miR-29及其靶基因COLI在增生性瘢痕形成中的作用及機制研究
發(fā)布時間：2018-07-27 15:52
【摘要】：背景:增生性瘢痕(HS)是皮膚創(chuàng)傷愈合過程中常見的并發(fā)癥,發(fā)生率高達4~16%,嚴(yán)重影響組織功能恢復(fù)和外觀。膠原的合成與降解代謝失衡是HS形成的根本原因,但導(dǎo)致膠原持續(xù)過度合成的病因?qū)W與分子機制尚不十分明確,臨床上對HS的治療是一大臨床挑戰(zhàn)。盡管采用了許多治療方法,但其治療效果卻不盡相同。因此,需要探尋新的治療靶點。在我們前期的研究中,基因芯片檢測顯示,正常皮膚(NS)組織和HS組織中micro RNA(miRNA)分子的表達存在明顯差異,這些miRNA分子所調(diào)節(jié)的靶基因涵蓋了細胞生命活動的多個方面,如細胞凋亡與增殖、周期與運動、合成與代謝,其中miR-29的差異最明顯。而目前有研究認(rèn)為,miR-29可以在轉(zhuǎn)錄后水平抑制膠原的合成,從而在多種纖維化疾病的發(fā)生中起重要的調(diào)控作用。但miR-29與HS形成關(guān)系的研究尚未見文獻報道。因此,我們推測,持續(xù)低水平表達的miR-29喪失了對COLI基因表達的有效調(diào)控,進而導(dǎo)致膠原為主的細胞外基質(zhì)(ECM)的過度沉積,最終形成HS。如果施用正義miR-29提高HS局部組織中miR-29表達水平,將有可能成功抑制組織的過度纖維化過程,為HS的臨床防治提供新的靶點。目的:通過miRNA表達譜芯片篩選出HS組織及NS組織中顯著差異表達的miRNA分子,探討miR-29通過在轉(zhuǎn)錄后水平調(diào)控其靶基因COLI表達從而影響HS形成的作用機制,以期為HS的生物學(xué)治療提供新的靶點。方法:1.收集HS組織和NS組織標(biāo)本,經(jīng)過HE染色后證明所取臨床標(biāo)本符合實驗需要。分離并培養(yǎng)人HS及NS原代成纖維細胞。通過miRNA基因芯片篩選出HS及NS中差異表達的miRNA分子,并采用生物信息學(xué)方法預(yù)測miR-29下游靶基因。2.進一步使用熒光定量PCR方法及western blot法明確人NS與HS中miR-29c及COLI基因的差異性表達,并采用卡方檢驗或Fisher精確檢驗進行分析,探討miR-29與COLI基因表達水平的相關(guān)性;3.構(gòu)建COLI A1-3’UTR及其3種突變體的重組熒光素酶報告基因載體,分析正常成纖維細胞和瘢痕成纖維細胞中COLI A1-3’UTR熒光素酶報告載體活性是否存在顯著差異,miR-29c模擬劑是否能顯著降低報告載體的活性,從而驗證COLI是否為miR-29c下游的有效靶基因。4.構(gòu)建和包裝人miR-29c過表達腺病毒,分別轉(zhuǎn)染培養(yǎng)的NS成纖維細胞及HS成纖維細胞,采用流式細胞分析法及CCK-8法檢測HS成纖維細胞在感染腺病毒過表達miR-29c后,細胞的增殖與凋亡是否受到顯著影響。結(jié)果:1.收集的HS和NS組織標(biāo)本經(jīng)過HE染色后均證明符合實驗需要。免疫熒光染色鑒定HS組織及NS組織原代培養(yǎng)所得的細胞確實為成纖維細胞;2.基因芯片技術(shù)篩選發(fā)現(xiàn),HS組織中miR-143、miR-32、miR-92b及miR-25等45個miRNA的表達水平與NS組織相比上調(diào),同時miR-29a、miR-29b、miR-29c、miR-713、miR-195等68個miRNA顯著表達下調(diào),提示它們可能在HS的發(fā)生發(fā)展過程中發(fā)揮作用。其中,miR-29的改變最為顯著,在12個HS樣本中miR-29分子表達均顯著低于NS組織(平均降低15.3倍)。3.熒光定量PCR方法對篩選出的miRNA-29在HS及NS組織中的表達情況進行檢測,結(jié)果發(fā)現(xiàn),無論是在組織中還是原代成纖維細胞中,miR-29c在HS中的表達顯著低于NS組織。而miRNA-29家族的其他成員在HS/NS中的表達量均無顯著差異。4.應(yīng)用生物信息學(xué)數(shù)據(jù)庫及軟件對miR-29靶基因進行預(yù)測,初步推測COLI是miR-29可能的靶基因。熒光定量PCR及Western Blot檢測表明COLI基因在NS組織中的表達量顯著低于HS組織,COLI在HS組織及NS組織原代培養(yǎng)所得的成纖維細胞中的表達量與組織中表達趨勢相符,卡方檢驗分析發(fā)現(xiàn),無論在HS及NS組織中還是在原代培養(yǎng)的成纖維細胞中,miR-29與COLI基因表達水平均具有顯著負(fù)相關(guān)性。5.構(gòu)建了COLI基因及miR-29c相應(yīng)結(jié)合區(qū)的突變體熒光素酶報告基因載體,酶切及測序證實構(gòu)建成功。熒光素酶活性的檢測結(jié)果顯示:瘢痕成纖維細胞中,COLI A1-3’UTR熒光素酶報告載體活性顯著高于正常成纖維細胞。在正常成纖維細胞及增生性瘢痕成纖維細胞中,miR-29c模擬劑均能有效降低COLI A1-3’UTR熒光素酶報告載體活性,卻不能有效降低其突變體的熒光素酶報告載體活性。進一步說明miR-29c可直接作用于COLI A1-3’UTR從而發(fā)揮作用。6.成功構(gòu)建和包裝人miR-29c過表達腺病毒后,分別轉(zhuǎn)染培養(yǎng)的NS成纖維細胞及HS成纖維細胞,采用定量PCR結(jié)合WB檢測發(fā)現(xiàn)miR-29過表達后COLI基因表達顯著受到抑制,細胞增殖實驗及流式細胞分析發(fā)現(xiàn)miR-29過表達后成纖維細胞的增殖率顯著提高而凋亡率顯著下降。結(jié)論:1.基因芯片技術(shù)篩選發(fā)現(xiàn),HS組織中miR-143、miR-32、miR-92b及miR-25等45個miRNA的表達水平與NS組織相比上調(diào),同時miR-29a、miR-29b、miR-29c、miR-713、miR-195等68個miRNA顯著表達下調(diào),提示它們可能在HS的發(fā)生發(fā)展過程中發(fā)揮作用。其中,miR-29的表達差異最為顯著。2.在無論是組織還是細胞,miR-29c在HS中的表達均顯著低于NS,降低HS成纖維細胞中miR-29c的表達可以促進其增殖并抑制其凋亡,提示miR-29在HS的發(fā)生發(fā)展中發(fā)揮負(fù)調(diào)節(jié)作用。3.COLI基因是miR-29c直接作用的下游靶基因,miR-29對COLI基因表達發(fā)揮負(fù)調(diào)控作用,miR-29在增生性瘢痕組織中的持續(xù)性低水平表達使其對靶基因COL1表達喪失了有效的調(diào)控,造成COL1基因過表達,導(dǎo)致膠原為主的ECM的過度沉積,最終促進了增生性瘢痕組織的形成。miR-29c有可能成為增生性瘢痕治療的新靶點。
[Abstract]:Background: hypertrophic scar (HS) is a common complication in the process of skin wound healing. The occurrence rate is up to 4~16%, which seriously affects the recovery and appearance of tissue function. The synthesis of collagen and the imbalance of degradation metabolism are the fundamental causes of the formation of HS. However, the etiology and molecular mechanism of the continuous hypersynthesis of collagen are not very clear. The treatment of HS is clinically. Therapy is a major clinical challenge. Although many treatments are used, the therapeutic effect is not the same. Therefore, new therapeutic targets need to be explored. In our previous study, gene chip detection showed that the expression of micro RNA (miRNA) subdivision in normal skin (NS) tissues and HS tissues was significantly different from those of miRNA molecules. The target gene covers many aspects of cell life activities, such as cell apoptosis and proliferation, cycle and movement, synthesis and metabolism, in which the difference of miR-29 is most obvious. At present, studies have suggested that miR-29 can inhibit the synthesis of collagen at post transcriptional level and thus play an important role in the development of various fibrotic diseases. But miR-29 and HS The study of the formation of the relationship has not yet been reported. Therefore, we speculate that the continuous low level expression of miR-29 has lost the effective regulation of the expression of COLI gene, leading to the excessive deposition of collagen based extracellular matrix (ECM), and eventually the formation of HS. may be successful if the miR-29 expression level in the HS local tissue is developed by the use of justice miR-29. The process of inhibiting the excessive fibrosis of the tissues provides new targets for the clinical prevention and control of HS. Objective: to screen out the significant differential miRNA molecules expressed in HS tissues and NS tissues by miRNA expression chip, and to explore the mechanism of miR-29 by regulating the expression of the target gene COLI in the post transcriptional level and affecting the formation of HS, so as to be a biological treatment for HS. New therapeutic targets were provided. Methods: 1. the specimens of HS and NS tissues were collected. After HE staining, it was proved that the clinical specimens were in conformity with the experimental needs. The human HS and NS primary fibroblasts were isolated and cultured. The miRNA molecules expressed differently in HS and NS were screened by miRNA gene chip, and the target gene.2. in the downstream of miR-29 was predicted by bioinformatics. Further using the fluorescence quantitative PCR method and the Western blot method to clarify the differential expression of miR-29c and COLI genes in human NS and HS, and using chi square test or Fisher accurate test to analyze the correlation between miR-29 and COLI gene expression level. 3. the recombinant luciferase reporter gene vector of COLI A1-3 'and its 3 mutants was constructed. Whether there are significant differences in the activity of COLI A1-3 'UTR luciferase reporter vector in normal fibroblasts and scar fibroblasts, whether miR-29c simulant can significantly reduce the activity of the reported carrier, thus verifying whether COLI is an effective target gene for the downstream of miR-29c,.4. construction and packaging of human miR-29c overexpressed adenovirus, respectively. NS fibroblasts and HS fibroblasts were cultured. The proliferation and apoptosis of HS fibroblasts were detected by flow cytometry and CCK-8 method. Results: 1. the specimens of HS and NS collected from HS and NS proved to be in accordance with the experimental needs. Immunofluorescence staining was used. The cells in the primary culture of HS tissue and NS tissue were fibroblasts. 2. gene chip technology screening showed that the expression levels of 45 miRNA in HS tissues were up regulated compared with NS tissues, while miR-29a, miR-29b, miR-29c, etc. In the process of the development of HS, the change of miR-29 is the most significant. In 12 HS samples, the expression of miR-29 molecules is significantly lower than that of NS tissue (averaging 15.3 times). The.3. fluorescence quantitative PCR method is used to detect the expression of the selected miRNA-29 in HS and NS tissues. The results have been found, both in the tissue and in the original generation. In the fibrous cells, the expression of miR-29c in HS was significantly lower than that of NS tissue. There was no significant difference in the expression of other members of the miRNA-29 family in HS/NS..4. applied bioinformatics database and software to predict the miR-29 target gene. It was preliminarily presumed that COLI was a possible target gene for miR-29. Fluorescent quantitative PCR and Western Blot test showed that COLI The expression of gene in NS tissues was significantly lower than that in HS tissue. The expression of COLI in the fibroblasts derived from HS and NS tissues was consistent with the expression trend in the tissue. The chi square test found that both miR-29 and COLI gene expression levels were significant in both HS and NS tissues and in the primary cultured fibroblasts. The negative correlation.5. was used to construct the COLI gene and the mutant luciferase reporter gene vector of the corresponding binding region of miR-29c. The enzyme activity was confirmed by enzyme digestion and sequencing. The results of luciferase activity showed that the activity of COLI A1-3 'UTR luciferase reporter vector was significantly higher than normal fibroblast in scar fibroblasts. In vascular and hypertrophic scar fibroblasts, miR-29c simulants can effectively reduce the activity of the COLI A1-3 'UTR luciferase reporter carrier, but it can not effectively reduce the activity of the luciferase reporter vector of the mutant, and further indicates that miR-29c can directly act on COLI A1-3' UTR to play a role of.6. successfully construct and package the miR-29 of the COLI A1-3 'UTR. After C overexpressed the adenovirus, the cultured NS fibroblasts and HS fibroblasts were transfected respectively. The quantitative PCR combined with WB detection showed that the expression of COLI gene was significantly inhibited after miR-29 overexpression. The proliferation test and flow cytometry showed that the proliferation rate of fibroblasts increased significantly and the apoptosis rate decreased significantly after miR-29 overexpression. Conclusion: 1. gene chip technology screening showed that the expression levels of 45 miRNA, such as miR-143, miR-32, miR-92b and miR-25 in HS tissues were up regulated compared with those of NS tissues, while miR-29a, miR-29b, miR-29c, miR-713, miR-195 and so on were down regulated, suggesting that they might play a role in the development of the miRNA. The most significant.2. in tissues and cells, the expression of miR-29c in HS is significantly lower than that of NS, and the reduction of miR-29c in HS fibroblasts can promote its proliferation and inhibit its apoptosis, suggesting that miR-29 plays a negative regulatory role in the development of HS, the.3.COLI gene is the downstream target gene directly acting on miR-29c, miR-29 on COLI genes. The expression of negative regulation in the expression of miR-29 in hypertrophic scar tissue makes it lose effective regulation on the expression of target gene COL1, resulting in overexpression of COL1 gene, resulting in the overdeposition of collagen based ECM, which ultimately promotes the formation of hypertrophic scar tissue that may become a hypertrophic scar treatment. New target.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R622
[Abstract]:Background: hypertrophic scar (HS) is a common complication in the process of skin wound healing. The occurrence rate is up to 4~16%, which seriously affects the recovery and appearance of tissue function. The synthesis of collagen and the imbalance of degradation metabolism are the fundamental causes of the formation of HS. However, the etiology and molecular mechanism of the continuous hypersynthesis of collagen are not very clear. The treatment of HS is clinically. Therapy is a major clinical challenge. Although many treatments are used, the therapeutic effect is not the same. Therefore, new therapeutic targets need to be explored. In our previous study, gene chip detection showed that the expression of micro RNA (miRNA) subdivision in normal skin (NS) tissues and HS tissues was significantly different from those of miRNA molecules. The target gene covers many aspects of cell life activities, such as cell apoptosis and proliferation, cycle and movement, synthesis and metabolism, in which the difference of miR-29 is most obvious. At present, studies have suggested that miR-29 can inhibit the synthesis of collagen at post transcriptional level and thus play an important role in the development of various fibrotic diseases. But miR-29 and HS The study of the formation of the relationship has not yet been reported. Therefore, we speculate that the continuous low level expression of miR-29 has lost the effective regulation of the expression of COLI gene, leading to the excessive deposition of collagen based extracellular matrix (ECM), and eventually the formation of HS. may be successful if the miR-29 expression level in the HS local tissue is developed by the use of justice miR-29. The process of inhibiting the excessive fibrosis of the tissues provides new targets for the clinical prevention and control of HS. Objective: to screen out the significant differential miRNA molecules expressed in HS tissues and NS tissues by miRNA expression chip, and to explore the mechanism of miR-29 by regulating the expression of the target gene COLI in the post transcriptional level and affecting the formation of HS, so as to be a biological treatment for HS. New therapeutic targets were provided. Methods: 1. the specimens of HS and NS tissues were collected. After HE staining, it was proved that the clinical specimens were in conformity with the experimental needs. The human HS and NS primary fibroblasts were isolated and cultured. The miRNA molecules expressed differently in HS and NS were screened by miRNA gene chip, and the target gene.2. in the downstream of miR-29 was predicted by bioinformatics. Further using the fluorescence quantitative PCR method and the Western blot method to clarify the differential expression of miR-29c and COLI genes in human NS and HS, and using chi square test or Fisher accurate test to analyze the correlation between miR-29 and COLI gene expression level. 3. the recombinant luciferase reporter gene vector of COLI A1-3 'and its 3 mutants was constructed. Whether there are significant differences in the activity of COLI A1-3 'UTR luciferase reporter vector in normal fibroblasts and scar fibroblasts, whether miR-29c simulant can significantly reduce the activity of the reported carrier, thus verifying whether COLI is an effective target gene for the downstream of miR-29c,.4. construction and packaging of human miR-29c overexpressed adenovirus, respectively. NS fibroblasts and HS fibroblasts were cultured. The proliferation and apoptosis of HS fibroblasts were detected by flow cytometry and CCK-8 method. Results: 1. the specimens of HS and NS collected from HS and NS proved to be in accordance with the experimental needs. Immunofluorescence staining was used. The cells in the primary culture of HS tissue and NS tissue were fibroblasts. 2. gene chip technology screening showed that the expression levels of 45 miRNA in HS tissues were up regulated compared with NS tissues, while miR-29a, miR-29b, miR-29c, etc. In the process of the development of HS, the change of miR-29 is the most significant. In 12 HS samples, the expression of miR-29 molecules is significantly lower than that of NS tissue (averaging 15.3 times). The.3. fluorescence quantitative PCR method is used to detect the expression of the selected miRNA-29 in HS and NS tissues. The results have been found, both in the tissue and in the original generation. In the fibrous cells, the expression of miR-29c in HS was significantly lower than that of NS tissue. There was no significant difference in the expression of other members of the miRNA-29 family in HS/NS..4. applied bioinformatics database and software to predict the miR-29 target gene. It was preliminarily presumed that COLI was a possible target gene for miR-29. Fluorescent quantitative PCR and Western Blot test showed that COLI The expression of gene in NS tissues was significantly lower than that in HS tissue. The expression of COLI in the fibroblasts derived from HS and NS tissues was consistent with the expression trend in the tissue. The chi square test found that both miR-29 and COLI gene expression levels were significant in both HS and NS tissues and in the primary cultured fibroblasts. The negative correlation.5. was used to construct the COLI gene and the mutant luciferase reporter gene vector of the corresponding binding region of miR-29c. The enzyme activity was confirmed by enzyme digestion and sequencing. The results of luciferase activity showed that the activity of COLI A1-3 'UTR luciferase reporter vector was significantly higher than normal fibroblast in scar fibroblasts. In vascular and hypertrophic scar fibroblasts, miR-29c simulants can effectively reduce the activity of the COLI A1-3 'UTR luciferase reporter carrier, but it can not effectively reduce the activity of the luciferase reporter vector of the mutant, and further indicates that miR-29c can directly act on COLI A1-3' UTR to play a role of.6. successfully construct and package the miR-29 of the COLI A1-3 'UTR. After C overexpressed the adenovirus, the cultured NS fibroblasts and HS fibroblasts were transfected respectively. The quantitative PCR combined with WB detection showed that the expression of COLI gene was significantly inhibited after miR-29 overexpression. The proliferation test and flow cytometry showed that the proliferation rate of fibroblasts increased significantly and the apoptosis rate decreased significantly after miR-29 overexpression. Conclusion: 1. gene chip technology screening showed that the expression levels of 45 miRNA, such as miR-143, miR-32, miR-92b and miR-25 in HS tissues were up regulated compared with those of NS tissues, while miR-29a, miR-29b, miR-29c, miR-713, miR-195 and so on were down regulated, suggesting that they might play a role in the development of the miRNA. The most significant.2. in tissues and cells, the expression of miR-29c in HS is significantly lower than that of NS, and the reduction of miR-29c in HS fibroblasts can promote its proliferation and inhibit its apoptosis, suggesting that miR-29 plays a negative regulatory role in the development of HS, the.3.COLI gene is the downstream target gene directly acting on miR-29c, miR-29 on COLI genes. The expression of negative regulation in the expression of miR-29 in hypertrophic scar tissue makes it lose effective regulation on the expression of target gene COL1, resulting in overexpression of COL1 gene, resulting in the overdeposition of collagen based ECM, which ultimately promotes the formation of hypertrophic scar tissue that may become a hypertrophic scar treatment. New target.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R622
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