miR-9、let-7b和let-7g调控Cthrc1基因影响胃癌增殖转移的实验研究
摘要
胃癌是最常见的肿瘤之一,亚洲发病率最高,尤其是韩国、日本和中国。胃癌在全世界肿瘤发病率中居第四位,导致的死亡中列第2位。据统计,2008年全球新发生的胃癌病例大约是989,000例,死亡738,000例。幽门螺杆菌(H.pylori, HP)被归为是胃癌的Ⅰ类致癌原。
微小RNA (miRNA, microRNA)是一类20-22碱基大小的小分子非编码RNA,在转录后水平调节目的基因的表达。MiRNA参与细胞生长、分化、凋亡和增殖的调控。越来越多的证据表明,miRNAs的差异表达在肿瘤的发生发展过程中发挥着重要的作用,一些表现为癌基因的作用,一些类似抑癌基因。
胶原三股螺旋重复蛋白1(collagen triple helix repeat containing1, Cthrc1)基因最早是在动脉损伤过程中筛选出的一种过表达基因,Cthrc1可减少胶原沉积,并促进细胞迁移。最近发现Cthrc1在人类癌症如乳腺癌和黑素瘤中过度表达,参与肿瘤的侵袭和转移过程,但是具体机制仍不明确。
本实验目的在于通过miRNA表达谱芯片技术,筛选胃癌组织中差异表达的miRNAs,并分析其和HP的相关性,为胃癌的诊断和治疗提供理论基础,然后进一步研究这些miRNAs调控胃癌细胞增殖转移的机制。
方法：
胃癌中miRNAs表达谱的筛选和验证
1、采用TaqMan microarray芯片技术筛选胃癌组织中384个miRNAs的表达情况,挑选出明显差异表达的miRNAs,在47对胃癌及对应正常胃粘膜组织采用real-time PCR验证芯片结果；
2、以HP为相关因素分析miRNAs的差异表达,通过HP刺激细胞来观察miRNAs的表达差异。
miR-9、let-7b、let-7g调控Cthrc1基因影响胃癌的增殖转移
1、生物信息学预测iR-9、let-7b、let-7g的靶基因,免疫组化、western-blot以及qRT-PCR检测靶基因在胃癌中的表达,分析靶基因表达与临床病理学参数之间的关系。
2、体外转染miR-9、let-7b、let-7g模拟物及抑制物,荧光素酶报告基因实验检验这三个miRNAs与Cthrcl3'-UTR的直接靶向关系,western blot检测其对Cthrc1蛋白水平的影响。
3、构建Cthrc1过表达/干扰的慢病毒载体,转染至胃癌细胞中,利用MTT研究Cthrc1对胃癌细胞增殖的影响,Transwell法检测Cthrc1对胃癌细胞迁移侵袭能力的影响。
结果：
胃癌中miRNAs表达谱的筛选和验证
1、芯片结果显示let-7b、let-7g、miR-9、miR-133a、miR-133b、miR-139-5p、 miR-141、miR-145、miR-204及miR-212在胃癌组织中低表达；
2、qRT-PCR进一步验证显示let-7b、let-7g、miR-9和miR-204在胃癌组织中显著下调,并且let-7b、let-7g、miR-9在HP阳性的组织中表达更低；
3、胃癌细胞SGC-7901中let-7b、let-7g和miR-9的表达较GES-1明显下调,HP刺激后下降更明显。
miR-9、let-7b、let-7g调控Cthrc1基因影响胃癌的增殖转移
1、生物信息学预测Cthrc1是miR-9、let-7b、let-7g的共同靶基因,免疫组化结果显示Cthrcl在胃癌组织中高表达,且与胃癌的淋巴结转移相关。Western-blot及qRT-PCR检测胃癌细胞株中Cthrc1的表达,发现胃癌细胞中Cthrc1表达升高,以SGC-7901最明显。
2、通过转染]miR-9、let-7b、let-7g的模拟物、抑制物以及荧光素酶报告基因实验,证实Cthrc1是miR-9、let-7b、let-7g共同的特异性靶基因之一
3、过表达Cthrc1的GES-1细胞增殖侵袭和迁移能力加强,干扰表达Cthrc1的SGC-7901细胞增殖侵袭和迁移能力减弱。
结论：
1、miR-9、let-7b和let-7g在胃癌组织中低表达,且HP阳性的胃癌组织和细胞表达更低。
2、Cthrc1是miR-9、let-7b和let-7g共同的特异性靶基因之一。
3、miR-9、let-7b和let-7g在胃癌组织和胃癌细胞株中低表达,导致对Cthrc1的抑制作用减弱,Cthrc1的表达升高,促进胃癌细胞的增殖、侵袭和迁移能力。
Gastric cancer is one of the most common cancer, especilly in Asia included Korea,Japan and China.And gastric cancer is the second leading cause of cancer-related death worldside,which is the first in China.In2008, the number of new cases of stomach cancer was989,000and the estimated death reached738,000worldside.H.pylori (HP) is condsided as the I class carcinogen of gastric cancer.
MicroRNAs (miRNAs) are small non-coding RNAs of20-22nucleotides that regulate expression of target mRNAs at post transcriptional level. MiRNAs are involved in crucial biological processes, including development, differentiation, apoptosis and proliferation.Accumulating evidence suggests that alterations of miRNAs expression may play various roles in the pathogenesis of many human cancers. Some miRNAs have been shown to possess oncogenic or tumor suppressor activity.
Collagen triple helix repeat protein (collagen triple helix repeat containing1,Cthrc1) gene was first found highexpresion in the process of arterial injury. Cthrcl can reduce the deposition of collagen and promote cell migration. Recently,Cthrc1was revealed overexpression in human cancers such as breast cancer and melanoma, involved in tumor invasion and metastasis, but the exact mechanism remains unclear.
In the current study, we screened the miRNAs expression profile in gastric cancer using Taqman Low Density Array (TLDA) chips followed by individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays in tissues and cells respectivelly.We further invesgated the mechanism of these miRNAs regulating the proliferation and metastasis of gastric cancer cell.
Methods:
Aberrant miRNAs in gastric cancer
1.384miRNAs expression profiles in8pairs of gastric cancer tissues were analyzed using microarray assay,the obviously dyregulation miRNAs were confirmed by real-time RT PCR in the gastric cancer tissues in47cases which29HP(+) cases and18HP(-) cases.
2. The expression levelsin SGC-7901and GES-1cells were further analyzed by real-time PCR,which in cells after HPstimulated were also examined.
miR-9, let-7b and let-7g suppress Cthrcl expression as tumor-suppressor gene in gastric cancer
1. We used three microRNA (miRNA) target prediction programs, and information from the existing literature to predict the target genes of miR-9, let-7b and let-7g. To test the Cthrcl expression in gastric cancer and adjacent normal tissues, immunohistochemistry was performed in47pairs of gastric tissue samples, and then we analyzed the clinicopathological characteristics in47gastric tumors;Then we investigated Cthrcl expression by qRT-PCR and western blot in gastric cancer cell lines.
2. Transfection in vitro with mimics or inhibitors of miR-9, let-7b and let-7gwas used to observe the binding relationship of the miRNAs with Cthrc13'-UTR via luciferase reporter assay test, western blot was detected Cthrcl protein levels.
3. After constructingCthrc1over-expression/RNA interference lentiviral vectors, we screened the stable Cthrcl-overexpressing and Cthrcl-knockdown gastric cancer cell lines.The cellular growth activity was measured by MTT assay.The cellular migration and invasion ability were detected by transwell assay.
Results:
Aberrant miRNAs in gastric cancer
1. Ten miRNAs were down regulated in gastric cancer(>2folds),included let-7b,let-7g,miR-9,miR-133a,miR-133b,miR-139-5p,miR-141,miR-145, miR-204and miR-212.
2. The miRNAs (miR-9, let-7b,let-7g and miR-204) were identified and consistently validated to be significantly down-regulatedin47paires of gastric cancer tissues. Meanwhile, we also found that the expression levels of miR-9,let-7b and let-7gwere decreased in H.pylori infection samples.
3. The levels of miR-9,let-7b and let-7g were also siginificantly decreased in SGC-7901cells,which were especially after being infected with HP.
miR-9, let-7b and let-7g suppress Cthrcl expression as tumor-suppressor gene in gastric cancer
1. Using bioinformatic tools, we predicted that Cthrc1might be the most potent target gene of themiR-9, let-7b and let-7g. In addition, we found that Cthrcl expression is dramatically increased both in gastric cancer tissues and cell lines, and is also related with lymph node metastasis.
2. Cthrc1was the direct target gene of miR-9, let-7b and let-7g via transfecting the miRNAs mimics, inhibitor and luciferase reporter assay.
3. Cthrcl knockdown and overexpression markedly affected the proliferative ability of gastriccancer cell lines.Overexpression of Cthrcl promoted the migration and invasion abilities of GES-1cells. Whereas knockdown of Cthrcl attenuated the migration and invasion ability ofSGC-7901cells.
Conclusion:
1. miR-9,let-7b and let-7gwere down expressed in gastric cancer tissuesandSGC-7901cells.ThesemiRNAswere low-regulated in HP infectionsamples providing a new direction to study the relationship between HPinfection and gastric cancer.
2. Cthrc1was the direct target of miR-9, let-7b and let-7g.
3. Down-regulated miR-9, let-7b and let-7g reduced the inhibition of Cthrcl,which inducedthe expression of Cthrcl and promoted themigration and invasion abilities of gastric cancer cells.
微小RNA (miRNA, microRNA)是一类20-22碱基大小的小分子非编码RNA,在转录后水平调节目的基因的表达。MiRNA参与细胞生长、分化、凋亡和增殖的调控。越来越多的证据表明,miRNAs的差异表达在肿瘤的发生发展过程中发挥着重要的作用,一些表现为癌基因的作用,一些类似抑癌基因。
胶原三股螺旋重复蛋白1(collagen triple helix repeat containing1, Cthrc1)基因最早是在动脉损伤过程中筛选出的一种过表达基因,Cthrc1可减少胶原沉积,并促进细胞迁移。最近发现Cthrc1在人类癌症如乳腺癌和黑素瘤中过度表达,参与肿瘤的侵袭和转移过程,但是具体机制仍不明确。
本实验目的在于通过miRNA表达谱芯片技术,筛选胃癌组织中差异表达的miRNAs,并分析其和HP的相关性,为胃癌的诊断和治疗提供理论基础,然后进一步研究这些miRNAs调控胃癌细胞增殖转移的机制。
方法：
胃癌中miRNAs表达谱的筛选和验证
1、采用TaqMan microarray芯片技术筛选胃癌组织中384个miRNAs的表达情况,挑选出明显差异表达的miRNAs,在47对胃癌及对应正常胃粘膜组织采用real-time PCR验证芯片结果；
2、以HP为相关因素分析miRNAs的差异表达,通过HP刺激细胞来观察miRNAs的表达差异。
miR-9、let-7b、let-7g调控Cthrc1基因影响胃癌的增殖转移
1、生物信息学预测iR-9、let-7b、let-7g的靶基因,免疫组化、western-blot以及qRT-PCR检测靶基因在胃癌中的表达,分析靶基因表达与临床病理学参数之间的关系。
2、体外转染miR-9、let-7b、let-7g模拟物及抑制物,荧光素酶报告基因实验检验这三个miRNAs与Cthrcl3'-UTR的直接靶向关系,western blot检测其对Cthrc1蛋白水平的影响。
3、构建Cthrc1过表达/干扰的慢病毒载体,转染至胃癌细胞中,利用MTT研究Cthrc1对胃癌细胞增殖的影响,Transwell法检测Cthrc1对胃癌细胞迁移侵袭能力的影响。
结果：
胃癌中miRNAs表达谱的筛选和验证
1、芯片结果显示let-7b、let-7g、miR-9、miR-133a、miR-133b、miR-139-5p、 miR-141、miR-145、miR-204及miR-212在胃癌组织中低表达；
2、qRT-PCR进一步验证显示let-7b、let-7g、miR-9和miR-204在胃癌组织中显著下调,并且let-7b、let-7g、miR-9在HP阳性的组织中表达更低；
3、胃癌细胞SGC-7901中let-7b、let-7g和miR-9的表达较GES-1明显下调,HP刺激后下降更明显。
miR-9、let-7b、let-7g调控Cthrc1基因影响胃癌的增殖转移
1、生物信息学预测Cthrc1是miR-9、let-7b、let-7g的共同靶基因,免疫组化结果显示Cthrcl在胃癌组织中高表达,且与胃癌的淋巴结转移相关。Western-blot及qRT-PCR检测胃癌细胞株中Cthrc1的表达,发现胃癌细胞中Cthrc1表达升高,以SGC-7901最明显。
2、通过转染]miR-9、let-7b、let-7g的模拟物、抑制物以及荧光素酶报告基因实验,证实Cthrc1是miR-9、let-7b、let-7g共同的特异性靶基因之一
3、过表达Cthrc1的GES-1细胞增殖侵袭和迁移能力加强,干扰表达Cthrc1的SGC-7901细胞增殖侵袭和迁移能力减弱。
结论：
1、miR-9、let-7b和let-7g在胃癌组织中低表达,且HP阳性的胃癌组织和细胞表达更低。
2、Cthrc1是miR-9、let-7b和let-7g共同的特异性靶基因之一。
3、miR-9、let-7b和let-7g在胃癌组织和胃癌细胞株中低表达,导致对Cthrc1的抑制作用减弱,Cthrc1的表达升高,促进胃癌细胞的增殖、侵袭和迁移能力。
Gastric cancer is one of the most common cancer, especilly in Asia included Korea,Japan and China.And gastric cancer is the second leading cause of cancer-related death worldside,which is the first in China.In2008, the number of new cases of stomach cancer was989,000and the estimated death reached738,000worldside.H.pylori (HP) is condsided as the I class carcinogen of gastric cancer.
MicroRNAs (miRNAs) are small non-coding RNAs of20-22nucleotides that regulate expression of target mRNAs at post transcriptional level. MiRNAs are involved in crucial biological processes, including development, differentiation, apoptosis and proliferation.Accumulating evidence suggests that alterations of miRNAs expression may play various roles in the pathogenesis of many human cancers. Some miRNAs have been shown to possess oncogenic or tumor suppressor activity.
Collagen triple helix repeat protein (collagen triple helix repeat containing1,Cthrc1) gene was first found highexpresion in the process of arterial injury. Cthrcl can reduce the deposition of collagen and promote cell migration. Recently,Cthrc1was revealed overexpression in human cancers such as breast cancer and melanoma, involved in tumor invasion and metastasis, but the exact mechanism remains unclear.
In the current study, we screened the miRNAs expression profile in gastric cancer using Taqman Low Density Array (TLDA) chips followed by individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays in tissues and cells respectivelly.We further invesgated the mechanism of these miRNAs regulating the proliferation and metastasis of gastric cancer cell.
Methods:
Aberrant miRNAs in gastric cancer
1.384miRNAs expression profiles in8pairs of gastric cancer tissues were analyzed using microarray assay,the obviously dyregulation miRNAs were confirmed by real-time RT PCR in the gastric cancer tissues in47cases which29HP(+) cases and18HP(-) cases.
2. The expression levelsin SGC-7901and GES-1cells were further analyzed by real-time PCR,which in cells after HPstimulated were also examined.
miR-9, let-7b and let-7g suppress Cthrcl expression as tumor-suppressor gene in gastric cancer
1. We used three microRNA (miRNA) target prediction programs, and information from the existing literature to predict the target genes of miR-9, let-7b and let-7g. To test the Cthrcl expression in gastric cancer and adjacent normal tissues, immunohistochemistry was performed in47pairs of gastric tissue samples, and then we analyzed the clinicopathological characteristics in47gastric tumors;Then we investigated Cthrcl expression by qRT-PCR and western blot in gastric cancer cell lines.
2. Transfection in vitro with mimics or inhibitors of miR-9, let-7b and let-7gwas used to observe the binding relationship of the miRNAs with Cthrc13'-UTR via luciferase reporter assay test, western blot was detected Cthrcl protein levels.
3. After constructingCthrc1over-expression/RNA interference lentiviral vectors, we screened the stable Cthrcl-overexpressing and Cthrcl-knockdown gastric cancer cell lines.The cellular growth activity was measured by MTT assay.The cellular migration and invasion ability were detected by transwell assay.
Results:
Aberrant miRNAs in gastric cancer
1. Ten miRNAs were down regulated in gastric cancer(>2folds),included let-7b,let-7g,miR-9,miR-133a,miR-133b,miR-139-5p,miR-141,miR-145, miR-204and miR-212.
2. The miRNAs (miR-9, let-7b,let-7g and miR-204) were identified and consistently validated to be significantly down-regulatedin47paires of gastric cancer tissues. Meanwhile, we also found that the expression levels of miR-9,let-7b and let-7gwere decreased in H.pylori infection samples.
3. The levels of miR-9,let-7b and let-7g were also siginificantly decreased in SGC-7901cells,which were especially after being infected with HP.
miR-9, let-7b and let-7g suppress Cthrcl expression as tumor-suppressor gene in gastric cancer
1. Using bioinformatic tools, we predicted that Cthrc1might be the most potent target gene of themiR-9, let-7b and let-7g. In addition, we found that Cthrcl expression is dramatically increased both in gastric cancer tissues and cell lines, and is also related with lymph node metastasis.
2. Cthrc1was the direct target gene of miR-9, let-7b and let-7g via transfecting the miRNAs mimics, inhibitor and luciferase reporter assay.
3. Cthrcl knockdown and overexpression markedly affected the proliferative ability of gastriccancer cell lines.Overexpression of Cthrcl promoted the migration and invasion abilities of GES-1cells. Whereas knockdown of Cthrcl attenuated the migration and invasion ability ofSGC-7901cells.
Conclusion:
1. miR-9,let-7b and let-7gwere down expressed in gastric cancer tissuesandSGC-7901cells.ThesemiRNAswere low-regulated in HP infectionsamples providing a new direction to study the relationship between HPinfection and gastric cancer.
2. Cthrc1was the direct target of miR-9, let-7b and let-7g.
3. Down-regulated miR-9, let-7b and let-7g reduced the inhibition of Cthrcl,which inducedthe expression of Cthrcl and promoted themigration and invasion abilities of gastric cancer cells.
引文
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