCR. The specimens include samples of colorectal cancer tissue and matched normal colonic mucosa of30patients. While colorectal cancer six human colonic carcinoma cell lines include HCT116, HT29, LS174T, SW480, SW620and LOVO.
2. Effect of miR-339-5p/3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined
(1) Using the cationic liposome method, the miR-339-5p/3p mimics was transiently transfected into colorectal cancer cell line SW620. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle and transwell migration, invasion assay.
(2) Using the cationic liposome method, the miR-339-5p/3p inhibitor was transiently transfected into colorectal cancer cell line HCT116. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle and transwell migration, invasion assay.
(3) A lentiviral expression vector pLVTHM-pre-miR-339was designed^and constructed. SW620cells were transduced with vector supernatant and subsequently FACS-sorted for green fluorescent protein (GFP). Confirmation of stable transfection of the plasmids was obtained using the miR qRT-PCR assay. The colorectal cancer cell line was called SW620-p1VTHM/pre-miR-339. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle, clone formation assay and transwell migration, invasion assay. The effect of tumorigenicity in nude mice subcutaneous was observed using the whole visualization animal model.
3. miR-339-5p/3p target genes were predicted, related pathways were verified.
(1) Three commonly used databases including TargetScan, Pictar and microRNA.org were used to forecast the target genes with the search terms of miR-339-5p/3p.
(2) PRL-1, the target gene of miR-339-5p, mRNA was detected in different colorectal cancer cell lines by qRT-PCR, and the correlation was analysis. The expression of PRL-1mRNA and protein was detected in SW620overexpressing miR-339-5p/3p and in HCT116transfected with miR-339-5p/3p inhibitor.
(3) The sequence containing the PRL-1gene3'UTR seed region fragment3'UTR seed region was subcloned into psiCHECK-2vector, and the recombinant vector psiCHECK-2-wt-PRL-1an psiCHECK-2-mut-PRL-1vector site-directed mutations were constructed. Combination of PRL-1and miR-339-5p/3p were detected by luciferase reporter system in order to determine whether miR-339-5p/3p targeted PRL-1gene directly.
(4) The relational colorectal cancer signal pathway was detected.
ERK1/2, p-ERK1/2protein expression was detected in SW620overexpressing by miR-339-5p/3p and in HCT116transfected with miR-339-5p/3p inhibitor using Western Blot.
4. Statistical analysis
SPSS13.0sorftware was used for statistical analysis. Data were presented as Mean±SEM of at least3independent experiments. Shapiro-Wilk test was used to verify the clinical samples'distribution. Differences were analyzed using the nonparametric test Mann-Whitney-Wilcoxon. Relative quantification value(2-ΔΔCt) of QPCR in cells were analyzed through One-way ANOVA, with the SNK, LSD or Dunnett's T3tests for multiple comparisons. It also analyzed through two-tailed Student's t test. Colony formation assay and transwell in vitro invasion assay were analyzed through two-tailed Student's t test. And it also was used for comparisons of relative luciferase activities experiments. The results of CCK8assay was analyzed by Factorial design analysis of variance. P values of <0.05were considered statistically significant.
Result
1. miR-339-5p/3p expression was detected in colorectal cancer tissue and cells.
(1) The expression of miR-339-5p/3p were detected in colorectal cancer tissues.
In30fresh colorectal cancer paired tissues, fluorescence quantitative PCR test results show that the expression of miR-339-5p/3p in colorectal cancer tissues was significantly lower than that in normal mucosa (z=-5.671, P<0.001)(z=-5.597, P<0.001). The result also showed significant difference between miR-339-5p/3p node metastasis in colorectal cancer group with lymph and without lymph node metastasis in colorectal cancer group (t=2.063, P=0.041). Clinical and pathological analysis showed that miR-339-5p/3p in30cases of colorectal carcinoma tissues has no relationship with matched with age, sex, the degree of tumor differentiation (P=0.853, P=0.179, P=0.397).
(2) miR-339-5p/3p expression was detected in colorectal cancer cell lines with different metastatic potential.
qRT-PCR results showed that there was significant difference between the6kinds of cells expressing miR-339-5p/3p (F=66.554, P<0.001)(F-74.603, P<0.001). The Dunnett's T3multiple comparison indicated the expression of miR-339-5p in HCT116cells was higher than that of the other6cells (P=0.03, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001). The expression of miR-339-3p in HT-29cells was higher than that of the other6cells (P<0.001, P=0.112, P<0.001, P<0.001, P<0.001).
2. Effect of miR-339-5p/3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined.
a. The biological behavior of of SW620cell lines was performed transfected with miR-339-5p/3p mimics.
(1) Compared with miR mimics control, increased miR-339-5p/3p expression levels were measured in SW620cells after transfected with miR-339-5p/3p mimics. The difference was statistically significant (t=-5.780, P=0.029)(t=-7.020, P=0.002) showed significant moderating effect.
(2) Compared with miR mimics control, decreased proliferation properties of SW620transfected with the miR-339-5p/3p mimics were also detected by CCK-8assay. The difference was statistically significant (F=60.735, P<0.001)(F=7.175, P<0.001) showed significant moderating effect.
(3) By analyzing the distribution of cell cycle by flow cytometry, compared with miR mimics control, miR-339-5p overexpression in SW620cells after S cells were decreased (t=8.516, P<0.001), and the corresponding G1phase increased (t=-13.178, P<0.001). miR-339-3p overexpression has no influence on SW620cells, S cells were not changed (t=-5.220, P=0.629), and the corresponding G1phase was not changed (t=0.508, P=0.6)
(4) The number of migratory SW620cells transfected with the miR-339-5p mimics was fewer than miR mimics control, the difference was statistically significant (t=14.219, P<0.001). The number of migratory SW620cells transfected with the miR-339-3p mimics was fewer than miR mimics control, the difference was statistically significant (t=10.862, P<0.001).
(5) The number of invasive SW620cells transfected with the miR-339-5p mimics was fewer than miR mimics control, the difference was statistically significant (t=8.817, P<0.001). The number of invasive SW620cells transfected with the miR-339-3p mimics was fewer than miR mimics control, the difference was statistically significant (t=9.014, P<0.001).
b. The biological behavior of of HCT116cell lines was performed transfected with miR-339-5p/3p inhibitor.
(1) Compared with miR inhibitor control, decreased miR-339-5p/3p expression level was measured in HCT116cells after transfected with miR-339-5p/3p inhibitor. The difference was statistically significant showed significant moderating effect (t=-36.847, P=0.001)(t=-6.924, P=0.02)
(2) Compared with miR inhibitor control, increased proliferation properties of HCT116transfected with the miR-339-5p/3p inhibitor were also detected by CCK-8assay. The difference was statistically significant showed significant moderating effect (F=33.604, P<0.001)(F=18.204, P<0.001)
(3) By analyzing the distribution of cell cycle by flow cytometry, compared with miR inhibitor control, inhibition of miR-339-5p expression in HCT116cells after S cells were increased (t=-8.335, P=0.001), and the corresponding G1phase decreased (t=7.393, P=0.002). Inhibition of miR-339-3p expression in HCT116cells, S cells were not changed (t=-0.518, P=0.632), and the corresponding G1phase was not changed (t=-13.917, P<0.001).
(4) The number of migratory HCT116cells transfected with the miR-339-5p inhibitor was more than miR inhibitor control, the difference was statistically significant (t=-17.882, P<0.001). The number of migratory HCT116cells transfected with the miR-339-3p inhibitor were more than miR inhibitor control, the difference was statistically significant (t=-13.917, P<0.001).
(5) The number of invasive HCT116cells transfected with the miR-339-5p inhibitor were more than miR inhibitor control, the difference was statistically significant (t=-7.178, P<0.001). The number of migratory HCT116cells transfected with the miR-339-3p inhibitor was more than miR inhibitor control, the difference was statistically significant (t=-8.243, P<0.001).
c. The biological behavior of of SW620lines was performed transfected with pLVTHM/pre-miR-339.
(1) The pLVTHM/pre-miR-339lentiviral expression vector was constructed.
(2) The established stable cell line SW620expressing of miR-339-5p and miR-339-3p was named SW620/pLVTHM-pre-miR-339.
(3) The expression of miR-339-5p and miR-339-3p in SW620/pLVTHM-pre-miR-339group was higher than that of SW620/group pLVTHM-NC (P=0.046, P=0.046). Compared with SW620/pLVTHM-NC, increased miR-339-5p/3p expression levels were measured in SW620/pLVTHM-pre-miR-339.The difference was statistically significant (t=-7.621, P=0.02)(t=-5.638, P=0.03) showed significant moderating effect.
(4) Compared with SW620/pLVTHM-NC, decreased proliferation properties of SW620transfected with the SW620/pLVTHM-pre-miR-339were also detected by CCK-8assay (F=12.189, P=0.001). The difference was statistically significant showed significant moderating effect.
(5) By analyzing the distribution of cell cycle by flow cytometry, compared with/pLVTHM-NC, S cells in SW620/pLVTHM-pre-miR-339were decreased (t=5.056, P=0.007), and the corresponding G1phase increased (t=-3.669, P=0.021).
(6) Colony formation assay showed that the ability of SW620cells to form colony were decreased after pLVTHM-pre-miR-339(t=11.841, P<0.001).
(7) The number of migratory SW620cells transfected with pLVTHM-pre-miR-339were fewer than with the control, the difference was statistically significant (t=11.841, P<0.001).
(8) The number of invasive SW620cells transfected with pLVTHM-pre-miR-339were fewer than with the control, the difference was statistically significant (t=7.606, P<0.001).
(9) SW620/pLVTHM-pre-miR-339cells or SW620/pLVTHM cells were injected subcutaneously to the blank of nude mice, respectively, into nude mice. The tumour growth rate of SW620/pLVTHM-pre-miR-339cells was slower than that of SW620/pLVTHM cells the difference was statistically significant (F=5.024, P=0.008).
4. Bioinformatics analysis of target gene of miR-339-5p and miR-339-3p function, detection of miR-339-5p target gene identification and the downstream signal molecules
(1) The bioinformatics prediction of target genes for miR-339-5p/3p was perdicted by software TargetScan, PicTar, MICRORNA.ORG. Considering the development and metastasis related genes of tumor, preliminary candidate PRL-1was studied as a target gene miR-339-5p. The miR-339-3p target gene was predicted, the final results will be performed in subsequent experiments certification.
(2) qRT-PCR detection in6kinds colon cell lines indicated the highest expression level in LOVO, and lowest expression level in HCT116(F=7.432, P=0.002). In6colorectal cancer cell lines, expression of miR-339-5p and PRL-1in the opposite trend.
(3) Compared with the miR mimics control, in SW620cells transfected with miR-339-5p mimics, mRNA and protein expression level of PRL-1decreased significantly (t=26.858,/P=0.001). However, compared with miR mimics control, in SW620cells transfected with miR-339-3p mimics, mRNA and protein expression level of PRL-1had no change (t=0.452, P=0.675). Also in the stable transfection experiments, compared with the SW620/group pLVTHM-NC, mRNA and PRL-1protein expression in SW620/pLVTHM-pre-miR-339cells was decreased. QRT-PCR and Western blot results showed that compared with miR inhibitor control, transfection of miR-339-5p inhibitor in HCT116cells, mRNA and protein expression level of PRL-1was increased (t=-9.036, P=0.012). Compared with miR inhibitor control, transfection of miR-339-3p inhibitor in HCT116cells, mRNA and protein expression level of PRL-1had no change (t=-1.501, P=0.272).
(4) After constructing psiCHECK-2/PRL-13'UTR vector and its mutation carriers. PsiCHECK-2/WT-3'UTR and miR-339-5p mimics, miR mimics control after transfection,293FT cells and HCT116cells luciferase activity was decreased respectively; by the statistical analysis, the differences were statistically significant (P<0.001, P=0.002). PsiCHECK-2/MUT-3'UTR and miR-339-5p mutant plasmid mimics, miR mimics control cotransfected luciferase activity were not changed (P=0.069, P=0.453), showed that miR-339-5p and PRL-13'UTR can specifically bind. PsiCHECK-2/WT-3'UTR and miR-339-3p mimics/miR mimics control after transfection,293FT cells and HCT116cells can not be changed in luciferase activity (P=0.466, P=0.243). The mutation plasmid psiCHECK-2/MUT-3'UTR and miR-339-3p mimics/miR mimics control were transfected into cells also did not change the luciferase activity (P=0.379, P=0.953), showed that miR-339-3p did not act on PRL-13'UTR.
(5) The Western blot results showed that compared with pLVTHM-NC/SW620, in SW620cells transfected with pLVTHM-pre-miR-339, ERK1/2expression levels did not change, and the decreased expression of p-ERK1/2was detected. Compared with miR inhibitor control, transfection of miR-339-5p inhibitor in HCT116cells following ERK1/2expression levels did not change, and the expression of p-ERKl/2increased. MiR-339-5p may inhibit the activity of p-ERK1/2.
Conclusion
1MiR-339-5p regulates the growth, colony formation and metastasis of colorectal cancer cells by targeting PRL-1
2MiR-339-3p inhibits colorectal cancer cell proliferation, invasion and metastasis ability, the identification of target genes will be carried out in a follow-up experiment
3MiR-339-5p may affect the activation of ERK signaling pathway.
结直肠癌是危害人类生命和健康的恶性肿瘤之一。在世界范围内,每年有近100万的新发病例,在西方国家的死亡率达33%。在我国,随着人们生活水平的提高,饮食习惯与结构的改变,结直肠癌发病率和死亡率均呈明显上升,且具有年轻化,家族聚集,遗传易感等特征。
结直肠癌与其他类型肿瘤一样,是复杂的基因表达异常性疾病,其中包括编码及非编码基因的结构和表达的异常。那么调控结直肠癌细胞特异的基因表达及其发展过程中基因表达变化的分子生物学机制究竟如何?
近年来,非编码RNA(non. coding RNAs),特别是微小RNA(microRNA, miRNA)调控基因表达研究的兴起为该问题的解决提供了新的视点。MicroRNA(miRNA)是一类大小约为19-22个核苷酸的小分子RNA。miRNA的加工成熟过程分为两步：pri-miRNA在核内被Drosha酶加工成前体：pre-miRNA。 Pre-miRNA转运到细胞质后在Dicer酶的作用下,通过剪辑加工后形成两种成熟序列,根据丰度多少,称作miRNA/miRNA*。而当前未通过实验确定丰度高低的,以诸如miR-5p(5‘臂)和miR-3p(3‘臂)这种命名形式来区分。最近越来越多的文献表明：位于同一发卡结构上的5p端和3p端可作用于相同或不同的靶基因,发挥相同或不同的功能。
人类的pre-miR-339可产生两种不同序列的剪接体：miR-339-5p和miR-339-3p。它们在结肠癌中的表达特点如何?它们又如何调控结肠癌细胞生物学过程?目前尚未见研究报道。为深入探讨这些问题,我们拟通过实时定量PCR技术的方法,检测miR-339-5p/3p在结直肠癌组织和细胞株中表达特性；利用RNA干扰技术及构建]miRNA过表达慢病毒载体,通过CCK-8、细胞周期、细胞迁移和侵袭、裸鼠皮下成瘤等实验检测miR-339-5p/3p对结肠癌细胞的增殖及转移能力的影响；根据生物信息学预测结果,通过实时定量PCR技术、蛋白质电泳、荧光素酶报告实验确定miR-339-5p的靶基因,而miR-339-3p靶基因的研究将在后续研究中进行。这些疑问的解答有助于明确miR-339-5p/3p在结直肠癌生长及转移过程中的作用,为探讨结直肠癌发生、转移机理提供重要实验依据,为探索新的治疗靶点提供线索和思路。
研究方法
1. miR-339-5p/3p在结直肠癌组织及细胞中的表达
采用荧光定量RT-PCR的方法检测了30例结直肠癌组织标本及其配对的正常组织中miR-339-5p/3p的表达。检测miR-339-5p/3p在SW480、SW620、HT29、 LOVO、HCT116、LS174T6种结直肠癌细胞的表达。
2. miR-339-5p/3p对结直肠癌细胞体内外生物学特性的影响
(1)利用阳离子脂质体法,将miR-339-5p/3p mimics瞬时转染至结直肠癌细胞株SW620中,利用CCK8法、细胞周期和Transwell迁移、侵袭实验,检测miR-339-5p/3p对体外癌细胞增殖、迁移和侵袭能力的影响。
(2)利用阳离子脂质体法,将miR-339-5p/3p inhibitor瞬时转染至结直肠癌细胞株HCT116中,利用CCK8法、细胞周期和Transwell迁移、侵袭实验,检测miR-339-5p/3p对体外癌细胞增殖、迁移和侵袭能力的影响。
(3)设计并构建慢病毒表达载体pLVTHM-pre-miR-339,病毒包装后感染SW620细胞,通过流式细胞仪分选鉴定稳定过表达niR-339-5p/3p的结直肠癌细胞株SW620-p1VTHM/pre-miR-339。通过CCK8、平板克隆形成实验和流式细胞术细胞周期检测pre-miR-339过表达对细胞体外增殖能力的影响,通过Transwell小室观察pre-miR-339过表达对结直肠癌细胞的运动、迁移能力的影响,利用整体可视化动物模型,进一步观察pre-miR-339对裸鼠皮下成瘤能力的影响。
3. miR-339-5p/3p靶基因的预测、验证及相关通路分子检测
(1)利用生物信息学在线网站TargetScan、Pictar和microRNA.ORG预测miR-339-5p/3p的靶基因,筛选miR-339-5p/3p的候选靶基因。
(2)利用荧光定量PCR检测miR-339-5p的候选靶基因PRL-1在6株不同结直肠癌细胞株中的表达；利用荧光定量PCR、Western Blot检测瞬时miR-339-5p/3p及稳定过表达miR-339-5p/3p的细胞SW620、干扰miR-339-5p/3p表达的HCT116中靶基因PRL-1的mRNA及蛋白表达变化。
(3)扩增包含PRL-13'UTR种子区域的基因片段并将其亚克隆至psiCHECK-2载体,并对此重组载体进行定点突变构建psiCHECK-2-PRL-1-Mut载体。利用荧光素酶报告系统检测PRL-1与miR-339-5p/3p的结合情况,明确PRL-1是否为miR-339-5p/3p直接作用的靶基因。
(4)结直肠癌中miR-339-5p参与结直肠癌信号通路分子的检测：利用WesternBlot检测过表达miR-339-5p的结直肠癌细胞株SW620-p1VTHM7pre-miR-339和干扰miR-339-5p表达的HCT116中ERK1/2、p-ERK1/2蛋白质的表达变化。
4.统计学分析
采用SPSS13.0统计软件进行数据分析。组织的荧光定量PCR结果比较2-△Ct采用非参数检验(nonparametric test Mann-Whitney-Wilcoxon),细胞荧光定量PCR结果比较2-ΔΔCt值,采用单因素方差分析(One-Way ANOVA),方差不齐时采用Welch近似方差分析；方差齐性的多重比较采用LSD法、SNK(Student-Newman-Keuls)法,方差不齐时采用Dunnett's T3法。亦采用两独立样本t检验(Independent-Samples T text),方差不齐时采用近似t检验。细胞周期检测、平板克隆形成实验、Transwell迁移实验,荧光素酶活性检测结果方差齐性时采用两独立样本t检验(Independent-Samples T text),方差不齐时采用近似t检验。荧光素酶活性检测结果方差齐性时采用两独立样本t检验(Independent-Samples T test),方差不齐时采用近似t检验。CCK8体外增殖实验采用析因设计的方差分析和单因素方差分析。P<0.05为差异有统计学意义。
研究结果
1. miR-339-5p/3p在结直肠癌组织及细胞中的表达
(1)miR-339-5p/3p在结直肠癌组织中的表达
在30对新鲜结直肠癌配对组织中,荧光定量RT-PCR检测结果显示：miR-339-5p/3p在结直肠癌组织中的表达低于正常粘膜组织(z=-5.671, P<0.001)(z=-5.597, P<0.001)。miR-339-5p/3p在伴有淋巴结转移的结直肠癌组和不伴有淋巴结转移的结直肠癌组之间的表达有统计学差异(t=2.063,P=0.041)；临床病理分析显示miR-339-5p/3p在30例结直肠癌配对组织中的表达与患者性别、年龄、以及肿瘤分化程度均无显著关系(P=0.853,P=0.179,P=0.397)。
(2) miR-339-5p/3p在不同转移潜能结直肠癌细胞系中的表达
荧光定量PCR结果显示,以正常组织作为对照,6种细胞之间miR-339-5p/3p的表达差异有统计学意义(F=66.554,P<0.001)(F=74.603,P<0.001)。经Dunnett'sT3多重比较发现,miR-339-5p在HCT116细胞中的表达高于其他6种细胞(P=0.03, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001)。而miR-339-3p在HT-29细胞中的表达高于其他6种细胞(P<0.001, P=0.112, P<0.001, P<0.001, P<0.001)。
2. miR-339-5p/3p对结直肠癌细胞体内外生物学特性的影响
a.转染miR-339-5p/3p mimics对结直肠癌细胞体外生物学特性的影响
(1)将商品化的miR-339-5p mimics分别转染结直肠癌SW620细胞,荧光定量PCR结果表明：和miR mimics control组相比,转染后的SW620细胞中miR-339-5p的表达水平上升,差异具有统计学意义(t=-5.780,P=0.029)。将商品化的niR-339-3p mimics转染结直肠癌SW620细胞,荧光定量PCR结果表明：和miR mimics control组相比,转染后的SW620细胞中miR-339-3p的表达水平上升,差异具有统计学意义(t=-7.020,P=0.002)说明干扰效果显著。
(2)CCK8法对细胞体外增殖能力的改变进行检测结果显示：与miR mimics control组相比,转染miR-339-5p mimics的SW620细胞增殖能力减弱(F=60.735,P<0.001)。与miR mimics control组相比,转染miR-339-3p mimics的SW620细胞增殖能力减弱(F=7.175,P<0.001)。
(3)采用流式细胞仪分析细胞周期分布,与miR mimics control组相比,过表达miR-339-5p后导致SW620细胞S期细胞数减少(t=8.516,P<0.001),而相应G1期则增多(t=-13.178,P<0.001)。过表达miR-339-3p对SW620细胞周期无明显影响,S期细胞数无明显变化(t=-5.220,P=0.629),相应G1期细胞数也无明显变化(t=0.598,P=0.582)。
(4) Transwell小室迁移实验发现与miR mimics control组相比,转染miR-339-5p mimics后的SW620细胞的运动迁移能力减弱,差异具有统计学意义(t=14.219, P<0.001)。Transwell小室迁移实验发现与miR mimics control组相比,转染miR-339-3p mimics后的SW620细胞的运动迁移能力减弱,差异具有统计学意义(t=10.862,P<0.001)。
(5)铺基层胶后Transwell小室侵袭实验发现与miR mimics control组相比,转染miR-339-5p mimics后的SW620细胞的侵袭能力减弱,差异具有统计学意义(t=8.817,P<0.001)。Transwell小室侵袭实验发现与miR mimics control组相比,转染miR-339-3p后的SW620细胞的侵袭能力减弱,差异具有统计学意义(t=9.014,P<0.001)。
b.转染miR-339-5p/3p inhibitor对结直肠癌细胞体外生物学特性的影响
(1)将商品化的niR-339-5p及miR-339-3p inhibitor分别转染结直肠癌HCT116细胞,荧光定量PCR结果表明：miR inhibitor control组相比,转染后HCT116细胞中miR-339-5p的表达水平下降,差异具有统计学意义(t=-36.847,P=0.001)。与miR inhibitor control组相比,转染后HCT116细胞中miR-339-3p的表达水平下降,差异具有统计学意义(t=-6.924,P=0.02),说明干扰效果明显。
(2)CCK8法对细胞体外增殖能力的改变进行检测结果显示：与miR inhibitorcontrol组相比,转染miR-339-5p inhibitor的HCT116增殖能力增强(F=33.604,P<0.001)。与miR inhibitor control组相比,转染miR-339-3p inhibitor的HCT116增殖能力增强(F=18.204,P<0.001)。
(3)采用流式细胞仪分析细胞周期分布,与miR inhibitor control组相比,抑制miR-339-5p表达后导致HCT116细胞S期细胞数增多(t=-8.335,P=0.001),而相应G1期则减少(t=7.393,P=0.002)。抑制miR-339-3p表达后对HCT116细胞细胞周期无明显影响,S期细胞数无明显变化(t=-0.518,P=0.632),G1期细胞数无明显变化(t=0.508,P=0.6)。
(4) Transwell小室迁移实验发现与niR inhibitor control组相比,转染miR-339-5p inhibitor后的HCT116细胞的运动迁移能力增强,差异具有统计学意义(t=-17.882, P<0.001)。Transwell小室迁移实验发现与miR inhibitor control组相比,转染miR-339-3p inhibitor后的HCT116细胞的运动迁移能力增强,差异具有统计学意义(t=-13.917,P<0.001)。
(5)铺基层胶后Transwell小室侵袭实验发现与miR inhibitor control组相比,转染miR-339-5p inhibitor后的HCT116细胞的侵袭能力增强,差异具有统计学意义(t=-7.178,P<0.001). Transwell小室侵袭实验发现与miR inhibitor control组相比,转染miR-339-3p inhibitor后的HCT116细胞的侵袭能力增强,差异具有统计学意义(t=-8.243,P<0.001)。
3.慢病毒表达载体pLVTHM/pre-miR-339对结直肠癌细胞体内外生物学特性的影响
(1)分子克隆技术构建慢病毒表达载体pLVTHM/pre-miR-339。
(2)流式细胞仪筛选稳定过表达miR-339-5p和miR-339-3p的细胞株SW620,命名为S W620/pLVTHM-pre-miR-339。
(3)荧光定量PCR鉴定miR-339-5p和miR-339-3p表达,与转染空载pLVTHM的SW620细胞(命名为SW620/pLVTHM-NC)相比,SW620/pLVTHM-pre-miR-339组中miR-339-5p和miR-339-3p的表达量高于SW620/pLVTHM-NC组(t=7.621, P=0.02)(t=-5.638, P=0.03);说明pre-miR-339转染成功。
(4)CCK8法对细胞体外增殖能力的改变进行检测结果显示：与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞增殖能力减弱(F=12.189,P=0.001)。
(5)采用流式细胞仪分析细胞周期分布,与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞细胞S期细胞数减少(t=5.056,P=0.007),而相应G1期则增多(t=-3.669,P=0.021)。
(6)克隆形成实验结果显示,与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞形成克隆的能力降低(t=11.841,P<0.001)。
(7) Transwell小室迁移实验发现与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞的运动迁移能力降低,差异具有统计学意义(t=7.606,P<0.001)。
(8)铺基层胶后Transwell小室侵袭实验发现与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞的侵袭能力降低,差异具有统计学意义(t=8.682,P<0.001)。
(9)裸鼠皮下成瘤实验显示,分别接种SW620/pLVTHM-NC组、SW620/pLVTHM-pre-miR-339细胞后,裸鼠皮下肿瘤生长具有明显组间差异(F=13.984, P<0.001); SW620/pLVTHM-pre-miR-339组与SW620/pLVTHM-NC组相比,皮下肿瘤生长速度明显减慢(F=5.024,P=0.008)。
4. miR-339-5p和miR-339-3p作用的靶基因的生物信息学分析,miR-339-5p作用靶基因的鉴定及下游信号分子检测
(1)生物信息学预测软件TargetScan、PicTar、MICRORNA.ORG对miR-339-5p/3p的靶基因进行预测,取交集获得与肿瘤的发生发展及转移过程相关的基因,通过查阅文献,初步候选PRL-1作为miR-339-5p的靶基因进行研究。miR-339-3p的靶基因只是进行了预测,最终的筛选结果将在后续的实验中得到认证。
(2)荧光定量PCR检测6种细胞株SW480、SW620、HT29、HCT116、LS174T、 LOVO中PRL-1的表达水平。在6种结直肠癌细胞株中,miR-339-5p和PRL-1的表达趋势相反。
(3)荧光定量PCR与Western blot结果显示：与miR mimics control组相比,转染miR-339-5p mimics的SW620细胞中,PRL-1的mRNA (t=26.858, P=0.001)及蛋白表达水平均明显下降；但是与miR mimics control组相比,转染miR-339-3p mimics的SW620细胞中,PRL-1的mRNA (t=0.452, P=0.675)及蛋白表达水平均未见变化。同样在稳定转染的细胞株检测实验中,与SW620/pLVTHM-NC组相比,SW620/pLVTHM-pre-miR-339细胞中PRL-1的mRNA及蛋白表达水平均下降。与miR inhibitor control组相比,转染miR-339-5p inhibitor的HCT116细胞中,PRL-1的mRNA (t=-9.036, P=0.012)及蛋白表达水平均上升;与miR inhibitor control组相比,转染miR-339-3p inhibitor的HCT116细胞中,PRL-1的：mRNA (t=-1.501, P=0.272)及蛋白表达水平均未见改变。
(4)构建psiCHECK-2/PRL-13'UTR载体及其突变载体。psiCHECK-2/WT-3'UTR和miR-339-5p mimics、miR mimics control共转染后,293FT细胞和HCT116细胞荧光素酶活性分别降低,经统计分析,差异均有统计学意义(P<0.001,P=0.002)。突变质粒psiCHECK-2/MUT-3'UTR与miR-339-5p mimics、miR mimics control共转染均不能改变荧光素酶活性(P=0.069,P=0.453),表明miR-339-5p与PRL-13'UTR可发生特异性结合。psiCHECK-2/WT-3'UTR和miR-339-3p mimics、miR mimics control共转染后,293FT细胞和HCT116均不能改变荧光素酶活性(P=0.466、P=0.243)。突变质粒psiCHECK-2/MUT-3'UTR与miR-339-3p mimics、miR mimics control共转染也均不改变荧光素酶活性(P=0.273,P=0.224),表明miR-339-3p与PRL-13'UTR未发生结合。
(5) Western blot结果显示：与miR inhibitor control组相比,转染miR-339-5p inhibitor后的HCT116细胞中的ERK1/2表达水平未见改变,而ERK1/2磷酸化水平升高,相反,与pLVTHM-NC组相比,转染pLVTHM-pre-miR-339的SW620细胞中的ERKl/2表达水平未见改变,而ERK1/2磷酸化水平降低。说明miR-339-5p能抑制p-ERK1/2的活性。
结论
1.MiR-339-5p通过靶基因PRL-1抑制结直肠癌细胞的增殖、侵袭和转移能力。
2. MiR-339-3p抑制结直肠癌细胞的增殖、侵袭和转移能力,其靶基因的鉴定将在后续实验中展开
3.初步验证MiR-339-5p影响ERK信号通路的活化,从而抑制结直肠癌细胞的增殖、侵袭和转移过程。
Backgroud and Objective
Colorectal cancer is one of the most prevalent carcinomas throughout the world. Every year, more than1million individuals will develop colorectal cancer, and the disease-specific mortality is nearly33%in the developed world. In our country, with the improvement of living standards and dietary changes, colorectal cancer has been increasing gradually year by year. With the change of dietary habits and structure, the morbidity and mortality were significantly increased. And younger, familial aggregation, genetic susceptibility are the characteristics of the disease in our country.
Colorectal cancer, like other tumor types, is a disease full of complex gene expression abnormalities, including abnormal and expression of coding and noncoding gene structure. Then the the molecular biological mechanism which controls node specific gene expression and gene expression in the process of development of the disease is need to study.
In recent years, non-coding RNA (non.coding RNAs), especially the microRNA (microRNA, miRNA) can regulate gene expression in the world, which offers a new perspective to solve this problem. MicroRNA (miRNA) is a class of small molecule RNA size of about19-22nucleotides. The processing of miRNA ripening process is divided into two steps:pri-miRNA Drosha enzyme processing into the precursor in the nucleus is called pre-miRNA. Pre-miRNA translocation to the cytosol in Dicer is under the action of the enzyme, the formation of two mature sequence by editing processing, which are called miRNA/miRNA*. And now the abundance of miRNAs does not be decided through experiments, are named miR-5p (5'arm) and miR-3p (3' arm). More recent literatures show that:5p and3p can be applied to the same or different target genes, play the same or different function.
Human pre-miR-339has two different sequences of the splicing:miR-339-5p and miR-339-3p. What is their expression in colon cancer? How do they control the biological cells process? There are no reports about it. To discuss these issues, we examined the expression level of miR-339-5p in human colon cancer cells and cancer tissues using qRT-PCR, and tested its effects on cell growth, cell-cycle distribution, and colony formation and invasion capacity in vitro using RNA interference technology and construction of miRNAs expression by lentiviral vector. We administered miR-339-5p precursor to a mouse colon cancer tumor xenograft model and further demonstrated that it could suppress colon tumor growth in vivo. We determined the target gene of miR-339-5p according to the prediction of biological information, real-time quantitative PCR, protein electrophoresis, luciferase reporter experiment. The solution of questions would indicate the function of miR-339-5p/3p in colorectal cancer growth and metastasis of colorectal cancer. These results indicate that miR-339-5p/3p can function as a tumor suppressor, regulating the maintenance and progression of cancers, providing the clue and thinking for exploring new therapeutic targets.
Methods
1. Mature miR-339-5p/3p expression in tissues and cells was detected using Real-time RT-PCR
Mature miR-339-5p/3p expression in tissues and cells was detected using Real-time RT-PCR. The specimens include samples of colorectal cancer tissue and matched normal colonic mucosa of30patients. While colorectal cancer six human colonic carcinoma cell lines include HCT116, HT29, LS174T, SW480, SW620and LOVO.
2. Effect of miR-339-5p/3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined
(1) Using the cationic liposome method, the miR-339-5p/3p mimics was transiently transfected into colorectal cancer cell line SW620. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle and transwell migration, invasion assay.
(2) Using the cationic liposome method, the miR-339-5p/3p inhibitor was transiently transfected into colorectal cancer cell line HCT116. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle and transwell migration, invasion assay.
(3) A lentiviral expression vector pLVTHM-pre-miR-339was designed^and constructed. SW620cells were transduced with vector supernatant and subsequently FACS-sorted for green fluorescent protein (GFP). Confirmation of stable transfection of the plasmids was obtained using the miR qRT-PCR assay. The colorectal cancer cell line was called SW620-p1VTHM/pre-miR-339. The cell proliferation, invasion and migration ability was evaluated using CCK8method, cell cycle, clone formation assay and transwell migration, invasion assay. The effect of tumorigenicity in nude mice subcutaneous was observed using the whole visualization animal model.
3. miR-339-5p/3p target genes were predicted, related pathways were verified.
(1) Three commonly used databases including TargetScan, Pictar and microRNA.org were used to forecast the target genes with the search terms of miR-339-5p/3p.
(2) PRL-1, the target gene of miR-339-5p, mRNA was detected in different colorectal cancer cell lines by qRT-PCR, and the correlation was analysis. The expression of PRL-1mRNA and protein was detected in SW620overexpressing miR-339-5p/3p and in HCT116transfected with miR-339-5p/3p inhibitor.
(3) The sequence containing the PRL-1gene3'UTR seed region fragment3'UTR seed region was subcloned into psiCHECK-2vector, and the recombinant vector psiCHECK-2-wt-PRL-1an psiCHECK-2-mut-PRL-1vector site-directed mutations were constructed. Combination of PRL-1and miR-339-5p/3p were detected by luciferase reporter system in order to determine whether miR-339-5p/3p targeted PRL-1gene directly.
(4) The relational colorectal cancer signal pathway was detected.
ERK1/2, p-ERK1/2protein expression was detected in SW620overexpressing by miR-339-5p/3p and in HCT116transfected with miR-339-5p/3p inhibitor using Western Blot.
4. Statistical analysis
SPSS13.0sorftware was used for statistical analysis. Data were presented as Mean±SEM of at least3independent experiments. Shapiro-Wilk test was used to verify the clinical samples'distribution. Differences were analyzed using the nonparametric test Mann-Whitney-Wilcoxon. Relative quantification value(2-ΔΔCt) of QPCR in cells were analyzed through One-way ANOVA, with the SNK, LSD or Dunnett's T3tests for multiple comparisons. It also analyzed through two-tailed Student's t test. Colony formation assay and transwell in vitro invasion assay were analyzed through two-tailed Student's t test. And it also was used for comparisons of relative luciferase activities experiments. The results of CCK8assay was analyzed by Factorial design analysis of variance. P values of <0.05were considered statistically significant.
Result
1. miR-339-5p/3p expression was detected in colorectal cancer tissue and cells.
(1) The expression of miR-339-5p/3p were detected in colorectal cancer tissues.
In30fresh colorectal cancer paired tissues, fluorescence quantitative PCR test results show that the expression of miR-339-5p/3p in colorectal cancer tissues was significantly lower than that in normal mucosa (z=-5.671, P<0.001)(z=-5.597, P<0.001). The result also showed significant difference between miR-339-5p/3p node metastasis in colorectal cancer group with lymph and without lymph node metastasis in colorectal cancer group (t=2.063, P=0.041). Clinical and pathological analysis showed that miR-339-5p/3p in30cases of colorectal carcinoma tissues has no relationship with matched with age, sex, the degree of tumor differentiation (P=0.853, P=0.179, P=0.397).
(2) miR-339-5p/3p expression was detected in colorectal cancer cell lines with different metastatic potential.
qRT-PCR results showed that there was significant difference between the6kinds of cells expressing miR-339-5p/3p (F=66.554, P<0.001)(F-74.603, P<0.001). The Dunnett's T3multiple comparison indicated the expression of miR-339-5p in HCT116cells was higher than that of the other6cells (P=0.03, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001). The expression of miR-339-3p in HT-29cells was higher than that of the other6cells (P<0.001, P=0.112, P<0.001, P<0.001, P<0.001).
2. Effect of miR-339-5p/3p on colorectal cancer cells in vitro and in vivo biological characteristics was examined.
a. The biological behavior of of SW620cell lines was performed transfected with miR-339-5p/3p mimics.
(1) Compared with miR mimics control, increased miR-339-5p/3p expression levels were measured in SW620cells after transfected with miR-339-5p/3p mimics. The difference was statistically significant (t=-5.780, P=0.029)(t=-7.020, P=0.002) showed significant moderating effect.
(2) Compared with miR mimics control, decreased proliferation properties of SW620transfected with the miR-339-5p/3p mimics were also detected by CCK-8assay. The difference was statistically significant (F=60.735, P<0.001)(F=7.175, P<0.001) showed significant moderating effect.
(3) By analyzing the distribution of cell cycle by flow cytometry, compared with miR mimics control, miR-339-5p overexpression in SW620cells after S cells were decreased (t=8.516, P<0.001), and the corresponding G1phase increased (t=-13.178, P<0.001). miR-339-3p overexpression has no influence on SW620cells, S cells were not changed (t=-5.220, P=0.629), and the corresponding G1phase was not changed (t=0.508, P=0.6)
(4) The number of migratory SW620cells transfected with the miR-339-5p mimics was fewer than miR mimics control, the difference was statistically significant (t=14.219, P<0.001). The number of migratory SW620cells transfected with the miR-339-3p mimics was fewer than miR mimics control, the difference was statistically significant (t=10.862, P<0.001).
(5) The number of invasive SW620cells transfected with the miR-339-5p mimics was fewer than miR mimics control, the difference was statistically significant (t=8.817, P<0.001). The number of invasive SW620cells transfected with the miR-339-3p mimics was fewer than miR mimics control, the difference was statistically significant (t=9.014, P<0.001).
b. The biological behavior of of HCT116cell lines was performed transfected with miR-339-5p/3p inhibitor.
(1) Compared with miR inhibitor control, decreased miR-339-5p/3p expression level was measured in HCT116cells after transfected with miR-339-5p/3p inhibitor. The difference was statistically significant showed significant moderating effect (t=-36.847, P=0.001)(t=-6.924, P=0.02)
(2) Compared with miR inhibitor control, increased proliferation properties of HCT116transfected with the miR-339-5p/3p inhibitor were also detected by CCK-8assay. The difference was statistically significant showed significant moderating effect (F=33.604, P<0.001)(F=18.204, P<0.001)
(3) By analyzing the distribution of cell cycle by flow cytometry, compared with miR inhibitor control, inhibition of miR-339-5p expression in HCT116cells after S cells were increased (t=-8.335, P=0.001), and the corresponding G1phase decreased (t=7.393, P=0.002). Inhibition of miR-339-3p expression in HCT116cells, S cells were not changed (t=-0.518, P=0.632), and the corresponding G1phase was not changed (t=-13.917, P<0.001).
(4) The number of migratory HCT116cells transfected with the miR-339-5p inhibitor was more than miR inhibitor control, the difference was statistically significant (t=-17.882, P<0.001). The number of migratory HCT116cells transfected with the miR-339-3p inhibitor were more than miR inhibitor control, the difference was statistically significant (t=-13.917, P<0.001).
(5) The number of invasive HCT116cells transfected with the miR-339-5p inhibitor were more than miR inhibitor control, the difference was statistically significant (t=-7.178, P<0.001). The number of migratory HCT116cells transfected with the miR-339-3p inhibitor was more than miR inhibitor control, the difference was statistically significant (t=-8.243, P<0.001).
c. The biological behavior of of SW620lines was performed transfected with pLVTHM/pre-miR-339.
(1) The pLVTHM/pre-miR-339lentiviral expression vector was constructed.
(2) The established stable cell line SW620expressing of miR-339-5p and miR-339-3p was named SW620/pLVTHM-pre-miR-339.
(3) The expression of miR-339-5p and miR-339-3p in SW620/pLVTHM-pre-miR-339group was higher than that of SW620/group pLVTHM-NC (P=0.046, P=0.046). Compared with SW620/pLVTHM-NC, increased miR-339-5p/3p expression levels were measured in SW620/pLVTHM-pre-miR-339.The difference was statistically significant (t=-7.621, P=0.02)(t=-5.638, P=0.03) showed significant moderating effect.
(4) Compared with SW620/pLVTHM-NC, decreased proliferation properties of SW620transfected with the SW620/pLVTHM-pre-miR-339were also detected by CCK-8assay (F=12.189, P=0.001). The difference was statistically significant showed significant moderating effect.
(5) By analyzing the distribution of cell cycle by flow cytometry, compared with/pLVTHM-NC, S cells in SW620/pLVTHM-pre-miR-339were decreased (t=5.056, P=0.007), and the corresponding G1phase increased (t=-3.669, P=0.021).
(6) Colony formation assay showed that the ability of SW620cells to form colony were decreased after pLVTHM-pre-miR-339(t=11.841, P<0.001).
(7) The number of migratory SW620cells transfected with pLVTHM-pre-miR-339were fewer than with the control, the difference was statistically significant (t=11.841, P<0.001).
(8) The number of invasive SW620cells transfected with pLVTHM-pre-miR-339were fewer than with the control, the difference was statistically significant (t=7.606, P<0.001).
(9) SW620/pLVTHM-pre-miR-339cells or SW620/pLVTHM cells were injected subcutaneously to the blank of nude mice, respectively, into nude mice. The tumour growth rate of SW620/pLVTHM-pre-miR-339cells was slower than that of SW620/pLVTHM cells the difference was statistically significant (F=5.024, P=0.008).
4. Bioinformatics analysis of target gene of miR-339-5p and miR-339-3p function, detection of miR-339-5p target gene identification and the downstream signal molecules
(1) The bioinformatics prediction of target genes for miR-339-5p/3p was perdicted by software TargetScan, PicTar, MICRORNA.ORG. Considering the development and metastasis related genes of tumor, preliminary candidate PRL-1was studied as a target gene miR-339-5p. The miR-339-3p target gene was predicted, the final results will be performed in subsequent experiments certification.
(2) qRT-PCR detection in6kinds colon cell lines indicated the highest expression level in LOVO, and lowest expression level in HCT116(F=7.432, P=0.002). In6colorectal cancer cell lines, expression of miR-339-5p and PRL-1in the opposite trend.
(3) Compared with the miR mimics control, in SW620cells transfected with miR-339-5p mimics, mRNA and protein expression level of PRL-1decreased significantly (t=26.858,/P=0.001). However, compared with miR mimics control, in SW620cells transfected with miR-339-3p mimics, mRNA and protein expression level of PRL-1had no change (t=0.452, P=0.675). Also in the stable transfection experiments, compared with the SW620/group pLVTHM-NC, mRNA and PRL-1protein expression in SW620/pLVTHM-pre-miR-339cells was decreased. QRT-PCR and Western blot results showed that compared with miR inhibitor control, transfection of miR-339-5p inhibitor in HCT116cells, mRNA and protein expression level of PRL-1was increased (t=-9.036, P=0.012). Compared with miR inhibitor control, transfection of miR-339-3p inhibitor in HCT116cells, mRNA and protein expression level of PRL-1had no change (t=-1.501, P=0.272).
(4) After constructing psiCHECK-2/PRL-13'UTR vector and its mutation carriers. PsiCHECK-2/WT-3'UTR and miR-339-5p mimics, miR mimics control after transfection,293FT cells and HCT116cells luciferase activity was decreased respectively; by the statistical analysis, the differences were statistically significant (P<0.001, P=0.002). PsiCHECK-2/MUT-3'UTR and miR-339-5p mutant plasmid mimics, miR mimics control cotransfected luciferase activity were not changed (P=0.069, P=0.453), showed that miR-339-5p and PRL-13'UTR can specifically bind. PsiCHECK-2/WT-3'UTR and miR-339-3p mimics/miR mimics control after transfection,293FT cells and HCT116cells can not be changed in luciferase activity (P=0.466, P=0.243). The mutation plasmid psiCHECK-2/MUT-3'UTR and miR-339-3p mimics/miR mimics control were transfected into cells also did not change the luciferase activity (P=0.379, P=0.953), showed that miR-339-3p did not act on PRL-13'UTR.
(5) The Western blot results showed that compared with pLVTHM-NC/SW620, in SW620cells transfected with pLVTHM-pre-miR-339, ERK1/2expression levels did not change, and the decreased expression of p-ERK1/2was detected. Compared with miR inhibitor control, transfection of miR-339-5p inhibitor in HCT116cells following ERK1/2expression levels did not change, and the expression of p-ERKl/2increased. MiR-339-5p may inhibit the activity of p-ERK1/2.
Conclusion
1MiR-339-5p regulates the growth, colony formation and metastasis of colorectal cancer cells by targeting PRL-1
2MiR-339-3p inhibits colorectal cancer cell proliferation, invasion and metastasis ability, the identification of target genes will be carried out in a follow-up experiment
3MiR-339-5p may affect the activation of ERK signaling pathway.
引文
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[5]Bandres E, Cubedo E, Agirre X, et al. Identification by Real—time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues [J]. Mol Cancer.2006,5:29.
[6]Guo J, Miao Y, Xiao B, et al.Differential expression of microRNA species in human gastric cancer versus nontumorous tissues [J]. J Gastroenterol Hepatol. 2009,24:652-657.
[7]Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human Cancers [J]. Nature.2005,435:834-838.
[8]Rosenfeld N, Aharonov R, Meiri E, et al. MicroRNAs accurately identify cancer tissue origin [J]. Nat Biotechnol.2008,26:462-469.
[9]Sun Y, Koo S, White N, et al. Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs [J]. Nucleic Acids Res.2004,32(22):e188.
[10]Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR [J]. Nucleic Acids Res.2005,33(20):e179.
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