乳腺癌细胞增殖与转移相关miRNA的功能研究
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摘要
微小RNA(micro-RNA,miRNA)是一类长约22nt的非编码功能小RNA,它在许多生物中都有表达。miRNA原初转录本(pri-miRNA)经过核内和胞质中一系列的加工修饰后形成成熟的miRNA。首先,miRNA基因在RNA polymeraseⅡ作用下,转录生成pri-miRNA;然后在内切酶Drosha作用下,pri-miRNA释放出60—70nt的前体(pre-miRNA),后者具有不完美的发卡结构;最后,pre-miRNA在exportin5作用下进入胞质,经DICER切割形成成熟miRNA。成熟的miRNA通过与靶基因3'非翻译区(3'UTR区)形成不完全配对,从而抑制靶基因翻译,或形成接近完全的配对,导致靶mRNA降解。最近发现在人类基因组中,大约有533个miRNA基因座位,数目大约为蛋白编码基因的1%,但可以调控大约30%的人类基因,说明miRNA是一个调控网络。
miRNA在很多生物的生理和病理过程中发挥重要作用,影响细胞发育、凋亡和增殖,并与疾病发生相关。越来越多的证据表明,许多肿瘤的发生与发展与miRNA的异常表达相关,例如乳腺癌、脑癌、慢性粒细胞白血病、结肠癌、肝癌和肺癌等。
本论文以乳腺癌表达异常的miRNA为研究对象,开展了下述的系列研究。
第一部分研究显示,某些在肿瘤组织中低表达的miRNA能够抑制癌基因或细胞生长相关的基因,因此具有抑癌基因的活性,例如let-7家族。
为了寻找具有抑癌活性的miRNA,根据过去对乳腺癌表达谱的报道和一些开放数据库,本研究挑选了10个在乳腺癌中低表达的miRNA,它们依次是:mir-30d、mir-99a、mir-100、mir-125b1、mir-130a、mir-126/126*、mir-145、mir-191、mir-199a和mir-214。
我们构建了上述10个候选miRNA的表达载体,通过在细胞中对它们进行过量表达,检测它们对细胞的影响。结果发现,mir-126可以显著抑制细胞的增殖。进一步实验表明,mir-126可以抑制细胞从G0/G1期进入S期。
通过生物信息软件预测,结合体外靶基因3'UTR luciferase报告基因验证,我们发现mir-126可以调控IRS-1的表达。Western blot和real-time PCR实验表明,mir-126并不影响IRS-1mRNA水平,而是通过影响翻译导致IRS-1蛋白表达量下降,结果抑制了HEK293和乳腺癌细胞MCF-7的增殖。定量分析显示,在HEK293、HUVEC和MDA-MB-231中,IRS-1和mir-126的表达呈负相关。
该部分研究确定了mir-126能够抑制细胞从G/G0进入S期,IRS-1是其靶基因。结合其它学者的研究成果,我们推测mir-126可能通过作用于IGF1R-PI3K/Akt信号通路,产生抑制细胞增殖的效应。
第二部分
成熟miRNA分子量较小,所以检测比较困难。目前用于检测miRNA的方法主要有以下三种:1)基于分子杂交的Northern Blot。该方法需要较多的总RNA,且经常要使用同位素,操作繁琐,不适用于大通量检测。2)基于Stem-loop RT-PCR的检测方法。这种方法要求每个miRNA都需要特异的反转录,操作更为繁琐,对样本和试剂的耗费较多,特异性也比较低。为了提高特异性,有些试剂盒使用Taqman探针进行检测,价格随之大幅提升。所以这个方法也不适用于大样本、多个miRNA的检测;3)microarray芯片检测。目前很多公司都推出相关产品,但花费高,操作也不够灵活机动。
经过多次摸索,我们建立了polyA RT-PCR检测miRNA的方法。该方法操作简单,适用于中等规模的检测,有望在临床中获得应用。
使用polyA RT-PCR,我们检测了侵袭能力不同的乳腺癌细胞中22种miRNA的表达情况,结果显示mir-29a/c、mir-99a、mir-100和mir-125b1在高侵袭性乳腺癌细胞中高表达,而mir-191则低表达。这些结果最终经real-time RT-PCR得到了确认。更多乳腺癌细胞中的检测结果显示,mir-29a/c、mir-99a、mir-100和mir-125b1同样在高侵袭性细胞中高表达,但mir-191被排除。同时,我们又引入taqman通用探针对其精确的定量,得到了同样的结果,通用taqman探针的引入也使得polyA RT-PCR检测方法更为精确。
为了探索这些miRNA与乳腺癌之间的关系,我们使用了miRNA inhibitor对miRNA进行功能封闭,实验结果显示,mir-125b封闭剂会明显抑制细胞增殖并降低高侵袭乳腺癌细胞MDA-MB-231和BT-549穿透martrigel的能力。另外,在低侵袭性乳腺痛细胞MCF7中种构建了mir-99a、mir-100和mir-125b1的稳定表达细胞株,这为我们下一步进行动物体内试验做好了准备。
这些只是初步的实验结果,我们还将通过一系列实验检测这些miRNA对乳腺癌细胞的影响。它们对肿瘤侵袭转移,以及成瘤能力的影响最终还要在动物实验中进一步确认。
第三部分
HOXA10是抑癌基因,它可以诱导P53表达,还能抑制癌基因SNAIL的表达。NOXA10在高侵袭乳腺癌中常因甲基化而低表达甚至不表达。但临床试验表明,HOXA10在高恶性乳腺组织中mRNA的表达量并不一定降低。我们的实验结果也表明,高侵袭乳腺癌细胞BT549的HOXA10mRNA表达水平较高。我们推测,HOXA10可能存在mRNA水平上的调控。
通过生物信息学分析,结合3'UTR luciferase报告基因检测方法,我们发现mir-135a可以显著下调HOXA10的表达。采用定点突变的方法,我们确定了mir-135a在HOXA10 3'-UTR区的作用位点。PCR检测显示,在高侵袭乳腺癌细胞中,mir-135a和HOXA10 mRNA有共分布现象。
miRNA inhibitor结果显示,mir-135a的封闭可以导致BT-549细胞侵袭能力的下降。
我们将进一步确认mir-135a在乳腺癌侵袭中的地位,并验证mir-135a对HOXA10的调控作用。
MicroRNAs(miRNAs) are a class of approximately 22-nuleotide-long noncoding RNAs which are expressed in many organisms.Mature miRNAs arise from one arm of endogenous hairpin transcripts by sequential processing in the nucleus and cytoplasm.First,miRNAs are transcribed by RNA polymeraseⅡas the long primary miRNAs(pri-miRNAs) in the nucleus. Second,the pri-miRNAs are cleaved by the endonuclease Drosba to release the shorter precursor miRNA(pre-miRNA),which are 60- to 70-nucleotide-long imperfect hairpin structure.Finally,pre-miRNAs are exported to cytoplasm by exportin-5 and are processed by the endonuclease DICER to generate the 22-nucleotide RNA duplexes,one strand of which is the mature miRNA,miRNAs regulate target genes post-transcriptionally, by inhibiting the translation through imperfect base-pairing interaction with the 3' -untranslated regions(3' -UTRs) of their respective target genes,or degrading their target mRNAs through perfect or near-perfect base pairing.Recent large-scale studies have revealed that there are about 533 miRNA loci in huamn.The number of miRNAs may be more than 1% of the total protein-coding genes,but about 30%of protein- coding genes are regulated by miRNAs.
miRNAs have important roles in many biological processes,such as development,cell death,proliferation,differentiation and disease.And increasing evidences show that mutation and differential expression of some specific miRNAs are associated with the formation and progress of many types of cancers,including brain cancer,chronic lymphocytic leukemia,colorectal neoplasia,hepatocellular carcinoma,lung cancer, lymphomas,papillary thyroid carcinoma,testicular germ cell tumors,etc.
Breast cancer is one of the most common cancers in adult females and studies showed that breast cancer was also associated with the deregulation of miRNAs.The expression of miRNAs have been examined in a wide range of breast cancer cell lines,and in clinical normal and cancer breast tissues,but less is knowed about their exact mechanism.We carried out our reseash in function of miRNA associated with breast cancer.
Part 1
Previous studies showed that the underexpressed miRNAs in cancers, such as let-7,may function as tumor suppressor genes and inhibit cancers by regulating oncogenes and genes that control cell differentiation or apoptosis.In order to investigate the tumor suppressor miRNAs in breast cancer,we focused on the miRNAs whose expression decrease in breast cancer.According to some miRNA databases and the published miRNAs profiles in breast cancer and other types of cancers,we chose 10 miRNA candidates,including mir-30d,99a,100,125b1,130a,126/126*,145,191, 199a and 214.
We assayed their effects on cell by overexpressing them.We found that mir-126 could strongly suppress cell growth in HEK293 by inhibiting the cell cycle transition from G1/G0 to S1 phase and had the same effect on breast cancer cell MCF-7,which was in accordance with the recent finding that mir-126 can reduce metastasis of malignant cells(CN34) obtained From the pleural fluid of a patient with metastatic breast cancer,by significantly suppressing growth of tumor volume in mice.We went on to investigate the mechanism of proliferation suppression of mir-126.
To identify the mRNA targets of mir-126,we performed a computational screen for genes with mir-126 complementary sites in their 3'-UTR using several open access database.Considering the the suppressing effect of mir-126 on cell cycle progression,5 genes which have a high prediction score and are associated with cell growth were chosen.3'-UTR luciferase report assay suggested that mir-126 negatively regulated IRS-1.Further studies showed that mir-126 suppressed translation of IRS-1 mRNA and had no effects on mRNA level.And knockdown of IRS-1 can also suppress cell growth in HEK293 and breast cnacer cell MCF-7,which recapitulate the effects of mir-126.Quatative assay revealed that expression of IRS-1 protein was negatively correlated with mir-126.
In a conclusion,this part of work revealed that mir-126 suppressed cell growth by inhibiting cell cycle transition from G0/G1 to S phase. Considering recent reports,we supposed that mir-126 supressed cell growth by intervening IGF1R-PI3K/Akt signal pathway.
Part 2
miRNAs are difficult to detect due to their small sizes(about 20 nt). Besides northern blot,most of traditional methods for miRNA detection are based on step-loop PCR.These methods are tedious and costly for high-throughput screening because each miRNA needs one specific revere transcription(RT) reaction.For example,if we used stem-loop RT-PCR to detect 30 miRNAs in 4 cell lines,120 reverse transcription reactions would be needed in addition to 4 reactions for reference gene,which is very sample-demanding.Because there are only 6 bp-complement between miRNA template and its specific RT primer,step-loop PCR often has low specificity and amplification efficiency.So Taqman probes are often used but it is costly.We developed a simple method called poly(A) RT PCR,which has many advantages:1) it is simple.One sample only needs one RT reaction to detect different miRNAs;2) the efficiency of RT reaction is high because of the 13 bp-complement between anchor RT primer and tailed RNA templates;3) it has high specificity because of the additional 12-(dT) distance between the binding sites of miRNA-specific forward primer and the universal reverse primer.
With this method,we detected expression of 22 miRNA in breast cancer cell lines with different metastasis potentials.Results showed that mir-29a/c,mir-99a,mir-100 and mir-125b1 were up-regulated in high metastasis breast cancer cell lines and mir-191 was down-regulated in high metastasis breast cancer cell line,which was confirmed by real-time RT-PCR.Except mir-191,the same results were recaptulated in other breast cancer cell lines.And we used universal Taqman probe to quantify the miRNAs and got the results accordant with previous ones.Use of the universal taqman probes made polyA RT-PCR more precise.
miRNA inhibitor assay revealed that inhibition of mir-125b can suppress cell growth and reduced the ability of penetrating martrigel in breast cancer cell lines MDA-MB-231 and BT-549.In order to investigate the role of the 3 miRNAs in breast cnacer cell metatasis,we construted the MCF-7 cell lines stably expressing mir-99a,mir-100 and mir-125b1.
In this part of our work,we constructed a new and simple method detecting miRNA.With this method,we found 4 miRNA was associated with breast cancer line metatastasis and mir-125b may be involed in cell proliferation an invision.Research of these 4 miRNAs is being procceded and the effects of the 4 miRNAs on metatasis will be determined by animal experiment in vivo.And we will detect their expression in breast cancer tissues with different metatasis.
We believe that our results will be meaningful to research of breast cancer miRNAs and miRNA application in diagnosis and treatment of breast cancer.Due to the advantages,polyA RT-PCR will find much application in future.
Part 3
HOXA10 induces expression of P53 and reduces expression of SNAIL,so HOXA10 acts as tumor suppressor.Previous studies showed that HOXA10 is often silenced by promoter methylation in high metastic breast cancer cells and tissues.But some clinical researches showed that there were no redcued expression of HOXA10 in some high metastic breast cancer samples,and we detected a high expression level of HOXA10 in high metastic breast cancer cell BT-549.So we suspected that,like many other members of HOX gene family,HOXA10 may be regulated at the translation level by miRNAs.
By computational screening,we selected two candidate miRNAs-mir-196a and mir-135a.With 3' UTR luciferase assay,we found mir-135a can significant reduced the report gene activity of HOXA10 3' UTR,and this result was confirmed by mutating the predicted mir-135a binding site at HOXA10 3' UTR.And RT-PCR results showed that mir-135a is highly co-expressed with HOXA10 in BT-549.
Inhibition of mir-135a reduced cell invision in breast cancer cell BT549.
A recent report showed mir-135,as a tumor suppressor,can regulate the Adenomatous Polyposis Coli(APC) gene in colorectal cancer.This part of our work first preliminariely revealed that mir-135a can suppressed HOXA10 and is asscociated with cell invision.We will investigate the function of mir-135a in progress of breast cancer and confirm the regulation of HOXA10 by mir-135a.
miRNA在很多生物的生理和病理过程中发挥重要作用,影响细胞发育、凋亡和增殖,并与疾病发生相关。越来越多的证据表明,许多肿瘤的发生与发展与miRNA的异常表达相关,例如乳腺癌、脑癌、慢性粒细胞白血病、结肠癌、肝癌和肺癌等。
本论文以乳腺癌表达异常的miRNA为研究对象,开展了下述的系列研究。
第一部分研究显示,某些在肿瘤组织中低表达的miRNA能够抑制癌基因或细胞生长相关的基因,因此具有抑癌基因的活性,例如let-7家族。
为了寻找具有抑癌活性的miRNA,根据过去对乳腺癌表达谱的报道和一些开放数据库,本研究挑选了10个在乳腺癌中低表达的miRNA,它们依次是:mir-30d、mir-99a、mir-100、mir-125b1、mir-130a、mir-126/126*、mir-145、mir-191、mir-199a和mir-214。
我们构建了上述10个候选miRNA的表达载体,通过在细胞中对它们进行过量表达,检测它们对细胞的影响。结果发现,mir-126可以显著抑制细胞的增殖。进一步实验表明,mir-126可以抑制细胞从G0/G1期进入S期。
通过生物信息软件预测,结合体外靶基因3'UTR luciferase报告基因验证,我们发现mir-126可以调控IRS-1的表达。Western blot和real-time PCR实验表明,mir-126并不影响IRS-1mRNA水平,而是通过影响翻译导致IRS-1蛋白表达量下降,结果抑制了HEK293和乳腺癌细胞MCF-7的增殖。定量分析显示,在HEK293、HUVEC和MDA-MB-231中,IRS-1和mir-126的表达呈负相关。
该部分研究确定了mir-126能够抑制细胞从G/G0进入S期,IRS-1是其靶基因。结合其它学者的研究成果,我们推测mir-126可能通过作用于IGF1R-PI3K/Akt信号通路,产生抑制细胞增殖的效应。
第二部分
成熟miRNA分子量较小,所以检测比较困难。目前用于检测miRNA的方法主要有以下三种:1)基于分子杂交的Northern Blot。该方法需要较多的总RNA,且经常要使用同位素,操作繁琐,不适用于大通量检测。2)基于Stem-loop RT-PCR的检测方法。这种方法要求每个miRNA都需要特异的反转录,操作更为繁琐,对样本和试剂的耗费较多,特异性也比较低。为了提高特异性,有些试剂盒使用Taqman探针进行检测,价格随之大幅提升。所以这个方法也不适用于大样本、多个miRNA的检测;3)microarray芯片检测。目前很多公司都推出相关产品,但花费高,操作也不够灵活机动。
经过多次摸索,我们建立了polyA RT-PCR检测miRNA的方法。该方法操作简单,适用于中等规模的检测,有望在临床中获得应用。
使用polyA RT-PCR,我们检测了侵袭能力不同的乳腺癌细胞中22种miRNA的表达情况,结果显示mir-29a/c、mir-99a、mir-100和mir-125b1在高侵袭性乳腺癌细胞中高表达,而mir-191则低表达。这些结果最终经real-time RT-PCR得到了确认。更多乳腺癌细胞中的检测结果显示,mir-29a/c、mir-99a、mir-100和mir-125b1同样在高侵袭性细胞中高表达,但mir-191被排除。同时,我们又引入taqman通用探针对其精确的定量,得到了同样的结果,通用taqman探针的引入也使得polyA RT-PCR检测方法更为精确。
为了探索这些miRNA与乳腺癌之间的关系,我们使用了miRNA inhibitor对miRNA进行功能封闭,实验结果显示,mir-125b封闭剂会明显抑制细胞增殖并降低高侵袭乳腺癌细胞MDA-MB-231和BT-549穿透martrigel的能力。另外,在低侵袭性乳腺痛细胞MCF7中种构建了mir-99a、mir-100和mir-125b1的稳定表达细胞株,这为我们下一步进行动物体内试验做好了准备。
这些只是初步的实验结果,我们还将通过一系列实验检测这些miRNA对乳腺癌细胞的影响。它们对肿瘤侵袭转移,以及成瘤能力的影响最终还要在动物实验中进一步确认。
第三部分
HOXA10是抑癌基因,它可以诱导P53表达,还能抑制癌基因SNAIL的表达。NOXA10在高侵袭乳腺癌中常因甲基化而低表达甚至不表达。但临床试验表明,HOXA10在高恶性乳腺组织中mRNA的表达量并不一定降低。我们的实验结果也表明,高侵袭乳腺癌细胞BT549的HOXA10mRNA表达水平较高。我们推测,HOXA10可能存在mRNA水平上的调控。
通过生物信息学分析,结合3'UTR luciferase报告基因检测方法,我们发现mir-135a可以显著下调HOXA10的表达。采用定点突变的方法,我们确定了mir-135a在HOXA10 3'-UTR区的作用位点。PCR检测显示,在高侵袭乳腺癌细胞中,mir-135a和HOXA10 mRNA有共分布现象。
miRNA inhibitor结果显示,mir-135a的封闭可以导致BT-549细胞侵袭能力的下降。
我们将进一步确认mir-135a在乳腺癌侵袭中的地位,并验证mir-135a对HOXA10的调控作用。
MicroRNAs(miRNAs) are a class of approximately 22-nuleotide-long noncoding RNAs which are expressed in many organisms.Mature miRNAs arise from one arm of endogenous hairpin transcripts by sequential processing in the nucleus and cytoplasm.First,miRNAs are transcribed by RNA polymeraseⅡas the long primary miRNAs(pri-miRNAs) in the nucleus. Second,the pri-miRNAs are cleaved by the endonuclease Drosba to release the shorter precursor miRNA(pre-miRNA),which are 60- to 70-nucleotide-long imperfect hairpin structure.Finally,pre-miRNAs are exported to cytoplasm by exportin-5 and are processed by the endonuclease DICER to generate the 22-nucleotide RNA duplexes,one strand of which is the mature miRNA,miRNAs regulate target genes post-transcriptionally, by inhibiting the translation through imperfect base-pairing interaction with the 3' -untranslated regions(3' -UTRs) of their respective target genes,or degrading their target mRNAs through perfect or near-perfect base pairing.Recent large-scale studies have revealed that there are about 533 miRNA loci in huamn.The number of miRNAs may be more than 1% of the total protein-coding genes,but about 30%of protein- coding genes are regulated by miRNAs.
miRNAs have important roles in many biological processes,such as development,cell death,proliferation,differentiation and disease.And increasing evidences show that mutation and differential expression of some specific miRNAs are associated with the formation and progress of many types of cancers,including brain cancer,chronic lymphocytic leukemia,colorectal neoplasia,hepatocellular carcinoma,lung cancer, lymphomas,papillary thyroid carcinoma,testicular germ cell tumors,etc.
Breast cancer is one of the most common cancers in adult females and studies showed that breast cancer was also associated with the deregulation of miRNAs.The expression of miRNAs have been examined in a wide range of breast cancer cell lines,and in clinical normal and cancer breast tissues,but less is knowed about their exact mechanism.We carried out our reseash in function of miRNA associated with breast cancer.
Part 1
Previous studies showed that the underexpressed miRNAs in cancers, such as let-7,may function as tumor suppressor genes and inhibit cancers by regulating oncogenes and genes that control cell differentiation or apoptosis.In order to investigate the tumor suppressor miRNAs in breast cancer,we focused on the miRNAs whose expression decrease in breast cancer.According to some miRNA databases and the published miRNAs profiles in breast cancer and other types of cancers,we chose 10 miRNA candidates,including mir-30d,99a,100,125b1,130a,126/126*,145,191, 199a and 214.
We assayed their effects on cell by overexpressing them.We found that mir-126 could strongly suppress cell growth in HEK293 by inhibiting the cell cycle transition from G1/G0 to S1 phase and had the same effect on breast cancer cell MCF-7,which was in accordance with the recent finding that mir-126 can reduce metastasis of malignant cells(CN34) obtained From the pleural fluid of a patient with metastatic breast cancer,by significantly suppressing growth of tumor volume in mice.We went on to investigate the mechanism of proliferation suppression of mir-126.
To identify the mRNA targets of mir-126,we performed a computational screen for genes with mir-126 complementary sites in their 3'-UTR using several open access database.Considering the the suppressing effect of mir-126 on cell cycle progression,5 genes which have a high prediction score and are associated with cell growth were chosen.3'-UTR luciferase report assay suggested that mir-126 negatively regulated IRS-1.Further studies showed that mir-126 suppressed translation of IRS-1 mRNA and had no effects on mRNA level.And knockdown of IRS-1 can also suppress cell growth in HEK293 and breast cnacer cell MCF-7,which recapitulate the effects of mir-126.Quatative assay revealed that expression of IRS-1 protein was negatively correlated with mir-126.
In a conclusion,this part of work revealed that mir-126 suppressed cell growth by inhibiting cell cycle transition from G0/G1 to S phase. Considering recent reports,we supposed that mir-126 supressed cell growth by intervening IGF1R-PI3K/Akt signal pathway.
Part 2
miRNAs are difficult to detect due to their small sizes(about 20 nt). Besides northern blot,most of traditional methods for miRNA detection are based on step-loop PCR.These methods are tedious and costly for high-throughput screening because each miRNA needs one specific revere transcription(RT) reaction.For example,if we used stem-loop RT-PCR to detect 30 miRNAs in 4 cell lines,120 reverse transcription reactions would be needed in addition to 4 reactions for reference gene,which is very sample-demanding.Because there are only 6 bp-complement between miRNA template and its specific RT primer,step-loop PCR often has low specificity and amplification efficiency.So Taqman probes are often used but it is costly.We developed a simple method called poly(A) RT PCR,which has many advantages:1) it is simple.One sample only needs one RT reaction to detect different miRNAs;2) the efficiency of RT reaction is high because of the 13 bp-complement between anchor RT primer and tailed RNA templates;3) it has high specificity because of the additional 12-(dT) distance between the binding sites of miRNA-specific forward primer and the universal reverse primer.
With this method,we detected expression of 22 miRNA in breast cancer cell lines with different metastasis potentials.Results showed that mir-29a/c,mir-99a,mir-100 and mir-125b1 were up-regulated in high metastasis breast cancer cell lines and mir-191 was down-regulated in high metastasis breast cancer cell line,which was confirmed by real-time RT-PCR.Except mir-191,the same results were recaptulated in other breast cancer cell lines.And we used universal Taqman probe to quantify the miRNAs and got the results accordant with previous ones.Use of the universal taqman probes made polyA RT-PCR more precise.
miRNA inhibitor assay revealed that inhibition of mir-125b can suppress cell growth and reduced the ability of penetrating martrigel in breast cancer cell lines MDA-MB-231 and BT-549.In order to investigate the role of the 3 miRNAs in breast cnacer cell metatasis,we construted the MCF-7 cell lines stably expressing mir-99a,mir-100 and mir-125b1.
In this part of our work,we constructed a new and simple method detecting miRNA.With this method,we found 4 miRNA was associated with breast cancer line metatastasis and mir-125b may be involed in cell proliferation an invision.Research of these 4 miRNAs is being procceded and the effects of the 4 miRNAs on metatasis will be determined by animal experiment in vivo.And we will detect their expression in breast cancer tissues with different metatasis.
We believe that our results will be meaningful to research of breast cancer miRNAs and miRNA application in diagnosis and treatment of breast cancer.Due to the advantages,polyA RT-PCR will find much application in future.
Part 3
HOXA10 induces expression of P53 and reduces expression of SNAIL,so HOXA10 acts as tumor suppressor.Previous studies showed that HOXA10 is often silenced by promoter methylation in high metastic breast cancer cells and tissues.But some clinical researches showed that there were no redcued expression of HOXA10 in some high metastic breast cancer samples,and we detected a high expression level of HOXA10 in high metastic breast cancer cell BT-549.So we suspected that,like many other members of HOX gene family,HOXA10 may be regulated at the translation level by miRNAs.
By computational screening,we selected two candidate miRNAs-mir-196a and mir-135a.With 3' UTR luciferase assay,we found mir-135a can significant reduced the report gene activity of HOXA10 3' UTR,and this result was confirmed by mutating the predicted mir-135a binding site at HOXA10 3' UTR.And RT-PCR results showed that mir-135a is highly co-expressed with HOXA10 in BT-549.
Inhibition of mir-135a reduced cell invision in breast cancer cell BT549.
A recent report showed mir-135,as a tumor suppressor,can regulate the Adenomatous Polyposis Coli(APC) gene in colorectal cancer.This part of our work first preliminariely revealed that mir-135a can suppressed HOXA10 and is asscociated with cell invision.We will investigate the function of mir-135a in progress of breast cancer and confirm the regulation of HOXA10 by mir-135a.
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