KR102216590B1 - Composition for treatment and diagnosis of pancreatic neuroendocrine tumors - Google Patents

Abstract

The present invention relates to a composition and method for diagnosing a pancreatic neuroendocrine tumor and determining the prognosis, and more specifically, by confirming the association of a pancreatic neuroendocrine tumor according to HuD expression, HuD is used to diagnose and determine the prognosis of a pancreatic neuroendocrine tumor. It is about using it.

Description

Composition for diagnosis of pancreatic neuroendocrine tumor {COMPOSITION FOR TREATMENT AND DIAGNOSIS OF PANCREATIC NEUROENDOCRINE TUMORS}

The present invention relates to a composition and method for diagnosis and prognosis of a pancreatic neuroendocrine tumor, and more particularly, to a method of diagnosing a pancreatic neuroendocrine tumor and predicting its prognosis by checking the expression level of HuD.

The Hu family includes HuB, HuC and HuD, and these Elav/Hu proteins are generally known to bind to the U or AU-rich RNA sites of the target transcript through conserved RNA recognition motifs (RRMs 1-3). Yes (Hinman and Lou, 2008; Pascale et al., 2008). HuD is known to promote neurite outgrowth (Kasashima et al., 1999; Abdelmohsen et al., 2010), but the physiological role of HuD in animals is known to be complex. HuD targets mRNAs encoding several proteins preferentially expressed in nerves, such as GAP-43, acetylcholinesterase, tau, PSD-95, neuroserpin and musashi-1. do.

On the other hand, for neuroendocrine tumors that mainly occur in the gastrointestinal tract, pancreas, and rectum, early treatment is important because symptoms and survival rates are different for each cancer site. Among them, pancreatic neuroendocrine tumors (PNETs) are known to be rare diseases with an overall prevalence of less than 1 per 100,000 people, so the clinical manifestations and prognostic factors related to survival are not accurately known (Halfdanarson TR, Rabe KG, Rubin J, Petersen GM.Pancreatic neu-roendocrine tumors (PNETs): incidence, prognosis and recent trend toward improved survival.Ann Oncol 2008;19:1727-33.), and the incidence is increasing recently (Fischer L, Bergmann F, Schimmack S, Hinz U, Priess S, Muller-Stich BP, et al. Outcome of surgery for pancreatic neuroendocrine neo-plasms.Br J Surg 2014;101:1405-12.). Pancreatic neuroendocrine tumors can be classified into functional and non-functional neuroendocrine tumors according to clinical manifestations, but they are difficult to differentiate microscopically and are classified based on the appearance of clinical symptoms related to active neuroendocrine hormones secreted by the tumor itself. . Functional neuroendocrine tumors are known to have clinical symptoms related to neuroendocrine hormones secreted by the tumor, but in actual clinical practice, even if such symptoms are complained, it is difficult to suspect and diagnose due to the rarity of the disease, so diagnosis and proper treatment are delayed. There are many cases. Among the neuroendocrine tumors of the pancreas, in the case of functional neuroendocrine tumors, the frequency of highly malignant tumors with invasion or distant metastasis to surrounding tissues is 10% for insulinoma, 60% for gastrinoma, and 50% for nonfunctional neuroendocrine tumors. It is known to be about 90% (Ectors N. Pancreatic endocrine tumors: diagnostic pitfalls. Hepa-togastroenterology 1999;46:679-90.), but the growth rate is slower than that of pancreatic adenocarcinoma, but most of them die from liver failure if malignant progresses. (Eriksson B, Oberg K. Neuroendocrine tumours of the pancreas. Br J Surg 2000;87:129-31.).

The stage classification of pancreatic neuroendocrine tumors is largely divided into AJCC 7th edition classification and World Health Organization (WHO) classification. In the 7th edition of the American Joint Committee on Cancer (AJCC), the stage of pancreatic neuroendocrine tumors, which had no separate classification until the 6th edition, was introduced, but the existing classification method for pancreatic cancer was applied. The WHO classification, revised in 2010, divided tumors into three classes by reflecting the nuclear meiosis and Ki-67 index presented at the European Neuroendocrine Oncology Association in 2005 (Rindi G, Kloppel G, Alhman H, Caplin M, Couvelard A, de Herder WW, et al. TNM staging of foregut (neuro) endocrine tumors: a consen-sus proposal including a grading system.Virchows Arch 2006;449: 395-401.) The size has been excluded, and its clinical value has not yet been fully understood.

Pancreatic neuroendocrine tumor is not well known yet, but it is one of the terrifying diseases that lead to death due to poor prognosis when it occurs in the pancreas, which is not well detected even by examination. In addition, since the incidence of cancer is increasing in recent years, it is necessary to develop a diagnostic method through research and interest from academia.

Rindi G, Kloppel G, Alhman H, Caplin M, Couvelard A, de Herder WW, et al. TNM staging of foregut (neuro)endocrine tumors: a consen-sus proposal including a grading system. Virchows Arch 2006;449: 395-401.

In the present invention, by confirming the correlation between the expression amount of HuD and the pancreatic neuroendocrine tumor, it is an object of the present invention to be used for diagnosis and prognosis of a pancreatic neuroendocrine tumor.

In order to achieve the above object, the present invention provides a composition for diagnosing a pancreatic neuroendocrine tumor, comprising an agent for measuring the expression level of the HuD gene.

In addition, the present invention provides a composition for diagnosing the prognosis of a pancreatic neuroendocrine tumor, comprising an antibody against the HuD protein.

In addition, the present invention provides a method of providing information for diagnosing a pancreatic neuroendocrine tumor, comprising measuring the amount of HuD expression in a sample isolated from the human body.

In addition, the present invention provides a method of providing information for prognosis diagnosis of a patient with pancreatic neuroendocrine tumor, comprising the step of measuring the amount of HuD expression in a sample isolated from the human body.

According to the present invention, when HuD expression decreases, the growth of pancreatic neuroendocrine tumors is promoted, pancreatic neuroendocrine tumor patients with reduced HuD expression have poor prognosis, and overexpression of HuD suppresses pancreatic neuroendocrine tumors, HuD Since it was confirmed that patients with pancreatic neuroendocrine tumors with increased expression have a high survival rate and a good prognosis, HuD can be used for diagnosis and prognosis of pancreatic neuroendocrine tumors.

1 is a diagram showing the degree of expression of HuD protein in tissues of a pancreatic-derived neuroendocrine patient, and accordingly, a mitotic count and a tumor size.
2 is a diagram illustrating the expression patterns of p27 and Ki-67 in the tissues of pancreatic-derived neuroendocrine patients in the HuD negative group and the HuD positive group.
3 is a diagram showing the survival rate through the disease-free survival (DFS) and progression free survival of human pancreatic-derived neuroendocrine tumor patients according to HuD expression (HuD-negative group and HuD-positive group).
4 is a diagram showing the survival rate of insulinoma cell lines according to inhibition of HuD expression.
5 is a diagram showing cell cycle changes and cell growth of an insulinoma cell line according to inhibition of HuD expression.
6 shows the growth of insulinoma cell lines according to inhibition of HuD expression It is a diagram confirmed through colony formation assay.
7 is a diagram confirming the formation of insulinoma through the size of the insulinoma according to inhibition of HuD expression.
8 is a diagram showing the survival rate of an insulinoma cell line overexpressing HuD.
9 is a diagram showing changes in the insulinoma cell cycle and cell growth in which HuD is overexpressed.
10 shows the growth of an insulinoma cell line according to HuD overexpression This is a diagram confirmed through colony formation analysis.

The present invention will be described in detail. However, the present invention may be changed or modified in various forms and implemented, and is not limited to the embodiments described herein.

The term "detection" or "measurement" as used in the present invention means to quantify the concentration of a detected or measured object.

The "agent for measuring the expression level of the HuD gene" used in the present invention means a molecule capable of detecting the mRNA amount of the HuD gene or the level of the HuD protein by checking the expression level of the HuD gene, and is preferably a HuD-specific It means an antibody, primer or probe.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3 hydroxyl group, which can form a complementary template and a base pair, and is used as a starting point for template strand copying. It refers to a short functional nucleic acid sequence. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerate or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. In the present invention, PCR amplification is performed using the sense and antisense primers of the HuD gene, and the degree of HuD expression can be determined by measuring whether a desired product is generated and the level thereof. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art, so the present invention is not particularly limited thereto.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to a few hundred bases for a specific binding to mRNA, and is labeled to confirm the presence or absence of a specific mRNA. I can. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. In the present invention, hybridization is performed using a probe that is complementary to the HuD gene, and the degree of expression of the HuD gene may be diagnosed through hybridization. Selection and hybridization conditions for suitable probes may be modified based on those known in the art, and thus are not particularly limited in the present invention.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, e.g., uncharged linkers (e.g. methyl phosphonate, phosphotriester, phosphoro Amidates, carbamates, etc.) or to charged linkers (eg phosphorothioate, phosphorodithioate, etc.).

In the present invention, the term "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a protein expressed in the HuD gene, which is a marker of the present invention, and the method for producing the antibody can be prepared using well-known methods. This includes partial peptides that can be made from these proteins. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, monoclonal antibody, or any one having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

In one aspect, the present invention relates to a composition for diagnosing a pancreatic neuroendocrine tumor, comprising an agent for measuring the expression level of a human antigen D (Hud) gene. In one embodiment, the measurement of the expression level of the HuD gene can be determined by checking the expression level of the mRNA of the HuD gene or the level of the HuD protein encoded by the gene. The amount of the mRNA of the HuD gene can be checked using a primer pair or a probe, and analysis methods for this include RT-PCR, competitive RT-PCR, and real-time RT-PCR. , RNase protection assay (RPA), Northern blotting, DNA chip, etc., but are not limited thereto. The amount of HuD protein encoded by the HuD gene can be determined using an antibody that specifically binds to the protein, and analysis methods for this include western blot, enzyme linked immunosorbent assay (ELISA), radiation Immunoassay (RIA: Radioimmunoassay), radioimmunodiffusion, Ouchterlony immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation assay), complement fixation assay ( Complement Fixation Assay), FACS, and protein chips, but are not limited thereto. In one embodiment, the pancreatic neuroendocrine tumor may be a functional tumor related to hormone secretion, may be insulin, depending on the type of hormone, insulin, gastrin, or glucagon, but insulin species is most preferred.

In one aspect, the present invention relates to a composition for diagnosing the prognosis of a pancreatic neuroendocrine tumor, comprising an antibody against HuD protein. In one embodiment, the pancreatic neuroendocrine tumor may be a functional tumor related to hormone secretion, may be insulin, depending on the type of hormone, insulin, gastrin, or glucagon, but insulin species is most preferred.

In one aspect, the present invention relates to a method of providing information for diagnosing a pancreatic neuroendocrine tumor, comprising measuring the amount of HuD expression in a sample isolated from a human body. In one embodiment, the pancreatic neuroendocrine tumor may be a functional tumor related to hormone secretion, may be insulin, depending on the type of hormone, insulin, gastrin, or glucagon, but insulin species is most preferred. In one embodiment, the measurement of the HuD expression level can be confirmed by measuring the mRNA level or the HuD protein level of the HuD gene. In one embodiment, the method comprises the steps of measuring the mRNA amount of the HuD gene or the expression level of the protein thereof in a sample isolated from the patient to provide information for diagnosing a pancreatic neuroendocrine tumor; And it may be a method of providing information for diagnosis of a pancreatic neuroendocrine tumor comprising the step of comparing the measured expression level of the mRNA or protein thereof with a control sample isolated from a patient not suffering from the disease. In one embodiment, the method may include determining that the risk of developing a pancreatic neuroendocrine tumor is high when the expression decreases by 50% or more compared to the expression level of HuD in a normal person who does not suffer from the disease.

The patient's sample includes, but is not limited to, a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine. In addition, a specific method of measuring the expression level of the mRNA or protein thereof of the gene can detect the expression of the gene at the mRNA level or protein level, and the isolation of mRNA or protein from a biological sample is performed using a known process. can do. The mRNA or HuD protein level of the HuD gene may be expressed as an absolute (eg, ug/ml) or relative (eg, relative intensity of a signal) difference. Preferably, immunohistochemical staining may be performed using one or more antibodies against the Hud protein. After collecting and fixing a sample from a patient, a paraffin-embedded block may be prepared by a method well known in the art. After these are made into sections having a thickness of several um and attached to a glass slide, the paraffin-embedded block can be reacted with any of the above antibodies by a known method. Subsequently, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and whether the antibody is labeled can be read on a microscope.

In one aspect, the present invention relates to a method of providing information for prognosis diagnosis of a patient with pancreatic neuroendocrine tumor, comprising the step of measuring the amount of HuD expression in a sample isolated from the human body. In one embodiment, the prognosis of the pancreatic neuroendocrine tumor patient may be determined by the risk of disease free survival (DFS) or progression free survival. In one embodiment, the measurement of the HuD expression level can be confirmed by measuring the mRNA level of the HuD gene or the HuD protein level. In one example, it was confirmed that the HuD-positive group significantly increased the number of cells remaining in the G1 phase compared to the negative group, and the cells in the mitotic phase significantly decreased, thereby significantly reducing cell division (see Table 1). In one embodiment, the pancreatic neuroendocrine tumor may be a functional tumor related to hormone secretion, may be insulin, depending on the type of hormone, insulin, gastrin, or glucagon, but insulin species is most preferred. In one embodiment, the method may include determining that the risk of prognosis is high when the expression decreases by 50% or more compared to the expression level of HuD in a normal person who does not suffer from the disease.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

Example 1. Confirmation of the correlation between HuD expression and pancreatic neuroendocrine tumor

1-1. Confirmation of HuD expression patterns in pancreatic-derived neuroendocrine tumors

After staining the tissues of 88 pancreatic neuroendocrine patients with an anti-HuD antibody (Santa Cruz Biotechnology, Inc.), analyze the intensity of the DAB signal by analyzing the brightness of the DAB signal with a slide scanner to determine the expression level of the HuD protein. Confirmed. Pancreatic tissues were classified into a HuD negative group and a HuD positive group according to the presence or absence of the brightness of the DAB signal reacted specifically to the HuD protein. Analysis of clinical data of human pancreatic-derived neuroendocrine tumor patients classified according to lesions through the TNM (tumor-Node-Metastasis) classification method, and the degree of HuD expression (HuD negative and HuD positive groups), tumor size, metastasis, and degree of differentiation. The correlation between the two was analyzed and described in Table 1 below.

HuD Negative (23) Positive (65) P Sex male 43 13 (56.5) 20 (46.2) 0.393 female 45 10 (43.5) 35 (53.8) Age (year) 54 53.4±13.5 52.3±11.4 0.488 Size (cm) 2.4 4.6±4.2 2.7 ± 2.8 0.006* Grade by 2010 WHO classification G1 64 11 (47.8) 53 (81.5) <0.001* G2 20 8 (34.8) 12 (18.5) G3 4 4 (17.4) 0 (0.0) Ki-67 index (%) 4.1±8.9 1.2±2.5 0.271 Mitotic count (10HPF) 5.9 ± 10.9 0.7 ± 2.1 0.001* Extent of invasion Limited in pancreas 63 11 (47.8) 52 (80.0) 0.003* Extended to other organ 25 12 (52.2) 13 (20.0) Angioinvasion Absent 58 11 (47.8) 47 (72.3) 0.033* Present 30 12 (52.2) 18 (27.7) pT ( AJCC 7 th ed) One 32 3 (13.0) 29 (44.6) 0.004* 2 31 8 (34.8) 23 (35.4) 3 25 12 (52.2) 13 (20.0) pN 0 80 21 (26.3) 59 (90.8) 0.980 One 8 2 (8.7) 6 (9.2) pM 0 81 21 (91.3) 60 (92.3) 1.000 One 7 2 (8.7) 5 (7.7) Associated syndrome Sporadic 80 18 (78.3) 62 (95.4) 0.009* Von - Hippel - Lindau syndrom ( VHL ) 5 2 (8.7) 3 (4.6) Multiple endocrine neoplasm (MEN) 3 3 (13.0) 0 (0.0)

meanSD, *P <0.05

As a result, it was observed that the HuD-negative group significantly increased mitotic count and tumor size, and increased the dividing ability of the tumor compared to the HuD-positive group (FIG. 1).

1-2. Confirmation of cell division through cell cycle analysis

In HuD-negative and HuD-positive tissues, assayed by immunostaining as in 1-1. using an antibody specific for the cell cycle regulator p27 protein and the cell division marker Ki-67 (Santa Cruz Biotechnology, Inc.) I did. After staining, the DAB signal was converted to a digital image, and then the difference in protein between the two groups was quantitatively analyzed using Image J software.

As a result, it was confirmed that the HuD-negative group showed the characteristics of a decrease in p27 protein and an increase in Ki-67 compared to the HuD-positive group (FIG. 2). In addition, the correlation between the expression of HuD and p27 was analyzed using this, and it was confirmed that there is a positive correlation between HuD and p27.

Example 2. HuD Expression and pancreatic origin Neuroendocrine tumor Analysis of the patient's prognosis

HuD expression and disease-free survival (DFS) and progression free survival using clinical data of human pancreatic-derived neuroendocrine tumor patients classified according to lesions through the TNM (tumor-node-metastasis) classification method. ) Was tracked and analyzed using Excel and Graphpad.

As a result, the survival rate in the HuD-negative group was significantly decreased compared to the positive group, and it was found that the HuD-negative group had a poor prognosis (FIG. 3). In addition, in order to further verify the specificity of HuD protein in pancreatic neuroendocrine tumors, univariate analysis and multivariate analysis were analyzed for the clinical indicators of pancreatic neuroendocrine tumor patients according to the presence or absence of HuD protein. As a result, both analysis results confirmed that the relative expression level of HuD was significantly different from the size of pancreatic neuroendocrine tumor.

Univariate analysis Multivariate analysis N progress
n(%) Mean(month)
meanSE P hazard ratio
[95% CI] P HuD Negative 23 14 (60.9) 70±11 <0.001* 3.506 [1.246-9.871] 0.018* Positive 65 9 (13.8) 181±11 One Sex male 43 14 (32.6) 134±18 0.042* One female 45 9 (20.0) 114±10 0.28 [0.095-0.827] 0.021* Age (year) 0.998 [0.962-1.036] 0.931 Size () 1.298 [1.178-1.431] <0.001* 1.209 [1.071-1.385] 0.002* Grade by 2010 WHO classification G1 64 12 (18.7) 158±17 0.001* G2 20 7 (35.0) 88±14 G3 4 4 (100.0) 38±29 Ki-67 index (%) 1.042 [1.005-1.080] 0.025* 1.01 [0.963-1.058] 0.694 Mitotic count (10HPF) 1.101 [1.059-1.145] <0.001* Extent of invasion Limited in pancreas 63 10 (15.9) 148±21 <0.001* One Extended to other organ 25 13 (52.0) 70±12 1.113 [0.332-3.726] 0.862 Angioinvasion Absent 58 7 (12.1) 170±19 <0.001* One Present 30 16 (53.3) 70±11 3.338 [1.128-9.8722] 0.029* Perineural invasion Absent 73 16 (21.9) 133±18 0.005* One Present 15 7 (46.7) 46±8 0.842 [0.210-3.384] 0.843 pT (AJCC 7th ed) One 32 2 (6.2) 192±16 <0.001* 2 31 8 (25.8) 94±11 3 25 13 (52.0) 70±12 pN 0 80 19 (23.7) 129±18 0.002* One One 8 5 (62.5) 36±11 3.097 0.680-14.119] 0.144 pM 0 81 18 (22.2) 128 0.003* One One 7 5 (71.4) 59 2.029 [0.435-9.458] 0.368

by Cox regression analysis, hazard ratio [95% confidence interval], * P <0.05

Example 3. HuD Through suppression of expression Insulinoma Growth check

3-1. HuD Suppression of expression Insulinoma Cell viability check

ΒTC6, a mouse insulinoma cell line, was cultured in DMEM media containing 10% FBS and 1% antibiotics. HuD-specific siRNA (GCAUCCUGGUUGAUCAAGU) (Genolution Inc.) was injected into the cultured cell line in the form of liposomes using Lipofectamine 2000 (Invitrogen). Thereafter, after adding a 20mg/ml MTT solution to the cells, the survival rate of the cell line was confirmed through an MTT assay comparing the degree of formazane formation 16 hours later.

As a result, it was inferred that the survival rate of the insulinoma cell line that suppressed HuD expression with siRNA increased, and that inhibition of HuD increased the number of insulinoma cells (FIG. 4 ).

3-2. HuD Suppression of expression Insulinoma Cell cell cycle and cell growth check

As in Example 3-1 above, the insulinoma cell line was transfected with HuD siRNA and cultured for 48 hours. After fixing the cultured cells in an ethanol solution, cell cycle changes were analyzed using FACS equipment. In addition, the control siRNA-treated control group and the HuD siRNA-treated experimental group cells were inoculated into a culture dish in the same amount, cultured for 3 days, and then the total number of cells was investigated with a FACS device to analyze cell growth.

As a result, it was confirmed that in cells treated with HuD siRNA, cells in the G2/M phase of the cell cycle increased compared to the control, while the number of cells in the G0G1 phase decreased.Through this, the decrease in expression of HuD is caused by the cells of the insulinoma cell line. It was confirmed that cell growth was promoted and the number of cells increased by increasing division (FIG. 5).

3-3. HuD Suppression of expression Insulinoma Confirmation of cell growth

As in Example 3-1, control siRNA and HuD siRNA were each transfected into an insulinoma cell line, and then 100 cells were dispensed into a 35 mm culture dish, and the number of colonies formed by incubating for 3 weeks was confirmed.

As a result, it was found that the growth of insulinoma cells suppressing HuD expression using siRNA significantly increased compared to the control group (FIG. 6).

3-4. HuD Suppression of expression Insulinoma tumor Formation ability Confirm

Diabetic model mice were 6 weeks old BALB/c, and streptozotocin was injected at a dose of 200 mg/Kg. For transplantation of insulinoma cells, matrigel and insulinoma cells were mixed and injected into the subcutaneous tissue of mice three times a week, and this process was performed a total of 10 times. Tumor formation was observed for about 5 weeks after inoculation. All animal experiments were conducted in compliance with the Animal Experimental Research Ethics Regulations of Ajou University and Catholic University.

As a result, it was confirmed that in the case of insulinoma in which the expression of HuD was suppressed, the tumorigenic ability was significantly increased compared to the control cells that did not (FIG. 7 ).

Example 4. HuD Through overexpression Insulinoma growth Inhibitory ability Confirm

4-1. HuD Overexpression Insulinoma Cell viability check

In contrast to Example 3-1., it was attempted to confirm the growth of insulin species due to overexpression of HuD. Specifically, the myc-tagged HuD plasmid was transfected into the insulinoma cell line βTC6 using lipofectamine to induce overexpression of HuD, and then the cell viability of the HuD overexpressing cell line was confirmed through MTT assay.

As a result, the survival rate of the insulinoma cell line overexpressing HuD decreased by 40% or more, and it was inferred that HuD causes the death of the insulinoma (FIG. 8 ).

4-2. HuD Overexpression Insulinoma Cell cell cycle and cell growth check

Insulinoma cell line βTC6 was transfected with myc-tagged HuD plasmid to overexpress HuD, and then cell cycle and cell growth changes were analyzed in the same manner as in Example 4-2 using a FACS equipment.

As a result, when HuD was overexpressed, it was confirmed that the cells in the G2/M phase decrease and the cells in the G0/G1 phase increase compared to the control group, and it was confirmed that overexpression of HuD inhibits the growth of insulinoma cells (Fig. ).

4-3. HuD Overexpression Insulinoma Confirmation of cell growth

After transfection of the myc-tagged HuD plasmid into the insulinoma cell line βTC6, 100 cells were dispensed into a 35mm culture dish. Cell lines were cultured for 3 weeks after dispensing, and the number of colonies formed was checked.

As a result, the growth of the insulinoma cell line was decreased compared to the control group by overexpression of HuD (FIG. 10).

Claims (12)

A composition for prognosis diagnosis of pancreatic neuroendocrine tumor, comprising an agent for measuring the expression level of the HuD gene. The composition of claim 1, wherein the measurement of the expression level of the HuD gene is a measurement of the mRNA level of the HuD gene or the level of the HuD protein. According to claim 1, wherein the pancreatic neuroendocrine tumor is insulinoma, the composition for prognosis diagnosis of a pancreatic neuroendocrine tumor. A composition for prognosing a pancreatic neuroendocrine tumor, comprising an antibody against the HuD protein. The composition for prognostic diagnosis of a pancreatic neuroendocrine tumor according to claim 4, wherein the pancreatic neuroendocrine tumor is insulinoma. delete delete delete A method of providing information for prognosis diagnosis of a patient with pancreatic neuroendocrine tumor, comprising the step of measuring the amount of HuD expression in a sample isolated from the human body. The method of claim 9, wherein the prognosis of the pancreatic neuroendocrine tumor patient is disease free survival (DFS) or a progression free survival rate (DFS). . The method of claim 9, wherein the HuD expression level is measured by measuring the mRNA level of the HuD gene or the level of the HuD protein. The method of claim 9, wherein the pancreatic neuroendocrine tumor is an insulinoma. The method of providing information for prognostic diagnosis of a patient with pancreatic neuroendocrine tumor.

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