BZAP45基因与黏液表皮样癌关系的研究
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摘要
人涎腺黏液表皮样癌(Mucoepidermoid Carcinoma,MEC)是涎腺最常见的恶性肿瘤,约占涎腺恶性肿瘤的30%,尽管有时表现像良性病变,生长比较慢,但是该肿瘤有时是高度恶性而且预后很差。低分化的涎腺黏液表皮样癌侵袭和转移能力强,5年的存活率不超过43%。
对于BZAP45(或BZW1)基因的相关研究的文献不多。至今仅知BZAP45是一种调节因子。而对于BZAP45基因是否有其他功能并没有文献报道,也未见有BZAP45基因与肿瘤相关的报道。
RNA干扰(RNA interference, RNAi)作用机制为应用小的双链RNA引发序列特异性的基因表达的“沉默”或“敲除”。在哺乳动物细胞中这种双链RNA可以是短发夹结构RNA (Small hairpin RNA,shRNA);也可以是3'-端带有游离碱基的简单的二聚体(Small interference RNA, siRNA)。转染合成的siRNA所得到的靶基因抑制效果往往时间比较短,而且局限于容易转染的细胞系。应用于RNA干扰的载体有很多种,而利用慢病毒构建载体应用于基因沉默,是研究基因功能很好的方法。
在本课题的前期研究中,我们初步发现在黏液表皮样癌组织和细胞中,BZAP45均高表达。在此基础上,本课题构建了BZAP45慢病毒干涉载体,将Mc3细胞中的BZAP45基因沉默,观察其体外和体内生物学特性的改变,并对其影响Mc3细胞生物学行为的机制进行初步探索。本课题主要的研究内容包括以下几个方面:
1.进一步确认BZAP45基因在黏黏液表皮样癌中高表达
为验证基因芯片结果正确与否并且明确BZAP45基因是否为黏液表皮样癌特异性的差异表达基因,我们设计了BZAP45基因实时定量PCR的引物,进行实时定量PCR检测;并预制了BZAP45基因的多克隆抗体,对4例肿瘤组织(制备成15个样本)和4例正常组织进行了免疫组化染色。结果显示,BZAP45基因在黏液表皮样癌细胞和组织中表达均比正常细胞和组织高。
2. BZAP 45基因RNA干扰慢病毒载载体的制备及黏黏液液表皮样癌细细胞胞感染
针对靶基因序列,利用公用网站按照RNA干扰序列设计原则,设计4个针对BZAP45基因的RNA干扰靶点序列;合成含干扰序列的双链DNA oligo,其两端含酶切位点粘端,直接连入酶切后的载体上,将连接好的产物转入制备好的感受态细胞,对长出的阳性克隆先进行PCR鉴定,再进行测序比对后,鉴定阳性的克隆即为构建成功的BZAP45基因RNA干扰慢病毒载体。选择干涉效果最好的序列,经过病毒的小量包装、大量包装、滴度检测及浓缩后获得足够滴度的慢病毒溶液,利用慢病毒载体感染Mc3细胞,利用有限稀释法挑选阳性单克隆,并利用时定量PCR对获得的细胞进行鉴定,从而获得了稳定感染的细胞株。
3. BZAP 45基因干涉后Mc3细胞体体体外外及及体体内内生生物物学学行为的改变
为了研究BZAP45基因在Mc3细胞中可能的作用,我们将BZAP45基因干涉,使其沉默后,利用MTT法、细胞计数法、平板克隆形成实验观、细胞划痕实验、transwell实验、HE染色、透射电镜等方法察Mc3细胞体外的生物学行为的变化情况,利用裸鼠移植瘤方法观察了Mc3细胞干涉前后在裸鼠体内的生长情况。结果显示,BZAP45基因沉默后,Mc3细胞体外增殖变缓,细胞的群体倍增时间由原来的约23 h增为约48 h;克隆形成能力下降,克隆形成率由原来的78%下降为20%;体外迁移能力下降,Mc3、Mc3-NC和Mc3-RNAi三种细胞迁移的距离分别为65.833±4.940μm、64.733±2.684μm和45.667±3.066μm;体外侵袭能力下降,Mc3、Mc3-NC和Mc3-RNAi三种细胞到达小室底面的细胞数分别为85±6.1、82.0±7.8和24.0±3.7个;细胞超微结构改变,干涉后细胞核染色质浓缩、碎裂、边集于核膜,呈境界清楚的块状或半月状;体内成瘤速度变缓,抑瘤率约为57.95%。
4. BZAP 45影响MMCC 3细细细胞胞胞生生生物物物学学行行为为的的机机制初探为研究BZAP45影响MC3细胞生物学行为的机制,我们利用流式细胞仪检测了干涉前后细胞周期分布的改变,并利用免疫荧光检测了细胞周期相关因子、凋亡相关因子以及细胞增殖相关因子的表达改变,结果显示,BZAP45基因干涉后,细胞出现G1期阻滞,干涉前后p53、c-myc和P21的表达无明显变化;干涉后,caspase3的表达有所升高,而cyclin-D1和PCNA的表达有所降低。
结论:
1. BZAP45基因在黏液表皮样癌组织和细胞中高表达。
2.成功构建了BZAP45基因干涉的慢病毒干涉载体。并成功将MC3细胞的BZAP45基因干涉,获得了稳定的细胞株。
3. BZAP45基因被干涉后,Mc3细胞的体内和体外的生物学特性发生改变,细胞的恶性程度下降。
4.BZAP45基因可能与黏液表皮样癌的发生和/或发展有关。
Mucoepidermoid carcinoma (MEC) is the most common malignacy in salivary glands and accounts for about 30% of malignant tumors in salivary glands in Chinese population. Although it sometimes shows a slow growth resembling a benign lesion, this neoplasm can be highly aggressive with a dismal prognosis. A 5-year disease-free survival rate between 0 and 43% has been demonstrated in the patients with high-grade tumors.
There is little reports about BZAP45, up to now, we only know that it serves as cell cycle regulator. Whether this gene has other funtions or its relationship with tumors has no reports.
RNA interference (RNAi) is a cellular mechanism in which double-stranded RNA triggers the sequence-specific“silencing”or“knockdown”of gene expression. In mammalian cells this double-stranded RNA may be either a small hairpin RNA (shRNA) or a simple RNA duplex with two unpaired nucleotides on the 3'-ends (siRNA). The transfection of synthetic siRNAs causes only transient suppression of target genes, which is often limited to the cell lines that are easy to be transfected. However, compared with siRNA, shRNA is more efficient to achieve stable long-term RNAi effects.There are many vectors used in RNAi, but it is a better way to study gene funtion by construction of lentivirus-RNAi vector.
Based on our previous work, in this study, we constructed lentivirus-RNAi vector to silence BZAP45 gene in Mc3 cells. The biofeatures changes of Mc3 cells in vivo and in vitro and the possible mechanisms were studied, including four parts as follows:
1. To confirm the expression level of BZAP45 in MEC tissue and cells was higher than that in normal tissue and cells
We designed specific primers of BZAP45 to verify the results of cDNA microarray by real time PCR. Rabbit antibody specific for BZAP45 was generated by immunizing rabbits with recombinant human BZAP45 protein and purified by using standard protocols. The expression level of BZAP45 in MEC and normal salivary gland tissue was detected by immunohistochemistry and real time PCR. The expression level of BZAP45 MEC cells was detected by real time PCR. The results showed that the expression level of BZAP45 in MEC tissue, Mc3 cells were much higher than that in normal fibroblast cells and normal salivary gland tissue
2. To construct BZAP45 siRNA expression lentivirus and to infect cells
To generate lentivirus expressing RNAi specific for the BZAP45 gene, the four siRNAs were designed based on two conservative cDNA fragments within the coding region of human BZAP45 gene. The most effective one was chosed for later study. After Mc3 cells were infected, limiting dilution assay was used to pick positive clone, and real time PCR was used to identify the infected cells. The cells expressed BZAP45 siRNA were got and were used for later study.
3. To study the changes of biofeatures in Mc3 cells after BZAP45 scilenced in vitro and in vivo
MTT assay, cell counting assay, colony formation assay, monolayer wound healing assay, transwell invasion assay, HE staining and transmission electron microscopy were used to detect the changes in Mc3 cells in vitro after BZAP45 scilenced, Tumorigenesis assay was used to to detect the changes in Mc3 cells in vivo after BZAP45 scilenced. The results showed that the proliferation, the ability of clone formation and migration were decreased notably. Cells doubling time of Mc3 and Mc3-RNAi was 23h and 48h, the clone forming efficiency of Mc3 and Mc3-RNAi was 78% and 20%. Following incubation of physically wounded cells for 48 h, the Mc3 and Mc3-NC migrated insignificantly at 65.833±4.940μm or 64.733±2.684μm, respectively. In contrast, the Mc3-RNAi only migrated about 45.667±3.066μm and the mobile distance of Mc3-RNAi was significantly shorter than that of controls. In transwell invasion assay, average 85±6.1 Mc3 and 82.0±7.8 Mc3-NC cells per high power field had migrated onto the filter surface while only 24.0±3.7 Mc3-RNAi cells reached on the filter. The development of Mc3-RNAi cell-related solid tumors grew slowly and the mean volume of solid MEC tumors in Mc3-RNAi group decreased by about 60%, as compared with that in control groups . As a result, the mean weight of tumors in Mc3-RNAi group was significantly lighter than that in control groups The ultrastructure of Mc3 cells also changed after BZAP45 silenced.
4. The possible mechanisms of BZAP45 effects the biofeatures of Mc3 cells
The cell cycle distribution was detected by flow cytometry. Also some factors related to cell cycle, apoptosis and cell proliferation were detected by immunofluorescence. The expression levels of P53, c-myc, P21 seem no change after BZAP45 silenced, but the expression level of caspase3 was higher in Mc3-RNAi cells than in the other two groups, while the expression levels of cyclin-D1 and PCNA seem lower in Mc3-RNAi cells than that in the other two groups.
Conclusions:
1. The expression level of BZAP45 in MEC tissues and cells was higher than that in normal salivary gland tissues and cells
2. BZAP45-RNAi-lentivirus was constructed and Mc3 cells were infected successfully.
3. The biofeatures in Mc3 cells after BZAP45 scilenced were changed in vitro and in vivo notably, the malignancy of Mc3 cells was decreased after BZAP45 gene scilenced.
4. BZAP45 gene may be related to the genesis and/or development of MEC
对于BZAP45(或BZW1)基因的相关研究的文献不多。至今仅知BZAP45是一种调节因子。而对于BZAP45基因是否有其他功能并没有文献报道,也未见有BZAP45基因与肿瘤相关的报道。
RNA干扰(RNA interference, RNAi)作用机制为应用小的双链RNA引发序列特异性的基因表达的“沉默”或“敲除”。在哺乳动物细胞中这种双链RNA可以是短发夹结构RNA (Small hairpin RNA,shRNA);也可以是3'-端带有游离碱基的简单的二聚体(Small interference RNA, siRNA)。转染合成的siRNA所得到的靶基因抑制效果往往时间比较短,而且局限于容易转染的细胞系。应用于RNA干扰的载体有很多种,而利用慢病毒构建载体应用于基因沉默,是研究基因功能很好的方法。
在本课题的前期研究中,我们初步发现在黏液表皮样癌组织和细胞中,BZAP45均高表达。在此基础上,本课题构建了BZAP45慢病毒干涉载体,将Mc3细胞中的BZAP45基因沉默,观察其体外和体内生物学特性的改变,并对其影响Mc3细胞生物学行为的机制进行初步探索。本课题主要的研究内容包括以下几个方面:
1.进一步确认BZAP45基因在黏黏液表皮样癌中高表达
为验证基因芯片结果正确与否并且明确BZAP45基因是否为黏液表皮样癌特异性的差异表达基因,我们设计了BZAP45基因实时定量PCR的引物,进行实时定量PCR检测;并预制了BZAP45基因的多克隆抗体,对4例肿瘤组织(制备成15个样本)和4例正常组织进行了免疫组化染色。结果显示,BZAP45基因在黏液表皮样癌细胞和组织中表达均比正常细胞和组织高。
2. BZAP 45基因RNA干扰慢病毒载载体的制备及黏黏液液表皮样癌细细胞胞感染
针对靶基因序列,利用公用网站按照RNA干扰序列设计原则,设计4个针对BZAP45基因的RNA干扰靶点序列;合成含干扰序列的双链DNA oligo,其两端含酶切位点粘端,直接连入酶切后的载体上,将连接好的产物转入制备好的感受态细胞,对长出的阳性克隆先进行PCR鉴定,再进行测序比对后,鉴定阳性的克隆即为构建成功的BZAP45基因RNA干扰慢病毒载体。选择干涉效果最好的序列,经过病毒的小量包装、大量包装、滴度检测及浓缩后获得足够滴度的慢病毒溶液,利用慢病毒载体感染Mc3细胞,利用有限稀释法挑选阳性单克隆,并利用时定量PCR对获得的细胞进行鉴定,从而获得了稳定感染的细胞株。
3. BZAP 45基因干涉后Mc3细胞体体体外外及及体体内内生生物物学学行为的改变
为了研究BZAP45基因在Mc3细胞中可能的作用,我们将BZAP45基因干涉,使其沉默后,利用MTT法、细胞计数法、平板克隆形成实验观、细胞划痕实验、transwell实验、HE染色、透射电镜等方法察Mc3细胞体外的生物学行为的变化情况,利用裸鼠移植瘤方法观察了Mc3细胞干涉前后在裸鼠体内的生长情况。结果显示,BZAP45基因沉默后,Mc3细胞体外增殖变缓,细胞的群体倍增时间由原来的约23 h增为约48 h;克隆形成能力下降,克隆形成率由原来的78%下降为20%;体外迁移能力下降,Mc3、Mc3-NC和Mc3-RNAi三种细胞迁移的距离分别为65.833±4.940μm、64.733±2.684μm和45.667±3.066μm;体外侵袭能力下降,Mc3、Mc3-NC和Mc3-RNAi三种细胞到达小室底面的细胞数分别为85±6.1、82.0±7.8和24.0±3.7个;细胞超微结构改变,干涉后细胞核染色质浓缩、碎裂、边集于核膜,呈境界清楚的块状或半月状;体内成瘤速度变缓,抑瘤率约为57.95%。
4. BZAP 45影响MMCC 3细细细胞胞胞生生生物物物学学行行为为的的机机制初探为研究BZAP45影响MC3细胞生物学行为的机制,我们利用流式细胞仪检测了干涉前后细胞周期分布的改变,并利用免疫荧光检测了细胞周期相关因子、凋亡相关因子以及细胞增殖相关因子的表达改变,结果显示,BZAP45基因干涉后,细胞出现G1期阻滞,干涉前后p53、c-myc和P21的表达无明显变化;干涉后,caspase3的表达有所升高,而cyclin-D1和PCNA的表达有所降低。
结论:
1. BZAP45基因在黏液表皮样癌组织和细胞中高表达。
2.成功构建了BZAP45基因干涉的慢病毒干涉载体。并成功将MC3细胞的BZAP45基因干涉,获得了稳定的细胞株。
3. BZAP45基因被干涉后,Mc3细胞的体内和体外的生物学特性发生改变,细胞的恶性程度下降。
4.BZAP45基因可能与黏液表皮样癌的发生和/或发展有关。
Mucoepidermoid carcinoma (MEC) is the most common malignacy in salivary glands and accounts for about 30% of malignant tumors in salivary glands in Chinese population. Although it sometimes shows a slow growth resembling a benign lesion, this neoplasm can be highly aggressive with a dismal prognosis. A 5-year disease-free survival rate between 0 and 43% has been demonstrated in the patients with high-grade tumors.
There is little reports about BZAP45, up to now, we only know that it serves as cell cycle regulator. Whether this gene has other funtions or its relationship with tumors has no reports.
RNA interference (RNAi) is a cellular mechanism in which double-stranded RNA triggers the sequence-specific“silencing”or“knockdown”of gene expression. In mammalian cells this double-stranded RNA may be either a small hairpin RNA (shRNA) or a simple RNA duplex with two unpaired nucleotides on the 3'-ends (siRNA). The transfection of synthetic siRNAs causes only transient suppression of target genes, which is often limited to the cell lines that are easy to be transfected. However, compared with siRNA, shRNA is more efficient to achieve stable long-term RNAi effects.There are many vectors used in RNAi, but it is a better way to study gene funtion by construction of lentivirus-RNAi vector.
Based on our previous work, in this study, we constructed lentivirus-RNAi vector to silence BZAP45 gene in Mc3 cells. The biofeatures changes of Mc3 cells in vivo and in vitro and the possible mechanisms were studied, including four parts as follows:
1. To confirm the expression level of BZAP45 in MEC tissue and cells was higher than that in normal tissue and cells
We designed specific primers of BZAP45 to verify the results of cDNA microarray by real time PCR. Rabbit antibody specific for BZAP45 was generated by immunizing rabbits with recombinant human BZAP45 protein and purified by using standard protocols. The expression level of BZAP45 in MEC and normal salivary gland tissue was detected by immunohistochemistry and real time PCR. The expression level of BZAP45 MEC cells was detected by real time PCR. The results showed that the expression level of BZAP45 in MEC tissue, Mc3 cells were much higher than that in normal fibroblast cells and normal salivary gland tissue
2. To construct BZAP45 siRNA expression lentivirus and to infect cells
To generate lentivirus expressing RNAi specific for the BZAP45 gene, the four siRNAs were designed based on two conservative cDNA fragments within the coding region of human BZAP45 gene. The most effective one was chosed for later study. After Mc3 cells were infected, limiting dilution assay was used to pick positive clone, and real time PCR was used to identify the infected cells. The cells expressed BZAP45 siRNA were got and were used for later study.
3. To study the changes of biofeatures in Mc3 cells after BZAP45 scilenced in vitro and in vivo
MTT assay, cell counting assay, colony formation assay, monolayer wound healing assay, transwell invasion assay, HE staining and transmission electron microscopy were used to detect the changes in Mc3 cells in vitro after BZAP45 scilenced, Tumorigenesis assay was used to to detect the changes in Mc3 cells in vivo after BZAP45 scilenced. The results showed that the proliferation, the ability of clone formation and migration were decreased notably. Cells doubling time of Mc3 and Mc3-RNAi was 23h and 48h, the clone forming efficiency of Mc3 and Mc3-RNAi was 78% and 20%. Following incubation of physically wounded cells for 48 h, the Mc3 and Mc3-NC migrated insignificantly at 65.833±4.940μm or 64.733±2.684μm, respectively. In contrast, the Mc3-RNAi only migrated about 45.667±3.066μm and the mobile distance of Mc3-RNAi was significantly shorter than that of controls. In transwell invasion assay, average 85±6.1 Mc3 and 82.0±7.8 Mc3-NC cells per high power field had migrated onto the filter surface while only 24.0±3.7 Mc3-RNAi cells reached on the filter. The development of Mc3-RNAi cell-related solid tumors grew slowly and the mean volume of solid MEC tumors in Mc3-RNAi group decreased by about 60%, as compared with that in control groups . As a result, the mean weight of tumors in Mc3-RNAi group was significantly lighter than that in control groups The ultrastructure of Mc3 cells also changed after BZAP45 silenced.
4. The possible mechanisms of BZAP45 effects the biofeatures of Mc3 cells
The cell cycle distribution was detected by flow cytometry. Also some factors related to cell cycle, apoptosis and cell proliferation were detected by immunofluorescence. The expression levels of P53, c-myc, P21 seem no change after BZAP45 silenced, but the expression level of caspase3 was higher in Mc3-RNAi cells than in the other two groups, while the expression levels of cyclin-D1 and PCNA seem lower in Mc3-RNAi cells than that in the other two groups.
Conclusions:
1. The expression level of BZAP45 in MEC tissues and cells was higher than that in normal salivary gland tissues and cells
2. BZAP45-RNAi-lentivirus was constructed and Mc3 cells were infected successfully.
3. The biofeatures in Mc3 cells after BZAP45 scilenced were changed in vitro and in vivo notably, the malignancy of Mc3 cells was decreased after BZAP45 gene scilenced.
4. BZAP45 gene may be related to the genesis and/or development of MEC
引文
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