WO2019153656A1 - piRNA-54265在结直肠癌的诊治和预后评价方面的应用 - Google Patents

piRNA-54265在结直肠癌的诊治和预后评价方面的应用 Download PDF

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WO2019153656A1
WO2019153656A1 PCT/CN2018/096398 CN2018096398W WO2019153656A1 WO 2019153656 A1 WO2019153656 A1 WO 2019153656A1 CN 2018096398 W CN2018096398 W CN 2018096398W WO 2019153656 A1 WO2019153656 A1 WO 2019153656A1
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colorectal cancer
pirna
pir
serum
expression
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林东昕
郑健
麦冬梅
谭莉萍
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中山大学
中山大学肿瘤防治中心
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Publication of WO2019153656A1 publication Critical patent/WO2019153656A1/zh
Priority to US16/576,746 priority Critical patent/US20200017906A1/en
Priority to US17/830,354 priority patent/US11674172B2/en

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  • the invention belongs to the field of biomedical technology. More specifically, it relates to the application of piRNA-54265 in the diagnosis and treatment and prognosis evaluation of colorectal cancer.
  • Colorectal cancer is a common gastrointestinal tumor in the world, ranking first among all kinds of malignant tumors in China. It is one of the main causes of death in the world, with high incidence and mortality, and there is a great clinical treatment. Difficulties. The diagnosis of colorectal cancer, especially early screening and early diagnosis, is of great significance for treatment.
  • Chemotherapy for colorectal cancer is also plagued by cancer resistance. According to estimates by the American Cancer Society, more than 90% of cancer patients die from different degrees of drug resistance, and the drug resistance problem has become a key factor in the success of tumor chemotherapy. The patient's chemosensitivity has an important influence on the effect of chemotherapy.
  • the technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing diagnosis and treatment techniques for colorectal cancer, and to provide a new diagnostic marker for colorectal cancer, piRNA-54265, which can be used for early diagnosis and early screening, and can be specifically sexual indications of chemotherapy sensitivity and prognosis in patients with colorectal cancer contribute to more precise individualized selection and improved prognosis of clinical treatment programs. They can also be used as therapeutic targets for colorectal cancer and for the treatment of colorectal cancer.
  • the first study of the present invention found that the expression of piRNA-54265 in colorectal cancer and adjacent tissues was statistically significant, and piRNA-54265 was highly expressed in cancer tissues.
  • the expression level of piRNA-54265 in cancer tissues is related to the survival prognosis of patients with colorectal cancer, and the prognosis of patients with colorectal cancer in piR-54365 high expression group is poor.
  • the expression of piRNA-54265 was detected in the serum of patients with colorectal cancer.
  • the expression level in serum was positively correlated with the expression level in cancer tissues and correlated with the survival prognosis of patients.
  • serum piR-54265 can effectively predict the pathogenesis of colorectal cancer, and can detect abnormally high serum piR-54265 expression for up to 3 years before the onset of cancer, and can perform early colorectal cancer in normal population. Screening and early diagnosis can effectively predict the incidence rate in three years.
  • piRNA-54265 as a diagnostic marker for colorectal cancer.
  • the diagnosis includes early diagnosis or early screening.
  • piRNA-54265 as a marker for evaluating the prognosis of colorectal cancer.
  • piRNA-54265 as a marker for evaluation of chemosensitivity in colorectal cancer patients.
  • the piRNA-54265 refers to piRNA-54265 in serum; it can be conveniently detected by blood samples.
  • piRNA-54265 as a therapeutic target for colorectal cancer.
  • piRNA-54265 in the preparation of therapeutic agents or drugs for colorectal cancer.
  • piRNA-54265 expression inhibitor or knockout reagent as a therapeutic drug for colorectal cancer.
  • piRNA-54265 expression inhibitor or knockout reagent as an auxiliary drug for improving the chemotherapy effect of colorectal cancer.
  • the above-mentioned drug is a drug capable of inhibiting proliferation, invasion, metastasis, and/or colonization of colorectal cancer cells.
  • piR-54265 is used as an inhibitor (Inhibitor), and the effective sequence is the reverse complement of the original sequence. Therefore, based on the sequence or as an active ingredient, any form of modification application should be protected, that is, the following The sequences set forth in SEQ ID NO. 1-3 and their use are all within the scope of the present invention.
  • SEQ ID NO. 2 UGGAG GUGAU GAACU GUCUG AGCCU GACC
  • SEQ ID NO. 3 5'-GGUCAGGCUCAGACAGUUCAUCACCUCCA-3'
  • piRNA-54265 can be used as a diagnostic marker for colorectal cancer for the diagnosis of colorectal cancer.
  • the present invention also found that the expression level of piRNA-54265 in cancer tissues is related to the survival prognosis of patients with colorectal cancer, and the prognosis of patients with colorectal cancer in the piR-54265 high expression group is poor, so piRNA-54265 can be used as colorectal cancer. Markers for prognostic evaluation were used for prognostic evaluation of colorectal cancer.
  • the present invention also found that the expression of piRNA-54265 can be detected in the serum of patients with colorectal cancer, and its expression level in serum is positively correlated with the expression level in cancer tissues, and is related to the survival prognosis of patients; Serum piR-54265 expression levels were extremely low in normal subjects, and the differences were significant and well differentiated compared with serum piR-54265 levels in colorectal cancer patients. And serum piR-54265 can effectively predict the incidence of colorectal cancer, and the abnormal expression of serum piR-54265 can be detected sensitively up to 3 years before the onset of cancer. Therefore, it can be directly and conveniently detected by blood samples. It can also be used for early diagnosis or primary screening.
  • the present inventors have also found that the proliferation and invasion ability of colorectal cancer cells are significantly reduced after knocking down piRNA-54265, and can inhibit the growth of subcutaneous xenografts in nude mice and the distant metastasis and colonization of cancer cells; thus piRNA-54265 It can be used as a therapeutic target for colorectal cancer for the treatment of colorectal cancer.
  • piRNA-54265 was disturbed, and colorectal cancer cells showed a certain degree of reduction or enhancement of chemosensitivity; patients with high expression of piRNA-54265 had poorer efficacy in chemotherapy. Therefore, based on piRNA-54265, it can be used as adjunctive therapy for chemotherapy.
  • Figure 1 shows TCGA data analysis and patient tissue sample validation of piR-54265 expression levels in colorectal cancer.
  • Figure 2 shows the expression of piR-54265 in cancer tissues affecting the survival prognosis of patients with colorectal cancer.
  • Figure 3 shows the effect of serum piR-54265 expression level on survival prognosis in patients with colorectal cancer.
  • Figure 4 is a series of cell phenotypes that are disturbed by the level of piR-54265.
  • Figure 5 shows the effect of disturbing piR-54265 levels on subcutaneous tumor formation and metastasis growth in nude mice.
  • Figure 6 shows that piR-54265 binds to PIWIL2 and promotes activation of STAT3 and its downstream signaling pathway.
  • Figure 7 shows the effect of perturbation piR-54265 on drug sensitivity of intestinal cancer cells.
  • Figure 8 shows that specific piR-54265 inhibitors inhibit xenograft growth.
  • Figure 9 shows the short-term effect of piR-54265 on chemotherapy in patients with colorectal cancer.
  • Figure 10 shows the expression of serum piR-54265 in the normal population and the long-term follow-up of colorectal cancer.
  • the identified piR-54265 was verified by qPCR again in 108 cases of colorectal cancer patients and adjacent tissues from Suzhou. Bioinformatics analysis of COAD sample gene copy number variation and DNA methylation modification data in TCGA.
  • piRNA-54265 (Accession: DQ587153) is a mature body formed by processing its precursor snoRNA57 in cells.
  • the sequence length is 29 nt, and the location of the genome is chr20: 2637585-2637613; the sequence is as follows:
  • SEQ ID NO. 2 UGGAG GUGAU GAACU GUCUG AGCCU GACC
  • SEQ ID NO. 3 5'-GGUCAGGCUCAGACAGUUCAUCACCUCCA-3'.
  • DNA methylation and SCNA results suggest that piRNA-54265 has reduced DNA methylation and gene copy number amplification in colorectal cancer patients, and the difference is statistically different from the normal group. Significance, which explains the increased expression of piRNA-54265 in colorectal cancer patients from the genomic level at the source.
  • SPSS (19.0) was used to analyze the expression of piR-54265 in cancer tissues and the prognosis of patients with colorectal cancer (such as the above-mentioned 110 cases of postoperative chemotherapy for colorectal cancer in Guangzhou and 108 cases in Suzhou).
  • the expression level of piR-54265 in cancer tissues was correlated with the survival prognosis of patients with colorectal cancer.
  • the prognosis of patients with colorectal cancer in piR-54265 high expression group was poor, and its 5-year overall survival and 5-year progression-free survival time were shorter.
  • the results of the two clinical sample centers were consistent (as shown in Figure 2).
  • the pre-treatment serum samples corresponding to the colorectal cancer cases used as before were retrieved, and 4 serum samples were randomly selected for Northern Blot test to directly determine the presence or absence of serum piR-54265 expression; then all samples were subjected to RNA extraction and RT-qPCR.
  • the expression level of piR-54265 was detected, and the correlation between serum piR-54265 and cancer tissue piR-54265 was analyzed. Survival analysis was performed on serum piR-54265 levels and corresponding patient prognosis. A total of 111 normal human serum samples were obtained.
  • RNA expression and RT-qPCR were used to detect the expression of piR-54265, and serum piR-54265 levels in colorectal cancer patients were analyzed by unpaired Student’s t-test.
  • the colorectal cancer cell lines stably overexpressing or knocking down piR-54265 were inoculated subcutaneously into nude mice to establish a subcutaneous xenograft model. The volume of subcutaneous xenografts was recorded weekly after successful modeling.
  • the colorectal cancer cell line was constructed to stably express Luciferase and overexpressed (OE) or knockdown (KD) piR-54265.
  • Luc cells were injected into the nude mice by tail vein to construct a metastatic tumor model. After successful modeling, the animals were used in vitro.
  • the in vivo imaging (IVIS) method periodically detects Luc photon values in nude mice and monitors the progression of metastases.
  • RNA of the cells was IP-expressed with the PIWI protein family antibody, and the IP RNA was eluted and verified by RT-qPCR.
  • biotinylated piR-54265 was incubated with streptomycin magnetic beads and incubated in freshly lysed proteins of HCT116 and lovo cells respectively. The pulled proteins were eluted and verified by Western Blot. The expression level of piR-54265 was disturbed, and the expression level of the protein of interest was detected by Western Blot.
  • Co-IP assay fresh cell lytic proteins were incubated with PIWIL2 or STAT3 antibodies, respectively, and the protein after IP was verified by Western Blot.
  • the two-way proof piR-54265 specifically binds to the PIWIL2 protein.
  • the expression level of piR-54265 was disturbed, and the levels of STAT3 and SRC protein were not changed, but the expression level of p-STAT3 was affected.
  • the expression of p-STAT3 protein was up-regulated when overexpressing piR-54265 (Fig. 6 c).
  • Perturbation of piR-54265 levels can affect the expression of apoptosis and metastasis-associated proteins in the downstream signaling pathway of STAT3 (Fig. 6 e).
  • IC50 experiments were performed on colorectal cancer cell lines stably overexpressing or knocking down piR-54265 for 5-FU and L-OHP.
  • the subcutaneous tumor formation model of nude mice was also constructed using the above-mentioned stable transgenic cell line.
  • intraperitoneal administration 5-FU or L-OHP was started, and the size of subcutaneous xenografts was measured periodically.
  • the colorectal cancer cell line stably overexpressing or knocking down piR-54265 was inoculated subcutaneously into nude mice to establish a subcutaneous xenograft model. After the subcutaneous transplantation tumor volume reached 50 mm 3 , multi-point injection of specific piR-54265 was started. The agent is regularly monitored and recorded for tumor volume (see Figure 8a for specific operating time points).
  • the colorectal cancer cell line was constructed to stably express Luciferase and overexpressed (OE) or knockdown (KD) piR-54265. Luc cells were injected into nude mice by tail vein to construct a metastatic tumor model. IVIS was used to monitor Luc luminescence value. After the value reached 1 ⁇ 10 6 , the specific piR-54265 inhibitor or the 5-FU combined with chemotherapy drug was administered intraperitoneally. IVIS monitored the progression of metastases (see Figure 8b for the specific operation time).
  • the piR-54265 qPCR expression was correlated with the patient's new post-evaluation evaluation results and the ROC curve was modeled to obtain the optimal cutoff value to help predict the efficacy of chemotherapy.
  • this study selected the expression of pre-treatment serum of patients with new auxiliary colorectal cancer with therapeutic evaluation, as shown in Fig. 9
  • patients with high serum piRNA-54265 expression had poorer short-term efficacy; the results of the two clinical samples were consistent.
  • the ROC curve was constructed based on the serum piR-54265 expression level in the short-term efficacy of chemotherapy. The results are shown in Figure c. In the training set, the serum piR-54265 expression level can accurately reflect the chemotherapy efficacy or chemotherapy of colorectal cancer patients.
  • piRNA-54265 will be a promising colorectal cancer-specific molecular marker that can be conveniently detected by blood samples, specifically indicating chemotherapy sensitivity and prognosis in patients with colorectal cancer, based on serum piR- The expression level of 54265 and Cutoff value will contribute to more precise individualized selection and improved prognosis of clinical treatment.
  • serum piR-54265 can be used for early screening and early diagnosis of colorectal cancer in a normal population, which can effectively predict the incidence in three years.

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Abstract

提供了piRNA-54265在结直肠癌的诊治和预后评价方面的应用。piRNA-54265可作为结直肠癌诊断标志物，特异性指示结直肠癌患者的化疗敏感性及预后判断；还可作为结直肠癌治疗靶点，用于制备结直肠癌治疗药物。piRNA-54265表达抑制剂或敲除试剂可作为结直肠癌治疗药物、或提高结直肠癌化疗效果的辅助药物，能够抑制结直肠癌细胞的增殖、侵袭、转移和/或定植。

Description

piRNA-54265在结直肠癌的诊治和预后评价方面的应用 技术领域
本发明属于生物医药技术领域。更具体地，涉及piRNA-54265在结直肠癌的诊治和预后评价方面的应用。
背景技术
结直肠癌是世界范围内常见的胃肠道肿瘤，在我国各种恶性肿瘤中居首位，是全球范围内致人死亡的主要病因之一，发生率及死亡率较高，并且临床治疗存在很大的困难。结直肠癌的诊断，尤其是早期筛查及早期诊断，对于治疗来说具有重要的意义。
结直肠癌的化疗方案也受肿瘤耐药问题的困扰。据美国癌症协会估计，死于不同程度的耐药的肿瘤患者占90％以上，肿瘤的耐药问题已经成为肿瘤化疗成功与否的关键因素。患者的化疗敏感性对于化疗效果具有重要的影响作用。
另外，结直肠癌治疗的预后判断与个体化精准治疗方案的选择也是至关重要的，不同个体的差异性会显著影响治疗效果。
发明内容
本发明要解决的技术问题是克服现有结直肠癌诊断及治疗技术的缺陷和不足，提供一种新的结直肠癌诊断标志物piRNA-54265，可用于早期诊断和早期筛查，并能够特异性指示结直肠癌患者的化疗敏感性及预后判断，有助于临床治疗方案更精准的个体化选择及改善预后；还可作为结直肠癌治疗靶点、用于制备结直肠癌治疗药物。
本发明上述目的通过以下技术方案实现：
本发明首次研究发现公开piRNA-54265在结直肠癌与癌旁组织中的表达具有显著统计学差异，piRNA-54265在癌组织中高表达。piRNA-54265在癌组织中表达量的高低与结直肠癌患者的生存预后相关，piR-54365高表达组结直肠癌患者预后较差。结直肠癌患者血清中能够检测到piRNA-54265的表达，其在血清中的表达水平与在癌组织中的表达水平呈正相关性，并与患者生存预后相关；而在正常人中血清piR-54265表达水平极低，与结直肠癌患者血清piR-54265水平相比，差异显著并区分良好。敲降piRNA-54265后，结直肠癌细胞的增殖及侵 袭能力等均明显减弱，能够抑制裸鼠皮下移植瘤的生长及癌细胞的远处转移及定植。扰动piRNA-54265的表达水平，结直肠癌细胞均表现出一定程度的化疗敏感性降低或增强；piRNA-54265高表达患者，化疗近期疗效较差。而且，血清piR-54265能有效预测结直肠癌的发病，最长可在癌发病前3年即可敏感地检测到异常的血清piR-54265高表达，能够在正常人群中进行结直肠癌的早期筛查及早期诊断，可有效预测三年发病率。
因此，以下应用均应在本发明的保护范围之内：
piRNA-54265在作为结直肠癌诊断标志物方面的应用。所述诊断包括早期诊断或早期筛查。
piRNA-54265在作为结直肠癌预后评价标志物方面的应用。
piRNA-54265在作为结直肠癌患者化疗敏感性评价标志物方面的应用。
其中，优选地，所述piRNA-54265是指血清中piRNA-54265；能方便地经血标本进行检测。
piRNA-54265在作为结直肠癌治疗靶点方面的应用。
piRNA-54265在制备结直肠癌治疗试剂或药物方面的应用。
piRNA-54265表达抑制剂或敲除试剂在作为结直肠癌治疗药物方面的应用。
piRNA-54265表达抑制剂或敲除试剂在作为提高结直肠癌化疗效果的辅助药物方面的应用。
其中，优选地，上述药物是能够抑制结直肠癌细胞的增殖、侵袭、转移和/或定植的药物。
另外，piR-54265作为抑制剂(Inhibitor)应用，有效序列为原序列的反向互补序列，因此，以该序列为基础或作为有效成分，任何形式的修饰应用，均应受到保护，即下述SEQ ID NO.1-3所示序列及其应用均应在本发明的保护范围之内。
SEQ ID NO.1：tggag gtgat gaact gtctg agcct gacc
SEQ ID NO.2：UGGAG GUGAU GAACU GUCUG AGCCU GACC
SEQ ID NO.3：5’-GGUCAGGCUCAGACAGUUCAUCACCUCCA-3’
本发明具有以下有益效果：
本发明首次发现公开piRNA-54265在结直肠癌与癌旁组织中的表达具有显著统计学差异，piRNA-54265在癌组织中高表达。因此piRNA-54265可作为结直肠癌的诊断标志物用于结直肠癌的诊断应用。
同时，本发明还发现piRNA-54265在癌组织中表达量的高低与结直肠癌患者的生存预后相关，piR-54265高表达组结直肠癌患者预后较差，因此piRNA-54265可作为结直肠癌预后评价的标志物用于结直肠癌预后评价。
再者，本发明还发现结直肠癌患者血清中能够检测到piRNA-54265的表达，其在血清中的表达水平与在癌组织中的表达水平呈正相关性，并与患者生存预后相关；而在正常人中血清piR-54265表达水平极低，与结直肠癌患者血清piR-54265水平相比，差异显著并区分良好。且血清piR-54265能有效预测结直肠癌的发病，最长可在癌发病前3年即可敏感地检测到异常的血清piR-54265高表达，因此，既可以直接方便地经血标本进行检测，又能用于进行早期诊断或初筛。
另外，本发明还发现敲降piRNA-54265后，结直肠癌细胞的增殖及侵袭能力等均明显减弱，能够抑制裸鼠皮下移植瘤的生长及癌细胞的远处转移及定植；因此piRNA-54265可用于结直肠癌治疗靶点用于结直肠癌的治疗。
最后，本发明还发现扰动piRNA-54265的表达水平，结直肠癌细胞均表现出一定程度的化疗敏感性降低或增强；piRNA-54265高表达患者，化疗近期疗效较差。因此基于piRNA-54265可作为化疗辅助治疗手段。
附图说明
图1为TCGA数据分析及病人组织样本验证piR-54265在结直肠癌中表达水平升高。
图2为癌组织piR-54265表达水平影响结直肠癌患者的生存预后。
图3为血清piR-54265表达水平影响结直肠癌患者的生存预后。
图4为扰动piR-54265水平影响系列细胞表型。
图5为扰动piR-54265水平影响裸鼠皮下成瘤及转移灶生长。
图6为piR-54265结合PIWIL2后促进激活STAT3及其下游信号通路。
图7为扰动piR-54265影响肠癌细胞药物敏感性。
图8为特异性piR-54265抑制剂抑制移植瘤生长。
图9为piR-54265影响结直肠癌患者化疗近期疗效。
图10为发病前血清piR-54265在正常人群及后期随访罹患结直肠癌时间远近各组中的表达情况。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1
1、实验方法
提取并分析TCGA及GEO表达谱数据，获得在结直肠癌中最高表达的前20个piRNA，对前20个piRNAs设计特异性引物，并在110例来自广州的结直肠癌患者的癌组织与癌旁组织样本中进行qPCR表达量检测。对鉴定出的piR-54265在苏州来源的108例结直肠癌患者癌与癌旁组织样本中进行再次qPCR验证。对TCGA中COAD样本基因拷贝数变异及DNA甲基化修饰数据进行生物信息学分析。
2、实验结果
如图1中a图和b图所示，只有piRNA-54265在结直肠癌与癌旁组织中的表达具有显著统计学差异，piRNA-54265在癌组织中高表达，两个临床中心(广州、苏州)样本得到同样结果。
piRNA-54265(Accession:DQ587153)是由其前体snoRNA57在细胞内经过加工后形成的成熟体，序列长度29nt，基因组所在位置chr20:2637585-2637613；其序列如下：
SEQ ID NO.1：tggag gtgat gaact gtctg agcct gacc
或SEQ ID NO.2：UGGAG GUGAU GAACU GUCUG AGCCU GACC
piRNA-54265的有效序列即原序列的反向互补序列为：
SEQ ID NO.3：5’-GGUCAGGCUCAGACAGUUCAUCACCUCCA-3’。
如图1中c图和d图所示，DNA methylation及SCNA结果提示，piRNA-54265在结直肠癌患者中DNA甲基化程度降低、基因拷贝数扩增，与正常组相比差异具有统计学意义，这从源头上基因组水平解释了piRNA-54265在结直肠癌患者中的表达升高。
实施例2
1、实验方法
使用SPSS(19.0)对piR-54265癌组织中表达量与结直肠癌患者预后情况(如 前述110例广州及108例苏州的结直肠癌术后化疗患者)进行生存分析。
2、实验结果
piR-54265在癌组织中表达量的高低与结直肠癌患者的生存预后相关，piR-54265高表达组结直肠癌患者预后较差，其5年总生存及5年无进展生存时间较短。两个临床样本中心结果一致(如图2所示)。
实施例3
1、实验方法
调取如前所用的结直肠癌病例对应的治疗前血清样本，随机选4例血清样本行Northern Blot实验检测，直接明确有无血清piR-54265表达；而后对全部样本进行RNA提取及RT-qPCR piR-54265表达量检测，并对血清piR-54265及癌组织piR-54265水平进行相关性分析。对血清piR-54265水平及对应的患者预后情况进行生存分析。申请调取111例正常人血清样本，行RNA提取及RT-qPCR检测piR-54265表达量，与结直肠癌患者血清piR-54265水平行unpaired Student’s t-test统计分析。
2、实验结果
如图3中a图所示，结直肠癌患者血清中能够检测到piRNA-54265的表达。图3中b图和c图，其在患者血清中的表达水平与在癌组织中的表达水平呈正相关性，并与患者生存预后负性相关。图3中d图，结直肠癌患者血清piR-54265表达量高于正常人群水平，差异具有显著统计学差异。上述结果表明，血清piR-54265表达水平在正常人、结直肠癌癌发生及预后的进程中具有一定线性关系，有作为结直肠癌早期诊断及预后判断重要标志物的潜能。
实施例4
1、实验方法
成功构建piR-54265稳定过表达及敲降的结直肠癌细胞株HCT116和LoVo，进行平板克隆实验、transwell细胞迁移或侵袭实验、CCK8细胞活力检测、Annexin-V/PI流式细胞凋亡及流式细胞周期检测。
2、实验结果
如图4所示，在两株结直肠癌细胞系中进行piRNA-54265体外功能验证，结果提示：与对照组相比，过表达piRNA-54265后能显著增强结直肠癌细胞的增殖(图4中a图)、克隆形成(图4中b图)、抗凋亡(图4中c图)及侵袭、 迁移能力(图4中d图)；反之，敲降piRNA-54265后，结直肠癌细胞的增殖及侵袭能力等均明显减弱。扰动piR-54265表达水平不影响细胞周期(未展示)。
实施例5
1、实验方法
将稳定过表达或敲降piR-54265的结直肠癌细胞株分别接种至裸鼠皮下建立皮下移植瘤模型，建模成功后每周记录皮下移植瘤体积大小。构建结直肠癌细胞株同时稳定表达Luciferase及过表达(OE)或敲降(KD)的piR-54265，Luc细胞经尾静脉注射入裸鼠体内构建转移瘤模型，建模成功后用小动物体外活体成像(IVIS)方法定期检测裸鼠体内Luc光子值，监察转移灶进展。
2、实验结果
如图5所示，动物实验结果同样提示：过表达piRNA-54265能够促进裸鼠皮下移植瘤的生长及癌细胞的远处转移及定植；敲降后则结果反之。
实施例6
1、实验方法
RIP实验，用PIWI蛋白家族抗体分别对细胞总RNA进行IP，对IP下来的RNA进行洗脱后行RT-qPCR验证。Pull Down实验，用生物素合成的piR-54265经链霉素磁珠孵育后分别在HCT116和lovo细胞新鲜裂解蛋白中进行孵育，拉下的蛋白经洗脱后行Western Blot验证。扰动piR-54265表达水平，Western Blot检测兴趣蛋白表达水平变化。Co-IP实验，在扰动piR-54265水平的情况下，分别用PIWIL2或STAT3抗体对新鲜细胞裂解蛋白进行孵育，IP后的蛋白进行Western Blot验证。
2、实验结果
如图6中a图和b图所示，双向证明piR-54265特异性与PIWIL2蛋白结合。扰动piR-54265表达水平，STAT3、SRC蛋白水平没有变化，但p-STAT3表达水平受到影响，过表达piR-54265时p-STAT3蛋白表达量上调(图6中c图)。
进而经Co-IP实验证明piR-54265、PIWIL2和STAT3三者相互结合，且过表达或敲降piR-54265能影响PIWIL2和STAT3的结合量(图6中d图)。
扰动piR-54265水平，经WB实验验证，能影响STAT3下游信号通路凋亡及转移相关蛋白的表达水平(图6中e图)。
本研究通过一系列分子生物学实验从分子机制上阐明了piRNA-54265的促 癌机制(信号通路及分子机制如图6中f图所示)，其可通过结合PIWIL2促进对STAT3的招募及磷酸化激活，从而促进了STAT3介导的抗凋亡、促转移通路。
实施例7
1、实验方法：
分别对稳定过表达或敲降piR-54265的结直肠癌细胞株进行肠癌一线化疗药物5-FU和L-OHP的IC50实验。同样用上述的稳转细胞株构建裸鼠皮下成瘤模型，当皮下移植瘤体积达250mm 3时开始腹腔给药(5-FU或L-OHP)进行治疗，定期测量皮下移植瘤大小变化。
2、实验结果：如图7中a图所示，细胞实验药物IC50结果提示扰动piRNA-54265表达水平能影响结直肠癌细胞系的药物敏感性，过表达piR-54265时肠癌细胞对5-FU和L-OHP的耐药性增强；敲降piR-54265后则敏感性提高。动物实验结果与前一致(图7中b图)：过表达piR-54265时，化疗药物治疗皮下移植瘤效果较差，敲降piR-54265后化疗敏感性增强，肿瘤体积显著缩小。
实施例8
1、实验方法：
将稳定过表达或敲降piR-54265的结直肠癌细胞株分别接种至裸鼠皮下建立皮下移植瘤模型，待皮下移植瘤体积达50mm 3后开始行瘤内多点注射特异性piR-54265抑制剂，定期监测及记录肿瘤体积(具体操作时间点见图8a)。构建结直肠癌细胞株同时稳定表达Luciferase及过表达(OE)或敲降(KD)的piR-54265，Luc细胞经尾静脉注射入裸鼠体内构建转移瘤模型，IVIS监测Luc发光值，待发光值达1×10 6后开始定期经尾静脉注射特异性piR-54265抑制剂或联合化疗药物5-FU腹腔给药，IVIS监察转移灶进展(具体操作时间点见图8b)。
2、实验结果：
结果如图8中c图-g图所示，使用特异性的piRNA-54265抑制剂或联合化疗药物进行裸鼠肿瘤治疗，能有效抑制皮下移植瘤及转移瘤的生长，实验组转移模型裸鼠总生存期显著延长。故，特异性piR-54265抑制剂有一定抑癌作用，并可辅助提高化疗药物疗效，piR-54265抑制剂有望成为新一代结直肠癌分子靶向药物。
实施例9
1、实验方法
选取广州(215例，训练集)及北京(102例，验证集)两个临床中心接受了术前新辅助化疗的结直肠癌患者治疗前取样的血清标本进行总RNA提取及随后RT-qPCR piRNA-54265表达水平检测。piR-54265 qPCR表达结果与患者新辅化后疗效评估结果进行相关性分析并进行ROC曲线建模，获取最优cutoff值以助预测化疗疗效。
2、实验结果
为更直接地研究piRNA-54265与化疗的关系，排除手术等多种复杂因素的干扰，本研究选取含疗效评价的新辅化结直肠癌患者治疗前血清进行表达量分析，如图9中a图和b图所示，血清piRNA-54265高表达患者，化疗近期疗效较差；两个临床样本中心结果一致。以血清piR-54265表达水平对患者的化疗近期疗效情况构建ROC曲线，结果如图9中c图所示，训练集中，血清piR-54265表达水平能较准确反映结直肠癌患者的化疗疗效或化疗敏感性(AUC＝0.819,P<0.0001,Guangzhou)，得到Cutoff值为0.01227(敏感度66.7％，特异度90.0％)；以该Cutoff值在验证集中进行模拟，敏感度53.3％，特异度93.1％，AUC＝0.808,P<0.0001。故，piRNA-54265将是一个十分有潜力有前景的结直肠癌特异性分子标志物，能方便地经血标本进行检测，特异性指示结直肠癌患者的化疗敏感性及预后判断，基于血清piR-54265表达水平及Cutoff值，将有助于临床治疗方案更精准的个体化选择及改善预后。
实施例10
1、实验方法：
2008～2013年间采集正常人群血清样本1160例，并长期随访并监测记录这批人群患癌情况。双盲条件下，用微滴式数字PCR绝对定量的方法在1160例正常人群血清样本中检测piR-54265的表达水平，揭盲后进行统计分析piR-54265能够提前预测正常人发生结直肠癌的效能。
2、实验结果：
结果如图10所示，越接近结直肠癌确诊时间，血清piR-54265的表达水平越高，血清piR-54265能有效预测结直肠癌的发病，而且最长可在癌发病前3年即可敏感地检测到异常的血清piR-54265高表达。
因此，血清piR-54265能够在正常人群中进行结直肠癌的早期筛查及早期诊断，可有效预测三年发病率。

Claims (10)

  1. piRNA-54265在作为结直肠癌诊断标志物方面的应用，其特征在于，所述piRNA-54265的序列为SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示。
  2. 根据权利要求1所述的应用，其特征在于，所述诊断包括早期诊断或早期筛查。
  3. piRNA-54265在作为结直肠癌预后评价标志物方面的应用。
  4. piRNA-54265在作为结直肠癌患者化疗敏感性评价标志物方面的应用。
  5. 根据权利要求1～4任一所述应用，其特征在于，所述piRNA-54265是指血清中piRNA-54265。
  6. piRNA-54265在作为结直肠癌治疗靶点方面的应用。
  7. piRNA-54265在制备结直肠癌治疗试剂或药物方面的应用。
  8. piRNA-54265表达抑制剂或敲除试剂在作为结直肠癌治疗药物方面的应用。
  9. piRNA-54265表达抑制剂或敲除试剂在作为提高结直肠癌化疗效果的辅助药物方面的应用。
  10. 根据权利要求7～9任一所述应用，其特征在于，所述药物是能够抑制结直肠癌细胞的增殖、侵袭、转移和/或定植的药物。
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