CN109486952A - MiR-495 is preparing the application in Pancreatic Neuroendocrine Tumors diagnostic tool - Google Patents

Abstract

The invention discloses the application of miR-495 in the preparation of a diagnostic tool for pancreatic neuroendocrine tumors. The present invention also provides a diagnostic kit for detecting pancreatic neuroendocrine tumors. Further, the present invention also provides a pharmaceutical composition for the prevention or treatment of pancreatic neuroendocrine tumors, the pharmaceutical composition comprising a mimetic of miR-495 as an active ingredient. The present invention finds that miR-495 has strong specificity and high sensitivity, can be effectively used for early detection of pancreatic neuroendocrine tumors, and can be used for clinical applications such as gene therapy and drug therapy.

Description

Application of miR-495 in the preparation of diagnostic tools for pancreatic neuroendocrine tumors

technical field

The invention relates to the field of biotechnology, in particular to the application of miR-495 in the preparation of a diagnostic tool for pancreatic neuroendocrine tumors.

Background technique

Neuroendocrine neoplasms (NENs) originate from pluripotent neuroendocrine stem cells, possess neuroendocrine markers, produce bioactive amines and/or polypeptide hormones, and have significant heterogeneity. According to their embryonic origin, NENs are generally classified into foregut, midgut, and hindgut tumors. Foregut tumors occur in the respiratory tract, thymus, stomach, duodenum, and pancreas. Midgut tumors occur in the small intestine, appendix, and ascending colon. Hindgut tumors occur in the transverse colon, descending colon, and rectum. Over the past 30 years, the prevalence of NENs has increased from 1.09/100,000 to 5.25/100,000. Compared with other tumors, the increase of NENs is more rapid, which may be related to the advancement of diagnostic technology, more frequent tumor screening and environmental factors. Pancreatic neuroendocrine tumors (PNETs) account for about 1/3 of all neuroendocrine tumors, and the percentage of pancreatic tumors is about 2% to 5%. Although PNETs are rare, they often act on multiple systems throughout the body due to their secretion of hormones, seriously affecting the health and quality of life of patients, and are prone to liver metastases, often resulting in death due to liver failure. With the advancement of imaging technology, the detection rate also showed an increasing trend. Due to the large differences in clinical manifestations, pathological features, malignancy and prognosis, the current clinical understanding of pNENs is still insufficient, and it is easy to be misdiagnosed and missed.

For various PNETs, the 2017 WHO classification of endocrine tumors is divided into: ① differentiated PNETs (neuroendocrine tumors), which are further divided into three grades: G1, G2 and G3; ② undifferentiated PNECs (neuroendocrine carcinomas), which are further divided into Small cell type and large cell type, highly malignant; ③ mixed neuroendocrine-non-neuroendocrine tumors. From the perspective of clinical manifestations, PNETs can be divided into functional and non-functional categories. Functional can produce typical clinical symptoms, and the most common ones are insulinoma and gastrinoma. Non-functioning has increased significantly in recent years, they only secrete polypeptide fragments, less or no hormones, and do not produce clinical signs of endocrine abnormalities. Functional PNETs can be treated in a timely manner because they can produce symptoms at an early stage; non-functional PNETs are often delayed in diagnosis, and the tumors often grow very large, resulting in local invasion or distant metastasis.

At present, the treatment of PNETs is mainly surgery, which is suitable for patients with relatively limited tumors, but the prognosis varies greatly. Drug therapy can be applied with somatostatin analogs such as octreotide and sanitidine, which can partially relieve symptoms. The molecularly targeted drug sunitinib has been approved for the treatment of PNETs, but clinical observations show that after a short-term benefit, the tumor invasive ability of sunitinib in the treatment of PNETs is significantly enhanced, and the tumor is more likely to metastasize. Targeted drug therapy against angiogenesis and anti-growth factors is a newly attempted treatment, and its efficacy remains to be determined.

miRNA (microRNA) is a class of non-coding small RNAs with a length of 20 to 25 nucleotides, encoded by higher eukaryotic genomes. miRNAs guide silencing through their own seed sequences and target gene mRNA base pairing. The complex degrades mRNA or hinders its translation. Studies have shown that miRNAs affect the occurrence and development of tumors by regulating the expression of target genes, and participate in the signaling network of tumor regulation.

Therefore, it is suggested to search for potential miRNA targets, which may provide new ideas for the early detection and treatment of PNETs.

SUMMARY OF THE INVENTION

In order to solve the technical problems mentioned above, the purpose of the present invention is to provide a use of miRNA in the diagnosis and treatment of pancreatic neuroendocrine tumors.

Based on this, the inventors found in the related research on pancreatic neuroendocrine tumors that the expression of miR-495 in pancreatic neuroendocrine tumor cells was significantly lower than that in normal cells. This indicates that abnormal expression of miR-495 is related to the occurrence and development of pancreatic neuroendocrine tumors. miR-495 plays an important role in the pathogenesis and progression of pancreatic neuroendocrine tumors, and can be used as a marker for the identification of pancreatic neuroendocrine tumors. Therefore, the level of miR-495 in the blood of patients with pancreatic neuroendocrine tumors can be used as the basis for the diagnosis of pancreatic neuroendocrine tumors, that is, miR-495 can be used as a molecular marker for the diagnosis of pancreatic neuroendocrine tumors.

First, the present invention provides the application of miRNA in the preparation of a diagnostic tool for pancreatic neuroendocrine tumors, the miRNA is selected from the following group: initial miRNA, precursor miRNA, mature miRNA; the initial miRNA can be cleaved and expressed in human cells as Mature miRNA; precursor miRNA can be cleaved and expressed as mature miRNA in human cells; the miRNA is miR-495. The nucleotide sequence of the miR-495 is shown in SEQ ID NO:10.

In some specific embodiments of the present invention, the miRNA is mature miR-495-5p; the nucleotide sequence of the miR-495-5p is shown in SEQ ID NO:11.

Although mature miRNAs are used in certain embodiments, those skilled in the art can expect that initial miRNAs, precursor miRNAs will achieve the same technical effect as mature miRNAs, because cells have the ability to further convert initial miRNAs, precursor miRNAs Somatic miRNAs are processed into mature miRNAs.

Preferably, the expression of miR-495 is down-regulated in pancreatic neuroendocrine tumor cells compared to normal pancreatic epithelial cells H6C7.

Preferably, the tools include, but are not limited to, kits, chips, test strips, and high-throughput sequencing platforms. The diagnostic tool includes reagents for detecting miR-495 expression levels.

Preferably, the kit includes primers and/or probes for miR-495; the chip includes a solid-phase carrier; and an oligonucleotide probe immobilized on the solid-phase carrier, the oligonucleotide The acid probe includes part or all of the sequence specifically corresponding to miR-495; the test paper includes primers and/or probes for miR-495; the high-throughput sequencing platform includes primers and/or probes for miR-495 or probe.

Further, the present invention provides a diagnostic kit for detecting pancreatic neuroendocrine tumors, the kit comprising primers and instructions for specifically amplifying pancreatic neuroendocrine tumor-related miR-495.

Preferably, the primers include a pair of cDNA amplification primers whose sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Preferably, the detection primers and/or probes of the above-mentioned miR-495 and various miRNAs can be placed in the same kit to diagnose pancreatic neuroendocrine tumors by detecting various miRNA indicators, which is also included in the protection scope of the present invention. Inside.

Further, the present invention provides the application of the miR-495 in the preparation of a pharmaceutical composition for preventing or treating pancreatic neuroendocrine tumors.

Preferably, the pharmaceutical composition includes an effective dose of a miR-495 promoter.

As used herein, an "effective dose" refers to an amount that produces function or activity in humans and/or animals and is acceptable to humans and/or animals. The effective dose of miR-495 described in the present invention may vary with the mode of administration and the severity of the disease to be treated. Selection of the preferred effective amount can be determined by one of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the miRNA promoter such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the administration way etc.

Preferably, the miR-495 promoter can promote the expression of miR-495 or can activate the function of miR-495.

Further, miR-495 promoters include oligonucleotides (mimetics), small molecule compounds, and oligonucleotide expression vectors.

Preferably, the vectors for oligonucleotide expression include viral vectors and eukaryotic vectors.

The viral vector can be any suitable vector, including but not limited to retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes virus vectors, alphavirus vectors, and the like.

The eukaryotic expression vector can be any suitable expression vector, including but not limited to pCMV-Myc expression vector, pcDNA3.0 expression vector, pcDNA3.1 expression vector, pEGFP expression vector, pEF Bos expression vector, pTet expression vector, pTRE expression vector, etc.

Preferably, the miR-495 promoter is an expression vector of a miR-495 mimic or a miR-495 sequence.

Preferably, the nucleotide sequence of the miR-495 mimic is shown in SEQ ID NO:5.

The pharmaceutical composition for treating pancreatic neuroendocrine tumors of the present invention further comprises a pharmaceutically acceptable carrier, which includes but is not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulations, excipients Excipients, fillers, binders, sprays, transdermal absorption agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

Preferably, the pharmaceutical composition can be prepared including, but not limited to, microinjection, dosage form, dosage form suitable for transfection, injection, tablet, powder, granule, and capsule. The medicines in the above-mentioned various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

Preferably, the pharmaceutical composition can be administered alone; or in combination with other drugs capable of treating pancreatic neuroendocrine tumors.

Preferably, the drug can be administered in vitro: the expression vector of miR-495 is introduced or transfected in vitro into human self or allogeneic cells (or xenogeneic cells), and after cell expansion in vitro, it is returned to the human body.

Preferably, the drug can be administered in vivo: the expression vector of miR-495 is directly introduced into the body. Such vectors can be viral or non-viral, or even naked DNA or RNA.

Preferably, the subject may be human or other mammals. More specifically, the subject is an organ, tissue, cell.

Further, the present invention also provides a method for screening potential substances for the treatment of pancreatic neuroendocrine tumors, comprising: treating the pancreatic neuroendocrine tumor system with a candidate substance, if the candidate substance can promote the expression or activity of miR-495, then It indicates that the candidate substance can be used as a potential substance for the treatment of pancreatic neuroendocrine tumors.

beneficial effect

The present invention provides a miR-495 related to pancreatic neuroendocrine tumor, and provides a diagnostic kit for detecting pancreatic neuroendocrine tumor, which can predict whether there is a pancreatic neuroendocrine tumor by detecting the expression of miR-495, thereby Provide a basis for targeted treatment of the disease. The present invention also provides a pharmaceutical composition for preventing or treating pancreatic neuroendocrine tumors. The pharmaceutical composition contains an effective amount of a miR-495 mimic as an active agent, so that it can treat patients with pancreatic neuroendocrine tumors whose miR-495 expression is down-regulated in vivo. An effective amount of the miR-495 mimetic is administered, respectively, so as to achieve the effect of preventing or treating pancreatic neuroendocrine tumors. The invention finds that miR-495 has strong specificity and high sensitivity, not only can achieve early detection quickly and effectively, but also provides a therapeutic target and an important basis for clinical applications such as gene therapy and drug therapy.

Description of drawings

Figure 1 shows the expression of miR-495 in pancreatic neuroendocrine tumor cells detected by QPCR;

Figure 2 shows the interference of miR-495 expression on miR-495 expression detected by QPCR.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The result data in the following examples are all expressed in the form of mean ± standard deviation. SPSS19.0 statistical software is used for statistical analysis. The difference between the two is carried out by t test. It is considered that when P<0.05, there is statistical significance study meaning.

In the examples of the present invention, normal pancreatic epithelial cells H6C7 were purchased from the National Cell Experiment Technology Platform; BON-1 and QGP-1 cells were purchased from ATCC.

H6C7 was cultured in serum-free medium at 37°C in an incubator containing 5% CO ₂ . When the cells reached 80% confluence after adherence, they were digested with 0.025% trypsin and passaged.

BON-1 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum and 20 mmol/L Gln (Sigma, USA); QGP-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, placed in in a 37 °C, 5% _CO2 incubator. Medium was purchased from Invitrogen.

Example 1 miR-495-5p was significantly abnormally expressed in pancreatic neuroendocrine tumor cells

1. Sample total RNA extraction

use Reagent (Invitrogen, Carlsbad, CA, USA) extracted RNA from normal pancreatic epithelial cells H6C7, pancreatic neuroendocrine tumor cells BON-1 and QGP-1 samples. The experimental operations were carried out according to the product instructions. The specific operations are as follows:

After collecting the samples, they were frozen and stored in liquid nitrogen. About 30 mg of cell samples were taken out and ground in a pre-cooled mortar. After the cell samples were ground to no particles, follow the steps below:

①Add Trizol and let stand for 10 minutes at room temperature;

②Add 0.2 mL of chloroform, shake the centrifuge tube vigorously, mix well, and let it stand at room temperature for 3 minutes;

③ After centrifugation at 4°C and 12000rpm for 15 minutes, suck the upper aqueous phase into another new centrifuge tube, being careful not to suck up the protein material between the two aqueous phases. Transfer to a new tube, add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 10 minutes;

④ After centrifugation at 4°C and 12000rpm for 15 minutes, carefully discard the supernatant, add 1 mL of 75% ethanol to wash the precipitate, shake and mix, and centrifuge at 7500g for 5 minutes at 4°C;

⑤ Discard the ethanol liquid, put it at room temperature for 10 minutes to fully dry the precipitate, and add DEPC water to dissolve the precipitate;

⑥Measure the RNA concentration and purity with NanoDrop One spectrophotometer, and store it at -80℃. Sample requirements for RNA-seq sequencing: OD260/OD280 of 1.8-2.2.

2. miRNA reverse transcription

One Step PrimeScript miRNA cDNA Synthesis Kit (Takara, CodeNo.D350A) was used for reverse transcription. The experimental operation was carried out according to the product instructions. The specific operations are as follows:

A 20 μL reaction system was used, and 1 μg of total RNA was taken from each sample as a template. The obtained cDNA was stored in a -20°C refrigerator for later use.

3. Real-Time PCR

The relative quantitative analysis of the data was carried out by the 2- ^ΔΔCt method using an ABI7500 fluorescence quantitative PCR instrument.

SYBR PrimeScipt miRNA RT-PCR Kit (Takara, Code No. RR716) was used for amplification, and the experimental operation was carried out according to the product instructions.

Table 1 RT-PCR reaction system of miRNA

component Adding amount SYBR Premix Ex Taq II (2×) 10μL PCR Forward Primer (10μM) 0.5μL PCR Reverse Primer (10μM) 0.5μL ROX Reference Dye II (50×) 2μL Template (cDNA solution) 2μL dH₂O to 20 μL

The expression detection of miRNAs was performed in 3 parallel tube reactions each time. The forward primer sequence for amplifying the cDNA of miR-495-5p is shown in SEQ ID NO: 1 (CGCCAGGGTTTTCCCAGTCACGAC), and the reverse primer sequence is shown in SEQ ID NO: 2 (CGCGAGGAGAGAATTAATACGACTC), with snRNA U6 as an internal reference, for The forward primer for cDNA amplification is shown in SEQ ID NO: 3 (GTGCTCGCTTCGGCAGCACATAT), and the reverse primer is shown in SEQ ID NO: 4 (AAAATATGGAACGCTTCACGAA). Amplification program: 95°C for 30s; 40x (95°C for 5s, 60°C for 34s).

4. Analysis of results

According to the relative quantitative formula of qRT-PCR: 2- ^ΔΔCt × 100%, the expression levels of miR-495-5p in pancreatic neuroendocrine tumor cells BON-1 and QGP-1 and control normal pancreatic epithelial cells H6C7 were compared, respectively.

The results showed that the qRT-PCR amplification results were stable. Compared with normal pancreatic epithelial cell H6C7, miR-495-5p was significantly lower in QGP-1 and BON-1 pancreatic neuroendocrine tumor cells, as shown in Figure 1.

Example 2 Effects of mimics of miR-495-5p on the expression of miR-495-5p in pancreatic neuroendocrine tumor cells

1. Design and synthesis of mimics against miR-495-5p

According to the sequence information of miR-495-5p, Guangzhou Ribo Biotechnology Co., Ltd. designed and synthesized the mimic sequence of miR-495-5p as shown in SEQ ID NO: 5.

SEQ ID NO: 5: GAAGUUGCCCAUGUUAUUUUCG.

2. Transfection

Transient transfection was carried out by cationic liposome method, and the operation was carried out according to the instructions of Lipofectamine ^TM 2000TransfectionReagent. 24h before transfection, the QGP-1 and BON-1 pancreatic neuroendocrine tumor cells with good growth status were inoculated into 6-well plates, and the cell count was about 5×10 ⁴ . % to test. Add 80nM miR-495-5p mimic to 100uL DMEM medium, mix gently; dilute 2uL Lipofectamin ^TM 2000 liposome with 100uL LDMEM medium, mix gently, incubate at room temperature for 5min; mix DMEM-liposome with DMEM-miRNAs were incubated at room temperature for 20 min to form transfection complexes; then the above mixture was added to the cell culture medium and mixed gently to mix them well. Among them, the non-specific sequence was used as a negative control (NC), and a blank control (Blank) group was set in the same period. After 48h of culture, the total RNA of cells was extracted for the next experiment.

3. Real-Time PCR detection of mimic effect of miR-495-5p on the expression of miR-495-5p in pancreatic neuroendocrine tumor cells QGP-1 and BON-1

Cell total RNA extraction and PCR steps were the same as in Example 1.

The Real-time PCR results are shown in Figure 2. The negative control (NC) group had no significant inhibitory effect on the expression of miR-495-5p in pancreatic neuroendocrine tumor cells QGP-1 and BON-1, and the blank control (Blank) group had no statistical effect. After transfection of miR-495-5p mimic in QGP-1 and BON-1 pancreatic neuroendocrine tumor cells for 48h, the expression of miR-495-5p in the cells was significantly increased (*P<0.05).

Example 3 Effects of mimics of miR-495-5p on S100P in pancreatic neuroendocrine tumor cells

The extraction procedure of total cell RNA was the same as that in Example 1.

1. mRNA reverse transcription

use III Reverse Transcriptase (invitrogen, product number 18080-044) is used for reverse transcription of cDNA. The experimental operation is carried out according to the product manual. The specific operations are as follows:

Using a reverse transcription kit, 1 μg of total RNA was reverse-transcribed to synthesize cDNA with reverse transcription buffer. A 25 μL reaction system was used, and 1 μg of total RNA was taken from each sample as template RNA. The obtained cDNA was stored in a -20°C refrigerator for later use.

2. Real-Time PCR

The relative quantitative analysis of the data was carried out by the 2- ^ΔΔCt method using an ABI7500 fluorescence quantitative PCR instrument.

Use Power Green PCR Master Mix (invitrogen, Cat. No. 4367659) was used for amplification, and the experimental operation was carried out according to the product instructions. The reaction system is shown in Table 2.

Table 2 RT-PCR reaction system of mRNA

component Adding amount 2×mix 10μL Forward primer (10uM) 0.5μL Reverse primer (10uM) 0.5μL template 2μL Add sterilized distilled water to 25 μL

The expression detection of mRNAs was performed in 3 parallel tube reactions each time. The forward primer sequence for amplifying S100P is shown in SEQ ID NO:6, and the reverse primer sequence is shown in SEQ ID NO:7. Taking GAPDH as an internal reference gene, the forward primer is shown in SEQ ID NO: 8, and the reverse primer is shown in SEQ ID NO: 9. The amplification program was: 95°C for 10 min, 45x (95°C for 15s, 60°C for 60s).

Table 3 Primer sequences

3. Analysis of results

The results showed that the qRT-PCR amplification results were stable, and the expression of S100P was not significantly affected by transfection of miR-495-5p mimic in two pancreatic neuroendocrine tumor cells. The above experiments show that miR-495-5p plays an important role in the pathogenesis and progression of pancreatic neuroendocrine tumors, not by regulating the S100P gene. The signaling regulation mechanism of miR-495-5p involved in pancreatic neuroendocrine tumors needs to be further studied.

Example 4 Preparation of the kit

Based on the primers obtained in Example 1, a kit for detecting pancreatic neuroendocrine tumors according to the present invention was assembled, the kit comprising (1) a reverse transcriptase buffer containing reverse transcriptase buffer, reverse transcription primers, reverse transcriptase, etc. Transcription reagent; (2) the forward primer that specifically amplifies miR-495 is shown in SEQ ID NO: 1, and the reverse primer is a universal primer such as SEQ ID NO: 2: and the specific amplification snRNA T6 (as an internal reference) The primer pairs are shown in SEQ ID NO: 3 and SEQ ID NO: 4; (3) also include SYBR Green polymerase chain reaction system, such as PCR buffer, SYBR Green fluorescent dye, dNTPs. The composition of the PCR buffer was 25 mM KCl, 2.5 mM MgCl ₂ , 200 mM (NH 4 ) ₂ SO ₄ .

Through the optimization of primer concentration and annealing temperature, the final reaction system is determined as shown in Table 4:

Table 4PCR reaction system

component volume/μL Final concentration cDNA 1 primer positive (20μM) 0.5 10μM primer reverse (20μM) 0.5 10μM SYBR Green polymerase chain reaction (2×) 10 1× ddH₂O 8

The optimal reaction conditions are:

Pre-denaturation at 48 °C for 30 min; denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 sec, annealing at 60 °C + extension for 1 sec, a total of 40 cycles.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

sequence listing

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Claims (10)

1. The application of miRNA in the preparation of a diagnostic tool for pancreatic neuroendocrine tumors, wherein the miRNA is selected from the following group: initial miRNA, precursor miRNA, mature miRNA; initial miRNA can be spliced in human cells and expressed as Mature miRNA; precursor miRNA can be cleaved and expressed as mature miRNA in human cells; the miRNA is miR-495. 2. The use of claim 1, wherein the miRNA is mature miR-495-5p. 3. The use according to claim 1 or 2, wherein the expression of miR-495 is down-regulated in pancreatic neuroendocrine tumor cells compared to normal pancreatic epithelial cells H6C7. 4. The application according to claim 1 or 2, wherein the tool comprises a kit, a chip, a test strip, and a high-throughput sequencing platform. 5. An early diagnosis kit for detecting pancreatic neuroendocrine tumors, characterized in that the kit comprises primers and instructions for specifically amplifying pancreatic neuroendocrine tumor-related miR-495. 6. The kit of claim 5, wherein the primers comprise a pair of cDNA amplification primers with sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. 7. The application of miR-495 according to claim 1 in the preparation of a pharmaceutical composition for preventing or treating pancreatic neuroendocrine tumors. 8. The use according to claim 7, wherein the pharmaceutical composition comprises a miR-495 promoter as an active ingredient. 9 . The use according to claim 8 , wherein the miR-495 promoter can promote the expression of miR-495 or activate the function of miR-495. 10 . 10. The use according to claim 9, wherein the miR-495 promoter is a miR-495 mimic or a small molecule compound.