人肝癌p16INK4a、p15INK4b基因突变及其肝癌恶性表型逆转的研究
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摘要
原发性肝癌是严重威胁人类健康的恶性肿瘤之一,研究和阐明肝癌发生、发展的分子机理,探索有效的早期诊断和基因治疗手段,对最终战胜肝癌具有重要意义。本研究为探讨细胞周期抑癌基因p16INK4a和p15INK4b基因的突变缺失与人原发性肝癌发生的关系,应用PCR-SSCP、序列分析、多重PCR方法检测人原发性肝癌中p16INK4a和p15INK4b基因第2外显子突变以及p16INK4a基因第1、2外显子和部分第1、2内含子缺失状况。为探索外源性p16INK4a和p15INK4b基因对肝癌恶性表型逆转的可能性,我们构建了pXJ-p16和pXJ-p15重组质粒,用脂质体法建立了稳定表达外源性p16、p15基因的肝癌细胞株,并在分析细胞系遗传背景基础上研究了这两个基因分别对肝癌细胞体外增殖、凋亡、恶性表型的影响。此外,本研究建立了裸鼠移植瘤模型,观察分析p15INK4b基因对肝癌细胞在体内成瘤性的抑制作用。
第一部分:人原发性肝癌中p16INK4a和p15INK4b基因突变和p16INK4a基因缺失的研究
结果:
1.在31例人原发性肝癌和癌旁肝硬化组织中p16INK4a基因第1内
Human hepatocarcinoma is one of the most malignant cancers, which is hazarding to the people's health. It is important to elucidate the mechanism of tumorigenesis and development of hepatocarcinoma. P16INK4a and P15INK4b are homologous cell cycle tumor suppressors, which are within 30kb of one another on chromosome 9p21 region and in the same transcription orientation. P16INK4a has three exons, whereas p15INK4b is encoded by two exons. They act as competitive inhibitors by binding directly to CDK4/6 preventing their association with cyclin, which in turn arrest the cell cycle in late Gl phase with pRB in a hypophosphorylated state. Although the alteration of pl6INK4a gene in human hepatocacinoma has been reported, there are few and discrepant reports regarding the state of p15INK4b gene in hepatocarcinoma. Moreover, the role of p15INK4b on hepatocacinoma remains to be investigated. To illustrate the mechanism of inactivation of p16INK4a and p15INK4b genes, in the present study, mutation of exon 1,2 and 3 of pl6INK4a and pl5INK4b exon2 as well as deletion of exon1 and 2 of p16INK4a in 31 cases of human Hepatocarcinoma (HCC) were analyzed with
PCR-SSCP, sequencing and comparative multiple PCR.Furthermore, to assess the effects of pl6INK4a and pl5INK4b genes on reversing hepatocacinoma cell lines, based on the identification of background of pl6INK4a, pl5INK4b and RB genes in two hepatocarcinoma cell lines, the constructed eukaryotic expression recombinant with pi6 or pi 5 was transfected into the two cell lines with liposome, and animal models transplanted with pi5 transfected and original hepatocarcinoma cell lines were established. The effects on the proliferation, apoptosis, malignant phenotype in hepatocarcinoma cells in vitro for pl6INK4a and pl5INK4b, as well as in vivo for p5INK4b were investigated.Part one: Study on mutation of pl6INK4a and pl5INK4b genes in humanprimary hepatocellular carcinoma Results:1. Among 31 cases of HCCs tumor and their corresponding adjacent noncacerous cirrhosis tissues, 168bp fragment of pl6INK4a intronl and exon2 exhibited three patterns (A, B, B' ). The B pattern(48%, 15/31) out numbered the B' pattern (26%, 8/31). A pattern was the least (13%, 4/31). Two pattern (B, B' ) at SSCP analyzing were observed in 8 cases of healthy human blood cells which frequency were 37.5 (3/8) and 62.5% (5/8) for B and B' pattern, respectively.2. Among the same samples described above, the fragment (204bp) containing pl6INK4a gene exonl and intronl was amplified by PCR. The PCR amplified 362bp fragment covering exon2 and 3 of pl6INK4a gene was cleaved into two fragments (114bp and 248bp) by digesting with Smal.. Neither band shift nor polymorphism at SSCP analysis was
observed.3. Among the same samples described above, a PCR amplified 345bp fragment of pl5INK4b exon2 exhibited abnormal migration single strand in one case of adjacent noncancerous cirrhosis rather than in its corresponding tumor tissue or other ? samples. No evidence of polymorphism was found in 8 cases of healthy human people. Sequence analyzing of the cloned abnormal single strand DNA showed an identical sequence to pl5INK4b exon2 and its upstream 60 nucleotides in other reports.4. Among another batch of 31 cases of HCCs tumor, adjacent and distal noncacerous cirrhosis tissues, deletion of pl6INK4a gene exonl rather than exon2 was detected in 4 cases (13%, 4/31) with comparative multiple PCR techniques.Summary: The above results indicate that among HCCs rare mutation of exonl, 2 and 3 of pl6INK4a gene and pl5IN4b exon2, low frequency of deletion of pl6INK4a exon2 occurred, suggesting mutation or deletion might be not the important mechanism involved in inactivating pl6INK4a andpl5INK4binHCC.Part two: Impact of pl6INK4a and pl5INK4b on human hepatocellular carcinomacell proliferation and apoptosis Results:1. With comparative multiple PCR method, the hepatocarcinoma cell lines, BEL7402 and SMMC7721 were identified as containing intact endogenous pl6INK4a and pl5INK4b genes, as well as no deletion of RB gene exon22-23. SMMC7721, rather than BEL7402, has shown deletion of
exonl4-16 of RB gene.2. With restriction enzyme analysis the constructs, pXJ-pl6 and pXJ-pl5, were confirmed containing an intact pi6, pi5 complete cDNA insert respectively. PXJ-pl6 exhibited an identical intact ORF sequence to that of reported.3. With PCR using the sense primer at the T7 promoter of vector and antisense primer at the pi6 or pi 5 insert, the ectopic pi6 cDNA and pl5INK4b cDNA were confirmed in BEL7402-pl6, MMC7721-pl6, BEL7402-pl5, respectively. Moreover, the expression of pi6 mRNA or pi 5 mRNA was observed increasing in corresponding gene transfected cells at the RNA dot blot analysis.4. MTT and colony formation analysis indicated that ectopic pi 5 inhibited proliferation of BEL7402 with intact RB gene compared with vector-transfected or mock cells. The effect of pi 5 was not influenced by endogenous pi5 gene. In contrast ectopic pi6 lightly stimulated the growth of BEL7202, which contained endogenous pi6 and RB gene, whereas it did not suppress the growth of SMMC7721 with intact endogenous pi6 gene and deficient RB gene.5. Flow cytometry analysis demonstrated that ectopic pi 5 significantly stimulated increasing proportion of GO/Gl and decreasing S phases in BEL7402 compared with vector-transfected or mock cells, and induced apoptosis in the cell line, whereas ectopic pi6 has no impact on cell cycle of the cell line.Summary: The above results indicate that exogenous pl5INK4b gene plays a role on suppressing proliferation and inducing cell cycle arrest in GO/Gl phase and apoptosis in hepatocarcinoma cell line BEL7402. The effect
was not influenced by endogenous pi5 gene. The function of ectopic pi6 on inhibiting growth of hepatocarcinoma cells might rely on intact RB gene.Part three: Animal experiment of reversing hepatocarcinoma malignantphenotype by overexpression of pl5INK4b gene Results:1. With PCR method, the ectopic pi 5 was confirmed integrated in the BEL7402-pl5 xenograft, suggesting the BEL7402 , BEL7402-pl5 xenografts model of nude mice was successfully established.2. Both weight and volume of BEL7402-pl5 xenograft were significantly smaller than that of mock cell xenograft in nude mice (P<0.05).3. Flow cytometry analysis showed increasing fraction of G0/G1 phase from 26.5% to 45.4%, decreasing fraction of S phases from 30.8% to 25.4%, decreasing fraction of G2 phase from 42.7 to 29.2% within BEL7402-pl5 xenograft compared with within mock cell xenograft in nude mice.Summary: The above results indicate that the exogenous pl5INK4b inhibits cell growth of hepatocarcinoma and induces cell cycle arrest in G0/G1 phase in vivo.Conclusions: (1 ) Our study first demonstrated that cell cycle tumor suppressor gene pl5INK4b induced apoptosis in human hepatocarcinoma cell line BEL7402 with intact RB gene in vitro. To our knowledge, there has been no report concerning pi 5 induces tumor cell apoptosis; (2 ) Ectopic pi5INK4b gene inhibited proliferation of human hepatocarcinoma in vitro and in vivo. The mechanism involved cell cycle arrest in G0/G1 phase and apoptosis. So
far as we know, there has been no report regarding exogenous pi5 inhibits xenograft tumor cell growth and cell cycle arrest in G0/G1 phase in nude mice; ( 3) The effect of p 15INK4b inhibiting hepatocarcinoma cell growth in vitro and in vivo was not influenced by endogenous pl5INK4b gene; (4) The cell growth inhibition effect of ectopic pl6INK4a gene on RB gene intact hepatocarcinoma cell line BEL7402 was influenced by endogenous pi6 gene;(5) The cell growth inhibition effect of ectopic pl6INK4a gene was not observed in RB deficient hepatocarcinoma cell line SMMC7721, suggesting the effect of ectopic pi 6 gene might rely on intact RB gene in the cells; (6) No mutation of pl6INK4a gene exon 1,2,3 and intronl, 2, as well as pl5INK4b gene exon 2 were detected in primary human hepatocarcinoma tumor tissues. Moreover, the deletion frequency of pl6INK4a gene exon 1 and partial intron 1 is low (13%).The data suggest that the mutation of pl6INK4a and pl5INK4b genes and deletion of pl6INK4a gene might be not the important event in hepatocarcinogenesis.
第一部分:人原发性肝癌中p16INK4a和p15INK4b基因突变和p16INK4a基因缺失的研究
结果:
1.在31例人原发性肝癌和癌旁肝硬化组织中p16INK4a基因第1内
Human hepatocarcinoma is one of the most malignant cancers, which is hazarding to the people's health. It is important to elucidate the mechanism of tumorigenesis and development of hepatocarcinoma. P16INK4a and P15INK4b are homologous cell cycle tumor suppressors, which are within 30kb of one another on chromosome 9p21 region and in the same transcription orientation. P16INK4a has three exons, whereas p15INK4b is encoded by two exons. They act as competitive inhibitors by binding directly to CDK4/6 preventing their association with cyclin, which in turn arrest the cell cycle in late Gl phase with pRB in a hypophosphorylated state. Although the alteration of pl6INK4a gene in human hepatocacinoma has been reported, there are few and discrepant reports regarding the state of p15INK4b gene in hepatocarcinoma. Moreover, the role of p15INK4b on hepatocacinoma remains to be investigated. To illustrate the mechanism of inactivation of p16INK4a and p15INK4b genes, in the present study, mutation of exon 1,2 and 3 of pl6INK4a and pl5INK4b exon2 as well as deletion of exon1 and 2 of p16INK4a in 31 cases of human Hepatocarcinoma (HCC) were analyzed with
PCR-SSCP, sequencing and comparative multiple PCR.Furthermore, to assess the effects of pl6INK4a and pl5INK4b genes on reversing hepatocacinoma cell lines, based on the identification of background of pl6INK4a, pl5INK4b and RB genes in two hepatocarcinoma cell lines, the constructed eukaryotic expression recombinant with pi6 or pi 5 was transfected into the two cell lines with liposome, and animal models transplanted with pi5 transfected and original hepatocarcinoma cell lines were established. The effects on the proliferation, apoptosis, malignant phenotype in hepatocarcinoma cells in vitro for pl6INK4a and pl5INK4b, as well as in vivo for p5INK4b were investigated.Part one: Study on mutation of pl6INK4a and pl5INK4b genes in humanprimary hepatocellular carcinoma Results:1. Among 31 cases of HCCs tumor and their corresponding adjacent noncacerous cirrhosis tissues, 168bp fragment of pl6INK4a intronl and exon2 exhibited three patterns (A, B, B' ). The B pattern(48%, 15/31) out numbered the B' pattern (26%, 8/31). A pattern was the least (13%, 4/31). Two pattern (B, B' ) at SSCP analyzing were observed in 8 cases of healthy human blood cells which frequency were 37.5 (3/8) and 62.5% (5/8) for B and B' pattern, respectively.2. Among the same samples described above, the fragment (204bp) containing pl6INK4a gene exonl and intronl was amplified by PCR. The PCR amplified 362bp fragment covering exon2 and 3 of pl6INK4a gene was cleaved into two fragments (114bp and 248bp) by digesting with Smal.. Neither band shift nor polymorphism at SSCP analysis was
observed.3. Among the same samples described above, a PCR amplified 345bp fragment of pl5INK4b exon2 exhibited abnormal migration single strand in one case of adjacent noncancerous cirrhosis rather than in its corresponding tumor tissue or other ? samples. No evidence of polymorphism was found in 8 cases of healthy human people. Sequence analyzing of the cloned abnormal single strand DNA showed an identical sequence to pl5INK4b exon2 and its upstream 60 nucleotides in other reports.4. Among another batch of 31 cases of HCCs tumor, adjacent and distal noncacerous cirrhosis tissues, deletion of pl6INK4a gene exonl rather than exon2 was detected in 4 cases (13%, 4/31) with comparative multiple PCR techniques.Summary: The above results indicate that among HCCs rare mutation of exonl, 2 and 3 of pl6INK4a gene and pl5IN4b exon2, low frequency of deletion of pl6INK4a exon2 occurred, suggesting mutation or deletion might be not the important mechanism involved in inactivating pl6INK4a andpl5INK4binHCC.Part two: Impact of pl6INK4a and pl5INK4b on human hepatocellular carcinomacell proliferation and apoptosis Results:1. With comparative multiple PCR method, the hepatocarcinoma cell lines, BEL7402 and SMMC7721 were identified as containing intact endogenous pl6INK4a and pl5INK4b genes, as well as no deletion of RB gene exon22-23. SMMC7721, rather than BEL7402, has shown deletion of
exonl4-16 of RB gene.2. With restriction enzyme analysis the constructs, pXJ-pl6 and pXJ-pl5, were confirmed containing an intact pi6, pi5 complete cDNA insert respectively. PXJ-pl6 exhibited an identical intact ORF sequence to that of reported.3. With PCR using the sense primer at the T7 promoter of vector and antisense primer at the pi6 or pi 5 insert, the ectopic pi6 cDNA and pl5INK4b cDNA were confirmed in BEL7402-pl6, MMC7721-pl6, BEL7402-pl5, respectively. Moreover, the expression of pi6 mRNA or pi 5 mRNA was observed increasing in corresponding gene transfected cells at the RNA dot blot analysis.4. MTT and colony formation analysis indicated that ectopic pi 5 inhibited proliferation of BEL7402 with intact RB gene compared with vector-transfected or mock cells. The effect of pi 5 was not influenced by endogenous pi5 gene. In contrast ectopic pi6 lightly stimulated the growth of BEL7202, which contained endogenous pi6 and RB gene, whereas it did not suppress the growth of SMMC7721 with intact endogenous pi6 gene and deficient RB gene.5. Flow cytometry analysis demonstrated that ectopic pi 5 significantly stimulated increasing proportion of GO/Gl and decreasing S phases in BEL7402 compared with vector-transfected or mock cells, and induced apoptosis in the cell line, whereas ectopic pi6 has no impact on cell cycle of the cell line.Summary: The above results indicate that exogenous pl5INK4b gene plays a role on suppressing proliferation and inducing cell cycle arrest in GO/Gl phase and apoptosis in hepatocarcinoma cell line BEL7402. The effect
was not influenced by endogenous pi5 gene. The function of ectopic pi6 on inhibiting growth of hepatocarcinoma cells might rely on intact RB gene.Part three: Animal experiment of reversing hepatocarcinoma malignantphenotype by overexpression of pl5INK4b gene Results:1. With PCR method, the ectopic pi 5 was confirmed integrated in the BEL7402-pl5 xenograft, suggesting the BEL7402 , BEL7402-pl5 xenografts model of nude mice was successfully established.2. Both weight and volume of BEL7402-pl5 xenograft were significantly smaller than that of mock cell xenograft in nude mice (P<0.05).3. Flow cytometry analysis showed increasing fraction of G0/G1 phase from 26.5% to 45.4%, decreasing fraction of S phases from 30.8% to 25.4%, decreasing fraction of G2 phase from 42.7 to 29.2% within BEL7402-pl5 xenograft compared with within mock cell xenograft in nude mice.Summary: The above results indicate that the exogenous pl5INK4b inhibits cell growth of hepatocarcinoma and induces cell cycle arrest in G0/G1 phase in vivo.Conclusions: (1 ) Our study first demonstrated that cell cycle tumor suppressor gene pl5INK4b induced apoptosis in human hepatocarcinoma cell line BEL7402 with intact RB gene in vitro. To our knowledge, there has been no report concerning pi 5 induces tumor cell apoptosis; (2 ) Ectopic pi5INK4b gene inhibited proliferation of human hepatocarcinoma in vitro and in vivo. The mechanism involved cell cycle arrest in G0/G1 phase and apoptosis. So
far as we know, there has been no report regarding exogenous pi5 inhibits xenograft tumor cell growth and cell cycle arrest in G0/G1 phase in nude mice; ( 3) The effect of p 15INK4b inhibiting hepatocarcinoma cell growth in vitro and in vivo was not influenced by endogenous pl5INK4b gene; (4) The cell growth inhibition effect of ectopic pl6INK4a gene on RB gene intact hepatocarcinoma cell line BEL7402 was influenced by endogenous pi6 gene;(5) The cell growth inhibition effect of ectopic pl6INK4a gene was not observed in RB deficient hepatocarcinoma cell line SMMC7721, suggesting the effect of ectopic pi 6 gene might rely on intact RB gene in the cells; (6) No mutation of pl6INK4a gene exon 1,2,3 and intronl, 2, as well as pl5INK4b gene exon 2 were detected in primary human hepatocarcinoma tumor tissues. Moreover, the deletion frequency of pl6INK4a gene exon 1 and partial intron 1 is low (13%).The data suggest that the mutation of pl6INK4a and pl5INK4b genes and deletion of pl6INK4a gene might be not the important event in hepatocarcinogenesis.
引文
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2. Kamb A, Gruis NA, Weaver-Feldhaus Q. et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science, 1994, 264:436-440
3.Kamb A Shattuck-Eidens D, Eeles R, et al. Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus.Nat. Genet, 1994, 8: 23-26
4.Hannon GJ, Beach D, p15INK4b is a potential effector of TGF - β -induced cell cycle arrest. Nature, 1994, 371: 257-261
5.Kaneto H, sasaki S, Yamamoto H, et al Detection of hypermethylation of p16INK4a gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut, 2001, 48: 372-377
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