靶向Hiwi基因的RNAi对膀胱癌细胞生物学行为的影响
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摘要
膀胱移行细胞癌约占膀胱恶性肿瘤的90%以上,是泌尿系统最常见的恶性肿瘤。经尿道膀胱肿瘤电切术是治疗浅表性膀胱移行细胞癌的最常用方法,但术后1年复发率高达70%以上,其中大约20%患者肿瘤进展为浸润性膀胱癌。浸润性膀胱癌的治疗以膀胱全切肠代膀胱或尿流改道,术后严重影响患者生活质量。目前,膀胱癌的治疗疗效仍不令人满意,主要因为膀胱癌的复发和转移率仍很高。因此,探索预防膀胱癌术后复发和治疗浸润性膀胱癌的有效方法是泌尿外科医生的重要课题。近年,随着分子生物学理论的发展、基因的发现和技术的进步及其在恶性肿瘤研究中的应用,发现恶性肿瘤的发生发展是癌基因的激活、抑癌基因失活以及凋亡抑制基因激活、凋亡基因失活共同作用的结果。因此,干扰肿瘤细胞中异常表达的基因的治疗方法,是目前研究的热点。
RNAi具有高效沉默目的基因的作用,是近年新兴的肿瘤基因治疗技术之一。Piwi基因首先在果蝇的生殖干细胞中发现的,Piwi基因是干细胞分裂的重要调控因子, Hiwi基因是来源于人的Piwi基因的同源物,在组织中广泛表达,并且发现其在肿瘤组织中高表达。Hiwi基因具有平衡干细胞的自我更新、分化的作用。
本文详细地研究了Hiwi基因在正常膀胱组织、膀胱癌组织及膀胱癌细胞株中的表达,探讨Hiwi基因的表达与膀胱癌临床分期和病理分级的关系,靶向Hiwi基因的shRNA表达载体的构建和干扰效果评价,并进一步以膀胱癌T24细胞为研究对象,利用RNAi技术,沉默Hiwi基因的表达,研究细胞相关生物学变化。结果显示,Hiwi基因高表达膀胱癌组织而非正常膀胱组织,与临床分期及病理分级可能关联。成功地构建了靶向Hiwi基因的shRNA表达载体,转染膀胱癌细胞后,能够抑制膀胱癌T24细胞的增殖,能够降低S期膀胱癌T24细胞百分比,增加G0/G1和G2/M期细胞百分比,造成膀胱癌T24细胞G1和G2期阻滞,提示Hiwi基因可能参与膀胱癌细胞周期调控。
本研究提示Hiwi基因在膀胱移行细胞癌中的高表达具有作为膀胱癌临床诊断标记物的可能,并且可以辅助判断膀胱移行细胞癌的恶性程度,RNAi可以作为沉默膀胱癌中Hiwi基因表达的有效手段,而靶向Hiwi基因治疗,可以为膀胱癌的治疗提供新的思考。
Transitional cell carcinoma of bladder is the most commen urologic malignance, and it accounts for 90% in bladder carcinoma. At present, the treatment in baldder carcinoma are mainly surgical resection with adjunctive chemotherapy, but the effects are not satisfied because of high recurrence and metastasis rate. Therefore, explore the effective methods of preventing postoperative recurrence and curing invasive bladder carcinoma are an important issue of urologic surgeon. Recently, with the development of molecular biology theory, the discovery of genes, the improvement of technology as well as its application in maligency reserach, the gene therapy of human disease is gradually becaming a reality. Through years of development, gene therapy is considered as an important complement of surgery and chemotherapy. It is expected as a radical method for bladder cancer. But the greatest obstacle of clinical applicaition is the choice of efficient gene targets and gene therapy. RNAi included post transcriptional gene silencing, transcriptional silencing and translation silencing. Post transcriptional gene silencing is a kind of phenomenon that double strand RNA molecules induced their complementary mRNA degradation. Short exongeous double strands RNA were introduced into cells, it reached the aim throμgh the targeted mRNA degradation. The simple operation and highly efficient inhibition of targeted gene made it become a strong research method of gene function, and the systemic and high-throughout functional gene screening become possible. RNAi technology application in bladder cancer therapy was relatively new. Piwi gene was first discovered in reproductive stem cell in drosophila, and it is an important regulating factor in stem cell division. Hiwi gene was a congener Piwi in human, and was first cloned from human testis cDNA library. Hiwi expressed in many kinds of tissues, including prostate, ovary, brain, liver, heart, kidney and bone muscle etc. It expressed high in human testis, and was found high expression in many kinds of tumours. Hiwi gene had the functions to balance the stem cells renewing and differentiation.. It reported that RNAi targeted Hiwi gene could inhibit gastric carcinoma cell proliferation and induce cell G2 /M phage arrest. These indicated that we could investigate Hiwi expression in bladder cancer and discover new marker for clinical bladder cancer diagnosis. We could also carry out RNAi targeted Hiwi and provide new idea for bladder cancer genetherapy.
The research measured Hiwi gene expression in normal bladder tissue, bladder cancer tissue and bladder cancer cells (T24、EJ、MGH-U1 and BIU-87) cultured in vitro applied with immunohistochemistry, immunocytochemisry, RT-PCR and Western blot. RT-PCR and Western Blot, to explorer the relations between Hiwi expressions and clinical stage and pathological classification; and to construct shRNA expression vectors targeted Hiwi gene, and the vectors were transfected into bladder cancer T24 cells to observe the efficiency of Hiwi mRNA and protein silencing; and then to measure the effects of T24 cell proliferation and cell phage biological behaviour changes caused by the silencing. Results showed that Hiwi expressed lowly in normal bladder tissue, but highly in bladder cancer tissue, and expressed highly in T24 and BIU-87 cultrued in vitro, but not expressed in EJ and MGH-U1; we analyzed the relations between Hiwi expression and clinical stage and pathological classification with statistical methods, found that there was some correlations between Hiwi high expression and clinical stage and pathological classification. We constructed shRNA expression vectors targeted Hiwi successfully, and after these vectors were transfected into T24 cells which expressed highly Hiwi, the shRNA could silence Hiwi expression efficiently, and then inhibited T24 cell proliferation, increased the cell ratio in G1 and G2 phage, reduced the cell ratio in S phage, and caused cell G1 and G2 arrest. These results provided experimental data for bladder cancer genetherapy.
1 Hiwi gene expression in human being bladder transitional cell cancer and relation to clinical stage and pathological classification
Hiwi expressed generally in tissues, and expressed highly in adult testis and many tumour tissues. This section of research intended to investigate Hiwi expressions in normal bladder tissue, bladder cancer tissue and cells cultured in vitro. To measure the expression with immunohistochemistry, immunocytochemisry, RT-PCR and Western Blot. RT-PCR and Western Blot results showed: Hiwi expressed lowly in 2 of 15 normal mucous membrane of urinary bladder specimen, the expression rate was 13.3%, but expressed highly in 39 of 50 bladder cancer tissue specimen, the expression rate was 78%. We analyzed the expressions in superficial bladder (Tis、Ta、T1) and invasion cancer (T2、T3、T4) with Pearsonχ2, and found that no difference existed in both of them (P > 0.05), but obvious difference existed in three of G1, G2 and G3 (P < 0.05), immunohistochemistry results showed that Hiwi expressions had obvious difference between 40 superficial bladder (Tis、Ta、T1) and invasion cancer (T2、T3、T4) specimens (P < 0.05), that the results was different with RT-PCR and Western Blot in bladder cancer might because of specimen case and experimental mean. RT-PCR and Western Blot results showed: Hiwi mRNA and protein expressed in T24 and BIU-87 cells, but not in EJ and MGH-U1. The four cells belonged to different phatological classification, and these further proved that Hiwi expressions related to bladder cancer pathological classification, and then we measured Hiwi protein expression in T24 and BIU-87 cells with immunocytochemistry. The results showed that Hiwi expressed highly in the both cells, and it was same to Western blotting. These results provided us essensial base for our next RNAi experiment.
2 Construction of shRNA expression vectors targeted Hiwi and silencing efficiency evaluation
To investigate further bladder cancer T24 cell biological behaviour changes after Hiwi gene was silenced, the section of essay silenced Hiwi expression specifically with RNAi technology. Two pairs of silencing sequence targeted Hiwi were designed with biological means, at same time, one pair of negative control sequence not targeted any genes, specific silencing Hiwi shRNA sequence was synthesized, and linkaged to pGenesil-2 vector, to construct two shRNA expression plasmid targeted Hiwi (pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263). Two shRNA expression plasmids were transfected into T24 cells with PEI, transfection efficiency was evalued with eukaryotic expression vector pGenesil-1 carried green fluorescence protein by flow cytometry and fluorescence microscope; after shRNA expression plasmid was trasfected into T24 cells, Hiwi mRNA and protein expressions were measured with RT-PCR and Western blot, and to evaluate interferencing efficiency. Results showed after SalⅠenzyme digested 400 bp fragments were produced, it was same to the design, and then plasmid was identified by sequencing, enzyme digesting and sequencing results showed theses accord with design, and it indicated construction was successful. pGenesil-1 plasmid was transfected into T24 cells with PEI, cells carried more green fluorescence than that in normal cells, and green fluorescence protein expression rate was (67.6±3.5)% with flow cytometry, transfection efficiency reached 62.5%, could meet experiment need. After pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 were transfected into T24 cells, Hiwi mRNA didn’t express, but there were not obvious difference among normal group, PEI and negative control group. Hiwi mRNA was silenced efficiently, and silencing efficiency reached 100%. Hiwi protein decreased obviously, and Inhibitory rates reached 74.2% and 64.5%, interferecing vectors targeted Hiwi gene could silence Hiwi gene efficiently from mRNA and protein levels.
3 Effects on bladder cancer cell biological behaviours caused by RNAi targeted Hiwi gene
In the section of essay, we observed after pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 were transfected into T24 cells, the proliferation changes at 24 h、48 h and 72 h, which were measured with MTT and clone formation essay,. A thet same time, cell cycle changes after 48 h transfection were observed with flow cytometry. MTT results showed that normal cells proliferated fast from 0 to 72 h, cells in PEI and negative control proliferated slightly slower than that in normal group, but cell proliferation capability in pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 plasmid groups was weaker , proliferation inhibitory rate increased obviously compared with negative control group (P < 0.01) at 24 h; clone formation essay results showed that 14 days after pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 transfection,the percentage of clone formation in T24 cells decreased obviously compared with normal group (P < 0.05), but it had not obviously changes in negative control. Flow cytometry results showed that 48 h after pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 transfection, T24 cell percentage in G0/G1 and G2/M phage increased obviously compared with normal group (P < 0.05, P < 0.01), but that in S phage decreased obviously (P < 0.05, P < 0.01), this indicated that T24 cells were arrested in G1 and G2 phage. Therefore, we could drawed the conclusions that RNAi targeted Hiwi could inhibit T24 cell proliferation with high efficiency,and induce T24 cell G1 and G2 phage arrest.
In one word, the research prompted that to investigate Hiwi mRNA and protein expression difference between normal bladder tissue,bladder cancer tissue and bladder cancer cells,and to approach the relations between Hiwi expressions and clinical stage and pathological classification. These could help to have adjuvant judgement for bladder transitional cell cancer malignancy degree, and guide further bladder cancer therapy; silencing directed to Hiwi gene expression might decrease bladder cancer cell malignant behaviour; RNAi technology as an effective mean for silencing bladder cancer Hiwi gene and provided a new thought for bladder cancer therapy. At present, there was no report about silencing Hiwi gene expressions in bladder cancer with RNAi technology at home and abroad. These experiment results and conclusions might provide essential data for bladder cancer genetherapy.
RNAi具有高效沉默目的基因的作用,是近年新兴的肿瘤基因治疗技术之一。Piwi基因首先在果蝇的生殖干细胞中发现的,Piwi基因是干细胞分裂的重要调控因子, Hiwi基因是来源于人的Piwi基因的同源物,在组织中广泛表达,并且发现其在肿瘤组织中高表达。Hiwi基因具有平衡干细胞的自我更新、分化的作用。
本文详细地研究了Hiwi基因在正常膀胱组织、膀胱癌组织及膀胱癌细胞株中的表达,探讨Hiwi基因的表达与膀胱癌临床分期和病理分级的关系,靶向Hiwi基因的shRNA表达载体的构建和干扰效果评价,并进一步以膀胱癌T24细胞为研究对象,利用RNAi技术,沉默Hiwi基因的表达,研究细胞相关生物学变化。结果显示,Hiwi基因高表达膀胱癌组织而非正常膀胱组织,与临床分期及病理分级可能关联。成功地构建了靶向Hiwi基因的shRNA表达载体,转染膀胱癌细胞后,能够抑制膀胱癌T24细胞的增殖,能够降低S期膀胱癌T24细胞百分比,增加G0/G1和G2/M期细胞百分比,造成膀胱癌T24细胞G1和G2期阻滞,提示Hiwi基因可能参与膀胱癌细胞周期调控。
本研究提示Hiwi基因在膀胱移行细胞癌中的高表达具有作为膀胱癌临床诊断标记物的可能,并且可以辅助判断膀胱移行细胞癌的恶性程度,RNAi可以作为沉默膀胱癌中Hiwi基因表达的有效手段,而靶向Hiwi基因治疗,可以为膀胱癌的治疗提供新的思考。
Transitional cell carcinoma of bladder is the most commen urologic malignance, and it accounts for 90% in bladder carcinoma. At present, the treatment in baldder carcinoma are mainly surgical resection with adjunctive chemotherapy, but the effects are not satisfied because of high recurrence and metastasis rate. Therefore, explore the effective methods of preventing postoperative recurrence and curing invasive bladder carcinoma are an important issue of urologic surgeon. Recently, with the development of molecular biology theory, the discovery of genes, the improvement of technology as well as its application in maligency reserach, the gene therapy of human disease is gradually becaming a reality. Through years of development, gene therapy is considered as an important complement of surgery and chemotherapy. It is expected as a radical method for bladder cancer. But the greatest obstacle of clinical applicaition is the choice of efficient gene targets and gene therapy. RNAi included post transcriptional gene silencing, transcriptional silencing and translation silencing. Post transcriptional gene silencing is a kind of phenomenon that double strand RNA molecules induced their complementary mRNA degradation. Short exongeous double strands RNA were introduced into cells, it reached the aim throμgh the targeted mRNA degradation. The simple operation and highly efficient inhibition of targeted gene made it become a strong research method of gene function, and the systemic and high-throughout functional gene screening become possible. RNAi technology application in bladder cancer therapy was relatively new. Piwi gene was first discovered in reproductive stem cell in drosophila, and it is an important regulating factor in stem cell division. Hiwi gene was a congener Piwi in human, and was first cloned from human testis cDNA library. Hiwi expressed in many kinds of tissues, including prostate, ovary, brain, liver, heart, kidney and bone muscle etc. It expressed high in human testis, and was found high expression in many kinds of tumours. Hiwi gene had the functions to balance the stem cells renewing and differentiation.. It reported that RNAi targeted Hiwi gene could inhibit gastric carcinoma cell proliferation and induce cell G2 /M phage arrest. These indicated that we could investigate Hiwi expression in bladder cancer and discover new marker for clinical bladder cancer diagnosis. We could also carry out RNAi targeted Hiwi and provide new idea for bladder cancer genetherapy.
The research measured Hiwi gene expression in normal bladder tissue, bladder cancer tissue and bladder cancer cells (T24、EJ、MGH-U1 and BIU-87) cultured in vitro applied with immunohistochemistry, immunocytochemisry, RT-PCR and Western blot. RT-PCR and Western Blot, to explorer the relations between Hiwi expressions and clinical stage and pathological classification; and to construct shRNA expression vectors targeted Hiwi gene, and the vectors were transfected into bladder cancer T24 cells to observe the efficiency of Hiwi mRNA and protein silencing; and then to measure the effects of T24 cell proliferation and cell phage biological behaviour changes caused by the silencing. Results showed that Hiwi expressed lowly in normal bladder tissue, but highly in bladder cancer tissue, and expressed highly in T24 and BIU-87 cultrued in vitro, but not expressed in EJ and MGH-U1; we analyzed the relations between Hiwi expression and clinical stage and pathological classification with statistical methods, found that there was some correlations between Hiwi high expression and clinical stage and pathological classification. We constructed shRNA expression vectors targeted Hiwi successfully, and after these vectors were transfected into T24 cells which expressed highly Hiwi, the shRNA could silence Hiwi expression efficiently, and then inhibited T24 cell proliferation, increased the cell ratio in G1 and G2 phage, reduced the cell ratio in S phage, and caused cell G1 and G2 arrest. These results provided experimental data for bladder cancer genetherapy.
1 Hiwi gene expression in human being bladder transitional cell cancer and relation to clinical stage and pathological classification
Hiwi expressed generally in tissues, and expressed highly in adult testis and many tumour tissues. This section of research intended to investigate Hiwi expressions in normal bladder tissue, bladder cancer tissue and cells cultured in vitro. To measure the expression with immunohistochemistry, immunocytochemisry, RT-PCR and Western Blot. RT-PCR and Western Blot results showed: Hiwi expressed lowly in 2 of 15 normal mucous membrane of urinary bladder specimen, the expression rate was 13.3%, but expressed highly in 39 of 50 bladder cancer tissue specimen, the expression rate was 78%. We analyzed the expressions in superficial bladder (Tis、Ta、T1) and invasion cancer (T2、T3、T4) with Pearsonχ2, and found that no difference existed in both of them (P > 0.05), but obvious difference existed in three of G1, G2 and G3 (P < 0.05), immunohistochemistry results showed that Hiwi expressions had obvious difference between 40 superficial bladder (Tis、Ta、T1) and invasion cancer (T2、T3、T4) specimens (P < 0.05), that the results was different with RT-PCR and Western Blot in bladder cancer might because of specimen case and experimental mean. RT-PCR and Western Blot results showed: Hiwi mRNA and protein expressed in T24 and BIU-87 cells, but not in EJ and MGH-U1. The four cells belonged to different phatological classification, and these further proved that Hiwi expressions related to bladder cancer pathological classification, and then we measured Hiwi protein expression in T24 and BIU-87 cells with immunocytochemistry. The results showed that Hiwi expressed highly in the both cells, and it was same to Western blotting. These results provided us essensial base for our next RNAi experiment.
2 Construction of shRNA expression vectors targeted Hiwi and silencing efficiency evaluation
To investigate further bladder cancer T24 cell biological behaviour changes after Hiwi gene was silenced, the section of essay silenced Hiwi expression specifically with RNAi technology. Two pairs of silencing sequence targeted Hiwi were designed with biological means, at same time, one pair of negative control sequence not targeted any genes, specific silencing Hiwi shRNA sequence was synthesized, and linkaged to pGenesil-2 vector, to construct two shRNA expression plasmid targeted Hiwi (pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263). Two shRNA expression plasmids were transfected into T24 cells with PEI, transfection efficiency was evalued with eukaryotic expression vector pGenesil-1 carried green fluorescence protein by flow cytometry and fluorescence microscope; after shRNA expression plasmid was trasfected into T24 cells, Hiwi mRNA and protein expressions were measured with RT-PCR and Western blot, and to evaluate interferencing efficiency. Results showed after SalⅠenzyme digested 400 bp fragments were produced, it was same to the design, and then plasmid was identified by sequencing, enzyme digesting and sequencing results showed theses accord with design, and it indicated construction was successful. pGenesil-1 plasmid was transfected into T24 cells with PEI, cells carried more green fluorescence than that in normal cells, and green fluorescence protein expression rate was (67.6±3.5)% with flow cytometry, transfection efficiency reached 62.5%, could meet experiment need. After pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 were transfected into T24 cells, Hiwi mRNA didn’t express, but there were not obvious difference among normal group, PEI and negative control group. Hiwi mRNA was silenced efficiently, and silencing efficiency reached 100%. Hiwi protein decreased obviously, and Inhibitory rates reached 74.2% and 64.5%, interferecing vectors targeted Hiwi gene could silence Hiwi gene efficiently from mRNA and protein levels.
3 Effects on bladder cancer cell biological behaviours caused by RNAi targeted Hiwi gene
In the section of essay, we observed after pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 were transfected into T24 cells, the proliferation changes at 24 h、48 h and 72 h, which were measured with MTT and clone formation essay,. A thet same time, cell cycle changes after 48 h transfection were observed with flow cytometry. MTT results showed that normal cells proliferated fast from 0 to 72 h, cells in PEI and negative control proliferated slightly slower than that in normal group, but cell proliferation capability in pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 plasmid groups was weaker , proliferation inhibitory rate increased obviously compared with negative control group (P < 0.01) at 24 h; clone formation essay results showed that 14 days after pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 transfection,the percentage of clone formation in T24 cells decreased obviously compared with normal group (P < 0.05), but it had not obviously changes in negative control. Flow cytometry results showed that 48 h after pGenesil-2-Hiwi1 and pGenesil-2-Hiwi2263 transfection, T24 cell percentage in G0/G1 and G2/M phage increased obviously compared with normal group (P < 0.05, P < 0.01), but that in S phage decreased obviously (P < 0.05, P < 0.01), this indicated that T24 cells were arrested in G1 and G2 phage. Therefore, we could drawed the conclusions that RNAi targeted Hiwi could inhibit T24 cell proliferation with high efficiency,and induce T24 cell G1 and G2 phage arrest.
In one word, the research prompted that to investigate Hiwi mRNA and protein expression difference between normal bladder tissue,bladder cancer tissue and bladder cancer cells,and to approach the relations between Hiwi expressions and clinical stage and pathological classification. These could help to have adjuvant judgement for bladder transitional cell cancer malignancy degree, and guide further bladder cancer therapy; silencing directed to Hiwi gene expression might decrease bladder cancer cell malignant behaviour; RNAi technology as an effective mean for silencing bladder cancer Hiwi gene and provided a new thought for bladder cancer therapy. At present, there was no report about silencing Hiwi gene expressions in bladder cancer with RNAi technology at home and abroad. These experiment results and conclusions might provide essential data for bladder cancer genetherapy.
引文
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