上皮性卵巢癌中自噬基因Beclin1的表达及其调控相关信号途径PI3K/PKB的实验研究
摘要
背景与研究目的卵巢癌是是妇科恶性肿瘤中死亡率最高的肿瘤，寻找有效的治疗方法一直是临床的迫切要求。在卵巢癌的治疗中，基因治疗治疗已成为手术、化疗、放疗三大治疗外新的治疗手段。
自噬在清除损伤或多余的细胞器，维持细胞内稳定方面起重要作用。自噬可清除损伤的细胞器，避免如自由基等有基因毒性的物质的产生，从而使突变率降低，抑制肿瘤的形成，而自噬的缺陷可导致肿瘤的发生。
Beclin1基因是第一个确认的哺乳动物的自噬基因，也是第一个确认的在自噬的溶酶体降解途径中起肿瘤抑制的基因，对自噬体的形成至关重要。有研究报道乳腺癌中Beclin1的表达下调，Beclin1缺陷明显增加肝癌、肺癌和淋巴瘤的发生率，但自噬基因Beclin1与卵巢癌的关系尚不清楚。P13K／PKB是目前涉及凋亡调控较清楚的细胞信号传导途径，有研究表明其亦是调控自噬的主要途径之一，ClassⅠP13K抑制自噬，而ClassⅢP13K刺激自噬的发生。
本研究通过检测上皮性卵巢癌组织中自噬基因Beclin 1的表达及其调控相关信号传导途径P13K／PKB中的成分ClassⅠP13K (p110α)、ClassⅢ P13K(hvps34)及其下游的磷酸化PKB(p-PKB)的表达变化，探讨自噬基因Beclin1及其调控相关信号传导途径P13K／PKB中的成分p110α、hvps34、p-PKB的表达与上皮性卵巢癌的发生发展之间的关系；研究自噬基因Beclin1在SKOV3细胞中过表达对肿瘤细胞在体内、体外增殖的影响及其相关调控信号途径P13K／PKB的变化，探讨自噬基因Beclin1表达载体作为卵巢癌基因治疗的可行性。方法1.用免疫组化的方法对25例正常卵巢组织、25例良性上皮性卵巢肿瘤、19例交界性上皮性卵巢肿瘤及69例上皮性卵巢癌组织进行Beclin1、ClassⅠP13K(p110α)、ClassⅢP13K(hvps34)及其下游的磷酸化PKB的表达水平进行检测；
2.通过RT-PCR方法，从人正常卵巢组织中获得目的基因Beclin1，将其插入真核表达载体pcDNA3.1(＋)中，构建真核表达载体pcDNA3.1／Beclin1；
3.脂质体法将重组质粒pcDNA3.1／Beclin1、空质粒pcDNA3.1分别转染人卵巢癌细胞株SKOV3。用荧光定量RT-PCR及western blot分别在mRNA和蛋白质水平检测转染前后Beclin1、hvps34、p110α和p-PKB的表达变化。细胞免疫组化方法观察pcDNA3.1／Beclin1转染后SKOV3中Beclin1蛋白表达变化；在电镜和荧光显微镜下观察自噬囊泡；流式细胞仪检测凋亡及自噬情况，并用MTT法分析外源性Beclin1过表达对SKOV3增殖的影响；
4.将重组质粒pcDNA3.1／Beclin1转染SKOV3细胞后，接种到裸鼠皮下，观察体内致瘤活性及生长情况；免疫组化检测瘤组织Beclin1蛋白的表达。
结果1.Beclin 1、hvps34在正常卵巢组织与良性卵巢肿瘤组织中表达较高，在交界性卵巢癌组织中开始下降，在上皮性卵巢癌中的表达最低；p110α、p-PKB在交界性卵巢癌组织中开始升高，在上皮性卵巢癌中的表达明显高于其它3组(P＜0.05)。卵巢癌Ⅰ～Ⅱ期、高中分化、淋巴结无转移的卵巢癌组织中Beclin 1的表达均高于Ⅲ～Ⅳ期、低分化、淋巴结有转移者(P＜0.05)，而p110α及p-PKB的表达均低于Ⅲ～Ⅳ期、低分化、淋巴结有转移者(P＜0.05)；
2.利用酶切、PCR分析、DNA测序证实，真核表达载体pcDNA3.1／Beclin1构建成功；
3.pcDNA3.1／Beclin1转染后SKOV3细胞Beclin1表达在mRNA和蛋白质水平明显高于转染空质粒pcDNA3.1和未转染细胞；pcDNA3.1／Beclin1转染后hvps34表达上调，而p110α、p-PKB的表达下调。pcDNA3.1／Beclin1转染SKOV3细胞后在在电镜下可见大量自噬囊泡形成。在荧光显微镜下见pcDNA3.1／Beclin1转染后SKOV3细胞中MDC阳性细胞明显增加。pcDNA3.1／Beclin1转染后SKOV3的凋亡率为(21.26±3.89)％，高于pcDNA3.1转染组和未转染组，差异有显著性(P＜0.05)；与未转染组相比，G1期细胞比例明显增多，S期细胞比例明显降低。流式细胞仪检测pcDNA3.1／Beclin1转染后SKOV3细胞MDC平均荧光强度高于pcDNA3.1转染组和未转染组。pcDNA3.1／Beclin1转染SKOV3后细胞的体外增殖能力明显弱于转染空质粒组和未转染组，细胞抑制率为58.68％(P＜0.05)。
4.重组质粒pcDNA3.1／Beclin1使卵巢癌SKOV3细胞裸鼠体内成瘤时间延长，瘤体体积及重量明显小于空质粒组和空白对照组，抑瘤率为50.27％。免疫组化显示SKOV3-Beclin1组Beclin1蛋白表达强于SKOV3-pcDNA3.1组和SKOV3组，肿瘤组织坏死增加。
结论自噬基因Beclin 1在上皮性卵巢癌组织中的表达下调，对自噬起调控作用的PI3K／PKB信号通路中的成分p110α、hvps34、p-PKB存在表达异常，可能与上皮性卵巢癌的发生、发展及临床预后有关；自噬基因Beclin1在人卵巢癌细胞SKOV3中过表达可使ClassⅢPI3K(hvps34)表达上调，ClassⅠPI3K(p110α)及其下游的p-PKB表达下调；自噬基因Beclin1在人卵巢癌细胞SKOV3中过表达可诱导肿瘤细胞自噬和凋亡的发生，抑制SKOV3在体内、体外的增殖，针对自噬基因Beclin1的卵巢癌基因治疗可能具有可行性。
Background & Objective Epithelial ovarian cancer is the first leading cause of gynecologic malignancy-related deaths. It is urgent to find effective treatment of ovarian carcinoma in clinical practice. Surgery, chemotherapy and radiation therapy are conventional treatment for ovarian carcinoma, and gene therapy is regard as a new treatment strategy for ovarian carcinoma.
Autophagy plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis. For example, by removing damaged organelles, autophagy may limit the exposure of cellular DNA to genotoxic- stresses such as free radicals. The removal of damaged organelles through autophagic degradation would thus decrease the basal mutation rate and suppress oncogenesis.
Beclin 1, the first identified mammalian autophagy gene product, is the first identified tumor suppressor protein that functions in the lysosomal degradation pathway of autophagy. Beclin1 is essential to formation of autophagosome. Some studies reported that Beclin1 expression was down-regulated in breast carcinoma, and loss of Beclin 1 would contribute to an increased incidence of cancer, such as heptocellular carcinoma, lung adenocarcinoma and lymphoma. It is unclear that correlation of autophagy Gene Beclinl to tumorigenesis and development of epithelial ovarian cancer.
It was well known that apoptosis was regulated by PI3K/PKB signaling pathway, and this pathway is invovlved in ihe control of autophagy. Some studies reported the classⅠPI3K inhibit autophagy and the classⅢPI3K stimulates autophagy.
This study was to investigate Beclinl expression and expression of ClassⅠPI3K (p110α), ClassⅢPI3K (hvps34) and p-PKB in PI3K/PKB signaling pathway in epithelial ovarian carcinoma., and to explore relationship between autophagy gene Beclinl and involved PI3K/PKB signaling pathway and the occurrence, development of epithelial ovarian carcinoma. We study the effect of Beclinl overexpression on the growth of ovarian carcinoma cell line SKOV3 in vitro and vivo, and on change of PI3K/PKB signaling pathway, which provide some theoretic basis for gene therapy that target at Beclinl.
Methods 1. The expression of Beclin 1, ClassⅠPI3K (p110α), ClassⅢPI3K (hvps34) and p-PKB was detected by immunohistochemistry in 25 normal ovarian tissues, 25 benign neoplasia tissues, 19 borderline tissues, and 69 epithelial ovarian carcinoma tissues.
2. The gene fragment coding for Beclinl was obtained from human normal ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1/Beclin1.
3. The vectors were transfected into SKOV3 cells by lipofectamine 2000. The expression levels of Beclin1, hvps34, p110αand p-PKB mRNA and protein were detected by real-time RT-PCR, western blot analysis after transfection. Expression of Beclinl in SKOV3 cells transfected with plasmid pcDNA3.1/Beclin1 was detected by immunohistochemistry. Autophagic vacuoles were observed by electron microscopy and fluorescence microscope. Cell cycles, apoptotic rates and autophagy were measured by flow cytometry (FCM). MTT was used to evaluate the effect of Beclinl overexpression on the inhibition of proliferation and growth of the transfected cells and SKOV3 cells,
4. SKOV3 cells transfected with plasmid pcDNA3.1/Beclinl and pcDNA3.1 were seeded hypopercutaneously on nude mice. The carcinogenic and growth activities of cancer cell in vivo were observed. Beclinl protein expression in tumor tissues was detected by immunohistochemisty.
Results 1. The higher expression level of Beclin 1 and hvps34 was found in normal and benign ovarian neoplasia tissues. The expression of Beclin 1 and hvps34 was reduced in the borderline lesions, and the lowest level was detected in the ovarian carcinoma tissues(P<0.05). The level of p110αand p-PKB expression increased slightly in the borderline ovarian tissues, and the expression of p110αand p-PKB was higher in the epithelial ovarian carcinoma tissues than that of the other three groups (P<0.05). Beclin 1 expressions in stageⅠ-Ⅱ, high and middle grade and negative lymph node metastasis epithelial ovarian cancer tissues were higher than that in stageⅢ-Ⅳ, low grade, positive lymph node metastasis ovarian cancer tissues, and the expressions of p110αand p-PKB in the former were lower than that in the later(P<0.05).
The expression of Beclinl, p110α, hvps34 and p-PKB has no significant difference in normal and benign ovarian neoplasia tissues. The higher expression level of Beclinl and hvps34 was found in normal and benign ovarian neoplasia tissues. The expression of Beclinl and hvps34 was reduced in the borderline lesions, and the lowest level was detected in the ovarian carcinoma tissues(P<0.05). The level of pllOaand p-PKB expression increased slightly in the borderline ovarian tissues, and the expression of p110αand p-PKB was higher in the epithelial ovarian carcinoma tissues than that of the other three groups ( P<0.05). Beclin 1 expressions in stageⅠ- Ⅱ, high and middle grade and negative lymph node metastasis epithelial ovarian cancer tissues were higher than that in stageⅢ-Ⅳ, low grade, positive lymph node metastasis ovarian cancer tissues, and the expressions of p100αand p-PKB in the former were lower than that in the later(P<0.05).
2.The eukaryotic expression vectors of pcDNA3.1/Beclinl were constructed successfully, and verified by PCR, restriction endonucleases digestion and DNA sequencing.
3.Beclinl mRNA and protein expression were increased in SKOV3 cells transfected with pcDNA3.1/Beclin1. Hvps34 expression was up-regulated and the expression level of p100αand p-PKB was down-regulated after transfection with pcDNA3.1/Beclin1. A lot of Autophagic vacuoles were observed in SKOV3-Beclinl cells by electron microscopy. MDC staining positive cells were increased after transfection with pcDNA3.1/Beclin1. FCM investigation showed the apoptotic rate was (21.26±3.89)% in SKOV3 cells after transfection with pcDNA3.1/Beclin1, which was higher than that in SKOV3 cells transfected with pcDNA3.1 and SKOV3 cells (P<0.05). After transfected by vector pcDNA3.1/Beclin1, the ratios of G1 phase of SKOV3 cells were increased and the ratios of S phase were decreased significantly. FCM showed that fluorescent intensity of SKOV3 cells transfected with pcDNA3.1/Beclin1 was higher than higher than that of SKOV3 cells transfected with pcDNA3.1 and SKOV3 cells. MTT assay revealed the cell proliferations of SKOV3 cells was inhibited after transfection with pcDNA3.1/Beclin1, and cell inhibitory rate was 58.68% (P<0.05) .
4. After transfected with vector pcDNA3.1/Beclin1, the carcinogenic activity of SKOV3 cells was decreased in nude mice, and the rate of inhibition was 50.27%. The expressions of Beclinl protein were increased in SKOV3- Beclin1 group.
Conclusions Beclin 1 expression was down-regulated in epithelial ovarian cancer tissues, and the expression of p100α, hvps34 and p-PKB is abnormal in PI3K/PKB pathway, which may be correlated with the occurrence and development of ovarian carcinoma. Beclin 1 overexpression stimulated the expression of ClassⅢPI3K (hvps34) , and inhibited the expression level of p100αand p-PKB. Beclin 1 overexpression can induce autophagy and apoptosis in SKOV3 cells, and inhibit tumorigenesis of SKOV3 cells in vitro and vivo. So it might be one of the ideal strategies for gene therapy of ovarian carcinoma.
自噬在清除损伤或多余的细胞器，维持细胞内稳定方面起重要作用。自噬可清除损伤的细胞器，避免如自由基等有基因毒性的物质的产生，从而使突变率降低，抑制肿瘤的形成，而自噬的缺陷可导致肿瘤的发生。
Beclin1基因是第一个确认的哺乳动物的自噬基因，也是第一个确认的在自噬的溶酶体降解途径中起肿瘤抑制的基因，对自噬体的形成至关重要。有研究报道乳腺癌中Beclin1的表达下调，Beclin1缺陷明显增加肝癌、肺癌和淋巴瘤的发生率，但自噬基因Beclin1与卵巢癌的关系尚不清楚。P13K／PKB是目前涉及凋亡调控较清楚的细胞信号传导途径，有研究表明其亦是调控自噬的主要途径之一，ClassⅠP13K抑制自噬，而ClassⅢP13K刺激自噬的发生。
本研究通过检测上皮性卵巢癌组织中自噬基因Beclin 1的表达及其调控相关信号传导途径P13K／PKB中的成分ClassⅠP13K (p110α)、ClassⅢ P13K(hvps34)及其下游的磷酸化PKB(p-PKB)的表达变化，探讨自噬基因Beclin1及其调控相关信号传导途径P13K／PKB中的成分p110α、hvps34、p-PKB的表达与上皮性卵巢癌的发生发展之间的关系；研究自噬基因Beclin1在SKOV3细胞中过表达对肿瘤细胞在体内、体外增殖的影响及其相关调控信号途径P13K／PKB的变化，探讨自噬基因Beclin1表达载体作为卵巢癌基因治疗的可行性。方法1.用免疫组化的方法对25例正常卵巢组织、25例良性上皮性卵巢肿瘤、19例交界性上皮性卵巢肿瘤及69例上皮性卵巢癌组织进行Beclin1、ClassⅠP13K(p110α)、ClassⅢP13K(hvps34)及其下游的磷酸化PKB的表达水平进行检测；
2.通过RT-PCR方法，从人正常卵巢组织中获得目的基因Beclin1，将其插入真核表达载体pcDNA3.1(＋)中，构建真核表达载体pcDNA3.1／Beclin1；
3.脂质体法将重组质粒pcDNA3.1／Beclin1、空质粒pcDNA3.1分别转染人卵巢癌细胞株SKOV3。用荧光定量RT-PCR及western blot分别在mRNA和蛋白质水平检测转染前后Beclin1、hvps34、p110α和p-PKB的表达变化。细胞免疫组化方法观察pcDNA3.1／Beclin1转染后SKOV3中Beclin1蛋白表达变化；在电镜和荧光显微镜下观察自噬囊泡；流式细胞仪检测凋亡及自噬情况，并用MTT法分析外源性Beclin1过表达对SKOV3增殖的影响；
4.将重组质粒pcDNA3.1／Beclin1转染SKOV3细胞后，接种到裸鼠皮下，观察体内致瘤活性及生长情况；免疫组化检测瘤组织Beclin1蛋白的表达。
结果1.Beclin 1、hvps34在正常卵巢组织与良性卵巢肿瘤组织中表达较高，在交界性卵巢癌组织中开始下降，在上皮性卵巢癌中的表达最低；p110α、p-PKB在交界性卵巢癌组织中开始升高，在上皮性卵巢癌中的表达明显高于其它3组(P＜0.05)。卵巢癌Ⅰ～Ⅱ期、高中分化、淋巴结无转移的卵巢癌组织中Beclin 1的表达均高于Ⅲ～Ⅳ期、低分化、淋巴结有转移者(P＜0.05)，而p110α及p-PKB的表达均低于Ⅲ～Ⅳ期、低分化、淋巴结有转移者(P＜0.05)；
2.利用酶切、PCR分析、DNA测序证实，真核表达载体pcDNA3.1／Beclin1构建成功；
3.pcDNA3.1／Beclin1转染后SKOV3细胞Beclin1表达在mRNA和蛋白质水平明显高于转染空质粒pcDNA3.1和未转染细胞；pcDNA3.1／Beclin1转染后hvps34表达上调，而p110α、p-PKB的表达下调。pcDNA3.1／Beclin1转染SKOV3细胞后在在电镜下可见大量自噬囊泡形成。在荧光显微镜下见pcDNA3.1／Beclin1转染后SKOV3细胞中MDC阳性细胞明显增加。pcDNA3.1／Beclin1转染后SKOV3的凋亡率为(21.26±3.89)％，高于pcDNA3.1转染组和未转染组，差异有显著性(P＜0.05)；与未转染组相比，G1期细胞比例明显增多，S期细胞比例明显降低。流式细胞仪检测pcDNA3.1／Beclin1转染后SKOV3细胞MDC平均荧光强度高于pcDNA3.1转染组和未转染组。pcDNA3.1／Beclin1转染SKOV3后细胞的体外增殖能力明显弱于转染空质粒组和未转染组，细胞抑制率为58.68％(P＜0.05)。
4.重组质粒pcDNA3.1／Beclin1使卵巢癌SKOV3细胞裸鼠体内成瘤时间延长，瘤体体积及重量明显小于空质粒组和空白对照组，抑瘤率为50.27％。免疫组化显示SKOV3-Beclin1组Beclin1蛋白表达强于SKOV3-pcDNA3.1组和SKOV3组，肿瘤组织坏死增加。
结论自噬基因Beclin 1在上皮性卵巢癌组织中的表达下调，对自噬起调控作用的PI3K／PKB信号通路中的成分p110α、hvps34、p-PKB存在表达异常，可能与上皮性卵巢癌的发生、发展及临床预后有关；自噬基因Beclin1在人卵巢癌细胞SKOV3中过表达可使ClassⅢPI3K(hvps34)表达上调，ClassⅠPI3K(p110α)及其下游的p-PKB表达下调；自噬基因Beclin1在人卵巢癌细胞SKOV3中过表达可诱导肿瘤细胞自噬和凋亡的发生，抑制SKOV3在体内、体外的增殖，针对自噬基因Beclin1的卵巢癌基因治疗可能具有可行性。
Background & Objective Epithelial ovarian cancer is the first leading cause of gynecologic malignancy-related deaths. It is urgent to find effective treatment of ovarian carcinoma in clinical practice. Surgery, chemotherapy and radiation therapy are conventional treatment for ovarian carcinoma, and gene therapy is regard as a new treatment strategy for ovarian carcinoma.
Autophagy plays a critical role in removing damaged or surplus organelles in order to maintain cellular homeostasis. For example, by removing damaged organelles, autophagy may limit the exposure of cellular DNA to genotoxic- stresses such as free radicals. The removal of damaged organelles through autophagic degradation would thus decrease the basal mutation rate and suppress oncogenesis.
Beclin 1, the first identified mammalian autophagy gene product, is the first identified tumor suppressor protein that functions in the lysosomal degradation pathway of autophagy. Beclin1 is essential to formation of autophagosome. Some studies reported that Beclin1 expression was down-regulated in breast carcinoma, and loss of Beclin 1 would contribute to an increased incidence of cancer, such as heptocellular carcinoma, lung adenocarcinoma and lymphoma. It is unclear that correlation of autophagy Gene Beclinl to tumorigenesis and development of epithelial ovarian cancer.
It was well known that apoptosis was regulated by PI3K/PKB signaling pathway, and this pathway is invovlved in ihe control of autophagy. Some studies reported the classⅠPI3K inhibit autophagy and the classⅢPI3K stimulates autophagy.
This study was to investigate Beclinl expression and expression of ClassⅠPI3K (p110α), ClassⅢPI3K (hvps34) and p-PKB in PI3K/PKB signaling pathway in epithelial ovarian carcinoma., and to explore relationship between autophagy gene Beclinl and involved PI3K/PKB signaling pathway and the occurrence, development of epithelial ovarian carcinoma. We study the effect of Beclinl overexpression on the growth of ovarian carcinoma cell line SKOV3 in vitro and vivo, and on change of PI3K/PKB signaling pathway, which provide some theoretic basis for gene therapy that target at Beclinl.
Methods 1. The expression of Beclin 1, ClassⅠPI3K (p110α), ClassⅢPI3K (hvps34) and p-PKB was detected by immunohistochemistry in 25 normal ovarian tissues, 25 benign neoplasia tissues, 19 borderline tissues, and 69 epithelial ovarian carcinoma tissues.
2. The gene fragment coding for Beclinl was obtained from human normal ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1/Beclin1.
3. The vectors were transfected into SKOV3 cells by lipofectamine 2000. The expression levels of Beclin1, hvps34, p110αand p-PKB mRNA and protein were detected by real-time RT-PCR, western blot analysis after transfection. Expression of Beclinl in SKOV3 cells transfected with plasmid pcDNA3.1/Beclin1 was detected by immunohistochemistry. Autophagic vacuoles were observed by electron microscopy and fluorescence microscope. Cell cycles, apoptotic rates and autophagy were measured by flow cytometry (FCM). MTT was used to evaluate the effect of Beclinl overexpression on the inhibition of proliferation and growth of the transfected cells and SKOV3 cells,
4. SKOV3 cells transfected with plasmid pcDNA3.1/Beclinl and pcDNA3.1 were seeded hypopercutaneously on nude mice. The carcinogenic and growth activities of cancer cell in vivo were observed. Beclinl protein expression in tumor tissues was detected by immunohistochemisty.
Results 1. The higher expression level of Beclin 1 and hvps34 was found in normal and benign ovarian neoplasia tissues. The expression of Beclin 1 and hvps34 was reduced in the borderline lesions, and the lowest level was detected in the ovarian carcinoma tissues(P<0.05). The level of p110αand p-PKB expression increased slightly in the borderline ovarian tissues, and the expression of p110αand p-PKB was higher in the epithelial ovarian carcinoma tissues than that of the other three groups (P<0.05). Beclin 1 expressions in stageⅠ-Ⅱ, high and middle grade and negative lymph node metastasis epithelial ovarian cancer tissues were higher than that in stageⅢ-Ⅳ, low grade, positive lymph node metastasis ovarian cancer tissues, and the expressions of p110αand p-PKB in the former were lower than that in the later(P<0.05).
The expression of Beclinl, p110α, hvps34 and p-PKB has no significant difference in normal and benign ovarian neoplasia tissues. The higher expression level of Beclinl and hvps34 was found in normal and benign ovarian neoplasia tissues. The expression of Beclinl and hvps34 was reduced in the borderline lesions, and the lowest level was detected in the ovarian carcinoma tissues(P<0.05). The level of pllOaand p-PKB expression increased slightly in the borderline ovarian tissues, and the expression of p110αand p-PKB was higher in the epithelial ovarian carcinoma tissues than that of the other three groups ( P<0.05). Beclin 1 expressions in stageⅠ- Ⅱ, high and middle grade and negative lymph node metastasis epithelial ovarian cancer tissues were higher than that in stageⅢ-Ⅳ, low grade, positive lymph node metastasis ovarian cancer tissues, and the expressions of p100αand p-PKB in the former were lower than that in the later(P<0.05).
2.The eukaryotic expression vectors of pcDNA3.1/Beclinl were constructed successfully, and verified by PCR, restriction endonucleases digestion and DNA sequencing.
3.Beclinl mRNA and protein expression were increased in SKOV3 cells transfected with pcDNA3.1/Beclin1. Hvps34 expression was up-regulated and the expression level of p100αand p-PKB was down-regulated after transfection with pcDNA3.1/Beclin1. A lot of Autophagic vacuoles were observed in SKOV3-Beclinl cells by electron microscopy. MDC staining positive cells were increased after transfection with pcDNA3.1/Beclin1. FCM investigation showed the apoptotic rate was (21.26±3.89)% in SKOV3 cells after transfection with pcDNA3.1/Beclin1, which was higher than that in SKOV3 cells transfected with pcDNA3.1 and SKOV3 cells (P<0.05). After transfected by vector pcDNA3.1/Beclin1, the ratios of G1 phase of SKOV3 cells were increased and the ratios of S phase were decreased significantly. FCM showed that fluorescent intensity of SKOV3 cells transfected with pcDNA3.1/Beclin1 was higher than higher than that of SKOV3 cells transfected with pcDNA3.1 and SKOV3 cells. MTT assay revealed the cell proliferations of SKOV3 cells was inhibited after transfection with pcDNA3.1/Beclin1, and cell inhibitory rate was 58.68% (P<0.05) .
4. After transfected with vector pcDNA3.1/Beclin1, the carcinogenic activity of SKOV3 cells was decreased in nude mice, and the rate of inhibition was 50.27%. The expressions of Beclinl protein were increased in SKOV3- Beclin1 group.
Conclusions Beclin 1 expression was down-regulated in epithelial ovarian cancer tissues, and the expression of p100α, hvps34 and p-PKB is abnormal in PI3K/PKB pathway, which may be correlated with the occurrence and development of ovarian carcinoma. Beclin 1 overexpression stimulated the expression of ClassⅢPI3K (hvps34) , and inhibited the expression level of p100αand p-PKB. Beclin 1 overexpression can induce autophagy and apoptosis in SKOV3 cells, and inhibit tumorigenesis of SKOV3 cells in vitro and vivo. So it might be one of the ideal strategies for gene therapy of ovarian carcinoma.
引文
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