A biomarker related to hedgehog activation of pancreatic cancer neural remodeling and its application

Abstract

The present application relates to the field of genetic engineering, and particularly relates to application of circular RNA (circ0011536) as a biomarker in prediction of nerve remodeling of Hedgehog-activated pancreatic cancer and development of a targeted drug. The present application provides a biomarker circ0011536 for predicting nerve remodeling of Hedgehog-activated pancreatic cancer, and discloses that inhibiting expression level of circ0011536 of cancer cells will effectively improve nerve-related pain of Hedgehog-activated pancreatic cancer, and provides a theoretical basis for later development of a targeted drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular, the application of circular RNA (circ0011536) as a biomarker in predicting nerve remodeling of Hedgehog-activated pancreatic cancer and developing targeted drugs. BACKGROUND

[0002] Peripheral nerve remodeling is one of the main reasons for early metastasis and postoperative recurrence of pancreatic tumors; it is worth further studying how tumor cells affect the peripheral nerves of tumors; Sonic Hedgehog (Hh) signaling pathway activation is a characteristic event in the early stage of pancreatic cancer; recent studies have shown that the activation of its transcription factor Gli1 is closely related to peripheral nerve remodeling and neuropathic pain.

[0003] Exosomes are vesicular structures with lipid bilayers secreted by tumor cells, which can carry proteins, nucleic acids, IncRNA and circRNA, etc. Signal molecules participate in the communication between tumor cells and distant microenvironment cells including peripheral nerve cells. Exosomal circRNA has been shown to play a crucial role in the development of human cancer. For example, circ0006156 is highly expressed in exosomes of thyroid tumor tissues and cell lines as well as serum samples, and its detection value is increased in the above-mentioned samples, which may be a new biomarker. In addition, exosomal circ-PDE8A released by tumor cells further regulates colon cancer metastasis by suppressing miR-338 and is closely related to the invasive growth of pancreatic ductal adenocarcinoma. In addition, exosomal circRNA0001445 acts as a suppressor of miRNA-127-5p to increase the expression of nexin 5, and then promotes the progression of glioma.

[0004] However, there is no report on the correlation between circular RNA (circ0011536) as a new biomarker and Hedgehog-Gli1-activated neuropathic pain in pancreatic cancer. SUMMARY

[0005] The present application aims to provide a biomarker circ0011536 that can predict the neuropathic pain of Hedgehog-activated pancreatic cancer and is closely related to its pathological characteristics, and reveals that inhibiting the expression level of circ0011536 in cancer cells will effectively improve the nerve remodeling of Hedgehog-activated pancreatic cancer.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] (1) In vivo and in vitro model to discover the function of circ0011536 in predicting Hedgehog-Gli1-activated pancreatic cancer nerve remodeling

[0008] ① In vitro construction of pancreatic cancer associated nerve cell remodeling model (PANR); ② The expression of circ0011536 in the plasma of pancreatic cancer patients and the exosomes secreted by pancreatic cancer cells is significantly increased; ③ The level of circ0011536 in serum as a diagnostic biomarker for hedgehog-Gli1 activation of pancreatic cancer mouse model nerve remodeling.

[0009] (2) Study on the regulatory mechanism of circ0011536 affecting dorsal root ganglion DRG nerve remodeling

[0010] ① Through the PANR model constructed in vitro, it is found that circ0011536 affects the neuron plasticity of DRG dorsal root ganglion, including the length of the axon of the nerve cell and the apoptosis rate of the cell; ② In terms of regulatory mechanism, circ0011536 regulates the expression of nerve growth factor-induced protein VGF by competitively inhibiting miR-451a endogenous up-regulation, thereby further affecting the nerve remodeling of DRG cells.

[0011] Based on the above technical scheme, the first aspect of the present application provides a biomarker for predicting Hedgehog-activated pancreatic cancer nerve remodeling, wherein the biomarker is a circular RNA: hsa_circ_0011536 (hereinafter referred to as circ0011536); the nucleotide sequence is shown as SEQ ID NO. 1.

[0012] The second aspect of the present application provides the use of circ0011536 as a biomarker in the preparation of a kit for predicting Hedgehog-activated pancreatic cancer nerve remodeling.

[0013] Further, the use of a reagent for detecting the expression amount of circ0011536 in serum in the preparation of a kit for predicting Hedgehog-activated pancreatic cancer nerve remodeling.

[0014] The third aspect of the present application provides the use of a reagent for detecting the expression amount of circ0011536 in the preparation of a kit for predicting Hedgehog-activated pancreatic cancer neuropathic pain.

[0015] Further, patients with high expression of circ0011536 in the primary tumor are more likely to have Hedgehog-activated pancreatic cancer neuropathic pain.

[0016] The fourth aspect of the present application provides the use of a reagent for detecting the expression amount of serum circ0011536 in the preparation of a kit for predicting the tumor size and clinical stage of Hedgehog-activated pancreatic cancer patients.

[0017] In a fifth aspect of the present application, there is provided use of circ0011536 in the preparation of a medicament for promoting axonal remodeling of nerve cells of Hedgehog-activated pancreatic cancer.

[0018] In a sixth aspect of the present application, there is provided use of an agent for inhibiting circ0011536 in the preparation of a medicament for improving neuropathic pain of Hedgehog-activated pancreatic cancer.

[0019] Further, inhibiting the expression level of circ0011536 of cancer cells will effectively improve the neuropathic pain of Hedgehog-Gli1-activated pancreatic cancer.

[0020] In a seventh aspect of the present application, there is provided a medicament for improving neuropathic pain of Hedgehog-activated pancreatic cancer, wherein the active ingredient of the medicament is an agent for inhibiting circ0011536.

[0021] Still further, the agent for inhibiting circ0011536 is an siRNA small interfering fragment:

[0022] si-circ0011536-1: 5'-ATGCTCAAGTGGTGGTATCAT-3' (SEQ ID NO. 2);

[0023] si-circ0011536-2: 5'-GACAATGCTCAAGTGGTGGTA-3' (SEQ ID NO. 3).

[0024] The present application has the following advantages:

[0025] 1. The present application discusses the regulation mechanism and significance of circ0011536 in Hedgehog-Gli1-activated pancreatic cancer cells through exosomes to regulate VGF protein of nerve cells;

[0026] 2. The conclusion of the present application can be used to explain the reason why high expression of circ0011536 is widespread in Hedgehog-Gli1-activated pancreatic cancer cells;

[0027] 3. The present application provides a theoretical basis for evaluating the value of circ0011536 in clinical samples as a new target for diagnosis, prognosis, and treatment of Hedgehog-Gli1-activated pancreatic cancer cells;

[0028] 4. The application relates to the application of circular RNA (circ0011536) in predicting nerve remodeling of Hedgehog-Gli1-activated pancreatic cancer and development of a targeted drug. Overexpression of circ0011536 in pancreatic cancer cells can be transported to nerve cells surrounding the pancreatic cancer through exosomes, and further promoted through a circ0011536-miR-451a-VGF axis to promote axon remodeling of the nerve cells. Patients with high expression of circ0011536 in primary tumors are more likely to have nerve pain of Hedgehog-activated pancreatic cancer. The application provides a biomarker circ0011536 for predicting nerve remodeling of Hedgehog-activated pancreatic cancer, and discloses that inhibiting the expression level of circ0011536 in cancer cells will effectively improve nerve-related pain of Hedgehog-Gli1-activated pancreatic cancer, and provides a theoretical basis for later targeted drug research and development. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Inducing effect of Gli1 on an in-vitro co-culture model of pancreatic tumor peripheral nerve invasion. Figure 1 A: The protein level of Gli1 lentivirus overexpression in cfpacl and panc-1 pancreatic cancer cells is detected by western blot technology; Figure 1 B and Figure 1 C: The mRNA expression level of Gli1 in cfpacl and panc-1 cells is quantitatively detected by RT-PCR; Figure 1 D and Figure 1 E: By using an in-vitro living cell experiment, the growth of axons of newborn mouse DRG cells in each treatment group is observed under a microscope by an in-vitro co-culture model; Figure 1 F and Figure 1 G: By using a flow cytometry apoptosis experiment, the apoptosis rate of mouse DRG cells in each group and the expression of apoptosis-related protein CASP3 are detected by an in-vitro co-culture model.

[0030] Figure 2 Effect of Gli1 on circrna of pancreatic cancer cells is analyzed by whole transcriptome sequencing. Figure 2 A: The distribution of cfpacl circrna is analyzed by whole transcriptome sequencing. Figure 2 B- Figure 2 F: The expression of different differential circrnas in each pancreatic cancer cell strain (*P<0.05, **P<0.01 vs. control group; n=3).

[0031] Figure 3 Exosome identification and verification of high expression and transfection efficiency of has_circ_0011536 circrna.Figure 3 A and Figure E: Identification of cfpacl and panc-1 cell and pancreatic cancer tissue and cancer exosome extraction purity by western blot. Figure 3 B and Figure 3 F: Further detection of has_circ_0011536 expression in exosomes secreted by human pancreatic cancer cells and tissues by RT-PCR. Figure 3 C and Figure 3 D: Overexpression of has_circ_0011536 by lentivirus and detection of its expression efficiency in cfpacl and panc-1 cells (**P <0.01 vs control group; n = 3).

[0032] Figure 4 .Gli1 affects peripheral nerve axon changes and apoptosis levels through has_circ_0011536 circrna expression levels. Figure 4 A: Effect of Gli1 different expression groups on has_circ_0011536 circrna expression levels. Figure 4 B: Apoptosis effect of different treatment groups on DRG cells. Figure 4 C and Figure 4 D: Regulation effect of different treatment groups on DRG cell axon morphology and length.

[0033] Figure 5 .has_circ_0011536 circrna inhibits nerve growth factor-induced protein VGF expression levels by targeting miR-451a. Figure 5 A: Use multiple databases to find the potential targeting miR-451a of has_circ_0011536 circrna. Figure 5 B: Respectively show the binding sites and mutation sites of has_circ_0011536 circrna and miR-451a; miR-451a and its target gene VGF, and design the mutation sites of miR and VGF target gene. Figure 5 C and Figure 5 D: Detect luciferase activity of wild type and mutant binding sites between different groups by luciferase experiment. Figure 5 E: Western blot detection of the effect of overexpression of has_circ_0011536 circrna on DRG cells on VGF protein levels. Figure 5 F: Effect of has_circ_0011536 circrna and miR-451a on DRG cells on VGF protein levels, β-actin as reference protein. DETAILED DESCRIPTION

[0034] The specific embodiments provided by the present application are described in detail below with reference to the accompanying examples.

[0035] Examples:

[0036] I. Experimental materials and methods

[0037] 1. To explore the regulatory mechanism of circ0011536 on the stable expression of nerve growth factor-induced protein VGF

[0038] (1) Transwell chamber was used to construct the in vitro model of pancreatic cancer nerve remodeling

[0039] ①Hydrated basement membrane: add 70ul of serum-free medium to each well, 37℃ for 30min, and remove the medium;

[0040] ②In vitro culture of CFPAC-1 and Panc-1 pancreatic cancer cells with different interference and overexpression vectors, using logarithmic growth phase pancreatic cancer cells, trypsinizing the cells, suspending in 10% serum-containing medium, counting, diluting to a density of 1×10 5 / ml, adding 200ul of cell suspension to each well;

[0041] ③The company provides in vitro primary extraction of milk mouse DRG nerve cells, 600ul of medium containing 10% newborn calf serum is added to the lower chamber of the 24-well plate, and 2-week-old primary milk mouse DRG nerve cells are inoculated to a density of 1×10 3 / ml;

[0042] ④Conduct continuous live cell imaging experiments under a microscope for 7 days, observe under a microscope every 4 hours, obtain representative pictures, use professional software to calculate the axon length of DRG nerve cells, and use an apoptosis kit to detect the cell activity of in vitro DRG in different groups.

[0043] (2) Lentivirus construction:

[0044] Lenti-Gli1 lentivirus overexpression vector and si-circ0011536 small RNA interference vector were purchased from a commercial company, and circ0011536 different expression human pancreatic cancer cell lines Lenti-Gli1-CFPAC-1 and Lenti-Gli1-Panc-1 were cultured in vitro; On this basis, CFPAC-1 (si-circ0011536) and Panc-1 (si-circ0011536) cell lines with low expression of circ0011536 were established, and the subsequent protein immunoblotting detection analyzed the regulatory effect of circ0011536 in pancreatic cancer cell secreted exosomes on the expression of nerve growth factor-induced protein VGF protein in DRG (dorsal root ganglion cells).

[0045] (3) Western blotting:

[0046] ① After the cell protein extraction, the glue is filled and loaded:

[0047] After the glass plate is aligned, it is clamped in the clamp;

[0048] Then vertically clamp on the shelf ready to fill the glue;

[0049] According to the previous method, prepare an appropriate amount of concentrated separation glue, add TEMED immediately after shaking evenly to fill the glue;

[0050] When filling the glue, use a 10mL syringe to take 5mL glue along the glass, and when the glue surface rises to the middle line height of the green belt, it is ready;

[0051] Then add a layer of water on the glue, and the glue will solidify faster after liquid sealing;

[0052] When there is a refractive line between water and glue, it means the glue has solidified;

[0053] Wait for 3 minutes to make the glue fully solidify, then pour away the water on the glue and use a water-absorbing paper to dry the water;

[0054] Prepare 4% concentrated glue according to the previous method, add TEMED immediately after shaking evenly to fill the glue;

[0055] Fill the remaining space with concentrated glue and then insert the comb into the concentrated glue;

[0056] When filling the glue, the glue should also flow down the glass plate to prevent air bubbles from forming in the glue;

[0057] When inserting the comb, make sure the comb is horizontal;

[0058] Because the volume of the glue will shrink when it solidifies, the loading volume of the sample well will decrease, so the glue should be replenished frequently on both sides during the solidification of the concentrated glue;

[0059] After the concentrated glue solidifies, hold the two sides of the comb vertically and gently pull it out;

[0060] Rinse the concentrated glue with water and place it in the electrophoresis tank;

[0061] According to the protein quantification results, calculate the volume of the solution containing 50mg of protein as the loading volume;

[0062] Take the loading sample to a 0.5mL centrifuge tube, add 5×SDS loading buffer to a final concentration of 1×.

[0063] Before loading, boil the sample in boiling water for 5min to denature the protein;

[0064] Load, electrophorese, the electrophoresis time is generally 1-3h, and the voltage is preferably 80V to 120V;

[0065] The electrophoresis is terminated when the bromophenol blue runs out, and the membrane is transferred.

[0066] ② Transfer membrane:

[0067] Cut the PVDF membrane and hydrate it in methanol for 30 minutes;

[0068] Put the clamp for transferring membrane, two sponge pads, a glass rod, filter paper and the soaked membrane in the porcelain dish with transfer solution;

[0069] Open the clamp and keep the black side horizontal;

[0070] Put a sponge pad on it, and peel off the glue after removing the glass plate. Gently scrape off the concentrated glue;

[0071] Carefully peel off the separating glue and cover it on the filter paper. Adjust it to be aligned with the filter paper, and gently roll it with a glass rod to remove air bubbles;

[0072] Cover the membrane on the glue, and make sure it covers the entire glue (do not move it after covering) and remove air bubbles;

[0073] Cover 3 filter papers on the membrane and remove air bubbles;

[0074] Finally, cover another sponge pad and roll it a few times to close the clamp;

[0075] The entire operation is carried out in the transfer solution;

[0076] Put the clamp into the transfer slot, and make sure the black side of the clamp faces the black side of the slot, and the white side of the clamp faces the red side of the slot;

[0077] Heat is generated during the electric transfer, so put an ice block on one side of the slot to cool it down;

[0078] Transfer for 30 minutes at 20V;

[0079] After the transfer, dye the membrane with 1x eosin solution for 5 minutes (shake on the decolorizing shaker);

[0080] Then wash off the dye that has not been dyed to see the proteins on the membrane;

[0081] Dry the membrane and use it.

[0082] ③ Immunoreaction:

[0083] After soaking the membrane with TBS from bottom to top, move it to the dish containing the blocking solution, and shake on the decolorizing shaker at room temperature for 1 hour;

[0084] Dilute the primary antibody with TBST to the appropriate concentration (in a 1.5 mL centrifuge tube);

[0085] Tear off a piece of plastic wrap of appropriate size and lay it on the experimental table, and soak the four corners with water to keep the plastic wrap flat.

[0086] Add the antibody solution to the plastic wrap;

[0087] Remove the membrane from the blocking solution, and place it on the antibody solution with the protein side down. Move the four corners of the membrane to remove the remaining bubbles.

[0088] Incubate at room temperature for 1-2 hours, and then wash twice with TBST at room temperature for 10 minutes each time.

[0089] Wash once more with TBS for 10 minutes.

[0090] Prepare the secondary antibody diluent in the same way as above, and contact it with the membrane. Incubate at room temperature for 1-2 hours, and then wash twice with TBST at room temperature for 10 minutes each time.

[0091] Wash twice with TBST at room temperature for 10 minutes each time.

[0092] Wash once more with TBS for 10 minutes.

[0093] Scan the membrane, and analyze the target band net optical density value using a related image processing system.

[0094] 2. Processing of tissue samples, tissue sectioning, extraction of tissue RNA and protein:

[0095] According to the different activation degrees of Hedgehog and whether there is combined nerve pain in patients with primary pancreatic cancer (100 cases), the plasma, cancer and paracancer tissues of the patients were obtained after surgery. The plasma part was extracted by a highly commercialized kit to extract exosomes, and the expression level of circ0011536 in each group of different patients was detected by PCR. In addition, the patient tissue part was quickly placed in liquid nitrogen for storage, and total RNA and total protein were extracted and quantified and stored at -20°C for Real time RT-PCR and Western Blotting detection. Another part was fixed with 4% paraformaldehyde, and subsequent ultra-thin paraffin tissue sections were made for pathological hematoxylin-eosin staining (HE staining) and immunohistochemical staining analysis. The above two parts of the experiment were used to detect the expression levels of circ0011536, miR-451a and VGF molecules, and to analyze the correlation between circ0011536 and the density of peripheral nerve invasion and the clinical pathological characteristics of Hedgehog-activated pancreatic cancer in each group.

[0096] II. Experimental results

[0097] 1. In vitro construction of pancreatic cancer-related nerve cell remodeling model (PANR).

[0098] To further investigate the potential molecular changes in neuronal plasticity associated with pancreatic cancer, we constructed an in vitro co-culture model. In addition to the intrapancreatic ganglia, the innervation of the pancreas is mainly performed by the autonomic nervous system (parasympathetic and sympathetic nerves), so according to the feasibility reported in previous literature [6Kiba, 2004; Salvioli et al., 2002], we used dorsal root ganglion (DRG) cells as neuronal elements to predict the molecular and functional changes in PANR.

[0099] To further investigate the functional effects of hedgehog-gli1 signaling in PANR neuronal plasticity, we generated gli1 overexpression constructs in CFPAC-1 and Panc-1 cell lines Figure 1 A-1C).

[0100] Transwell co-culture system showed that, similar to PDAC cells stimulated with nerve growth factor (NGF; Figure 1 D and 1E), PDAC cells with Gli1 overexpression promoted axon extension in DRG; in addition, Lenti-Gli1-CFPAC-1 and Lenti-Gli1-PANC-1 cells also protected DRG from serum starvation-induced apoptosis, as shown by Annexin V / PI test Figure 1 F). At the same time, the expression of activated caspase-3 in DRG cells was significantly reduced when co-cultured with Lenti-Gli1-CFPAC-1 and Lenti-Gli1-PANC-1 cells, which was comparable to the effect of NGF Figure 1 G). The above results showed that PDAC cells with high expression of Gli1 could change the plasticity of neurons in DRG cells.

[0101] 2. The expression of circ0011536 in the plasma of pancreatic cancer patients and the exosomes secreted by pancreatic cancer cells was significantly increased.

[0102] Tumor cells can affect the function of target cells by secreting exosomes. Exosomal circular RNA can play a communication role and promote the development of nerve cells and myelin synapses. Therefore, we further investigated the molecular mechanism of Gli1 overexpressed PDAC cells acting on the neuronal plasticity of DRG cells through circular RNA sequencing. In this sequencing experiment, we found that 86.14% of CircRNA came from exons Figure 2A). Among them, 23 down-regulated and 16 up-regulated CircRNAs were identified in the exosomes from Lenti-Gli1-CFPAC-1 cells compared with control cells. To verify the sequencing results, we selected 5 of them and further found that circ0011536 was significantly up-regulated in Lenti-Gli1-CFPAC-1 and Lenti-Gli1-PANC-1 cells by RT-PCR experiment Figure 2 B-2F).

[0103] Next, we detected the expression level of each circ0011536 in the exosomes of Gli1 differentially expressed pancreatic cancer cells. First, we isolated the exosomes from the culture medium of Lenti EV and Lenti-Gli1 PDAC cells (CFPAC-1 and PANC-1). The purity of the identified exosomes was further verified by Western blotting. Western blotting analysis showed that CD81 and CD63 were enriched in exosome vesicles Figure 3 A). Second, qRT-PCR data detected the expression of circ0011536 in the corresponding exosomes Figure 3 B). The expression of circ0011536 in the exosomes of Lenti-Gli1 cells was much higher than that in the exosomes of Lenti-EV Figure 3 C). In addition, overexpression of circ0011536 in Lenti-Gli1-CFPAC-1 and Lenti-Gli1-PANC-1 cells can significantly up-regulate the expression level of circ0011536 in exosomes Figure 3 D). The data showed that changing the level of circ0011536 in cells can affect the concentration of circ0011536 in exosomes.

[0104] In addition, we extracted the plasma exosomes of pancreatic cancer patients (PDCA, T-exo) and compared them with healthy controls (n-exo, n = 45, Figure 3 E and 3F). Compared with n-exo, the expression of circ0011536 in serum exosomes of PDCA patients was significantly increased. In addition, the expression of serum exosome circ0011536 was related to the tumor size and clinical stage of PDCA patients (Table 1). Therefore, these results suggest that the exosomes derived from PDCA cells are rich in circ0011536 and can exert its special function in the form of exosomes.

[0105] Table 1. Correlation of plasma exosome circ0011536 of PDAC patients with clinicopathological features of pancreatic cancer tissue (n = 44)

[0106]

[0107] Abbreviations: HBsAg, hepatitis B surface antigen; NA, not available; AFP, alpha-fetoprotein; a P<0.05.

[0108] 3. Circ0011536 affects DRG cell neuron plasticity

[0109] First, circ0011536 in the overexpression group of circ0011536-DRG cells (oe-circ0011536-DRG) was significantly higher than that in oe-NC DRG cells. At the same time, in DRG cells co-cultured with exosomes derived from Lenti-Gli1, (oe-NC-Exo) was significantly higher than that in oe-NC DRG cells Figure 4 A). Next, we studied the effect of circ0011536 on DRG cell survival and neuron plasticity. oe-NC DRG cells compared with oe-circ0011536, prevented DRG cell survival and its axon extension Figure 4 B-4D). However, Lenti-Gli1-PDAC cell-induced oe-NC DRG cells were significantly enhanced in cell survival and axon extension after overexpression of circ0011536 Figure 4 B-4D), indicating that Gli1 overexpressed pancreatic cancer cell exosomes can affect DRG neuron plasticity and DRG cell survival through circ0011536.

[0110] 4. Circ0011536 endogenously up-regulates VGF expression by competitively inhibiting miR-451a

[0111] To further study the molecular mechanism of Circ0011536 affecting DRG cell neuroplasticity, we used StarBase and Circinteractome to predict miRNAs that may be complementary to Circ0011536, Figure 5 A shows that circ0011536 binds to miR-451a. In addition, StarBase, miRTABASE and miRDB can predict that the downstream mRNA target of miR-451a may be VGF Figure 5 A). Subsequently, luciferase reporter assay confirmed that miR-451a is the targeted mirna of Circ0011536 Figure 5 B). At the same time, the luciferase signal of VGF vector can be significantly inhibited by miR-451a, but has no effect on VGF-MUT vectorFigure 5 C). In addition, Western blotting and qRT-PCR further verified the expression changes of miR-451a and VGF after overexpression of oe-Circ0011536 in DRG cells Figure 5 D and 5E). In addition, miR-451a and Circ0011536 were co-transfected into DRG cells. We found that overexpression of miR-451a inhibited VGF levels in DRG cells when co-transfected with Circ0011536, while the Circ0011536 overexpression vector attenuated the inhibitory function of mir-451a on VGF Figure 5 F). These results identified Circ0011536 by competitively inhibiting miR-451a to promote the expression of VGF in DRG cells.

[0112] 5. Circ0011536 level in serum as a diagnostic biomarker for nerve remodeling in hedgehog-Gli1 activated pancreatic cancer mouse model

[0113] To determine whether circ0011536 is a key factor for hedgehog-Gli1 pancreatic cancer nerve remodeling, we detected the expression of circ0011536 in the serum of pancreatic cancer mice by constructing a Lenti-Gli1-CFPAC-1 mouse subcutaneous pancreatic tumor model. We found that the expression of circ0011536 in the serum of Lenti-Gli1 pancreatic cancer mice was significantly up-regulated compared with normal and acute pancreatitis mice. In addition, we found that the expression level of circ0011536 was positively correlated with the intratumoral nerve density (R=0.757, P=0.004). Finally, we evaluated whether circ0011536 directly affects intratumoral nerve density in vivo. By interfering with the expression level of circ0011536 in mice in si-circ0011536, in the uninterfered group, the intratumoral nerve invasion density of the circ0011536 high expression group was higher than that of the circ0011536 low expression group. When the level of circ0011536 in the circ0011536 high expression group was reduced, the intratumoral nerve invasion density of the mouse was significantly reduced. These data determined that circ0011536 directly promoted the intratumoral nerve density in Lenti-Gli1-CFPAC-1 pancreatic cancer cells. The amount of circ0011536 in serum can be used as a biomarker to predict the nerve remodeling of hedgehog-GLI1 activated pancreatic cancer.

[0114] The preferred embodiments of the present application have been disclosed with the above particularity, but the application is not limited to the embodiments disclosed, and variations and modifications can be made by those skilled in the art without deviating from the spirit of the application, and such variations and modifications are intended to be included within the scope of the application as defined in the following claims.

Claims

1. Use of an agent for inhibiting circ0011536 in the preparation of a drug for improving neuropathic pain of Hedgehog-Gli1 activated pancreatic cancer, wherein the agent for inhibiting circ0011536 is a small interfering RNA fragment, and the nucleotide sequence of the small interfering RNA fragment is shown in SEQ ID NO. 2 or SEQ ID NO. 3.

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