Role of microRNA-21 in uveal melanoma cell invasion and metastasis by regulating p53 and its downstream protein

AIM： To reveal the insight mechanism of liver metastasis in uveal melanoma, we investigated cell functions of microRNA-21 in three different uveal melanoma cell lines and analyze the relationship of target gene p53 and its downstream targets which been found significant expression in our previous study.METHODS： Quantitative real-time polymerase chain reaction （qRT-PCR） was used to detect microRNA-21 expression in normal uveal tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct OCM-1, MuM-2B and M619 cell line with stable overexpression and inhibition of microRNA-21. In vitro cell function tests such as cell proliferation, cell apoptosis, cell circle and abilities of migration and invasion were examined by MTT, BrdU assay, flow cytometry, transwell assay and Matrigel invasion assay respectively. The target gene was predicted by bioinformatics and confirmed by using a dual luciferase reporter assay. The expression of p53 and its suspected downstream targets LIM and SH3 protein 1 （LASP1） and Glutathione S Transferase pi （GST-Pi） were determined by qRT-PCR in mRNA level and western blotting analysis in protein level. Finally, the effect of microRNA-21 in a xenograft tumor model was assessed in four-week-old BALB/c nude mice. RESULTS： Compared to normal uveal melanoma, expressions of microRNA-21 were significantly higher in uveal melanoma cell lines. Overexpression of microRNA-21 promoted proliferation, migration, and invasion of OCM-1, M619 and MuM-2B cells, while inhibition of microRNA-21 reveal opposite effects. Wild type p53 was identified as a target gene of microRNA-21-3p, and proved by dual luciferase reporter assay. Up-regulated microRNA-21 inhibited the expression of wild type p53 gene, and the increased expression of LASP1 in mRNA level and protein level, while down-regulated microRNA-21 presented opposite way. However, GST-pi showed the potential pattern as expected, but relative mRNA level showed no statistically significant difference in OCM-1 cells. Furthermore, the mRNA expression of GST-pi was decreased in microRNA-21 overexpressing MuM-2B, and increased in M619 cells with inhibition of microRNA-21. In vivo, inhibition of microRNA-21 reduced tumor growth with statistically significant difference.CONCLUSION： These findings provide novel insight into molecular etiology of microRNA-21 in uveal melanoma cell lines, and suggest that microRNA-21 might be a potential candidate for the diagnosis and prognostic factor of human uveal melanoma in future.

INHIBITION OF FARNESYL PROTEIN TRANSFERASE AND P21^(RAS) MEMEBRANE ASSOCIATION BY D-LIMONENE IN HUMAN PANCREAS TUMOR CELLS IN VITRO

The monoterpene d limonene inhibit the plasma membrane associated P21 ras expression and the posttranslational isoprenylation of P21 ras , a mechanism that may contribute to its efficacy in the chemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study,the relative abilities of d limonene to inhibit membrane associated P21 ras expression in pancreas tumor cell(PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FTPase) activity during the Ras protein isoprenylation and cell proliferation were determined.Concomitantly,the effects of d limonene on P21 ras localization by immunohistochemistry and H ras oncogene expression in PaCa tumor cell line by Northern blotting were observed. The results showed that d limonene inhibited FPTase activity, thus to reduce P21H ras isoprenylation. d limonene could decrease P21 ras membrane association and increase cytosolic accumulation of P21 ras . This phenomenon was also noted when d limonene treated PaCa cells were stained immunohistochemically with anti P21 ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21 ras membrane association and the alteration of P21 ras localization. Inhibition of farnesylation of P21 ras altered their intracellular localization and, hence, disrupted their biological activity,but no relationship with H ras oncogene expression was found.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

Objective： We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Methods： Fluorescence in situ hybridisation （FISH） and immunohistochemistry （IHC） techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. Results： FISH detection of HER2 gene amplification rate was 16.9% （10/59）, HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% （29/59）. HER2 protein expression was 42.4% （25/59）, HER2 gene amplification rates in patients with ＋＋＋, ＋＋ HER2 protein expression were 3/3 and 5/8, while in patients with ＋ HER2 protein expression, it was 2/14, there was significant difference （P 0.05）. p21 protein expression rate was 49.2% （29/59）, HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth, lymph node metastasis （P 0.05）; had no statistical significance in histological type, age, gender differences （P 0.05）. Conclusion： HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.

Detection and Its Implication of Heat Stress Protein 27 and P21 in Nasopharyngeal Carcinoma

To study the expression of heat stress protein 27 (HSP27) and P21 in nasopharyngeal car- cinoma (NPC) tissue, and to evaluate the significance of both HSP27 and P21 in the pathogenesis, development and prognosis of NPC. Indirect immunofluorescence method combined with SABC was applied. Our results showed that (1) the positive rates of HSP27 and P21 expressed in NPC tissue in 36 cases were 88. 9 % and 94. 4%; (2) while in 10 hyperplastic nasopharyngitis tissues, the positive rate of HSP27 and P21 were both 5; (3) all the 5 normal tissues were negatively stained. It is obvi- ous that a co-expressing tendency of HSP27 and P21 could be identified, and it was associated with the degree of malignancy and the clinical stage of NPC. It is concluded that the positive expression of HSP27 and P21 may have clinical significance in the evaluation of the occurring, development and prognosis of NPC, and in NPC treatment.

THE STUDY ON RELATIONSHIP BETWEEN CIGARETTE SMOKING AND THE p53 PROTEIN AND P21 PROTEIN EXPRESSION IN NON-SMALL LUNG CANCER

This paper discusses the relationship between cigarette smoking and the p53 protein and P21 protein expression by the immunohistochemical analysis in 93 cases with lung cancer in which squamous cell carcinoma accounted for 45 cases, adenocarcinoma 48 cases. The results showed that positive proportion of p53 protein expression was 74.20% (28 of 37 squamous cell carcinoma, 21 of 30 adenocarcinomas) in cigarette smoking group with lung cancers, and 38.46% (3 of 8 squamous cell carcinoma, 7 of 18 adenocarcinomas) in nonsmoking group with lung cancers. The difference was statistically significant. Odds ratio was 4.14 and confidence limits for OR was 1.42-12.52. A dose-related presents in the p53 protein expression for the smoking amount and smoking years. The positive proportion of P21 protein expression was 79.31% (21 of 28 squamous cell carcinoma, 25 of 30 adenocarcinomas) in cigarette smoking group with lung cancers, and 82.75% (10 of 11 squamous, 14 of 18 adenocarcinomas) in nonsmoking group with lung cancers, the difference was not statistically significant. But their positive proportion of P21 protein expression were very high in both groups. It was indicated that no relationship between cigarette smoking and the P21 protein expression. We suggest that the p53 gene could be a common target of tobacco-associated carcinogenesis in lung cancer.

Expression of p53 and p21 proteins in rat brain tissue after reperfusion following forebrain ischemia

Objective: To investigate the relationship between p53, p21 proteins and delayed neuronal death (DND) after reperfusion following forebrain ischemia in rats. Methods With four-vessel occlusion model of rats, the expression of p53, p21 proteins in brain tissue using labeled streptavindin-biotin immunohistochemical (LAAB) suming were observed. Re sults: The expression of p53, p21 proteins in brain was upregulated after reperfusion following 15 min forebrain ischemia and their distribution was similar. p53 and p21 proteins in brian sections was detected earlier in the white matter of hippocampal formation, thalamus, hypothalamus (6 h following reperfusion) than in the neuronal nuclei in cerebral cortex and CA1 region (24h), and the maximal induction was observed at 72 h following reperfusion. CA1 region suffered the most serious injury, where the positive expression of p53 and off proteins was most. Conclusion: Reperfusion following forebrain ischemia could upregulate the expression of p53 and p21 proteins in the brain region, suggesting that p53 and p21 proteins participate in and possibly promote the apoptosis of ’DND.

c-Ha-ras and c-myc antisense oligodeoxynucleotides inhibit the proliferation and DNA synthesis in human gastric carcinoma cell lines

The effects of two antisense oligodeoxynucleotides on the expression of c-Ha-ras proto-oncogene and the growth of human gastric carcinoma cell lines were observed. Synthetic 15-mer directed at the region of the translational initiation site of c-Ha-ras proto-oncogene (ASO-r) greatly inhibited the proliferation (55. 61%,P<0. 05) and DNA synthesis (76. 79%,P<0. 05) of MGc-803 cell line. It also inhibited the proliferation (62. 02%,P<0. 05) and DNA synthesis (76. 78%, P<0. 05) of SGc-7901 cell line. A reduction in intracellular P21 ras protein levels in MGc-803 cell line was observed 6 h after the treatment with ASO-r and maintained over 12 h. Another synthetic 15-mer targeted against the initiation codon and downstream 4 codons of c-myc proto-oncogene (ASOm) inhibited only DNA synthesis of MGc-803 cell line (71. 37%, P<0. 05). The control 15-mer did not inhibit the expression of P21 protein and proliferation of these cell lines. These experiments seemed to provide evidence that ASO-r could be effective in inhibiting the expression of c-Ha-ras proto-oncogene and controlling the growth of human gastric carcinoma cells,and that the over-expression of c-Ha-ras proto-oncogene might mainly be associated with the malignant proliferation of human gastric carcinoma cells.

P21^(ras)、P16在大肠癌中的表达及临床意义

目的探讨癌基因P21^(ras)及抑癌基因P16与大肠癌的关系。方法应用免疫组化SP法分析、研究了P21^(ras)蛋白、P16蛋白在80例大肠癌、20例大肠腺瘤及20例正常大肠组织中的表达。结果①P21^(ras)在前二者中的阳性表达率分别为70.1％(57/80)和65％(13/20),明显高于在正常组织中的30％(6/20),其比较具有显著性差异(x^2=17.66、P<0.005;x^2=5.23、P<0.05);P16在大肠癌中的阳性表达率为36.3％(29/80)低于在腺瘤及正常组织中的65％(13/20)和75％(15/20),其比较亦具有显著性差异。②P21^(ras)、P16的阳性表达率在大肠癌患者的年龄、性别及肿瘤大小方面无显著性差异,而与肿瘤的临床Dukes分期(x^2=6.20、P<0.025;x^2=6.25、P<0.05)、有无淋巴结转移及术后5a生存率密切相关。③P21^(ras)在不同的大肠癌组织病理类型中,其表达率无显著性差异;P16的阳性表达率随着肿瘤浸润深度的增加有下降趋势,但无统计学意义。④在80例大肠癌患者中,P21^(ras)阳性而P16阴性者比率高于其他三者,而且这类大肠癌患者的术后5a生存率下降。结论大肠癌的发生、发展,是由包括癌基因、抑癌基因在内的多种因素作用的结果;在临床工作中联合检测P21^(ras)及P16蛋白在大肠癌中的表达水平,对我们估计患者的病情、推测其预后有指导性意义。

大鼠全脑缺血再灌流后脑组织P^(53)及P^(21)蛋白的表达

目的 探讨大鼠全脑缺血再灌流后Ｐ５３、Ｐ２１蛋白的表达及其与迟发性神经元死亡（ＤＮＤ）的关系。方法 在４ 血管闭塞法全脑缺血模型上，采用ＨＥ及ＬＳＡＢ染色法，观察脑组织病理改变，检测脑组织Ｐ５３、Ｐ２１蛋白的表达，以及蛋白合成抑制剂放线菌酮对其的影响。结果 全脑缺血１５ ｍ ｉｎ 再灌流后，脑组织Ｐ５３、Ｐ２１蛋白表达增加，且两者分布接近。海马结构、丘脑、下丘脑等白质区（再灌流后６ ｈ）较皮层、海马的神经细胞核（２４ ｈ）先检测到Ｐ５３、Ｐ２１蛋白，７２ ｈ 表达达高峰。并且以缺血损伤最严重的海马ＣＡ１ 区Ｐ５３、Ｐ２１蛋白表达为强。另外，放线菌酮可抑制脑组织Ｐ５３、Ｐ２１蛋白的表达，并对ＤＮＤ具有一定的保护作用。结论 全脑缺血再灌流损伤后，脑组织Ｐ５３、Ｐ２１蛋白表达增加，放线菌酮可抑制其表达，并对ＤＮＤ起保护作用，提示Ｐ５３、Ｐ２１蛋白参与了全脑缺血后ＤＮＤ的凋亡机制。

贲门癌组织P53、P21和CD_(44V6)蛋白的表达及其意义

目的 探讨P53 、P2 1和CD44V6蛋白的表达情况与贲门癌生物学行为的关系。方法 采用免疫组化法对 4 1例贲门癌组织进行P53 、P2 1和CD44V6蛋白的测定。结果 贲门癌组织中P53 、P2 1和CD44V6蛋白的阳性率分别为 75 6 %、5 3 6 %和 4 1 4 % ,其表达与患者性别、年龄、临床分期和病理类型无明显关系。CD44V6蛋白阳性表达与肿瘤浸润深度有关 (P <0 0 5 ) ;P53 蛋白表达与P2 1蛋白表达呈正相关 ,但无统计学意义。P53 、P2 1和CD44V6蛋白阳性者预后比其阴性者差。结论 P53 、P2 1和CD44V6蛋白的测定可作为判断贲门癌患者预后的参考指标 。

洛伐他汀对NB4细胞ras基因p21^(Ras)膜结合作用影响的体外试验

目的 :探讨洛伐他汀 (LOV)对人白血病NB4细胞的作用及其机制。方法 :以MTT比色法首先观察LOV对NB4细胞增殖的影响 ;利用逆转录 -聚合酶链反应半定量测定LOV作用于NB4细胞不同时间H -ras、K -ras、N -ras癌基因mRNA表达水平 ;同时采用流式细胞术测定NB4细胞p2 1Ras总蛋白、膜蛋白的表达。结果 :①LOV抑制NB4细胞增殖 ,IC5 0为 12 5 9μmol/L。②NB4细胞H、K、N -ras基因表达均为阳性。③LOV处理不同时间的NB4细胞H、K、N -ras基因转录水平无明显变化 ;p2 1Ras总蛋白水平也无变化 ,而细胞膜表面p2 1Ras蛋白水平随时间进行性下降。结论 :LOV抑制NB4细胞增殖。LOV靶向HMG -CoA还原酶 ,抑制p2 1Ras蛋白异戊二烯化、阻滞p2 1Ras蛋白与细胞膜结合 ;不影响ras癌基因以及p2 1Ras总蛋白的表达。

口腔白斑及鳞癌病理组织中ras癌基因P^(21)蛋白表达

口腔白斑及鳞癌为口腔临床中常见的癌前病损和恶性病变。本研究应用免疫组化技术（ＡＢＣ法）检测口腔鳞状细胞癌及白斑组织中ｒａｓ癌基因产物Ｐ２１蛋白的表达。检测结果表明：在４０例口腔鳞癌中Ｐ２１蛋白阳性表达率为９２．５０％；２１例口腔白斑病变中其阳性率为１９．０５％。Ｐ２１阳性表达位于分化差的癌细胞膜及胞浆内，且阳性表达与临床病理分级呈正相关，提示Ｐ２１蛋白检测可以作为癌及癌前病损预后观察的客观指标。

腺苷三磷酸对人横纹肌肉瘤细胞系P^(21)和c-myc蛋白表达的影响

为进一步研究腺苷三磷酸抑制癌细胞增殖诱导分化的作用机理，用ＡＴＰ作用于人横纹肌肉瘤单细胞克隆亚系ＲＤＬ６细胞，用免疫荧光细胞化学方法观察ＡＴＰ作用后的ＲＤＬ６细胞核内Ｐ２１蛋白表达增强，ｃ－ｍｙｃ蛋白表达降低，膜下胞质内微丝束－应力纤维组装改善，粘着斑内的主要粘附蛋白——纽蛋白（ｖｉｎｃｕｌｉｎ）表达增强，正常小鼠成肌细胞Ｃ２Ｃ１２细胞核内Ｐ２１蛋白高表达，ｃ－ｍｙｃ蛋白不表达。结果表明：ＲＤＬ６细胞Ｐ２１表达的丢失和ｃ－ｍｙｃ表达的活化与细胞的增殖和分化异常相关，从而表明Ｐ２１表达增强和ｃ－ｍｙｃ表达的降低在ＲＤＬ６细胞表型逆转过程中起重要作用。

肺鳞癌细胞p^(53)蛋白及ras p^(21)的免疫组织化学定量研究

应用免疫组织化学方法检测２２例肺鳞癌组织标本的ｐ５３及ｒａｓｐ２１表达，用显微图像分析仪测定阳性细胞百分率Ｐ／Ａ（％）和平均光密度（ＡＯＤ），并采用阳性水平指数（ｐｏｓｉｔｉｖｅｌｅｖｅｌｉｎｄｅｘ，ＰＬＩ）作为免疫组化反应水平指标，对癌基因蛋白进行比较研究。结果显示：ｐ５３表达阳性率为５０％，ｒａｓｐ２１表达阳性率为６３６％。ｐ５３蛋白表达在非角化型鳞癌组高于角化型鳞癌组；ｒａｓｐ２１的表达随组织分化程度的增高而增高，ｒａｓｐ２１与ｐ５３的表达水平无直线相关关系。

胃癌术后硫酸性肠化切缘P^（53）和ras P^（21）蛋白的表达及其意义

目的探讨硫酸性肠化切缘Ｐ５３和ｒａｓＰ２１蛋白的表达，进一步从分子生物学水平证实硫酸性肠化与胃癌术后切缘复发的关系。方法应用流式细胞术和细胞免疫荧光染色技术，对１８例硫酸性和１７例非硫酸性肠化切缘粘膜上皮细胞的Ｐ５３和ｒａｓＰ２１蛋白的表达量进行了定量研究．结果硫酸性肠化切缘上皮的Ｐ５３和ｒａｓｐ２１蛋白的表达量显著高于非硫酸性肠化切缘和正常粘膜上皮，而低于胃癌细胞．结论硫酸性肠化切缘的ｐ５３和ｒａｓＰ２１蛋白有过量表达，可能与胃癌术后复发有关。

rasP^(21)表达早期诊断大肠腺瘤的临床价值

采用流式细胞术（ＦＣＭ）对４４ 例散发性大肠腺瘤组织、腺癌组织和２３ 例正常组织进行了Ｐ２１表达、ＤＮＡ倍体分析。结果为Ｐ２１标记率、ＤＮＡ异倍体检出率在正常大肠粘膜－腺瘤－腺癌的顺序中依次增高，各组间有显著性差异（Ｐ＜ ０．０１）；随着腺瘤的增大，二指标亦趋增高（Ｐ＜ ０．０５），且呈同相变化趋势；Ｐ２１标记率在异倍体腺癌中明显高于二倍体腺癌。说明用流式细胞术检测Ｐ２１标记率结合ＤＮＡ倍体分析，

鼻腔鼻窦恶性肿瘤P^(16)、P^(21(WAF1/CIP1))基因表达的研究

目的 :探讨抑癌基因 P1 6 和 P2 1 的表达在鼻腔鼻窦恶性肿瘤中的意义。方法 :用免疫组化 SP法检测了 6 0例鼻腔鼻窦恶性肿瘤 ,10例良性肿瘤 ,2 0例无瘤组织的 P1 6、P2 1基因的表达情况。结果 :鼻腔鼻窦恶性肿瘤组织的 P1 6、P2 1蛋白阳性率低于良性肿瘤及无瘤组织相应的阳性率 ,差异有统计学意义 (P <0 .0 5 )。 P1 6 、 P2 1 蛋白的改变为非相关性事件。结论 :P1 6 、 P2 1基因表达与鼻腔鼻窦恶性肿瘤的发生密切相关 ,且作用是相互独立的 ,与该肿瘤患者性别、年龄、组织来源、局部浸润及转移等因素无关。

蛋白激酶C抑制剂对SACC-83系P_(21)^(ras)、c-myc表达的影响

本实验采用ＰＫＣ抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ作用于ＳＡＣＣ－８３系，观察对Ｐ２１ｒａｓ、ｃ－ｍｙｃ表达的影响．结果表明，Ｓｔａｕｒｏｐｏｒｉｎｅ可明显降低Ｈ－ｒａｓ、ｃ－ｍｙｃ表达程度（ｐ＜０．０１），这提示ＰＫＣ对ｒａｓ、ｃ－ｍｙｃ基因表达具有调控作用，ＰＫＣ抑制剂可能通过调控癌基因的表达来表现抗肿瘤作用的．

云南个旧非小细胞肺癌ras癌基因产物P_(21)蛋白的表达

应用ＡＢＣ免疫组化法对来自云南个旧的２０例非小细胞肺癌标本进行了ｒａｓ癌基因产物Ｐ２１蛋白的表达检测结果阳性１５例，阳性率７５０％，表达率较高其中１６例鳞癌中。

ras-P_(21)过表达和 P_(53)蛋白蓄积对肺癌预后的研究

应用Ｈａ－ｒａｓＰ２１和Ｐ５３（Ｄｏ－１）单克隆抗体，采用Ｓ－Ｐ免疫组化法对７７例原发性肺癌进行研究。结果表明：Ｐ２１强阳性（）率在细支气管肺泡癌的表达明显高于腺泡状腺癌和大细胞癌（Ｐ＜０．０５），分化程度越高，染色越强。Ｐ５３蛋白蓄积与肺癌的分型、分化、ＴＮＭ分期无关，但Ｐ５３染色阳性与阴性患者的术后平均存活月数间有显著差异（Ｐ＜０．０５），Ｐ５３染色越强术后存活时间越短，提示Ｐ５３蛋白蓄积是判定肺癌预后的指标之一。