microRNA-133b在结直肠癌中的表达及功能研究
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摘要
结直肠癌是消化道最常见的恶性肿瘤之一,其发病率和死亡率在全球范围内呈逐年上升的趋势。结直肠癌的发生发展过程涉及基因及蛋白质层面众多肿瘤相关因子的异常表达及功能改变,对这些异质性的分子事件进行深入研究是发现新的肿瘤治疗手段及治疗靶点的重要方法。最近的研究结果证实,microRNA的异常表达在结直肠癌等众多恶性肿瘤的发生发展过程中扮演重要的角色,microRNA是一类内源性的非编码RNA,由22个左右核苷酸组成,通过与靶mRNA的3'UTR碱基互补配对,抑制mRNA翻译或直接使其降解,使靶基因的表达在转录后水平受到抑制,从而调控基因表达。尽管目前对microRNA生物学功能的了解仍然十分有限,但是可以肯定的是,microRNA在生物体的发育、生长、分化、凋亡及肿瘤形成等众多方面具有非常重要的调节作用,特别是在一些恶性肿瘤中,microRNA通过调控不同的靶基因而起到类似于原癌基因或抑癌基因的作用。
miR-133b最初被认为是一种心肌特异性的microRNA,可靶向调节一些关键性的心肌发育及肌信号通路相关基因。近年来,应用基因芯片对结直肠癌的microRNA表达谱进行分析后发现,miR-133b在结直肠癌中的表达水平发生了显著的下调,并与结直肠癌的分化程度等一些病理指标密切相关。但是,miR-133b在结直肠癌中表达改变的实际意义及其生物学功能目前仍不明确。
本文拟展开miR-133b在结直肠癌中的实验研究,由“miR-133b在结直肠癌中的表达水平发生了显著下调”这一现象,我们提出miR-133b在结直肠癌中是否作为一种肿瘤抑制因子而存在?是否是一种潜在的肿瘤治疗靶点?由此,我们设计实验研究如下：
1.研究miR-133b在不同恶性程度的人结直肠癌细胞系及正常结肠细胞中的表达特点,探讨其表达水平与肿瘤细胞分化水平及转移能力的关系。
2.构建miR-133b的真核表达载体,并稳定转染低表达miR-133b的人结直肠癌细胞,筛选并建立高表达miR-133b的结直肠癌细胞系。
3.研究高表达miR-133b对人结直肠癌细胞的增殖、凋亡、迁移及侵袭能力的影响,并对miR-133b可能的靶基因进行初步预测。
4.建立结直肠癌裸鼠皮下种植瘤模型,研究miR-133b对荷瘤裸鼠种植瘤的生长抑制作用,动物体内实验进一步证实miR-133b对结直肠癌生物学行为的影响。
目的
研究miR-133b在不同恶性程度的结直肠癌细胞及正常结肠细胞中的表达特点。
方法
抽提细胞总RNA,采用实时定量PCR (Real time quantitative PCR)检测miR-133b在人正常结肠细胞系(CCD-18Co)及四种不同恶性程度的结直肠细胞系(SW-480、HT-29、LoVo、SW-620)中的表达,并分析其表达特点。
结果
正常结肠细胞系CCD-18Co中miR-133b的表达显著高于四种结直肠癌细胞(SW-480、SW-620、HT-29、LoVo) (P＜0.05),且随着癌细胞恶性程度的增加,miR-133b的表达渐次降低(SW-480＞HT-29＞LoVo＞SW-620)。
结论
1.miR-133b在结直肠癌细胞中的表达显著降低,并且随着细胞恶性程度的增高而降低。
2.SW-620细胞中miR-133b表达量最低,选择SW-620细胞进行后继实验。
目的
构建miR-133b真核表达载体并稳定转染人结直肠癌细胞系SW-620。
方法
1.根据人miR-133b前体序列设计并合成双链核苷酸Pre-miR-133b,构建重组质粒pGenesi1-1-miR-133b,并对质粒测序。
2.将质粒pGenesi1-1-miR-133b及对照质粒pGenesi1-1-HK转染人结直肠癌细胞SW-620,在浓度为800μg／mL的G418培养液中筛选出稳定高表达miR-133b及对照序列的细胞系SW-620-133b和SW-620-HK。
3.实时定量PCR检测SW-620、SW-620-HK及SW-620—133b中miR-133b的表达,证明稳定转染成功。
结果
1.构建miR-133b真核表达载体pGenesi1-1-miR-133b,测序结果正确。
2.将pGenesi1-1-miR-133b及对照质粒pGenesi1-1-HK转染SW-620细胞后,经浓度为800μg／mL的G418-RPMI-1640培养基筛选4周后得到稳定表达miR-133b的细胞系SW-620-133b及稳定表达对照序列的细胞系SW-620-HK。
3.实时定量PCR检测miR-133b在各组细胞中的表达,SW-620及SW-620-HK中未检测出miR-133b,SW-620-133b中miR-133b的表达显著升高,是LoVo细胞中miR-133b表达的39.4倍(P＜0.05)。
结论
成功构建miR-133b的真核表达载体,转染人结直肠癌细胞SW-620并筛选得到稳定高表达miR-133b及对照序列的细胞系SW-620-133b和SW-620-HK。
目的
研究高表达miR-133b对结直肠癌细胞SW-620增殖、凋亡、迁移及侵袭等生物行为的影响,并对miR-133b能的作用靶点进行预测。
方法
1.实验分为三组：SW-620细胞组、SW-620-HK细胞组、SW-620-133b细胞组。
2.MTT实验绘制各组细胞增殖曲线,平板克隆形成实验比较各组细胞克隆形成能力,综合评价miR-133b对SW-620细胞增殖能力的影响。
3. Annexin V-PI流式细胞学检测各组细胞的细胞凋亡情况,评价miR-133b对SW-620细胞凋亡的影响。
4.细胞划痕实验检测各组细胞的细胞迁移能力,评价miR-133b对SW-620细胞的迁移能力的影响。
5. Transwell侵袭实验检测各组细胞侵袭能力,评价miR-133b对SW-620细胞侵袭能力的影响。
6.通过microRNA靶点预测网站预测miR-133b可能的作用靶点,并用RT-PCR及Western blot进行初步验证。
结果
1.MTT法绘制细胞生长曲线,SW-620组与SW-620-HK组间生长曲线差异无显著性(P>0.05)；SW-620-133b与SW-620及SW-620-HK组间增殖曲线有明显差异,SW-620—133b组细胞增殖显著低于SW-620组及SW-620-HK组(P＜0.05)。
2.平板克隆形成实验,SW-620组平均克隆形成数为396.6±43.1、SW-620-HK组平均克隆形成数为385.4±40.8,显著高于SW-620-133b组的平均克隆形成数106.9±13.6(P<0.05)；SW-620组与SW-620-HK组间差异无显著性(P＞0.05)。
3. Annexin V-PI流式细胞结果示,SW-620、SW-620-HK及SW-620-133b三组细胞平均凋亡率分别为：(4.26±0.65)%、(4.98±0.71)%及(16.75±2.35)%。SW-620-133b细胞组凋亡率高于对照组SW-620-HK及空白组SW-620,组间差异具有显著性(P＜0.05)；对照组SW-620-HK及空白组SW-620间的细胞凋亡无显著性差异(P＞0.05)。
4.细胞划痕实验：SW-620组平均向划痕区迁移率为(77.9±5.6)%,SW-620-HK组平均迁移率为(74.6±5.2)%；两组间差异无显著性(P70.05)。而高表达miR-133b的SW-620-133b组细胞平均向划痕区迁移率为(42.1±3.7)%,显著低于SW-620组和SW-620-HK组(P＜0.05)。
5. Tanswell侵袭实验中各组侵袭细胞数为：SW-620组, (420±31.5)／HP；SW-620-HK组,(396士30.2)／HP；SW-620-133b组,(108士12.5)／HP.SW-620-133b组侵袭细胞数显著低于SW-620组及SW-620-HK组(P<0.05),SW-620组及SW-620-HK组间侵袭细胞数无显著差异(P>0.05)。
6.经microRNA靶点预测软件预测,MET和MMP9可能是miR-133b的靶基因；Western blot结果示：MET蛋白在SW-620-133b组中的表达较SW-620及SW-620-HK中分别下降了(62.5±4.36)%及(59.8±5.21)%(P<0.05)；MMP9蛋白在SW-620-133b组中的表达较SW-620及SW-620-HK中分别下降了(35.6±3.72)%及(37.1±4.23)%(P<0.05)；MET和MMP9 mRNA表达水平在三组细胞中无明显改变。
结论
1.体外细胞实验中,外源性高表达miR-133b可有效抑制结直肠癌细胞SW-620增殖,促进细胞凋亡；并对细胞迁移及侵袭能力有显著的抑制作用。
2. MET及MMP9可能是miR-133b作用的靶基因,miR-133b可能在转录后的翻译水平抑制MET和MMP9的表达。
目的
探讨miR-133b对人结直肠癌裸鼠皮下种植瘤生长的抑制作用,体内实验进一步验证miR-133b是一种新的抑癌基因。
方法
1.实验分组：30只裸鼠随机分为3组。分别为接种SW-620细胞组、接种SW-620-HK细胞组、接种SW-620-133b细胞组。
2.建立人结直肠癌裸鼠皮下种植瘤模型,计算成瘤率。
3.致瘤成功后每5天测量裸鼠皮下种植瘤的体积,绘制肿瘤增长曲线。
4.30天后处死裸鼠,获取肿瘤,记录并比较各组瘤重。
5. TUNEL法检测各组种植瘤肿瘤细胞的凋亡情况。
6.免疫组化检测MET及MMP9在各组种植瘤中的表达水平。
结果
1.各组裸鼠皮下接种肿瘤细胞5天后均成功致瘤,成瘤率100%。
2.绘制各组种植瘤生长曲线,各组种植瘤持续生长,未出现致瘤裸鼠死亡,SW-620-133b组种植瘤生长速度显著低于SW-620与SW-620-HK组(P<0.05)；终点时间第30天时,SW-620组裸鼠皮下种植瘤的体积为(3.56 ±0.79)cm3,SW-620-HK组为(3.24±0.68)cm3,显著大于SW-620-133b组肿瘤体积(0.85±0.14)cm3(P<0.05)；SW-620-133b组种植瘤平均质量为(0.92±0.16)g,明显低于SW-620组(4.12±0.64)g及SW-620-HK组(3.95±0.61)g。
3. TUNEL法检测各组种植瘤肿瘤细胞的凋亡情况,高表达miR-133b的细胞SW-620-133b组AI值为(30.28±3.47)%,显著高于对照组SW-620-HK(7.26±0.84)%及空白组SW-620(7.96±0.92)%(P<0.05)；SW-620-HK组与SW-620组间AI值无显著性差异(P＞0.05)。
4.免疫组化结果证实MET和MMP9蛋白在SW-620-133b组种植瘤中的表达明显下调,与SW-620及SW-620-HK组有显著差别(P＜0.05),结果与体外细胞试验相符合。
结论
1.外源性高表达miR-133b可显著抑制人结直肠癌裸鼠皮下种植瘤的生长,并促进肿瘤细胞凋亡。
2.MET及MMP9在高表达miR-133b组种植瘤中表达下调,miR-133b可能通过MET及MMP9发挥抑瘤作用。
Colorectal cancer (CRC) is one of the leading causes of cancer related mortality worldwide. Analysis of the CRC genome and proteome has demonstrated that focusing on molecular heterogeneity within CRC may be a viable approach to identify and develop novel therapeutics. Recent data suggest that dysregulation of microRNAs is an important step in the development of many cancers, including CRC. MicroRNAs consist of-22 nucleotides and regulate gene expression in a posttranscriptional manner by pairing with complementary nucleotide sequences in 3'-UTRs of target mRNAs. Although at present the precise biological effects are not well understood, miRNAs appear to be crucial factors in diverse regulation pathways, including development, tumorigenesis, cell differentiation, proliferation, and apoptosis. Recent studies have shown that some miRNAs play roles as tumor suppressors or oncogenes in cancers, depending on whether they specifically target tumor suppressor genes or oncogenes, respectively.
miR-133b was initially considered to be a muscle-specific miRNA, Through targeting critical genes involved in cardiac development and cardiac channel expression, miR-133b is implicated in the regulation of cardiac myogenesis and development and cardiac ion channel expression. Recently, investigators determined that miR-133b harbored anti-tumorigenic properties in tongue squamous cell carcinoma through direct targeting of pyruvate kinase type M2. Studies have demonstrated that miR-133b was significantly downregulated in several CRC cell lines, as well as in colorectal tumors, compared with nonneoplastic tissues. However, the precise role of miR-133b in CRC remains largely unknown.
This article intended to study "The relationship between miR-133b and colorectal cancer". Inspired by the phenomenon that "miR-133b was significantly downregulated in CRC tissues compared with nonneoplastic tissues", we assumed that whether miR-133b as a tumor suppressor or potential cancer therapeutic target in colorectal cancer? Accordingly, the design of this experiment was as follows:
1. To investigate the expression levels of miR-133b in normal human colon cells (CCD-18Co) and four colorectal cancer cell lines with different malignant potential, and exploring the relationships between the expression level of miR-133b and tumor differentiation and metastasis.
2. miR-133b genomic sequence would be constructed into the eukaryotic expression vector. CRC cells with low miR-133b expression level were stable tansfected with the identified plasmid by Effectene, and then, the cell line with stable expression of miR-133b would be obtained.
3. To explore the effects of miR-133b on proliferation, apoptosis, migration and invasion activities in colorectal cancer cells, and then the targets genes of miR-133b would be predicted by a web-based computer program.
4. Establishing the subcutaneous tumor model of colorectal cancer in nude mice. Research on the therapeutic effects of miR-133b on bearing-tumor node mice of human coloretal cancer, which can further confirm the role of miR-133b on biological behaviors of colorectal cancer in vivo.
Objective
To explore the expression pattern of miR-133b in colorectal carcinoma cell lines with different malignant potential.
Methods
The expression of miR-133b in normal human colon cells (CCD-18Co) and four colorectal cancer cell lines (SW-480、HT-29、LoVo、SW-620) with different malignant potential were examined by real-time PCR.
Results
The miR-133b expression level in CCD-18Co was much higher than in the four cancer cells (SW-480, HT-29, LoVo, and SW-620), with the lowest showing a 4-fold increase in expression (p<0.05). This downregulation of miR-133b is associated with malignant potential of colorectal cancer cell.
Conclusion
1. miR-133b expression shown to be greatly downregulated in human colorectal cancer cells compared to normal colon cells and this downregulation is associated with malignant potential of colorectal cancer.
2. The expression of miR-133b was lowest in SW-620 cells, and this cell line was selected for further experiment.
Objective
To construct eukaryotic expression vector of miR-133b and stable tansfection into human colorectal cancer cell SW-620.
Methods
1. According to the sequence of pre-miR-133b, design and synthesis the double-stranded nucleotide sequence of Pre-miR-133b. To construct the recombinant plasmid pGenesi 1-1-miR-133b, then get plasmid sequencing.
2. SW-620 cells were transfected with pGenesil-1-HK or pGenesil-l-miR-133b plasmid, stable transfectants were then selected by incubation with geneticin G418 and maintained in medium containing 800μg/mL G418, SW-620 cell lines with stable expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.
3. The expression of miR-133b in SW-620、SW-620-HK and SW-620-133b were detected by real-time quantitative PCR.
Results
1. Construction of miR-133b eukaryotic expression vector pGenesil-l-miR-133b, sequencing result is correct.
2. After a 4-week selection period in medium supplemented with G418 (800μg/mL), SW-620 cell lines with stable expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.
3. The expression of miR-133b was dramatically greater in SW-620-133b compared with SW-620-HK and SW-620. Since miR-133b was not detected in either SW-620 or SW-620-HK, LoVo cells were selected as a standard, in which case a 39.4-fold greater expression was observed in SW-620-133b compared with this standard (p<0.05).
Conclusion
The eukaryotic expression vector of miR-133b was successfully constructed. SW-620 cell lines with high expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.
Objective
Research on the effects of high expression miR-133b on the cell proliferation, apoptosis, migration and invasion ability in SW-620 cells and predict the possible targets of miR-133b.
Methods
1. The experiments were consists of three groups:SW-620 cell group, SW-620-HK cell group and SW-620-133b cell group.
2. MTT assay and plat clone formation assay were employed to measure cell proliferation of SW-620, SW-620-HK and SW-620-133b cells.
3. We assessed for the induction of apoptosis in each group through the measurement of flow cytometry for Annexin/PI.
4. The effect of miR-133b on migration of SW-620 cells was investigated using a wound-healing assay to evaluate cell migration.
5. Matrigel invasion assay in Transwell culture chambers to determine the effect of miR-133b on in vitro SW-620 invasion.
6. The possible targets of miR-133b were predicted by a web-based computer program and initially verified by RT-PCR and Western blot.
Results
1. MTT assay shown that markedly retarded cell proliferation rates were observed in SW-620-133b cells compared to SW-620 and SW-620-HK (p<0.05). Plat clone formation assay was performed to confirm this observation. The SW-620 and SW-620-HK cells grew and formed large colonies than SW-620-133b within 4 wks. The number of colonies in SW-620-133b (106.9±13.6) cells was significantly lower than SW-620 (396.6±43.1) and SW-620-HK (385.4±40.8) cells (p<0.05).
2. Fluorescence-activated cell sorting assay:the result showed that as comparing to the SW-620 (4.26±0.65)% and SW-620-HK (4.98±0.71)% group, the percentage of apoptosis cells in SW-620-133b (16.75±2.35)% was markedly increased (P＜0.05), there was no significant difference between SW-620 and SW-620-HK group.
3. The wound-healing assay:SW-620-133b (42.1±3.7)%cells exhibited a notable decrease in closure or migrating ability compared with SW-620-HK (74.6±5.2)% or SW-620 (77.9±5.6)% cells (p<0.05), suggesting a role for miR-133b in the migration of SW-620 cells.
4. Tanswell invasion assay:the average invasion cells of each group was as follows:SW-620 group, (420±31.5)/HP; SW-620-HK group, (396±30.2)/HP; SW-620-133b group, (108±12.5)/HP. The number of SW-620-133b cells that passed through Matrigel was much lower compared with SW-620 and SW-620-HK cells (p<0.05).
5. MET and MMP9 are listed as targets candidate for miR-133b interaction according to a prediction by the web-based microRNA targets prediction program. As confirmed by Western blot and RT-PCR, MET and MMP9 expression was significantly less in SW-620-133b cells compared with SW-620-HK or SW-620 cells (p<0.05); but MET and MMP9 mRNA concentrations were unaffected in these three groups.
Conclusion
1. In cancer SW-620 cells, ectopic expression of miR-133b potently inhibits tumor cell proliferation, migration and invasion, meanwhile promotes apoptosis in vitro.
2. MET and MMP9 are the candidate targets of miR-133b. miR-133b might have negatively regulated MET and MMP9 at the posttranscriptional level.
Objective
To explore the growth inhibition effects of miR-133b on planted subcutaneous tumors in human colorectal tumor-bearing nude mice. Further validation of miR-133b to be a new tumor suppressor in vivo.
Methods
1.30 nude mice were randomized into 3 groups and each group contained 10:SW-620 cell group, SW-620-HK cell group and SW-620-133b cell group.
2. To establish the animal model of human colorectal tumor-bearing mice and calculate the tumor-formation rate.
3. The volumes of planted subcutaneous tumors were measured per 5 days after tumorigenesis for mapping tumors growth curves.
4. Nude mice were sacrificed to obtain tumor 30 days after injection, recorded and compared the tumor weight in each group.
5. The cell apoptosis of planted tumor were detected by TUNEL assay.
6. The expression levels of MET and MMP9 in tumor were measured by immunohistochemisty.
Results
1. The model animals of human colorectal tumor-bearing mice were successfully established, the tumor-formation rate was 100%.
2. The growth curves of planted tumor were drawed, as compared to SW-620 and SW-620-HK group, the tumor growth rate of SW-620-133b group was much slower (p<0.05). At the end point time, the average tumor volume of SW-620-133b group (0.85±0.14)cm3 was lower than SW-620 (3.56±0.79)cm3 and SW-620-HK group (3.24±0.68)cm3 (p<0.05); the average tumor weight of SW-620-133b group (0.92±0.16)g was lower than SW-620 (4.12±0.64)g and SW-620-HK group (3.95±0.61)g (p<0.05);
3. As shown by TUNEL assay, the AI value of SW-620-133b group (30.28±3.47)%was significantly higher than that of SW-620 (7.96±0.92)%and SW-620-HK group (7.26±0.84) (p<0.05), but with no significant difference between SW-620 and SW-620-HK group.
4. The results of immunohistochemisty showed that the expression of MET and MMP9 protein was significantly down-regulated in SW-620-133b group compared with SW-620 and SW-620-HK group (p<0.05).
Conclusion
1. The ectopic expression of miR-133b can effectively inhibite the growth of tumor and promote apoptosis in human colorectal tumor-bearing nude mice.
2. The expression of MET and MMP9 was significantly down regulated in SW-620-133b group, miR-133b might suppress tumor growth through modulation of the MET and MMP9 signaling pathway.
miR-133b最初被认为是一种心肌特异性的microRNA,可靶向调节一些关键性的心肌发育及肌信号通路相关基因。近年来,应用基因芯片对结直肠癌的microRNA表达谱进行分析后发现,miR-133b在结直肠癌中的表达水平发生了显著的下调,并与结直肠癌的分化程度等一些病理指标密切相关。但是,miR-133b在结直肠癌中表达改变的实际意义及其生物学功能目前仍不明确。
本文拟展开miR-133b在结直肠癌中的实验研究,由“miR-133b在结直肠癌中的表达水平发生了显著下调”这一现象,我们提出miR-133b在结直肠癌中是否作为一种肿瘤抑制因子而存在?是否是一种潜在的肿瘤治疗靶点?由此,我们设计实验研究如下：
1.研究miR-133b在不同恶性程度的人结直肠癌细胞系及正常结肠细胞中的表达特点,探讨其表达水平与肿瘤细胞分化水平及转移能力的关系。
2.构建miR-133b的真核表达载体,并稳定转染低表达miR-133b的人结直肠癌细胞,筛选并建立高表达miR-133b的结直肠癌细胞系。
3.研究高表达miR-133b对人结直肠癌细胞的增殖、凋亡、迁移及侵袭能力的影响,并对miR-133b可能的靶基因进行初步预测。
4.建立结直肠癌裸鼠皮下种植瘤模型,研究miR-133b对荷瘤裸鼠种植瘤的生长抑制作用,动物体内实验进一步证实miR-133b对结直肠癌生物学行为的影响。
目的
研究miR-133b在不同恶性程度的结直肠癌细胞及正常结肠细胞中的表达特点。
方法
抽提细胞总RNA,采用实时定量PCR (Real time quantitative PCR)检测miR-133b在人正常结肠细胞系(CCD-18Co)及四种不同恶性程度的结直肠细胞系(SW-480、HT-29、LoVo、SW-620)中的表达,并分析其表达特点。
结果
正常结肠细胞系CCD-18Co中miR-133b的表达显著高于四种结直肠癌细胞(SW-480、SW-620、HT-29、LoVo) (P＜0.05),且随着癌细胞恶性程度的增加,miR-133b的表达渐次降低(SW-480＞HT-29＞LoVo＞SW-620)。
结论
1.miR-133b在结直肠癌细胞中的表达显著降低,并且随着细胞恶性程度的增高而降低。
2.SW-620细胞中miR-133b表达量最低,选择SW-620细胞进行后继实验。
目的
构建miR-133b真核表达载体并稳定转染人结直肠癌细胞系SW-620。
方法
1.根据人miR-133b前体序列设计并合成双链核苷酸Pre-miR-133b,构建重组质粒pGenesi1-1-miR-133b,并对质粒测序。
2.将质粒pGenesi1-1-miR-133b及对照质粒pGenesi1-1-HK转染人结直肠癌细胞SW-620,在浓度为800μg／mL的G418培养液中筛选出稳定高表达miR-133b及对照序列的细胞系SW-620-133b和SW-620-HK。
3.实时定量PCR检测SW-620、SW-620-HK及SW-620—133b中miR-133b的表达,证明稳定转染成功。
结果
1.构建miR-133b真核表达载体pGenesi1-1-miR-133b,测序结果正确。
2.将pGenesi1-1-miR-133b及对照质粒pGenesi1-1-HK转染SW-620细胞后,经浓度为800μg／mL的G418-RPMI-1640培养基筛选4周后得到稳定表达miR-133b的细胞系SW-620-133b及稳定表达对照序列的细胞系SW-620-HK。
3.实时定量PCR检测miR-133b在各组细胞中的表达,SW-620及SW-620-HK中未检测出miR-133b,SW-620-133b中miR-133b的表达显著升高,是LoVo细胞中miR-133b表达的39.4倍(P＜0.05)。
结论
成功构建miR-133b的真核表达载体,转染人结直肠癌细胞SW-620并筛选得到稳定高表达miR-133b及对照序列的细胞系SW-620-133b和SW-620-HK。
目的
研究高表达miR-133b对结直肠癌细胞SW-620增殖、凋亡、迁移及侵袭等生物行为的影响,并对miR-133b能的作用靶点进行预测。
方法
1.实验分为三组：SW-620细胞组、SW-620-HK细胞组、SW-620-133b细胞组。
2.MTT实验绘制各组细胞增殖曲线,平板克隆形成实验比较各组细胞克隆形成能力,综合评价miR-133b对SW-620细胞增殖能力的影响。
3. Annexin V-PI流式细胞学检测各组细胞的细胞凋亡情况,评价miR-133b对SW-620细胞凋亡的影响。
4.细胞划痕实验检测各组细胞的细胞迁移能力,评价miR-133b对SW-620细胞的迁移能力的影响。
5. Transwell侵袭实验检测各组细胞侵袭能力,评价miR-133b对SW-620细胞侵袭能力的影响。
6.通过microRNA靶点预测网站预测miR-133b可能的作用靶点,并用RT-PCR及Western blot进行初步验证。
结果
1.MTT法绘制细胞生长曲线,SW-620组与SW-620-HK组间生长曲线差异无显著性(P>0.05)；SW-620-133b与SW-620及SW-620-HK组间增殖曲线有明显差异,SW-620—133b组细胞增殖显著低于SW-620组及SW-620-HK组(P＜0.05)。
2.平板克隆形成实验,SW-620组平均克隆形成数为396.6±43.1、SW-620-HK组平均克隆形成数为385.4±40.8,显著高于SW-620-133b组的平均克隆形成数106.9±13.6(P<0.05)；SW-620组与SW-620-HK组间差异无显著性(P＞0.05)。
3. Annexin V-PI流式细胞结果示,SW-620、SW-620-HK及SW-620-133b三组细胞平均凋亡率分别为：(4.26±0.65)%、(4.98±0.71)%及(16.75±2.35)%。SW-620-133b细胞组凋亡率高于对照组SW-620-HK及空白组SW-620,组间差异具有显著性(P＜0.05)；对照组SW-620-HK及空白组SW-620间的细胞凋亡无显著性差异(P＞0.05)。
4.细胞划痕实验：SW-620组平均向划痕区迁移率为(77.9±5.6)%,SW-620-HK组平均迁移率为(74.6±5.2)%；两组间差异无显著性(P70.05)。而高表达miR-133b的SW-620-133b组细胞平均向划痕区迁移率为(42.1±3.7)%,显著低于SW-620组和SW-620-HK组(P＜0.05)。
5. Tanswell侵袭实验中各组侵袭细胞数为：SW-620组, (420±31.5)／HP；SW-620-HK组,(396士30.2)／HP；SW-620-133b组,(108士12.5)／HP.SW-620-133b组侵袭细胞数显著低于SW-620组及SW-620-HK组(P<0.05),SW-620组及SW-620-HK组间侵袭细胞数无显著差异(P>0.05)。
6.经microRNA靶点预测软件预测,MET和MMP9可能是miR-133b的靶基因；Western blot结果示：MET蛋白在SW-620-133b组中的表达较SW-620及SW-620-HK中分别下降了(62.5±4.36)%及(59.8±5.21)%(P<0.05)；MMP9蛋白在SW-620-133b组中的表达较SW-620及SW-620-HK中分别下降了(35.6±3.72)%及(37.1±4.23)%(P<0.05)；MET和MMP9 mRNA表达水平在三组细胞中无明显改变。
结论
1.体外细胞实验中,外源性高表达miR-133b可有效抑制结直肠癌细胞SW-620增殖,促进细胞凋亡；并对细胞迁移及侵袭能力有显著的抑制作用。
2. MET及MMP9可能是miR-133b作用的靶基因,miR-133b可能在转录后的翻译水平抑制MET和MMP9的表达。
目的
探讨miR-133b对人结直肠癌裸鼠皮下种植瘤生长的抑制作用,体内实验进一步验证miR-133b是一种新的抑癌基因。
方法
1.实验分组：30只裸鼠随机分为3组。分别为接种SW-620细胞组、接种SW-620-HK细胞组、接种SW-620-133b细胞组。
2.建立人结直肠癌裸鼠皮下种植瘤模型,计算成瘤率。
3.致瘤成功后每5天测量裸鼠皮下种植瘤的体积,绘制肿瘤增长曲线。
4.30天后处死裸鼠,获取肿瘤,记录并比较各组瘤重。
5. TUNEL法检测各组种植瘤肿瘤细胞的凋亡情况。
6.免疫组化检测MET及MMP9在各组种植瘤中的表达水平。
结果
1.各组裸鼠皮下接种肿瘤细胞5天后均成功致瘤,成瘤率100%。
2.绘制各组种植瘤生长曲线,各组种植瘤持续生长,未出现致瘤裸鼠死亡,SW-620-133b组种植瘤生长速度显著低于SW-620与SW-620-HK组(P<0.05)；终点时间第30天时,SW-620组裸鼠皮下种植瘤的体积为(3.56 ±0.79)cm3,SW-620-HK组为(3.24±0.68)cm3,显著大于SW-620-133b组肿瘤体积(0.85±0.14)cm3(P<0.05)；SW-620-133b组种植瘤平均质量为(0.92±0.16)g,明显低于SW-620组(4.12±0.64)g及SW-620-HK组(3.95±0.61)g。
3. TUNEL法检测各组种植瘤肿瘤细胞的凋亡情况,高表达miR-133b的细胞SW-620-133b组AI值为(30.28±3.47)%,显著高于对照组SW-620-HK(7.26±0.84)%及空白组SW-620(7.96±0.92)%(P<0.05)；SW-620-HK组与SW-620组间AI值无显著性差异(P＞0.05)。
4.免疫组化结果证实MET和MMP9蛋白在SW-620-133b组种植瘤中的表达明显下调,与SW-620及SW-620-HK组有显著差别(P＜0.05),结果与体外细胞试验相符合。
结论
1.外源性高表达miR-133b可显著抑制人结直肠癌裸鼠皮下种植瘤的生长,并促进肿瘤细胞凋亡。
2.MET及MMP9在高表达miR-133b组种植瘤中表达下调,miR-133b可能通过MET及MMP9发挥抑瘤作用。
Colorectal cancer (CRC) is one of the leading causes of cancer related mortality worldwide. Analysis of the CRC genome and proteome has demonstrated that focusing on molecular heterogeneity within CRC may be a viable approach to identify and develop novel therapeutics. Recent data suggest that dysregulation of microRNAs is an important step in the development of many cancers, including CRC. MicroRNAs consist of-22 nucleotides and regulate gene expression in a posttranscriptional manner by pairing with complementary nucleotide sequences in 3'-UTRs of target mRNAs. Although at present the precise biological effects are not well understood, miRNAs appear to be crucial factors in diverse regulation pathways, including development, tumorigenesis, cell differentiation, proliferation, and apoptosis. Recent studies have shown that some miRNAs play roles as tumor suppressors or oncogenes in cancers, depending on whether they specifically target tumor suppressor genes or oncogenes, respectively.
miR-133b was initially considered to be a muscle-specific miRNA, Through targeting critical genes involved in cardiac development and cardiac channel expression, miR-133b is implicated in the regulation of cardiac myogenesis and development and cardiac ion channel expression. Recently, investigators determined that miR-133b harbored anti-tumorigenic properties in tongue squamous cell carcinoma through direct targeting of pyruvate kinase type M2. Studies have demonstrated that miR-133b was significantly downregulated in several CRC cell lines, as well as in colorectal tumors, compared with nonneoplastic tissues. However, the precise role of miR-133b in CRC remains largely unknown.
This article intended to study "The relationship between miR-133b and colorectal cancer". Inspired by the phenomenon that "miR-133b was significantly downregulated in CRC tissues compared with nonneoplastic tissues", we assumed that whether miR-133b as a tumor suppressor or potential cancer therapeutic target in colorectal cancer? Accordingly, the design of this experiment was as follows:
1. To investigate the expression levels of miR-133b in normal human colon cells (CCD-18Co) and four colorectal cancer cell lines with different malignant potential, and exploring the relationships between the expression level of miR-133b and tumor differentiation and metastasis.
2. miR-133b genomic sequence would be constructed into the eukaryotic expression vector. CRC cells with low miR-133b expression level were stable tansfected with the identified plasmid by Effectene, and then, the cell line with stable expression of miR-133b would be obtained.
3. To explore the effects of miR-133b on proliferation, apoptosis, migration and invasion activities in colorectal cancer cells, and then the targets genes of miR-133b would be predicted by a web-based computer program.
4. Establishing the subcutaneous tumor model of colorectal cancer in nude mice. Research on the therapeutic effects of miR-133b on bearing-tumor node mice of human coloretal cancer, which can further confirm the role of miR-133b on biological behaviors of colorectal cancer in vivo.
Objective
To explore the expression pattern of miR-133b in colorectal carcinoma cell lines with different malignant potential.
Methods
The expression of miR-133b in normal human colon cells (CCD-18Co) and four colorectal cancer cell lines (SW-480、HT-29、LoVo、SW-620) with different malignant potential were examined by real-time PCR.
Results
The miR-133b expression level in CCD-18Co was much higher than in the four cancer cells (SW-480, HT-29, LoVo, and SW-620), with the lowest showing a 4-fold increase in expression (p<0.05). This downregulation of miR-133b is associated with malignant potential of colorectal cancer cell.
Conclusion
1. miR-133b expression shown to be greatly downregulated in human colorectal cancer cells compared to normal colon cells and this downregulation is associated with malignant potential of colorectal cancer.
2. The expression of miR-133b was lowest in SW-620 cells, and this cell line was selected for further experiment.
Objective
To construct eukaryotic expression vector of miR-133b and stable tansfection into human colorectal cancer cell SW-620.
Methods
1. According to the sequence of pre-miR-133b, design and synthesis the double-stranded nucleotide sequence of Pre-miR-133b. To construct the recombinant plasmid pGenesi 1-1-miR-133b, then get plasmid sequencing.
2. SW-620 cells were transfected with pGenesil-1-HK or pGenesil-l-miR-133b plasmid, stable transfectants were then selected by incubation with geneticin G418 and maintained in medium containing 800μg/mL G418, SW-620 cell lines with stable expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.
3. The expression of miR-133b in SW-620、SW-620-HK and SW-620-133b were detected by real-time quantitative PCR.
Results
1. Construction of miR-133b eukaryotic expression vector pGenesil-l-miR-133b, sequencing result is correct.
2. After a 4-week selection period in medium supplemented with G418 (800μg/mL), SW-620 cell lines with stable expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.
3. The expression of miR-133b was dramatically greater in SW-620-133b compared with SW-620-HK and SW-620. Since miR-133b was not detected in either SW-620 or SW-620-HK, LoVo cells were selected as a standard, in which case a 39.4-fold greater expression was observed in SW-620-133b compared with this standard (p<0.05).
Conclusion
The eukaryotic expression vector of miR-133b was successfully constructed. SW-620 cell lines with high expression of miR-133b or control were obtained, designated SW-620-133b and SW-620-HK, respectively.
Objective
Research on the effects of high expression miR-133b on the cell proliferation, apoptosis, migration and invasion ability in SW-620 cells and predict the possible targets of miR-133b.
Methods
1. The experiments were consists of three groups:SW-620 cell group, SW-620-HK cell group and SW-620-133b cell group.
2. MTT assay and plat clone formation assay were employed to measure cell proliferation of SW-620, SW-620-HK and SW-620-133b cells.
3. We assessed for the induction of apoptosis in each group through the measurement of flow cytometry for Annexin/PI.
4. The effect of miR-133b on migration of SW-620 cells was investigated using a wound-healing assay to evaluate cell migration.
5. Matrigel invasion assay in Transwell culture chambers to determine the effect of miR-133b on in vitro SW-620 invasion.
6. The possible targets of miR-133b were predicted by a web-based computer program and initially verified by RT-PCR and Western blot.
Results
1. MTT assay shown that markedly retarded cell proliferation rates were observed in SW-620-133b cells compared to SW-620 and SW-620-HK (p<0.05). Plat clone formation assay was performed to confirm this observation. The SW-620 and SW-620-HK cells grew and formed large colonies than SW-620-133b within 4 wks. The number of colonies in SW-620-133b (106.9±13.6) cells was significantly lower than SW-620 (396.6±43.1) and SW-620-HK (385.4±40.8) cells (p<0.05).
2. Fluorescence-activated cell sorting assay:the result showed that as comparing to the SW-620 (4.26±0.65)% and SW-620-HK (4.98±0.71)% group, the percentage of apoptosis cells in SW-620-133b (16.75±2.35)% was markedly increased (P＜0.05), there was no significant difference between SW-620 and SW-620-HK group.
3. The wound-healing assay:SW-620-133b (42.1±3.7)%cells exhibited a notable decrease in closure or migrating ability compared with SW-620-HK (74.6±5.2)% or SW-620 (77.9±5.6)% cells (p<0.05), suggesting a role for miR-133b in the migration of SW-620 cells.
4. Tanswell invasion assay:the average invasion cells of each group was as follows:SW-620 group, (420±31.5)/HP; SW-620-HK group, (396±30.2)/HP; SW-620-133b group, (108±12.5)/HP. The number of SW-620-133b cells that passed through Matrigel was much lower compared with SW-620 and SW-620-HK cells (p<0.05).
5. MET and MMP9 are listed as targets candidate for miR-133b interaction according to a prediction by the web-based microRNA targets prediction program. As confirmed by Western blot and RT-PCR, MET and MMP9 expression was significantly less in SW-620-133b cells compared with SW-620-HK or SW-620 cells (p<0.05); but MET and MMP9 mRNA concentrations were unaffected in these three groups.
Conclusion
1. In cancer SW-620 cells, ectopic expression of miR-133b potently inhibits tumor cell proliferation, migration and invasion, meanwhile promotes apoptosis in vitro.
2. MET and MMP9 are the candidate targets of miR-133b. miR-133b might have negatively regulated MET and MMP9 at the posttranscriptional level.
Objective
To explore the growth inhibition effects of miR-133b on planted subcutaneous tumors in human colorectal tumor-bearing nude mice. Further validation of miR-133b to be a new tumor suppressor in vivo.
Methods
1.30 nude mice were randomized into 3 groups and each group contained 10:SW-620 cell group, SW-620-HK cell group and SW-620-133b cell group.
2. To establish the animal model of human colorectal tumor-bearing mice and calculate the tumor-formation rate.
3. The volumes of planted subcutaneous tumors were measured per 5 days after tumorigenesis for mapping tumors growth curves.
4. Nude mice were sacrificed to obtain tumor 30 days after injection, recorded and compared the tumor weight in each group.
5. The cell apoptosis of planted tumor were detected by TUNEL assay.
6. The expression levels of MET and MMP9 in tumor were measured by immunohistochemisty.
Results
1. The model animals of human colorectal tumor-bearing mice were successfully established, the tumor-formation rate was 100%.
2. The growth curves of planted tumor were drawed, as compared to SW-620 and SW-620-HK group, the tumor growth rate of SW-620-133b group was much slower (p<0.05). At the end point time, the average tumor volume of SW-620-133b group (0.85±0.14)cm3 was lower than SW-620 (3.56±0.79)cm3 and SW-620-HK group (3.24±0.68)cm3 (p<0.05); the average tumor weight of SW-620-133b group (0.92±0.16)g was lower than SW-620 (4.12±0.64)g and SW-620-HK group (3.95±0.61)g (p<0.05);
3. As shown by TUNEL assay, the AI value of SW-620-133b group (30.28±3.47)%was significantly higher than that of SW-620 (7.96±0.92)%and SW-620-HK group (7.26±0.84) (p<0.05), but with no significant difference between SW-620 and SW-620-HK group.
4. The results of immunohistochemisty showed that the expression of MET and MMP9 protein was significantly down-regulated in SW-620-133b group compared with SW-620 and SW-620-HK group (p<0.05).
Conclusion
1. The ectopic expression of miR-133b can effectively inhibite the growth of tumor and promote apoptosis in human colorectal tumor-bearing nude mice.
2. The expression of MET and MMP9 was significantly down regulated in SW-620-133b group, miR-133b might suppress tumor growth through modulation of the MET and MMP9 signaling pathway.
引文
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