CN114774547B - Molecular marker USP13 related to human osteosarcoma and application thereof - Google Patents

Abstract

The invention discloses a molecular marker USP13 related to human osteosarcoma, and the amino acid sequence of the marker is shown as SEQ ID No. 1. The inventor finds out through experimental research that: USP13 is highly expressed in osteosarcoma, and the poor prognosis is prompted, the expression of USP13 in osteosarcoma cells is knocked down, so that the in vitro proliferation, clone formation capability and invasion and transfer capability of the osteosarcoma cells can be reduced, and the in vivo proliferation and invasion and transfer capability of the osteosarcoma cells can be reduced; USP13 proteins can bind to METTL3 protein and reduce its level of ubiquitination; METTL3 is highly expressed in osteosarcoma and has obvious positive correlation with the expression level of USP 13. The results show that USP13 can be used as a molecular marker of osteosarcoma, and can be used for osteosarcoma diagnosis and curative effect evaluation by quantitative detection of the molecular marker, which has important significance for early diagnosis of osteosarcoma and can improve the overall survival rate of patients.

Description

Molecular marker USP13 related to human osteosarcoma and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a human osteosarcoma related molecular marker USP13 and application thereof.

Background

Osteosarcoma (OS) is a malignant bone tumor derived from mesenchymal cells, mainly occurring in children and adolescents, accounting for about 2.4% of all malignant tumors of pediatric patients, and having a very high mortality rate. Osteosarcoma often occurs in the metaphyseal region of the proximal tibia, proximal humerus and distal femur, and is highly invasive, with pulmonary metastases often associated in the middle and late stages. Despite the rapid development of medical techniques including surgical approaches, concepts and chemoradiotherapy techniques in the past decades, the five-year survival rate of osteosarcoma remains low due to lung metastasis and drug resistance. In fact, since there is still no effective treatment currently available, the overall survival rate of osteosarcoma patients with metastases is rather low, approximately between 10% and 20%. Therefore, the deep biological mechanism of the development of osteosarcoma is fully understood, and the early screening and diagnosis of osteosarcoma are of great significance.

The current method for determining osteosarcoma is a puncture biopsy or a biopsy obtained by surgical excision, the osteosarcoma is mainly determined according to cellular heterogeneity, chromosome number and structural abnormality, the classical osteosarcoma has a wide immunohistochemical expression profile, the diagnosis significance is limited, and the main detection indexes comprise ostocelcin, osteonnectin, ostopontin, vim, S100, actin, SMA, CK, NSE, CD99, SATB2, MDM2, CDK4, ki67 and P53, but the specificity is lacked, so that the development of a new treatment means and a molecular diagnosis marker with high specificity are urgent requirements for preventing and treating the osteosarcoma.

Ubiquitination modification is one of the most common post-translational modifications of proteins, which are involved in a range of biological activities by regulating protein stability, signaling, and other processes. Like other post-translational modifications, ubiquitination is also a reversible process that can be reversed by certain proteases, collectively known as Deubiquitinases (DUBs), and currently over 100 DUBs have been identified, of which ubiquitin-specific proteases (USPs) are the largest family of deubiquitinases. Currently, studies have demonstrated that DUBs are aberrantly expressed in several malignant tumors and play an important role in their development by regulating a range of biological actions of tumor cells. However, to date, there has been little research associated with DUBs in osteosarcomas.

The research aims at providing a molecular marker related to human osteosarcoma, and the molecular pathological change of a patient with osteosarcoma can be detected, so that early screening and auxiliary diagnosis can be realized, and the early diagnosis and the overall survival rate of the patient with osteosarcoma can be improved.

Disclosure of Invention

The invention aims to solve the defects of osteosarcoma diagnosis technology in the prior art, and provides a molecular marker related to human osteosarcoma and application thereof in preparing an osteosarcoma early diagnosis or curative effect evaluation product.

Technical scheme

A molecular marker related to human osteosarcoma is USP13, and the amino acid sequence of the USP13 is shown as SEQ ID No. 1.

SEQ ID No.1:

MQRRGALFGMPGGSGGRKMAAGDIGELLVPHMPTIRVPRSGDRVYKNECAFSYDSPNSEGGLYVCMNTFLAFGREHVERHFRKTGQSVYMHLKRHVREKVRGASGGALPKRRNSKIFLDLDTDDDLNSDDYEYEDEAKLVIFPDHYEIALPNIEELPALVTIACDAVLSSKSPYRKQDPDTWENELPVSKYANNLTQLDNGVRIPPSGWKCARCDLRENLWLNLTDGSVLCGKWFFDSSGGNGHALEHYRDMGYPLAVKLGTITPDGADVYSFQEEEPVLDPHLAKHLAHFGIDMLHMHGTENGLQDNDIKLRVSEWEVIQESGTKLKPMYGPGYTGLKNLGNSCYLSSVMQAIFSIPEFQRAYVGNLPRIFDYSPLDPTQDFNTQMTKLGHGLLSGQYSKPPVKSELIEQVMKEEHKPQQNGISPRMFKAFVSKSHPEFSSNRQQDAQEFFLHLVNLVERNRIGSENPSDVFRFLVEERIQCCQTRKVRYTERVDYLMQLPVAMEAATNKDELIAYELTRREAEANRRPLPELVRAKIPFSACLQAFSEPENVDDFWSSALQAKSAGVKTSRFASFPEYLVVQIKKFTFGLDWVPKKFDVSIDMPDLLDINHLRARGLQPGEEELPDISPPIVIPDDSKDRLMNQLIDPSDIDESSVMQLAEMGFPLEACRKAVYFTGNMGAEVAFNWIIVHMEEPDFAEPLTMPGYGGAASAGASVFGASGLDNQPPEEIVAIITSMGFQRNQAIQALRATNNNLERALDWIFSHPEFEEDSDFVIEMENNANANIISEAKPEGPRVKDGSGTYELFAFISHMGTSTMSGHYICHIKKEGRWVIYNDHKVCASERPPKDLGYMYFYRRIPS。

The application of the molecular marker USP13 as a detection target in preparing osteosarcoma diagnostic reagent or curative effect evaluation products.

The application of the reagent for detecting the expression level of the molecular marker USP13 in preparing osteosarcoma diagnostic reagents or curative effect evaluation products. The reagent for detecting the expression level of the molecular marker USP13 comprises specific primers, wherein the specific primers are USP13-F and USP13-R:

USP13-F:5’-CAAAAGATCGCCTGATGAACCA-3’

USP13-R:5’-TGATCCAGTTGAAGGCCACC-3’。

a kit for diagnosing or evaluating curative effect of osteosarcoma comprises a reagent for detecting the expression level of a molecular marker USP13, wherein the amino acid sequence of the USP13 is shown as SEQ ID No. 1. The reagent for detecting the expression level of the molecular marker USP13 comprises specific primers, wherein the specific primers are USP13-F and USP13-R:

USP13-F:5’-CAAAAGATCGCCTGATGAACCA-3’

USP13-R:5’-TGATCCAGTTGAAGGCCACC-3’。

the invention has the beneficial effects that:

the invention discovers a molecular marker USP13 related to osteosarcoma diagnosis and curative effect evaluation, and can be used for osteosarcoma diagnosis and curative effect evaluation by quantitatively detecting the molecular marker. Compared with the existing osteosarcoma diagnosis method, the method has the advantages of convenience, rapidness, repeatability, high specificity and the like, enriches the diagnosis method and histological typing of osteosarcoma, helps to predict the prognosis of a patient, and truly achieves early discovery, early diagnosis and early treatment of osteosarcoma.

Drawings

FIG. 1 shows the results of immunohistochemical detection of USP13 expression in osteosarcoma patient tissues and normal bone tissues;

FIG. 2 shows the Western Blot results of the expression levels of USP13 protein in osteosarcoma patient tissues and normal bone tissues;

FIG. 3 shows the results of qRT-PCR detection of USP13 mRNA expression levels in osteosarcoma tissue and normal bone tissue;

FIG. 4 shows the results of Westernblot assay for USP13 expression in osteosarcoma cell lines and normal osteoblast cell lines;

FIG. 5 shows the Western blot analysis of the protein level expression of osteosarcoma cell line 143B after specific knockdown of USP 13;

FIG. 6 shows the result of CCK-8 assay to knock down the proliferative capacity of osteosarcoma cells after USP13 in osteosarcoma cell line 143B;

FIG. 7 shows the result of EDU assay for knocking down the proliferation potency of osteosarcoma cells after USP13 in osteosarcoma cell line 143B;

FIG. 8 shows the results of the detection of the clonogenic capacity of osteosarcoma cells after knockdown of USP13 in osteosarcoma cell line 143B;

FIG. 9 is a statistical result of tumor volume and mass formed 28 days after injecting 143B cells after knocking down USP13 subcutaneously into nude mice;

FIG. 10 shows the results of the invasive motility test of osteosarcoma cells after USP13 in knockdown osteosarcoma cell line 143B;

FIG. 11 shows statistical results of H & E staining of lung metastasis and the number of lung metastases after 28 days of tail vein injection of 143B cells knocked-down USP13 into nude mice;

FIG. 12 is a graph showing mass spectrometric identification of METTL 3-specific peptide fragments in USP13 immunoprecipitation complexes in osteosarcoma cell line 143B;

FIG. 13 shows the results of forward-reverse co-immunoprecipitation identification of USP13 and METTL3 proteins in osteosarcoma cell line 143B;

FIG. 14 is a test result of the level of endogenous METTL3 ubiquitination after knockdown of USP13 in osteosarcoma cell line 143B;

FIG. 15 shows the results of Westernblot detection of METTL3 protein expression in osteosarcoma tissues and normal tissues;

FIG. 16 shows the results of qRT-PCR detection of METTL3 mRNA expression in osteosarcoma and normal tissues;

FIG. 17 shows the Western blot analysis of METTL3 expression in osteosarcoma cell lines and normal osteoblast cell lines.

Detailed Description

The technical scheme of the invention is further explained by combining the drawings and the specific embodiments.

EXAMPLE 1 analysis of USP13 expression levels in clinical samples of osteosarcoma

The osteosarcoma and normal bone tissue clinical samples adopted by the invention are from the first subsidiary hospital of Nanjing medical university. The project was solicited patient consent and approved by the ethical committee of the first clinical medical school of the university of medical, nanjing.

(1) Source of tissue specimen

A total of 100 clinical samples of osteosarcoma and normal bone tissue were collected at the first subsidiary hospital of Nanjing medical university. Immediately placing the tissue in liquid nitrogen for freezing after the tissue is separated in the operation, and then transferring and storing the tissue in a refrigerator at the temperature of minus 80 ℃.

(2) Tissue section and immunohistochemistry

a) Tissue fixation in 4% paraformaldehyde for 24 hours;

b) Preparing a paraffin-embedded tissue wax block from the fixed tissue, cutting the tissue wax block into a wax block with the thickness of 6 mu m by using a slicer, and putting the cut tissue piece in an oven at 37 ℃ for one night;

c) Sequentially carrying out xylene dewaxing and gradient alcohol hydration on the cut paraffin sections, and flushing with running water for 3-5min;

d) Immersing the slices in citrate antigen retrieval solution, carrying out antigen retrieval in boiling water bath for 15 minutes, and taking out and naturally cooling;

e) Putting the slices into a film washing box soaked with PBS, and washing for 5 minutes each time on a shaking table for 3 times;

f) Dropwise adding closed serum onto the slices, and sealing for 1 hour at normal temperature;

g) Blocking sera were gently shaken off, and 0.5% of bsa-diluted USP13 antibody (Proteintech, 1:200 dilution) are added on the slices dropwise and incubated overnight at 4 ℃;

h) Taking out the slices the next day, washing the slices with PBS for 3 times, 5 minutes each time;

i) The HRP-labeled secondary antibody diluted 0.5% bsa (petunia, dilution ratio 1:1000 Dropwise adding the mixture on the slices, incubating for 1 hour at normal temperature, and washing the slices with PBS for 3 times for 5 minutes each time;

j) Dropwise adding DAB developing solution on the slices, reacting at room temperature, observing under a mirror, stopping reaction in water according to the color development condition, recording the color development time, and flushing for 5 minutes by running water;

k) Staining the core with hematoxylin for 30-60 seconds, washing with running water for 5 minutes, and observing the staining condition under a mirror;

l) sequentially placing the slices in 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 100% alcohol I, 100% alcohol II for two minutes respectively, and xylene I, xylene II for 2 minutes respectively;

m) sealing the neutral resin, airing in a fume hood, and taking a picture under a microscope for recording.

The results of immunohistochemical experimental detection of USP13 expression in osteosarcoma patient tissues and normal bone tissues are shown in FIG. 1, and it can be seen that the expression of USP13 protein in osteosarcoma tissues is significantly higher than that in normal bone tissues. Statistics is carried out on the expression of USP13 protein in clinical samples of osteosarcoma, and 54 USP13 strong positive cases, 29 positive cases, 11 weak positive cases and 6 negative cases in osteosarcoma tissues are found.

(3)Western Blot

a) And (3) total protein extraction: 12 pairs of osteosarcoma and normal bone tissues were randomly selected, and protein lysates were prepared from 1ml of lysate, 10. Mu.l of phosphatase inhibitor, 10. Mu.l of PMSF and 1. Mu.l of protease inhibitor. Placing the tissue into a homogenizer for fully homogenizing at 4 ℃, then cracking for 20 minutes on ice, collecting lysate in an EP tube, centrifuging at 4 ℃,12000rpm for 5 minutes, and transferring the supernatant into a new EP tube; absorbing part of protein lysate for BCA protein concentration determination; adding 5 XLoading Buffer into the rest volume according to the proportion of 4; after cooling at room temperature, the sample is placed at-20 ℃ for storage or Western Blot experiment according to the experimental arrangement.

b) Electrophoresis and membrane transfer: preparing separation glue and concentrated glue with different concentrations according to experimental requirements; adding the sample, running concentrated gel at 80V; adjusting the voltage to 120V when the protein sample reaches the separation gel; and stopping electrophoresis when the protein is at the bottom. Cutting a PVDF film with a proper size, sequentially stacking filter paper, gel and the PVDF film to prepare a film transfer sandwich, and removing air bubbles in the film transfer sandwich; clamping the film transferring clamp and placing the film transferring clamp into a film transferring groove; and (3) putting the film transferring groove into an ice box, adding pre-cooled film transferring liquid, and transferring the film at a constant current of 300mA for a certain time according to conditions. After the membrane conversion, the PVDF membrane was removed and placed in 5% BSA blocking solution for blocking for 2 hours.

c) Antibody incubation and detection: incubation of USP13 primary antibody (Proteintech, 1 dilution 2000) at 4 ℃ overnight after blocking; the next day, the membrane was washed three times with TBST, and the corresponding secondary antibody (Jackson, 1, 10000 dilution) was incubated at room temperature for 2 hours; washing the membrane, preparing exposure liquid, uniformly coating the exposure liquid on the PVDF membrane, putting the PVDF membrane into a gel imaging system, and taking pictures for analysis.

The Western Blot detection results of the USP13 protein expression in the tissues of osteosarcoma patients and normal bone tissues are shown in FIG. 2, and it can be seen that the USP13 protein expression in the tissues of osteosarcoma patients is significantly higher than that in the normal bone tissues.

(4)qRT-PCR

a) Extracting total RNA: randomly selecting 40 pairs of osteosarcomas and normal bone tissues, transferring into a homogenate tube, adding an RNA lysate, fully homogenizing by a homogenizer at 4 ℃, then cracking on ice for 15 minutes, and transferring into an EP tube; adding 0.2ml of chloroform into 1ml of RNA lysate, violently shaking for 15 seconds, adding isopropanol with the same volume, uniformly mixing, cleaning and precipitating with 75% ethanol, centrifuging at 12000rpm for 5 minutes at 4 ℃, and removing supernatant; after being dried at room temperature, adding 50 mu l of DEPC water, uniformly mixing by blowing, and measuring the RNA concentration by using Nanodrop 2000; the samples are stored at-80 ℃ or subjected to reverse transcription according to the experiment requirements.

b) Reverse transcription of RNA: the reverse transcription kit used in this study was purchased from Takara, japan, and the reverse transcription system is shown in Table 1:

TABLE 1

Carrying out reverse transcription reaction, setting the program at 50 ℃,15 minutes, 85 ℃ and 5 seconds; the reverse transcribed cDNA was purified using DEPC water 1: after 20 dilution, the mixture is stored at-20 ℃ or subjected to qRT-PCR experiment according to the experiment requirement.

c)qRT-PCR

The PCR amplification system is shown in Table 2:

TABLE 2

The two-step amplification conditions are as follows: 5 minutes at 95 ℃; the reaction is circulated for 40 cycles of reaction at 95 ℃,10 seconds, 60 ℃,30 seconds; post dissolution profile phase.

d) The expression level of the target gene was corrected to GAPDH and then adjusted to 2 ^-ΔΔCT And (4) calculating by using the method. The gene primer sequences are respectively as follows:

USP13-F:5’-CAAAAGATCGCCTGATGAACCA-3’

USP13-R:5’-TGATCCAGTTGAAGGCCACC-3’

GAPDH forward:5’-GACAGTCAGCCGCATCTTCT-3’

GAPDH reverse:5’-GCGCCCAATACGACCAAATC-3’。

the qRT-PCR detection results of USP13 mRNA expression level in osteosarcoma tissue and normal bone tissue are shown in FIG. 3, and it can be seen that USP13 mRNA expression in osteosarcoma tissue is significantly higher than that in normal bone tissue (P < 0.001).

Example 2 analysis of USP13 expression levels in osteosarcoma cell lines

(1) Cell source and culture

Osteosarcoma cell lines include HOS, saos-2, U-2OS, 143B and MG63, and the normal osteoblast line hFOB 1.19 cell line was purchased from the cell bank of Chinese academy of sciences (Shanghai). Osteosarcoma cell line and osteoblast cell line were cultured in DMEM (Gibco) medium containing 10% fetal bovine serum (Gibco) and 1% triple antibody (Gibco).

(2)Western Blot

a) And (3) total protein extraction: the original cell culture medium was discarded, washed three times with PBS, and 500. Mu.l of lysate was added per 10cm dish. The protein lysate was prepared as 1ml lysate, 10. Mu.l phosphatase inhibitor, 10. Mu.l PMSF and 1. Mu.l protease inhibitor. Placing the cell and protein lysate on ice for cracking for 10 minutes, scraping off the lysate by using a cell scraper, and collecting the lysate in an EP (EP) tube; centrifuging at 12000rpm for 5min at 4 deg.C, and transferring the supernatant into a new EP tube; absorbing part of protein lysate for BCA protein concentration determination; adding 5 XLoading Buffer into the rest volume according to the proportion of 4; after cooling at room temperature, the sample is placed at-20 ℃ for storage or Western Blot experiment according to the experimental arrangement.

b) Electrophoresis and membrane transfer: preparing separation glue and concentrated glue with different concentrations according to experimental requirements; adding the sample, running concentrated gel at 80V; adjusting the voltage to 120V when the protein sample reaches the separation gel; and stopping electrophoresis when the protein reaches the bottom. Cutting a PVDF film with a proper size, sequentially stacking filter paper, gel and the PVDF film to prepare a film transfer sandwich, and removing bubbles in the film transfer sandwich; clamping the film transferring clamp and placing the film transferring clamp into a film transferring groove; and (3) putting the film transferring groove into an ice box, adding pre-cooled film transferring liquid, and transferring the film at a constant current of 300mA for a certain time according to conditions. After the membrane conversion, the PVDF membrane was removed and placed in 5% BSA blocking solution for blocking for 2 hours.

c) Antibody incubation and detection: incubation of USP13 primary antibody (1 dilution 2000) at 4 ℃ overnight after blocking; the next day, the membrane was washed three times with TBST, and the corresponding secondary antibody (Jackson, 1 diluted 10000) was incubated for 2 hours at room temperature; washing the membrane, preparing exposure liquid, uniformly coating the exposure liquid on the PVDF membrane, putting the PVDF membrane into a gel imaging system, and taking pictures for analysis.

The Western blot detection results of the expression of USP13 in the osteosarcoma cell line and the normal osteoblast cell line are shown in FIG. 4, and it can be seen that the protein expression level of USP13 in the osteosarcoma cell line (HOS, saos-2, U-2OS, 143B and MG 63) is obviously higher than that of the normal osteoblast cell line hFOB 1.19.

Example 3 the Effect of USP13 on the proliferative and clonogenic potency of osteosarcoma cells

As USP13 has the highest expression level in 143B cells of osteosarcoma cell line, in order to further clarify the effect of USP13 in osteosarcoma, we used shRNA to knock down USP13 gene in 143B cells, and examined the knocking efficiency and the influence of USP13 on the proliferation and clonogenic capacity of osteosarcoma cells in vitro and in vivo

(1) Knockdown of USP13 in osteosarcoma cells

Three shRNAs with different sequences are used for knocking down the USP13 gene, and the knocking down sequences are USP13 shRNA1:

5’-CCGGGCTCTGTCCTGTGTGGAAAGTCTCGAGACTTTCCACACAGGACAGAGCTTTTTG-3’，USP13 shRNA2:

5’-CCGGGCACGAAACTGAAGCCAATGTCTCGAGACATTGGCTTCAGTTTCGTGCTTTTTG-3’，USP13 shRNA3：

5’-CCGGGCCTTTAGATCCAACACAAGACTCGAGTCTTGTGTTGGATCTAAAGGCTTTTTG-3’。

and (3) extracting cell proteins of the shNC control group, the USP13 shRNA1 group, the USP13shRNA2 group and the USP13 shRNA3 group according to the Western Blot method, and detecting the expression quantity of the USP13 proteins in different groups by the Western Blot to verify the knockout efficiency. The results are shown in fig. 5, and it can be seen that the expression of USP13 in osteosarcoma cells can be effectively knocked down by using shRNA, and the knocking efficiency of shRNA1 and shRNA2 is higher.

(2) In vitro cell proliferation and clonal formation

a) CCK-8 experiment

Cells of the shNC control group, the USP13 shRNA1 and the shRNA2 group are respectively inoculated into a 96-well plate according to the cell amount of 2000 cells/well, and are placed into a cell culture box for more than 12 hours until the cells adhere to the wall. CCK-8 reagent (syngen) was added to the well plate and incubated for 24, 48, 72, 96 and 120 hours as required by the instructions. The absorbance was measured at 450nm using a microplate reader, and the cell proliferation ability was expressed by the increase times compared with the absorbance at 0 hour.

The test results are shown in fig. 6, and it can be seen that the proliferation capacity of osteosarcoma cells is obviously reduced after the USP13 is knocked down (shNC control VS USP13 shRNA1 group, P <0.001, shNC control VS USP13shRNA2 group, P < 0.001).

b) EDU experiment

Cells of the shNC control group, USP13 shRNA1 and shRNA2 group were inoculated into 24-well plates according to the amount of 10 ten thousand cells per well, and placed in a cell incubator for more than 12 hours until the cells adhere to the wall. The analytical assay was performed using the EDU cell proliferation kit (petit sky) according to the instructions and photographed under an inverted fluorescence microscope. Red fluorescence is EDU positive and blue fluorescence is nucleus. The number of red fluorescent cells/the number of blue fluorescent cells was the EDU positive rate.

The results are shown in fig. 7, the EDU positive rate of osteosarcoma cells decreased significantly after knockdown of USP13, representing a significant decrease in cell proliferation capacity (shNC control VS USP13 shRNA1 group, P <0.01 shNC control VS USP13shRNA2 group, P < 0.01.

c) Clone formation assay

Cells of the shNC control group, USP13 shRNA1 and shRNA2 group were planted in a six-well plate at a density of 500 cells/well, cultured for 2 weeks, and then fixed and stained with crystal violet, photographed by a camera, counted for the number of colony formations and analyzed.

The results are shown in fig. 8, and it can be seen that there is a significant reduction in the number of osteosarcoma cell clones formed following knockdown of USP13, representing a significant reduction in proliferative capacity (shNC control VS USP13 shRNA1 group, P <0.01 shNC control VS USP13shRNA2 group, P < 0.01.

(3) Knockdown USP13 inhibition of osteosarcoma in vivo tumor growth

The shNC control group and USP13 shRNA1 group cells are inoculated under the skin of 4-week-old female nude mice (Nanjing university of medicine laboratory animal center) in 200 ten thousand, the diameter of the tumor is measured every 4 days, the volume of the tumor tissue is calculated, and the tumor tissue is taken out, weighed and photographed after the experiment is finished on the 28 th day.

The results are shown in fig. 9, and it can be seen that the tumor growth rate of the osteosarcoma cells knocked down USP13 is slower than that of the osteosarcoma cells in the control group, the tumor volume is small (P < 0.001), and the tumor mass is small (P < 0.001), indicating that knocking down USP13 inhibits the growth of the osteosarcoma in vivo.

Example 4 Effect of USP13 on the invasive metastatic potential of osteosarcoma cells

(1) Transwell experiment

Osteosarcoma cell invasion-metastasis-associated assay was performed using a 24-well Transwell chamber (Ibidi). Approximately 2 million osteosarcoma cell line 143B cells (cells of the shNC control group, USP13 shRNA1 and shRNA2 group) suspended in fetal bovine serum-free medium were planted in the upper chamber, and 10% fetal bovine serum-containing complete medium was placed in the lower chamber. After 24 hours, uninfected upper cells were swabbed with a cotton swab. And fixing and crystal violet staining the lower layer cells invaded and transferred, taking pictures by a microscope, counting the number of the lower layer cells and analyzing.

The results are shown in fig. 10, and it can be seen that there is a significant decrease in the number of invasive metastases of osteosarcoma cells following knockdown of USP13, representing a significant decrease in invasive metastatic capacity (shNC control VS USP13 shRNA1 group, P <0.01 shNC control VS USP13shRNA2 group, P < 0.01.

(2) Knockdown of USP13 inhibits lung metastasis in osteosarcoma

The cells of the shNC control group and the USP13 shRNA1 group were suspended in an amount of 200 ten thousand in 100 μ l DMEM medium and injected into 4-week-old female nude mice (laboratory animal center of tokyo medical university) through tail vein to evaluate the lung transfer ability of the osteosarcoma cells in vivo. Lung tissue was taken at the end of the experiment on day 28, sectioned, H & E stained, photographed under microscope and tumor metastasis numbers recorded, statistically analyzed.

As shown in fig. 11, it can be seen that the number of lung metastases in vivo was reduced in osteosarcoma cells knocked down for USP13 compared to control osteosarcoma cells (P < 0.001), suggesting that knocking down for USP13 inhibits lung metastasis in vivo in osteosarcoma.

Example 5 binding of USP13 protein to METTL3 protein

(1) USP13 binding to METTL3

a) Immunoprecipitation mass spectrometry (IP/MS)

A500. Mu.g sample of the Protein was taken, 1. Mu.g of IgG of the same genus as the subsequent IP and 20. Mu.l of well resuspended Protein A/G-agarose beads (Santa Cruz) were added, shaken slowly at 4 ℃ for 1 hour, and then centrifuged at 2500rpm for 5 minutes, and the supernatant was collected for the subsequent immunoprecipitation. Add 5. Mu.g USP13 antibody for IP (Proteitech, 1, 50 dilution) and shake slowly overnight at 4 ℃. The following day 20. Mu.l of well resuspended ProteinA/G-agarose beads were added and shaken slowly at 4 ℃ for 2 hours. After 5 minutes centrifugation at 2500rpm, the supernatant was carefully aspirated, leaving a pellet, which was washed 5 times with lysis solution. After the last washing is finished, adding a proper amount of 5 × Loading Buffer according to the rest volume in proportion, and boiling at 100 ℃; after cooling at room temperature, the sample is placed at-20 ℃ for storage or Western Blot experiment according to the experimental arrangement. The gel was run and stained with Coomassie Brilliant blue (Byunnan), and mass-spectrometrically detected and analyzed by the analytical testing center of Nanjing university of medical science.

FIG. 12 shows the identification of specific peptide fragment mass spectra of METTL 3. METTL3 may be combined with USP13 by comparing the amino acid sequence of METTL3 protein and combining the mass spectra results.

b) Protein Co-immunoprecipitation (Co-IP)

The Co-IP experiment further verified whether USP13 binds to METTL 3. IP procedure as described above, USP13 antibody (Proteintech, 1 diluted by 50) was used as IP, the METTL3 antibody (Proteintech, 1 diluted by 200) was analyzed by IB (western blot); IP was followed by METTL3 antibody (Proteintech, 1, 50 dilution) and USP13 antibody (Proteintech, 1, 200 dilution) for IB analysis.

The results are shown in FIG. 13, and the Co-IP results suggest that the USP13 protein binds to the METTL3 protein in osteosarcoma cells.

Example 6 USP13 protein Deubiquitinated METTL3 protein

Since USP13 is a deubiquitinase, METTL3 ubiquitinase levels in osteosarcoma cells of the shNC control group, USP13 shRNA1 group, and shRNA2 group were next evaluated. METTL3 antibody (Proteintech, 1, 50 dilution) for IP, ub antibody (CST, 1, 200 dilution) for IB analysis.

Fig. 14 is a test result of endogenous METTL3 ubiquitination level after USP13 in osteosarcoma cell line 143B is knocked down, and the result shows that endogenous ubiquitination levels of osteosarcoma cells in USP13 shRNA1 group and shRNA2 group are significantly increased compared to those in shNC control group, which indicates that USP13 regulates and controls METTL3 protein ubiquitination level, and METTL3 protein ubiquitination level is increased after USP13 is knocked down.

Example 7 analysis of METTL3 expression level in osteosarcoma clinical samples

A Western Blot method is used for detecting 12 the expression quantity of the METTL3 protein in an osteosarcoma clinical sample and a normal bone tissue, and the detection result is shown in figure 15, so that the expression of the METTL3 protein in the osteosarcoma tissue is obviously higher than that in the normal bone tissue.

Statistics were performed on the expression of METTL3 protein in 100 clinical samples of osteosarcoma, and correlations between USP13 and METTL3 expression were analyzed, with the results shown in table 3:

TABLE 3

USP13 and METTL3 expression correlation analysis (spearman method)

As shown in Table 3, the results of 43 cases of strong positive METTL3, 29 cases of positive, 19 cases of weak positive and 9 negative cases in osteosarcoma tissues indicate that the expression levels of the USP13 protein and the METTL3 protein in the osteosarcoma are obviously and positively correlated (R =0.471 and P-woven 0.001).

qRT-PCR detection is performed on expression quantity of METTL3 mRNA in 40 pairs of osteosarcoma clinical samples and normal bone tissues, the method is the same as the part (4) in the example 1, and the gene primer sequences are respectively as follows:

METTL3 forward:5’-TTTTCCGGTTAGCCTTCGGG-3’

METTL3 reverse:5’-GATAGAGCTCCACGTGTCCG-3’

GAPDH forward:5’-GACAGTCAGCCGCATCTTCT-3’

GAPDH reverse:5’-GCGCCCAATACGACCAAATC-3’。

the results are shown in fig. 16, and it can be seen that the expression of METTL3 mRNA in osteosarcoma tissue is significantly higher than that in normal bone tissue (P < 0.001).

Example 8 analysis of METTL3 expression level in osteosarcoma cell line

Western Blot was used to detect the expression of METTL3 in osteosarcoma cell lines and normal osteoblast cell lines as described in section (3) of example 1.

As shown in FIG. 17, it can be seen that the protein expression level of METTL3 in osteosarcoma cell line (HOS, saos-2, U-2OS, 143B and MG 63) is significantly higher than that of normal osteoblast cell line hFOB 1.19.

Taken together, USP13 was found to be highly expressed in osteosarcoma and suggested a poor prognosis. The expression of USP13 in osteosarcoma cells is knocked down, so that the in-vitro proliferation, clone formation capacity and invasion and transfer capacity of the osteosarcoma cells can be reduced; and can reduce the proliferation and invasion and metastasis abilities of osteosarcoma in vivo. USP13 proteins can bind to METTL3 protein and reduce its ubiquitination levels. METTL3 is highly expressed in osteosarcoma and has obvious positive correlation with the expression level of USP 13. These results suggest that USP13 can be used as a molecular marker of osteosarcoma, and can be applied to the auxiliary diagnosis, curative effect prediction and prognosis judgment of osteosarcoma.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art can obviously make simple changes or equivalent substitutions of the technical solution within the technical scope of the present invention.

Sequence listing

<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

<120> molecular marker USP13 related to human osteosarcoma and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 863

<212> PRT

<213> Osteosarcoma (Osteosarcoma)

<400> 1

Met Gln Arg Arg Gly Ala Leu Phe Gly Met Pro Gly Gly Ser Gly Gly

1 5 10 15

Arg Lys Met Ala Ala Gly Asp Ile Gly Glu Leu Leu Val Pro His Met

20 25 30

Pro Thr Ile Arg Val Pro Arg Ser Gly Asp Arg Val Tyr Lys Asn Glu

35 40 45

Cys Ala Phe Ser Tyr Asp Ser Pro Asn Ser Glu Gly Gly Leu Tyr Val

50 55 60

Cys Met Asn Thr Phe Leu Ala Phe Gly Arg Glu His Val Glu Arg His

65 70 75 80

Phe Arg Lys Thr Gly Gln Ser Val Tyr Met His Leu Lys Arg His Val

85 90 95

Arg Glu Lys Val Arg Gly Ala Ser Gly Gly Ala Leu Pro Lys Arg Arg

100 105 110

Asn Ser Lys Ile Phe Leu Asp Leu Asp Thr Asp Asp Asp Leu Asn Ser

115 120 125

Asp Asp Tyr Glu Tyr Glu Asp Glu Ala Lys Leu Val Ile Phe Pro Asp

130 135 140

His Tyr Glu Ile Ala Leu Pro Asn Ile Glu Glu Leu Pro Ala Leu Val

145 150 155 160

Thr Ile Ala Cys Asp Ala Val Leu Ser Ser Lys Ser Pro Tyr Arg Lys

165 170 175

Gln Asp Pro Asp Thr Trp Glu Asn Glu Leu Pro Val Ser Lys Tyr Ala

180 185 190

Asn Asn Leu Thr Gln Leu Asp Asn Gly Val Arg Ile Pro Pro Ser Gly

195 200 205

Trp Lys Cys Ala Arg Cys Asp Leu Arg Glu Asn Leu Trp Leu Asn Leu

210 215 220

Thr Asp Gly Ser Val Leu Cys Gly Lys Trp Phe Phe Asp Ser Ser Gly

225 230 235 240

Gly Asn Gly His Ala Leu Glu His Tyr Arg Asp Met Gly Tyr Pro Leu

245 250 255

Ala Val Lys Leu Gly Thr Ile Thr Pro Asp Gly Ala Asp Val Tyr Ser

260 265 270

Phe Gln Glu Glu Glu Pro Val Leu Asp Pro His Leu Ala Lys His Leu

275 280 285

Ala His Phe Gly Ile Asp Met Leu His Met His Gly Thr Glu Asn Gly

290 295 300

Leu Gln Asp Asn Asp Ile Lys Leu Arg Val Ser Glu Trp Glu Val Ile

305 310 315 320

Gln Glu Ser Gly Thr Lys Leu Lys Pro Met Tyr Gly Pro Gly Tyr Thr

325 330 335

Gly Leu Lys Asn Leu Gly Asn Ser Cys Tyr Leu Ser Ser Val Met Gln

340 345 350

Ala Ile Phe Ser Ile Pro Glu Phe Gln Arg Ala Tyr Val Gly Asn Leu

355 360 365

Pro Arg Ile Phe Asp Tyr Ser Pro Leu Asp Pro Thr Gln Asp Phe Asn

370 375 380

Thr Gln Met Thr Lys Leu Gly His Gly Leu Leu Ser Gly Gln Tyr Ser

385 390 395 400

Lys Pro Pro Val Lys Ser Glu Leu Ile Glu Gln Val Met Lys Glu Glu

405 410 415

His Lys Pro Gln Gln Asn Gly Ile Ser Pro Arg Met Phe Lys Ala Phe

420 425 430

Val Ser Lys Ser His Pro Glu Phe Ser Ser Asn Arg Gln Gln Asp Ala

435 440 445

Gln Glu Phe Phe Leu His Leu Val Asn Leu Val Glu Arg Asn Arg Ile

450 455 460

Gly Ser Glu Asn Pro Ser Asp Val Phe Arg Phe Leu Val Glu Glu Arg

465 470 475 480

Ile Gln Cys Cys Gln Thr Arg Lys Val Arg Tyr Thr Glu Arg Val Asp

485 490 495

Tyr Leu Met Gln Leu Pro Val Ala Met Glu Ala Ala Thr Asn Lys Asp

500 505 510

Glu Leu Ile Ala Tyr Glu Leu Thr Arg Arg Glu Ala Glu Ala Asn Arg

515 520 525

Arg Pro Leu Pro Glu Leu Val Arg Ala Lys Ile Pro Phe Ser Ala Cys

530 535 540

Leu Gln Ala Phe Ser Glu Pro Glu Asn Val Asp Asp Phe Trp Ser Ser

545 550 555 560

Ala Leu Gln Ala Lys Ser Ala Gly Val Lys Thr Ser Arg Phe Ala Ser

565 570 575

Phe Pro Glu Tyr Leu Val Val Gln Ile Lys Lys Phe Thr Phe Gly Leu

580 585 590

Asp Trp Val Pro Lys Lys Phe Asp Val Ser Ile Asp Met Pro Asp Leu

595 600 605

Leu Asp Ile Asn His Leu Arg Ala Arg Gly Leu Gln Pro Gly Glu Glu

610 615 620

Glu Leu Pro Asp Ile Ser Pro Pro Ile Val Ile Pro Asp Asp Ser Lys

625 630 635 640

Asp Arg Leu Met Asn Gln Leu Ile Asp Pro Ser Asp Ile Asp Glu Ser

645 650 655

Ser Val Met Gln Leu Ala Glu Met Gly Phe Pro Leu Glu Ala Cys Arg

660 665 670

Lys Ala Val Tyr Phe Thr Gly Asn Met Gly Ala Glu Val Ala Phe Asn

675 680 685

Trp Ile Ile Val His Met Glu Glu Pro Asp Phe Ala Glu Pro Leu Thr

690 695 700

Met Pro Gly Tyr Gly Gly Ala Ala Ser Ala Gly Ala Ser Val Phe Gly

705 710 715 720

Ala Ser Gly Leu Asp Asn Gln Pro Pro Glu Glu Ile Val Ala Ile Ile

725 730 735

Thr Ser Met Gly Phe Gln Arg Asn Gln Ala Ile Gln Ala Leu Arg Ala

740 745 750

Thr Asn Asn Asn Leu Glu Arg Ala Leu Asp Trp Ile Phe Ser His Pro

755 760 765

Glu Phe Glu Glu Asp Ser Asp Phe Val Ile Glu Met Glu Asn Asn Ala

770 775 780

Asn Ala Asn Ile Ile Ser Glu Ala Lys Pro Glu Gly Pro Arg Val Lys

785 790 795 800

Asp Gly Ser Gly Thr Tyr Glu Leu Phe Ala Phe Ile Ser His Met Gly

805 810 815

Thr Ser Thr Met Ser Gly His Tyr Ile Cys His Ile Lys Lys Glu Gly

820 825 830

Arg Trp Val Ile Tyr Asn Asp His Lys Val Cys Ala Ser Glu Arg Pro

835 840 845

Pro Lys Asp Leu Gly Tyr Met Tyr Phe Tyr Arg Arg Ile Pro Ser

850 855 860

Claims (2)

1. The application of a molecular marker USP13 as a detection target in preparing osteosarcoma diagnostic reagent or curative effect evaluation products is provided, and the amino acid sequence of the USP13 is shown as SEQ ID No. 1.

2. The application of the reagent for detecting the expression level of the molecular marker USP13 in the preparation of osteosarcoma diagnostic reagents or curative effect evaluation products comprises specific primers, wherein the specific primers are USP13-F and USP13-R:

USP13-F: 5’-CAAAAGATCGCCTGATGAACCA-3’，

USP13-R: 5’-TGATCCAGTTGAAGGCCACC-3’。

Images