PRR11在胃癌细胞增殖与体内生长中的作用及其机制研究

发布时间：2018-07-03 15:03

本文选题：PRR11 + 胃癌　；参考：《第三军医大学》2015年博士论文

【摘要】：研究背景和目的胃癌(Gastric cancer,GC)是世界范围内最常见的恶性肿瘤之一,其发病率居全部恶性肿瘤的第四位。2008年全球胃癌患者新发病例约100万例,当年因胃癌死亡的患者大约为73万例,约占全部恶性肿瘤总发病的8%,及总死亡的10%。其中,超过70%的新发胃癌患者和死亡患者出现在发展中国家,主要分布在东欧、南美洲及亚洲地区。即使在过去数十年里,胃癌的诊断和治疗已经取得了巨大的进展,患者的死亡有所改善,但在全世界范围内,其仍然是恶性肿瘤相关死亡的第二大原因。造成这种情况的主要原因是大多数胃癌患者在诊断时已处于胃癌晚期,失去手术根治的机会,得不到有效的治疗。目前,已经报道了几个新的影响胃癌患者预后和/或治疗效果的组织蛋白标志物,然而,仅有少量的蛋白标志物被用于临床实践。因此,发现可精确预测胃癌患者预后或预测治疗效果的新分子标志物,对其进行深入研究,制备出相应的分子靶向药物,对于胃癌患者来说是非常迫切的需要。富含脯氨酸蛋白11(Proline-rich protein 11,PRR11)基因是一个新近鉴定的具有癌基因潜能基因。Ji Ying等于2013年初步研究了其在肺癌中的作用,并揭示其在肺癌细胞周期和肺癌形成中具有关键性的作用,可能在肺癌中作为一个新的潜在的诊断和/或治疗靶点。目前,PRR11基因在胃癌中的表达模式及其对胃癌影响的认识尚未见报道,需要进行研究,可能为胃癌的诊断与治疗提供新的理论思路和临床实践。在这个研究中,我们收集了216例胃癌患者的胃癌组织及其正常胃粘膜组织进行了PRR11蛋白表达状态评估,分析了PRR11蛋白表达与胃癌临床病理参数之间的关系,判断PRR11蛋白表达是否可以预测胃癌患者的预后及其与哪些胃癌临床病例参数相关。在PRR11蛋白高表达的胃癌细胞中沉默PRR11基因后,分析其对胃癌细胞增殖、克隆形成及裸鼠体内移植瘤生长的影响。基因表达谱分析PRR11基因沉默后引起一些基因表达变化显著,筛选表达变化显著的基因做相关性分析。我们筛选出表达显著降低的基因三螺旋重复胶原蛋白1(Collagen triple helix repeat containing 1,CTHRC1)及表达显著增加的基因羧肽酶A抑制因子(Latexin,LXN),免疫组化检测PRR11与CTHRC1及LXN共表达情况,分析PRR11与CTHRC1及LXN表达的相关性。研究方法1.组织样本及患者信息从病理资料库中选择符合以下条件的胃癌患者216例:2001年1月至2005年12月期间,在解放军上海长海医院普通外科进行手术切除治疗的胃癌患者,随访日期至2010年12月。并从组织标本库中挑出HE染色切片及组织蜡块,经病理科两位经验丰富的病例专家独立确定为胃癌。胃癌患者临床病理参数包括年龄、性别、肿瘤大小、T分期、N分期、TNM分期及肿瘤分化程度。本研究所有组织样本的获得,均得到患者的同意,并签订知情同意书,并得到长海医院伦理委员会许可。2.组织芯片(Tissue microarray,TMA)及免疫组化(Immunohistochemistry,IHC)由两位经验丰富的病理学专业人员对所有患者的HE染色组织玻片均进行了会诊及鉴定,确定为胃癌组织,并在具有代表性的部位进行标记。从每个供体蜡块上打孔取出直径为1.5mm的组织柱,放入受体石蜡中。4um厚的切片被放到包被3-氨基丙基三乙氧基硅烷(3-aminopropyltriethoxysilane,APTS)的玻片上。抗体稀释如下:抗-PRR11(1:100稀释),抗-LXN(1:100稀释);抗-CTHRC1(1:100稀释)。链霉亲和素—过氧化物酶法(Streptavidin-perosidase,S-P法)免疫组化染色使抗体结合组织显色,苏木素复染。PRR11、LXN、CTHRC1免疫组化染色由两个独立的专家在奥林帕斯CX31显微镜下观察。肿瘤细胞PRR11蛋白染色(胞浆呈棕色)数量10%即为阳性。3.细胞系及培养条件在5个GC细胞系(SGC7901,MKN45,MKN28,HGC27,MGC803)中发现PRR11蛋白均有表达。GC细胞系培养于杜尔伯克改良伊格尔培养基(Dulbecco modified Eagle medium,DMEM)加上10%的胎牛血清(Fetal calf serum,FBS)中,并置于37℃,含5%CO2的培养箱中培养,细胞覆盖率达80%左右传代培养。4.通过载有sh RNA的慢病毒敲低胃癌细胞中的PRR11蛋白表达包含靶向PRR11的短发夹RNA(Short hairpin RNA,sh RNA)(5’-ACG CAG GCC UUA AGG AGA ATT-3’)的慢病毒由GENECHEM公司构建,含6ug/ml聚凝胺,用来转染胃癌细胞。转染后的胃癌细胞用嘌呤霉素进行选择,利用蛋白质免疫印迹(Immunoblotting,Western blot)方法检测PRR11敲低的效果。5.细胞增殖分析和克隆形式分析以每孔5000个细胞的密度,将稳定转染的PRR11-sh RNA细胞(PRR11-KO细胞)或空载体阴性对照细胞(PRR11-NC细胞)接种于96孔板中,各设三个副孔,利用细胞计数盒(Cell counting Kit-8,CCK8),通过计算24小时及48小时的吸光度,检测细胞增殖率。同上,以每孔500个细胞的密度,将PRR11-KO细胞和PRR11-NC细胞接种于6孔板中,各三孔。孵育两周后,甲醇/丙酮(1:1)固定,结晶紫染色,计算超过50个细胞的克隆数量。6. Western blot分别准备5种胃癌细胞系总蛋白,利用兔抗人PRR11、CTHRC1、LNX的多克隆抗体(1:1000稀释),及辣根过氧化物酶(Horse radish peroxidase,HRP)结合的山羊抗兔Ig G抗体(1:10000稀释)作为二抗,β-actin作为内参。根据生产厂家的说明,利用Amersham增强化学发光系统进行蛋白显影及灰度值检测。7.实时定量逆转录PCR(q RT-PCR)分析根据说明,利用SYBR1 Premix Ex Taq TM试剂盒进行实时定量逆转录聚合酶链式反应(Quantitative Real-time reverse transcription PCR,q RT-PCR),甘油醛-3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase,GAPDH)作为内参。PCR程序为95℃1分钟,然后在95℃15秒,60℃31秒,共40个循环。利用ΔCt方法计算倍增表达。利用ABI PRISM 7300系统进行PCR检测及分析。引物利用如下:PRR11:前5’-CGT ATC TGC CAC CGA GAA CTT-3’,反:5’-GAG ATG GTC TTC AGT GCT TCC T-3’;GAPDH:前5’-TGA CTT CAA CAG CGA CAC CCA-3’,反:5’-CAC CCT GTT GCT GTA GCC AAA-3。8.动物模型通过裸鼠皮下细胞移植瘤模型评估PRR11敲低后的SGC7901细胞即PRR11-KO细胞体内成瘤及生长能力变化。PRR11-KO细胞和PRR11-NC细胞(1×106个细胞0.1ml PBS)分别注射于4周龄雌性Balb/c裸鼠右侧腋下。每三天测量肿瘤直径。移植后2周,处死所有裸鼠。收集肿瘤并测量肿瘤体积,V=0.52(长×宽×高)。9.统计分析统计分析利用SPSS 13.0软件。PRR11蛋白表达和胃癌临床病理参数之间的关系分析采用χ2检验(chi-squared分布)。根据PRR11蛋白表达情况对胃癌患者分层,利用Kaplan-Meier方法进行生存分析。单因素和多因素分析基于Cox比例风险回归模型。实验计量资料以平均值±标准差的方式呈现,计量资料分析采用Student’t-检验。p㩳0.05认为有统计学意义。研究结果1.胃癌组织及正常胃粘膜组织中PRR11蛋白表达差异显著,PRR11蛋白表达与胃癌临床病理参数相关我们对216例确诊为胃癌的患者样本进行免疫组化评估。结果显示PRR11蛋白在正常胃粘膜组织中的免疫染色为低强度,而在部分胃癌组织中的免疫染色为高强度。在216例胃癌组织样本中有107例(49.5%)PRR11蛋白表达阳性,109例(50.5%)PRR11蛋白表达阴性。q RT-PCR及Western blot分析进一步证明,相对于相应的正常胃粘膜组织,PRR11 m RNA及蛋白在胃癌组织样本中高表达。另外在SGC7901,MKN45,MKN28,HGC27和MGC803等5个胃癌细胞系中,我们发现PRR11蛋白均有表达。PRR11蛋白表达水平与多个胃癌临床病理参数之间关系的分析显示:PRR11蛋白表达与患者年龄、性别、肿瘤大小、N分期无明显相关性,而与T分期、肿瘤分化程度、TNM分期显著相关。该结果提示PRR11蛋白高表达可能与胃癌的侵袭及转移相关。2.PRR11表达上调和胃癌患者生存降低相关216位胃癌患者中,122例在随访期间死亡,中位生存期(Median survival time,MST)为51.5个月。PRR11蛋白表达阴性的胃癌患者中位生存期为74.5个月,而PRR11蛋白表达阳性的胃癌患者中位生存期为46.4个月。另外,单变量COX回归分析显示肿瘤大小、肿瘤T分期、区域淋巴结转移、TNM分期、肿瘤分化程度及PRR11表达与患者总生存(Overall survival,OS)显著相关。多变量分析显示肿瘤大小、肿瘤分化程度、PRR11表达是胃癌患者独立的预后因素。把患者分成I/II期和III/IV期两个亚群,分析PRR11表达对各亚群患者生存时间的影响,结果显示在I/II期与III/IV期两个亚群中,相对于PRR11不表达患者,PRR11高表达患者预后较差(P0.05)。3.PRR11低表达与SGC7901胃癌细胞体外增殖、克隆形成减少以及体内肿瘤生长缓慢相关利用慢病毒载荷PRR11 sh RNA稳定沉默SGC7901细胞系中PRR11表达。Western blot方法检测确证PRR11低表达SGC7901细胞系(PRR11-KO)成功建立,PRR11-KO细胞中PRR11表达显著降低,PRR11低表达与SGC7901细胞系体外增殖及克隆形成明显下降相关。同样,PRR11表达下调导致SGC7901细胞系裸鼠体内生长延缓。4.PRR11表达敲低后导致CTHRC1表达下调及LXN表达上调我们利用基因表达谱进行差异表达分析发现,PRR11表达下降导致CTHRC1m RNA表达下调及LXN m RNA表达上调。进一步利用CTHRC1蛋白和LXN蛋白抗体进行了Western blot分析,来探索这些蛋白是否随着PRR11表达降低而改变。结果显示:与PRR11-NC细胞相比较,在PRR11-KO细胞中CTHRC1蛋白表达下调,LXN蛋白表达上调。通过胃癌组织免疫组化检测进行共表达分析。数据显示胃癌组织中CTHRC1蛋白表达与PRR11蛋白表达正相关(r=0.299,p0.001),LNX蛋白表达和PRR11蛋白表达负相关(r=-0.188,p=0.005)。而CTHRC1蛋白表达和LXN蛋白表达无相关性(r=-0.042,p=0.539)。结论1.PRR11蛋白在胃癌组织中高表达,在正常胃粘膜组织中低表达,且与胃癌分化程度、T分期、TNM分期等临床病理参数相关。这提示PRR11蛋白高表达可能与胃癌细胞的侵袭与转移相关。2.多因素分析显示胃癌肿块大小、分化程度与PRR11蛋白表达是胃癌患者总生存的独立预后因素。3.PRR11蛋白在胃癌细胞系中表达,PRR11蛋白低表达降低了胃癌细胞体外增殖、克隆形成能力及体内肿瘤生长能力。4.PRR11表达敲低后引起胃癌细胞中CTHRC1表达下调以及LXN表达上调。5.进一步研究PRR11蛋白对胃癌发生、发展的作用机制,可为临床防治胃癌提供新的作用靶点。
[Abstract]:Background and objective Gastric cancer (GC) is one of the most common malignant tumors in the world, with the incidence of fourth new cases of global cancer in the world, about 1 million cases of global gastric cancer, and about 730 thousand patients died of gastric cancer in those years, accounting for 8% of the total incidence of all malignant tumors and the 10%. of total death. Among them, more than 70% of new and dead patients were found in developing countries, mainly in Eastern Europe, South America and Asia. Even in the past few decades, the diagnosis and treatment of gastric cancer had made great progress and the death of the patients improved, but in the world, it was still associated with the death of malignant tumors. Second major reasons. The main reason for this is that most gastric cancer patients are in the advanced stage of gastric cancer at the time of diagnosis, lose the chance of radical operation, and have no effective treatment. At present, several new egg white markers have been reported to affect the prognosis and / or treatment effect of gastric cancer patients. However, only a small amount of protein markers are used. It is used in clinical practice. Therefore, it is very urgent to find a new molecular marker to predict the prognosis of gastric cancer and predict the therapeutic effect of gastric cancer accurately. It is very urgent for the patients with gastric cancer to prepare the corresponding molecular targeted drugs. The gene of proline protein 11 (Proline-rich protein 11, PRR11) is a new approach. The identification of the oncogene potential gene.Ji Ying is equal to the preliminary study of its role in lung cancer in 2013, and reveals its key role in the cell cycle of lung cancer and the formation of lung cancer. It may be a new potential diagnostic and / or therapeutic target in lung cancer. At present, the expression pattern and its expression of PRR11 gene in gastric cancer The understanding of the influence of gastric cancer has not yet been reported. It needs to be studied, which may provide new theoretical ideas and clinical practice for the diagnosis and treatment of gastric cancer. In this study, we collected 216 cases of gastric cancer and normal gastric mucosa to evaluate the expression of PRR11 protein, and analyzed the expression of PRR11 protein and gastric cancer. The relationship between the clinicopathological parameters to determine whether the expression of PRR11 protein can predict the prognosis of gastric cancer patients and which of the clinical case parameters of gastric cancer. After the silencing of PRR11 gene in the gastric cancer cells with high expression of PRR11 protein, the effects of the gene expression on the proliferation of gastric cancer cells, clonogenic formation and the growth of xenografts in nude mice. A significant change in gene expression was caused by PRR11 gene silencing, and a significant gene was screened for correlation analysis. We screened three spiral repeat collagen 1 (Collagen triple helix repeat containing 1, CTHRC1) and a significant increase in expression of the gene carboxypeptidase A inhibitor (Latexin, LXN), the co expression of PRR11, CTHRC1 and LXN was detected by immunohistochemistry. The correlation between PRR11 and CTHRC1 and LXN expression was analyzed. Methods 1. tissue samples and patient information were selected from the pathological database to select 216 cases of gastric cancer patients in accordance with the following conditions: from January 2001 to December 2005, surgery was performed in the general surgery of the PLA Changhai Hospital of Shanghai. Patients with gastric cancer treated by resection were followed up to December 2010. HE staining sections and tissue paraffin blocks were selected from the tissue specimen bank. Two experienced experts in science were independently identified as gastric cancer. The clinicopathological parameters of the patients were age, sex, tumor size, T staging, N staging, TNM staging and tumor differentiation. All the samples were obtained, all received the consent of the patient, signed the informed consent, and obtained the Tissue microarray, TMA and Immunohistochemistry (Immunohistochemistry, IHC) of the ethics committee of Changhai Hospital (.2., Immunohistochemistry, IHC), which were performed by two experienced pathological professionals on all the HE stained tissue slides in all patients. Diagnosis and identification, identified as gastric cancer tissue, and marked on a representative site. A tissue column with a diameter of 1.5mm was removed from each of the donor wax blocks, and the.4um thick slices in the receptor paraffin were placed on the slides coated with 3- amino propyl triethoxy silane (3-aminopropyltriethoxysilane, APTS). The antibody was diluted as follows: Anti - PRR11 (1:100 dilution), anti -LXN (1:100 dilution); anti -CTHRC1 (1:100 dilution). Streptomycin peroxidase (Streptavidin-perosidase, S-P) immunohistochemical staining made the antibody binding tissue color, hematoxylin re dyeing.PRR11, LXN, CTHRC1 immunohistochemical staining was observed by two independent experts under Olympus CX31 microscope. The number of PRR11 protein staining (cytosolic Brown) was 10% that was positive.3. cell line and culture conditions found in 5 GC cell lines (SGC7901, MKN45, MKN28, HGC27, MGC803) and found that PRR11 protein expressed.GC cell lines in dur Burke modified Igor medium (Dulbecco modified) plus 10% fetal bovine serum Serum, FBS), and at 37 C, cultured in a incubator containing 5%CO2, the cell coverage rate is about 80%, and the.4. through the SH RNA lentivirus knockdown gastric cancer cells express the short hairpin containing the target PRR11 (Short hairpin RNA, 5 '). Constructed, 6ug/ml polyamines were used to transfect gastric cancer cells. The transfected gastric cancer cells were selected with purinamycin, and Immunoblotting (Western blot) was used to detect the effect of PRR11 knockdown on.5. cell proliferation analysis and cloning form analysis for the density of 5000 cells per pore, and the stable transfected PRR11-sh RNA thin. Cell (PRR11-KO cells) or empty carrier negative control cells (PRR11-NC cells) were inoculated in 96 orifice plates, each set of three vice pores, using cell count box (Cell counting Kit-8, CCK8), to calculate the cell proliferation rate by calculating the absorbance of 24 hours and 48 hours. With the density of 500 cells per pore, PRR11-KO cells and PRR11-NC cells were inoculated with the density of PRR11-KO cells and PRR11-NC cells. In the 6 orifice, each three holes. After two weeks of incubation, methanol / acetone (1:1) was fixed, crystal violet was stained, and the number of more than 50 cell clones,.6. Western blot, was prepared to prepare 5 kinds of gastric cancer cell lines, using Rabbit anti human PRR11, CTHRC1, LNX polyclonal antibody (1:1000 dilute release), and the combination of horseradish peroxidase (Horse radish peroxidase, HRP). The Goat anti rabbit Ig G antibody (1:10000 dilution) was used as two resistance and beta -actin as the internal parameter. According to the manufacturer's instructions, the Amersham enhanced chemiluminescence system was used for protein development and.7. real-time quantitative reverse transcriptase PCR (Q RT-PCR) analysis. The real-time quantitative reverse transcriptase was carried out by SYBR1 Premix Ex. The synthase chain reaction (Quantitative Real-time reverse transcription PCR, Q RT-PCR), glyceraldehyde -3- phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase, GAPDH) as the internal parameter.PCR program is 95 centigrade 1 minutes, then 95 centigrade 15 seconds, 60 centigrade 31 seconds, a total of 40 cycles. PCR detection and analysis. Primers are used as follows: PRR11: before 5 '-CGT ATC TGC CAC CGA GAA CTT-3', 5 '-GAG ATG GTC. C7901 cells, PRR11-KO cells, tumor formation and growth ability change.PRR11-KO and PRR11-NC cells (1 x 106 cells 0.1ml PBS) were injected into the right axillary of female Balb/c nude mice in 4 weeks of age respectively. The tumor diameter was measured every three days. All nude mice were killed at 2 weeks after transplantation. The tumor was collected and the tumor volume was measured, and V=0.52 (long x width * high).9. statistics Analysis and statistical analysis used the analysis of the relationship between SPSS 13 software.PRR11 protein expression and the clinicopathological parameters of gastric cancer using the chi 2 test (chi-squared distribution). According to the expression of PRR11 protein, the gastric cancer patients were stratified and the Kaplan-Meier method was used for survival analysis. The single factor and multifactor analysis were based on the Cox proportional risk regression model. The measurement data were presented with mean standard deviation. The measurement data were analyzed by Student 't- test.P? 0.05. The results were statistically significant. 1. the expression of PRR11 protein in gastric carcinoma and normal gastric mucosa was significantly different. The expression of PRR11 protein was related to the clinicopathological parameters of gastric cancer, and 216 cases of gastric cancer were diagnosed as gastric cancer. The results showed that the immune staining of PRR11 protein in normal gastric mucosa was low, and the immune staining in some gastric carcinoma tissues was high. In 216 cases of gastric carcinoma, 107 cases (49.5%) PRR11 protein expression was positive, 109 cases (50.5%) PRR11 protein expression negative.Q RT-PCR and Western blot The analysis further demonstrated that PRR11 m RNA and protein were highly expressed in gastric cancer tissue samples compared with the corresponding normal gastric mucosa. In 5 gastric cancer cell lines, such as SGC7901, MKN45, MKN28, HGC27 and MGC803, we found that PRR11 protein expressed the relationship between the expression of.PRR11 protein expression and the clinicopathological parameters of multiple gastric cancer. The expression of PRR11 protein was not associated with age, sex, tumor size and N staging, but was significantly related to T staging, degree of tumor differentiation, and TNM staging. The results suggest that the high expression of PRR11 protein may be associated with the up regulation of.2.PRR11 expression related to the invasion and metastasis of gastric cancer and the decrease of the survival of 216 patients with gastric cancer, and 122 cases are in the patients with gastric cancer. During the follow-up period, the median survival period (Median survival time, MST) was 74.5 months for gastric cancer patients with negative.PRR11 protein expression for 51.5 months, while the median survival period of the gastric cancer patients with PRR11 positive expression was 46.4 months. In addition, the single variable COX regression analysis showed the size of tumor, the T staging of the tumor, regional lymph node metastasis, TN. M staging, tumor differentiation and PRR11 expression were significantly related to the total survival of patients (Overall survival, OS). Multivariate analysis showed that tumor size, tumor differentiation, and PRR11 expression were independent prognostic factors of gastric cancer patients. The patients were divided into two subgroups of I/II phase and III/IV phase, and the effect of PRR11 expression on the survival time of each subgroup was analyzed. The results showed that in the two subgroups of I/II and III/IV phase, compared with PRR11 non expression patients, the prognosis of PRR11 high expression patients was poor (P0.05).3.PRR11 low expression and proliferation of SGC7901 gastric cancer cells in vitro, the decrease of clone formation and the slow growth of tumor in vivo was associated with the lentivirus PRR11 sh RNA stable silent SGC7901 cell line in the SGC7901 cell line. .Western blot method confirmed that PRR11 low expression SGC7901 cell line (PRR11-KO) was successfully established. The expression of PRR11 in PRR11-KO cells decreased significantly. The low expression of PRR11 was correlated with the proliferation and cloning of SGC7901 cell lines in vitro. Similarly, the downregulation of PRR11 led to the growth of SGC7901 cell lines in nude mice to delay the low expression of.4.PRR11. After the downregulation of CTHRC1 expression and up regulation of LXN expression, we found that the decrease of PRR11 expression led to the downregulation of CTHRC1m RNA expression and the up regulation of LXN m RNA expression. Further, Western blot analysis was used to explore whether these proteins decreased with the expression of CTHRC1 protein and LXN protein antibody. The results showed that the expression of CTHRC1 protein in PRR11-KO cells was down regulated and the expression of LXN protein was up-regulated in the PRR11-NC cells. The expression of CTHRC1 protein in gastric carcinoma tissue was positively correlated with the expression of PRR11 protein (r=0.299, p0.001), LNX protein expression and PRR11 protein table in gastric carcinoma. Negative correlation (r=-0.188, p=0.005). There was no correlation between the expression of CTHRC1 protein and the expression of LXN protein (r=-0.042, p=0.539). Conclusion the high expression of 1.PRR11 protein in gastric cancer tissues is low in normal gastric mucosa, and is related to the pathological parameters of gastric cancer differentiation, T staging, TNM staging and so on. This suggests that the high expression of PRR11 protein may be associated with gastric cancer. The.2. multivariate analysis showed that the size, differentiation and PRR11 protein expression of gastric cancer were the total number of gastric cancer patients.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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