CN110951873A

CN110951873A - Bone and sarcoma marker, application thereof and kit

Info

Publication number: CN110951873A
Application number: CN201911217922.7A
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: 2020-04-03

Abstract

The invention discloses an osteosarcoma marker HOXB8 and its application. HOXB8 is significantly highly expressed in osteosarcoma tissues, and its expression level is significantly correlated with clinical grade, with the highest expression in osteosarcoma with lung metastases; knockdown of HOXB8 expression can inhibit the growth of osteosarcoma cells, suggesting that HOXB8 can become osteosarcoma target of tumor therapy. The present invention includes the application of HOXB8 protein in the preparation of kits or medicines with the following functions: (1) as a new marker of osteosarcoma for early diagnosis and prognosis judgment including grading; (2) as a drug target to inhibit bone and flesh tumor growth. The invention provides a new idea for osteosarcoma treatment by using HOXB8 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

Bone and sarcoma marker, application thereof and kit

Technical Field

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

Background

The osteosarcoma is the most common primary bone malignant tumor, the osteosarcoma is the most common primary malignant bone tumor of teenagers, comprehensive treatment combining surgery and new adjuvant chemotherapy can cure about 60% of patients, 40% of patients with the osteosarcoma have far metastasis treatment failure, particularly lung metastasis is the main cause of death of the osteosarcoma, a lung metastasis focus is insensitive to the existing chemotherapeutic drugs, the targeted drugs do not have breakthrough progress, 5-year survival rate of the patients with the lung metastasis is lower than 20%, the pulmonary sarcoma pulmonary metastasis becomes a bottleneck restricting prognosis improvement, so that the curative effect is not long in thirty years, how to effectively discover and inhibit the pulmonary metastasis of the osteosarcoma is a clinical problem to be solved, a series of discussions on the onset of the osteosarcoma and the like exist in the past, the defects of low sensitivity and low specificity of commonly-used markers are found, the molecular limitation mechanism of the pulmonary metastasis of the osteosarcoma is deeply researched and clarified, a new osteosarcoma prevention and treatment target is found and can effectively intervene in controlling the pulmonary metastasis of the osteosarcoma, the further improvement of the curative effect of the osteosarcoma is the lung metastasis of the low grade of the benign metastatic tumor, the malignant tumor is represented by the international grade of the international grade T No. 7, the international grade No. 7. the international grade No.1, the international grade No. 7. the benign metastatic tumor is represented by the international grade No.1, the international grade No. 8. the international grade of the international grade No.1, the international grade of the intraspecific malignant tumor, the international grade No.1, the international grade of the intraspecific malignant tumor is represented by the international grade No.1, the international grade of the international grade No.1, the international grade No. 8. the international grade of the intraspecific malignant tumor is represented by the international grade of the intraspecific malignant tumor, the international grade No. 1.

The most common part of the hematogenous metastasis of osteosarcoma is the lung, so postoperative follow-up chest imaging examination of osteosarcoma patients is extremely important. Once lung metastasis occurs, the prognosis of the patient is poor and the survival rate is far low. However, even so, for patients with lung metastasis, surgical resection of metastasis is still claimed at present, and conventional chemotherapy is required before and after resection of metastasis, so that development of a new targeted therapeutic approach is an urgent need for osteosarcoma prevention and treatment. The tumor marker kit is used for detecting the molecular pathological changes of patients, and early screening or auxiliary diagnosis is realized, so that effective tumor markers need to be found. Specific treatment by new means such as gene therapy, immunotherapy, molecular targeted therapy requires finding specific markers for osteosarcoma cells.

The HOX (Homeobox) gene is a development related gene, and the encoded protein plays a role in regulating transcription factors in the process of embryonic development. It was first discovered to play an important role in the embryonic development of drosophila melanogaster. HOXB8(NCBI accession No. NM-024016.4, GeneID:3218) is one of the HOX gene family members and is located on chromosome 17. A recent study has shown that HOXB8 is aberrantly expressed in a variety of tumor cells and is associated with tumor development and chemotherapeutic effects. The research finds that HOXB8 in the osteosarcoma patient is abnormally high expressed, the expression is more obvious along with the increase of the level of the osteosarcoma, and the HOXB8 high expression is obviously related to the metastasis and poor prognosis of the osteosarcoma patient; by constructing a HOXB8 lentiviral vector, a HOXB8 knockdown cell model is established, and the influence of the HOXB8 knockdown cell model on the growth and transfer of osteosarcoma cells is researched. Aims at discussing the relation between the expression of HOXB8 and the generation and transfer of osteosarcoma and lays a foundation for further researching the targeted therapy and regulation mechanism of HOXB8 on osteosarcoma. And knockdown of HOXB8 expression with shRNA significantly inhibited osteosarcoma growth. The application shows that the HOXB8 can be used as a biological marker and a therapeutic target of osteosarcoma and is used for auxiliary diagnosis, curative effect detection, prognosis judgment and the like of osteosarcoma. Through retrieval, no relevant report and patent application aiming at HOXB8 in osteosarcoma exists at present.

HOXB8 sequence information:

disclosure of Invention

The invention aims to screen out a tumor marker related to osteosarcoma, wherein the marker is a DNA sequence HOXB8 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 a specific primer of a HOXB8 gene, an upstream primer and a downstream primer of the specific primer of the HOXB8 gene, wherein the sequence of the specific upstream primer of the HOXB8 gene is shown as SEQ ID No.1, and the sequence of the downstream primer of the HOXB8 gene is shown as SEQ ID No. 2.

The kit comprises a real-time fluorescent quantitative PCR detection kit and an immunohistochemical 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 immunohistochemical detection kit comprises a standard substance control, an HOXB8 antibody and a reagent related to immunohistochemical detection.

The reference value level is the expression level of the HOXB8 gene in normal cells, i.e. normal human osteoblasts, of the same line as the test cells and free from canceration. The assay cell is known or suspected to comprise a tumor cell.

The inventor finds that the primer for detecting the HOXB8 has good specificity, has high accuracy for diagnosing osteosarcoma, finds a novel gene which can be used as a tumor marker, has high expression in tumor cells and tissues, has the expression quantity remarkably related to clinical pathological parameters of tumors, and shows that the HOXB8 is a specific tumor marker. The present invention has been completed based on this finding.

In order to verify the diagnostic effectiveness of the invention, the invention verifies the expression characteristics of the cancer marker and the influence of the expression characteristics on the growth function of osteosarcoma cells by adopting a RT-PCR (reverse transcription-polymerase chain reaction), immunohistochemistry and gene knock-down method. The experimental method mainly comprises the following parts:

1. gene expression differences were verified in osteosarcoma and paracarcinoma samples using RT-PCR: extracting total RNA of osteosarcoma and paratissue thereof; designing a primer, and carrying out PCR reaction; HOXB8 expression in osteosarcoma tissues was significantly higher than in paracarcinoma tissues.

2. The immunohistochemistry technology is applied to 60 osteosarcoma sample sections for dyeing to verify that the expression difference of the HOXB8 in osteosarcoma patients with different degrees of malignancy is as follows: the samples are osteosarcomas with different degrees of malignancy collected clinically, and the survival curve of osteosarcoma patients is analyzed according to the HOXB8 staining score and clinical follow-up data, so that the prognosis of the patients with high expression HOXB8 is worse than that of the patients with low expression, and the expression of HOXB8 in the patients with lung metastasis is higher than that of the patients without lung metastasis.

2. The influence of the gene on the growth and transfer functions of osteosarcoma cells is verified at a cellular level: expressing shRNA knock-down gene; the effect of HOXB8 on cell growth was verified. The result shows that the osteosarcoma cell growth capacity of knocking down the HOXB8 gene is obviously reduced.

3. The influence of the gene on the growth function of osteosarcoma is verified at the global level: the HOXB8 gene is knocked down, and is transplanted to the tibia of a nude mouse in situ after verification, so that the influence of the gene on the growth and transfer of osteosarcoma is verified. The result shows that the osteosarcoma growth and transfer capability of knocking down the HOXB8 gene are obviously reduced.

The inventor finds that the primer for detecting the HOXB8 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 comprises a real-time fluorescent quantitative PCR detection kit and an immunohistochemical 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 immunohistochemical detection kit comprises a standard substance control, an HOXB8 antibody and a reagent related to immunohistochemical detection. 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 test sample is fresh, frozen or paraffin-fixed embedded tissue.

The reference value level is the expression level of the HOXB8 gene in normal bone tissue.

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

the application of the HOXB8 gene as a biological marker of osteosarcoma is characterized in that the HOXB8 gene is highly expressed in osteosarcoma tissues.

The HOXB8 gene can be used as a detection target of a detection kit for the auxiliary diagnosis, the curative effect prediction and the prognosis judgment of osteosarcoma.

3. The reagent for detecting the expression of the HOXB8 can be used as a reagent for auxiliary diagnosis, curative effect prediction and prognosis judgment of osteosarcoma, and the reagent for detecting the expression of the HOXB8 is a probe or a primer for specifically detecting HOXB8 mRNA or an antibody specifically binding with HOXB8 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 the expression of HOXB8 can be used for preparing a medicine for treating osteosarcoma, wherein the substance for inhibiting the expression of HOXB8 is miRNA, siRNA, dsRNA or shRNA, or an over-expression plasmid vector or lentivirus containing the miRNA, siRNA, dsRNA or shRNA.

The HOXB8 protein as a target can be used for screening anti-osteosarcoma drugs.

Drawings

FIG. 1 shows the comparison of the expression level of HOXB8 in the GEO database in the normal osteoblasts and in the osteosarcoma tissues (A), and the expression level of HOXB8 in the tissues of the non-relapsed and relapsed osteosarcomas (B).

FIG. 2 is a comparison (A) of the expression levels of HOXB8 detected by RT-qPCR in clinical osteosarcoma samples and in paracancerous tissue samples, the expression level of HOXB8 in different encocking grades shown by immunohistochemistry, the expression level difference between metastatic and non-metastatic osteosarcoma (B), the relation (C) between the expression level of HOXB8 and the survival curve of osteosarcoma patients shown by immunohistochemistry, and the statistical table (D) of the staining score of HOXB8 and various clinical indexes.

FIG. 3 shows the effect of different shRNAs on osteosarcoma cell proliferation and metastatic capacity. Shows the effect of shRNA interference on the growth of nude mouse orthotopic tumor graft (C) and the difference of metastasis (D) on the number of cell growth changes (A) and invasiveness changes (B) of two tumor cell lines after the knockdown of HOXB 8.

Detailed Description

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

Example 1 analysis of the data from the GEO database reveals the difference in expression of HOXB8 gene between osteosarcoma tissue and para-carcinoma tissue, and the relationship between HOXB8 and osteosarcoma patient recurrence.

RNA-seq sequencing data and clinical 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 difference of HOXB8 in osteosarcoma tissues and tissues beside the osteosarcoma tissues and the expression difference of HOXB8 in patients with recurrent and non-recurrent osteosarcoma.

As a result: referring to fig. 1A, analysis found that HOXB8 expression in osteosarcoma was significantly increased compared to normal osteoblasts, and fig. 1B shows that HOXB8 expression in relapsed osteosarcoma was higher than in non-relapsed patients, considering its use as an early diagnostic indicator of osteosarcoma.

Example 2: RT-qPCR verified the expression difference of the HOXB8 gene in osteosarcoma tissues and para-carcinoma tissues.

Osteosarcoma tissue was obtained by surgical excision and confirmed by pathological diagnosis by measuring the expression status of HOXB8 gene in 9 pairs of osteosarcoma tissue and paracarcinoma tissue.

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).

And thirdly, quantitatively carrying out PCR (polymerase chain reaction) to identify the expression quantity of the HOXB8 gene, wherein the specific operation steps comprise that all operations and related reagents are put on ice for operation, the reagents and the concentration shown in the table 1 are added into a PCR reaction tube, and finally, a cDNA sample is added, reaction conditions are set in a PCR instrument, wherein the reaction conditions comprise that the temperature is 94 ℃ for pre-denaturation for 10s, the temperature is 94 ℃ for denaturation for 5s, the temperature is 60 ℃ for annealing/extension for 34s, 40 cycles are totally, β -Actin is used as a relative quantitative internal reference, and PCR primers are shown in the table 1.

Table 1 method for identifying expression quantity of HOXB8 gene 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 primer sequences for RT-PCR identification of the HOXB8 gene

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. 2A, we examined the mRNA expression of HOXB8 in osteosarcoma tissue and para-carcinoma tissue, found that HOXB8 mRNA was expressed in tumor tissue, and validated the database.

Example 3: by immunohistochemistry, the relationship between HOXB8 expression and overall survival of osteosarcoma patients was explored.

Placing a clinically collected osteosarcoma sample in 10% neutral buffered formalin, fixing the osteosarcoma sample for 8-24 h at normal temperature, dehydrating, slicing, adhering a slide glass with the thickness of 2 mu m, setting the temperature of the baking sheet at 65-68 ℃ for 2h, and dewaxing the xylene I and II for 12 minutes respectively, wherein the amount of a general fixing solution is 4-10 times of the volume of a tissue block; 3 minutes for each of the absolute ethyl alcohol I and the absolute ethyl alcohol II; (xylene wash) 95% ethanol for 3 minutes; 85% ethanol for 3 minutes; rinsing with tap water for 3 minutes, washing completely, soaking with 0.1% Triton x-100 for 10min, soaking with PBS for 5min × 2 times, heating EDTA antigen repairing solution to 92-98 deg.C (microwave oven middle fire for 5 min), and then low fire at 92-98 deg.C for 5min × 4 times (guaranteed temperature). Cooling to room temperature, soaking in PBS, and soaking the repaired antigen slice in tap water for 2 min. The samples were placed in endogenous peroxidase blocker 3% H2O2 (methanol) for 4 minutes at room temperature. The washing was carried out with tap water for 2 minutes and PBS buffer for 2 minutes. The sections were taken out, wiped to dry the liquid surrounding the tissue, 10% BSA blocking solution was added dropwise, and incubated at 37 ℃ for 40 minutes. Dropping primary antibody (concentration ratio, PBS is diluted by 1:50, 20ul, tissue is kept in a wet state but no liquid can be on the surface, putting the tissue into a special incubation box, taking out the tissue after overnight staying in a refrigerator at 4 ℃, taking out the tissue, washing the tissue for 3 minutes and 3 times in PBS buffer solution, wiping the liquid around the tissue, dropping secondary antibody, incubating the tissue in an incubator at 37 ℃ for 40 minutes, washing the tissue with PBS buffer solution for 3 minutes and 3 times, dropping a ready-prepared DAB color developing agent for room temperature color development, washing the tissue with tap water for 3 minutes, dyeing the tissue in hematoxylin dyeing solution for 40 seconds, washing the tissue for 1 minute, differentiating the tissue with 1% hydrochloric acid alcoholic solution for 3 seconds, rinsing with running water, dehydrating, placing the tissue in sequence in 85% ethanol, 95% ethanol for 1 minute, absolute ethanol for 2 minutes in sequence I and II, and placing the tissue in sequence in xylene.

As a result: referring to fig. 2B, immunohistochemical staining showed that HOBX8 was expressed in higher amounts in metastatic osteosarcoma than in metastatic osteosarcoma, and the expression was also increased with increasing grade. Statistical analysis revealed that HOXB8 expression had a significant correlation with survival in osteosarcoma patients, and that overall survival in patients with high HOXB8 expression was significantly lower than in patients with HOXB8 low expression, suggesting that HOXB8 is likely to have an effect in promoting cancer progression (fig. 2C). Fig. 2D shows how HOXB8 relates to various clinical indicators.

Example 4: effect of inhibition of HOXB8 expression levels using shRNA on osteosarcoma cell proliferation. (FIG. 3A)

Knockdown of the HOXB8 gene was performed using two different shrnas (shRNA sequences are shown in table 3), and the degree of down-regulation of HOXB8 expression was compared in both cases. Dividing osteosarcoma cells 143B and SJSA1 into three groups, wherein group I is a control group without gene knock-down and expression inhibitor; group II is HOXB8 shRNA1 knockdown group; group III is the HOXB8 shRNA2 knockdown group. The proliferation of the tumor cells was observed 12h, 24h, 36h, 48h, 60h and 72h after the culture under the same culture conditions.

TABLE 3 ShRNA sequences for knockdown of the HOXB8 Gene

Numbering	Name of nucleic acid sequence	Sequence of
			SEQ ID NO 3	HOXB8 shRNA1	AGTACGCAGACTGCAAGCTTG
SEQ ID NO 4	HOXB8 shRNA2	GAGTTCCTATTTAATCCCTAT

As a result: in both the shRNA1 and shRNA2 groups, the number of cell lines was significantly lower than the control group compared to the control group (fig. 3A). These results demonstrate that knockdown HOXB8 is capable of inhibiting the growth of osteosarcoma cell line 143B, SJSA 1.

Example 5: effect of inhibition of HOXB8 expression levels using shRNA on the ability of osteosarcoma cells to invade. (FIG. 3B)

Knockdown of the HOXB8 gene was performed using two different shrnas (shRNA sequences are shown in table 3), and the degree of down-regulation of HOXB8 expression was compared in both cases. Dividing osteosarcoma cells 143B and SJSA1 into three groups, wherein group I is a control group without gene knock-down and expression inhibitor; group II is HOXB8 shRNA1 knockdown group; group III is the HOXB8 shRNA2 knockdown group. Each group of cells was inoculated into a transwell chamber (40000 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 6: blocking osteosarcoma metastasis in vivo after inhibiting HOXB8 expression level by shRNA

143B cells obtained in the three groups of example 5 were transformed with luciferase with lentivirus at a dose of 1.5X 10⁶The tibia of a 5-week-old female nude mouse is inoculated in situ, the first in-vivo imaging shooting is started after 1 week, the shooting is performed every 7 days later, the fluorescence intensity of a tumor part is calculated, and the experiment is ended after 5 weeks.

As a result: the fluorescence intensity of the orthotopic transplantation tumor is shown in FIG. 3C, and the fluorescence intensity of the metastasis is shown in FIG. 3D. Both HOXB8 knockdown 143B cells had significantly less tumor volume than the control cells. Osteosarcoma lung metastases also disappeared after inhibition of HOXB 8.

In combination with the above results, HOXB8 was significantly highly expressed in osteosarcoma, and the expression level in osteosarcoma with lung metastasis was higher than that in osteosarcoma without lung metastasis. And the conclusion is verified on the functional level. The high expression of the HOXB8 is reflected to have the capability of marking osteosarcoma metastasis, and the HOXB8 can be used as an osteosarcoma marker and 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.

Claims

The application of HOXB8 gene as osteosarcoma biomarker, characterized in that HOXB8 gene is highly expressed in osteosarcoma tissue.
2. An osteosarcoma detection kit comprises a reagent for detecting the expression of HOXB8, 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 the expression of HOXB8 is a probe or primer for specifically detecting HOXB8 mRNA, or an antibody specifically binding to HOXB8 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 application of the substance for inhibiting the expression of HOXB8 in preparing the medicine for treating osteosarcoma is that the substance for inhibiting the expression of HOXB8 is miRNA, siRNA, dsRNA or shRNA, or an over-expression plasmid vector or lentivirus containing the miRNA, siRNA, dsRNA or shRNA.
7. The substance that inhibits the expression of HOXB8 according to claim 6, comprising the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.
The application of the HOXB8 protein as a target in screening anti-osteosarcoma drugs.