RNA聯(lián)合干擾STAT3及Survivin對(duì)膀胱癌細(xì)胞的抑制作用研究
發(fā)布時(shí)間：2018-08-22 20:47
【摘要】：研究背景： 膀胱癌是指發(fā)生在膀胱粘膜上的惡性腫瘤。在西方，膀胱癌發(fā)病率僅次于前列腺癌，居第2位；在我國(guó)，膀胱癌是十大常見(jiàn)腫瘤之一，是泌尿系統(tǒng)發(fā)病率和死亡率雙第一的惡性腫瘤。膀胱癌可發(fā)生于任何年齡，甚至于兒童，2012年全國(guó)腫瘤登記地區(qū)膀胱癌的發(fā)病率為6.61/10萬(wàn)，列惡性腫瘤發(fā)病率的第9位。男性發(fā)病率為女性的3～4倍，2013年男性膀胱癌發(fā)病率為10/10萬(wàn)，列惡性腫瘤的第六位。 膀胱癌具有多灶性、多基因參與、多階段異質(zhì)性生長(zhǎng)、高發(fā)病率、高復(fù)發(fā)率等惡性生物學(xué)特性，治療難度大，膀胱癌目前的治療方法主要包括手術(shù)、放射性治療和術(shù)后膀胱腔內(nèi)灌注抗癌藥物，該方法對(duì)患者損傷大，病人生活質(zhì)量嚴(yán)重下降，而且該方法無(wú)法解決高復(fù)發(fā)率（60%以上）和高耐藥性的難題。在這種情況下，小干擾RNA基因靶向療法引起了科研人員的極大關(guān)注。 RNA干擾(RNA interference)是一種轉(zhuǎn)錄后的基因沉默，RNA經(jīng)切割后，觸發(fā)某種轉(zhuǎn)錄后監(jiān)控程序，識(shí)別有同源序列的mRNA，對(duì)其產(chǎn)生特異性切割，從而阻斷其翻譯功能，該方法能夠從基因水平上抑制癌基因的過(guò)度表達(dá)，卻不干擾正常細(xì)胞的生理活動(dòng)。利用siRNA進(jìn)行特異性基因抑制己被證實(shí)在病毒感染性疾病及腫瘤治療中具有極大的潛力，是極有希望根治膀胱癌的最新方法。 基因治療的關(guān)鍵是靶點(diǎn)，我們選擇了STAT3和Survivin作為實(shí)驗(yàn)對(duì)象。 STAT3是腫瘤多條信號(hào)傳導(dǎo)通路的連接點(diǎn)與調(diào)控環(huán)節(jié)，它在多種腫瘤中持續(xù)性激活且高量表達(dá)，并全程參與了腫瘤的侵襲轉(zhuǎn)移、血 III管發(fā)生、凋亡抵抗和免疫逃避等過(guò)程，是已知的最重要的癌基因之一。 Survivin是凋亡抑制蛋白家族的新成員，它通過(guò)多個(gè)途徑抑制效應(yīng)性蛋白酶，從而促進(jìn)轉(zhuǎn)化細(xì)胞的異常增殖。Survivin在所有常見(jiàn)惡性腫瘤中表達(dá)，在正常組織中無(wú)表達(dá)。作為一種廣泛的腫瘤抗原，，Survivin可以作為抗腫瘤免疫治療的一個(gè)廣泛適用的靶基因。 目的： 有越來(lái)越多的證據(jù)表明，信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3（STAT3）和凋亡抑制基因Survivin在膀胱癌的發(fā)生發(fā)展中起到了重要作用，但是沉默這兩種基因?qū)Π螂装┘?xì)胞T24的影響是未知的。因此，我們觀察在體內(nèi)、體外沉默STAT3和Survivin基因后，T24細(xì)胞的生長(zhǎng)情況，為膀胱癌基因治療的提供理論依據(jù)和靶點(diǎn)。 方法： 1網(wǎng)絡(luò)在線設(shè)計(jì)兩組小干擾RNA，分別靶向抑制STAT3、Survivin兩種基因，生物信息學(xué)方法驗(yàn)證siRNA的特異性和有效性； 2采用Invitrogen試劑盒制備表達(dá)載體，瞬時(shí)轉(zhuǎn)染T24細(xì)胞； 3用實(shí)時(shí)熒光定量PCR檢測(cè)轉(zhuǎn)染前后STAT3和Survivin的基因表達(dá)情況；Western blot比較轉(zhuǎn)染前后表達(dá)水平的差異；MTT實(shí)驗(yàn)測(cè)定細(xì)胞的增殖及細(xì)胞活性； 4將兩條干擾效果最好的siRNA串聯(lián)建立復(fù)合干擾RNA，用上述方法檢驗(yàn)干擾效果； 5建立裸鼠膀胱癌移植瘤模型，測(cè)定干擾對(duì)腫瘤體積和重量的影響，以分析siRNA的體內(nèi)干擾效果。 數(shù)據(jù)均用SPSS軟件分析v17.0（IBM，阿蒙克，NY，USA）。兩組間比較使用t檢驗(yàn)。采用單向方差分析確定多組間統(tǒng)計(jì)學(xué)意義（方差分析）。數(shù)據(jù)均值±標(biāo)準(zhǔn)偏差（SD）。P＜0.05被認(rèn)為有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1.成功構(gòu)建并合成了四個(gè)STAT3siRNA和四個(gè)Survivin siRNA。 2.四個(gè)siRNA中的三個(gè)顯著降低了STAT3的表達(dá)，其中STAT3-3是最有效的STAT3抑制劑。所有的Survivin siRNA有效的下調(diào)Survivin表達(dá)，核酸與蛋白含量檢測(cè)結(jié)果表明，Survivin-4是最有效的Survivin抑制劑。而且，質(zhì)粒STAT3-3和Survivin-4分別能顯著降低STAT3和Survivin的表達(dá)，兩者串聯(lián)的復(fù)合干擾RNA也能顯著降低STAT3和Survivin的表達(dá)，三者在核酸和細(xì)胞水平上的干擾效果沒(méi)有顯著差異。 3.體內(nèi)檢測(cè)結(jié)果表明，上述質(zhì)粒干擾顯著降低裸鼠膀胱癌移植瘤模型的腫瘤體積和重量，差異有統(tǒng)計(jì)學(xué)意義。 結(jié)論： 研究結(jié)果表明，我們?cè)O(shè)計(jì)的siRNA能有效抑制STAT3和Survivin基因在T24細(xì)胞中的表達(dá)，從而抑制膀胱癌細(xì)胞T24的增殖。將兩種siRNA串聯(lián)在同一質(zhì)粒中，抑制效果同樣明顯，為基因藥物的研制探索了新的途徑。同時(shí)抑制這兩種基因不能顯著提高在T24細(xì)胞中的抗癌效果，這表明它們可能在T24細(xì)胞同一信號(hào)傳導(dǎo)通路發(fā)揮作用。這些結(jié)果為研究膀胱癌的發(fā)生發(fā)展提供了寶貴的材料，也為膀胱癌臨床基因治療指明了靶向目標(biāo)。
[Abstract]:Research background:
Bladder cancer is a malignant tumor that occurs on the bladder mucosa. In the west, the incidence of bladder cancer is second only to prostate cancer, ranking second; in our country, bladder cancer is one of the ten common tumors, is the first malignant tumor in the incidence and mortality of the urinary system. Bladder cancer can occur at any age, even in children, in 2012 the national cancer. The incidence of bladder cancer was 6.61/100,000, ranking ninth in the incidence of malignant tumors. Male incidence was 3-4 times that of females. Male incidence of bladder cancer was 10/100,000 in 2013, ranking sixth in the incidence of malignant tumors.
Bladder cancer has many malignant biological characteristics, such as multi-focal, multi-gene participation, multi-stage heterogeneous growth, high incidence, high recurrence rate, and so on. The treatment of bladder cancer is very difficult. Current treatment methods mainly include surgery, radiotherapy and postoperative intravesical instillation of anticancer drugs. This method is harmful to patients, and the quality of life of patients is seriously reduced. In this case, small interfering RNA gene targeting therapy has attracted great attention of researchers.
RNA interference is a kind of gene silencing after transcription. After RNA is cut, it triggers a post-transcriptional monitoring program, identifies the mRNA with homologous sequence and produces specific cleavage to it, thus blocking its translation function. This method can inhibit the overexpression of oncogenes at the gene level, but does not interfere with the physiological activities of normal cells. Specific gene suppression with siRNA has been demonstrated to have great potential in the treatment of viral infectious diseases and tumors, and is a promising new method for radical treatment of bladder cancer.
The key of gene therapy is target. We chose STAT3 and Survivin as experimental objects.
STAT3 is the junction and regulation link of multiple signal transduction pathways in tumors. It is persistently activated and overexpressed in various tumors and participates in the invasion and metastasis of tumors.
III tube formation, apoptosis resistance and immune escape are one of the most important oncogenes known.
Survivin is a new member of the inhibitor of apoptosis protein family. It inhibits effector proteases through multiple pathways and promotes the abnormal proliferation of transformed cells. Survivin is expressed in all common malignant tumors but not in normal tissues. As a wide range of tumor antigens, Survivin can be used as a wide range of anti-tumor immunotherapy. Suitable target genes.
Objective:
There is increasing evidence that signal transduction and activator of transcription 3 (STAT3) and apoptosis suppressor gene Survivin play an important role in the development of bladder cancer, but the effect of silencing these two genes on bladder cancer cell T24 is unknown. The growth condition provides theoretical basis and target for gene therapy of bladder cancer.
Method:
1. Two groups of small interfering RNA were designed on-line, targeting STAT3 and Survivin, respectively. Bioinformatics methods were used to verify the specificity and effectiveness of siRNA.
2 the expression vector was prepared by Invitrogen kit and transiently transfected into T24 cells.
The expression of STAT 3 and Survivin was detected by real-time fluorescence quantitative PCR, the expression of STAT 3 and Survivin was compared by Western blot, and the proliferation and cell viability were measured by MTT assay.
4 establish two interference siRNA with the best interference in series to establish Compound Jamming RNA.
5 To establish a nude mouse model of bladder cancer xenograft and determine the effect of interference on tumor volume and weight in order to analyze the effect of siRNA interference in vivo.
The data were analyzed with SPSS software v17.0 (IBM, Amund, NY, USA). The t test was used for comparison between the two groups. The one-way ANOVA was used to determine the statistical significance among the groups (ANOVA). The data mean (+) standard deviation (SD). P < 0.05 was considered statistically significant.
Result:
1. four STAT3siRNA and four Survivin siRNA. were successfully constructed and synthesized.
2. Three of the four siRNA significantly reduced the expression of STAT3, of which STAT3-3 was the most effective STAT3 inhibitor. All of the survivivin siRNA effectively down-regulated the expression of Survivin. Nucleic acid and protein assays showed that Survivin-4 was the most effective Survivin inhibitor. Moreover, plasmid STAT3-3 and Survivin-4 significantly decreased the expression of STAT3 and Surivin-4, respectively. The expression of vivin and the expression of STAT3 and Survivin were also significantly decreased by the combined interfering RNA in series. There was no significant difference between the interfering effect of the three on nucleic acid and cell level.
3. The results of in vivo detection showed that the above plasmid interference significantly reduced the tumor volume and weight of nude mice bladder cancer xenograft tumor model, the difference was statistically significant.
Conclusion:
The results showed that the siRNA designed by us could effectively inhibit the expression of STAT3 and Survivin genes in T24 cells and thus inhibit the proliferation of bladder cancer cell line T24. The inhibition effect of two siRNAs in the same plasmid was also obvious, which explored a new way for the development of gene drugs. These results provide valuable materials for the study of the genesis and development of bladder cancer and point out the target of gene therapy for bladder cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14
[Abstract]:Research background:
Bladder cancer is a malignant tumor that occurs on the bladder mucosa. In the west, the incidence of bladder cancer is second only to prostate cancer, ranking second; in our country, bladder cancer is one of the ten common tumors, is the first malignant tumor in the incidence and mortality of the urinary system. Bladder cancer can occur at any age, even in children, in 2012 the national cancer. The incidence of bladder cancer was 6.61/100,000, ranking ninth in the incidence of malignant tumors. Male incidence was 3-4 times that of females. Male incidence of bladder cancer was 10/100,000 in 2013, ranking sixth in the incidence of malignant tumors.
Bladder cancer has many malignant biological characteristics, such as multi-focal, multi-gene participation, multi-stage heterogeneous growth, high incidence, high recurrence rate, and so on. The treatment of bladder cancer is very difficult. Current treatment methods mainly include surgery, radiotherapy and postoperative intravesical instillation of anticancer drugs. This method is harmful to patients, and the quality of life of patients is seriously reduced. In this case, small interfering RNA gene targeting therapy has attracted great attention of researchers.
RNA interference is a kind of gene silencing after transcription. After RNA is cut, it triggers a post-transcriptional monitoring program, identifies the mRNA with homologous sequence and produces specific cleavage to it, thus blocking its translation function. This method can inhibit the overexpression of oncogenes at the gene level, but does not interfere with the physiological activities of normal cells. Specific gene suppression with siRNA has been demonstrated to have great potential in the treatment of viral infectious diseases and tumors, and is a promising new method for radical treatment of bladder cancer.
The key of gene therapy is target. We chose STAT3 and Survivin as experimental objects.
STAT3 is the junction and regulation link of multiple signal transduction pathways in tumors. It is persistently activated and overexpressed in various tumors and participates in the invasion and metastasis of tumors.
III tube formation, apoptosis resistance and immune escape are one of the most important oncogenes known.
Survivin is a new member of the inhibitor of apoptosis protein family. It inhibits effector proteases through multiple pathways and promotes the abnormal proliferation of transformed cells. Survivin is expressed in all common malignant tumors but not in normal tissues. As a wide range of tumor antigens, Survivin can be used as a wide range of anti-tumor immunotherapy. Suitable target genes.
Objective:
There is increasing evidence that signal transduction and activator of transcription 3 (STAT3) and apoptosis suppressor gene Survivin play an important role in the development of bladder cancer, but the effect of silencing these two genes on bladder cancer cell T24 is unknown. The growth condition provides theoretical basis and target for gene therapy of bladder cancer.
Method:
1. Two groups of small interfering RNA were designed on-line, targeting STAT3 and Survivin, respectively. Bioinformatics methods were used to verify the specificity and effectiveness of siRNA.
2 the expression vector was prepared by Invitrogen kit and transiently transfected into T24 cells.
The expression of STAT 3 and Survivin was detected by real-time fluorescence quantitative PCR, the expression of STAT 3 and Survivin was compared by Western blot, and the proliferation and cell viability were measured by MTT assay.
4 establish two interference siRNA with the best interference in series to establish Compound Jamming RNA.
5 To establish a nude mouse model of bladder cancer xenograft and determine the effect of interference on tumor volume and weight in order to analyze the effect of siRNA interference in vivo.
The data were analyzed with SPSS software v17.0 (IBM, Amund, NY, USA). The t test was used for comparison between the two groups. The one-way ANOVA was used to determine the statistical significance among the groups (ANOVA). The data mean (+) standard deviation (SD). P < 0.05 was considered statistically significant.
Result:
1. four STAT3siRNA and four Survivin siRNA. were successfully constructed and synthesized.
2. Three of the four siRNA significantly reduced the expression of STAT3, of which STAT3-3 was the most effective STAT3 inhibitor. All of the survivivin siRNA effectively down-regulated the expression of Survivin. Nucleic acid and protein assays showed that Survivin-4 was the most effective Survivin inhibitor. Moreover, plasmid STAT3-3 and Survivin-4 significantly decreased the expression of STAT3 and Surivin-4, respectively. The expression of vivin and the expression of STAT3 and Survivin were also significantly decreased by the combined interfering RNA in series. There was no significant difference between the interfering effect of the three on nucleic acid and cell level.
3. The results of in vivo detection showed that the above plasmid interference significantly reduced the tumor volume and weight of nude mice bladder cancer xenograft tumor model, the difference was statistically significant.
Conclusion:
The results showed that the siRNA designed by us could effectively inhibit the expression of STAT3 and Survivin genes in T24 cells and thus inhibit the proliferation of bladder cancer cell line T24. The inhibition effect of two siRNAs in the same plasmid was also obvious, which explored a new way for the development of gene drugs. These results provide valuable materials for the study of the genesis and development of bladder cancer and point out the target of gene therapy for bladder cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14
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