WO2018219264A1 - 一种长链非编码rna作为前列腺癌分子标志物的应用 - Google Patents

一种长链非编码rna作为前列腺癌分子标志物的应用 Download PDF

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WO2018219264A1
WO2018219264A1 PCT/CN2018/088809 CN2018088809W WO2018219264A1 WO 2018219264 A1 WO2018219264 A1 WO 2018219264A1 CN 2018088809 W CN2018088809 W CN 2018088809W WO 2018219264 A1 WO2018219264 A1 WO 2018219264A1
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lnclox5
prostate cancer
expression
prostate
patients
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French (fr)
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孙颖浩
任善成
施晓磊
刘飞
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上海长海医院
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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  • the invention belongs to the field of cancer molecular diagnosis, and particularly relates to the application of long-chain non-coding RNA IncLOX5-1 as a marker for early diagnosis of prostate cancer and prognosis of prostate cancer, and also relates to a corresponding IncRNA chip and a diagnostic kit.
  • PCa Prostatic Cancer
  • PSA Prostate specific antigen
  • the present invention provides a long-chain non-coding RNA lncRNA (lncLOX5-1) having a nucleotide sequence as shown in SEQ ID NO.
  • the invention provides an application of the lncLOX5-1 as a molecular marker for early diagnosis and prognosis of prostate cancer, and improves the accuracy and specificity of prostate cancer diagnosis by detecting lncLOX5-1.
  • early diagnosis and prognosis of prostate cancer can be performed by changing the expression level of lncLOX5-1.
  • the present invention also provides an isolated polynucleotide (DNA corresponding to lncLOX5-1) which can be transcribed by a cell (e.g., a human cell) into lncLOX5-1 as described above, and the multinucleus
  • a cell e.g., a human cell
  • the nucleotide homology is 80%, 85%, 90%, 95%, and 99% of the sequence, and can also be used as a marker for prostate cancer.
  • sequence having the nucleotide sequence homology of the lncLOX5-1 as shown in SEQ ID NO. 1 is 80%, 85%, 90%, 95% and 99%, and can also be used as prostate cancer. landmark.
  • the invention also provides an isolated polynucleotide which is transcribed by human cells into lncLOX5-1 of claim 1.
  • the invention also provides the use of a detection reagent of lncLOX5-1 for preparing a product for early diagnosis of prostate cancer, and the detection reagent of the lncLOX5-1 can be, but is not limited to, a nucleic acid probe for specifically detecting lncLOX5-1.
  • Early diagnosis of prostate cancer is performed by quantifying the expression level of the gene lncLOX5-1.
  • the invention also provides the use of a detection reagent of lncLOX5-1 for preparing a product for prognosis of prostate cancer, and the detection reagent of the lncLOX5-1 can be, but is not limited to, a nucleic acid probe for specifically detecting lncLOX5-1. Prognosis is judged for prostate cancer by quantifying the expression level of the gene lncLOX5-1.
  • the expression level of the lncLOX5-1 can be determined by the following methods: microarray technology, Northern blotting and quantitative PCR, in situ hybridization (ISH), etc.; the quantitative PCR is real-time quantitative PCR or Multiplex PCR, etc.
  • the invention also provides an oligonucleotide primer, the sequence of which is positive:
  • TCCTCCTAAGCCGTATCCCATCTG (SEQ ID NO. 2), reverse:
  • this primer can be used to amplify the lncLOX5-1.
  • the present invention also provides a reverse transcription PCR system for detecting lncLOX5-1 for the diagnosis of prostate cancer, the system for detecting lncLOX5-1 comprising the primers as described above.
  • the present invention also provides a prostate cancer in vitro diagnostic product comprising an agent that specifically detects lncLOX5-1.
  • the prostate cancer in vitro diagnostic product can be used for early diagnosis of prostate cancer and prognosis of prostate cancer.
  • the reagent for specifically detecting lncLOX5-1 can be, but is not limited to, a nucleic acid probe capable of specifically recognizing the lncLOX5-1.
  • the prostate cancer in vitro diagnostic product comprises a kit, a gene chip, a solid support, and the like; the solid support comprises an array, a micro array, and the like.
  • the invention also provides the use of the detection reagent of lncLOX5-1 for predicting the degree of tumor malignancy in prostate cancer patients.
  • the expression of lncLOX5-1 is associated with malignant progression of prostate cancer cells.
  • the invention also provides the use of a detection reagent of lncLOX5-1 for predicting whether a tumor of a prostate cancer patient progresses or not.
  • the invention also provides the use of the detection reagent of lncLOX5-1 for predicting the positive rate of a patient undergoing prostate puncture.
  • the present invention also provides a lncLOX5-1 clinical diagnosis model (Model-lncLOX5-1) constructed based on lncLOX5-1, which is constructed by age, PSA, %fPSA, prostate volume, and digital rectal examination ( DRE), lncLOX5-1 expression, and PCA3 expression were analyzed by single factor logistic regression to determine independent risk factors for predicting prostate puncture; independent risk factors for age, PSA, %fPSA, and prostate were determined by one-way logistic regression analysis.
  • lncLOX5-1 clinical diagnosis model Model-lncLOX5-1 clinical diagnosis model (Model-lncLOX5-1) constructed based on lncLOX5-1, which is constructed by age, PSA, %fPSA, prostate volume, and digital rectal examination ( DRE), lncLOX5-1 expression, and PCA3 expression were analyzed by single factor logistic regression to determine independent risk factors for predicting prostate puncture; independent risk factors for age, PSA, %f
  • the volume, lncLOX5-1 expression, and PCA3 expression were included in multivariate logistic regression analysis to construct the lncLOX5-1 clinical diagnosis model (Model-lncLOX5-1) based on lncLOX5-1 and constructed based on PCA3.
  • PCA3 clinical diagnosis model Model-PCA3; wherein the AUC of the lncLOX5-1 clinical diagnosis model reached 0.909.
  • the invention also provides the use of the lncLOX5-1 clinical diagnostic model (Model-lncLOX5-1) for predicting the positive rate of prostate puncture.
  • the invention also provides a method for early diagnosis of prostate cancer, the method comprising the steps of:
  • ROC receiver operating curve
  • AUC area under the curve
  • the method for early diagnosis of prostate cancer comprises the following steps:
  • b' The expression level of lncLOX5-1 measured by a') is divided into high expression group and low expression group by the expression level of lncLOX5-1 equal to 68. If the expression level of lncLOX5-1 is less than 68, The positive probability of prostate puncture is lower; if the expression level of lncLOX5-1 is greater than or equal to 68, it indicates that the probability of positive prostate puncture is higher;
  • the expression level of lncLOX5-1 measured by a') is bounded by the expression level of lncLOX5-1 equal to 68, and combined with the patient's age, PSA, %fPSA and prostate volume specific values, and the application is based on the lncLOX5-1 clinical diagnosis model.
  • the nomogram calculates the risk score of the individual risk factors of the patient, and adds the cumulative risk score of the patient, corresponding to the probability of puncture positive of the patient.
  • step a' the expression level of lncLOX5-1 is determined as follows.
  • Urine RNA was extracted by the TRIzol method. 200 ⁇ L of chloroform was added to each 1 mL of TRIzol-dissolved EP tube, mixed by inversion, placed in an ice box for 5 min, and centrifuged at 14,000 rpm for 15 min at 4 °C. The upper transparent water sample layer is visible, with a layer of white floc protein in the middle, and the red liquid below is a phenol-chloroform layer. Carefully pipet approximately 500 ⁇ L of the upper aqueous phase into a new EP tube. Add 500 ⁇ L of isopropanol to each EP tube, place it in the ice box for 10 min to precipitate RNA, and centrifuge at 14000 rpm for 10 min at 4 °C.
  • the bottom of the tube is clear or white jelly-like precipitate. Carefully discard the supernatant and add 1 mL of 75% ethanol. Rinse again, centrifuge again at 14000 rpm for 5 min at 4 ° C, carefully discard the supernatant, dry thoroughly, and add 10-20 ⁇ L of enzyme-free water to dissolve the RNA. The extracted RNA was applied to the Nanodrop 2000c assay concentration and the 260/280 absorbance evaluation quality. The measurement was repeated twice, and the 260/280 absorbance ratio should be between 1.80 and 2.00.
  • WTA2 18°C10min ⁇ 25°C10min ⁇ 37°C30min ⁇ 42°C10min ⁇ 70°C20min ⁇ 4°C
  • WTA3 94 ° C 2 min ⁇ (94 ° C 30 s ⁇ 70 ° C 5 min) ⁇ 17 cycles ⁇ 4 ° C.
  • Library synthesis 0.5 ⁇ L of Library Synthesis Solution was added to 100 ng of total RNA, and the enzyme-free water was added to 3.32 ⁇ L. After pipetting and mixing, incubate using the PCR instrument WTA1 program.
  • Detection of the expression level of the target gene was performed using an ABI StepOnePlus real-time quantitative fluorescent PCR instrument.
  • 20 ⁇ L of each well contained 10 ⁇ L of SYBR Green Master Mix, 1 ⁇ L of upstream primer, 1 ⁇ L of downstream primer, 0.4 ⁇ L of 50 ⁇ Rox Dye, 2 ⁇ L of cDNA and 5.6 ⁇ L of enzyme-free water.
  • the reaction conditions were 10 minutes at 95 ° C for 10 minutes, 95 ° C for 15 s and 60 ° C for 60 s. Data were statistically analyzed and exported using StepOne Software version v2.1 (Applied BioSystems, USA) software.
  • lncLOX5-1 expression level 2 Ct(PSA)-Ct(lncLOX5-1)
  • PSA was used as an internal reference for urinary slag RNA. For samples with a Ct value greater than 28 for PSA, the RNA content was insufficient, so the sample was rejected.
  • the invention also provides a method for prognosis of prostate cancer, the method comprising the following steps:
  • the prostate cancer patients are divided into low expression group and high expression group by the expression amount measured in a). If the expression level of lncLOX5-1 in the pathological sample is higher than the mean value of lncLOX5-1 in the prostate cancer patient population, it is high expression.
  • the group indicates that the tumor has a high degree of malignancy and invasiveness, and the survival prognosis level is poor; otherwise, it is a low expression group.
  • the DAB coloration in tissue in situ hybridization was taken as an example, and the comprehensive staining intensity and the proportion of positive cells in the high power microscope were semi-quantitatively determined.
  • the staining intensity score standard low expression-no coloration or Light yellow, high expression - brown or dark brown.
  • the present invention also provides the use of an inhibitor of lncLOX5-1 for inhibiting proliferation, invasion and migration of prostate cancer cells.
  • the beneficial effects of the present invention are that the novel marker for diagnosis and prediction of prostate cancer provided by the present invention, that is, the long-chain non-coding RNA lncLOX5-1, which can perform early diagnosis of prostate cancer and prognosis of prostate cancer, has High accuracy, high specificity and high sensitivity; the invention provides a prostate cancer in vitro diagnostic product comprising the detection reagent lncLOX5-1, which is convenient to use, has high accuracy, high specificity and high sensitivity. .
  • Figure 1 shows the expression of IncLOX5-1 in patients with prostate cancer, benign prostatic hyperplasia, renal cancer and bladder cancer, and in healthy human urine. Among them, the expression of IncLOX5-1 in the urine of patients with prostate cancer is significantly higher than other populations.
  • Figure 2 shows the expression of lncLOX5-1 in the urine of 243 patients undergoing prostate puncture.
  • the expression of lncLOX5-1 in the urine of patients with positive prostate puncture is significantly higher than that of patients with negative prostate puncture.
  • A is the general population and B is PSA4- In the 10 ng/mL population, C was a population of PSA 4-20 ng/mL, and D was a population of PSA 10-20 ng/mL.
  • Figure 3 shows the detection rate of prostate cancer with different expression levels of IncLOX5-1 in a population with positive prostate puncture; among them, the detection rate of prostate cancer is increased in a population with high expression of IncLOX5-1.
  • Figure 4 shows the diagnostic performance of PSA, IncLOX5-1, PCA3, fPSAratio and other indicators in the work curve of the subjects; among them, A is the general population, B is the PSA 4-10ng/mL population, and C is the PSA 4-20ng/mL population. .
  • Figure 5 shows the base model of the receiver's work curve analysis based on clinical diagnostic indicators, the IncLOX5-1 clinical diagnostic model based on IncLOX5-1 (Model-IncLOX5-1), and the PCA3 based on PCA3 score. Diagnostic efficacy of the diagnostic model (Model-PCA3), etc.: population (A), PSA 4-10 ng/mL population (B), and PSA 4-20 ng/mL population (C).
  • Figure 6 is a nomogram and internal verification of the IncLOX5-1 clinical diagnosis model (Model-IncLOX5-1) constructed based on IncLOX5-1 (calibration curve test of the nomogram): A application age, PSA, %fPSA, prostate Volume, the nomogram drawn by the diagnostic model constructed by IncLOX5-1; the calibration curve of the B-column diagram.
  • Figure 7 shows the expression of ImpLOX5-1 in patients with prostate cancer associated with tumor progression metastasis and malignancy; among them, A is the expression of urine slag IncLOX5-1 in patients with localized prostate cancer and locally advanced/metastatic prostate cancer; B is different Gleason score The amount of urine slag IncLOX5-1 expression in patients with prostate cancer.
  • Figure 8 shows the amount of IncLOX5-1 expression in tumor tissues of patients with localized and metastatic prostate cancer.
  • Figure 9 is the overall survival curve of the population of patients with high and low expression of IncLOX5-1 in the TCGA database.
  • Figure 10 shows changes in tumor cell proliferation after exogenous changes in the expression of IncLOX5-1: in LNCaP (A) and PC3 (B) cells, the ability of tumor cells to proliferate after interference with the expression of IncLOX5-1; LNCaP (C) and When the expression level of IncLOX5-1 was overexpressed in PC3(D) cells, the tumor cell proliferation ability was enhanced; wherein the legends of Fig. 10A and Fig. 10B were the same.
  • Figure 11 shows changes in tumor cell migration and invasion after exogenous changes in the expression of IncLOX5-1: increased migration and invasion of tumor cells in overexpressing IncLOX5-1 levels in PC3(A) cells; interference with IncLOX5 in LNCaP(B) cells -1 level tumor cell migration and invasion ability is weakened.
  • Figure 12 is a demonstration of the role of IncLOX5-1 in prostate cancer in vivo; wherein AB is a subcutaneous tumor model of prostate cancer cell line PC3 which interferes with the expression of IncLOX5-1, and the tumor volume of prostate cancer is significantly lower than that of the normal control group; C is the expression of IncLOX5-1 in prostate cancer PC3 implanted tumors that interfere with the expression of IncLOX5-1, which is significantly lower than that of the control group; D is immunohistochemistry (IHC), in the tumor of prostate cancer cell PC3 that interferes with IncLOX5-1, cells The expression levels of the proliferation markers Ki-67 and PCNA were significantly reduced.
  • AB is a subcutaneous tumor model of prostate cancer cell line PC3 which interferes with the expression of IncLOX5-1, and the tumor volume of prostate cancer is significantly lower than that of the normal control group
  • C is the expression of IncLOX5-1 in prostate cancer PC3 implanted tumors that interfere with the expression of IncLOX5-1, which is significantly lower than that of the control group
  • Example 1 Expression of lncLOX5-1 in urine of patients with prostate cancer, benign prostatic hyperplasia, renal cancer and bladder cancer and normal humans
  • the prostate cancer in vitro diagnostic reagents as described above such as a kit, a gene chip, and the like, may be used for detecting the expression level of lncLOX5-1.
  • the present inventors found that the expression level of lncLOX5-1 in the urine residue of prostate cancer patients was significantly higher than that in the urine of benign prostatic hyperplasia patients, renal cancer patients, bladder cancer patients and healthy persons (Fig. 1). And the expression of lncLOX5-1 in urine slag of patients with prostate cancer and benign prostatic hyperplasia is higher than other populations, lncLOX5-1 can be used as a prostate specific index.
  • the expression level of lncLOX5-1 in urine slag of prostate cancer patients was significantly higher than that of lncLOX5-1 in urine slag of other populations.
  • the expression of lncLOX5-1 in urine slag after prostate massage was measured by including 243 patients with prostate puncture, including 146 puncture-negative patients and 97 puncture-positive patients. It was found that the expression level of lncLOX5-1 in urine samples of patients with puncture-positive patients was significantly higher than that of lncLOX5-1 in urine samples of puncture-negative patients (Fig. 2A).
  • the invention detects the expression of lncLOX5-1 in the urine residue of 97 patients with positive prostate puncture, and finds that the higher the expression level of lncLOX5-1, the higher the detection rate of prostate cancer in the corresponding population.
  • lncLOX5-1 was sorted from small to large, 0-25% was the lowest 25% of lncLOX5-1 expression, 75-100% was the highest expression of 1% of lncLOX5-1, and the lowest expression of lncLOX5-1 was found.
  • the detection rate of prostate cancer (0-25%) was lower than that of the higher expression group (75-100%); in the general population, PSA ⁇ 10 ng/mL population, PSA 10-20 ng/mL population and PSA> The same trend was observed in the 20 ng/mL population ( Figure 3).
  • Urine slag lncLOX5-1 score as an auxiliary diagnostic indicator for prostate cancer
  • Figure 4 shows the diagnostic performance of each indicator in the receiver's working curve analysis.
  • the receiver operating curve (ROC) was used to analyze the diagnostic efficacy of the urine residue IncLOX5-1.
  • the diagnostic power of the IncLOX5-1 score was also significantly better than the PCA3 score (0.719 vs 0.600, P ⁇ 0.001) in the PSA 4-20 ng/mL patient population (Fig. 4C).
  • the independent risk factors determined by single factor logistic regression analysis included age, prostate volume, PSA, and %fPSA in multivariate logistic regression analysis to construct a diagnostic prediction model for predicting prostate biopsy results.
  • the basic diagnostic model constructed by clinical diagnostic indicators is first applied separately.
  • the clinical diagnosis model (Model-PCA3) based on the PCA3 score had no statistically significant difference in AUC and PA compared with the baseline diagnosis model (Fig. 5).
  • the present invention draws a nomogram, assigning each independent risk factor, each clinical indicator and lncLOX5- The scores of the 1 scores are given corresponding scores, and the total scores obtained by adding the scores of the indicators can quickly calculate the probability that the model predicts positive prostate puncture (Fig. 6A).
  • the present invention tests the calibration curve of the nomogram, and the abscissa is the nomogram prediction probability of the prediction model constructed by the invention, the ordinate is the real occurrence probability, and the oblique 45° curve represents the ideal curve, indicating the nomogram prediction probability and the true probability.
  • the bias correction curve of the present invention fits closely with the ideal curve (Fig. 6B).
  • the nomogram can be used simply and effectively in clinical practice to predict the probability of puncture positive in patients undergoing prostate puncture.
  • the present invention compares the expression levels of urine lncLOX5-1 expression in different grades of prostate cancer patients, and finds that the expression level of lncLOX5-1 based on urine residue detection is expressed in patients with localized prostate cancer and locally advanced/metastatic prostate cancer. There was a significant difference in the expression of Gleason scores (GS) ⁇ 7 points and ⁇ 7 points in prostate cancer populations (Fig. 7). As the tumor progresses and the pathological malignancy increases, the expression of lncLOX5-1 increases, suggesting that the lncLOX5-1 score can be used to accurately diagnose high-grade prostate cancer.
  • Gleason scores ⁇ 7 points and ⁇ 7 points in prostate cancer populations
  • the present invention detects the expression of lncLOX5-1 in tumor tissue samples of 41 patients with prostate cancer, and found that the expression of lncLOX5-1 in patients with metastatic prostate cancer is significantly higher than that in patients with localized prostate cancer (P ⁇ 0.01). 8).
  • the present invention analyzes the expression level of lncLOX5-1 in tumor tissues of prostate cancer patients in the public database (TCGA) by means of bioinformatics, and finds that the prognosis of the high expression group and the low expression group of lncLOX5-1 is significantly different (Fig. 9). ).
  • TCGA public database
  • HR Hazard rate
  • Example 2 The role of lncLOX5-1 in the progression of malignant prostate cancer
  • the inventors found that the expression level of lncLOX5-1 is closely related to the degree of tumor progression metastasis and malignancy. Infinite proliferation and invasion and migration are important features of tumors.
  • the present invention studies the correlation between lncLOX5-1 and proliferation, invasion and migration of prostate cancer cells.
  • the present invention overexpresses IncLOX5-1 in prostate cancer cell lines LNCaP and PC3 by means of exogenous overexpression plasmid and siRNA transfection, and detects changes in cell proliferation ability by CCK8 assay (Fig. 10), and is detected by Transwell assay.
  • CCK8 assay Fig. 10
  • Transwell assay The change of tumor cell invasion and migration ability (Fig. 11) showed that after up-regulating the expression level of IncLOX5-1, the cell proliferation ability increased, and the cell invasion and migration ability also increased. After down-regulating the expression of IncLOX5-1, The proliferative capacity of the cells decreases, and the ability of cells to invade and migrate also decreases.
  • Subcutaneous injection of prostate cancer cell PC3 into nude mice When PC3 cells were grown to logarithmic growth phase, ie, the state was optimal, each nude mouse was injected with 1 ⁇ 10 6 number of cells, and the cell suspension was mixed with Matrigel (BD). After being mixed in a volume ratio of 1:1, it was injected into the outer side of the thigh of a nude mouse to facilitate intratumoral injection and measurement of tumor condition. After half a week, the nude mice were able to access the hard mass under the skin. The nude mice were randomly divided into two groups according to body weight and mass size. Intratumoral injection of IncLOX5-1 siRNA and control siRNA-NC were performed every two days, before each injection. The tumor size was measured. After 6 weeks, the tumor growth was more obvious.
  • BD Matrigel
  • Immunohistochemistry (IHC) analysis showed that the expression of cell proliferation markers Ki-67 and PCNA was significantly reduced in tumors of prostate cancer cell line PC3-siRNA-lncLOX5-1 that interfered with lncLOX5-1 (Fig. 12d).
  • the non-coding RNA lncLOX5-1 proposed by the present invention can be effectively used as a marker for early diagnosis and prognosis of prostate cancer; the lncLOX5-1 can be used for predicting the degree of malignancy of prostate cancer patients and predicting prostate cancer patients. Whether the tumor progresses and metastasizes, predicts the positive rate of patients who are going to undergo prostate puncture, etc., has the characteristics of high accuracy, high specificity and high sensitivity.

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Abstract

一种前列腺癌早期诊断和预后判断的标志物长链非编码RNA lncLOX5-1及其应用。该lncLOX5-1可用于前列腺癌的早期诊断和预后判断，具有高准确度、高特异性和高灵敏度的特点。

Description

一种长链非编码RNA作为前列腺癌分子标志物的应用 技术领域
本发明属于癌症分子诊断领域，具体涉及长链非编码RNA IncLOX5-1作为前列腺癌早期诊断以及对前列腺癌进行预后判断的标志物中的应用，还涉及相应的IncRNA芯片和诊断试剂盒。
背景技术
前列腺癌(Prostatic Cancer，PCa)是一种严重威胁男性健康的恶性肿瘤，是发达国家发病率最高的男性恶性肿瘤，占全球肿瘤发病率第二位、死亡率第六位。我国PCa的发病率近年来一直处于显著的上升趋势。在北京、上海、广州等医学、经济发达城市，PCa发病率已位于当地十大常见肿瘤之列。流行病学数据显示，中国PCa发病率已从1993年的1.71/10万男性人口增加到2005年的7.9/10万男性人口，年增幅约13％。依此推算，预计2020年，我国PCa发病率将超过40/10万男性人口，接近欧美国家水平，成为危害男性健康的主要肿瘤“杀手”。由此可见，随着我国普遍医疗水平的提高，以及中国正在步入老龄化社会，前列腺癌的防治、研究工作已经进入刻不容缓的阶段。由于PCa症状隐蔽，导致国内初诊时处于中晚期的患者居多，且超过46.3％的患者已发生局部进展或远处转移。前列腺癌患者一旦发生转移不仅预后差，而且严重影响其生活质量。SEER(Surveillance Epidemiology and End Results，SEER)数据库2004-2010年的资料显示，局限性前列腺癌的5年生存率为100％，而转移性前列腺癌仅为28％。前列腺特异性抗原(Prostate specific antigen，PSA)是目前最广泛应用于前列腺癌的分子诊断标记物，但其特异性较差，导致大量的不必要穿刺和医疗资源的浪费。因此亟需找到PCa高特异性的分子诊断标记物和相关检测技术。
发明内容
本发明提供了一种长链非编码RNA lncRNA(lncLOX5-1)，其核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种所述lncLOX5-1在作为对前列腺癌进行早期诊断及预后判断的分子标志物的应用，通过lncLOX5-1的检测，提高对前列腺癌诊断的准确性和特异性。
本发明中，可通过lncLOX5-1的表达量变化，从而对前列腺癌进行早期诊断以及预后判断。
本发明还提供了一种分离的多核苷酸(lncLOX5-1所对应的DNA)，所述多核苷酸能被细胞(如，人细胞)转录成如上所述的lncLOX5-1，与所述多核苷酸的同源性为80％、85％、 90％、95％及99％的序列，也可作为前列腺癌的标志物。
本发明中，与所述lncLOX5-1如SEQ ID NO.1所示的核苷酸序列同源性为80％、85％、90％、95％及99％的序列，也可作为前列腺癌的标志物。
本发明还提供了一种分离的多核苷酸，所述多核苷酸能被人细胞转录成权利要求1所述的lncLOX5-1。
本发明还提供了lncLOX5-1的检测试剂在制备对前列腺癌进行早期诊断的产品中的应用，所述lncLOX5-1的检测试剂可以但不限于是特异性检测lncLOX5-1的核酸探针。通过对所述基因lncLOX5-1的表达量进行定量，来对前列腺癌进行早期诊断。
本发明还提供了lncLOX5-1的检测试剂在制备对前列腺癌进行预后判断的产品中的应用，所述lncLOX5-1的检测试剂可以但不限于是特异性检测lncLOX5-1的核酸探针。通过对所述基因lncLOX5-1的表达量进行定量，对前列腺癌进行预后判断。
本发明中，所述lncLOX5-1的表达量可通过以下方法确定：微阵列技术、RNA印迹和定量PCR、组织原位杂交(In situ hybridization，ISH)等；所述定量PCR为实时定量PCR或多重PCR等。
本发明还提供了一种寡核苷酸引物，所述引物的序列为正向：
TCCTCCTAAGCCGTATCCCATCTG(SEQ ID NO.2)，反向：
CCAGGTGAGTTGAACAGTCCGATT(SEQ ID NO.3)，该引物能够用来扩增所述lncLOX5-1。
本发明还提供了一种诊断前列腺癌的检测lncLOX5-1的反转录PCR系统，该检测lncLOX5-1的系统包括如上所述的引物。
本发明还提供了一种前列腺癌体外诊断产品，所述体外诊断产品包括特异性检测lncLOX5-1的试剂。所述前列腺癌体外诊断产品可用于对前列腺癌进行早期诊断以及对前列腺癌进行预后判断。所述特异性检测lncLOX5-1的试剂可以但不限于是核酸探针，所述核酸探针能特异性识别所述lncLOX5-1。
其中，所述前列腺癌体外诊断产品包括试剂盒、基因芯片、固体支持体等；所述固体支持体包括阵列、微阵列等。
本发明还提供了lncLOX5-1的检测试剂在预测前列腺癌患者肿瘤恶性程度中的应用。lncLOX5-1的表达与前列腺癌细胞恶性进展相关。
本发明还提供了lncLOX5-1的检测试剂在预测前列腺癌患者肿瘤是否进展转移中的应用。
本发明还提供了lncLOX5-1的检测试剂在预测拟行前列腺穿刺患者的阳性率中的应用。
本发明还提供了一种基于lncLOX5-1构建的lncLOX5-1临床诊断模型 (Model-lncLOX5-1)，所述模型的构建过程为，将年龄、PSA、％fPSA、前列腺体积、直肠指诊(DRE)、lncLOX5-1表达量、PCA3表达量进行单因素logistic回归分析，确定能作为预测前列腺穿刺结果的独立危险因素；根据单因素logistic回归分析确定的独立危险因素年龄、PSA、％fPSA、前列腺体积、lncLOX5-1表达量、PCA3表达量，将其纳入多因素logistic回归分析中，构建所述基于lncLOX5-1构建的lncLOX5-1临床诊断模型(Model-lncLOX5-1)，和基于PCA3构建的PCA3临床诊断模型(Model-PCA3)；其中，所述lncLOX5-1临床诊断模型的AUC达到0.909。
本发明还提供了所述lncLOX5-1临床诊断模型(Model-lncLOX5-1)在预测前列腺穿刺的阳性率中的应用。
本发明还提供了一种前列腺癌进行早期诊断的方法，所述方法包括以下步骤：
a)检测PSA异常患者行前列腺按摩后尿渣中lncLOX5-1的表达量；
b)通过a)中测定的lncLOX5-1的表达量，采用受试者工作曲线(ROC)分析lncLOX5-1表达量的曲线下面积(AUC)，选择敏感性和特异性最大的点作为临界点，将患者分为高表达组和低表达组，若lncLOX5-1表达量小于临界点，提示其前列腺穿刺阳性概率较低；若lncLOX5-1表达量大于等于临界点，提示其前列腺穿刺阳性概率较高；
或，通过a)中测定的lncLOX5-1的表达量，采用受试者工作曲线(ROC)分析lncLOX5-1表达量的曲线下面积(AUC)，选择敏感性和特异性最大的点作为临界点，联合患者年龄、PSA、％fPSA和前列腺体积具体数值，应用基于lncLOX5-1临床诊断模型绘制的列线图计算患者单个风险因素的风险评分，相加得到患者的累积风险评分，对应得出患者的穿刺阳性概率。
在一个具体的实施方式中，本发明提供的前列腺癌进行早期诊断的方法，包括以下步骤：
a’)检测PSA异常患者行前列腺按摩后尿渣中lncLOX5-1的表达量；
b’)通过a’)中测定的lncLOX5-1的表达量，以lncLOX5-1表达量等于68为界，将患者分为高表达组和低表达组，若lncLOX5-1表达量小于68，提示其前列腺穿刺阳性概率较低；若lncLOX5-1表达量大于等于68，提示其前列腺穿刺阳性概率较高；
或，通过a’)中测定的lncLOX5-1的表达量，以lncLOX5-1表达量等于68为界，联合患者年龄、PSA、％fPSA和前列腺体积具体数值，应用基于lncLOX5-1临床诊断模型绘制的列线图计算患者单个风险因素的风险评分，相加得到患者的累积风险评分，对应得出患者的穿刺阳性概率。
其中，步骤a’)中，lncLOX5-1的表达量测定方法如下，
(1)尿液RNA抽提：
尿液RNA采用TRIzol法进行抽提。每1mL TRIzol溶解的EP管中加入200μL三氯甲烷，颠倒混匀后置于冰盒中放置5min，以4℃14000rpm离心15min。可见上层透明水样层，中间为一层白色絮状蛋白，下面红色液体为苯酚-氯仿层。小心吸取上层水相约500μL转移至新的EP管中。每个EP管中加入500μL的异丙醇，冰盒中放置10min以沉淀RNA，再以4℃14000rpm离心10min，可见管底透明或白色胶冻样沉淀，小心弃去上清加入1mL 75％乙醇冲洗一遍，再次以4℃14000rpm离心5min，小心弃去上清，充分干燥后加入10-20μL无酶水溶解RNA。抽提出来的RNA应用Nanodrop 2000c检测浓度和260/280吸光度评价质量，重复测量2次，260/280吸光度比值应介于1.80-2.00。
(2)反转录扩增：
反转录扩增采用Sigma公司的完整全转录组扩增试剂盒(
Figure PCTCN2018088809-appb-000001
Complete Whole Transcriptome Amplification Kit)。冰盒上解冻Library Synthesis Buffer，Library Synthesis Solution，Library Synthesis Enzyme，Amplification Mix，10mM dNTP Mix，Amplification Enzyme和无酶水。
PCR仪设置程序：
WTA1：70℃，5min→18℃
WTA2：18℃10min→25℃10min→37℃30min→42℃10min→70℃20min→4℃
WTA3：94℃2min→(94℃30s→70℃5min)×17个循环→4℃。
文库合成：100ng全RNA中加入0.5μL Library Synthesis Solution，加入无酶水补齐至3.32μL。吹打混匀后使用PCR仪WTA1程序孵育。
向管中加入0.5μL Library Synthesis Buffer，0.4μL Library Synthesis Enzyme和0.78μL无酶水。吹打混匀后，使用PCR仪WTA2程序进行孵育。
文库扩增：向管中加入60.2μL无酶水，7.5μL Amplification Mix，1.5μL dNTP Mix和0.75μL Amplification Enzyme，使用PCR仪WTA3程序进行孵育。反应完成后，将PCR产物cDNA储存于-20℃以备后续检测。
(3)实时定量PCR(qRT-PCR)：
目的基因表达水平的检测采用ABI StepOnePlus实时定量荧光PCR仪器进行检测。应用TOYOBO公司THUNDERBIRD SYBR real-time qPCR试剂盒，每个孔20μL反应体系中含有10μL SYBR Green Master Mix，1μL上游引物，1μL下游引物，0.4μL 50×Rox Dye，2μL cDNA和5.6μL无酶水。反应条件为，95℃10min，95℃15s和60℃60s连续40个循环。数据采用StepOne Software version v2.1(Applied BioSystems，USA)软件进行统计分析和导出。lncLOX5-1表达量计算公式为：lncLOX5-1表达量＝2 Ct(PSA)-Ct(lncLOX5-1)，PCA3表达量计算公 式为：PCA3表达量＝2 Ct(PSA)-Ct(PCA3)，以PSA作为尿渣RNA的内参，对于PSA的Ct值大于28的样本提示其RNA含量不足，故剔除样本。
qRT-PCR反应体系
Figure PCTCN2018088809-appb-000002
qRT-PCR扩增反应条件
Figure PCTCN2018088809-appb-000003
qRT-PCR引物序列
Figure PCTCN2018088809-appb-000004
本发明还提供了一种对前列腺癌进行预后判断的方法，所述方法包括以下步骤：
a)检测前列腺癌患者病理样本中lncLOX5-1的表达量；
b)通过a)中测定的表达量，将前列腺癌患者分为低表达组和高表达组，若病理样本中lncLOX5-1表达量高于前列腺癌患者人群lncLOX5-1的均值，则为高表达组，预示肿瘤恶性程度、侵袭性较高，生存预后水平较差；否则为低表达组。
所述lncLOX5-1高表达组，以组织原位杂交中DAB显色为例，将高倍镜下综合染色强度和阳性细胞所占比例进行半定量测定，染色强度评分标准：低表达-不着色或浅黄色，高表达-棕黄色或深黄褐色。
本发明还提供了一种lncLOX5-1的抑制剂在抑制前列腺癌中的应用，所述抑制剂用于抑制前列腺癌细胞的增殖、侵袭和迁移。
本发明的有益效果在于，本发明提供的诊断和预测前列腺癌的新标志物，即长链非编码RNA lncLOX5-1，该标志物能对前列腺癌进行早期诊断以及对前列腺癌进行预后判断，具有高准确度，高特异性和高灵敏度的特点；本发明提供的包含标志物lncLOX5-1的检测试剂的前列腺癌体外诊断产品，其使用方便，具有高准确度、高特异性和高灵敏度的特点。
附图说明
图1为前列腺癌、良性前列腺增生、肾癌和膀胱癌患者以及健康人尿渣中IncLOX5-1的表达量；其中，前列腺癌患者尿渣中IncLOX5-1表达量显著高于其他人群。
图2为243例行前列腺穿刺患者人群尿渣lncLOX5-1的表达量，前列腺穿刺阳性患者尿渣中lncLOX5-1表达量显著高于前列腺穿刺阴性患者；其中，A为总体人群，B为PSA4-10ng/mL人群，C为PSA4-20ng/mL人群，D为PSA 10-20ng/mL人群。
图3为前列腺穿刺阳性患者人群中不同IncLOX5-1表达量的前列腺癌检出率；其中，IncLOX5-1表达量高的人群中前列腺癌检出率增高。
图4为受试者工作曲线分析PSA、IncLOX5-1、PCA3、fPSAratio等指标的诊断效能；其中，A为总体人群、B为PSA 4-10ng/mL人群，C为PSA 4-20ng/mL人群。
图5为受试者工作曲线分析完全基于临床诊断指标构建的基础模型(Base Model)、基于IncLOX5-1构建的IncLOX5-1临床诊断模型(Model-IncLOX5-1)、基于PCA3评分构建的PCA3临床诊断模型(Model-PCA3)等的诊断效能：总体人群(A)、PSA 4-10ng/mL人群(B)和PSA 4-20ng/mL人群(C)。
图6为基于IncLOX5-1构建的IncLOX5-1临床诊断模型(Model-IncLOX5-1)绘制的列线图及内部验证(列线图的校正曲线检验)：A应用年龄、PSA、％fPSA、前列腺体积、IncLOX5-1构建的诊断模型绘制的列线图；B列线图的校正曲线检验。
图7为前列腺癌患者尿渣IncLOX5-1表达量与肿瘤进展转移和恶性程度相关；其中，A为局限性前列腺癌与局部进展/转移前列腺癌患者尿渣IncLOX5-1表达量；B不同Gleason评分前列腺癌患者尿渣IncLOX5-1表达量。
图8为局限性和转移性前列腺癌患者肿瘤组织的IncLOX5-1表达量。
图9为TCGA数据库中IncLOX5-1高表达和低表达前列腺癌患者人群总体生存曲线。
图10为外源性改变IncLOX5-1表达量后肿瘤细胞增殖能力的变化：LNCaP(A)和PC3(B)细胞中干扰IncLOX5-1表达量后，肿瘤细胞增殖能力减弱；LNCaP(C)和PC3(D)细胞中过 表达IncLOX5-1表达量后，肿瘤细胞增殖能力增强；其中，图10A和图10B的图例相同。
图11为外源性改变IncLOX5-1表达量后肿瘤细胞迁移和侵袭能力的变化：PC3(A)细胞中过表达IncLOX5-1水平肿瘤细胞迁移和侵袭能力增强；LNCaP(B)细胞中干扰IncLOX5-1水平肿瘤细胞迁移和侵袭能力减弱。
图12为体内实验证实IncLOX5-1在前列腺癌中的作用；其中，A-B为干扰IncLOX5-1表达的前列腺癌细胞PC3皮下荷瘤裸鼠模型，前列腺癌肿瘤体积与正常对照组相比明显降低；C为干扰IncLOX5-1表达的前列腺癌PC3种植瘤中IncLOX5-1表达量较对照组显著降低；D为免疫组化(IHC)显示，在干扰IncLOX5-1的前列腺癌细胞PC3的肿瘤中，细胞增殖标记物Ki-67和PCNA表达量明显降低。
(全文中星号含义相同，即，*：P＜0.05；**：P＜0.01；***：P＜0.001)。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。
实施例1 前列腺癌、良性前列腺增生、肾癌和膀胱癌患者以及正常人尿液中lncLOX5-1的表达
以下实施例中，检测lncLOX5-1表达量时可采用上文所述的前列腺癌体外诊断试剂，如试剂盒、基因芯片等。
(1.1)前列腺癌、良性前列腺增生、肾癌和膀胱癌患者以及正常人尿液中lncLOX5-1的表达量有显著差异
本发明发现前列腺癌患者的尿渣中，lncLOX5-1表达量显著高于良性前列腺增生患者、肾癌患者、膀胱癌患者和健康人的尿渣中lncLOX5-1表达量(图1)。且前列腺癌患者及良性前列腺增生患者尿渣中lncLOX5-1表达量高于其他人群，lncLOX5-1可作为前列腺特异性指标。
(1.2)前列腺穿刺阳性患者尿渣中lncLOX5-1表达量显著高于前列腺穿刺阴性患者
如上所述，lncLOX5-1在前列腺癌患者尿渣中表达量显著高于其他人群的尿渣中lncLOX5-1表达量。通过纳入243名行前列腺穿刺患者，包括146名穿刺阴性患者和97名穿刺阳性患者，检测患者前列腺按摩后尿渣中lncLOX5-1表达量。发现穿刺阳性患者尿渣中lncLOX5-1表达量显著高于穿刺阴性患者尿渣中lncLOX5-1的表达量(图2A)。在总体人群、PSA4-10ng/mL人群、PSA4-20ng/mL人群和PSA 10-20ng/mL人群中均发现lncLOX5-1表达量在穿刺阳性患者的尿渣中显著提高(图2)。
(1.3)lncLOX5-1表达量高的人群中前列腺癌检出率增高
本发明通过检测97名前列腺穿刺阳性的患者尿渣中lncLOX5-1表达量，发现lncLOX5-1表达量越高，其对应人群中前列腺癌检出率越高。将lncLOX5-1按照从小到大进行排序，0-25％是lncLOX5-1表达量最低的25％人群，75-100％是lncLOX5-1表达量最高的25％人群，发现lncLOX5-1表达量最低的群体(0-25％)前列腺癌检出率要低于表达量较高的群体(75-100％)；在总体人群、PSA＜10ng/mL人群、PSA 10-20ng/mL人群和PSA＞20ng/mL人群中均发现相同的趋势(图3)。
(1.4)尿渣lncLOX5-1评分作为前列腺癌的辅助诊断指标
图4为受试者工作曲线分析各指标诊断效能。运用受试者工作曲线(ROC)分析尿渣IncLOX5-1的诊断效能，发现尿渣IncLOX5-1的曲线下面积(AUC)达到了0.782，PSA的AUC为0.798，两者之间的差异无统计学意义(P＝0.696)(图4A)。而PCA3评分的AUC0.662，显著低于IncLOX5-1和PSA(P lncLOX5-1＜0.001，P PSA＝0.003)。在PSA 4-10ng/mL诊断灰区的患者人群中，IncLOX5-1评分的诊断效能要优于PCA3评分(0.709vs 0.601，P＝0.046)(图4B)。在PSA4-20ng/mL的患者人群中，IncLOX5-1评分的诊断效能也显著优于PCA3评分(0.719vs0.600，P＜0.001)(图4C)。
(1.5)基于尿渣lncLOX5-1诊断模型建立
单因素logistic回归分析确定的独立危险因素，本发明将年龄、前列腺体积、PSA、％fPSA纳入多因素logistic回归分析中，构建预测前列腺穿刺活检结果的诊断预测模型。首先单独应用临床诊断指标构建的基础诊断模型(Base Model)。基于IncLOX5-1构建的IncLOX5-1临床诊断模型(Model-IncLOX5-1)其AUC达到了0.909，显著优于基于临床诊断指标构建的基础诊断模型(P＝0.009)。而基于PCA3评分构建的临床诊断模型(Model-PCA3)其AUC和PA与基础诊断模型相比，差异均无显著的统计学意义(图5)。
(1.6)基于尿渣lncLOX5-1诊断预测模型绘制列线图
基于尿渣lncLOX5-1构建的lncLOX5-1临床诊断模型(Model-lncLOX5-1)，本发明绘制了列线图(nomogram)，将每个独立危险因素进行了赋值，每个临床指标以及lncLOX5-1评分的数值都给予了相应的分数，将各指标分数相加得到的总分可以迅速计算出模型预测前列腺穿刺阳性的概率(图6A)。接下来本发明对nomogram进行了校正曲线的检验，横坐标为本发明构建的预测模型的nomogram预测概率情况，纵坐标为真实发生概率，斜45°曲线代表理想曲线，表明nomogram预测概率与真实概率完全吻合；本发明的偏倚校正曲线与理想曲线贴合比较密切(图6B)。该列线图可简单、有效应用于临床，预测拟行前列腺穿刺患者的穿刺阳性概率。
(1.7)lncLOX5-1高表达与前列腺癌患者的肿瘤进展转移及恶性程度相关
本发明对于尿液lncLOX5-1表达量在不同级别的前列腺癌患者的表达量进行了比较，发现基于尿渣检测的lncLOX5-1表达量在局限性前列腺癌和局部进展/转移前列腺癌患者人群表达存在显著差别，Gleason评分(GS)＜7分和≥7分的前列腺癌人群中表达有显著差别(图7)。随着肿瘤进展转移和病理恶性程度增加，lncLOX5-1表达量增加，提示lncLOX5-1评分可用于精准诊断高级别前列腺癌。
(1.8)局限性和转移性前列腺癌患者肿瘤组织的lncLOX5-1表达量
本发明对41例前列腺癌患者的肿瘤组织样本进行lncLOX5-1表达量的检测，发现转移前列腺癌患者的lncLOX5-1表达量显著高于局限性前列腺癌患者肿瘤组织样本(P＜0.01)(图8)。
(1.9)lncLOX5-1高表达与前列腺癌患者的总体生存率相关
本发明通过生物信息学的方法，分析公开数据库(TCGA)中前列腺癌患者肿瘤组织中lncLOX5-1的表达量，发现lncLOX5-1的高表达组和低表达组患者的预后有显著差异(图9)。在333例随访时间达到15年的前列腺癌患者人群，发现lncLOX5-1高表达是其预后较差的危险因素，风险率(Hazard rate，HR)等于3.736。提示lncLOX5-1高表达在前列腺癌预后判断方面具有重要价值。
实施例2 lncLOX5-1在前列腺癌恶性进展过程中的作用
(2.1)lncLOX5-1与前列腺癌细胞的增殖、迁移及侵袭性的相关性
既往并无lncLOX5-1与前列腺癌恶性进展相关的研究报道。
在本发明的前述内容中，发明人发现lncLOX5-1的表达量与肿瘤进展转移和恶性程度密切相关。而无限增殖以及侵袭迁移是肿瘤的重要特征，本发明对lncLOX5-1与前列腺癌细胞的增殖、侵袭迁移的相关性进行了研究。
本发明通过外源性过表达质粒和siRNA转染的方式，在前列腺癌细胞系LNCaP和PC3中过表达IncLOX5-1，通过CCK8实验检测了细胞增殖能力的变化(图10)，通过Transwell实验检测了肿瘤细胞侵袭和迁移能力的变化(图11)，发现在上调IncLOX5-1表达量后，细胞的增殖能力出现上升，细胞的侵袭和迁移能力也发生增强；在下调IncLOX5-1表达量后，细胞的增殖能力出现下降，细胞的侵袭和迁移能力也发生减弱。
(2.2)裸鼠荷瘤实验
向裸鼠皮下注射前列腺癌细胞PC3：待PC3细胞生长至对数增长期，即状态最佳时，每只裸鼠注射1×10 6数量的细胞，将细胞悬液与基质胶(BD公司)按照1∶1的体积比混合后注射至裸鼠大腿外侧皮下，便于进行瘤内注射与测量肿瘤情况。半周后，裸鼠皮下可触及质硬包块，将裸鼠按照体重和包块大小随机分为两组；每两天进行瘤内注射IncLOX5-1的siRNA 和对照siRNA-NC，每周一注射前测量肿瘤大小。6周后，肿瘤生长较为明显。
结果表明，干扰lncLOX5-1表达的前列腺癌细胞PC3-siRNA-lncLOX5-1的裸鼠，前列腺癌肿瘤体积明显降低(图12a-b)，干扰后，qPCR检测肿瘤中lncLOX5-1表达量显著下降(图12c)。
免疫组化(IHC)分析显示，在干扰lncLOX5-1的前列腺癌细胞PC3-siRNA-lncLOX5-1的肿瘤中，细胞增殖标记物Ki-67和PCNA表达量明显降低(图12d)。
以上研究均表明，lncLOX5-1的过表达可以促进前列腺癌细胞的增殖、侵袭和迁移能力。
综上所述，本发明提出的非编码RNA lncLOX5-1可有效作为前列腺癌早期诊断和预后判断的标志物；所述lncLOX5-1能够用于预测前列腺癌患者肿瘤的恶性程度，预测前列腺癌患者肿瘤是否进展转移，预测拟行前列腺穿刺的患者的阳性率等，具有高准确度、高特异性和高灵敏度的特点。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。
Figure PCTCN2018088809-appb-000005
Figure PCTCN2018088809-appb-000006

Claims (16)

  1. 一种长链非编码RNA lncLOX5-1，其特征在于，所述lncLOX5-1的核苷酸序列如SEQ ID NO.1所示。
  2. 一种分离的多核苷酸，其特征在于，所述多核苷酸能被细胞转录成如权利要求1所述的lncLOX5-1。
  3. 一种寡核苷酸引物，其特征在于，所述引物的序列为正向：
    TCCTCCTAAGCCGTATCCCATCTG，反向：CCAGGTGAGTTGAACAGTCCGATT，该引物能够用来扩增如权利要求1所述的lncLOX5-1。
  4. 一种诊断前列腺癌的检测lncLOX5-1的反转录PCR系统，其特征在于，该检测lncLOX5-1的系统包括如权利要求3所述的引物。
  5. 如权利要求1所述的长链非编码RNA lncLOX5-1的用途，其特征在于，所述lncLOX5-1的检测试剂用于制备前列腺癌早期诊断和预后判断的前列腺癌体外诊断产品。
  6. 如权利要求5所述的用途，其特征在于，所述前列腺癌体外诊断产品包括试剂盒、基因芯片、固体支持体。
  7. 一种lncRNA芯片，其特征在于，所述的lncRNA芯片包括：
    固相载体：以及有序固定在所述固相载体上的寡核苷酸探针，所述的寡核苷酸探针特异性地对应于如权利要求1所述的lncLOX5-1的部分或全部序列。
  8. 一种试剂盒，其特征在于，所述试剂盒中含有如权利要求1所述的lncLOX5-1的检测试剂。
  9. 如权利要求1所述的长链非编码RNA lncLOX5-1的检测试剂在预测前列腺癌患者肿瘤恶性程度中的应用。
  10. 如权利要求1所述的长链非编码RNA lncLOX5-1的检测试剂在预测前列腺癌患者肿瘤是否进展转移中的应用。
  11. 如权利要求1所述的长链非编码RNA lncLOX5-1的检测试剂在对拟行前列腺穿刺患者的阳性率预测中的应用。
  12. 一种基于lncLOX5-1构建的lncLOX5-1临床诊断模型Model-lncLOX5-1，其特征在于，所述模型的构建过程为，将年龄、PSA、％fPSA、前列腺体积、如权利要求1所述的lncLOX5-1表达量进行单因素logistic回归分析，确定能作为预测前列腺穿刺结果的独立危险因素；根据单因素logistic回归分析确定的独立危险因素，将其纳入多因素logistic回归分析中，构建所述基于lncLOX5-1构建的lncLOX5-1临床诊断模型Model-lncLOX5-1。
  13. 一种如权利要求12所述的基于lncLOX5-1构建的lncLOX5-1临床诊断模型Model-lncLOX5-1的应用，其特征在于，所述Model-lncLOX5-1用于预测前列腺穿刺的阳性率。
  14. 一种对前列腺癌进行早期诊断的方法，其特征在于，所述方法包括以下步骤：
    a)检测PSA异常患者行前列腺按摩后尿渣中lncLOX5-1的表达量；
    b)通过a)中测定的lncLOX5-1的表达量，采用受试者工作曲线(ROC)分析lncLOX5-1表达量的曲线下面积(AUC)，选择敏感性和特异性最大的点作为临界点，将患者分为高表达组和低表达组，若lncLOX5-1表达量小于临界点，提示其前列腺穿刺阳性概率较低；若lncLOX5-1表达量大于等于临界点，提示其前列腺穿刺阳性概率较高；
    或，通过a)中测定的lncLOX5-1的表达量，采用受试者工作曲线(ROC)分析lncLOX5-1表达量的曲线下面积(AUC)，选择敏感性和特异性最大的点作为临界点，联合患者年龄、PSA、％fPSA和前列腺体积具体数值，应用基于lncLOX5-1临床诊断模型绘制的列线图计算患者单个风险因素的风险评分，相加得到患者的累积风险评分，对应得出患者的穿刺阳性概率。
  15. 一种对前列腺癌进行预后判断的方法，其特征在于，所述方法包括以下步骤：
    a)检测前列腺癌患者病理样本中如权利要求1所述的lncLOX5-1的表达量；
    b)通过a)中测定的表达量，将前列腺癌患者分为低表达组和高表达组，若病理样本中lncLOX5-1表达量高于前列腺癌患者人群lncLOX5-1的均值，则为高表达组，预示肿瘤恶化、侵袭性较高，易发生局部进展或转移，生存预后水平较差；否则为低表达组。
  16. 一种如权利要求1所述的lncLOX5-1的抑制剂在抑制前列腺癌中的应用，其特征在于，所述抑制剂用于抑制前列腺癌细胞的增殖、侵袭和迁移。
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CN105154448A (zh) * 2015-09-16 2015-12-16 复旦大学 Rp11-1023l17.1作为前列腺癌分子靶标及其在诊断试剂盒中的应用
CN105316341A (zh) * 2015-12-08 2016-02-10 浙江理工大学 一种LncRNA及其在作为前列腺癌检测标记物或前列腺癌预后复发标记物中的应用
CN106148561A (zh) * 2016-09-28 2016-11-23 北京致成生物医学科技有限公司 前列腺癌的诊疗标记物
CN106967719A (zh) * 2017-06-01 2017-07-21 上海长海医院 一种长链非编码rna作为前列腺癌分子标志物的应用

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WO2021114137A1 (zh) * 2019-12-11 2021-06-17 清华大学 长非编码rna letn作为肿瘤标志物及治疗靶点
JP7406278B2 (ja) 2019-12-11 2023-12-27 清華大学 腫瘍マーカー及び治療ターゲットとしての長鎖ノンコーディングｒｎａ ｌｅｔｎ
CN113355411A (zh) * 2020-03-02 2021-09-07 中山大学孙逸仙纪念医院 一种基于lncRNA标记物的肿瘤免疫分型方法
CN113355411B (zh) * 2020-03-02 2022-05-10 中山大学孙逸仙纪念医院 一种基于lncRNA标记物的肿瘤免疫分型方法

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