连接蛋白32在人原发性肝癌转移中的作用及其机制研究
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摘要
研究背景与目的
原发性肝细胞癌(hepatocellular carcinoma, HCC)是我国高发的恶性肿瘤之一目前全球每年新增HCC患者约56万,其中约一半病例出现在我国,且其死亡率从我国恶性肿瘤死亡率的第三位跃居至第二位。目前尚无有效的治疗手段,即使行手术、化疗等综合治疗,预后仍然极差。据统计,即使是小于5cm的小肝癌行手术治疗,术后3年的复发率仍高达50%以上。其预后差的主要原因是：HCC具有极强的侵袭转移能力。侵袭转移是恶性肿瘤最重要的生物学行为。肿瘤的侵袭转移是一个多环节、多阶段的过程,它包括肿瘤细胞与周围细胞和细胞外基质分离、游出,穿过血管基底膜进入血循环,随血液到达靶器官,再次从血管壁渗出进入靶器官,局部增殖成为转移瘤,是涉及到多个信号通路功能、多个基因激活／失活的复杂过程。对肝癌侵袭转移相关分子机制的研究,有助于寻找新的干预靶点,改善肝癌的疗效和预后。
细胞与周围细胞以及细胞外基质之间信号传递异常,导致二者之间黏附特性发生改变,是促进肿瘤侵袭转移启始的重要因素之一。连接蛋白(connexin,Cx)由多基因家族编码的一类膜蛋白家族,哺乳动物中发现二十种此类膜蛋白,是细胞间缝隙连接(gap junction, GJ)的基本结构单位。其中连接蛋白32 (Connexin 32, Cx32)是人类肝脏中表达的主要连接蛋白,也是肝细胞缝隙连接的主要组分。缝隙连接是细胞膜上的特殊结构,也是相邻细胞间唯一能直接进行物质交换和信息传递的通道。细胞能通过缝隙连接胞间通讯(gap junction intercellular communication, GJIC)进行细胞间信息和能量的传递,调控细胞的生长和分化,并维持内环境的稳定。有研究表明,Cx32蛋白在多种肿瘤组织中的表达下调,其表达下调及相关信号传导通路的紊乱可能导致相邻细胞间GJIC异常,学者们认为这可能是肿瘤发生的重要机制之一。另有学者采用显性负相技术在动物实验中证实Cx32对于致癌物二乙基亚硝胺(DEN)诱导的肝癌早期和晚期均有抑制作用。这提示Cx32可能调控肝癌细胞的侵袭转移。但目前尚未在细胞水平得到验证,且关于Cx32调控肿瘤细胞侵袭的分子机制研究甚少。
因此,本课题选择Cx32作为分子靶,拟从组织和细胞水平深入研究Cx32在HCC侵袭转移中的作用及其相关机制,为充实肝癌侵袭转移的理论提供实验基础。
方法
收集同济医院肝外科HCC手术切除标本(经术后病理证实)20例,采用RT-PCR、Western blot方法检测人肝癌和癌旁组织中Cx32基因核酸和蛋白表达的相对水平,探讨Cx32基因与人HCC发生发展的关系。培养三株不同转移潜能的人肝癌细胞株Hep G2、MHCC-97L以及MHCC-97H,采用RT-PCR、Western blot方法检测细胞株中Cx32基因核酸和蛋白表达的相对水平,并分析其表达与肝癌细胞株转移潜能的相关性。用pcDNA3.1(-)质粒构建Cx32的真核表达载体,应用脂质体将其转染至低表达Cx32的人肝癌细胞株MHCC-97H,用pGensil-2质粒构建Cx32-shRNA的真核表达载体,应用脂质体将其转染至相对高表达Cx32的人肝癌细胞株Hep G2,通过G418筛选出稳定高表达Cx32的MHCC-97H细胞株、Cx32表达受抑制的Hep G2细胞株,采用Western blot检测细胞株中Cx32基因的蛋白表达水平,采用划痕标记荧光传输试验显示细胞胞间通讯功能的变化,采用Transwell试验检测细胞侵袭能力的改变。采用Western blot法检测Cx32表达受抑制的Hep G2细胞株中细胞膜蛋白Occludin、Claudin-1、ZO-1表达的变化。用pGensil-2质粒构建Notchl-shRNA的真核表达载体,应用脂质体将其转染至人肝癌细胞株MHCC-97H,通过G418筛选出Notchl表达受抑制的MHCC-97H细胞株,采用Westernblot方法检测细胞株中Notchl基因的蛋白表达水平,同时给予缝隙连接阻滞剂甘琥酸孵育,采用划痕标记荧光传输试验显示细胞胞间通讯功能的变化,采用Transwell试验检测细胞侵袭能力的改变。
结果
1. RT-PCR、Western blot检测结果显示,Cx32基因mRNA的相对表达量在人肝癌和癌旁组织中分别为0.1600±0.0793、0.3169±0.0821,蛋白的相对表达量在癌和癌旁组织中分别为0.5564±0.1735、0.8720±0.1321,Cx32基因mRNA和蛋白的表达水平在肝癌组织较癌旁组织明显降低,差异有统计学意义(P<0.05)；在三株不同转移潜能的人肝癌细胞株Hep G2、MHCC-97L以及MHCC-97H中,Cx32基因mRNA表达的相对水平分别为0.3023±0.0401、0.1829±0.02760、0.0746±0.0106,蛋白表达的相对水平分别为0.5972±0.08831、0.2866±0.03280、0.1544±0.02354,均有显著性差异(P<0.05),且随人肝癌细胞株转移潜能的降低而增高。
2.成功构建pcDNA 3.1(-)/Cx32真核表达载体,稳定转染pcDNA 3.1(-)/Cx32真核表达载体可使MHCC-97H细胞中Cx32蛋白表达(1.2996±0.1641)较空载体转染组(0.5097±0.04927)和空白对照组(0.4964±0.08969)明显增强(P<0.05)。与对照组和转染空质粒组细胞相比,转染pcDNA 3.1(-)/Cx32真核表达载体、过表达Cx32的细胞胞间通讯功能增强,细胞迁移和侵袭实验中穿膜细胞数分别为159.3±14.0个、69.7±6.8个,均较对照组(213.3±17.6个、102.0±12.5个)和空载体转染组(209.0±16.5个、96.0±10.5个)减少(P<0.05)
3.成功构建Cx32-shRNA真核表达载体,转染Cx32-shRNA真核表达载体,可使Hep G2细胞的Cx32蛋白表达较对照组和空载体转染组降低67.4%。与对照组和转染空质粒组细胞相比,转染Cx32-shRNA真核表达载体、Cx32表达受抑制的细胞胞间通讯功能增强,而细胞迁移和侵袭实验的穿膜细胞数分别为198.3±18.5个、116.0±10.5个,均高于对照组(148.7±14.0个、79.3±8.5个)和空载体转染组(137.3±14.6个、76.3±6.5个) (P<0.05)。
4.成功构建Cx32-shRNA真核表达载体,转染Cx32-shRNA真核表达载体,使得Hep G2细胞的Cx32蛋白表达下调67.4%。转染Cx32-shRNA真核表达载体、Cx32表达受抑细胞的胞膜上紧密连接蛋白Occludin、Claudin-1以及ZO-1的相对量分别为0.2675±0.03958、0.4213±0.02933、0.1319±0.00857,均低于对照组(0.414±0.03677、1.0472±0.1095、0.4573±0.03507)和空载体转染组(0.4279±0.03958、1.1145±0.1325、0.4682±0.04186：P<0.05)
5.成功构建Notchl-shRNA真核表达载体,Notchl-shRNA真核表达载体转染至MHCC-97H细胞后,Notchl蛋白表达受抑制(抑制率60.78%)。与空白对照组和转染空载体组细胞相比,转染Notchl-shRNA真核表达载体、Notchl表达受抑制的细胞胞间通讯功能增强,而细胞迁移和侵袭实验的穿膜细胞数分别为85.7±6.0个、45.7±5.5个,低于对照组(215.7±15.6个、112.0±12.1个)和空载体转染组(204.7±15.5个、106.0±9.5个)(P<0.05)。同时给予缝隙连接阻滞剂孵育细胞,则穿膜细胞数升至119.3±10.1个、63.3±6.1个,但仍低于空白对照组(P<0.05)。
结论
Cx32基因在人HCC癌组织和人肝癌细胞株中均出现低表达,并随人肝癌细胞株转移潜能的降低而增高。过表达Cx32能使人肝癌细胞株的细胞通讯功能增强,侵袭能力下调；抑制人肝癌细胞株Cx32的表达则可抑制其细胞通讯功能,而促进细胞的侵袭能力。验证了Cx32可调控人肝癌细胞的侵袭能力。
进一步探讨Cx32调控人肝癌细胞侵袭能力的分子机制时,我们发现,抑制人肝癌细胞株Cx32的表达使得紧密连接蛋白Occludin、Claudin-1、ZO-1在细胞膜的分布减少,表明Cx32可能通过影响紧密连接蛋白调控人肝癌细胞的侵袭能力。抑制人肝癌细胞株Notchl的表达,可使细胞通讯功能恢复,细胞侵袭能力降低;而同时孵育缝隙连接阻滞剂甘琥酸则可部分逆转Notchl下调导致的细胞侵袭能力受抑。这提示Cx32可能对Notch信号通路调控的肝癌细胞侵袭迁移有一定影响作用。
综上所述,Cx32基因可能是通过缝隙连接抑制肝癌细胞的侵袭能力,并对Notch信号通路调控的细胞侵袭迁移有一定作用。这提示我们Cx32基因也可能是影响肝癌侵袭能力的一个有效分子靶。
Human hepatocellular carcinoma (HCC) is one of malignant tumors with high incidence in China. Every year there comes out about fifty six thousand new HCC patients in the world, of which about half emerged in China. The death rate of HCC in China jumped from third to second rank among cancer mortality. There exists no effective treatment, even with combined treatment of surgery and chemotherapy, the prognosis is still poor. Three-year relapse rate of small HCC less than 5 cm after sugery was higher than 50%. The main reason is that HCC possesses strong metastasis potential. Metastasis is the most important biological behavior of malignant tumors. It is a multi-stage process, which includes the tumor cells separating with surrounding cells and extracellular matrix, drilling through the vascular basement membrane into the blood circulation, reaching the target organ with the blood flow, colonying to a metastatic tumor. It involved in multiple signaling pathways and a number of gene activation or inactivation. Research on the molecular mechanism of HCC invasion and metastasis will help us to find new targets for intervention to improve the HCC treatment efficacy and prognosis.
Aberrant signal transmission between cells, cells and stroma leads to changes in adhesion characteristics, which is an important factor to switch on tumor invasion and metastasis. Connexin (Cx) is a class of membrane proteins encoded by multigene family, including at least 20 kinds in mammals, which is the basic structural unit of gap junction (GJ). Connexin 32 (Cx32) is the major connexin expressing in human liver and main component of hepatocyte gap junctions. Gap junction is a special structure in cell membrane and the only direct channel for material exchange and information trasmission. Cells can transfer energy and information through gap junction intercellular communication (GJIC) to regulate cell growth, differentiation and maintain homeostasis. Recent studies showed that the protein expression of Cx32 was down-regulated in a variety of tumor tissue, moreover, the reduction and the disorder of associated signaling pathway may lead to GJIC dysfunction between cells, which was thought to be an important mechanism of carcinogenesis.
Therefore, we chose Cx32 as a potential molecular target to further investigate its effect on HCC invasion and related mechanism in order to provide an experimental basis for the theory of HCC invasion and metastasis. Methods
Twenty cases of HCC resection specimens proven by pathology were collected from liver surgery department of Tongji Hospital and detected mRNA and protein related expression level of Cx32 gene by RT-PCR and Western blot analysis to explore the relationship between Cx32 gene and carcinogenesis of HCC. Three human hepatocellular carcinoma cell lines with different metastatic potential, Hep G2, MHCC-97L and MHCC-97H were cultured and detected mRNA and protein related expression level of Cx32 gene by RT-PCR and Western blot analysis to investigate the correlation between Cx32 gene and metastasis of HCC. The pcDNA 3.1(-)/Cx32 eukaryotic expression vector was constructed and transfected into MHCC-97H cells. Cells with Cx32 overexpression were screened with G418 and validated by Western blot analysis. The gap junction intercellular communication between cells was detected by scrape-loading and dye transfer assay. The invasion of cells was detected by Transwell assay. The Cx32-shRNA eukaryotic expression vector was constructed and transfected into Hep G2 cells. Cells with Cx32 depression were screened with G418 and validated by Western blot analysis. The gap junction intercellular communication between cells was detected by scrape-loading and dye transfer assay. The invasion of cells was detected by Transwell assay. Moreover, the expressions of membrane protein Occludin、Claudin-1、ZO-1 were detected by Western blot assay in cells with Cx32 depression. Furthermore, the Notchl-shRNA was constructed and transfected into MHCC-97H cells. Cells with Notchl depression were screened with G418 and validated by Western blot analysis. Scrape-loading and dye transfer assay and transwell assay were performed on cells with or without gap junction blocker, carbenoxolone.
Results
1. RT-PCR and Western blot analysis showed that the expression level of mRNA in tumor and adjacent tissue were 0.1600±0.0793 and 0.3169±0.0821, while the expression level of protein in them were 0.5564±0.1735 and 0.8720±0.1321, respectively. Compared with adjacent tissue, the mRNA and protein expression of Cx32 in human HCC tissue was significantly depressed(P<0.05). Furthermore, the expression level of mRNA in three HCC cell lines were 0.3023±0.0401,0.1829±0.02760,0.0746±0.0106 and the protein level in them were 0.5972±0.08831,0.2866±0.03280,0.1544±0.02354, respectively. There were significant difference in mRNA and protein expression of Cx32 gene in human HCC cell lines with different metastatic potential (P <0.05). The expression level was down-regulated with the upgrade of metastatic potential.
2. The pcDNA 3.1(-)/Cx32 eukaryotic expression vector was constructed successfully. The protein level of cells transfected with pcDNA 3.1(-)/Cx32 plasmid was 1.2996±0.1641, higher than blank(0.4964±0.08969) and empty vector group(0.5097±0.04927). Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Cx32 overexpression was enhanced. The migration cell count of Cx32 plasmid group were 159.3±14.0,69.7±6.8, lower than blank (213.3±17.6,102.0±12.5) and empty vector group (209.0±16.5,96.0±10.5).
3. We constructed Cx32-shRNA eukaryotic expression vector successfully. The Cx32 expression of cells transfected with Cx32-shRNA plasmid was decreased by 67.4%. Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Cx32 depression was weaken. The migration cell count of Cx32-shRNA group were 198.3±18.5 and 116.0±10.5, higher than blank (148.7±14.0,79.3±8.5) and empty vector group (137.3±14.6,76.3±6.5).
4. Western blot analysis detecting the expressions of membrane proteins including Occludin, Claudin-1 and ZO-1 showed that the expression level of tight junction protein Occludn、Claudin-1 and ZO-1 in cells with Cx32 depression were 0.2675±0.03958, 0.4213±0.02933,0.1319+0.00857, lower than blank (0.414±0.03677,1.0472±0.1095,0.4573±0.03507) and empty vector group (0.4279±0.03958,1.1145±0.1325, 0.4682±0.04186).
5. Notchl-shRNA eukaryotic expression vector was constructed successfully and transfected to MHCC-97H to obtain cell line with stable low expression of Notchl which was decreased by 60.78%. Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Notchl depression was enhanced. The migration cell count were 85.7±6.0,45.7±5.5, lower than blank (215.7±15.6,112.0±12.1) and empty vector group (204.7±15.5,106.0±9.5; P<0.05), while incubating cells with carbenoxolone could increase the cell count to 119.3±10.1 and 63.3±6.1, partially reversed the depression of invasion potential (P <0.05).
Conclusion
The expression of Cx32 gene was low in both human HCC tissues and cell lines and was decreased with upgrade of the metastasis potential of cell lines. Cx32 overexpression could promote the gap junction intercellular communication of HCC cells, but reduce the invasion potential of cells. On the other hand, inhibiting Cx32 expression could depress the gap junction intercellular communication, but promote cell invasion. This verified that Cx32 gene may regulate the metastatic potential of human HCC cell lines.
Further exploring the molecular mechanism of Cx32 regulating the invasion of human HCC, we found that inhibiting the expression of Cx32 depressed the expression of Occludin、Claudin-1、ZO-1 in the cell membrane, which suggested that Cx32 could control the invasion of human HCC through tight junction. Furthermore, Notchl depression would up-regulate the gap junction intercellular communication and down-regulate the invasion potential of cells, while incubating with gap junction blocker may partially reverse the down-regulation. This suggested that Cx32 may be involved in Notch signaling pathway on the regulation of cell invasion.
In summary, Cx32 gene may inhibit the invasion potential of human HCC through gap junction, while it was involved in the regulation of HCC invasion by Notch pathway. It suggested that Cx32 may be an effective molecular target of human HCC treatment.
原发性肝细胞癌(hepatocellular carcinoma, HCC)是我国高发的恶性肿瘤之一目前全球每年新增HCC患者约56万,其中约一半病例出现在我国,且其死亡率从我国恶性肿瘤死亡率的第三位跃居至第二位。目前尚无有效的治疗手段,即使行手术、化疗等综合治疗,预后仍然极差。据统计,即使是小于5cm的小肝癌行手术治疗,术后3年的复发率仍高达50%以上。其预后差的主要原因是：HCC具有极强的侵袭转移能力。侵袭转移是恶性肿瘤最重要的生物学行为。肿瘤的侵袭转移是一个多环节、多阶段的过程,它包括肿瘤细胞与周围细胞和细胞外基质分离、游出,穿过血管基底膜进入血循环,随血液到达靶器官,再次从血管壁渗出进入靶器官,局部增殖成为转移瘤,是涉及到多个信号通路功能、多个基因激活／失活的复杂过程。对肝癌侵袭转移相关分子机制的研究,有助于寻找新的干预靶点,改善肝癌的疗效和预后。
细胞与周围细胞以及细胞外基质之间信号传递异常,导致二者之间黏附特性发生改变,是促进肿瘤侵袭转移启始的重要因素之一。连接蛋白(connexin,Cx)由多基因家族编码的一类膜蛋白家族,哺乳动物中发现二十种此类膜蛋白,是细胞间缝隙连接(gap junction, GJ)的基本结构单位。其中连接蛋白32 (Connexin 32, Cx32)是人类肝脏中表达的主要连接蛋白,也是肝细胞缝隙连接的主要组分。缝隙连接是细胞膜上的特殊结构,也是相邻细胞间唯一能直接进行物质交换和信息传递的通道。细胞能通过缝隙连接胞间通讯(gap junction intercellular communication, GJIC)进行细胞间信息和能量的传递,调控细胞的生长和分化,并维持内环境的稳定。有研究表明,Cx32蛋白在多种肿瘤组织中的表达下调,其表达下调及相关信号传导通路的紊乱可能导致相邻细胞间GJIC异常,学者们认为这可能是肿瘤发生的重要机制之一。另有学者采用显性负相技术在动物实验中证实Cx32对于致癌物二乙基亚硝胺(DEN)诱导的肝癌早期和晚期均有抑制作用。这提示Cx32可能调控肝癌细胞的侵袭转移。但目前尚未在细胞水平得到验证,且关于Cx32调控肿瘤细胞侵袭的分子机制研究甚少。
因此,本课题选择Cx32作为分子靶,拟从组织和细胞水平深入研究Cx32在HCC侵袭转移中的作用及其相关机制,为充实肝癌侵袭转移的理论提供实验基础。
方法
收集同济医院肝外科HCC手术切除标本(经术后病理证实)20例,采用RT-PCR、Western blot方法检测人肝癌和癌旁组织中Cx32基因核酸和蛋白表达的相对水平,探讨Cx32基因与人HCC发生发展的关系。培养三株不同转移潜能的人肝癌细胞株Hep G2、MHCC-97L以及MHCC-97H,采用RT-PCR、Western blot方法检测细胞株中Cx32基因核酸和蛋白表达的相对水平,并分析其表达与肝癌细胞株转移潜能的相关性。用pcDNA3.1(-)质粒构建Cx32的真核表达载体,应用脂质体将其转染至低表达Cx32的人肝癌细胞株MHCC-97H,用pGensil-2质粒构建Cx32-shRNA的真核表达载体,应用脂质体将其转染至相对高表达Cx32的人肝癌细胞株Hep G2,通过G418筛选出稳定高表达Cx32的MHCC-97H细胞株、Cx32表达受抑制的Hep G2细胞株,采用Western blot检测细胞株中Cx32基因的蛋白表达水平,采用划痕标记荧光传输试验显示细胞胞间通讯功能的变化,采用Transwell试验检测细胞侵袭能力的改变。采用Western blot法检测Cx32表达受抑制的Hep G2细胞株中细胞膜蛋白Occludin、Claudin-1、ZO-1表达的变化。用pGensil-2质粒构建Notchl-shRNA的真核表达载体,应用脂质体将其转染至人肝癌细胞株MHCC-97H,通过G418筛选出Notchl表达受抑制的MHCC-97H细胞株,采用Westernblot方法检测细胞株中Notchl基因的蛋白表达水平,同时给予缝隙连接阻滞剂甘琥酸孵育,采用划痕标记荧光传输试验显示细胞胞间通讯功能的变化,采用Transwell试验检测细胞侵袭能力的改变。
结果
1. RT-PCR、Western blot检测结果显示,Cx32基因mRNA的相对表达量在人肝癌和癌旁组织中分别为0.1600±0.0793、0.3169±0.0821,蛋白的相对表达量在癌和癌旁组织中分别为0.5564±0.1735、0.8720±0.1321,Cx32基因mRNA和蛋白的表达水平在肝癌组织较癌旁组织明显降低,差异有统计学意义(P<0.05)；在三株不同转移潜能的人肝癌细胞株Hep G2、MHCC-97L以及MHCC-97H中,Cx32基因mRNA表达的相对水平分别为0.3023±0.0401、0.1829±0.02760、0.0746±0.0106,蛋白表达的相对水平分别为0.5972±0.08831、0.2866±0.03280、0.1544±0.02354,均有显著性差异(P<0.05),且随人肝癌细胞株转移潜能的降低而增高。
2.成功构建pcDNA 3.1(-)/Cx32真核表达载体,稳定转染pcDNA 3.1(-)/Cx32真核表达载体可使MHCC-97H细胞中Cx32蛋白表达(1.2996±0.1641)较空载体转染组(0.5097±0.04927)和空白对照组(0.4964±0.08969)明显增强(P<0.05)。与对照组和转染空质粒组细胞相比,转染pcDNA 3.1(-)/Cx32真核表达载体、过表达Cx32的细胞胞间通讯功能增强,细胞迁移和侵袭实验中穿膜细胞数分别为159.3±14.0个、69.7±6.8个,均较对照组(213.3±17.6个、102.0±12.5个)和空载体转染组(209.0±16.5个、96.0±10.5个)减少(P<0.05)
3.成功构建Cx32-shRNA真核表达载体,转染Cx32-shRNA真核表达载体,可使Hep G2细胞的Cx32蛋白表达较对照组和空载体转染组降低67.4%。与对照组和转染空质粒组细胞相比,转染Cx32-shRNA真核表达载体、Cx32表达受抑制的细胞胞间通讯功能增强,而细胞迁移和侵袭实验的穿膜细胞数分别为198.3±18.5个、116.0±10.5个,均高于对照组(148.7±14.0个、79.3±8.5个)和空载体转染组(137.3±14.6个、76.3±6.5个) (P<0.05)。
4.成功构建Cx32-shRNA真核表达载体,转染Cx32-shRNA真核表达载体,使得Hep G2细胞的Cx32蛋白表达下调67.4%。转染Cx32-shRNA真核表达载体、Cx32表达受抑细胞的胞膜上紧密连接蛋白Occludin、Claudin-1以及ZO-1的相对量分别为0.2675±0.03958、0.4213±0.02933、0.1319±0.00857,均低于对照组(0.414±0.03677、1.0472±0.1095、0.4573±0.03507)和空载体转染组(0.4279±0.03958、1.1145±0.1325、0.4682±0.04186：P<0.05)
5.成功构建Notchl-shRNA真核表达载体,Notchl-shRNA真核表达载体转染至MHCC-97H细胞后,Notchl蛋白表达受抑制(抑制率60.78%)。与空白对照组和转染空载体组细胞相比,转染Notchl-shRNA真核表达载体、Notchl表达受抑制的细胞胞间通讯功能增强,而细胞迁移和侵袭实验的穿膜细胞数分别为85.7±6.0个、45.7±5.5个,低于对照组(215.7±15.6个、112.0±12.1个)和空载体转染组(204.7±15.5个、106.0±9.5个)(P<0.05)。同时给予缝隙连接阻滞剂孵育细胞,则穿膜细胞数升至119.3±10.1个、63.3±6.1个,但仍低于空白对照组(P<0.05)。
结论
Cx32基因在人HCC癌组织和人肝癌细胞株中均出现低表达,并随人肝癌细胞株转移潜能的降低而增高。过表达Cx32能使人肝癌细胞株的细胞通讯功能增强,侵袭能力下调；抑制人肝癌细胞株Cx32的表达则可抑制其细胞通讯功能,而促进细胞的侵袭能力。验证了Cx32可调控人肝癌细胞的侵袭能力。
进一步探讨Cx32调控人肝癌细胞侵袭能力的分子机制时,我们发现,抑制人肝癌细胞株Cx32的表达使得紧密连接蛋白Occludin、Claudin-1、ZO-1在细胞膜的分布减少,表明Cx32可能通过影响紧密连接蛋白调控人肝癌细胞的侵袭能力。抑制人肝癌细胞株Notchl的表达,可使细胞通讯功能恢复,细胞侵袭能力降低;而同时孵育缝隙连接阻滞剂甘琥酸则可部分逆转Notchl下调导致的细胞侵袭能力受抑。这提示Cx32可能对Notch信号通路调控的肝癌细胞侵袭迁移有一定影响作用。
综上所述,Cx32基因可能是通过缝隙连接抑制肝癌细胞的侵袭能力,并对Notch信号通路调控的细胞侵袭迁移有一定作用。这提示我们Cx32基因也可能是影响肝癌侵袭能力的一个有效分子靶。
Human hepatocellular carcinoma (HCC) is one of malignant tumors with high incidence in China. Every year there comes out about fifty six thousand new HCC patients in the world, of which about half emerged in China. The death rate of HCC in China jumped from third to second rank among cancer mortality. There exists no effective treatment, even with combined treatment of surgery and chemotherapy, the prognosis is still poor. Three-year relapse rate of small HCC less than 5 cm after sugery was higher than 50%. The main reason is that HCC possesses strong metastasis potential. Metastasis is the most important biological behavior of malignant tumors. It is a multi-stage process, which includes the tumor cells separating with surrounding cells and extracellular matrix, drilling through the vascular basement membrane into the blood circulation, reaching the target organ with the blood flow, colonying to a metastatic tumor. It involved in multiple signaling pathways and a number of gene activation or inactivation. Research on the molecular mechanism of HCC invasion and metastasis will help us to find new targets for intervention to improve the HCC treatment efficacy and prognosis.
Aberrant signal transmission between cells, cells and stroma leads to changes in adhesion characteristics, which is an important factor to switch on tumor invasion and metastasis. Connexin (Cx) is a class of membrane proteins encoded by multigene family, including at least 20 kinds in mammals, which is the basic structural unit of gap junction (GJ). Connexin 32 (Cx32) is the major connexin expressing in human liver and main component of hepatocyte gap junctions. Gap junction is a special structure in cell membrane and the only direct channel for material exchange and information trasmission. Cells can transfer energy and information through gap junction intercellular communication (GJIC) to regulate cell growth, differentiation and maintain homeostasis. Recent studies showed that the protein expression of Cx32 was down-regulated in a variety of tumor tissue, moreover, the reduction and the disorder of associated signaling pathway may lead to GJIC dysfunction between cells, which was thought to be an important mechanism of carcinogenesis.
Therefore, we chose Cx32 as a potential molecular target to further investigate its effect on HCC invasion and related mechanism in order to provide an experimental basis for the theory of HCC invasion and metastasis. Methods
Twenty cases of HCC resection specimens proven by pathology were collected from liver surgery department of Tongji Hospital and detected mRNA and protein related expression level of Cx32 gene by RT-PCR and Western blot analysis to explore the relationship between Cx32 gene and carcinogenesis of HCC. Three human hepatocellular carcinoma cell lines with different metastatic potential, Hep G2, MHCC-97L and MHCC-97H were cultured and detected mRNA and protein related expression level of Cx32 gene by RT-PCR and Western blot analysis to investigate the correlation between Cx32 gene and metastasis of HCC. The pcDNA 3.1(-)/Cx32 eukaryotic expression vector was constructed and transfected into MHCC-97H cells. Cells with Cx32 overexpression were screened with G418 and validated by Western blot analysis. The gap junction intercellular communication between cells was detected by scrape-loading and dye transfer assay. The invasion of cells was detected by Transwell assay. The Cx32-shRNA eukaryotic expression vector was constructed and transfected into Hep G2 cells. Cells with Cx32 depression were screened with G418 and validated by Western blot analysis. The gap junction intercellular communication between cells was detected by scrape-loading and dye transfer assay. The invasion of cells was detected by Transwell assay. Moreover, the expressions of membrane protein Occludin、Claudin-1、ZO-1 were detected by Western blot assay in cells with Cx32 depression. Furthermore, the Notchl-shRNA was constructed and transfected into MHCC-97H cells. Cells with Notchl depression were screened with G418 and validated by Western blot analysis. Scrape-loading and dye transfer assay and transwell assay were performed on cells with or without gap junction blocker, carbenoxolone.
Results
1. RT-PCR and Western blot analysis showed that the expression level of mRNA in tumor and adjacent tissue were 0.1600±0.0793 and 0.3169±0.0821, while the expression level of protein in them were 0.5564±0.1735 and 0.8720±0.1321, respectively. Compared with adjacent tissue, the mRNA and protein expression of Cx32 in human HCC tissue was significantly depressed(P<0.05). Furthermore, the expression level of mRNA in three HCC cell lines were 0.3023±0.0401,0.1829±0.02760,0.0746±0.0106 and the protein level in them were 0.5972±0.08831,0.2866±0.03280,0.1544±0.02354, respectively. There were significant difference in mRNA and protein expression of Cx32 gene in human HCC cell lines with different metastatic potential (P <0.05). The expression level was down-regulated with the upgrade of metastatic potential.
2. The pcDNA 3.1(-)/Cx32 eukaryotic expression vector was constructed successfully. The protein level of cells transfected with pcDNA 3.1(-)/Cx32 plasmid was 1.2996±0.1641, higher than blank(0.4964±0.08969) and empty vector group(0.5097±0.04927). Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Cx32 overexpression was enhanced. The migration cell count of Cx32 plasmid group were 159.3±14.0,69.7±6.8, lower than blank (213.3±17.6,102.0±12.5) and empty vector group (209.0±16.5,96.0±10.5).
3. We constructed Cx32-shRNA eukaryotic expression vector successfully. The Cx32 expression of cells transfected with Cx32-shRNA plasmid was decreased by 67.4%. Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Cx32 depression was weaken. The migration cell count of Cx32-shRNA group were 198.3±18.5 and 116.0±10.5, higher than blank (148.7±14.0,79.3±8.5) and empty vector group (137.3±14.6,76.3±6.5).
4. Western blot analysis detecting the expressions of membrane proteins including Occludin, Claudin-1 and ZO-1 showed that the expression level of tight junction protein Occludn、Claudin-1 and ZO-1 in cells with Cx32 depression were 0.2675±0.03958, 0.4213±0.02933,0.1319+0.00857, lower than blank (0.414±0.03677,1.0472±0.1095,0.4573±0.03507) and empty vector group (0.4279±0.03958,1.1145±0.1325, 0.4682±0.04186).
5. Notchl-shRNA eukaryotic expression vector was constructed successfully and transfected to MHCC-97H to obtain cell line with stable low expression of Notchl which was decreased by 60.78%. Compared with blank and cells transfected with empty vector, the gap junction intercellular communication of cells with Notchl depression was enhanced. The migration cell count were 85.7±6.0,45.7±5.5, lower than blank (215.7±15.6,112.0±12.1) and empty vector group (204.7±15.5,106.0±9.5; P<0.05), while incubating cells with carbenoxolone could increase the cell count to 119.3±10.1 and 63.3±6.1, partially reversed the depression of invasion potential (P <0.05).
Conclusion
The expression of Cx32 gene was low in both human HCC tissues and cell lines and was decreased with upgrade of the metastasis potential of cell lines. Cx32 overexpression could promote the gap junction intercellular communication of HCC cells, but reduce the invasion potential of cells. On the other hand, inhibiting Cx32 expression could depress the gap junction intercellular communication, but promote cell invasion. This verified that Cx32 gene may regulate the metastatic potential of human HCC cell lines.
Further exploring the molecular mechanism of Cx32 regulating the invasion of human HCC, we found that inhibiting the expression of Cx32 depressed the expression of Occludin、Claudin-1、ZO-1 in the cell membrane, which suggested that Cx32 could control the invasion of human HCC through tight junction. Furthermore, Notchl depression would up-regulate the gap junction intercellular communication and down-regulate the invasion potential of cells, while incubating with gap junction blocker may partially reverse the down-regulation. This suggested that Cx32 may be involved in Notch signaling pathway on the regulation of cell invasion.
In summary, Cx32 gene may inhibit the invasion potential of human HCC through gap junction, while it was involved in the regulation of HCC invasion by Notch pathway. It suggested that Cx32 may be an effective molecular target of human HCC treatment.
引文
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4. Paul DL. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol, 1986,103:123-134.
5. Willecke K, Eiberger J, Degen J, et al. Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem,2002,383:725-737.
6. Beyer EC, Paul DL, Goodenough DA. Connexin43:a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol,1987,105:2621-2629.
7. Alexander DB, Goldberg GS. Transfer of biologically important molecules between cells through gap junction channels. Curr Med Chem,2003,10:2045-205.
8. Kumar NM, Gilula NB. The gap junction communication channel. Cell,1996, 84:381-388.
9. White TW, Paul DL. Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol,1999,61:283-310.
10. Valiunas V, Polosina YY, Miller H, et al. Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions. J Physiol,2005,568:459-68.
11. Krutovskikh V, Yamasaki H. Connexin gene mutations in human genetic diseases. Mutat Res,2000,462:197-207.
12. Vinken M, Vanhaecke T, Papeleu P, et al. Connexins and their channels in cell growth and cell death. Cell Signal,2006,18:592-600.
13. Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol,2004,36:1171-1186.
14. Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases. Biol Cell,2002,94:433-443.
15. White TW, Paul DL. Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol,1999,61:283-310.
16. King TJ, Lampe PD. Temporal regulation of connexin phosphorylation in embryonic and adult tissues. Biochim Biophys Acta,2005,1719:24-35.
17. Laird DW. Life cycle of connexins in health and disease. Biochem. J,2006,394: 527-543.
18. Musil LS, Goodenough DA. Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell,1993, 74:1065-1077.
19. Harris AL. Emerging issues of connexin channels:biophysics fills the gap. Q Rev Biophys,2001,34:325-472.
20. Sarma JD, Wang F, Koval M. Targeted gap junction protein constructs reveal connexinspecific differences in oligomerization. J Biol Chem,2002,277:20911-20918.
21. Evans WH, De VE, Leybaert L. The gap junction cellular internet:connexin hemichannels enter the signalling limelight. Biochem J,2006,397:1-14.
22. Gaietta G, Deerinck TJ, Adams SR, et al. Multicolor and electron microscopic imaging of connexin trafficking. Science,2002,296:503-507.
23. Bukauskas FF, Kreuzberg MM, Rackauskas M, et al. Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels:implications for atrioventricular conduction in the heart. Proc Natl Acad Sci USA,2006,103:9726-9731.
24. Kretz M, Euwens C, Hombach S, et al. Altered connexin expression and wound healing in the epidermis of connexin-deficient mice. J Cell Sci,2003,116:3443-3452.
25. Goldberg GS, Moreno AP, Lampe PD. Gap junctions between cells expressing connexin 43 or 32 show inverse permselectivity to adenosine and ATP. J Biol Chem,2002, 277:36725-30.
26. Segretain D, Falk MM. Regulation of connexin biosynthesis, assembly, gap junction formation, and removal. Biochim Biophys Acta,2004,1662:3-21.
27. Weber PA, Chang HC, Spaeth KE, et al. The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities. Biophys J,2004,87:958-73.
28. Bedner P, Niessen H, Odermatt B, et al. Selective permeability of different connexin channels to the second messenger cyclic AMP. J Biol Chem,2006,281:6673-6681.
29. Qin H, Shao Q, Curtis H, et al. Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication. J Biol Chem,2002,277: 29132-29138.
30. Dang X, Jeyaraman M, Kardami E. Regulation of connexin-43-mediated growth inhibition by a phosphorylatable amino-acid is independent of gap junction-forming ability. Mol. Cell Biochem,2006,289:201-207.
31. Yamasaki H, Naus CC. Role of connexin genes in growth control. Carcinogenesis,1996, 17:1199-1213.
32. Alexander DB, Ichikawa H, Bechberger JF, et al. Normal cells control the growth of neighboring transformed cells independent of gap junctional communication and SRC activity. Cancer Res,2004,64:1347-1358.
33. Plante I, Charbonneau M,. Cyr DG. Decreased gap junctional intercellular communication in hexachlorobenzene-induced genderspecific hepatic tumor formation in the rat. Carcinogenesis,2002,23:1243-1249.
34. Avanzo JL, Mesnil M, Hernandez-Blazquez FJ, et al. Increased susceptibility to urethane-induced lung tumors in mice with decreased expression of connexin43. Carcinogenesis,2004,25:1973-1982.
35. Zhang YW, Morita I, Ikeda M, et al. Connexin43 suppresses proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of p27. Oncogene, 2001,20:4138-4149.
36. Zhang YW, Kaneda M, Morita I. The gap junction-independent tumor-suppressing effect of connexin 43. J Biol Chem,2003,278:44852-44856.
37. Nakase T, Naus CG. Gap junctions and neurological disorders of the central nervous system. Biochim Biophys Acta,2004,1662:215-224.
38. Zhang YW, Nakayama K, Nakayama K, et al. A novel route for connexin 43 to inhibit cell proliferation:negative regulation of S-phase kinase-associated protein (Skp 2). Cancer Res,2003,63:1623-1630.
39. Goldberg GS, Bechberger JF, Tajima Y, et al. Connexin43 suppresses MFG-E8 while inducing contact growth inhibition of glioma cells. Cancer Res,2000,60:6018-6026.
40. Naus CC, Bond SL, Bechberger JF, et al. Identification of genes differentially expressed in C6 glioma cells transfected with connexin43. Brain Res Brain Res Rev,2000,32: 259-266.
41. Iacobas DA, Urban-Maldonado M, Iacobas S, et al. Array analysis of gene expression in connexin-43 null astrocytes. Physiol. Genomics,2003,15:177-190.
42. Huang R, Lin Y, Wang CC, et al. Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology. Cancer Res,2002,62:2806-2812.
43. Tate AW, Lung T, Radhakrishnan A, et al. Changes in gap junctional connexin isoforms during prostate cancer progression. Prostate,2006,66:19-31.
44. Iacobas DA, Scemes E, Spray DC. Gene expression alterations in connexin null mice extend beyond the gap junction. Neurochem Int,2004,45:243-250.
45. Shao Q, Wang H, McLachlan E, et al. Down-regulation of Cx43 by retroviral delivery of small interfering RNA promotes an aggressive breast cancer cell phenotype. Cancer Res,2005,65:2705-2711.
46. Saunders MM, Seraj MJ, Li Z, et al. Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication. Cancer Res,2001,61:1765-1767.
47. Jongen WM, Fitzgerald DJ, Asamoto M, et al. Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+in mouse epidermal cells is controlled by E-cadherin. J Cell Biol,1991,114:545-555.
48. Jamieson S, Going JJ, ArcyRD, et al. Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours. J Pathol,1998,184: 37-43.
49. Pollmann MA, Shao Q, Laird DW, et al. Connexin 43 mediated gap junctional communication enhances breast tumor cell diapedesis in culture. Breast Cancer Res, 2005,7:R522-R534.
50. Carystinos G, Bier A, Batist G. The role of connexin-mediated cell-cell communication in breast cancer metastasis. J Mammary Gland Biol Neoplasia,2001,6:431-440.
51. Ito A, Katoh F, Kataoka TR, et al. A role for heterologous gap junctions between melanoma and endothelial cells in metastasis. J Clin Invest,2000,105:1189-1197.
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53. El-Sabban ME, Merhi RA, Haidar HA, et al. Human Tcell lymphotropic virus type 1-transformed cells induce angiogenesis and establish functional gap junctions with endothelial cells. Blood,2002,99:3383-3389.
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56. Bazarbachi A, Abou Merhi R, Gessain A, et al. Human T-cell lymphotropic virus type I-infected cells extravasate through the endothelial barrier by a local angiogenesis-like mechanism. Cancer Res,2004,64:2039-2046.
57. Chen JT, Cheng YW, Chou MC, et al. The correlation between aberrant connexin 43 mRNA expression induced by promoter methylation and nodal micrometastasis in nonsmall cell lung cancer. Clin Cancer Res,2003,9:4200-4204.
58. Stuhlmann D, Ale-Agha N, Reinehr R, et al. Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells. Carcinogenesis,2003,24:1737-1748.
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1. Harris AL. Emerging issues of connexin channels:biophysics fills the gap. Q Rev Biophys,2001,34:325-472.
2. Iacobas DA, Scemes E, and Spray DC. Gene expression alterations in connexin null mice extend beyond the gap junction. Neurochem Int,2004,45:243-250.
3. Kumar NM, Gilula NB. Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J Cell Biol,1986,103:767-776.
4. Paul DL. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol, 1986,103:123-134.
5. Willecke K, Eiberger J, Degen J, et al. Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem,2002,383:725-737.
6. Beyer EC, Paul DL, Goodenough DA. Connexin43:a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol,1987,105:2621-2629.
7. Alexander DB, Goldberg GS. Transfer of biologically important molecules between cells through gap junction channels. Curr Med Chem,2003,10:2045-205.
8. Kumar NM, Gilula NB. The gap junction communication channel. Cell,1996, 84:381-388.
9. White TW, Paul DL. Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol,1999,61:283-310.
10. Valiunas V, Polosina YY, Miller H, et al. Connexin-specific cell-to-cell transfer of short interfering RNA by gap junctions. J Physiol,2005,568:459-68.
11. Krutovskikh V, Yamasaki H. Connexin gene mutations in human genetic diseases. Mutat Res,2000,462:197-207.
12. Vinken M, Vanhaecke T, Papeleu P, et al. Connexins and their channels in cell growth and cell death. Cell Signal,2006,18:592-600.
13. Lampe PD, Lau AF. The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol,2004,36:1171-1186.
14. Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases. Biol Cell,2002,94:433-443.
15. White TW, Paul DL. Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol,1999,61:283-310.
16. King TJ, Lampe PD. Temporal regulation of connexin phosphorylation in embryonic and adult tissues. Biochim Biophys Acta,2005,1719:24-35.
17. Laird DW. Life cycle of connexins in health and disease. Biochem. J,2006,394: 527-543.
18. Musil LS, Goodenough DA. Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell,1993, 74:1065-1077.
19. Harris AL. Emerging issues of connexin channels:biophysics fills the gap. Q Rev Biophys,2001,34:325-472.
20. Sarma JD, Wang F, Koval M. Targeted gap junction protein constructs reveal connexinspecific differences in oligomerization. J Biol Chem,2002,277:20911-20918.
21. Evans WH, De VE, Leybaert L. The gap junction cellular internet:connexin hemichannels enter the signalling limelight. Biochem J,2006,397:1-14.
22. Gaietta G, Deerinck TJ, Adams SR, et al. Multicolor and electron microscopic imaging of connexin trafficking. Science,2002,296:503-507.
23. Bukauskas FF, Kreuzberg MM, Rackauskas M, et al. Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels:implications for atrioventricular conduction in the heart. Proc Natl Acad Sci USA,2006,103:9726-9731.
24. Kretz M, Euwens C, Hombach S, et al. Altered connexin expression and wound healing in the epidermis of connexin-deficient mice. J Cell Sci,2003,116:3443-3452.
25. Goldberg GS, Moreno AP, Lampe PD. Gap junctions between cells expressing connexin 43 or 32 show inverse permselectivity to adenosine and ATP. J Biol Chem,2002, 277:36725-30.
26. Segretain D, Falk MM. Regulation of connexin biosynthesis, assembly, gap junction formation, and removal. Biochim Biophys Acta,2004,1662:3-21.
27. Weber PA, Chang HC, Spaeth KE, et al. The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities. Biophys J,2004,87:958-73.
28. Bedner P, Niessen H, Odermatt B, et al. Selective permeability of different connexin channels to the second messenger cyclic AMP. J Biol Chem,2006,281:6673-6681.
29. Qin H, Shao Q, Curtis H, et al. Retroviral delivery of connexin genes to human breast tumor cells inhibits in vivo tumor growth by a mechanism that is independent of significant gap junctional intercellular communication. J Biol Chem,2002,277: 29132-29138.
30. Dang X, Jeyaraman M, Kardami E. Regulation of connexin-43-mediated growth inhibition by a phosphorylatable amino-acid is independent of gap junction-forming ability. Mol. Cell Biochem,2006,289:201-207.
31. Yamasaki H, Naus CC. Role of connexin genes in growth control. Carcinogenesis,1996, 17:1199-1213.
32. Alexander DB, Ichikawa H, Bechberger JF, et al. Normal cells control the growth of neighboring transformed cells independent of gap junctional communication and SRC activity. Cancer Res,2004,64:1347-1358.
33. Plante I, Charbonneau M,. Cyr DG. Decreased gap junctional intercellular communication in hexachlorobenzene-induced genderspecific hepatic tumor formation in the rat. Carcinogenesis,2002,23:1243-1249.
34. Avanzo JL, Mesnil M, Hernandez-Blazquez FJ, et al. Increased susceptibility to urethane-induced lung tumors in mice with decreased expression of connexin43. Carcinogenesis,2004,25:1973-1982.
35. Zhang YW, Morita I, Ikeda M, et al. Connexin43 suppresses proliferation of osteosarcoma U2OS cells through post-transcriptional regulation of p27. Oncogene, 2001,20:4138-4149.
36. Zhang YW, Kaneda M, Morita I. The gap junction-independent tumor-suppressing effect of connexin 43. J Biol Chem,2003,278:44852-44856.
37. Nakase T, Naus CG. Gap junctions and neurological disorders of the central nervous system. Biochim Biophys Acta,2004,1662:215-224.
38. Zhang YW, Nakayama K, Nakayama K, et al. A novel route for connexin 43 to inhibit cell proliferation:negative regulation of S-phase kinase-associated protein (Skp 2). Cancer Res,2003,63:1623-1630.
39. Goldberg GS, Bechberger JF, Tajima Y, et al. Connexin43 suppresses MFG-E8 while inducing contact growth inhibition of glioma cells. Cancer Res,2000,60:6018-6026.
40. Naus CC, Bond SL, Bechberger JF, et al. Identification of genes differentially expressed in C6 glioma cells transfected with connexin43. Brain Res Brain Res Rev,2000,32: 259-266.
41. Iacobas DA, Urban-Maldonado M, Iacobas S, et al. Array analysis of gene expression in connexin-43 null astrocytes. Physiol. Genomics,2003,15:177-190.
42. Huang R, Lin Y, Wang CC, et al. Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology. Cancer Res,2002,62:2806-2812.
43. Tate AW, Lung T, Radhakrishnan A, et al. Changes in gap junctional connexin isoforms during prostate cancer progression. Prostate,2006,66:19-31.
44. Iacobas DA, Scemes E, Spray DC. Gene expression alterations in connexin null mice extend beyond the gap junction. Neurochem Int,2004,45:243-250.
45. Shao Q, Wang H, McLachlan E, et al. Down-regulation of Cx43 by retroviral delivery of small interfering RNA promotes an aggressive breast cancer cell phenotype. Cancer Res,2005,65:2705-2711.
46. Saunders MM, Seraj MJ, Li Z, et al. Breast cancer metastatic potential correlates with a breakdown in homospecific and heterospecific gap junctional intercellular communication. Cancer Res,2001,61:1765-1767.
47. Jongen WM, Fitzgerald DJ, Asamoto M, et al. Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+in mouse epidermal cells is controlled by E-cadherin. J Cell Biol,1991,114:545-555.
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