ezrin蛋白介导幽门螺杆菌促胃癌侵袭转移作用及其机制初探
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摘要
胃癌目前仍是我国死亡人数最多的恶性肿瘤之一,转移是肿瘤最具破坏性的本质,也是胃癌患者死亡的主要原因。深刻理解胃癌转移的分子机制对指导治疗十分重要。H.pylori感染是胃癌的重要危险因素,世界卫生组织已将H.pyori列为I类致癌原,对于H.pylori感染对胃癌的侵袭转移力有无影响,尚存在争论。H.pylori感染胃癌细胞可使肿瘤转移关键蛋白ezrin蛋白表达及功能发生变化,H.pylori感染是否能通过影响ezrin及其相关分子影响胃癌的侵袭转移,是本课题的需验证的设想。
目的:1、明确H.pylori感染与胃癌侵袭、转移是否相关;2、如果二者相关,探讨ezrin是否H.pylori感染与与胃癌侵袭、转移中起重要的作用;3、初步探讨ezrin在胃癌侵袭转移中可能分子机制,为H.pylori感染促胃癌进展提供新的实验依据。
方法:1、利用手术切除人体胃癌标本,联合血清H.pylori抗体检测及快速尿素酶法检测H.pylori感染状态,探讨H.pylori感染与胃癌淋巴结转移、胃癌浸润深度、病理分型的相关性;利用H.pylori与胃腺癌细胞AGS体外共培养,扫描电镜观察H.pylori感染AGS细胞形态学的改变,transwell小室检测H.pylori对AGS细胞侵袭力的影响;划痕实验观察H.pylori对AGS细胞迁移力的影响;MTT法检测H.pylori感染对AGS细胞失巢存活力的影响;2、免疫组化检测ezrin在胃癌组织中的表达,并探讨其表达强弱与胃癌浸润深度,有无淋巴结转移、是否有H.pylori感染的关系;免疫组化检测胃癌组织中CD44及E-cadherin的表达,探讨与H.pylori感染是否相关。选取AGS、MKN28、BGC823三种胃癌细胞株,western blot检测三种细胞株ezrin的表达,transwell小室检测侵袭力的高低,并探讨ezrin表达和侵袭力强弱的关系;以侵袭力最弱的MKN28细胞株为研究对象,设立H.pylori感染组与PBS对照组,观察两组细胞侵袭力是否有差异,并用western blot检测ezrin及其相关蛋白CD44、E-cadhrin的表达和ezrin上游调控因子Sixl的表达。3、构建VIL-2(ezrin编码基因)的RNA干涉真核表达载体以及阴性对照载体。将干涉载体利用脂质体转染侵袭力最强的AGS细胞,经G418抗性筛选获得稳定表达的细胞株AGSezrin。半定量RT-PCR及western blot检测沉默效应。分别将AGS+PBS、AGS+H.pylori、AGSezrin+PBS、AGSezrin+H.pylori四组细胞为研究对象,扫描电镜细胞观察形态特征的变化,通过MTT、侵袭小室实验、细胞迁移实验、黏附实验观察细胞的失巢存活、侵袭和迁移、黏附能力。western blot检测ezrin、E-cadherin、CD44表达的变化。
结果:1.①103例患者中,血清抗H.pylori抗体阳性71例,快速尿酶法阳性67例,二者均阳性60例,二者均阴性23例。共83例患者入组, H.pylori阳性患者淋巴结转移36例(60%),H.pylori阴性患者淋巴结转移5例(21.7%),H.pylori阳性患者与H.pylori阴性患者发生淋巴结转移率显著差异(p<0.05);H.pylori感染与肿瘤浸润深度评分无关(p>0.05),肠型胃癌45例,其中H.pylori阳性率为75.6%(34 /45) ,高于弥漫型胃癌癌灶H.pylori阳性率为42.1% ( 16 /38) ,差异显著(p<0.05)。②H. pylori与AGS细胞共培养,观察到细胞变形,相互分散,出现“蜂鸟表型”及空泡样变。电镜下观察H. pylori感染AGS细胞24小时,细胞变为长梭形,伸出细长伪足。③AGS细胞与H. pylori共孵育后行transwell实验,每200倍镜视野下穿膜细胞数130.4±11.5,高于PBS对照组87.1±9.3,差异具有统计学意义(P < 0.05)。④迁移实验中加入H. pylori的AGS细胞迁移距离均明显大于PBS对照组细胞。⑤H.pylori感染的细胞失巢培养24小时,细胞生存率高于PBS对照组(P<0.05)。2.①H.pylori阳性患者胃癌组织中ezrin蛋白表达强度高于H.pylori阴性患者(P<0.05),ezrin蛋白与胃癌浸润深度无关(P>0.05),有淋巴结转移的患者胃癌组织ezrin的免疫活性显著强于无淋巴结转移的患者(P<0.05),H.pylori感染与胃癌组织中CD44表达无关(P>0.05),H.pylori阳性患者较H.pylori阴性患者胃癌组织中E-cadherin蛋白表达显著降低(P<0.05)。②检测三种细胞系侵袭力,侵袭力最强的是AGS,其次为BGC823,最弱为MKN28。ezrin蛋白在三种细胞系中表达从高到低依次为AGS,BGC823, MKN28,在此三种细胞系中,ezrin的表达强弱与侵袭力高低一致。③以侵袭力最弱的MKN28为研究对象,加入H.pylori的感染组细胞,24小时穿膜数量为51.5±5.5 ,加入同体积的PBS的阴性对照组细胞,24小时穿膜数量为32.7±6.2。二者比较,H.pylori感染组显著多于阴性对照组,差异具有统计学意义(P<0.05),④H.pylori对MKN28细胞侵袭力的促进作用大于对AGS细胞的作用(P<0.05)。H.pylori感染促进MKN28细胞中ezrin、CD44蛋白表达,且差异显著(P<0.05,P<0.05)降低E-cadherin在MKN28中的表达(P<0.05),增加ezrin上游调控蛋白six1的表达。3、①成功构建针对ezrin转录基因(VIL2)的3种特异性干扰质粒,通过western blot及RT-PCR筛选感染效果最强的质粒转染侵袭力最强、ezrin表达最高的AGS细胞,经G418筛选建立稳定转染该感染质粒的AGSezrin细胞系;②扫描电子显微镜观察细胞表面形态学变化,AGS细胞呈椭圆形或长梭形,细胞表面粗糙,分布粗短的叶状伪足;加入100:1(细菌/细胞)H.pylori后,可见H.pylori黏附于细胞表面,细胞表面粗短突起减少,但细胞沿长轴伸出两细长突起,长度大于2倍细胞直径,AGSezrin细胞呈多边形或类圆形,细胞表面向四周伸出较多细长丝状伪足,可见较丰富的细胞间连接,亦见凋亡细胞;加入100:1(细菌/细胞)H.pylori后,细胞表面几乎被黏附的H.pylori覆盖,细胞仍呈多边形,细胞向周边辐射状伸出丝状伪足。扫描电镜放大4000倍,AGS表面黏附的H.pylori数量为:24.2±11.5,AGSezrin细胞表面黏附的H.pylori数量为:51.4±13.1,显著多于AGS细胞(P<0.05);③AGS细胞的黏附率为(76.2±11.7)%,AGSezrin细胞的黏附率为(90.5±7.6)%,二者比较(P<0.05),差异显著,10:1比例H.pylori感染AGS及AGSezrin细胞,黏附率分别为(61.8±8.5)%,(92.4±7.3)%,H.pylori感染AGS组细胞黏附率与AGS组比较,显著下降(P<0.05);⑤侵袭实验中200倍镜下平均每视野AGSEzrin细胞的跨膜数为27.67±4.50,与AGS细胞125.50±8.33比较差异显著( P< 0.05), AGSezrin细胞+H.pylori,跨膜细胞数为31.25±6.10,与AGSEzrin细胞比较,无显著差异; AGSezrin的迁移距离明显小于AGS细胞的迁移距离。而H.pylori感染对AGSezrin细胞迁移无明显影响;H.pylori感染的AGSezrin细胞在失巢生存率略低于PBS对照组,但无显著性差异(P>0.05);⑥H.pylori感染增加AGS细胞ezrin、CD44的表达,但对AGSezrin细胞无明显影响。
结论:1、H.pylori可促进胃癌的侵袭和淋巴结转移;2、ezrin蛋白在胃癌的侵袭转移中具有重要的作用,并在多个环节中参与了H.pylori促胃癌侵袭过程;3、H.pylori可通过促进Sixl的表达而上调ezrin在胃癌细胞中的表达,从而促进胃癌侵袭。
In China, gastric carcinoma is one of the most common malignant tumor with high fatality rate. The metastasis of gastric carcinoma is fatal. In order to increase the survival rate of patients with gastric carcinoma, it is very helpful for us to understand the molecular mechanism of the metastasis of gastric carcinoma. Helicobacter pylori (H.pylori) infection, Class I carcinogen defined by the World Health Organization, is a major risk factor for gastric carcinoma. However, the correlation between Helicobacter pylori (H.pylori) infection and metastasis of gastric carcinoma remains unclear. It was reported that the expression and function of ezrin protein( a key protein in tumor metastasis) were changed after gastric cancer cells was infected by H.pylori. In this research, we wanted to confirm whether H.pylori. infection influenced the metastasis and invasion of gastric carcinoma by regulating ezrin expression.
Objective: 1. To investigate the correlation between invasion and metastasis of gastric carcinoma and H.pylori infection; 2. To explore whether ezrin plays an important role in mediating invasion and metastasis of H.pylori infection-related gastric carcinoma and its molecular mechanism.
Methods: 1. By rapid urease test and serum antibody determination, we identified whether the patients were infected H.pylori. H.pylori and AGS cells of gastric adenocarcinoma were cultivated together in vitro. Then the AGS cell morphology was obversed by scanning electron microscopy, the invasion of AGS cell was detected by transwell chamber, AGS cell migration was obversed by scratch test. MTT assay was used to analyze the impact about loss of nests of AGS cells.
2. The expression of ezrin, CD44 and E-cadherin in tissues of gastric carcinoma were detected by immunohistochemistry. The express levels of ezrin protein in AGS, MKN28, BGC823 cells were determined by western blot and the invasion of the three cells was analyzed by transwell chamber. MKN28 cell lines possessing weak metastasis ability in above-mentioned cells were observed. MKN28 cell lines were divided into two groups, HP infected group and PBS control group. The invasion of two groups were determined by transwell chamber. The expressions of ezrin, CD44, E-cadhrin and Sixl in two groups were detected by western blot.
3. The VIL-2 of RNA interference plasmid vector and control plasmid vector were constructed by gene recombinant technique. AGS cells were transfected by these plasmid vectors. The stable expression cell lines AGSezrin was obtainted by G418 resistance screening. The expressions of Ezrin mRNA and protein were detected by semi-quantitative RT-PCR and western blot. The morphology of four groups, including AGS+PBS, AGS+H.pylori, AGSezrin+PBS, AGSezrin+H.pylori was detected by scanning electron microscope. Meanwhile,the ability of cell invasion, migration and adhesion was determined respectively. In addition, the expressions of ezrin, CD44 and E-cadherin protein in four groups were detected by western blot.
Results: 1.①Among 103 subjects, 71 cases were positive for serum H.pylori antibody, 67 cases were positive for rapid enzymatic urine analysis. 60 cases were both positive and 23 cases were both negative in serum H.pylori antibody and rapid enzymatic urine analysis. Among 83 patients enrolled in this research, 36 cases (60%) of H.pylori-positive showed lymph node metastasis and 5 cases (21.7%) of H.pylori-negative showed lymph node metastasis. The rate of lymph node metastasis was significantly different between H.pylori-positive cases and H.pylori-negative cases(P<0.05). The depths of tumor invasion between H.pylori positive cases and negative cases did not show significant difference(P>0.05). The rate of H.pylori infection in intestinal type gastric carcinoma (75.6%,34/45) was significantly higher than that in diffuse-type gastric carcinoma (42.1% ,16/ 38,P<0.05).②After incubated by H. pylori,AGS cells showed deformation, a "hummingbird phenotype" and vacuolated changes. The cells exhibited a long spindle-shaped, long-stretched thin pseudopodia after 24 hours of incubation by H. pylori.③The count of the transmembrane AGS cells was 130.4±11.5 from each version under microscope at 200 times after AGS cells were incubated by H. pylori, significantly higher than that of AGS cells without being incubated by H. pylori(87.1±9.3, P <0.05).④Migrate distance of AGS cells incubated by H. pylori was significantly higher than that of AGS cells without being incubated by H. pylori.⑤The survival rate of nest-losing AGS cells incubated by H. pylori was significantly higher than that of cells incubated without being incubated by H. pylori (P <0.05) after 24 hours of incubation.
2.①The expression of ezrin protein and E-cadherin in cases of H.pylori-positive gastric carcinoma were higher than those in cases of H.pylori-negative gastric carcinoma (P <0.05). The expressions of Ezrin in different invasion depths in tissues of gastric carcinoma did not show significant difference (P> 0.05).The immunological activity of ezrin protein in gastric carcinoma patients with lymph node metastasis was significantly stronger than that in patients without lymph node metastasis (P <0.05). The expressions of CD44 in H.pylori positive and H.pylori negative group did not show significant difference(P> 0.05).②Among three cell lines, AGS cell showed the strongest invasion ability, followed by BGC823 and MKN28. The expression of ezrin protein in AGS cells was highest in three cell lines, followed by BGC823 and MKN28 cells.③The count of transmembrane MKN28 cells incubated by H.pylori for 24 hours were 51.5±5.5, higher than that without being incubated H.pylori(32.7±6.2, P<0.05).④The promotion to MKN28 cells invasion caused by H.pylori infection was higher than that to AGS cells. (P<0.05). H.pylori infection can significantly promote the expression of ezrin,CD44 and six1 protein in MKN28 cells (P <0.05). H.pylori infection can also lower the expression of E-cadherin in MKN28(P <0.05).
3.①The three kinds of specific interference plasmids of ezrin gene transcription (VIL2) were successfully constructed. The stable expression of interference plasmids of ezrin gene cell lines AGSezrin was obtainted by G418 resistance screening. The expression of ezrin mRNA and protein were detected by semi-quantitative RT-PCR and western blot.②The morphology of cell surface was observed under scanning electron microscopy. AGS cells were oval-shaped or long-spindle. AGS cells were rough in cell surface and possessed stumpy lobed pseudopodia. After being added 100:1 (bacteria / cell) H.pylori, the attachment of H.pylori can be observed. AGS cells exhibited a“hummingbird”phenotype with two slender dendritic processes along their long axis, and the length of the processes is more than 2 times of the cellular diameter. AGSezrin cells appeared polygonal or round with a lot of slender filipodium extending around cell surface. Moreover, cell-cell junction and cell apoptosis were also observed. After AGS cells were added H. pylori at the ratio of 100:1(H. pylori/cells), cell surface was almost covered by the attachment of H. pylori. Filipodium was radially arranged from the cell surface. Cells remained polygonal in shape.Under scanning electron microscope, the adhesion numbers of H.pylori were 24.2±11.5 in AGS cells, 51.4±13.1 in AGSezrin cells. The adhesion number of H.pylori in AGSezrin cells was significantly higher than that in AGS cells (P <0.05).③The adhesion rate was ( 76.2±11.7)% in AGS cells, which was significantly lower than that in AGSezrin cells[ (90.5±7.6)%]. When AGS cells were added 10:1 ratio of H.pylori in AGS and AGSezrin cells, the adhesion rate was (61.8±8.5 )% and (92.4±7.3)% respectively. The adhesion rate of H.pylori infection in AGS and AGSezrin cells showed significant difference(P <0.05).④The average number of transmembrane of AGSezrin cells was 27.67±4.50 , which was signicantly lower than that of AGS cells(125.50±8.33, P <0.05). The number of transmembrane of AGSezrin cells infected with H.pylori were 31.25±6.10. H.pylori infection did not affect the number of transmembrane AGSezrin cells.
The migration distance of AGSezrin cells were significantly less than that of AGS cells. H.pylori did not affect migration distance of AGSezrin cells.⑤The percentages of anoikis in AGSezrin cells infected with H.pylori at 6-, 12-, 18- and 24-hour was slightly lower than those of cells infected without H.pylori (P>0.05).⑥The expressions of ezrin and CD44 were promoted in AGS cells infected with H.pylori but no significant change was observed in AGSezrin cells.
Conclusion: 1. H.pylori can promote the invasion and lymph node metastasis of gastric carcinoma. 2. Ezrin protein plays an important role in invasion and metastasis of gastric carcinoma and also participates the process of invasion of gastric carcinoma mediated by H.pylori. 3. H.pylori can regulate the expression of Sixl to promote the expression of ezrin in gastric carcinoma cells, resuting in the gastric cancer invasion.
目的:1、明确H.pylori感染与胃癌侵袭、转移是否相关;2、如果二者相关,探讨ezrin是否H.pylori感染与与胃癌侵袭、转移中起重要的作用;3、初步探讨ezrin在胃癌侵袭转移中可能分子机制,为H.pylori感染促胃癌进展提供新的实验依据。
方法:1、利用手术切除人体胃癌标本,联合血清H.pylori抗体检测及快速尿素酶法检测H.pylori感染状态,探讨H.pylori感染与胃癌淋巴结转移、胃癌浸润深度、病理分型的相关性;利用H.pylori与胃腺癌细胞AGS体外共培养,扫描电镜观察H.pylori感染AGS细胞形态学的改变,transwell小室检测H.pylori对AGS细胞侵袭力的影响;划痕实验观察H.pylori对AGS细胞迁移力的影响;MTT法检测H.pylori感染对AGS细胞失巢存活力的影响;2、免疫组化检测ezrin在胃癌组织中的表达,并探讨其表达强弱与胃癌浸润深度,有无淋巴结转移、是否有H.pylori感染的关系;免疫组化检测胃癌组织中CD44及E-cadherin的表达,探讨与H.pylori感染是否相关。选取AGS、MKN28、BGC823三种胃癌细胞株,western blot检测三种细胞株ezrin的表达,transwell小室检测侵袭力的高低,并探讨ezrin表达和侵袭力强弱的关系;以侵袭力最弱的MKN28细胞株为研究对象,设立H.pylori感染组与PBS对照组,观察两组细胞侵袭力是否有差异,并用western blot检测ezrin及其相关蛋白CD44、E-cadhrin的表达和ezrin上游调控因子Sixl的表达。3、构建VIL-2(ezrin编码基因)的RNA干涉真核表达载体以及阴性对照载体。将干涉载体利用脂质体转染侵袭力最强的AGS细胞,经G418抗性筛选获得稳定表达的细胞株AGSezrin。半定量RT-PCR及western blot检测沉默效应。分别将AGS+PBS、AGS+H.pylori、AGSezrin+PBS、AGSezrin+H.pylori四组细胞为研究对象,扫描电镜细胞观察形态特征的变化,通过MTT、侵袭小室实验、细胞迁移实验、黏附实验观察细胞的失巢存活、侵袭和迁移、黏附能力。western blot检测ezrin、E-cadherin、CD44表达的变化。
结果:1.①103例患者中,血清抗H.pylori抗体阳性71例,快速尿酶法阳性67例,二者均阳性60例,二者均阴性23例。共83例患者入组, H.pylori阳性患者淋巴结转移36例(60%),H.pylori阴性患者淋巴结转移5例(21.7%),H.pylori阳性患者与H.pylori阴性患者发生淋巴结转移率显著差异(p<0.05);H.pylori感染与肿瘤浸润深度评分无关(p>0.05),肠型胃癌45例,其中H.pylori阳性率为75.6%(34 /45) ,高于弥漫型胃癌癌灶H.pylori阳性率为42.1% ( 16 /38) ,差异显著(p<0.05)。②H. pylori与AGS细胞共培养,观察到细胞变形,相互分散,出现“蜂鸟表型”及空泡样变。电镜下观察H. pylori感染AGS细胞24小时,细胞变为长梭形,伸出细长伪足。③AGS细胞与H. pylori共孵育后行transwell实验,每200倍镜视野下穿膜细胞数130.4±11.5,高于PBS对照组87.1±9.3,差异具有统计学意义(P < 0.05)。④迁移实验中加入H. pylori的AGS细胞迁移距离均明显大于PBS对照组细胞。⑤H.pylori感染的细胞失巢培养24小时,细胞生存率高于PBS对照组(P<0.05)。2.①H.pylori阳性患者胃癌组织中ezrin蛋白表达强度高于H.pylori阴性患者(P<0.05),ezrin蛋白与胃癌浸润深度无关(P>0.05),有淋巴结转移的患者胃癌组织ezrin的免疫活性显著强于无淋巴结转移的患者(P<0.05),H.pylori感染与胃癌组织中CD44表达无关(P>0.05),H.pylori阳性患者较H.pylori阴性患者胃癌组织中E-cadherin蛋白表达显著降低(P<0.05)。②检测三种细胞系侵袭力,侵袭力最强的是AGS,其次为BGC823,最弱为MKN28。ezrin蛋白在三种细胞系中表达从高到低依次为AGS,BGC823, MKN28,在此三种细胞系中,ezrin的表达强弱与侵袭力高低一致。③以侵袭力最弱的MKN28为研究对象,加入H.pylori的感染组细胞,24小时穿膜数量为51.5±5.5 ,加入同体积的PBS的阴性对照组细胞,24小时穿膜数量为32.7±6.2。二者比较,H.pylori感染组显著多于阴性对照组,差异具有统计学意义(P<0.05),④H.pylori对MKN28细胞侵袭力的促进作用大于对AGS细胞的作用(P<0.05)。H.pylori感染促进MKN28细胞中ezrin、CD44蛋白表达,且差异显著(P<0.05,P<0.05)降低E-cadherin在MKN28中的表达(P<0.05),增加ezrin上游调控蛋白six1的表达。3、①成功构建针对ezrin转录基因(VIL2)的3种特异性干扰质粒,通过western blot及RT-PCR筛选感染效果最强的质粒转染侵袭力最强、ezrin表达最高的AGS细胞,经G418筛选建立稳定转染该感染质粒的AGSezrin细胞系;②扫描电子显微镜观察细胞表面形态学变化,AGS细胞呈椭圆形或长梭形,细胞表面粗糙,分布粗短的叶状伪足;加入100:1(细菌/细胞)H.pylori后,可见H.pylori黏附于细胞表面,细胞表面粗短突起减少,但细胞沿长轴伸出两细长突起,长度大于2倍细胞直径,AGSezrin细胞呈多边形或类圆形,细胞表面向四周伸出较多细长丝状伪足,可见较丰富的细胞间连接,亦见凋亡细胞;加入100:1(细菌/细胞)H.pylori后,细胞表面几乎被黏附的H.pylori覆盖,细胞仍呈多边形,细胞向周边辐射状伸出丝状伪足。扫描电镜放大4000倍,AGS表面黏附的H.pylori数量为:24.2±11.5,AGSezrin细胞表面黏附的H.pylori数量为:51.4±13.1,显著多于AGS细胞(P<0.05);③AGS细胞的黏附率为(76.2±11.7)%,AGSezrin细胞的黏附率为(90.5±7.6)%,二者比较(P<0.05),差异显著,10:1比例H.pylori感染AGS及AGSezrin细胞,黏附率分别为(61.8±8.5)%,(92.4±7.3)%,H.pylori感染AGS组细胞黏附率与AGS组比较,显著下降(P<0.05);⑤侵袭实验中200倍镜下平均每视野AGSEzrin细胞的跨膜数为27.67±4.50,与AGS细胞125.50±8.33比较差异显著( P< 0.05), AGSezrin细胞+H.pylori,跨膜细胞数为31.25±6.10,与AGSEzrin细胞比较,无显著差异; AGSezrin的迁移距离明显小于AGS细胞的迁移距离。而H.pylori感染对AGSezrin细胞迁移无明显影响;H.pylori感染的AGSezrin细胞在失巢生存率略低于PBS对照组,但无显著性差异(P>0.05);⑥H.pylori感染增加AGS细胞ezrin、CD44的表达,但对AGSezrin细胞无明显影响。
结论:1、H.pylori可促进胃癌的侵袭和淋巴结转移;2、ezrin蛋白在胃癌的侵袭转移中具有重要的作用,并在多个环节中参与了H.pylori促胃癌侵袭过程;3、H.pylori可通过促进Sixl的表达而上调ezrin在胃癌细胞中的表达,从而促进胃癌侵袭。
In China, gastric carcinoma is one of the most common malignant tumor with high fatality rate. The metastasis of gastric carcinoma is fatal. In order to increase the survival rate of patients with gastric carcinoma, it is very helpful for us to understand the molecular mechanism of the metastasis of gastric carcinoma. Helicobacter pylori (H.pylori) infection, Class I carcinogen defined by the World Health Organization, is a major risk factor for gastric carcinoma. However, the correlation between Helicobacter pylori (H.pylori) infection and metastasis of gastric carcinoma remains unclear. It was reported that the expression and function of ezrin protein( a key protein in tumor metastasis) were changed after gastric cancer cells was infected by H.pylori. In this research, we wanted to confirm whether H.pylori. infection influenced the metastasis and invasion of gastric carcinoma by regulating ezrin expression.
Objective: 1. To investigate the correlation between invasion and metastasis of gastric carcinoma and H.pylori infection; 2. To explore whether ezrin plays an important role in mediating invasion and metastasis of H.pylori infection-related gastric carcinoma and its molecular mechanism.
Methods: 1. By rapid urease test and serum antibody determination, we identified whether the patients were infected H.pylori. H.pylori and AGS cells of gastric adenocarcinoma were cultivated together in vitro. Then the AGS cell morphology was obversed by scanning electron microscopy, the invasion of AGS cell was detected by transwell chamber, AGS cell migration was obversed by scratch test. MTT assay was used to analyze the impact about loss of nests of AGS cells.
2. The expression of ezrin, CD44 and E-cadherin in tissues of gastric carcinoma were detected by immunohistochemistry. The express levels of ezrin protein in AGS, MKN28, BGC823 cells were determined by western blot and the invasion of the three cells was analyzed by transwell chamber. MKN28 cell lines possessing weak metastasis ability in above-mentioned cells were observed. MKN28 cell lines were divided into two groups, HP infected group and PBS control group. The invasion of two groups were determined by transwell chamber. The expressions of ezrin, CD44, E-cadhrin and Sixl in two groups were detected by western blot.
3. The VIL-2 of RNA interference plasmid vector and control plasmid vector were constructed by gene recombinant technique. AGS cells were transfected by these plasmid vectors. The stable expression cell lines AGSezrin was obtainted by G418 resistance screening. The expressions of Ezrin mRNA and protein were detected by semi-quantitative RT-PCR and western blot. The morphology of four groups, including AGS+PBS, AGS+H.pylori, AGSezrin+PBS, AGSezrin+H.pylori was detected by scanning electron microscope. Meanwhile,the ability of cell invasion, migration and adhesion was determined respectively. In addition, the expressions of ezrin, CD44 and E-cadherin protein in four groups were detected by western blot.
Results: 1.①Among 103 subjects, 71 cases were positive for serum H.pylori antibody, 67 cases were positive for rapid enzymatic urine analysis. 60 cases were both positive and 23 cases were both negative in serum H.pylori antibody and rapid enzymatic urine analysis. Among 83 patients enrolled in this research, 36 cases (60%) of H.pylori-positive showed lymph node metastasis and 5 cases (21.7%) of H.pylori-negative showed lymph node metastasis. The rate of lymph node metastasis was significantly different between H.pylori-positive cases and H.pylori-negative cases(P<0.05). The depths of tumor invasion between H.pylori positive cases and negative cases did not show significant difference(P>0.05). The rate of H.pylori infection in intestinal type gastric carcinoma (75.6%,34/45) was significantly higher than that in diffuse-type gastric carcinoma (42.1% ,16/ 38,P<0.05).②After incubated by H. pylori,AGS cells showed deformation, a "hummingbird phenotype" and vacuolated changes. The cells exhibited a long spindle-shaped, long-stretched thin pseudopodia after 24 hours of incubation by H. pylori.③The count of the transmembrane AGS cells was 130.4±11.5 from each version under microscope at 200 times after AGS cells were incubated by H. pylori, significantly higher than that of AGS cells without being incubated by H. pylori(87.1±9.3, P <0.05).④Migrate distance of AGS cells incubated by H. pylori was significantly higher than that of AGS cells without being incubated by H. pylori.⑤The survival rate of nest-losing AGS cells incubated by H. pylori was significantly higher than that of cells incubated without being incubated by H. pylori (P <0.05) after 24 hours of incubation.
2.①The expression of ezrin protein and E-cadherin in cases of H.pylori-positive gastric carcinoma were higher than those in cases of H.pylori-negative gastric carcinoma (P <0.05). The expressions of Ezrin in different invasion depths in tissues of gastric carcinoma did not show significant difference (P> 0.05).The immunological activity of ezrin protein in gastric carcinoma patients with lymph node metastasis was significantly stronger than that in patients without lymph node metastasis (P <0.05). The expressions of CD44 in H.pylori positive and H.pylori negative group did not show significant difference(P> 0.05).②Among three cell lines, AGS cell showed the strongest invasion ability, followed by BGC823 and MKN28. The expression of ezrin protein in AGS cells was highest in three cell lines, followed by BGC823 and MKN28 cells.③The count of transmembrane MKN28 cells incubated by H.pylori for 24 hours were 51.5±5.5, higher than that without being incubated H.pylori(32.7±6.2, P<0.05).④The promotion to MKN28 cells invasion caused by H.pylori infection was higher than that to AGS cells. (P<0.05). H.pylori infection can significantly promote the expression of ezrin,CD44 and six1 protein in MKN28 cells (P <0.05). H.pylori infection can also lower the expression of E-cadherin in MKN28(P <0.05).
3.①The three kinds of specific interference plasmids of ezrin gene transcription (VIL2) were successfully constructed. The stable expression of interference plasmids of ezrin gene cell lines AGSezrin was obtainted by G418 resistance screening. The expression of ezrin mRNA and protein were detected by semi-quantitative RT-PCR and western blot.②The morphology of cell surface was observed under scanning electron microscopy. AGS cells were oval-shaped or long-spindle. AGS cells were rough in cell surface and possessed stumpy lobed pseudopodia. After being added 100:1 (bacteria / cell) H.pylori, the attachment of H.pylori can be observed. AGS cells exhibited a“hummingbird”phenotype with two slender dendritic processes along their long axis, and the length of the processes is more than 2 times of the cellular diameter. AGSezrin cells appeared polygonal or round with a lot of slender filipodium extending around cell surface. Moreover, cell-cell junction and cell apoptosis were also observed. After AGS cells were added H. pylori at the ratio of 100:1(H. pylori/cells), cell surface was almost covered by the attachment of H. pylori. Filipodium was radially arranged from the cell surface. Cells remained polygonal in shape.Under scanning electron microscope, the adhesion numbers of H.pylori were 24.2±11.5 in AGS cells, 51.4±13.1 in AGSezrin cells. The adhesion number of H.pylori in AGSezrin cells was significantly higher than that in AGS cells (P <0.05).③The adhesion rate was ( 76.2±11.7)% in AGS cells, which was significantly lower than that in AGSezrin cells[ (90.5±7.6)%]. When AGS cells were added 10:1 ratio of H.pylori in AGS and AGSezrin cells, the adhesion rate was (61.8±8.5 )% and (92.4±7.3)% respectively. The adhesion rate of H.pylori infection in AGS and AGSezrin cells showed significant difference(P <0.05).④The average number of transmembrane of AGSezrin cells was 27.67±4.50 , which was signicantly lower than that of AGS cells(125.50±8.33, P <0.05). The number of transmembrane of AGSezrin cells infected with H.pylori were 31.25±6.10. H.pylori infection did not affect the number of transmembrane AGSezrin cells.
The migration distance of AGSezrin cells were significantly less than that of AGS cells. H.pylori did not affect migration distance of AGSezrin cells.⑤The percentages of anoikis in AGSezrin cells infected with H.pylori at 6-, 12-, 18- and 24-hour was slightly lower than those of cells infected without H.pylori (P>0.05).⑥The expressions of ezrin and CD44 were promoted in AGS cells infected with H.pylori but no significant change was observed in AGSezrin cells.
Conclusion: 1. H.pylori can promote the invasion and lymph node metastasis of gastric carcinoma. 2. Ezrin protein plays an important role in invasion and metastasis of gastric carcinoma and also participates the process of invasion of gastric carcinoma mediated by H.pylori. 3. H.pylori can regulate the expression of Sixl to promote the expression of ezrin in gastric carcinoma cells, resuting in the gastric cancer invasion.
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