CN111020030A

CN111020030A - An osteosarcoma stem cell marker and its application and kit

Info

Publication number: CN111020030A
Application number: CN201911220182.2A
Authority: CN
Inventors: 赵蔚; 邹昌业; 鲁冰
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2020-04-17

Abstract

The invention discloses an osteosarcoma stem cell marker LIF and its application. LIF is significantly highly expressed in osteosarcoma tissues, and its expression level is significantly correlated with the stemness of osteosarcoma cells; LIF can promote the growth of osteosarcoma cells, suggesting that LIF can be a target for osteosarcoma therapy. The present invention includes the application of LIF protein: (1) as a new marker of osteosarcoma stem cells for prediction including metastasis; (2) as a drug target to inhibit the growth of osteosarcoma stem cells. The invention provides a new idea for osteosarcoma treatment by using LIF protein as a biological marker and targeted therapy, and also provides an auxiliary detection index for early diagnosis and prognosis judgment, and has important clinical application value.

Description

Osteosarcoma stem cell marker, application thereof and kit

Technical Field

The invention belongs to the technical field of biological medicines. Relates to a tumor marker related to osteosarcoma stem cells and application thereof.

Background

Osteosarcoma is the most common primary malignant bone tumor of teenagers, about 60% of patients can be cured by the comprehensive treatment of surgery combined with neoadjuvant chemotherapy, 40% of patients with osteosarcoma have the failure of remote metastasis treatment, and lung metastasis is the main cause of death of osteosarcoma. The 5-year survival rate of patients with pulmonary metastasis is lower than 20%, the pulmonary metastasis is insensitive to the existing chemotherapy drugs, and the targeted drugs do not make a breakthrough progress. Therefore, osteosarcoma lung metastasis becomes a bottleneck restricting the improvement of prognosis. How to effectively and early discover and restrain osteosarcoma lung metastasis is an urgent clinical problem to be solved. A series of researches on the occurrence of osteosarcoma and the like are carried out, but the defects of low sensitivity, low specificity and the like of the found markers exist. Therefore, the molecular mechanism of the lung metastasis of the osteosarcoma is deeply researched and clarified, a new sarcomatosis marker and a new prevention target are found, and the effective intervention has extremely important value for controlling the lung metastasis of the osteosarcoma and further improving the curative effect of the osteosarcoma.

Numerous studies have shown that only a small fraction of cancer cells with tumor initiating ability are the core source of tumorigenesis, a subset of which is termed tumor stem cells. Tumor stem cells have some of the characteristics of normal stem cells, and they can self-renew through cell division to form the same daughter cells and differentiate into various types of progeny. While patients with osteosarcoma may still develop distant metastases decades after the first treatment, this may further highlight the tumorigenic character of tumor stem cells. Osteosarcoma tissue samples and cell lines present a subpopulation of cells capable of growing meat or bone balls under serum-free culture conditions, which cells exhibit resistance to chemotherapy and radiation and exhibit stem cell-like properties. Tumor stem cells are closely related to the occurrence, recovery, drug resistance, invasion and metastasis of malignant tumors. Thus, targeted therapy of osteosarcoma stem cells offers a promising strategy for the treatment of osteosarcoma. However, there is no clear marker for specific separation of osteosarcoma stem cells at present, and osteosarcoma stem cells can be separated from non-osteosarcoma stem cells, so that an effective osteosarcoma stem cell marker needs to be found for research of specific treatment.

LIF (LIF interfukin 6family cytokine) gene is a gene participating in multi-system action, and the encoded protein is a pleiotropic cytokine and plays different functions in different systems. LIF (NCBI accession No. NM-002309.5, GeneID:3976) is one of the members of the interleukin family and is located on chromosome 22. Recent research finds that LIF is osteosarcoma cells and a gene related to a clinical sample specific super enhancer, so that LIF has an important regulation effect on tumorigenesis and tumor development, and an LIF stimulated cell model is established by treating the osteosarcoma cells with LIF recombinant protein, and the influence of LIF on the growth and metastasis of the osteosarcoma cells is researched. Aims to discuss the relationship between LIF expression and osteosarcoma generation, self-renewal and metastasis, and lays a foundation for further research on LIF-based targeted therapy and regulation mechanism of osteosarcoma. The LIF is suggested to be used as a biological marker and a therapeutic target of osteosarcoma stem cells and used for auxiliary diagnosis, curative effect detection, prognosis judgment and the like of osteosarcoma. Through retrieval, no relevant report and patent application aiming at LIF in osteosarcoma exist at present.

The LIF sequence is as follows:

61atgaag gtcttggcgg caggagttgt gcccctgctg ttggttctgc actggaaaca

121tggggcgggg agccccctcc ccatcacccc tgtcaacgcc acctgtgcca tacgccaccc

181atgtcacaac aacctcatga accagatcag gagccaactg gcacagctca atggcagtgc

241caatgccctc tttattctct attacacagc ccagggggag ccgttcccca acaacctgga

301caagctatgt ggccccaacg tgacggactt cccgcccttc cacgccaacg gcacggagaa

361ggccaagctg gtggagctgt accgcatagt cgtgtacctt ggcacctccc tgggcaacat

421cacccgggac cagaagatcc tcaaccccag tgccctcagc ctccacagca agctcaacgc

481caccgccgac atcctgcgag gcctccttag caacgtgctg tgccgcctgt gcagcaagta

541ccacgtgggc catgtggacg tgacctacgg ccctgacacc tcgggtaagg atgtcttcca

601gaagaagaag ctgggctgtc aactcctggg gaagtataag cagatcatcg ccgtgttggc

661ccaggccttc tag

notch-1 is one of the members of the Notch gene family, and the Notch-1 signaling pathway plays an important role in the proliferation, differentiation, survival and apoptosis of cells, and the abnormal expression of the Notch-1 signaling pathway is closely related to the occurrence and development of tumors. The Notch-1 gene is involved in regulating the signal path of cell differentiation, development and regulation and has influence on the effects of cell apoptosis, proliferation, differentiation and vascularization. Expression of Notch-1 receptor and its ligand Jagged-1 is also commonly used in the diagnosis and prognosis of cancer. Activated Notch-1 converts normal cells to malignancy and is aberrantly expressed in many solid tumors (e.g., cervical, head and neck, endometrial, renal, lung, breast, pleural mesothelioma, and salivary gland tumors) and hematologic tumors in humans. Aberrant Notch-1 signaling is closely associated with the development of some tumors, and disruption of Notch-1 signaling can not only directly cause tumor development, but can ultimately induce tumor formation in a direct or indirect manner through interaction with many other signaling pathways. Given that the abnormal expression of Notch-1 is closely related to the occurrence and development of many tumors, the signaling pathway can be used as a new target for treating tumors.

Disclosure of Invention

The invention aims to screen out a tumor marker related to osteosarcoma stem cells, wherein the marker is a DNA sequence LIF gene. The sequence can also be applied to auxiliary diagnosis, metastasis prediction and prognosis judgment and treatment of osteosarcoma.

The kit for auxiliary diagnosis, curative effect prediction and prognosis of osteosarcoma comprises specific primers of LIF genes, and an upstream primer and a downstream primer of the specific primers of the LIF genes, wherein the sequence of the specific upstream primer of the LIF genes is shown as SEQ ID No.1, and the sequence of the LIF downstream primer is shown as SEQ ID No. 2.

The kit is a real-time fluorescent quantitative PCR detection kit. Wherein the primers in the real-time fluorescent quantitative PCR detection kit are suitable for detecting SYBR Green. In addition, the kit also comprises a standard DNA template and a PCR reaction system, wherein the PCR reaction solution in the PCR reaction system is real-time fluorescent quantitative PCR reaction solution and further comprises fluorescent dye. The real-time fluorescent quantitative PCR reaction solution comprises dNTP, Mg2+, Taq enzyme and buffer solution, wherein the fluorescent dye is SYBR Green II, and the Taq enzyme is hot-start enzyme.

The reference level is the expression level of the LIF gene in normal cells, i.e., normal human osteoblasts, which are of the same line as the test cells and are not cancerated. The assay cell is known or suspected to comprise a tumor cell.

The inventor finds that the primer for detecting the LIF has good specificity, has high accuracy for diagnosing osteosarcoma stem cell-like characteristics, finds a novel gene capable of being used as a tumor stem cell marker, has high expression in tumor cells and tissues, and has the expression quantity remarkably related to the stem cell-like characteristics, and the result shows that the LIF is a specific tumor dryness marker. The present invention has been completed based on this finding.

In order to verify the diagnostic effectiveness of the invention, the invention is verified by adopting the following methods, namely RT-PCR, cell invasion and drug treatment methods to verify the expression characteristics of the cancer marker and the influence of the expression characteristics on the growth function of osteosarcoma cells. The experimental method mainly comprises the following parts:

1. exploring specific enhancers in osteosarcoma using ChIP-seq technology; the RT-PCR technology is applied to verify the gene expression difference in normal bone tissues and osteosarcoma tissues: extracting total RNA of osteosarcoma and tissues beside the osteosarcoma; designing a primer, and carrying out PCR reaction; LIF expression in osteosarcoma tissue is significantly higher than in paracarcinoma tissue; the correlation of the enhancer and the sternness gene is verified by applying an RNA-seq technology.

2. The influence of the gene on the growth and transfer functions of osteosarcoma cells is verified at a cellular level: verifying the influence of LIF protein on the cell dry balling capacity; verifying the effect of LIF protein on the classic osteosarcoma stem cell marker CD 133; verifying the influence of LIF protein on the number of osteosarcoma stem cells; and verifying the change of LIF protein on the invasion capacity of osteosarcoma. The result shows that the growth capacity and the drying capacity of the osteosarcoma cells are obviously improved by increasing the LIF gene expression protein.

3. The influence of the gene on the growth function of osteosarcoma is verified at the in vivo level: taking osteosarcoma tissue of a patient, treating the osteosarcoma tissue with LIF, and verifying the action mechanism of LIF on osteosarcoma cell dryness. The results show that the LIF protein can activate the NOTCH1 signaling pathway of the classical stem cells.

The inventor finds that the primer for detecting LIF has good specificity and has high accuracy for diagnosing osteosarcoma. Therefore, the invention provides a kit for auxiliary diagnosis, curative effect prediction and prognosis judgment of osteosarcoma.

The kit is a real-time fluorescent quantitative PCR detection kit. Wherein the primers in the real-time fluorescent quantitative PCR detection kit are suitable for detecting SYBRGreen. In addition, the kit also comprises a standard DNA template and a PCR reaction system, wherein the PCR reaction solution in the PCR reaction system is real-time fluorescent quantitative PCR reaction solution and further comprises fluorescent dye. The real-time fluorescent quantitative PCR reaction solution comprises dNTP and Mg2⁺The fluorescent dye is SYBRGreenII, and the Taq enzyme is hot-start enzyme.

The use of the kit comprises obtaining a test sample from a tissue or cell; determining the expression level of a biomarker in the test sample; analyzing the expression level to generate a risk score, wherein the risk score can be used to provide a hierarchical diagnosis and prognosis of the subject.

The reference value level is the expression level of the LIF gene in normal bone tissue.

Based on the findings of the present invention, one skilled in the art can determine:

the LIF gene can be used as an osteosarcoma biomarker and is characterized in that the LIF gene is highly expressed in osteosarcoma tissues.

The LIF gene can be used as a detection target of a detection kit for auxiliary diagnosis, curative effect prediction and prognosis judgment of osteosarcoma.

3. The reagent for detecting LIF expression can be used as a reagent for auxiliary diagnosis, curative effect prediction and prognosis judgment of osteosarcoma, and the reagent for detecting LIF expression is a probe or primer for specifically detecting LIF mRNA or an antibody specifically binding LIF protein.

4. Conventional test samples such as fresh, frozen or paraffin-fixed embedded tissue can be used for the above tests.

5. The substance for inhibiting LIF expression can be used for preparing a medicine for treating osteosarcoma, and the substance for inhibiting LIF expression is miRNA, siRNA, dsRNA or shRNA, or an over-expression plasmid vector or lentivirus containing the miRNA, siRNA, dsRNA or shRNA.

The LIF protein as a target can be used for screening anti-osteosarcoma drugs.

Drawings

FIG. 1 shows that LIF is a gene related to super enhancer in osteosarcoma cells and clinical samples (A), LIF expression levels in normal osteoblasts and osteosarcoma tissues in GEO database are compared (B), LIF expression levels in normal osteoblasts and osteosarcoma tissues are compared (C), and LIF expression level in osteosarcoma is positively correlated with dry gene expression level (D)

FIG. 2 is a graph showing the effect on osteosarcoma stem cells after LIF treatment. Comprises the detection of the balling capacity, the dry related marker and the invasion capacity of osteosarcoma cells in a dry culture medium. After LIF recombinant protein treatment, the cell balling quantity and the balling diameter of two osteosarcoma cell lines are influenced (A), the expression quantity of marker CD133(B) on the surface of osteosarcoma stem cells after balling and related dry genes is changed (C), the quantity of the osteosarcoma stem cells is evaluated (D) by adopting a limiting dilution method under the stimulation of LIF with different concentrations, and the influence (E) of the LIF recombinant protein on the invasion capacity of the osteosarcoma cells is evaluated (E),

FIG. 3 shows the changes and mechanism of the relevant sternness genes after LIF treatment. The expression level of NOTCH1 signal pathway-related gene was changed in osteosarcoma cells after LIF treatment (A), and in osteosarcoma clinical specimens after LIF treatment (B).

Detailed Description

The invention is further described with reference to the following figures and examples.

Example 1 enhancers common to osteosarcoma cell lines and osteosarcoma tissue were determined by chromatin co-immunoprecipitation.

Osteosarcoma cell lines (143B, SJSA1, ZOS, ZOSM) and osteosarcoma tissues obtained by surgical excision and confirmed by pathological diagnosis are fixed by formaldehyde and then lysed, and the lysate is processed into DNA fragments in the range of 100-300 base pairs by ultrasonic disruption. The corrected and purified chromatin was incubated overnight with H3K27ac antibody. And (3) immunoprecipitating the antibody protein compound and A/G magnetic beads, washing, eluting and reversely crosslinking to obtain a product. And (3) sequencing the product, wherein the sequencing result is consistent with UCSC hg19 human genome reference, and performing visualization processing to obtain the graph 1A.

As a result: in FIG. 1A, LIF was found to be present as an enhancer in both the osteosarcoma cell line and the osteosarcoma tissue in each group of samples, and the signal intensity of H3K27ac was high.

Example 2 expression differences of LIF gene in normal bone tissue and osteosarcoma tissue were mined by data analysis of GEO database.

And (3) downloading RNA-seq sequencing data of two groups of osteosarcoma tissue samples by adopting a GEO standard method, and making expression difference of LIF in osteosarcoma tissues and tissues beside the carcinoma by using statistical software.

As a result: FIG. 1B shows that LIF expression level in osteosarcoma is significantly increased compared with that in normal osteoblasts, and is considered as an early diagnosis index of osteosarcoma.

Example 3: RT-qPCR verifies the expression difference of LIF gene in osteosarcoma tissue and normal bone tissue.

By determining the expression status of LIF gene in 12 pairs of osteosarcoma tissue and normal bone tissue, osteosarcoma tissue was obtained by surgical excision and confirmed by pathological diagnosis.

First, total RNA extraction of cells was performed. All manipulations and related reagents were placed on ice for manipulation. Extracting total RNA of cells by using Trizol reagent, which comprises the following steps: total RNA extraction is prepared. Cells were directly digested with Trizol, lysed, and Trizol was added. After adding Trizol, the cells were left at room temperature for 5min to be sufficiently lysed. The mixture was centrifuged at 12000rpm at 4 ℃ for 5min using a high-speed refrigerated centrifuge, and the precipitate was discarded. Adding chloroform into 200ul chloroform/ml Trizol, shaking and mixing for 15min, and standing at room temperature for 15 min. The mixture was again centrifuged at 12000g at 4 ℃ for 15min using a high-speed refrigerated centrifuge. The upper aqueous phase was then aspirated into another centrifuge tube. 0.5ml of isopropanol Trizol is added into the isopropanol and mixed evenly, and the mixture is placed for 10min at room temperature. The mixture was centrifuged again at 12000g at 4 ℃ for 10min by using a high-speed refrigerated centrifuge, and the supernatant was discarded, and RNA was deposited on the bottom of the tube. 1ml of 75% ethanol was added, the tube was gently shaken and the pellet suspended. Centrifuging at 4 deg.C and 8000g for 5min with a high-speed refrigerated centrifuge, discarding supernatant as much as possible, air drying residual RNA on a clean bench for 3min, and adding 30ul H2O to dissolve RNA sample. The quality and concentration of RNA are determined by measuring the O.D value, and the purity of RNA can be ensured when the A260/A280 value is 1.6-1.8.

Second step, use

III cDNA was synthesized using the reverse transcription kit (Invitrogen) according to the following protocol: all manipulations and related reagents were worked on ice. Reagents were added to the PCR reaction tube at concentrations according to the instructions and finally the RNA sample was added. The reaction conditions in the PCR instrument were set as follows: the cDNA samples were obtained at 42 ℃ for 45min (reverse transcription) and at 75 ℃ for 10min (heat shock termination reaction).

Thirdly, identifying the expression quantity of the LIF gene by quantitative PCR, and the specific operation steps are as follows: all manipulations and related reagents were worked on ice. The reagents and concentrations shown in Table 1 were added to the PCR reaction tube and the cDNA sample was added last. The reaction conditions in the PCR instrument were set as follows: pre-denaturation 94 ℃ for 10s, denaturation 94 ℃ for 5s, annealing/extension 60 ℃ for 34s, for 40 cycles, with GAPDH as the relative quantitative internal control, and PCR primers shown in Table 1.

TABLE 1 method for identifying LIF gene expression level by RT-PCR

Reactants	Reaction amount
		SYBR Green qPCR Master mix(2X)	10μl
Forward Primer(10μM)	1μl
		Reverse Primer(10μM)	1μl
H2O	7.5μl
		cDNA	0.5μl
Total amount of	20μl

TABLE 2 RT-PCR identification of primer sequences of LIF genes

Numbering	Primer name	Sequence of
			SEQ ID NO 1	LIF Forward Primer	CCAACGTGACGGACTTCCC
SEQ ID NO 2	LIF Reverse Primer	TACACGACTATGCGGTACAGC
			SEQ ID NO 3	GAPDH Forward Primer	CTGGGCTACACTGAGCACC
SEQ ID NO 4	GAPDH Reverse Primer	AAGTGGTCGTTGAGGGCAATG

Data processing and analysis: the relative expression amount of the gene is calculated by a 2-delta-Ct method.

As a result: referring to FIG. 1C, the mRNA expression level of LIF in osteosarcoma tissue and normal bone tissue was determined, and the LIF mRNA expression level in tumor tissue was found to be increased, thereby validating the database conclusion.

Example 4: through data analysis of a GEO database, the expression correlation of the LIF gene and the sternness genes SOX2 and NANOG is mined.

RNA-seq sequencing data of two groups of osteosarcoma tissue samples are downloaded by adopting a GEO standard method, and statistical software is used for making the expression correlation of LIF in osteosarcoma tissues and SOX2 and NANOG in osteosarcoma tissues.

As a result: see fig. 1D for an analysis showing that SOX2, NANOG gene and LIF gene have a certain correlation in expression in osteosarcoma tissues and are statistically significant.

Example 5: by cell balling experiment, the relationship between LIF protein level and osteosarcoma cell growth and self-renewal is observed

Digesting osteosarcoma cells (143B, SJSA1) into single cells, and dividing the osteosarcoma cells 143B and SJSA1 into three groups respectively, wherein the group I is a blank control group without treatment; group II is an experimental group of 50ng/ml recombinant protein; group III experiment group of 100ng/ml recombinant protein. 1000 cells were individually plated on low-adhesion cell culture plates and cultured for 5 days in DMEM/F12 medium containing 10ng/ml EGF, 10ng/ml bFGF and B27 cell culture supplement. The spheronization of the osteosarcoma cells after culture was observed and photographed.

As a result: referring to FIG. 2A, the spheronization results show that LIF has higher spheronization capacity in the LIF-treated osteosarcoma cells than the blank control group and increases with increasing protein concentration.

Example 6: changes in marker CD133 expression on the surface of osteosarcoma stem cells after LIF treatment were observed by flow cytometry.

Dividing osteosarcoma cells into two groups, wherein the group I is a blank control group without treatment; group II is an experimental group of 50ng/ml recombinant protein. And (3) respectively planting a certain amount of cells into the cell culture plates, and culturing for 48 h. The osteosarcoma cells were digested into single cells, stained with CD133-PE antibody for 30min, and the staining of the cells was examined by flow cytometry.

As a result: as shown in FIG. 2B, the expression level of CD133 was significantly increased in the LIF protein-treated group compared to the blank control group.

Example 7: influence of LIF protein on expression ability of sternness gene after treatment of osteosarcoma cells

And (3) treating the osteosarcoma cells by using the LIF recombinant protein, and comparing the expression degree of the sternness gene under the condition of existence of the LIF recombinant protein. Dividing osteosarcoma cells 143B into two groups, wherein group I is a blank control group without treatment; group II is LIF recombinant protein treated group. The cells of each group are respectively inoculated into a six-hole plate culture plate, 50ng/ml of LIF recombinant protein is added into the treated group, and the culture is carried out for 48 h.

Total RNA extraction from cells was performed as described in example 2 and used

III reverse transcription kit (Invitrogen) synthesizes cDNA, quantitative PCR identifies expression level of sternness genes (SOX2, OCT4, NANOG, CD133), GAPDH is used as relative quantitative internal reference, and PCR primers are shown in Table 3.

TABLE 3 primer sequences for RT-PCR identification of expression of sternness genes

As a result: in the LIF recombinant protein-treated group, the expression level of the relevant xerogene in osteosarcoma cells was significantly increased compared to the control group (FIG. 2C). These results demonstrate that an increase in LIF protein expression can promote expression of osteosarcoma sternness gene.

Example 8: the number of osteosarcoma stem cells at different LIF protein concentrations was assessed by limiting dilution.

The data obtained in example 5 were processed by limiting dilution. Different cultures (assays) were tested for the same proportion of active cells. Resulting in fig. 2D.

Example 9: through invasion experiments, the influence of LIF protein on the invasion capacity of osteosarcoma cells is observed. (FIG. 2E)

And (3) treating the osteosarcoma cells by using the LIF recombinant protein, and comparing the invasion capacity of the osteosarcoma cells under the condition of existence of the LIF recombinant protein. Dividing osteosarcoma cells 143B into two groups, wherein group I is a blank control group without treatment; group II is LIF recombinant protein treated group. Each group of cells was inoculated into a transwell chamber (30000 cells/well) plated with Matrigel and cultured for 24 hours, the chamber was taken out and washed with PBS, fixed with methanol, stained with 0.5% crystal violet, and the invasion of cells was observed under a microscope after washing.

Example 10: LIF protein enhances drygene expression by activating NOTCH1 signaling pathway. (FIG. 3A)

Osteosarcoma cells were treated with LIF recombinant protein, and the degree of expression of the NOTCH1 signal pathway gene was compared in the presence or absence of LIF recombinant protein (the primer sequence of NOTCH1 signal pathway gene is shown in Table 4). Dividing osteosarcoma cells 143B into two groups, wherein group I is a blank control group without treatment; group II is LIF recombinant protein treated group. The cells of each group are respectively inoculated into a six-hole plate culture plate, 50ng/ml of LIF recombinant protein is added into the treated group, and the culture is carried out for 48 h.

III reverse transcription kit (Invitrogen) cDNA was synthesized, the expression level of NOTCH1 signal pathway gene (NOTCH1, HEY1, HEYL, HES, KRT19) was identified by quantitative PCR with GAPDH as a relative quantitative internal reference, and PCR primers are shown in Table 4.

TABLE 4 primer sequences for identifying NOTCH1 signaling pathway gene expression by RT-PCR

Numbering	Primer name	Sequence of
			SEQ ID NO 13	NOTCH1 Forward Primer	TCCACCAGTTTGAATGGTCA
SEQ ID NO 14	NOTCH1 Reverse Primer	AGCTCATCATCTGGGACAGG
			SEQ ID NO 15	HEY1 Forward Primer	GAAACTTGAGTTCGGCTCTAGG
SEQ ID NO 16	HEY1 Reverse Primer	GCTTAGCAGATCCTTGCTCCAT
			SEQ ID NO 17	HES1 Forward Primer	TCAACACGACACCGGATAAAC
SEQ ID NO 18	HES1 Reverse Primer	GCCGCGAGCTATCTTTCTTCA
			SEQ ID NO 19	HEYL Forward Primer	GGCTGCTTACGTGGCTGTT
SEQ ID NO 20	HEYL Reverse Primer	GACCCAGGAGTGGTAGAGCAT
			SEQ ID NO 21	KRT19 Forward Primer	AACGGCGAGCTAGAGGTGA
SEQ ID NO 22	KRT19 Reverse Primer	GGATGGTCGTGTAGTAGTGGC

As a result: compared with the control group, the expression level of the related NOTCH1 signaling pathway gene in osteosarcoma cells was significantly increased in the LIF recombinant protein-treated group (FIG. 3A). These results demonstrate that elevated levels of LIF protein can promote activation of the NOTCH1 signaling pathway in osteosarcoma cells.

Example 11: LIF protein enhances drygene expression by activating NOTCH1 signaling pathway. (FIG. 3B)

Osteosarcoma samples collected clinically were minced into 1 to 2 mm pieces, which were then washed and digested with trypsin. The tissue suspension includes undigested osteosarcoma tissue, free osteosarcoma cells, and osteosarcoma stem cells. Dividing the tissue suspension into four groups, wherein group I is a blank control group without treatment; group II is 50ng/ml LIF recombinant protein treatment group; group III is 2uM NOTCH1 signal pathway inhibitor crediganestat processing group; group IV is LIF recombinant protein and 2uM Crenigacestat treatment group. The cell suspensions of each group were inoculated into six-well plates, and treated in groups for 48 h.

As a result: compared with each group, the LIF recombinant protein can save the influence caused by the NOTCH1 signal pathway inhibitor in the LIF recombinant protein and the Crenigacestat-treated group. The expression level of the related sternness gene in osteosarcoma cells was significantly increased (FIG. 3B). These results demonstrate that elevated levels of LIF protein can promote activation of NOTCH1 signaling pathway in osteosarcoma cells, thereby promoting expression of sternness genes.

Combining the above results, the expression level of LIF in osteosarcoma is high, which significantly affects the self-renewal and transfer ability of osteosarcoma, and the expression level of stem gene in osteosarcoma with high expression of LIF protein is higher than that of osteosarcoma with low expression of LIF protein. And the conclusion is verified on the functional level. The LIF is highly expressed, has the capacity of marking osteosarcoma stem cells, can be used as an osteosarcoma stem cell marker, and is applied to auxiliary diagnosis, curative effect prediction and prognosis judgment of osteosarcoma. In addition, the above description is only for the preferred embodiment of the present invention and should not be taken as limiting the invention, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhongshan university

<120> osteosarcoma stem cell marker, application thereof and kit

<130>201911220182.2

<160>23

<170>PatentIn version 3.5

<210>1

<211>19

<212>DNA

<213> Artificial sequence (artificial)

<400>1

ccaacgtgac ggacttccc 19

<210>2

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>2

tacacgacta tgcggtacag c 21

<210>3

<211>19

<212>DNA

<213> Artificial sequence (artificial)

<400>3

ctgggctaca ctgagcacc 19

<210>4

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>4

aagtggtcgt tgagggcaat g 21

<210>5

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>5

tacagcatgt cctactcgca g 21

<210>6

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>6

gaggaagagg taaccacagg g 21

<210>7

<211>23

<212>DNA

<213> Artificial sequence (artificial)

<400>7

gggagattga taactggtgt gtt 23

<210>8

<211>23

<212>DNA

<213> Artificial sequence (artificial)

<400>8

gtgtatatcc cagggtgatc ctc 23

<210>9

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>9

tttgtgggcc tgaagaaaac t 21

<210>10

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>10

agggctgtcc tgaataagca g 21

<210>11

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>11

agtcggaaac tggcagatag c 21

<210>12

<211>22

<212>DNA

<213> Artificial sequence (artificial)

<400>12

ggtagtgttg tactgggcca at 22

<210>13

<211>20

<212>DNA

<213> Artificial sequence (artificial)

<400>13

tccaccagtt tgaatggtca 20

<210>14

<211>20

<212>DNA

<213> Artificial sequence (artificial)

<400>14

agctcatcat ctgggacagg 20

<210>15

<211>22

<212>DNA

<213> Artificial sequence (artificial)

<400>15

gaaacttgag ttcggctcta gg 22

<210>16

<211>22

<212>DNA

<213> Artificial sequence (artificial)

<400>16

gcttagcaga tccttgctcc at 22

<210>17

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>17

tcaacacgac accggataaa c 21

<210>18

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>18

gccgcgagct atctttcttc a 21

<210>19

<211>19

<212>DNA

<213> Artificial sequence (artificial)

<400>19

ggctgcttac gtggctgtt 19

<210>20

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>20

gacccaggag tggtagagca t 21

<210>21

<211>19

<212>DNA

<213> Artificial sequence (artificial)

<400>21

aacggcgagc tagaggtga 19

<210>22

<211>21

<212>DNA

<213> Artificial sequence (artificial)

<400>22

ggatggtcgt gtagtagtgg c 21

<210>23

<211>609

<212>DNA

<213>Homo sapiens

<400>23

atgaaggtct tggcggcagg agttgtgccc ctgctgttgg ttctgcactg gaaacatggg 60

gcggggagcc ccctccccat cacccctgtc aacgccacct gtgccatacg ccacccatgt 120

cacaacaacc tcatgaacca gatcaggagc caactggcac agctcaatgg cagtgccaat 180

gccctcttta ttctctatta cacagcccag ggggagccgt tccccaacaa cctggacaag 240

ctatgtggcc ccaacgtgac ggacttcccg cccttccacg ccaacggcac ggagaaggcc 300

aagctggtgg agctgtaccg catagtcgtg taccttggca cctccctggg caacatcacc 360

cgggaccaga agatcctcaa ccccagtgcc ctcagcctcc acagcaagct caacgccacc 420

gccgacatcc tgcgaggcct ccttagcaac gtgctgtgcc gcctgtgcag caagtaccac 480

gtgggccatg tggacgtgac ctacggccct gacacctcgg gtaaggatgt cttccagaag 540

aagaagctgg gctgtcaact cctggggaag tataagcaga tcatcgccgt gttggcccag 600

gccttctag 609

Claims

The application of LIF gene as osteosarcoma biomarker is characterized in that LIF gene is highly expressed in osteosarcoma tissues.
2. An osteosarcoma detection kit comprises a reagent for detecting LIF expression, and is a kit for auxiliary diagnosis, curative effect prediction and prognosis judgment of osteosarcoma.
3. The osteosarcoma detection kit of claim 2, wherein said reagent for detecting LIF expression is a probe or primer that specifically detects LIF mRNA, or an antibody that specifically binds to LIF protein.
4. The detection kit as claimed in claim 3, wherein the kit comprises an upstream detection primer and a downstream detection primer, the upstream detection primer has the sequence of SEQ ID NO 1, and the downstream detection primer has the sequence of SEQ ID NO 2.
5. The test kit according to claim 3, which is a kit for prognosis of osteosarcoma, wherein the test sample is fresh, frozen or paraffin-fixed embedded tissue.
6. The LIF expression inhibiting substance is miRNA, siRNA, dsRNA or shRNA, or an overexpression plasmid vector or lentivirus containing the miRNA, siRNA, dsRNA or shRNA.
Application of LIF protein or gene as target in screening anti-osteosarcoma drugs.