调控胃癌BGC823细胞PTEN和Livin基因表达的生物学效应实验研究
摘要
背景与目的
胃癌的发病率在恶性肿瘤中位居第四位,仅次于肺癌、乳腺癌和肠癌,是严重威胁我国人民生命健康的主要疾病之一,其发病机制迄今为止仍不明确。胃癌在发病早期症状不典型,部分患者就诊时已进入进展期。尽管在手术、化疗、放疗等方而取得了长足进步,但是进展期胃癌患者术后5年生存率仍无明显提高。
大量研究表明,胃癌在发生、发展过程中有多种因素的参与,包括抑癌基因失活、癌基因突变、DNA损伤修复功能的降低、信号转导通路和凋亡过程的异常等,提示胃癌的发生、发展是一个多因素、多阶段、多步骤的过程。
第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosphatase and tensin homology deleted on chromosome ten, PTEN)是具有双特异性磷酸酶活性的抑癌基因,可以负调节PI3K-Akt信号通路的信号转导,具有抑制细胞增殖和迁移以及促进凋亡的效应。PTEN基因的突变、缺失以及甲基化可以导致其抑癌功能丧失,引起肿瘤的发生。研究显示,胃癌组织中PTEN基因表达显著降低,并且PTEN蛋白表达水平与肿瘤大小、Borrmann分型、淋巴结转移和肿瘤分期呈负相关,表明PTEN基因失活可能是胃癌发生、发展的原因之一。
Livin是凋亡抑制蛋白(inhibitor of apoptosis proteins, IAP)家族新成员之一,它可以和半胱氨酸天冬氨酸特异性蛋白酶(Caspase)直接结合而抑制细胞凋亡,进而起到促进肿瘤细胞生长的作用。研究发现Livin蛋白在包括胃癌在内的多种肿瘤组织中呈现高表达,沉默肿瘤细胞中Livin基因可以促进细胞凋亡、抑制肿瘤增殖。表明Livin的高表达与肿瘤的发生、发展及预后相关。
本课题采用分子生物学技术,分别建立过表达PTEN基因、沉默Livin基因表达以及过表达PTEN同时沉默Livin基因表达的胃癌细胞株；通过在体和离体实验,观察调控PTEN和Livin基因表达对胃癌细胞生长、增殖、凋亡和迁移等方面的影响,为胃癌的治疗提供新的靶点和策略。课题共分三部分：第一部分,PTEN基因表达载体、Livin基因siRNA载体的构建和鉴定；第二部分,体外调控PTEN、Livin基因表达对胃癌BGC823细胞生物学行为的影响；第三部分,调控PTEN/Livin基因表达对裸鼠移植瘤生长的影响。
第一部分PTEN基因表达载体、Livin基因siRNA载体的构建和鉴定
方法
设计带有EcoRI和SalI酶切位点的PTEN基因全编码区cDNA扩增引物；PCR扩增pCMV6-PTEN得到PTEN基因；扩增产物经EcoRI和SalI酶切后与表达载体pCL-neo重组；用EcoRI和salI双酶切鉴定转化重组子,并对重组子插入序列进行DNA序列分析,得到PTEN基因表达载体pCL-neo-PTEN。设计3个针对Livin基因的siRNA巴序列,合成发卡样DNA单链；退火成双链后与siRNA载体pRNAT-U6.1重组；用PCR扩增筛选转化重组于,DNA序列测定鉴定插入序列,得到3个针对Livin基因的siRNA载体。分别用脂质体LipofectamineTM2000包裹转染BGC823细胞,G418筛选得到对应的稳定转染细胞。采用荧光定量RT-PCR和Western-Blot分别检测转染的BGC823细胞Livin和PTEN mRNA和蛋白表达水平。
结果
1.PCR扩增得到1212bp的PTEN cDNA ORF区段目的基因；转化后得到多个重组子,经EcoRI/SalI双酶切和DNA测序鉴定证实获得的重组子pCL-neo-PTEN正确。
2. RNAi Explorer和siDESIGN Center扫描人Livin cDNA编码序列(NM_139317),初步筛选得到50个侯选片段,经BLAST同源性分析,选择确定3个19个碱基的siRNA靶序列,分别为178-196位(GACCTAAAGACAG TGCCAA)、539-557位(GAGGTGCTTCTTCTGCTAT)、648-666(GGAAGAGACTTTGTCCACA)。
3.双链发卡样DNA sil78、si539和si648与pRNAT-U6.1重组后,都得到多个重组子,PCR扩增筛选鉴定和DNA测序分析证实得到的阳性重组子pRNAT-U6.1-si178、pRNAT-U6.1-si539、pRNAT-U6.1-si648与设计完全一致。
4.过表达PTEN组(转染pCL-neo-PTEN)、沉默Livin 1组(转染pRNAT-U6.1-sil78)、沉默Livin 2组(转染pRNAT-U6.1-si539)、沉默Livin3组(转染pRNAT-U6.1-si648)、空载体1组(转染pCL-neo)和空载体2组(转染pRNAT-U6.1)细胞经G418筛选3w,均得到稳定转染细胞株。
5.与空白对照相比,空载体1组和空载体2组的Livin mRNA和蛋白表达水平无明显变化,差异无显著性(P>0.05)；与3个对照组比较,过表达PTEN组、沉默Livin 1组、沉默Livin 2组和沉默Livin3组细胞Livin mRNA和蛋白表达水平均显著降低,差异有显著性(P<0.05),其中沉默Livin 2组的相对表达量最低。
6.过表达PTEN组细胞的PTEN mRNA和蛋白表达水平显著高于其它各组(P<0.01)
第二部分体外调控PTEN基因和Livin基因表达对胃癌BGC823细胞生物学行为的影响
方法
切下沉默Livin效果最佳siRNA载体中的Livin siRNA单元,重组入PTEN基因表达载体pCL-neo-PTEN,得到表达PTEN基因、同时沉默Livin基因的调控载体pCL-neo-PTEN-siLivin,用LipofectamineTM2000包裹,然后将其转染BGC823细胞,G418筛选得到过表达PTEN同时沉默Livin基因的稳定转染细胞。与第一部分获得的单一过表达PTEN的胃癌BGC823细胞株、单一沉默Livin表达的胃癌BGC823细胞株一起作为实验对象,采用RT-PCR和Western-blot测定各组细胞Livin和PTEN mRNA和蛋白表达水平、MTT方法检测细胞增殖、流式细胞法检测细胞周期和细胞凋亡、EILSA检测Caspase-3和Caspase-9蛋白酶活性、Transwell方法检测细胞侵袭转移能力。
结果
1. EcoRI酶切鉴定重组子,得到2个条带(3.4Kb和3.8Kb),证实Livin siRNA单元成功插入；DNA序列测定确认得到阳性重组子。
2.经G418筛选得到稳定转染pCL-neo-PTEN-siLivin的胃癌BGC823细胞株。
3.与空白对照组和空载体对照组比较,调控组(稳定转染pCL-neo-PTEN-siLivin的BGC823细胞)、沉默Livin组(稳定转染pRNAT-U6.1-siLivin的BGC823细胞)和过表达PTEN组(稳定转染pCL-neo-PTEN的BGC823细胞)的Livin mRNA和蛋白表达水平均明显降低(P<0.05),其中调控组Livin mRNA和蛋白表达水平最低。
4.与空白对照组和空载体对照组比较,过表达PTEN组和调控组细胞PTEN mRNA和蛋白表达水平显著升高,差异有显著性(P<0.01)。而过表达PTEN组和调控组细胞PTEN mRNA和蛋白表达水平无显著性差异(P>0.05),说明在PTEN表达载体pCL-neo-PTEN中插入Livin siRNA单元不影响PTEN表达的水平。
5.在3d、4d和5d MTT检测细胞,与空白对照组和空载体对照组比较,调控组细胞OD值显著降低,差异有显著性(P<0.05)。
6.与空白对照组和空载体对照组比较,调控组、沉默Livin组和过表达PTEN组细胞的Caspase-3和Caspase-9活性显著升高,差异有显著性(P<0.05),其中以调控组的提高更为显著。
7.与空白对照组和空载体对照组比较,调控组、沉默Livin组和过表达PTEN组的细胞凋亡率显著升高(P<0.05)。与沉默Livin组和过表达PTEN组比较,调控组细胞凋亡率明显升高,差异有显著性(P<0.05)。
8.与空白对照组和空载体对照组比较,调控组、沉默Livin组和过表达PTEN组穿透Matrigel的平均细胞数显著降低(P<0.05)。与沉默Livin组和过表达PTEN组比较,调控组穿透Matrigel的平均细胞数明显减少,差异有显著性(P<0.05)。
第三部分调控PTEN/Livin的基因表达对裸鼠移植瘤生长的影响
方法
取调控PTEN/Livin基因表达的胃癌BGC823细胞、过表达PTEN的胃癌BGC823细胞、沉默Livin表达的胃癌BGC823细胞、空载体对照的胃癌BGC823细胞和胃癌BGC823细胞,调整细胞浓度至1×108/ml,在裸鼠右侧腋部皮下接种0.1ml。在注射接种后的第7d开始测定绘制肿瘤生长曲线,第28天测量肿瘤大小后处死裸鼠,取肿瘤组织称重。荧光定量RT-PCR和Western-blot检测PTEN和Livin mRNA和蛋白表达水平。
结果
1.裸鼠皮下细胞接种7d后均出现可见肿瘤块形成；调控组、过表达PTEN组和沉默Livin组移植瘤生长速度明显慢于空白对照组和空载体对照组(P<0.05)。
2.调控组移植瘤平均瘤重215.42±35.15mg、过表达PTEN组移植瘤平均瘤重461.73±58.17mg、沉默Livin组移植瘤平均瘤重368.23±53.72mg,均显著低于空白对照组和空载体对照组移植瘤平均瘤重(1051.29±175.95mg和968.88±194.39mg),差异有显著性(P<0.05),其中以调控组移植瘤平均瘤重最小。
3.与空白对照组、空载体组和沉默Livin组比较,调控组、过表达PTEN组的PTEN mRNA和蛋白表达水平显著升高,差异有显著性(P<0.01)。与空白对照组、空载体组比较,调控组、沉默Livin组、过表达PTEN组的Livin mRNA和蛋白表达水平显著降低,差异有显著性(P<0.05),其中以调控组最低。
结论
1.筛选得到过表达PTEN的胃癌BGC823细胞株、沉默Livin表达的胃癌BGC823细胞株和过表达PTEN同时又沉默Livin表达的胃癌BGC823细胞株。
2.建立一种表达一个基因同时又沉默另一个基因的载体构建方法。
3.过表达PTEN同时沉默Livin的调控可以有效抑制胃癌BGC823细胞的生长增殖、侵袭转移和促进细胞凋亡,比单一过表达PTEN或降低Livin表达的效果更为显著。
4.在胃癌细胞中过表达PTEN同时沉默Livin的复合调控方法有可能成为胃癌治疗的有效策略。
Background and Objective
Gastric cancer, as the fourth most commonly occurring cancer after lung, breast and colorectal cancers in order of incidence, is the main cause of cancer deaths for Chinese. However, the mechanisms for gastric cancer remain unknown by now. Many patients with gastric cancer express nonspecific symptoms in the early stage and some patients reach advanced stage before visiting the hospital. Therefore, although great progresses have been made in surgery, chemotherapy and radiation therapy, the five year survival rate of advanced gastric cancer is still very poor.
Many studies indicated that a lot of factors were involved in the development and progression of gastric cancer, including the loss of anti-oncogene, oncogene mutation, decrease in DNA damage and repair function, abnormality of signaling pathways and apoptosis, indicating that the development and progression of gastric cancer is a process with multifactor, multistage and multistep.
Phosphatase and tensin homology deleted on chromosome ten (PTEN), as an anti-oncogene possessing lipid and protein phosphatase activities, could inhibit cell proliferation and migration, and promote cell apoptosis by inhibiting phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway. It is reported that PTEN deletion, mutation and methylation result in tumorigenesis due to the lack of anti-cancer function. Some studies proved that tissue from gastric cancer developed decreased PTEN and the expression of PTEN was negatively correlated with the size of tumor, Borrmann type, lymph node metastasis and cancer staging, indicating that the loss of PTEN was a possible mechanism for gastric cancer.
Livin, a novel member of inhibitor of apoptosis protein (IAP) family, inhibits apoptosis and promotes the growth of cancer cells by binding to cysteine aspartate-specific protease. Over-expression of livin in many tumor cells including gastric cancer has been reported, and many studies indicated that silencing of livin gene inhibited the proliferation of cancer cells as a result of promoting apoptosis, demonstrating that the over-expression of livin is related to the development, progression and prognosis of tumor.
In this study, three kinds of gastric tumor cell lines with different expression of PTEN and livin were established by using molecular biological techniques, including over-expression of PTEN, silencing of livin, and over-expression of PTEN with silencing of livin simultaneously. In vivo and in vitro experiments were employed to detect the effects of PTEN and livin on growth, proliferation, apoptosis and migration of gastric cancer cells, which provided new targets and methods for therapy of gastric cancer. Generally, this study included three parts as follows:Part 1 included the construction and identification of PTEN gene expression vector and livin gene siRNA vector; part 2 effects of PTEN and livin on biological behaviors of gastric cancer BGC823 cells were studied In vitro; Part 3 revealed the in vivo effects of PTEN and livin genes on growth of cancer in nude mice.
Part 1 Construction and identification of PTEN gene expression vector and Livin gene SiRNA vector
Methods
The amplification primer with restriction enzyme cutting sites of EcoRI and SalI for all coding regions in PTEN gene was designed and used for amplification of pCMV6-PTEN by PCR. After digestion by EcoRI and SalI, PCR products were inserted into pCL-neo vector. Subsequently, the recombinants were identified by EcoRI and SalI digestion and the inserted fragments were sequenced. Three designed siRNA target sequences for livin gene transformed into hairpin-shaped single-chain DNA and subsequent double-chain DNA after annealing, and then was inserted into pRNAT-U6.1 vector. The recombinants were amplified, selected and transformed by PCR and the inserted fragments were sequenced. pCL-neo-PTEN and livin siRNA vectors were transfected into BGC823 cell line by LipofectamineTM2000, and stable transfected cell lines were selected by G418. Fluorescence quantitative RT-PCR and Western-Blot were used to detect mRNA and protein expression level of livin and PTEN in transfected BGC823 cells.
Results
1. PTEN cDNA ORF target gene with 1212bp was acquired by PCR. EcoRI/SalI digestion and DNA sequence analysis indicated that the recombinants of pCL-neo-PTEN was successfully constructed.
2. Fifty candidate fragments were obtained after first selection through scanning of human livin cDNA (NM_139317) by RNAi explorer and siDESIGN center, and subsequent BLAST homological analysis confirmed three siRNA target sequences with 19 bp,178-196nt (GACCTAAAGACAGTGCCAA),539-557 nt (GAGGTG CTTCTTCTGCTAT) and 648-666 nt (GGAAGAGACTTTGTCC ACA).
3. Amplification and selection by PCR and DNA sequence analysis indicated that the recombinants, which were acquired after recombination of hairpin-shaped double-chain DNA (si178, si539 and si648) with pRNAT-U6.1, were in accordance with expected.
4. After selection for three weeks by G418, stable transfected cell lines were acquired in over-expression of PTEN group (transfection of pCL-neo-PTEN), silencing of livin group 1 (transfection of pRNAT-U6.1-si178), silencing of livin group 2 (transfection of pRNAT-U6.1-si539), silencing of livin group 3 (transfection of pRNAT-U6.1-si648), empty vector group 1 (transfection of pCL-neo) and empty vector group 2 (transfection of pRNAT-U6.1).
5. There was no significant difference in mRNA and protein expression level of livin between blank group and empty vector groups (P>0.05). However, mRNA and protein expression level of livin in over-expression of PTEN group, silencing of livin group 1,2 and 3 decreased significantly compared with the above three control groups (P<0.05), and silencing of livin group 2 showed the lowest expression.
6. The mRNA and protein expression level of PTEN in over-expression of PTEN group increased significantly in comparison with other groups (P<0.01).
Part 2 Effects of PTEN and Livin on biological behaviors of gastric cancer BGC823 cells in vitro
Methods
Livin siRNA was cut from livin siRNA vector with the best silencing effect and then was recombined with pCL-neo-PTEN. Thus, pCL-neo-PTEN-siLivin which over-expressed PTEN and silenced livin simultaneously was obtained, and then was transfected into BGC823 cells by LipofectamineTM2000 subsequently. After stable transfected cell line was selected by G418, RT-PCR and Western-blot techniques were used to detect mRNA and protein expression level of livin and PTEN in over-expression of PTEN group, silencing of livin group and over-expression of PTEN with silencing of livin simultaneously group (regulation group). MTT, flow cytometry method, ELISA and Transwell methods were employed to study cell proliferation, cell cycle and apoptosis, activities of caspase-3 and caspase-9 proteinases and cell invasion respectively.
Results
1. After EcoRI digestion of recombinants, two fragments (3.4kb and 3.8kb) were acquired, indicating successful insertion of livin siRNA. Positive recombinants were confirmed by DNA sequence analysis.
2. Gastric cancer BGC823 cell line with stable transfection of pCL-neo-PTEN- siLivin was acquired by G418 selection.
3. The mRNA and protein expression level of livin in regulation group (BGC823 cell line with stable transfection of pCL-neo-PTEN-siLivin), silencing of livin group (BGC823 cell line with stable transfection of pRNAT-U6.1-siLivin) and over-expression of PTEN group (BGC823 cell line with stable transfection of pCL-neo-PTEN) decreased significantly compared with blank group and empty vector group (P<0.05), and regulation group developed the lowest mRNA and expression of livin.
4. Compared with blank group and empty vector group, mRNA and protein expression level of PTEN in over-expression of PTEN group and regulation group increased significantly (P<0.01). There was no significant difference between over-expression of PTEN group and regulation group (P>0.05), indicating that the insertion of livin siRNA into pCL-neo-PTEN did not affect the expression of PTEN.
5. MTT technique revealed that OD value in regulation group decreased significantly at 3,4 and 5 days compared with control groups including blank group and empty vector group (P<0.05).
6. The activities of caspase-3 and caspase-9 in regulation group, silencing of livin group and over-expression of PTEN group increased significantly in comparison with control groups including blank group and empty vector group (P<0.05), and regulation group showed the highest effects.
7. Apoptosis in regulation group, silencing of livin group and over-expression of PTEN group elevated significantly compared with control groups including blank group and empty vector group (P<0.05), and regulation group expressed higher apoptosis compared with silencing of livin group and over-expression of PTEN group (P<0.05).
8. The average cell numbers which passed through matrigel in regulation group, silencing of livin group and over-expression of PTEN group decreased significantly compared with control groups including blank group and empty vector group (P<0.05), and regulation group showed reduced cell invasion compared with silencing of livin group and over-expression of PTEN group (P<0.05).
Part 3 Effects of PTEN/Livin expression on implanted tumor in nude mice
Methods
Different gastric cancer BGC823 cells, including regulation group, over-expression of PTEN group, silencing of livin group, empty vectors group and blank group, were injected subcutaneously into the right oxter of nude mice (0.1mL, 1×108/mL). Tumor-growth curve was found 7 days after injection. Tumors were measured, isolated and weighed on the 28th days. Fluorescence quantitative RT-PCR and Western-blot were used to detect mRNA and protein expression of PTEN and livin.
Results
1. Implanted tumors were observed apparently 7 days after injection. The growth of tumors in regulation group, over-expression of PTEN group and silencing of livin group were significantly lower than those of blank group and empty vector group (P<0.05).
2. The average tumor weight in regulation group, over-expression of PTEN group and silencing of livin group were 215.42±35.15,461.73±58.17 and 368.23±53.72mg respectively, which were significantly lower than those of blank group and empty vector group (1051.29±175.95 and 968.88±194.39mg respectively, P<0.05). Regulation group developed the lowest tumor weight.
3. PTEN mRNA and protein expression level in regulation group and over-expression of PTEN group increased significantly compared with blank group, empty vector group and silencing of livin group (P<0.01). mRNA and protein expression level of livin in regulation group, silencing of livin group and over-expression of PTEN group decreased significantly in comparison with blank group and empty vector group (P<0.05), and regulation group showed the lowest expression.
Conclusions
1. Different gastric cancer BGC823 cell lines, including over-expression of PTEN, silencing of livin, over-expression of PTEN and silencing of livin simultaneously, were established successfully.
2. The technique about how to constructing a vector which expressed one gene and silenced another gene simultaneously was founded.
3. Over-expression of PTEN and silencing of livin simultaneously obviously inhibited growth, proliferation, invasion and migration of gastric cancer cells, which was more effective than that of single over-expression of PTEN or silencing of livin.
4. The complex of over-expression of PTEN and silencing of livin in gastric cancer cells was a potential method for gastric cancer therapy.
胃癌的发病率在恶性肿瘤中位居第四位,仅次于肺癌、乳腺癌和肠癌,是严重威胁我国人民生命健康的主要疾病之一,其发病机制迄今为止仍不明确。胃癌在发病早期症状不典型,部分患者就诊时已进入进展期。尽管在手术、化疗、放疗等方而取得了长足进步,但是进展期胃癌患者术后5年生存率仍无明显提高。
大量研究表明,胃癌在发生、发展过程中有多种因素的参与,包括抑癌基因失活、癌基因突变、DNA损伤修复功能的降低、信号转导通路和凋亡过程的异常等,提示胃癌的发生、发展是一个多因素、多阶段、多步骤的过程。
第10号染色体缺失的磷酸酶和张力蛋白同源基因(phosphatase and tensin homology deleted on chromosome ten, PTEN)是具有双特异性磷酸酶活性的抑癌基因,可以负调节PI3K-Akt信号通路的信号转导,具有抑制细胞增殖和迁移以及促进凋亡的效应。PTEN基因的突变、缺失以及甲基化可以导致其抑癌功能丧失,引起肿瘤的发生。研究显示,胃癌组织中PTEN基因表达显著降低,并且PTEN蛋白表达水平与肿瘤大小、Borrmann分型、淋巴结转移和肿瘤分期呈负相关,表明PTEN基因失活可能是胃癌发生、发展的原因之一。
Livin是凋亡抑制蛋白(inhibitor of apoptosis proteins, IAP)家族新成员之一,它可以和半胱氨酸天冬氨酸特异性蛋白酶(Caspase)直接结合而抑制细胞凋亡,进而起到促进肿瘤细胞生长的作用。研究发现Livin蛋白在包括胃癌在内的多种肿瘤组织中呈现高表达,沉默肿瘤细胞中Livin基因可以促进细胞凋亡、抑制肿瘤增殖。表明Livin的高表达与肿瘤的发生、发展及预后相关。
本课题采用分子生物学技术,分别建立过表达PTEN基因、沉默Livin基因表达以及过表达PTEN同时沉默Livin基因表达的胃癌细胞株；通过在体和离体实验,观察调控PTEN和Livin基因表达对胃癌细胞生长、增殖、凋亡和迁移等方面的影响,为胃癌的治疗提供新的靶点和策略。课题共分三部分：第一部分,PTEN基因表达载体、Livin基因siRNA载体的构建和鉴定；第二部分,体外调控PTEN、Livin基因表达对胃癌BGC823细胞生物学行为的影响；第三部分,调控PTEN/Livin基因表达对裸鼠移植瘤生长的影响。
第一部分PTEN基因表达载体、Livin基因siRNA载体的构建和鉴定
方法
设计带有EcoRI和SalI酶切位点的PTEN基因全编码区cDNA扩增引物；PCR扩增pCMV6-PTEN得到PTEN基因；扩增产物经EcoRI和SalI酶切后与表达载体pCL-neo重组；用EcoRI和salI双酶切鉴定转化重组子,并对重组子插入序列进行DNA序列分析,得到PTEN基因表达载体pCL-neo-PTEN。设计3个针对Livin基因的siRNA巴序列,合成发卡样DNA单链；退火成双链后与siRNA载体pRNAT-U6.1重组；用PCR扩增筛选转化重组于,DNA序列测定鉴定插入序列,得到3个针对Livin基因的siRNA载体。分别用脂质体LipofectamineTM2000包裹转染BGC823细胞,G418筛选得到对应的稳定转染细胞。采用荧光定量RT-PCR和Western-Blot分别检测转染的BGC823细胞Livin和PTEN mRNA和蛋白表达水平。
结果
1.PCR扩增得到1212bp的PTEN cDNA ORF区段目的基因；转化后得到多个重组子,经EcoRI/SalI双酶切和DNA测序鉴定证实获得的重组子pCL-neo-PTEN正确。
2. RNAi Explorer和siDESIGN Center扫描人Livin cDNA编码序列(NM_139317),初步筛选得到50个侯选片段,经BLAST同源性分析,选择确定3个19个碱基的siRNA靶序列,分别为178-196位(GACCTAAAGACAG TGCCAA)、539-557位(GAGGTGCTTCTTCTGCTAT)、648-666(GGAAGAGACTTTGTCCACA)。
3.双链发卡样DNA sil78、si539和si648与pRNAT-U6.1重组后,都得到多个重组子,PCR扩增筛选鉴定和DNA测序分析证实得到的阳性重组子pRNAT-U6.1-si178、pRNAT-U6.1-si539、pRNAT-U6.1-si648与设计完全一致。
4.过表达PTEN组(转染pCL-neo-PTEN)、沉默Livin 1组(转染pRNAT-U6.1-sil78)、沉默Livin 2组(转染pRNAT-U6.1-si539)、沉默Livin3组(转染pRNAT-U6.1-si648)、空载体1组(转染pCL-neo)和空载体2组(转染pRNAT-U6.1)细胞经G418筛选3w,均得到稳定转染细胞株。
5.与空白对照相比,空载体1组和空载体2组的Livin mRNA和蛋白表达水平无明显变化,差异无显著性(P>0.05)；与3个对照组比较,过表达PTEN组、沉默Livin 1组、沉默Livin 2组和沉默Livin3组细胞Livin mRNA和蛋白表达水平均显著降低,差异有显著性(P<0.05),其中沉默Livin 2组的相对表达量最低。
6.过表达PTEN组细胞的PTEN mRNA和蛋白表达水平显著高于其它各组(P<0.01)
第二部分体外调控PTEN基因和Livin基因表达对胃癌BGC823细胞生物学行为的影响
方法
切下沉默Livin效果最佳siRNA载体中的Livin siRNA单元,重组入PTEN基因表达载体pCL-neo-PTEN,得到表达PTEN基因、同时沉默Livin基因的调控载体pCL-neo-PTEN-siLivin,用LipofectamineTM2000包裹,然后将其转染BGC823细胞,G418筛选得到过表达PTEN同时沉默Livin基因的稳定转染细胞。与第一部分获得的单一过表达PTEN的胃癌BGC823细胞株、单一沉默Livin表达的胃癌BGC823细胞株一起作为实验对象,采用RT-PCR和Western-blot测定各组细胞Livin和PTEN mRNA和蛋白表达水平、MTT方法检测细胞增殖、流式细胞法检测细胞周期和细胞凋亡、EILSA检测Caspase-3和Caspase-9蛋白酶活性、Transwell方法检测细胞侵袭转移能力。
结果
1. EcoRI酶切鉴定重组子,得到2个条带(3.4Kb和3.8Kb),证实Livin siRNA单元成功插入；DNA序列测定确认得到阳性重组子。
2.经G418筛选得到稳定转染pCL-neo-PTEN-siLivin的胃癌BGC823细胞株。
3.与空白对照组和空载体对照组比较,调控组(稳定转染pCL-neo-PTEN-siLivin的BGC823细胞)、沉默Livin组(稳定转染pRNAT-U6.1-siLivin的BGC823细胞)和过表达PTEN组(稳定转染pCL-neo-PTEN的BGC823细胞)的Livin mRNA和蛋白表达水平均明显降低(P<0.05),其中调控组Livin mRNA和蛋白表达水平最低。
4.与空白对照组和空载体对照组比较,过表达PTEN组和调控组细胞PTEN mRNA和蛋白表达水平显著升高,差异有显著性(P<0.01)。而过表达PTEN组和调控组细胞PTEN mRNA和蛋白表达水平无显著性差异(P>0.05),说明在PTEN表达载体pCL-neo-PTEN中插入Livin siRNA单元不影响PTEN表达的水平。
5.在3d、4d和5d MTT检测细胞,与空白对照组和空载体对照组比较,调控组细胞OD值显著降低,差异有显著性(P<0.05)。
6.与空白对照组和空载体对照组比较,调控组、沉默Livin组和过表达PTEN组细胞的Caspase-3和Caspase-9活性显著升高,差异有显著性(P<0.05),其中以调控组的提高更为显著。
7.与空白对照组和空载体对照组比较,调控组、沉默Livin组和过表达PTEN组的细胞凋亡率显著升高(P<0.05)。与沉默Livin组和过表达PTEN组比较,调控组细胞凋亡率明显升高,差异有显著性(P<0.05)。
8.与空白对照组和空载体对照组比较,调控组、沉默Livin组和过表达PTEN组穿透Matrigel的平均细胞数显著降低(P<0.05)。与沉默Livin组和过表达PTEN组比较,调控组穿透Matrigel的平均细胞数明显减少,差异有显著性(P<0.05)。
第三部分调控PTEN/Livin的基因表达对裸鼠移植瘤生长的影响
方法
取调控PTEN/Livin基因表达的胃癌BGC823细胞、过表达PTEN的胃癌BGC823细胞、沉默Livin表达的胃癌BGC823细胞、空载体对照的胃癌BGC823细胞和胃癌BGC823细胞,调整细胞浓度至1×108/ml,在裸鼠右侧腋部皮下接种0.1ml。在注射接种后的第7d开始测定绘制肿瘤生长曲线,第28天测量肿瘤大小后处死裸鼠,取肿瘤组织称重。荧光定量RT-PCR和Western-blot检测PTEN和Livin mRNA和蛋白表达水平。
结果
1.裸鼠皮下细胞接种7d后均出现可见肿瘤块形成；调控组、过表达PTEN组和沉默Livin组移植瘤生长速度明显慢于空白对照组和空载体对照组(P<0.05)。
2.调控组移植瘤平均瘤重215.42±35.15mg、过表达PTEN组移植瘤平均瘤重461.73±58.17mg、沉默Livin组移植瘤平均瘤重368.23±53.72mg,均显著低于空白对照组和空载体对照组移植瘤平均瘤重(1051.29±175.95mg和968.88±194.39mg),差异有显著性(P<0.05),其中以调控组移植瘤平均瘤重最小。
3.与空白对照组、空载体组和沉默Livin组比较,调控组、过表达PTEN组的PTEN mRNA和蛋白表达水平显著升高,差异有显著性(P<0.01)。与空白对照组、空载体组比较,调控组、沉默Livin组、过表达PTEN组的Livin mRNA和蛋白表达水平显著降低,差异有显著性(P<0.05),其中以调控组最低。
结论
1.筛选得到过表达PTEN的胃癌BGC823细胞株、沉默Livin表达的胃癌BGC823细胞株和过表达PTEN同时又沉默Livin表达的胃癌BGC823细胞株。
2.建立一种表达一个基因同时又沉默另一个基因的载体构建方法。
3.过表达PTEN同时沉默Livin的调控可以有效抑制胃癌BGC823细胞的生长增殖、侵袭转移和促进细胞凋亡,比单一过表达PTEN或降低Livin表达的效果更为显著。
4.在胃癌细胞中过表达PTEN同时沉默Livin的复合调控方法有可能成为胃癌治疗的有效策略。
Background and Objective
Gastric cancer, as the fourth most commonly occurring cancer after lung, breast and colorectal cancers in order of incidence, is the main cause of cancer deaths for Chinese. However, the mechanisms for gastric cancer remain unknown by now. Many patients with gastric cancer express nonspecific symptoms in the early stage and some patients reach advanced stage before visiting the hospital. Therefore, although great progresses have been made in surgery, chemotherapy and radiation therapy, the five year survival rate of advanced gastric cancer is still very poor.
Many studies indicated that a lot of factors were involved in the development and progression of gastric cancer, including the loss of anti-oncogene, oncogene mutation, decrease in DNA damage and repair function, abnormality of signaling pathways and apoptosis, indicating that the development and progression of gastric cancer is a process with multifactor, multistage and multistep.
Phosphatase and tensin homology deleted on chromosome ten (PTEN), as an anti-oncogene possessing lipid and protein phosphatase activities, could inhibit cell proliferation and migration, and promote cell apoptosis by inhibiting phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway. It is reported that PTEN deletion, mutation and methylation result in tumorigenesis due to the lack of anti-cancer function. Some studies proved that tissue from gastric cancer developed decreased PTEN and the expression of PTEN was negatively correlated with the size of tumor, Borrmann type, lymph node metastasis and cancer staging, indicating that the loss of PTEN was a possible mechanism for gastric cancer.
Livin, a novel member of inhibitor of apoptosis protein (IAP) family, inhibits apoptosis and promotes the growth of cancer cells by binding to cysteine aspartate-specific protease. Over-expression of livin in many tumor cells including gastric cancer has been reported, and many studies indicated that silencing of livin gene inhibited the proliferation of cancer cells as a result of promoting apoptosis, demonstrating that the over-expression of livin is related to the development, progression and prognosis of tumor.
In this study, three kinds of gastric tumor cell lines with different expression of PTEN and livin were established by using molecular biological techniques, including over-expression of PTEN, silencing of livin, and over-expression of PTEN with silencing of livin simultaneously. In vivo and in vitro experiments were employed to detect the effects of PTEN and livin on growth, proliferation, apoptosis and migration of gastric cancer cells, which provided new targets and methods for therapy of gastric cancer. Generally, this study included three parts as follows:Part 1 included the construction and identification of PTEN gene expression vector and livin gene siRNA vector; part 2 effects of PTEN and livin on biological behaviors of gastric cancer BGC823 cells were studied In vitro; Part 3 revealed the in vivo effects of PTEN and livin genes on growth of cancer in nude mice.
Part 1 Construction and identification of PTEN gene expression vector and Livin gene SiRNA vector
Methods
The amplification primer with restriction enzyme cutting sites of EcoRI and SalI for all coding regions in PTEN gene was designed and used for amplification of pCMV6-PTEN by PCR. After digestion by EcoRI and SalI, PCR products were inserted into pCL-neo vector. Subsequently, the recombinants were identified by EcoRI and SalI digestion and the inserted fragments were sequenced. Three designed siRNA target sequences for livin gene transformed into hairpin-shaped single-chain DNA and subsequent double-chain DNA after annealing, and then was inserted into pRNAT-U6.1 vector. The recombinants were amplified, selected and transformed by PCR and the inserted fragments were sequenced. pCL-neo-PTEN and livin siRNA vectors were transfected into BGC823 cell line by LipofectamineTM2000, and stable transfected cell lines were selected by G418. Fluorescence quantitative RT-PCR and Western-Blot were used to detect mRNA and protein expression level of livin and PTEN in transfected BGC823 cells.
Results
1. PTEN cDNA ORF target gene with 1212bp was acquired by PCR. EcoRI/SalI digestion and DNA sequence analysis indicated that the recombinants of pCL-neo-PTEN was successfully constructed.
2. Fifty candidate fragments were obtained after first selection through scanning of human livin cDNA (NM_139317) by RNAi explorer and siDESIGN center, and subsequent BLAST homological analysis confirmed three siRNA target sequences with 19 bp,178-196nt (GACCTAAAGACAGTGCCAA),539-557 nt (GAGGTG CTTCTTCTGCTAT) and 648-666 nt (GGAAGAGACTTTGTCC ACA).
3. Amplification and selection by PCR and DNA sequence analysis indicated that the recombinants, which were acquired after recombination of hairpin-shaped double-chain DNA (si178, si539 and si648) with pRNAT-U6.1, were in accordance with expected.
4. After selection for three weeks by G418, stable transfected cell lines were acquired in over-expression of PTEN group (transfection of pCL-neo-PTEN), silencing of livin group 1 (transfection of pRNAT-U6.1-si178), silencing of livin group 2 (transfection of pRNAT-U6.1-si539), silencing of livin group 3 (transfection of pRNAT-U6.1-si648), empty vector group 1 (transfection of pCL-neo) and empty vector group 2 (transfection of pRNAT-U6.1).
5. There was no significant difference in mRNA and protein expression level of livin between blank group and empty vector groups (P>0.05). However, mRNA and protein expression level of livin in over-expression of PTEN group, silencing of livin group 1,2 and 3 decreased significantly compared with the above three control groups (P<0.05), and silencing of livin group 2 showed the lowest expression.
6. The mRNA and protein expression level of PTEN in over-expression of PTEN group increased significantly in comparison with other groups (P<0.01).
Part 2 Effects of PTEN and Livin on biological behaviors of gastric cancer BGC823 cells in vitro
Methods
Livin siRNA was cut from livin siRNA vector with the best silencing effect and then was recombined with pCL-neo-PTEN. Thus, pCL-neo-PTEN-siLivin which over-expressed PTEN and silenced livin simultaneously was obtained, and then was transfected into BGC823 cells by LipofectamineTM2000 subsequently. After stable transfected cell line was selected by G418, RT-PCR and Western-blot techniques were used to detect mRNA and protein expression level of livin and PTEN in over-expression of PTEN group, silencing of livin group and over-expression of PTEN with silencing of livin simultaneously group (regulation group). MTT, flow cytometry method, ELISA and Transwell methods were employed to study cell proliferation, cell cycle and apoptosis, activities of caspase-3 and caspase-9 proteinases and cell invasion respectively.
Results
1. After EcoRI digestion of recombinants, two fragments (3.4kb and 3.8kb) were acquired, indicating successful insertion of livin siRNA. Positive recombinants were confirmed by DNA sequence analysis.
2. Gastric cancer BGC823 cell line with stable transfection of pCL-neo-PTEN- siLivin was acquired by G418 selection.
3. The mRNA and protein expression level of livin in regulation group (BGC823 cell line with stable transfection of pCL-neo-PTEN-siLivin), silencing of livin group (BGC823 cell line with stable transfection of pRNAT-U6.1-siLivin) and over-expression of PTEN group (BGC823 cell line with stable transfection of pCL-neo-PTEN) decreased significantly compared with blank group and empty vector group (P<0.05), and regulation group developed the lowest mRNA and expression of livin.
4. Compared with blank group and empty vector group, mRNA and protein expression level of PTEN in over-expression of PTEN group and regulation group increased significantly (P<0.01). There was no significant difference between over-expression of PTEN group and regulation group (P>0.05), indicating that the insertion of livin siRNA into pCL-neo-PTEN did not affect the expression of PTEN.
5. MTT technique revealed that OD value in regulation group decreased significantly at 3,4 and 5 days compared with control groups including blank group and empty vector group (P<0.05).
6. The activities of caspase-3 and caspase-9 in regulation group, silencing of livin group and over-expression of PTEN group increased significantly in comparison with control groups including blank group and empty vector group (P<0.05), and regulation group showed the highest effects.
7. Apoptosis in regulation group, silencing of livin group and over-expression of PTEN group elevated significantly compared with control groups including blank group and empty vector group (P<0.05), and regulation group expressed higher apoptosis compared with silencing of livin group and over-expression of PTEN group (P<0.05).
8. The average cell numbers which passed through matrigel in regulation group, silencing of livin group and over-expression of PTEN group decreased significantly compared with control groups including blank group and empty vector group (P<0.05), and regulation group showed reduced cell invasion compared with silencing of livin group and over-expression of PTEN group (P<0.05).
Part 3 Effects of PTEN/Livin expression on implanted tumor in nude mice
Methods
Different gastric cancer BGC823 cells, including regulation group, over-expression of PTEN group, silencing of livin group, empty vectors group and blank group, were injected subcutaneously into the right oxter of nude mice (0.1mL, 1×108/mL). Tumor-growth curve was found 7 days after injection. Tumors were measured, isolated and weighed on the 28th days. Fluorescence quantitative RT-PCR and Western-blot were used to detect mRNA and protein expression of PTEN and livin.
Results
1. Implanted tumors were observed apparently 7 days after injection. The growth of tumors in regulation group, over-expression of PTEN group and silencing of livin group were significantly lower than those of blank group and empty vector group (P<0.05).
2. The average tumor weight in regulation group, over-expression of PTEN group and silencing of livin group were 215.42±35.15,461.73±58.17 and 368.23±53.72mg respectively, which were significantly lower than those of blank group and empty vector group (1051.29±175.95 and 968.88±194.39mg respectively, P<0.05). Regulation group developed the lowest tumor weight.
3. PTEN mRNA and protein expression level in regulation group and over-expression of PTEN group increased significantly compared with blank group, empty vector group and silencing of livin group (P<0.01). mRNA and protein expression level of livin in regulation group, silencing of livin group and over-expression of PTEN group decreased significantly in comparison with blank group and empty vector group (P<0.05), and regulation group showed the lowest expression.
Conclusions
1. Different gastric cancer BGC823 cell lines, including over-expression of PTEN, silencing of livin, over-expression of PTEN and silencing of livin simultaneously, were established successfully.
2. The technique about how to constructing a vector which expressed one gene and silenced another gene simultaneously was founded.
3. Over-expression of PTEN and silencing of livin simultaneously obviously inhibited growth, proliferation, invasion and migration of gastric cancer cells, which was more effective than that of single over-expression of PTEN or silencing of livin.
4. The complex of over-expression of PTEN and silencing of livin in gastric cancer cells was a potential method for gastric cancer therapy.
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