CN111944904B - Application of PTTG3P in preparation of pancreatic cancer diagnostic reagent or kit - Google Patents
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Abstract
The invention relates to the technical field of medical biological detection, in particular to application of PTTG3P in preparation of a pancreatic cancer diagnostic reagent or kit. The invention also provides a kit for diagnosing pancreatic cancer by utilizing the quantitative PCR detection of PTTG3P, and application of PTTG3P in preparing a medicament for treating pancreatic cancer. The kit and the detection method are simple, convenient, reliable, short in period, high in specificity and easy to popularize clinically.
Description
Technical Field
The invention relates to the technical field of medical biological detection, in particular to application of PTTG3P in lncRNA in preparation of a pancreatic cancer diagnostic reagent or kit.
Background
Pancreatic cancer is one of the major disease burdens in our country. The current common treatment means for pancreatic cancer include surgery, radiotherapy, chemotherapy, targeted therapy and the like, but the treatment of advanced pancreatic cancer by the above treatment methods is still limited.
The basic discovery of molecular biology in the process of researching pancreatic cancer occurrence and development shows that corresponding molecular markers for diagnosis and treatment have important clinical significance. The long-chain non-coding RNA is one of non-coding RNAs and is defined as non-coding RNA with the length of more than 200 bp. lncRNA has not been regarded as important by researchers in the past, and is considered to be "noise" in the transcription process. In recent years, there is growing research evidence that lncRNA plays an important role in a variety of diseases including tumors, where significance is increasingly recognized.
Studies have demonstrated that LncRNA can regulate cellular function through multiple mechanisms. lncRNA can directly influence the expression of downstream genes as cis-acting elements, or influence the expression process of target genes by forming RNA-RNA and RNA-DNA complexes, or degrade mRNA or protein through RNA interference. Through the mechanism, the lncRNA influences the expression and protein activity of genes, thereby changing the biological state of cells and influencing the generation and development of tumors.
Compared with normal cells, the lncRNA expression spectrum in the tumor cells has obvious difference, and participates in various malignant biological behaviors such as tumor cell proliferation, invasion, metastasis, drug resistance and the like. In recent years, LncRNA is proved to have high species and organ specificity, which suggests the potential of LncRNA as a tumor diagnosis marker (such as DD3 in prostatic cancer) and is expected to become a new tumor treatment target. However, the study of lncRNA as a diagnostic marker is still in its primary stage relative to miRNA.
Many secreted and circulating lncRNA were found to be significantly elevated in the expression levels in body fluids (e.g., urine, plasma) of tumor patients. The study found that HOTAIR was significantly highly expressed in the plasma of colorectal cancer patients, and that the expression level of HOTAIR was highly consistent in plasma samples and tumor tissues. Similar conclusions have been drawn in patients with esophageal cancer: HOTAIRs were significantly elevated in the plasma of tumor patients compared to healthy people, but expression levels were significantly down-regulated after surgical treatment. The above findings suggest that plasma HOTAIR can reflect its expression level in tumors, which is indicative of tumor development. After researchers detect the PCA3 level in the urine of a prostate cancer patient, the PCA3 has good diagnosis potential of the prostate cancer, and the diagnosis efficiency is obviously superior to that of the traditional PSA index. A study involving 57 patients showed that HULC is highly expressed in the blood of liver cancer patients and has significant organ and tumor specificity.
PTTG3P (Pituitary tumor-transforming 3, pseudogene) was located in genome GRCh38.p13 with GENE ID 26255 and was verified to be long-chain RNA without protein-encoding ability. There are currently very few studies on PTTG 3P.
Therefore, the research on the marker of lncRNA in early pancreatic cancer is of great significance for further understanding the biological function of lncRNA in early pancreatic cancer lesions. At present, no literature report that PTTG3P is applied to pancreatic cancer diagnosis exists.
Disclosure of Invention
The invention aims to provide a new application of PTTG3P in LncRNA, namely an application in preparing a pancreatic cancer diagnostic kit.
In a first aspect of the present invention, there is provided the use of PTTG3P as a diagnostic marker for pancreatic cancer.
In a second aspect of the invention, the application of PTTG3P in the preparation of a pancreatic cancer diagnostic reagent or kit is provided.
Furthermore, the diagnostic reagent or the kit is a reagent or a kit for detecting the content of PTTG3P in the biological sample.
Further, the diagnostic kit comprises a reagent for detecting the amount of PTTG3P in the biological sample.
Further, the reagent for detecting the content of PTTG3P in the biological sample is selected from the group consisting of: PCR primers with detection specificity for PTTG 3P.
Further, the nucleotide sequences of the PCR primers with detection specificity to PTTG3P are shown as SEQ ID NO.1 and SEQ ID NO. 2;
an upstream primer: AATCTGGTTGAGAGCGGCAA (SEQ ID NO. 1);
a downstream primer: CAGCCCATCCTTTGTAGCCA (SEQ ID NO. 2).
Further, the biological sample is selected from the group consisting of: peripheral blood obtained from a subject.
Further, the invention also provides application of the reagent for detecting PTTG3P in preparation of a pancreatic cancer diagnosis kit.
In a third aspect of the present invention, there is provided a pancreatic cancer diagnostic kit comprising a reagent for detecting the amount of PTTG3P in a biological sample.
Further, the pancreatic cancer diagnostic kit consists of a reverse transcription system, a primer system and an amplification system, wherein the primer system comprises PCR primers shown as SEQ ID NO.1 and SEQ ID NO. 2.
In a fourth aspect of the present invention, there is provided a method for pancreatic cancer detection using the diagnostic kit as described above, comprising the steps of:
A. centrifuging a blood sample to be detected at normal temperature to obtain plasma, extracting total RNA in the plasma and determining the purity of the plasma;
B. carrying out reverse transcription on the total RNA in the step A to obtain cDNA;
C. the copy number of PTTG3P is quantitatively detected by adopting a real-time quantitative PCR technology, and primers used in the detection process are shown as SEQ ID NO.1 and SEQ ID NO. 2.
Furthermore, data during detection are processed by SPSS16.0 and expressed in a mean value plus or minus a standard deviation manner.
By bioinformatic analysis, PTTG3P was found to be significantly highly expressed in pancreatic cancer. Experiments prove that the over-expression of PTTG3P can obviously improve the proliferation and invasion and metastasis capacities of pancreatic cancer cells, and the inhibition of the in-vivo expression of PTTG3P can inhibit the malignant phenotype of tumor cells, thereby achieving the effect of inhibiting pancreatic cancer. The results indicate that PTTG3P has high specificity and sensitivity in pancreatic cancer cells, has a clear cancer-promoting biological function, and can be applied to pancreatic cancer diagnosis or pancreatic cancer treatment.
In a fifth aspect of the invention, there is provided the use of PTTG3P in the manufacture of a medicament for the treatment of pancreatic cancer.
Further, the drug is an agent for inhibiting or silencing expression of PTTG 3P.
Furthermore, the reagent for inhibiting or silencing the expression of PTTG3P is specifically interfering RNA of PTTG 3P. In a preferred embodiment of the invention, the sequence of the interfering RNA of PTTG3P is shown as SEQ ID NO.5 or SEQ ID NO. 6;
siRNA#1，CTCAAGTTTCAATATCATGTTTT(SEQ ID NO.5)；
siRNA#2，ATCATGTTTTGGCAAAACATTCG(SEQ ID NO.6)。
the invention has the advantages that:
1. the applicant obtains a large number of peripheral blood samples of pancreatic cancer patients in Shanghai Changhai hospitals, and provides a powerful guarantee for the research of the invention.
2. In terms of technology, the detection of PTTG3P is essentially a quantitative PCR detection of blood genome, has the characteristics of simple operation, sensitive detection, good specificity, high repeatability and the like, and is increasingly applied to clinical examination technology nowadays. The basic detection method adopted by the people is quantitative PCR, the technology is proved to be a high-sensitivity and high-accuracy detection method in modern experimental diagnostics, the experimental technology is mature, and the standard curve quantitative method in the technology is adopted by the people, so that the characteristic nucleic acid molecules in various samples can be accurately quantified.
3. The diagnostic marker PTTG3P related by the invention is obviously up-regulated in cancer tissues, the difference has statistical significance (P <0.05), the clinical reference value and the reliability are higher, the detection rate and the accuracy of familial pancreatic cancer can be improved, the diagnostic marker has great significance for the clinical treatment of familial pancreatic cancer, the detection result can be obtained only by collecting blood of a detector, and the trouble that a patient needs to prepare in advance according to the requirement of detection equipment and the risk of radiation hazard of an instrument when an imaging means is adopted for detection is avoided.
4. The invention provides a pancreatic cancer detection kit based on quantitative PCR, which can diagnose pancreatic cancer by detecting the expression abundance of PTTG3P in the genomic RNA of peripheral blood of a patient. The detection method is particularly simple and convenient, has short period and high sensitivity, and is an effective supplement of the existing detection reagent.
Drawings
FIG. 1 is a graph showing the results of the expression of PTTG3P gene in pancreatic cancer and non-pancreatic cancer tissues detected by qPCR in the present invention;
FIG. 2 is a graph showing the results of the expression of PTTG3P gene in normal pancreatic ductal epithelial cells and pancreatic cancer cells measured by qPCR in the present invention;
FIG. 3 is a graph showing the results of the transfection efficiency of PTTG3P gene siRNA in pancreatic cancer cells detected by qPCR in the present invention;
FIG. 4 is a graph showing the effect of PTTG3P gene on pancreatic cancer cell proliferation measured by the CCK8 method according to the present invention;
FIG. 5 is a graph showing the effect of the present invention in detecting the ability of amplifying or knocking down PTTG3P on the tumorigenicity in pancreatic cancer cell animals.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions such as Sambrook et al molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), either according to conventional conditions or according to the manufacturer's recommendations. Percentages and parts are by volume unless otherwise indicated.
Example 1: detection of expression level of PTTG3P in pancreatic and non-cancerous tissues
1. Sample collection
60 pairs of pancreatic cancer and para-cancer tissue samples were collected and placed in a-80 ℃ refrigerator. All cases received no chemotherapy prior to surgery and all patients signed an informed consent, and the protocol was approved by the ethics committee of the unit.
2. Total RNA extraction
1) Clinical specimens were removed from the-80 ℃ freezer and placed on ice. Weighing 25mg of tissue, placing the tissue in a 2ml centrifuge tube, placing the centrifuge tube on ice, adding 1ml of Trizol into the centrifuge tube, processing the tissue by using a handheld automatic tissue homogenizer until no solid component exists, and standing the tissue for 10min at room temperature;
2) centrifuging at 13000rpm for 10min at 4 ℃;
3) the supernatant is sucked by a pipette into a clean 1.5mL centrifuge tube of RNase-Free;
4) adding 0.2ml of trichloromethane, shaking by a vortex mixer, and standing at room temperature for 10 min;
5) centrifuging at 4 deg.C and 3000rpm for 10 min;
6) taking the aqueous phase by using a pipettor and transferring the aqueous phase into a 1.5ml centrifuge tube of 1.5ml RNase-Free;
7) adding isopropanol with equal volume, reversing, mixing, and standing at-40 deg.C for 30 min;
8) centrifuging at 4 deg.C and 13000rpm for 10min, removing supernatant, adding 75% ethanol 0.5ml, and mixing;
9) centrifuging at 4 deg.C and 13000rpm for 10min, and removing supernatant;
10) the residual liquid was removed by low-speed centrifugation and aspiration, and 20. mu.l RNase-Free water was added to dissolve the precipitate sufficiently.
3. Total RNA quantitation and purity analysis
Determination of optical Density values at 260nm and 280nm, as OD, of RNA Using UV Spectrophotometer260/OD280The purity of RNA was considered reliable in the range of 1.8-2.0.
4. RT-qPCR method is adopted to verify the expression difference of PTTG3P in pancreatic cancer and paracarcinoma tissues
4.1 reverse transcription reaction
The reaction system is shown in table 1:
TABLE 1 reverse transcription reaction System
The reaction conditions are as follows: 15min at 37 ℃; 5s at 85 ℃; the reaction was terminated at 4 ℃. The product cDNA was subjected to the next step or stored at-20 ℃.
4.2 Synthesis and design of primers
The sequences of the primers used during the assay of PTTG3P are shown in table 2:
TABLE 2 summary of primer sequences for PTTG3P
4.3 qPCR reaction
The reaction system is shown in table 3:
TABLE 3 RT-PCR reaction System
The reaction conditions are as follows: at 95 ℃ for 30 s; 95 ℃ for 5 s; 30s at 60 ℃; 72 ℃ for 30 s; for a total of 45 cycles.
4.4 statistical analysis
All experiments were repeated three times and data were processed using SPSS16.0 and expressed as mean ± standard deviation. Paired comparison of cancer with paracarcinoma tissues using t-test, statistical differences were considered when p < 0.05.
4.5 results
As a result, as shown in fig. 1, the expression of PTTG3P was significantly up-regulated in cancer tissues compared to tissues adjacent to pancreatic cancer, and the difference was statistically significant (P < 0.05).
Example 2: expression of PTTG3P in pancreatic cancer cells
1. Cell culture
Human immortalized pancreatic ductal epithelial cells HPNE, human pancreatic cancer cell lines AsPC-1, BXPC-3, CaPAN-2, MiaPANC-2, PANC-1 and SW1990 all using 10% fetal bovine serum in DMEM at 37 ℃ with 5% CO2Cultured in an incubator.
2. RNA extraction
The culture medium is discarded, washed for 2 times by PBS, added with a proper amount of Trizol, and repeatedly blown by a pipette to accelerate cell lysis. The rest of the procedure was the same as in example 1.
3、RT-qPCR
The specific procedure was the same as in example 1.
4. Results
As shown in FIG. 2, the expression levels of PTTG3P were significantly up-regulated in the pancreatic cancer cell lines AsPC-1, BXPC-3, CaPAN-2, MiaPANC-2, PANC-1 and SW1990 compared to the pancreatic mucosal epithelial cells, with statistical differences (all P < 0.05).
Example 3: expression inhibition of PTTG3P
1. Cell culture
The specific procedure is the same as in example 2.
2. Design of siRNA
Two sirnas for PTTG3P were designed using an online siRNA design tool:
siRNA#1，CTCAAGTTTCAATATCATGTTTT(SEQ ID NO.5)；
siRNA#2，ATCATGTTTTGGCAAAACATTCG(SEQ ID NO.6)。
3. transfection
Pancreatic cancer cells ASPC-1 were divided into three groups, blank control group (transfection nonsense siRNA), siRNA group#1(transfection of siRNA#1) And siRNA group#2(transfection of siRNA#2)。
siRNA concentration was 50nM and the solution was changed 6 hours after transfection.
4. Total RNA extraction and expression detection and analysis of PTTG3P
After 48 hours, a cell sample was collected, followed by the same procedures as in example 2.
5. Results
As a result, as shown in fig. 3, PTTG3P was significantly down-regulated in the group transfected with specific siRNA compared to the control group, and the difference was statistically significant (P < 0.05).
Example 4: the capacity of reducing the expression of PTTG3P to obviously inhibit the proliferation of pancreatic cancer cells
The effect of PTTG3P on the proliferative capacity of pancreatic cancer cells was examined by CCK-8(Dojindo, cat # CK04-11) as follows:
1. the cell culture and transfection procedures were the same as in example 3.
2. After each group of cells are subjected to trypsinization and heavy suspension, the cell concentration is adjusted after counting, pancreatic cancer cells AsPC-1 are inoculated into a 96-well plate according to the total amount of 3000 cells per well, and 5 auxiliary wells are established for each group of cells.
3. After the cells reached the corresponding time points (1d, 2d, 3d, 4d, 5d), the cells were treated according to the instructions and the absorbance was measured at a wavelength of 450nm using a microplate reader.
4. Statistical analysis
The same as in example 3.
5. Results
As a result, as shown in fig. 4, the proliferation ability of pancreatic cancer cells in the siRNA group was significantly inhibited compared to the control group.
Example 5:
detecting the influence of the amplification or the knockdown of PTTG3P on the tumorigenicity capacity of the pancreatic cancer cell animal, and the steps are as follows:
1. cell culture and transfection procedures in example 3, PTTG3P was expanded in the PANC-1 cell line and PTTG3P was knocked down in the AspC-1 cell line.
2. The cell concentration of each group of cells was adjusted after trypsinization, resuspension and counting, pancreatic cancer cells (1X 10)6) Dissolved in 0.1ml Hank's balanced salt solution injected subcutaneously into nude mice.
3. Tumor volumes were measured weekly and all nude mice were sacrificed 4 weeks after inoculation.
4. Statistical analysis
The same as in example 3.
5. Results
The results are shown in fig. 5, and by comparison of the controls of each group, PTTG3P had a significant effect of promoting tumor proliferation in the pancreatic cancer cell line nude mouse subcutaneous tumorigenicity experiment.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Sequence listing
<110> Shanghai Changhai Hospital
Application of <120> PTTG3P in preparation of pancreatic cancer diagnostic reagent or kit
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Claims (3)
1. Application of a reagent for detecting the expression level of PTTG3P in preparation of a pancreatic cancer diagnostic kit.
2. The application of the reagent for detecting the expression level of PTTG3P in the preparation of a pancreatic cancer diagnostic kit according to claim 1, wherein the reagent for detecting the expression level of PTTG3P is a PCR primer having detection specificity to PTTG3P, and the nucleotide sequence of the PCR primer is shown as SEQ ID No.1 and SEQ ID No. 2.
3. The use of the reagent for detecting the expression level of PTTG3P in the preparation of a pancreatic cancer diagnostic kit according to claim 1, wherein the diagnostic kit comprises a reagent for detecting the expression level of PTTG3P in a biological sample selected from the group consisting of: peripheral blood obtained from a subject.
The use of interfering RNA of PTTG3P in the preparation of a medicament for the treatment of pancreatic cancer; the sequence of the interference RNA of PTTG3P is shown in SEQ ID NO.5 or SEQ ID NO. 6.
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| Long non-coding RNA PTTG3P functions as an oncogene by sponging miR-383 and up-regulating CCND1 and PARP2 in hepatocellular carcinoma;Zhou, Q等;《BMC Cancer》;20190724;第19卷(第1期);摘要 * |
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