CN106967719B - Application of a long non-coding RNA as a molecular marker for prostate cancer - Google Patents
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Abstract
本发明公开了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。本发明还公开了所述lncLOX5‑1在预测前列腺癌患者肿瘤恶性程度中的应用，在预测前列腺癌患者肿瘤是否进展转移中的应用，在预测拟行前列腺穿刺的患者的阳性率中的应用等。将lncLOX5‑1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。
The invention discloses a marker long-chain non-coding RNA lncLOX5-1 for early diagnosis and prognosis judgment of prostate cancer and its application. The invention also discloses the application of the lncLOX5-1 in predicting the malignant degree of the tumor in the prostate cancer patient, the application in predicting whether the tumor progresses and metastasis in the prostate cancer patient, the application in predicting the positive rate of the patient who is going to undergo prostate puncture, etc. . The use of lncLOX5‑1 for the early diagnosis and prognosis of prostate cancer has the characteristics of high accuracy, high specificity and high sensitivity.
Description
Technical Field
The invention belongs to the field of cancer molecular diagnosis, and particularly relates to application of long-chain non-coding RNA lncLOX5-1 as a marker for early diagnosis of prostate cancer and prognosis judgment of the prostate cancer, and also relates to a corresponding lncRNA chip and a diagnosis kit.
Background
Prostate Cancer (PCa) is a malignant tumor that seriously threatens the health of men, is the highest incidence male malignant tumor in developed countries, and accounts for the second place of tumor incidence and the sixth place of mortality in the world. The incidence of PCa in China has been in a remarkable rising trend in recent years. In the medically and economically developed cities of Beijing, Shanghai, Guangzhou and the like, the incidence rate of PCa is in the ten local common tumors. Epidemiological data show that the incidence of Chinese PCa has increased from 1.71/10 million men in 1993 to 7.9/10 million men in 2005, with an annual increase of about 13%. By calculation, in 2020, the PCa incidence rate in China is estimated to exceed 40/10 ten thousands of male population, which is close to the European and American national level and becomes a main tumor killer harmful to male health. Therefore, with the improvement of the general medical level in China and the step of China into the aging society, the prevention and the research work of the prostatic cancer are already in a reluctant stage. Due to the concealment of PCa symptoms, a majority of patients at the middle and advanced stages of home first visit are present, and over 46.3% of patients have developed local progression or distant metastasis. Prostate cancer patients not only have a poor prognosis once they develop metastases, but also have a severe impact on their quality of life. The data in SEER (Surveillance epidemic and End Results, SEER) database 2004-. Prostate Specific Antigen (PSA) is currently the most widely used molecular diagnostic marker for Prostate cancer, but its specificity is poor, resulting in a large number of unnecessary punctures and waste of medical resources. Therefore, the finding of a molecular diagnostic marker with high specificity to PCa and a related detection technology are urgently needed.
Disclosure of Invention
The invention provides a long-chain non-coding RNAlncRNA (lncLOX5-1), and the nucleotide sequence of the long-chain non-coding RNAlncRNA is shown in SEQ ID NO. 1.
The invention provides application of lncLOX5-1 as a molecular marker for early diagnosis and prognosis judgment of prostate cancer, and the accuracy and specificity of diagnosis of the prostate cancer are improved by detecting lncLOX 5-1.
In the invention, early diagnosis and prognosis judgment of the prostate cancer can be carried out by changing the expression level of the lncLOX 5-1.
The invention also provides an isolated polynucleotide (DNA corresponding to inclox 5-1) that can be transcribed by a cell (e.g., a human cell) into inclox 5-1 as described above, sequences that have 80%, 85%, 90%, 95%, and 99% homology to the polynucleotide, and that can also serve as a marker for prostate cancer.
In the invention, the sequences with 80%, 85%, 90%, 95% and 99% homology with the nucleotide sequence shown in SEQ ID NO.1 of the lncLOX5-1 can also be used as markers of prostate cancer.
The invention also provides an isolated polynucleotide that is transcribed by a human cell into lncLOX5-1 of claim 1.
The invention also provides application of a detection reagent of lncLOX5-1 in preparing a product for early diagnosis of prostate cancer, wherein the detection reagent of lncLOX5-1 can be but is not limited to a nucleic acid probe for specifically detecting lncLOX 5-1. Early diagnosis of prostate cancer is carried out by quantifying the expression level of the gene lncLOX 5-1.
The invention also provides application of a detection reagent of lncLOX5-1 in preparing a product for prognosis judgment of prostate cancer, wherein the detection reagent of lncLOX5-1 can be but is not limited to a nucleic acid probe for specifically detecting lncLOX 5-1. The expression level of the gene lncLOX5-1 is quantified to determine the prognosis of prostate cancer.
In the invention, the expression level of lncLOX5-1 can be determined by the following method: microarray technology, northern blot and quantitative PCR, tissue hybridization In Situ (ISH), etc.; the quantitative PCR is real-time quantitative PCR or multiplex PCR and the like.
The invention also provides an oligonucleotide primer, wherein the sequence of the primer is as follows:
TCCTCCTAAGCCGTATCCCATCTG (SEQ ID NO.2), reverse:
CCAGGTGAGTTGAACAGTCCGATT (SEQ ID NO.3), which can be used to amplify the lncLOX 5-1.
The invention also provides a reverse transcription PCR system for detecting the lncLOX5-1 for diagnosing the prostatic cancer, and the system for detecting the lncLOX5-1 comprises the primer.
The invention also provides an in vitro diagnosis product for the prostatic cancer, which comprises a reagent for specifically detecting the lncLOX 5-1. The prostate cancer in-vitro diagnosis product can be used for early diagnosis of prostate cancer and prognosis judgment of prostate cancer. The reagent for specifically detecting lncLOX5-1 can be, but is not limited to, a nucleic acid probe that specifically recognizes lncLOX 5-1.
Wherein, the prostate cancer in-vitro diagnosis product comprises a kit, a gene chip, a solid support and the like; the solid support includes an array, a microarray, and the like.
The invention also provides application of the detection reagent of lncLOX5-1 in predicting the malignancy degree of the tumor of a prostate cancer patient. The expression of lncLOX5-1 is correlated with the malignant progression of prostate cancer cells.
The invention also provides application of the detection reagent of lncLOX5-1 in predicting whether the tumor of a prostate cancer patient progresses and metastasizes.
The invention also provides application of the detection reagent of lncLOX5-1 in predicting the positive rate of a patient with proposed prostate puncture.
The invention also provides an lncLOX5-1 clinical diagnosis Model (Model-lncLOX5-1) constructed based on lncLOX5-1, wherein the construction process of the Model comprises the steps of carrying out single-factor logistic regression analysis on age, PSA,% fPSA, prostate volume, Digital Rectal Examination (DRE), lncLOX5-1 expression quantity and PCA3 expression quantity to determine independent risk factors capable of being used for predicting the prostate puncture result; constructing the lncLOX5-1 clinical diagnosis Model (Model-lncLOX5-1) constructed based on lncLOX5-1 and the PCA3 clinical diagnosis Model (Model-PCA3) constructed based on PCA3 by incorporating the independent risk factors of age, PSA,% fPSA, prostate volume, lncLOX5-1 expression level and PCA3 expression level determined by the one-factor logistic regression analysis into the multifactor logistic regression analysis; wherein the AUC of the lncLOX5-1 clinical diagnosis model reaches 0.909.
The invention also provides application of the lncLOX5-1 clinical diagnosis Model (Model-lncLOX5-1) in predicting the positive rate of prostate puncture.
The invention also provides a method for early diagnosis of prostate cancer, which comprises the following steps:
a) detecting the expression quantity of lncLOX5-1 in the urine sediment of a PSA abnormal patient after prostate massage;
b) analyzing the area under the curve (AUC) of the lncLOX5-1 expression quantity by using a Receiver Operating Curve (ROC) according to the lncLOX5-1 expression quantity measured in the step a), selecting a point with the highest sensitivity and specificity as a critical point, and dividing the patient into a high expression group and a low expression group, wherein if the lncLOX5-1 expression quantity is less than the critical point, the probability of positive prostate puncture is low; if the expression level of lncLOX5-1 is more than or equal to the critical point, the positive probability of prostate puncture is high;
or, by measuring the expression level of lncLOX5-1 in a), analyzing the area under the curve (AUC) of the expression level of lncLOX5-1 by using a Receiver Operating Curve (ROC), selecting the point with the highest sensitivity and specificity as a critical point, combining the specific values of age, PSA,% fPSA and prostate volume of the patient, calculating the risk score of the individual risk factors of the patient by using a nomogram drawn based on an lncLOX5-1 clinical diagnosis model, and adding to obtain the cumulative risk score of the patient, thereby correspondingly obtaining the puncture positive probability of the patient.
In a specific embodiment, the present invention provides a method for early diagnosis of prostate cancer, comprising the steps of:
a') detecting the expression level of lncLOX5-1 in the urine sediment of a PSA abnormal patient after prostate massage;
b ') dividing the patient into a high expression group and a low expression group by the expression level of lncLOX5-1 determined in a') and taking the expression level of lncLOX5-1 equal to 68 as a boundary, wherein if the expression level of lncLOX5-1 is less than 68, the probability of prostate puncture positivity is low; if the expression level of lncLOX5-1 is more than or equal to 68, the positive probability of prostate puncture is high;
or, calculating the risk score of a single risk factor of the patient by using a nomogram drawn based on a lonclinico 5-1 clinical diagnosis model by taking the expression quantity of lncLOX5-1 determined in a') and taking lncLOX5-1 expression quantity equal to 68 as a boundary, and adding to obtain the cumulative risk score of the patient, so as to correspondingly obtain the puncture positive probability of the patient.
Wherein, in the step a'), the expression amount of lncLOX5-1 is measured by the following method,
(1) urine RNA extraction:
urine RNA was extracted by TRIzol method. Add 200. mu.L of chloroform to each 1mL of dissolved EP tube, mix the mixture by inversion, place the mixture in an ice box for 5min, and centrifuge the mixture at 14000rpm for 15min at 4 ℃. The upper transparent water sample layer is provided, the middle white flocculent protein layer is provided, and the lower red liquid layer is a phenol-chloroform layer. Carefully pipette approximately 500. mu.L of the upper aqueous phase into a new EP tube. Adding 500 mu L of isopropanol into each EP tube, placing the tube in an ice box for 10min to precipitate RNA, centrifuging the tube for 10min at the temperature of 4 ℃ and 14000rpm to see transparent or white jelly-like precipitates at the tube bottom, carefully discarding the supernatant, adding 1mL of 75% ethanol for washing, centrifuging the tube for 5min at the temperature of 4 ℃ and 14000rpm again, carefully discarding the supernatant, fully drying the tube, and adding 10-20 mu L of non-enzyme water to dissolve the RNA. The quality of the extracted RNA is evaluated by using the Nanodrop 2000c detection concentration and 260/280 absorbance, the quality is repeatedly measured for 2 times, and the ratio of 260/280 absorbance should be between 1.80 and 2.00.
(2) Reverse transcription amplification:
reverse transcription amplification Using the complete Whole transcriptome amplification kit from Sigma Ltd
Complete wheel transfer Amplification Kit). On ice boxes, Library Synthesis Buffer, Library Synthesis Solution, Library Synthesis Enzyme, Amplification Mix, 10mM dNTP Mix, Amplification Enzyme and Enzyme-free water were thawed.
Setting program of PCR instrument:
WTA1：70℃，5min→18℃
WTA2：18℃10min→25℃10min→37℃30min→42℃10min→70℃20min→4℃
WTA 3: 94 ℃ 2min → (94 ℃ 30s → 70 ℃ 5min) × 17 cycles → 4 ℃.
Library synthesis: 0.5. mu.L of Library Synthesis Solution was added to 100ng of total RNA, and 3.32. mu.L of enzyme-free water was added. After being blown and evenly mixed, the mixture is incubated by a PCR instrument WTA1 program.
To the tube were added 0.5. mu.L of Library Synthesis Buffer, 0.4. mu.L of Library Synthesis Enzyme and 0.78. mu.L of Enzyme-free water. After pipetting and mixing, incubation was performed using the PCR machine WTA2 program.
Library amplification: mu.L of Enzyme-free water, 7.5. mu.L of Lamplification Mix, 1.5. mu.L of dNTP Mix and 0.75. mu.L of Lamplification Enzyme were added to the tube and incubated using the PCR machine WTA3 program. After the reaction was complete, the PCR product cDNA was stored at-20 ℃ for subsequent detection.
(3) Real-time quantitative PCR (qRT-PCR):
the detection of the expression level of the target gene is carried out by adopting an ABI StepOnePlus real-time quantitative fluorescent PCR instrument. Using TOYOBO THUNDER SYBR real-time qPCR kit, 10. mu.L SYBR Green Master Mix, 1. mu.L forward primer, 1. mu.L reverse primer, 0.4. mu.L 50 XRox Dye, 2. mu.L cDNA and 5.6. mu.L enzyme-free water were contained in 20. mu.L reaction per well. The reaction conditions were 95 ℃ for 10min, 95 ℃ for 15s and 60 ℃ for 60s for 40 consecutive cycles. Data were statistically analyzed and derived using StepOne Software version v2.1(Applied BioSystems, USA) Software. The expression quantity of lncLOX5-1 is calculated by the formula: lncLOX5-1 expression quantity 2Ct(PSA)-Ct(lncLOX5-1)The PCA3 expression quantity is calculated by the formula: PCA3 expression level 2Ct(PSA)-Ct(PCA3)PSA is used as an internal reference of urinary sediment RNA, and samples with the Ct value of PSA larger than 28 indicate that the RNA content is insufficient, so that the samples are rejected.
qRT-PCR reaction system
qRT-PCR amplification reaction conditions
qRT-PCR primer sequences
The invention also provides a method for prognosis of prostate cancer, which comprises the following steps:
a) detecting the expression level of lncLOX5-1 in a pathological sample of a prostate cancer patient;
b) dividing the prostate cancer patients into a low expression group and a high expression group through the expression quantity determined in the step a), wherein if the expression quantity of lncLOX5-1 in a pathological sample is higher than the mean value of lncLOX5-1 of the prostate cancer patients, the pathological sample is the high expression group, which indicates that the malignancy degree and the invasiveness of the tumor are higher, and the survival prognosis level is poorer; otherwise, it is a low expression group.
The lncLOX5-1 high expression group takes DAB color development in tissue in-situ hybridization as an example, the comprehensive staining intensity under a high-power microscope and the proportion of positive cells are semi-quantitatively determined, and the staining intensity score is standard: low expression-no coloration or light yellow, high expression-tan or dark tan.
The invention also provides application of an inhibitor of lncLOX5-1 in inhibiting prostate cancer, wherein the inhibitor is used for inhibiting proliferation, invasion and migration of prostate cancer cells.
The novel marker for diagnosing and predicting the prostate cancer, namely the long-chain non-coding RNAlncLOX5-1, can be used for early diagnosis of the prostate cancer and prognosis judgment of the prostate cancer, and has the characteristics of high accuracy, high specificity and high sensitivity; the prostate cancer in-vitro diagnosis product containing the detection reagent of the marker lncLOX5-1 provided by the invention is convenient to use and has the characteristics of high accuracy, high specificity and high sensitivity.
Drawings
FIG. 1 shows the expression level of lncLOX5-1 in the urine of patients with prostate cancer, benign prostatic hyperplasia, renal cancer and bladder cancer, and healthy persons; wherein, the expression level of lncLOX5-1 in the urine sediment of the prostate cancer patients is obviously higher than that of other people.
FIG. 2 shows the expression level of IncLOX 5-1 in the urine sediment of 243 patients with routine prostate puncture, wherein the expression level of IncLOX 5-1 in the urine sediment of patients with positive prostate puncture is significantly higher than that of patients with negative prostate puncture; wherein, A is the general population, B is the population with PSA 4-10ng/mL, C is the population with PSA4-20ng/mL, and D is the population with PSA 10-20 ng/mL.
FIG. 3 shows the prostate cancer detection rates for different lncLOX5-1 expression levels in a population of patients positive for prostate puncture; among them, the detection rate of prostate cancer is increased in the population with high lncLOX5-1 expression level.
FIG. 4 is a graph of the diagnostic efficacy of the subject's working curve in analyzing the PSA, lnCLOX5-1, PCA3, fPSAratio, etc. indicators; wherein, A is the general population, B is the population with PSA 4-10ng/mL, and C is the population with PSA4-20 ng/mL.
FIG. 5 shows the diagnostic efficacy of the basic Model (Base Model) constructed by analyzing the working curve of the subject based on the clinical diagnostic index, the lncLOX5-1 clinical diagnostic Model (Model-lncLOX5-1) constructed based on lncLOX5-1, the PCA3 clinical diagnostic Model (Model-PCA3) constructed based on the PCA3 score, and the like: population at population A, population at PSA 4-10ng/mL (B) and population at PSA4-20ng/mL (C).
FIG. 6 is a nomogram and internal verification (calibration curve test of nomogram) drawn based on a lonclone 5-1 clinical diagnosis Model (Model-lonclone 5-1) constructed based on lonclone 5-1: alignment plots drawn using diagnostic models constructed of age, PSA,% fPSA, prostate volume, lncLOX 5-1; and (4) checking a correction curve of the B alignment chart.
FIG. 7 shows that the expression level of lncLOX5-1 in the urine sediment of prostate cancer patients is correlated with the tumor progression metastasis and malignancy degree; wherein A is the expression level of lncLOX5-1 in the urine residues of patients with localized prostate cancer and local progression/metastatic prostate cancer; b different Gleason scores urine sediment lncLOX5-1 expression of prostate cancer patients.
FIG. 8 shows the expression levels of IncLOX 5-1 in tumor tissues of patients with localized and metastatic prostate cancer.
FIG. 9 is a graph of the overall survival curves for a population of patients with high-and low-expression lncLOX5-1 prostate cancer in the TCGA database.
FIG. 10 shows the change in the proliferation potency of tumor cells following exogenous alteration of the expression level of lncLOX 5-1: after the lncap (a) and PC3(B) cells interfere with the expression level of lncllox 5-1, the proliferation capacity of tumor cells is weakened; after the lncap (C) and PC3(D) cells over-express the lncLOX5-1 expression level, the proliferation capacity of the tumor cells is enhanced; fig. 10A and 10B are the same in illustration.
FIG. 11 shows the change in the migration and invasion capacity of tumor cells following exogenous alteration of the expression level of IncLOX 5-1: the cell migration and invasion capacity of the tumor cells with the level of lncLOX5-1 overexpressed in PC3(A) cells is enhanced; lncap (b) cells have reduced ability to interfere with lncllox 5-1 level tumor cell migration and invasion.
FIG. 12 is an in vivo experiment demonstrating the effect of lncLOX5-1 in prostate cancer; wherein, A-B is a prostate cancer cell PC3 subcutaneous tumor-bearing nude mouse model interfering with the expression of lncLOX5-1, and the tumor volume of the prostate cancer is obviously reduced compared with that of a normal control group; c is that the expression level of lncLOX5-1 in prostate cancer PC3 plantarfoma interfering with lncLOX5-1 expression is obviously reduced compared with that in a control group; d is Immunohistochemistry (IHC) and shows that the expression levels of cell proliferation markers Ki-67 and PCNA are obviously reduced in the tumor of prostate cancer cell PC3 interfering with lncLOX 5-1.
(the asterisks are the same throughout, i.e., P < 0.05;. P < 0.01;. P < 0.001).
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1 expression of lncLOX5-1 in urine of patients with prostate cancer, benign prostatic hyperplasia, renal cancer and bladder cancer, and normal humans
In the following examples, the prostate cancer in vitro diagnostic reagent described above, such as a kit, a gene chip, etc., can be used to detect the expression level of lncLOX 5-1.
(1.1) significant differences in the expression level of lncLOX5-1 in urine of patients with prostate cancer, benign prostatic hyperplasia, renal cancer, bladder cancer and normal humans
According to the invention, the expression level of lncLOX5-1 in the urine sediment of prostate cancer patients is obviously higher than the expression level of lncLOX5-1 in the urine sediment of benign prostatic hyperplasia patients, renal cancer patients, bladder cancer patients and healthy people (figure 1). And the expression level of lncLOX5-1 in the urine sediment of the prostate cancer patients and benign prostatic hyperplasia patients is higher than that of other people, and lncLOX5-1 can be used as a prostate specific index.
(1.2) the expression level of lncLOX5-1 in the urine sediment of the patient with positive prostate puncture is obviously higher than that of the patient with negative prostate puncture
As described above, the expression level of lncLOX5-1 in the urinary residue of prostate cancer patients is significantly higher than that of lncLOX5-1 in the urinary residue of other people. The expression level of lncLOX5-1 in the urine sediment of patients after prostate massage was determined by inclusion of 243 prostate puncture patients, including 146 puncture-negative patients and 97 puncture-positive patients. The expression level of lncLOX5-1 in the urine sediment of the puncture-positive patients was found to be significantly higher than the expression level of lncLOX5-1 in the urine sediment of the puncture-negative patients (FIG. 2A). Significant increases in lncLOX5-1 expression were found in the urine sediment of puncture-positive patients in the general population, the PSA 4-10ng/mL population, the PSA4-20ng/mL population, and the PSA 10-20ng/mL population (FIG. 2).
(1.3) the detection rate of prostatic cancer is increased in the population with high lncLOX5-1 expression level
According to the invention, the higher the expression level of lncLOX5-1 is, the higher the detection rate of prostatic cancer in the corresponding population is found by detecting the expression level of lncLOX5-1 in the urine residue of 97 patients with positive prostate puncture. Sorting lnCLOX5-1 from small to large, wherein 0-25% of the population is 25% of the population with the lowest expression level of lnCLOX5-1, and 75-100% of the population is 25% of the population with the highest expression level of lnCLOX5-1, and the detection rate of the prostate cancer of the population with the lowest expression level of lnCLOX5-1 (0-25%) is lower than that of the population with higher expression level (75-100%); the same trend was found in the general population, the PSA <10ng/mL population, the PSA 10-20ng/mL population and the PSA >20ng/mL population (FIG. 3).
(1.4) urinary residue lncLOX5-1 score as an auxiliary diagnostic index for prostate cancer
FIG. 4 is a test subject working curve analysis of the diagnostic efficacy of each index. The diagnostic efficacy of the urine sediment inclox 5-1 was analyzed using the Receiver Operating Curve (ROC), and it was found that the area under the curve (AUC) of the urine sediment inclox 5-1 reached 0.782 and the AUC of the PSA reached 0.798, and the difference between the two was not statistically significant (P ═ 0.696) (fig. 4A). While the PCA3 scored AUC0.662, significantly lower than lncLOX5-1 and PSA (P)lncLOX5‐1<0.001,PPSA0.003). The diagnostic efficacy of the lncLOX5-1 score was better than the PCA3 score (0.709vs 0.601, P ═ 0.046) in the patient population with PSA 4-10ng/mL diagnostic gray zone (fig. 4B). The diagnostic efficacy of the lncLOX5-1 score was also significantly better than the PCA3 score (0.719vs 0.600, P) in a patient population with PSA4-20ng/mL<0.001) (fig. 4C).
(1.5) establishing a diagnostic model based on the urinary sediment lncLOX5-1
According to the independent risk factors determined by the single-factor logistic regression analysis, the age, the prostate volume, the PSA and the percent fPSA are included in the multi-factor logistic regression analysis, and a diagnosis prediction model for predicting the prostate puncture biopsy result is constructed. Firstly, a basic diagnosis Model (Base Model) constructed by clinical diagnosis indexes is independently applied. The AUC of a lonCLOX 5-1 clinical diagnosis Model (Model-lonCLOX 5-1) constructed based on lonCLOX 5-1 reaches 0.909, and is remarkably superior to a basic diagnosis Model (P ═ 0.009) constructed based on clinical diagnosis indexes. In contrast, the clinical diagnosis Model (Model-PCA3) constructed based on the PCA3 score has no significant statistical significance in the differences of AUC and PA compared with the basic diagnosis Model (FIG. 5).
(1.6) plotting alignment chart based on urine sediment IncLOX 5-1 diagnostic prediction model
Based on a lncLOX5-1 clinical diagnosis Model (Model-lncLOX5-1) constructed by urinary residue lncLOX5-1, the invention draws a nomogram, assigns each independent risk factor, gives corresponding scores to each clinical index and the numerical value of lncLOX5-1 score, and can quickly calculate the probability of positive prostate puncture prediction of the Model (figure 6A) by adding the scores of the indexes. Then, the invention carries out calibration curve test on the nomogram, the abscissa is the nomogram prediction probability condition of the prediction model constructed by the invention, the ordinate is the real occurrence probability, and an inclined 45-degree curve represents an ideal curve, which indicates that the nomogram prediction probability is completely consistent with the real probability; the bias correction curve of the present invention was closely fitted to the ideal curve (fig. 6B). The nomogram can be simply and effectively applied to clinic to predict the puncture positive probability of a patient with a proposed prostate puncture.
(1.7) high expression of lncLOX5-1 was associated with tumor progression metastasis and malignancy in prostate cancer patients
According to the invention, the expression levels of urine IncLOX 5-1 in prostate cancer patients with different grades are compared, and the expression level of IncLOX 5-1 based on urine residue detection is found to have significant difference in the expression of patients with localized prostate cancer and locally advanced/metastatic prostate cancer, and significant difference in the expression of prostate cancer populations with Gleason Scores (GS) of less than 7 and not less than 7 (figure 7). As the tumor progresses and the degree of pathological malignancy is increased, the expression level of lncLOX5-1 is increased, and the suggestion that the lncLOX5-1 score can be used for accurately diagnosing high-grade prostate cancer.
(1.8) lncLOX5-1 expression level of tumor tissue of patients with localized and metastatic prostate cancer
The invention detects the expression level of lncLOX5-1 in tumor tissue samples of 41 prostate cancer patients, and finds that the expression level of lncLOX5-1 in metastatic prostate cancer patients is obviously higher than that in tumor tissue samples of localized prostate cancer patients (P <0.01) (figure 8).
(1.9) high expression of lncLOX5-1 correlates with overall survival of prostate cancer patients
According to the invention, the expression level of lncLOX5-1 in the tumor tissue of prostate cancer patients in a public database (TCGA) is analyzed by a bioinformatics method, and the significant difference of the prognosis of the high-expression group and the low-expression group of lncLOX5-1 patients is found (FIG. 9). In a population of 333 prostate cancer patients with a follow-up time of up to 15 years, it was found that high expression of lncLOX5-1 is a risk factor for a poorer prognosis, and the risk ratio (Hazardrate, HR) was equal to 3.736. The high expression of lncLOX5-1 is suggested to have important value in the aspect of prognosis judgment of prostate cancer.
Example 2 role of lncLOX5-1 in the progression of prostate cancer malignancy
(2.1) correlation of lncLOX5-1 with proliferation, migration, and invasiveness of prostate cancer cells
No study on the correlation between lncLOX5-1 and the malignant progression of the prostate cancer has been reported.
In the foregoing context of the present invention, the inventors found that the expression level of lncLOX5-1 is closely related to the degree of metastasis and malignancy of tumor progression. While unlimited proliferation and invasion and migration are important characteristics of the tumor, the invention researches the correlation between lncLOX5-1 and the proliferation and invasion and migration of prostate cancer cells.
According to the invention, by means of transfection of exogenous overexpression plasmids and siRNA, lncLOX5-1 is overexpressed in prostate cancer cell lines LNCaP and PC3, the change of cell proliferation capacity is detected by a CCK8 experiment (figure 10), the change of tumor cell invasion and migration capacity is detected by a Transwell experiment (figure 11), and the increase of cell proliferation capacity and the enhancement of cell invasion and migration capacity are found after the lncLOX5-1 expression amount is up-regulated; after the lncLOX5-1 expression is down-regulated, the proliferation capacity of the cells is reduced, and the invasion and migration capacity of the cells is weakened.
(2.2) tumor-bearing experiment in nude mice
Nude mice were injected subcutaneously with prostate cancer cells PC 3: when PC3 cells grow to logarithmic growth stage, i.e. optimum state, 1X 10 cells per nude mouse are injected6Number of cells, cell suspension and matrigel (BD company) were mixed according to 1: 1 is injected to the subcutaneous side of the thigh of a nude mouse after being mixed, so as to be convenient for intratumoral injection and measurement of tumor conditions. After half a week, the nude mice can touch the hard coating blocks subcutaneously, and the nude mice are randomly divided into two groups according to the weight and the size of the coating blocks; intratumoral injections of lncLOX5-1 siRNA and control siRNA-NC were performed every two days, and tumor sizes were measured before each Monday injection. After 6 weeks, tumor growth was evident.
The results show that the tumor volume of the prostate cancer of nude mice of the prostate cancer cell PC3-siRNA-lncLOX5-1 interfering with the expression of lncLOX5-1 is obviously reduced (figure 12A-B), and the expression level of lncLOX5-1 in the tumor is obviously reduced by qPCR detection after the interference (figure 12C).
Immunohistochemistry (IHC) analysis showed that the expression levels of cell proliferation markers Ki-67 and PCNA were significantly reduced in tumors interfering with the prostate cancer cell PC3-siRNA-lncLOX5-1 of lncLOX5-1 (FIG. 12D).
The above studies all show that the over-expression of lncLOX5-1 can promote the proliferation, invasion and migration ability of prostate cancer cells.
In conclusion, the non-coding RNAlncLOX5-1 provided by the invention can be effectively used as a marker for early diagnosis and prognosis judgment of the prostate cancer; the lncLOX5-1 can be used for predicting the malignancy of the tumor of the prostate cancer patient, predicting whether the tumor of the prostate cancer patient is advanced and metastasized, predicting the positive rate of the patient to be subjected to prostate puncture and the like, and has the characteristics of high accuracy, high specificity and high sensitivity.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
<110> Shanghai Changhai Hospital
<120> application of long-chain non-coding RNA as prostate cancer molecular marker
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Claims (12)
1. A long non-coding RNA, lncLOX5-1, wherein the nucleotide sequence of lncLOX5-1 is shown in SEQ ID NO. 1.
2. An isolated polynucleotide capable of being transcribed by a cell into lncLOX5-1 of claim 1.
3. The use of the long non-coding RNA, lncLOX5-1, according to claim 1, wherein lncLOX5-1 is used as a marker for the preparation of an in vitro prostate cancer diagnosis product for early diagnosis and prognosis of prostate cancer.
4. The use according to claim 3, wherein the prostate cancer in vitro diagnostic product is a kit.
5. The use of claim 3, wherein the prostate cancer in vitro diagnostic product is a gene chip.
6. The use of claim 3, wherein the prostate cancer in vitro diagnostic product is a solid support.
7. The lncRNA chip is characterized by comprising:
solid phase carrier: and oligonucleotide probes immobilized on the solid support in an ordered manner, the oligonucleotide probes specifically corresponding to part or all of the sequence of lncLOX5-1 according to claim 1.
8. A kit comprising a detection reagent specific to lncLOX5-1 according to claim 1.
9. The use of the long non-coding RNA of claim 1, lncLOX5-1, as a marker in the manufacture of a product for predicting the malignancy of a tumor in a patient with prostate cancer.
10. The use of the long non-coding RNA of claim 1, lncLOX5-1, as a marker in the manufacture of a product for predicting the progression or metastasis of a tumor in a prostate cancer patient.
11. Use of the long non-coding RNA, lncLOX5-1, as a marker in the manufacture of a product for positive rate prediction in patients with prostate puncture.
12. A lncLOX5-1 clinical diagnosis Model-lncLOX5-1 constructed based on lncLOX5-1, wherein the Model is constructed by performing one-way logistic regression analysis on age, PSA,% fPSA, prostate volume, and lncLOX5-1 expression level according to claim 1 to determine independent risk factors capable of being used as a prediction of prostate puncture results; and (3) constructing the lncLOX5-1 clinical diagnosis Model-lncLOX5-1 constructed based on lncLOX5-1 by taking the independent risk factors determined by the one-factor logistic regression analysis into the multi-factor logistic regression analysis.
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| KR20220113999A (en) * | 2019-12-11 | 2022-08-17 | 칭화대학교 | Long non-coding RNA LETN as tumor marker and therapeutic target |
| CN113355411B (en) * | 2020-03-02 | 2022-05-10 | 中山大学孙逸仙纪念医院 | Tumor immunotyping method based on lncRNA marker |
| CN114292918A (en) * | 2021-12-31 | 2022-04-08 | 刘飞 | Early diagnosis method for prostate cancer based on LncRNA546 marker |
| NL2036754B1 (en) * | 2024-01-05 | 2025-07-22 | Cancer Hospital Cams | Application of biomarker combination in diagnosing prostate cancer, and kit for diagnosing prostate cancer |
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