CN114606315A

CN114606315A - Papillary thyroid carcinoma biomarker and application thereof

Info

Publication number: CN114606315A
Application number: CN202011450689.XA
Authority: CN
Inventors: 朱欣
Original assignee: Zhejiang Cancer Hospital
Current assignee: Zhejiang Cancer Hospital
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2022-06-10

Abstract

The invention relates to a thyroid papillary carcinoma biomarker and application of a PIM1 gene inhibitor in preparation of an anti-thyroid papillary carcinoma preparation, wherein the biomarker is PIM1 protein regulated and controlled by BRAF V600E mutation. The invention researches the functions of PIM1 gene and BRAF V600E mutation in the process of PTC pathological development, finds that PIM1 possibly plays an important carcinogenic role in the generation and development of PTC, and the function of PIM1 in PTC is regulated and controlled by upstream BRAF V600E mutation and NOX4, namely, the BRAF V600E mutation regulates NOX4, and then the NOX4 regulates PIM 1. Therefore, the invention has important significance and value for the theoretical research of the pathogenesis of the PTC, and the biomarker in the invention can be used as the marker for PTC diagnosis, thereby improving the accuracy of the PTC diagnosis.

Description

Papillary thyroid carcinoma biomarker and application thereof

Technical Field

The invention relates to the field of biotechnology and oncology, in particular to a papillary thyroid cancer biomarker and application thereof.

Background

Papillary Thyroid Carcinoma (PTC) is one of the thyroid diseases, is relatively common in thyroid diseases, and accounts for about 80% of thyroid cancer (ATC). Papillary thyroid carcinoma is commonly seen in children or female patients under the age of 40, and the papillary thyroid carcinoma has slow tumor growth and can be limited in the thyroid gland for several years, but can also metastasize from the origin to other parts of the gland or lymph nodes.

In recent years, PIM1 has been found to be an oncogene that exerts a cancer-promoting effect in various tumors such as breast cancer, prostate cancer, skin cancer, esophageal cancer, and liver cancer. The protooncogene PIM1 is positioned on the long arm of chromosome 6 (6p 21.1-p 21.31), the expression product PIM1 protein has serine/threonine protein kinase activity, and PIM1 can exert carcinogenic effect by phosphorylating a series of downstream substrates. The substrates of PIM1 that have been suggested so far include BAD, Skp-2, p21AWF, CXCR4, STAT3, Eif4B, RUNX3, and the like, but the mechanism of action of PIM1 has not yet been fully elucidated.

The pathogenesis of papillary thyroid cancer is the focus of research in the field of oncology in recent years, and for example, the disclosures of patents such as a chinese patent invention with patent number ZL 201510574162.0(CN105039581B), a SNP marker related to the risk of onset of female PTC, and applications thereof, a chinese patent application with application number CN 201911348184.X (publication number CN110982901A), a circulating circular RNA marker and applications for invasive papillary thyroid cancer diagnosis, and a chinese patent invention with application number CN 201911415563.6 (publication number CN 111079862a), a thyroid papillary cancer pathological image classification method based on deep learning, and the like.

In addition, BRAF mutations are one of the most important molecular events in thyroid cancer, with BRAF mutations in about 60% of PTCs. Normally, a BRAF protein has kinase activity only after phosphorylation by RAS kinase, but after activation mutation of BRAF gene, the mutated BRAF protein is always in an activated state. The most common mutation in all BRAF oncogenes, the 600 th amino acid residue of BRAF, changes from valine (V) to glutamic acid (E), i.e., the V600E mutation, which leaves the BRAF protein in an activated, kinase-active state at all times. In addition, research finds that the mutation of BRAF V600E can play an important role in the occurrence and development of PTC by regulating NOX 4.

Disclosure of Invention

The first technical problem to be solved by the invention is to provide a papillary thyroid cancer biomarker aiming at the prior art.

The second technical problem to be solved by the present invention is to provide an application of the above biomarker in papillary thyroid cancer in view of the prior art.

The technical scheme adopted by the invention for solving the first technical problem is as follows: a papillary thyroid carcinoma biomarker which is a PIM1 protein regulated by a BRAF V600E mutation.

PIM1 is an oncogene (PIM1Human 5292 NM-001243186) that plays a cancer promoting role in various tumors such as breast cancer, prostate cancer, skin cancer, esophageal cancer, and liver cancer. The protooncogene PIM1 is positioned on the long arm of chromosome 6 (6p 21.1-p 21.31), the expression product PIM1 protein has serine/threonine protein kinase activity, and PIM1 can exert carcinogenic effect by phosphorylating a series of downstream substrates. The substrates of PIM1 that have been proposed so far include BAD, Skp-2, p21AWF, CXCR4, STAT3, Eif4B, RUNX3, and the like.

Further, the mutation of BRAF V600E regulates the expression of NOX4, while the expression of PIM1 protein is regulated by NOX 4.

Further, the expression level of the PIM1 protein is positively correlated with the NOX4 protein level.

The technical solution adopted to further solve the second technical problem is as follows: an application of PIM1 gene inhibitor in preparing anti-thyroid papillary carcinoma preparation is provided.

Further, the inhibitor is PIM1siRNA for resisting PIM1 gene expression, and the sequence of the siRNA is as follows:

SEQ ID NO.1(5‘-(CAAGAUCUCUUCGACUUCA)dTdT-3’)、

SEQ IDNO.2(5‘-(UGAAGUCGAAGAGAUCUUG)dTdT-3’)。

further, the inhibitor is NOX4iRNA expressed by anti-NOX 4 gene, and the sequence is as follows:

SEQ ID NO.3(5‘-(CAUCUGUUCUUAACCUCAA)dTdT-3’)、

SEQ IDNO.4(5‘-(UUGAGGUUAAGAACAGAUG)dTdT-3’)。

further, the inhibitor is at least one of SGI-1776, GLX351322 or PLX 4032.

The research of the invention finds that PIM1 is over-expressed in PTC tissues, and the expression of the PIM1 is obviously related to clinical parameters such as lymph node metastasis, envelope infiltration, tumor size and the like. Meanwhile, the results of analysis of 501 patients with PTC in TCGA database show that the total survival rate (OS) and the progression-free survival rate (DFS) of the patients with high PIM1 expression are obviously lower than those of the patients with low PIM1 expression. It can be seen that the expression of PIM1 is closely related to the malignant phenotype and prognosis of PTC, and PIM1 plays an important role in PTC.

In vitro tests of the invention find that the PIM1 levels of two PTC cell lines (BCPAP and TPC-1) are obviously higher than those of normal thyroid cells, and the BCPAP cells and TPC-1 cells are treated by using a PIM1 inhibitor SGI-1776, which shows that SGI-1776 has inhibition effect on the proliferation and movement capacity of the two PTC cells and can induce apoptosis, and PIM1 plays an important carcinogenic role in the generation and development of PTC.

PIM1 also plays an important role in oxidative stress, and the increase of the production of active oxygen is detected after the SGI-1776 is adopted to treat BCPAP cells and TPC-1 cells, so that PIM1 can be involved in the generation and development of PTC by regulating ROS.

Further, the present study found that NOX4is significantly more expressed in PTC tissues than in neighboring normal tissues. The expression level of NOX4 was also associated with various clinical pathological features, with higher expression of NOX4 in patients with late T stage, lymph node metastasis and regional invasion than in patients with early T stage, no lymph node metastasis and non-regional invasion, further suggesting a carcinogenic role for NOX4 in PTC.

Furthermore, analysis of 501 PTC patients in the TCGA database found significant correlation between PIM1 and NOX4 expression. Dividing 501 patients with PTC into in situ PTC 493 and transfer PTC 8, expression of PIM1 and NOX4 were significantly correlated in both groups. Furthermore, the results of treatment of BCPAP and TPC-1 cells with NOX4 inhibitor (GLX351322) showed that PIM1 expression was down-regulated in PTC cells, and similar results were obtained with PTC cells treated with NOX4siRNA, and histological results also showed that PIM1 was associated with NOX 4. PIM1 is known to be regulated by NOX4 in PTC, thereby playing a role in oxidative damage.

Increased ROS production has been detected in a variety of malignancies and can activate tumorigenic signals. PIM1 kinase has been reported to reduce cellular ROS levels by promoting antioxidant gene expression resulting from enhanced NRF2/ARE activity, thereby avoiding tumor cell death. The present study found that cells lacking PIM1 kinase or treated with PIM1 inhibitors can lead to the accumulation of lethal ROS, ultimately leading to cellular intolerance and death, suggesting that PIM1 kinase is an important regulator of cellular redox signaling. In the present invention, increased ROS production following SGI-1776 treatment of PTC cells suggests that PIM1 may down-regulate ROS.

The oncology database shows that the expression of SOD2 and GPX2 in the thyroid cancer antioxidant enzyme system is higher than that in normal thyroid tissue. The immunohistochemical results of the invention show that the expression of SOD2 and GPX2 in PTCs tissues is obviously higher than that in normal thyroid tissues. Therefore, SOD2 and GPX2 in PTC may play a major role in the removal of ROS. In the invention, after PIM1 in PTC cells is knocked out by PIM1siRNA, expression of SOD2 and GPX2 is found to be reduced. In addition, NOX4 was knocked out in PTC cells with NOX4siRNA, and PIM1, SOD2 and GPX2 were all found to be down-regulated. In addition, histological results also showed that PIM1 was associated with SOD2 and GPX 2. It can be seen that PIM1 can be regulated by NOX4, which in turn regulates SOD2 and GPX2, thereby avoiding the accumulation of lethal ROS leading to tumor progression.

In the invention, PLX4032 is adopted to treat four thyroid cancer cell strains (TPC-1, BCPAP, CAL-62 and 8505C), and the result shows that the expression of PIM1 in the BRAF V600E mutant cell strains (BCPAP and 8505C) is obviously reduced, but PLX4032 has no obvious influence on the expression of PIM1 in the BRAF wild type cell strains (TPC-1 and CAL-62). It can be seen that BRAF V600E in PTC cells and ATC cells regulates the expression of PIM 1.

It is well known that BRAF V600E mutations lead to constitutive activation of BRAF kinase, which in turn activates the MAPK pathway through phosphorylated MEK and ERK. In the invention, after BCPAP cell strain is treated by using inhibitor U0126 of MEK, the expression of PIM1 and ERK also shows a trend of being remarkably reduced, so that the inhibition of BRAF mutation and MEK can cause the reduction of PIM 1.

From the above, BRAF mutation activates MAPK pathway to cause up-regulation of PIM1, and PIM1 is a key molecule of MAPK pathway and may play a very important role in malignant evolution of thyroid cancer mediated by BRAF V600E mutation.

Moreover, the prior art shows that the mutation of BRAF V600E plays an important role in the occurrence and development of PTC by regulating NOX4, and the PIM1 is regulated and controlled by NOX4 in PTC, and PMI and NOX4 protein level in PTC tissues are positively correlated, so that the function of the PIM1 in PTC is regulated and controlled by the upstream mutation of BRAF V600E and NOX4, namely the mutation of BRAF V600E regulates NOX4, and then the NOX4 regulates PIM 1.

Compared with the prior art, the invention has the advantages that: the invention researches the functions of PIM1 gene and BRAF V600E mutation in the process of PTC pathological development, finds that PIM1 possibly plays an important carcinogenic role in the generation and development of PTC, and the function of PIM1 in PTC is regulated and controlled by upstream BRAF V600E mutation and NOX4, namely, the BRAF V600E mutation regulates NOX4, and then the NOX4 regulates PIM 1. Therefore, the invention has important significance and value for theoretical research on pathogenesis of PTC, and is helpful for further perfecting the action mechanism of MAPK signal transduction pathway and PIM 1. In addition, the biomarker can be used as a marker for PTC diagnosis, so that the accuracy of PTC diagnosis is improved, a theoretical basis is provided for the research of PTC diagnosis products, and the PIM1 gene inhibitor can provide a theoretical basis for the research of papillary thyroid cancer preparations, so that the curative effect of PTC treatment is improved.

Drawings

FIG. 1 is a Western blot of PIM1 expression in Nthy-Ori-3 cells, BCPAP cells, and TPC-1 cells in an example of the invention;

FIG. 2 is a bar graph of PIM1 expression in Nthy-Ori-3 cells, BCPAP cells, and TPC-1 cells in accordance with an embodiment of the present invention;

FIG. 3 is a Western blot of PIM1 expression in BCPAP cells and TPC-1 cells after SGI-1776 treatment in accordance with an embodiment of the present invention;

FIG. 4 shows the cell viability of BCPAP cells and TPC-1 cells after PIM1siRNA transfection in the examples of the present invention;

FIG. 5 is a graph showing the relationship between the cell viability of TPC-1 cells and the SGI-1776 dose in the examples of the present invention;

FIG. 6 is a graphical representation of cell viability of BCPAP cells in relation to SGI-1776 dosage in accordance with an embodiment of the present invention;

FIG. 7 shows the results of colony formation assay of BCPAP and TPC-1 cells after 2.5. mu.M and 5. mu.M SGI-1776 action in the examples of the present invention;

FIG. 8 is a graph showing the clonal formation of TPC-1 cells and BCPAP cells after the action of 2.5. mu.M and 5. mu.M SGI-1776 in accordance with one embodiment of the present invention;

FIG. 9 is a graph showing the rate of apoptosis of TPC-1 cells and BCPAP cells after 2.5. mu.M and 5. mu.M SGI-1776 action in examples of the present invention;

FIG. 10 is a graph showing the wound healing rates of TPC-1 cells and BCPAP cells after 2.5. mu.M and 5. mu.M SGI-1776 action in accordance with an embodiment of the present invention;

FIG. 11 is a graph of overall survival for patients with high expression of PIM1 and patients with low expression of PIM1, in accordance with an embodiment of the present invention;

FIG. 12 is a graph showing progression free survival for patients with high PIM1 expression and patients with low PIM1 expression in accordance with an embodiment of the present invention;

FIG. 13 is a graph of the expression of PIM1 and NOx4 in a TCGA database analysis in accordance with an embodiment of the present invention;

FIG. 14 is a graph of the expression of PIM1 and NOx4 in an in situ PTC and a transferred PTC in a TCGA database analysis in accordance with an embodiment of the present invention;

FIG. 15 is a Western blot of PIM1 expression in BCPAP cells and TPC-1 cells after 5. mu.M and 10. mu.M GLX351322 treatment in accordance with examples of the present invention;

FIG. 16 is a bar graph of PIM1 expression in BCPAP cells after 5. mu.M and 10. mu.M GLX351322 treatment in accordance with the present invention;

FIG. 17 is a bar graph of PIM1 expression in TPC-1 cells after 5. mu.M and 10. mu.M GLX351322 treatment in accordance with example of the present invention;

FIG. 18 is a Western blot of PIM1, SOD2, GPX2, CDK4, and Cyclin D1 expression in BCPAP cells and TPC-1 cells following NOX4 knock-out in accordance with examples of the invention;

FIG. 19 is an electron micrograph of immunohistochemical staining of PTC tissue and adjacent normal tissue (thyroid) from a patient according to an embodiment of the present invention;

FIG. 20 shows the results of flow cytometry of BCPAP cells after 2.5. mu.M and 5. mu.M SGI-1776 action in accordance with one embodiment of the present invention;

FIG. 21 shows the flow cytometry results of TPC-1 cells after 2.5. mu.M and 5. mu.M SGI-1776 action in examples of the present invention;

FIG. 22 is a bar graph of the flow cytometry results of BCPAP cells and TPC-1 cells after 2.5. mu.M and 5. mu.M SGI-1776 action in accordance with the present invention;

FIG. 23 is a bar graph of PIM1, SOD2, GPX2, CDK4, and Cyclin D1 expression in TPC-1 cells following NOX4 knock-out in accordance with embodiments of the present invention;

FIG. 24 is a bar graph of PIM1, SOD2, GPX2, CDK4, and Cyclin D1 expression in BCPAP cells following NOX4 knockdown in accordance with example invention;

FIG. 25 is a bar graph of SOD2, GPX2, CDK4 and CyclinD1 protein expression in TPC-1 cells following PIM1siRNA transfection in accordance with an embodiment of the invention;

FIG. 26 is a bar graph of SOD2, GPX2, CDK4, and CyclinD1 protein expression in BCPAP cells following PIM1siRNA transfection in accordance with an embodiment of the present invention;

FIG. 27 is a Western blot of the expression of SOD2, GPX2, CDK4 and CyclinD1 proteins in BCPAP cells and TPC-1 cells after PIM1siRNA transfection in accordance with an embodiment of the present invention;

FIG. 28 is a graph of the distribution of various mutations in PIM1 in patients with high and low expression of PTC according to an embodiment of the present invention;

FIG. 29 is a graph of the distribution of various mutations in patients with high and low expression of PTC to PIM1 in accordance with an embodiment of the present invention;

fig. 30 is a graph of PIM1mRNA levels in BRAF wild type and mutant PTC patients according to an embodiment of the invention;

FIG. 31 is a Western blot of PIM1 and C-myc expression in thyroid cancer cell lines in accordance with examples of the present invention;

FIG. 32 is a histogram of PIM1 and C-myc expression in thyroid cancer cell lines according to the present invention;

FIG. 33 is a Western blot of PIM1 expression in thyroid cancer cell lines after PLX4032 treatment in examples of the present invention;

FIG. 34 is a bar graph of PIM1 expression in thyroid cancer cell lines after PLX4032 treatment in accordance with the present invention;

FIG. 35 is a graph of BCPAP cell proliferation levels following administration of the BRAF V600E inhibitor PLX4032 and the PIM1 inhibitor SGI-1776, alone and in combination, in accordance with an embodiment of the present invention;

FIG. 36 is a graph of 8505C cell proliferation levels following the BRAF V600E inhibitor PLX4032 and the PIM1 inhibitor SGI-1776 alone and in combination in an example of the invention;

FIG. 37 is a graph of BCPAP and 8505C clonogenic after the BRAF V600E inhibitor PLX4032 and the PIM1 inhibitor SGI-1776, alone and in combination, in accordance with an embodiment of the present invention;

FIG. 38 is a Western blot of the expression of the BRAF V600E inhibitor PLX4032 and MEK inhibitor U0126, respectively, on BCPAP cells after treatment of the cells with the inhibitors;

fig. 39 is a bar graph of the expression of each protein after treatment of BCPAP cells with the BRAF V600E inhibitor PLX4032 and MEK inhibitor U0126, respectively, in accordance with an embodiment of the present invention.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.

Example 1: cell culture

Nthy-Ori-3 cells (normal thyroid cell line), TPC-1 cells (PTC/BRAF WT), BCPAP cells (PTC/BRAF MT), CAL-62 cells (ATC/BRAF WT) and 8505C cells (ATC/BRAF MT) are all purchased from cell resources of Shanghai bioscience research institute of Chinese academy of sciences. The Nthy-Ori-3 and BCPAP cell lines were placed in RPMI-1640(Gibco, USA) medium to which 10% fetal bovine serum (Gibco, Carlsad, CA) was added at 37 ℃ in 5% CO₂Incubate in incubator with humidified conditions. Among them, ATC cells (thyroid undifferentiated cancer cells) are formed by further progression and dedifferentiation of PTC cells, and their malignancy is much higher than that of PTC cells.

Example 2: transfection of Small interfering RNAs

NOX4 and PIM1siRNA sequences and their negative controls (Jiangsu, Baiaomaike Biotechnology, Inc., China) were designed and obtained as shown below. By using

3000 transfection kits (Thermo Fisher Science) were incubated for 6h and then mixed with the respective siRNA oligonucleotides or corresponding negative controls, respectively. After transfection, all cells were placed in 5% CO₂Incubating in an incubator at 37 ℃ for 48 h.

NOX4siRNA sequence:

positive: 5 '- (CAUCUGUUCUUAACCUCAA) dTdT-3',

and (3) carrying out the following steps: 5 '- (UUGAGGUUAAGAACAGAUG) dTdT-3';

PIM1siRNA sequence:

positive: 5 '- (CAAGAUCUCUUCGACUUCA) dTdT-3',

and (3) carrying out the following steps: 5 '- (UGAAGUCGAAGAGAUCUUG) dTdT-3'.

Example 3: SGI-1776 treatment

SGI-1776(Selleckchem) was dissolved in DMSO to prepare a SGI-1776 solution having a concentration of 5mmol/L, and the prepared SGI-1776 solution was stored in a refrigerator at-80 ℃ for further use.

BCPAP and TPC1 cells were treated with SGI-1776 solutions at final concentrations of 2.5. mu.M and 5. mu.M for 24h, 48h and 72h, respectively, and SGI-1776 at a final concentration of 0. mu.M was used as a control. In the experiment, the highest concentration of dimethyl sulfoxide in the culture medium, which had no significant effect on cell viability, was 0.05%.

Example 4: cell proliferation assay

Cell proliferation was detected using a cell counting kit (CCK-8) (BestBio, BB-4202-2) as follows:

BCPAP and TPC-1 cells were seeded in 96-well plates for 24h, and then treated with SGI-1776 solutions at different final concentrations for 48h and 72h, respectively. SGI-1776 was used at final concentrations of 1.25. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M in BCPAP cells, and 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M and 5. mu.M in TPC-1 cells, with 0. mu.M SGI-1776 as a control in the experiment. At the end of the experiment, the original medium was discarded and replaced with 100. mu.l of fresh medium containing 10% CCK-8. After incubation at 37 ℃ for 4h, the absorbance of the cells was measured at a wavelength of 450nm using a microplate reader, and the measurement was repeated at least three times.

Example 5: colony formation assay

BCPAP and TPC-1 cells were cultured in 6-well plates at a density of 1000 cells per well, and then the cells were exposed to 2.5. mu.M, 5. mu.M or 10. mu.M SGI-1776 for 72 h. After the fresh culture medium is washed, the cells grow for 5-7 d, and the colony growth condition is evaluated by 0.5% crystal violet staining (WB0026, produced by Davin Biotech, China).

Example 6: scratch test

BCPAP and TPC-1 cells were cultured in 6-well plates, and the concentration of the culture medium containing 3% fetal bovine serum was 80% -90% per well. 24h after inoculation, the wound was scratched with a sterile micropipette tip and replaced by rinsing with media containing SGI-1776 (0. mu.M, 2.5. mu.M, 5. mu.M). Cell migration was observed and recorded at 0h, 24h, 48h and 72h under a 100 × Magnifi cation microscope (IX45-3, olympus, japan), and finally the distance of the scratched area was measured in three fi areas randomly selected.

Example 7: determination of intracellular reactive oxygen species

For quantitative determination of intracellular ROS levels, 1X 10 wells of 6-well plates were incubated⁶And (4) one cell. After overnight cell incubation, cells were pretreated with SGI-1776 (2.5. mu.M, 5. mu.M) or 0.1% dimethyl sulfoxide for 2 hours, incubated with 10. mu.M DCFH-DA (Beyotime, S0033S, China) at 37 ℃ in the dark for 30min, and finally the Dcfffl fluorescence intensity was measured with a fl flow cytometer (FACSCalibur, BD FACSCVia, produced by FACSCalibur, USA).

Example 8: apoptosis assay

1×10⁶Cells were cultured in 6-well culture plates for 24 h. Apoptosis was detected by double staining with Annexin V-FITC and Propidium Iodide (PI) in the dark at room temperature after 72h exposure to SGI-1776(2.5, 5. mu.M) or 0.1% dimethylsulfoxide. Cells were collected and analyzed using a FACSCalibur flow cytometer (BD FACSVia, usa).

Example 9: GLX351322 treatment

GLX351322(835598-94-2, MedChemexpress) was dissolved in dimethyl sulfoxide to obtain a solution of MedChemexpress at a concentration of 20 mmol/L, and the prepared GLX351322 solution was stored in a refrigerator at-80 ℃ for later use.

BCPAP and TPC-1 cells were treated with 5. mu.M or 10. mu.M GLX351322 solution for 24h and 72h, respectively, and 0. mu.M GLX351322 solution was used as a control. In the experiment, the maximum concentration of dimethyl sulfoxide in the medium that had no significant effect on cell viability was 0.05%.

Example 10: PLX4032 and SGI-1776 processing

The above TPC-1 cells, BCPAP cells, CAL-62 cells and 8505C cells were treated with 10uM of BRAF V600E mutation inhibitor PLX4032(CAS No.:918504-65-1, MCE) for 72h, and PIM1 protein was collected.

BCPAP cells and 8505C cells were treated with 10uM PLX4032 and 5uM of the PIM1 inhibitor SGI-177, and absorbance was measured by the CCK8 method at

treatments

1, 2, and 3 d.

Example 11: TCGA database analysis

mRNA expression of PIM1 and NOX4 was collected from 501 PTC patients in TCGA (tumor genome map) database and analyzed by Kaplan-Meier. Meanwhile, correlation between PIM1mRNAs and BRAF mutations in 370 patients with PTC in the tumor genome map (TCGA) database was analyzed using Pearson correlation analysis.

Example 12: western blot analysis.

Cells treated or interfered with in the above examples were collected and lysed in RIPA lysis buffer (G2002, Servicebio) containing phosphatase inhibitor cocktail (CW2383, CWBIO) and PMSF (T0789, TargetMol). The lysate was centrifuged at 12000g at 4 ℃ for 10min to remove debris, according to the specifications. Protein concentrations were determined using the BCA protein assay kit (P0010, Beyotime) for all samples, which were stored in a refrigerator at-30 ℃ until use.

Each protein sample was separated by 12% SDS-PAGE, and all protein samples on the gel were transferred to PVDF membrane (PAL, 4406TC), and the membrane was blocked with 5% skim milk powder in TBST (P004E, AURAGENE) for 2h at room temperature to prevent non-specific binding. Blots were incubated with PIM1 primary antibody (working dilution 1: 2000); NOX4(14347-1-AP, Proteintech, 1: 500 dilution), SOD2(24127-1-AP, Proteitech, (1: 5000 dilution), GPX2(GTX100292, Genetex, 1: 1000 dilution), CDK4(ET1612-1, Huabiao, 1: 3000 dilution), Cyclin D1(ET1601-31, Huashi, 1: 3000 dilution), and β -actin (AC004, Abconal, 1: 5000 dilution), followed by reaction with horseradish peroxidase-labeled secondary antibody (SA009, AURAGENE) at room temperature for 2 h.

After washing with TBST, it was exposed to ECL Kit (FD8020, FDbio-Dura ECL Kit). Finally, Image acquisition and analysis were performed using Bio-Rad GelDoc XR, and density was measured using Image J (MD, national institute of health, USA).

Statistical analysis of the experimental results of the above examples statistical analysis was performed using IBM SPSS software (version 22.0, IBM Corp, Armonk, NY). Specifically, chi-square test, Fisher precise test or popularization of classification variables are adopted, in-vitro experimental results are expressed by mean values +/-standard deviation, single-factor analysis of variance (ANOVA) is adopted in statistical analysis, correlation between mRNAs in a tumor genome map (TCGA) database is analyzed by a Pearson correlation analysis method, Kaplan-Meier method is adopted in survival analysis, logarithmic rank sum test is adopted in significance evaluation, and P <0.05 is defined as difference and has statistical significance.

The test results of the above examples are as follows:

1. effect of SGI-1776 on expression of BCPAP and TPC-1 cellular PIM1 proteins

Expression of PIM1 in PTC cell lines (BCPAP and TPC-1 cells) and normal thyroid cell lines (Nthy-Ori-3 cells) was analyzed by Western blot. The results showed that the expression level of PIM1 was significantly higher in BCPAP and TPC-1 cells than in Nthy-Ori-3 cells, as shown in FIGS. 1 and 2. After SGI-1776 treatment, expression level of PIM1 protein in BCPAP and TPC-1 cells was detected by Western blot, compared with control group, PIM1 protein level was significantly reduced after exposure to SGI-1776 at 2.5. mu.M or 5. mu.M, as shown in FIG. 3, indicating that SGI-1776 can effectively inhibit expression of PIM1 in two cells.

2. Effect of SGI-1776 on BCPAP and TPC-1 cell proliferation, colony formation, migration and apoptosis

After transfecting BCPAP and TPC-1 cells with PIM1 or control siRNA for 48h, cell viability was measured by CCK-8 method. The results indicate that viability of both cells transfected with PIM1siRNA was reduced compared to the control siRNA, as shown in figure 4. Furthermore, SGI-1776 can inhibit the growth of TPC-1 and BCPAP cells in a dose-dependent manner, as shown in FIGS. 5 and 6. Furthermore, PIM1 protein levels were significantly reduced after SGI-1776 treatment at 2.5 μ M and 5 μ M (as shown in figure 3) as described above. Therefore, 2.5M SGI-1776 and 5. mu. MSGI-1776 concentrations were chosen for subsequent experiments.

Clonogenic analysis confirmed that SGI-1776 significantly inhibited clonogenic TPC-1 and BCPAP cells at 2.5 μ M and 5 μ M levels, as shown in fig. 7. As shown in fig. 8 and 9, SGI-1776 induced a significant increase in TPC-1 and BCPAP apoptosis, and was dose-dependent. In addition, wound healing experiments were used to evaluate the effect of SGI-1776 on cell migration, as shown in fig. 10, after SGI-1776 at 2.5 μ M or 5 μ M for 24h, 48h or 72h, the wound healing rate of both cell lines was significantly lower than that of the control group at each time point. It is shown that SGI-1776 can effectively inhibit the proliferation, colony formation and migration of BCPAP and TPC-1 cells and promote apoptosis.

3. PIM1 is related to NOx4 in TCGA databases

mRNA expression of PIM1 and NOX4 was collected in 501 PTC patients, and Kaplan-Meier results showed that both Overall Survival (OS) and progression free survival (DFS) were lower in patients with high PIM1 expression than in patients with low PIM1 expression, indicating that PIM1 was significantly associated with prognosis in PTC patients, as shown in FIG. 11 and FIG. 12. As shown in fig. 13, expression of PIM1 and NOX4 were significantly correlated, as shown in fig. 14, 493 of 501 PTC were in-situ PTC, 8 were shifted PTC, and expression of NOX4 and PIM1 in both groups was also highly correlated.

4. Expression of NOX4, PIM1, SOD2, GPX2, CDK4 and Cyclin D1 in BCPAP and TPC-1 cells after GLX351322 treatment

In order to determine the relationship between NOX4 and PIM1 in PTC, two PTC cells were treated with NOX4 inhibitor GLX351322, and then changes in PIM1 protein were detected using Western blot. The results show that, as shown in figure 15, expression of PIM1 was significantly reduced in both cells after treatment with 5 μ M and 10 μ M GLX 351322. Furthermore, GLX351322 was dose-dependent on PIM1 expression, suggesting that PIM1 is regulated by NOX4 in PTC. The semi-quantitative analysis was consistent with the Western blot results described above, as shown in FIGS. 16 and 17.

As shown in fig. 18, expression of PIM1, SOD2, GPX2, CDK4, and Cyclin D1 was down-regulated in both cells after NOX4 was knocked out, indicating that expression of PIM1, SOD2, GPX2, CDK4, and Cyclin D1 proteins was associated with NOX 4. Semi-quantitative analysis further demonstrated that SGI-1776 increased BCPAP and ROS levels in TPC-1 cells, as shown in FIG. 19.

As shown in fig. 20, 21, and 22, SGI-1776 significantly induced a dose-dependent increase in fluorescence intensity compared to untreated cells. Furthermore, as shown in fig. 23 and 24, it was suggested that PIM1 inhibitor SGI-1776 could increase ROS levels in BCPAP and TPC-1 cells.

5. Expression of PIM1, SOD2, GPX2, CDK4 and Cyclin D1 after transfection of BCPAP and TPC-1 cells with PIM1siRNA

Studies have shown that overexpression of PIM1 is closely associated with tumor resistance to ROS accumulation and promotes progression of PTC cells. Furthermore, expression of PIM1 and antioxidant proteins (SOD2 and GPX2) were down-regulated after NOX4 was knocked down. Expression of SOD2, GPX2, CDK4 and CyclinD1 proteins after PIM1siRNA transfection was examined by Western blot to determine whether SOD2 and GPX2 were altered by alteration of PIM 1.

As shown in fig. 27, the protein levels of SOD2, GPX2, CDK4, and Cyclin D1 were down-regulated in both cells, and in addition, the results were further confirmed by semi-quantitative analysis as shown in fig. 25 and fig. 26.

6. Expression of PIM1, NOX4, SOD2, GPX2 in cancer tissues and paracancerous normal tissues.

Cancer tissues and paracancer normal tissues were selected for immunohistochemical analysis (treated by the tumor hospital in Zhejiang province using conventional methods) to detect the expression levels of PIM1, NOx4, SOD2 and GPX2, which were mainly expressed in the nucleus and cytoplasm of cancer and thyroid tissues, as shown in FIG. 19. The high expression levels of PIM1, NOX4, SOD2 and GPX2 in cancer tissues are respectively 50.8% (61/120), 64.2% (77/120), 57.5% (69/120) and 21.7% (26/120), and the high expression levels of the above proteins in cancer tissues are respectively 15.8% (19/120), 3.3% (4/120), 5.8% (7/120) and 0.8% (1/120), which have statistical significance (P <0.001), as shown in table 1.

7. Clinical pathological relevance of PIM1, NOX4, SOD2, and GPX2 in PTC.

The relationship between PIM1, NOX4, SOD2, and GPX2 gene expression and various clinical pathological features of PTC was analyzed as described in table 2. As can be seen from table 2, NOX4 expression was associated with T stage, late T stage was higher than early T stage (P <0.05), PIM1 and NOX4 expression was associated with lymph node metastasis, and PIM1 and NOX4 expression were both higher in lymph node metastasized than in non lymph node metastasized (P <0.001, P <0.05, respectively). Expression of PIM1 was associated with envelope invasion, with expression of PIM1 higher in patients with envelope invasion than in patients without envelope invasion (P < 0.05).

Furthermore, as can be seen from table 2, NOX4 expression was associated with localized infiltration, with patients with localized infiltration expressing higher levels than those without (P < 0.05). The difference in expression levels of PIM1 was statistically significant depending on tumor size.

Expression of ≦ 1 was significantly higher in patients with tumor diameters >2cm (P <0.05) compared to patients with tumor diameters greater than 2cm, with the difference statistically significant (P < 0.05). However, it is noteworthy that patients with tumors >2cm express NOX4 more frequently (P ═ 0.051) than patients with tumors >2 cm. The expression level of SOD2 was only age-related. Expression of PIM1 was higher in young patients (<55 years) than in elderly patients (<55 years) (P <0.05), as described in table 2, whereas expression of GPX2 had no clear correlation with various clinical pathological features (P > 0.05).

8. Correlation of PIM1 with NOX4, SOD2, GPX2 in PTC.

Correlation of PIM1 with NOX4, SOD2, GPX2 expression levels in PTC chi-square analysis results are shown in table 3. As can be seen from table 3, high expression of PIM1 is closely related to high expression of oxidoreductase, and higher expression of NOX4, SOD2, and GPX2 (P <0.05) was observed in patients with higher expression of PIM1 than in patients with lower expression of PIM 1.

TABLE 1 correlation of PIM1, NOX4, SOD2, GPX2 in PTC cancer tissues with paracancerous normal tissues

^*Significantly different by the χ2test

TABLE 3 correlation of PIM1 with NOX4, SOD2 and GPX2 in PTC

The difference has statistical significance

9. Relationship of BRAF 6000E mutation in PTC tissue to PIM1

The results of clinical data analysis of 370 patients with PTC in the TCGA database showed that patients with high expression of PIM1 developed mainly BRAF mutations, significantly higher than the mutation rate in patients with low expression of PIM1, as shown in fig. 28 and 29. The 370 patients were divided into BRAF wild type group and BRAF V6000E mutant group for analysis, and it can be seen from fig. 30 that the PIM1 level of the BRAF 6000E mutant group patients was significantly higher than that of the BRAF wild type group.

10. Correlation between BRAF mutation and PIM1 on thyroid cancer cell line

Four thyroid cancer cells were selected: TPC-1(PTC/BRAF WT), BAPAB (PTC/BRAF MT), CAL-62(ATC/BRAF WT), 8505C (ATC/BRAF MT), the expression of PIM1 in four cells was examined. As shown in fig. 31 and fig. 32, the expression level of PIM1 in the BRAF MT group was significantly higher than that in the BRAF WT group in thyroid cancer cells of the same pathological type: BCPAP expression is higher than TPC-1, 8505C expression is higher than CAL-62; in the same BRAF WT or BRAF MT group, the level of PIM1 on ATC cells was significantly higher than that on PTC, 8505C was higher than that on BCPAP, and CAL-62 was higher than that on TPC-1.

As shown in fig. 33 and 34, after treatment of cells with the BRAF V600E inhibitor PLX4032, the results showed that PIM1 levels were significantly reduced (P <0.001) in BRAF mutant cell lines BCPAP and 8505C, whereas PIM1 levels were not significantly changed in BRAF wild-type cell lines CAL-62 and TPC-1.

11. Effect of Each inhibitor on RAF V600E mutant thyroid carcinoma cells

The cell strains BCPAP and 8505C with the mutation of BRAF V600E were selected and treated with the BRAF V600E inhibitor PLX4032 and the PIM1 inhibitor SGI-1776, and the CCK8 results show that the combination of the two inhibitors has a significantly enhanced effect on the cell viability compared with the single inhibitor, as shown in FIG. 35 and FIG. 36. As shown in fig. 37, the clonogenic results also show that the number of cell clones in the two inhibitor combination groups is significantly lower than that in the single inhibitor combination group, and the combination of PLX4032 and SGI-1776 has a better killing effect on the BRAF V600E mutant thyroid cancer cell line. As shown in fig. 38 and 39, after treatment of BCPAP cells with PLX4032 and MEK inhibitor U0126, respectively, the expression levels of each protein of PIM1, p-PIM1, C-myc, ERK, p-ERK, Cyclin D1 and CDK4 were significantly reduced relative to the control group, suggesting that PIM1 is a target gene downstream of BRAF V600E and MEK.

In vitro tests of the invention find that the level of PIM1 of two PTC cell lines (BCPAP and TPC-1) is obviously higher than that of normal thyroid cells, and the BCPAP cells and TPC-1 cells are treated by using a PIM1 inhibitor SGI-1776, which shows that SGI-1776 has an inhibition effect on the proliferation and the motility of the two PTC cells and can induce apoptosis, and PIM1 plays an important carcinogenic role in the generation and development of PTC.

Further, the present study found that NOX4is significantly more expressed in PTC tissues than in neighboring normal tissues. The expression level of NOX4 was also associated with various clinical pathological features, with higher expression of NOX4 in patients with late stage T, lymph node metastasis and regional invasion than in patients with early stage T, no lymph node metastasis and non-regional invasion, further suggesting a carcinogenic role for NOX4 in PTC.

Increased ROS production has been detected in a variety of malignancies and can activate tumorigenic signals. PIM1 kinase has been reported to reduce cellular ROS levels by promoting antioxidant gene expression resulting from enhanced NRF2/ARE activity, thereby avoiding tumor cell death. The present study found that cells lacking PIM1 kinase or treated with PIM inhibitors can lead to the accumulation of lethal ROS, ultimately leading to cellular intolerance and death, suggesting that PIM1 kinase is an important regulator of cellular redox signaling. In the present invention, increased ROS production following SGI-1776 treatment of PTC cells suggests that PIM1 may down-regulate ROS.

The oncology database shows that the expression of SOD2 and GPX2 in the thyroid cancer antioxidant enzyme system is higher than that in normal thyroid tissue. The immunohistochemical results of the present invention indicate that the expression of SOD2 and GPX2 in PTCs tissues is significantly higher than that in normal thyroid tissues. Therefore, SOD2 and GPX2 in PTC may play a major role in the removal of ROS. In the invention, after PIM1 in PTC cells is knocked out by PIM1siRNA, expression of SOD2 and GPX2 is found to be reduced. In addition, NOX4 was knocked out in PTC cells with NOX4siRNA, and PIM1, SOD2 and GPX2 were all found to be down-regulated. In addition, histological results also showed that PIM1 was associated with SOD2 and GPX 2. It can be seen that PIM1 can be regulated by NOX4, which in turn regulates SOD2 and GPX2, thereby avoiding the accumulation of lethal ROS leading to tumor progression.

In the invention, PLX4032 is adopted to treat four thyroid cancer cell strains (TPC-1, BCPAP, CAL-62 and 8505C), and the result shows that the expression of PIM1 in the BRAF V600E mutant cell strains (BCPAP and 8505C) is obviously reduced, but PLX4032 has no obvious influence on the expression of PIM1 in the BRAF wild type cell strains (TPC-1 and CAL-62). It can be seen that BRAF V600E regulates expression of PIM1 in PTC cells and ATC cells.

Furthermore, it is known from the prior art that the mutation of BRAF V600E can play an important role in the occurrence and development of PTC by regulating NOX4 (see: NADPH oxide NOX4Is a Critical media of BRAF (V600E) -Induced descending regulation of the Sodium/Iodi Symporter in Papilary Thyroid cartinosas, Artificial Redox Signal.26,864-877, 2017, Azouzi, N etc.), whereas from the above, PIM1 is regulated by NOX4 in PTC, and levels of PMI and NOX4 in PTC tissue are positively correlated, therefore, the function of PIM1 in PTC is regulated by upstream mutations of BRAF V600E and NOX4, namely, the mutation of BRAF V600E regulates NOX4, and then the mutation of NOX4 regulates the occurrence and development of PIM 1.

The PIM1 protein can be used as a biomarker of papillary thyroid carcinoma and can be applied to papillary thyroid carcinoma diagnosis and treatment products such as papillary thyroid carcinoma detection kits and papillary thyroid carcinoma targeted drugs.

Sequence listing

<110> Zhejiang province tumor hospital

<120> thyroid papillary carcinoma biomarker and application thereof

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<170> SIPOSequenceListing 1.0

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<212> RNA

<213> Artificial Sequence (Artificial Sequence)

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<210> 2

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<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ugaagucgaa gagaucuug 19

<210> 3

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<212> RNA

<213> Artificial Sequence (Artificial Sequence)

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caucuguucu uaaccucaa 19

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<213> Artificial Sequence (Artificial Sequence)

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Claims

1. A biomarker of papillary thyroid cancer, wherein the biomarker is PIM1 protein regulated by BRAF V600E mutation.

2. The biomarker of claim 1, wherein the BRAF V600E mutation modulates expression of NOX4, and expression of PIM1 protein is modulated by NOX 4.

3. The biomarker of claim 2, wherein the expression level of PIM1 protein is positively correlated with NOX4 protein level.

4. Use of an inhibitor of a biomarker according to any of claims 1 to 3 in the preparation of an anti-papillary thyroid cancer formulation.

5. The use of claim 4, wherein the inhibitor is a PIM1siRNA directed against the expression of PIM1 gene, having the sequence: SEQ ID NO.1 and EQ ID NO. 2.

6. The use of claim 4, wherein the inhibitor is a NOX4siRNA directed against the expression of NOX4 gene, having the sequence: SEQ ID NO.3, Q ID NO. 4.

7. The use of claim 4, wherein the inhibitor is at least one of SGI-1776, GLX351322 or PLX 4032.