CN114686486B

CN114686486B - Application of reagent for detecting MYOD1 gene expression level in preparation of osteosarcoma transformed rhabdomyosarcoma or osteosarcoma prognosis preparation

Info

Publication number: CN114686486B
Application number: CN202210437713.9A
Authority: CN
Inventors: 黎志宏; 杨眉; 刘忠越; 王嘉杰
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2022-04-25
Filing date: 2022-04-25
Publication date: 2024-03-26
Anticipated expiration: 2042-04-25
Also published as: CN114686486A

Abstract

The invention discloses an application of a reagent for detecting MYOD1 gene expression level in preparation of osteosarcoma transformed rhabdomyosarcoma or osteosarcoma prognosis preparation. The expression level of the MYOD1 gene in osteosarcoma patients can be detected, a powerful molecular biological basis is provided for transformation of rhabdomyosarcoma of osteosarcoma patients and prognosis prediction of osteosarcoma, and the method has profound clinical significance and popularization.

Description

Application of reagent for detecting MYOD1 gene expression level in preparation of osteosarcoma transformed rhabdomyosarcoma or osteosarcoma prognosis preparation

Technical field:

the invention belongs to the field of tumor molecular biology, and particularly relates to application of a reagent for detecting MYOD1 gene expression level in preparation of osteosarcoma transformed rhabdomyosarcoma or osteosarcoma prognosis preparation.

The background technology is as follows:

osteosarcoma is the primary bone tumor with highest incidence, has a few double-high incidence age groups, is about 5% of pediatric tumors in teenagers or childhood and senior, and is a disease with high mortality rate of children and adolescents malignant tumors. At present, diagnosis and treatment means for osteosarcoma are still quite conserved. Since the last eighties of the century chemotherapy was widely used, the prognosis and clinical strategy of osteosarcoma have been hardly changed in recent decades, surgical resection combined with traditional chemotherapy remains the main means of treatment, and the long-term survival rate of patients without metastasis is about 70%, while the survival rate of patients with metastasis is only about 20-30%. At present, the prognosis judgment of the osteosarcoma patient has no reference standard or specific index, and can not meet the requirement of clinical prognosis judgment of the osteosarcoma patient. Therefore, prognosis of osteosarcoma patients is judged so as to formulate better treatment schemes, and survival rate of the patients is remarkably improved, so that the method becomes an important subject to be solved in the field of bone tumors.

Furthermore, we have occasionally found by high throughput sequencing that rhabdomyosarcoma transformation occurs in some osteosarcoma patients. In subsequent expanded sample size assays, we found that patients with osteosarcoma in which MYOD1 expression was present had a significant proportion and had a poor prognosis. In subsequent experimental validation we found that overexpression of MYOD1 gene alone in osteosarcoma cells enabled osteosarcoma cells to induce differentiation into myofibers, resulting in rhabdomyosarcoma transformation. Therefore, the MYOD1 gene can be used as a specific detection molecule for judging whether rhabdomyosarcoma transformation occurs in osteosarcoma, and provides a detection target for prognosis and future personalized treatment of osteosarcoma patients.

The invention comprises the following steps:

the invention aims to overcome the defects of the prior art and provide application of a reagent for detecting the expression quantity of MYOD1 genes in preparation of a preparation for converting osteosarcoma into rhabdomyosarcoma and a preparation for detecting osteosarcoma prognosis, wherein the CDS sequence of the MYOD1 genes is shown in SEQ ID NO.1. The gDNA sequence of the MYOD1 gene is shown in SEQ ID NO.2.

The application of the method comprises the step of detecting the MYOD1 gene expression quantity by using a PCR detection reagent.

The application of the PCR detection reagent comprises a MYOD1 gene real-time fluorescence quantitative PCR specific primer: forward primer: 5'-CGCCATCCGCTATATCGAGG-3', SEQ ID NO.3; reverse primer: 5'-CTGTAGTCCATCATGCCGTCG-3', SEQ ID NO.4.

The application of the PCR detection reagent also comprises a GAPDH internal reference real-time fluorescence quantitative PCR specific primer: 5'-ACAACTTTGGTATCGTGGAAGG-3', SEQ ID NO.5; reverse primer: 5'-GCCATCACGCCACAGTTTC-3', SEQ ID NO.6.

The invention also provides application of the reagent for detecting the MYOD1 gene expression quantity in preparation of a preparation for detecting the conversion of rhabdomyosarcoma of osteosarcoma patients, wherein the CDS sequence of the MYOD1 gene is shown in SEQ ID NO.1.

The invention also provides application of the agent for over-expressing the MYOD1 gene in preparing preparations for inducing differentiation of osteosarcoma cells into muscle fibers.

The human MYOD1 gene is located on chromosome 11p15.1, gene ID in NCBI: 4654; only one transcript (NM-002478.5) and one encoded protein (NP-002469.2) is currently found in the GeneBank sequence. In the early research work, the inventor extracts RNA from osteosarcoma tissue, carries out real-time fluorescence quantitative PCR detection on the expression of MYOD1 genes after reverse transcription, and discovers that obvious difference exists between prognosis of osteosarcoma patients with MYOD1 gene expression or not: in the detected samples, the three-year recurrence rate of the osteosarcoma patient with MYOD1 gene expression is found to be 100% by statistical analysis, and the three-year recurrence rate of the osteosarcoma patient without MYOD1 gene expression is about 60% (P=0.016) (fig. 2), and the risk rate of MYOD1 gene expression is found to be 0.104 by ratio risk model prognosis analysis, which indicates that the prognosis of the patient with MYOD1 gene expression is relatively poor, and the expression of MYOD1 gene is an independent risk factor for prognosis risk prediction of osteosarcoma patient. Therefore, the MYOD1 gene can be used as a judging standard of osteosarcoma prognosis and used as a biomarker for preparing a prognosis preparation of osteosarcoma patients, and further a osteosarcoma prognosis kit with high cost performance and easy popularization and application can be provided.

The detection method for applying the MYOD1 gene to prognosis of osteosarcoma patients is as follows: (1) Collecting bone and meat tumor tissues obtained by biopsy or postoperative excision of an individual to be detected, and extracting total RNA; (2) reverse transcription of MYOD1 gene into cDNA using total RNA as template; (3) Performing real-time fluorescence quantitative PCR amplification on the detection sample and positive/negative control by using MYOD1 gene specific primers and internal reference GAPDH primers; and (4) judging the result: judging whether the experimental result is valid according to the following steps:

step one: the internal reference GAPDH primer in the negative control sample and the positive control sample has no amplification curve and Ct value of >38, and the MYOD1 gene specific primer in the positive control sample has amplification curve and Ct of <38, which indicates that the PCR reaction reagent has no pollution and the MYOD1 gene specific primer is effective;

step two: the internal reference GAPDH primer in the sample to be detected has an amplification curve with Ct <38, the MYOD1 gene specific primer has an amplification curve with Ct <38, and the sample has MYOD1 gene expression;

step three: the internal reference GAPDH primer in the sample to be detected has an amplification curve with Ct <38, the MYOD1 gene specific primer has no amplification curve with Ct value >38, and the sample has no MYOD1 gene expression;

step four: the internal reference GAPDH primer in the sample to be detected has no amplification curve and Ct value is more than 38, no matter how the MYOD1 gene specific primer is amplified, the sample preparation is problematic, the result is unreliable, and the sample needs to be prepared again;

step five: the internal reference GAPDH primer in the negative control has an amplification curve and Ct <38, no matter how the MYOD1 gene specific primer is amplified, the reagent or the sample has pollution, the result is unreliable, and the kit needs to be replaced and the sample needs to be prepared again;

the detection reagent used a humanized MYOD1 plasmid (pCDH-MYOD-puro, available from Hunan Fenghui Biotechnology Co., ltd.) as a positive control, and the negative control was RNase-Free TE buffer.

In conclusion, the detection preparation provided by the invention can be used for rapidly judging whether the MYOD1 gene is expressed in osteosarcoma of a patient, provides a powerful molecular biological basis for predicting prognosis of the patient, and has profound clinical significance and popularization.

Description of the drawings:

FIG. 1 shows the real-time fluorescent quantitative PCR analysis of the expression of MYOD1 gene in different osteosarcoma samples;

FIG. 2 is a relationship between MYOD1 gene and prognosis of osteosarcoma patients.

FIG. 3 is the morphology of osteosarcoma cell line (MG 63-MYOD 1) and its control cell line (MG 63-V) after specific induction, which stably overexpresses the MYOD1 gene.

FIG. 4 shows immunofluorescence staining results of myofiber-specific proteins MHC and nuclear DAPI of osteosarcoma cell lines (MG 63-MYOD 1) and control cell lines (MG 63-V) stably overexpressing MYOD1 gene after specific induction.

Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.

Detailed Description

The following examples are intended to further illustrate the invention, but not to limit it.

Example 1 preparation of MYOD1 Gene for osteosarcoma prognosis assay kit (50 reactions)

RNA stabilization solution 50ml

2. Isopropyl alcohol 100ml

3. Trichloromethane 100ml

4.Trizol 50ml

5. Enzyme-free water 10ml

6.1 uM random reverse transcription primer 50ul

7.5 X200 ml of reverse transcription buffer

8. Triphosphates deoxynucleotide (10 mM)

9.40U/ul RNase inhibitor 500ul

10.200U/ul MMLV reverse transcriptase 50ul

11.Premix Ex Taq 50ul

12.10 uM MYOD1 gene real-time fluorescence quantitative PCR specific primer:

forward primer: 5'-CGCCATCCGCTATATCGAGG-3'

Reverse primer: 5'-CTGTAGTCCATCATGCCGTCG-3'

13.10 uM reference GAPDH real-time fluorescent quantitative PCR specific primers:

forward primer: 5'-ACAACTTTGGTATCGTGGAAGG-3'

Reverse primer: 5'-GCCATCACGCCACAGTTTC-3'

14.2 Xfluorescent PCR reaction solution

Example 2 MYOD1 Gene detection in osteosarcoma

1) Preservation of osteosarcoma tissue: collecting osteosarcoma tissue to be detected, storing in a freezing tube, and placing in a refrigerator at-80deg.C;

2) Purification of RNA in tissues: grinding a proper amount of sample into powder in liquid nitrogen, and adding 1ml of Trizol to dissociate the sample at room temperature for 5min; adding 200 μl/ml chloroform into a centrifuge tube, vibrating at room temperature for 15-30s, standing for 5min until the liquid is layered, and centrifuging at 12000g for 15min at 4deg.C; carefully taking the upper water phase into a new centrifuge tube, adding 0.5ml of isopropanol, uniformly mixing, standing at room temperature for 20min, and centrifuging at 4 ℃ for 10min with 12000 g; removing the supernatant, adding 1-2ml of ethanol diluted by 75% enzyme-free water, mixing, centrifuging at 4deg.C for 5min at 7500g, removing the supernatant as much as possible, drying at room temperature for 5-10min, and adding 10-20ul of ribozyme-free water to dissolve RNA. Spectrophotometry is used for measuring concentration and quality of RNA, OD260/280 ratio is 1.8-2.0, and the RNA is preserved at-80 ℃.

3) Reverse transcription of RNA: a20 ul reverse transcription reaction system was used with the reverse transcription kit from Thermo company as shown in Table 1:

TABLE 1

Reverse transcription first step conditions: the reaction system was reconfigured at 65℃for 5 minutes as shown in Table 2:

TABLE 2

Composition of the components	Dose/tube
		5 Xreverse transcription buffer	4ul
Triphosphates deoxynucleotide (10 mM)	2ul
		RNase inhibitor (40U/ul)	1ul
MMLV reverse transcriptase (200U/ul)	1ul
		Reverse transcription product of the first step	12ul

Reverse transcription second step procedure: 25 ℃ for 5 minutes; 42 ℃ for 60 minutes; 70℃for 5 min.

4) Fluorescent quantitative PCR was performed with MYOD1 gene specific primers: the MYOD1 gene specific primer sequences were synthesized by Shanghai Biotechnology Inc.

20ul reaction system is as in Table 3:

TABLE 3 Table 3

Fluorescent quantitative PCR reaction procedure: 95 ℃ for 3 minutes; 95 ℃,10 seconds, 60 ℃,30 seconds, 40 cycles.

5) Qualitative analysis of whether MYOD1 gene was expressed or not in the samples: and judging the expression of the MYOD1 genes in the sample according to the data judging conditions. Analysis found that of the 25 osteosarcoma samples, 4 samples had expression of MYOD1 gene.

6) And (3) prognosis judgment: by follow-up of 25 patients or families with osteosarcoma, we have studied the time of first onset, treatment, recurrence and death of these patients in detail, with follow-up time of 1-60 months. Of the 25 osteosarcoma patients examined, 4 patients whose MYOD1 expression was positive were detected by fluorescent quantitative PCR and 21 patients whose expression was negative were compared with disease-free survival by Kaplan-Meier survival analysis, and patients whose MYOD1 expression was positive were poor in prognosis, with statistical significance (p=0.016). The ratio risk model prognosis analysis finds that the risk rate of MYOD1 gene expression is >1 (HR=9.62), the disease-free survival time of a patient with MYOD1 gene expression is obviously shortened, and the gene expression is an independent risk factor for prognosis risk prediction of osteosarcoma patients. The research also shows that the MYOD1 gene can be used as a specific molecular marker for prognosis of osteosarcoma patients.

Example 3: myofibrosis of osteosarcoma cells MG63-MYOD1 overexpressing the human MYOD1 gene:

MG63-MYOD1 stable over-expression cell strain and establishment of control cell strain MG63-V thereof

1. Lentivirus package

(1) Cell plating: and selecting 293T cells with good cell states for lentiviral packaging. The day before transfection, a proper amount of trypsin is used for digesting 293T cells, the cells are evenly spread in a 10cm cell culture dish, and the cell fusion degree is about 90% in the next day of transfection;

(2) Transfected cells: the cell culture fluid was replaced. 2 sterile centrifuge tubes were taken and 500. Mu.l of serum-free medium Opti-MEM was added to each tube. One tube was added with 30. Mu.l Lipofectamine 2000 reagent shaking; another tube was filled with pSPAX vector 2.5. Mu.g, pMD2 vector 2.5. Mu.g (both vectors purchased from Hunan Feng Hui Biotechnology Co., ltd.) and then with the over-expression plasmid pCDH-MYOD-puro 5. Mu.g or an equivalent amount of empty vector pCDH-puro (both vectors purchased from Hunan Feng Hui Biotechnology Co.); after incubation for 5 minutes at room temperature, the two tubes are mixed and inverted and mixed evenly; after incubation for 20min at room temperature, the transfection material forming the complex of DNA and Lipofectamine 2000 was added to 293T cells, mixed well at 37℃with 5% CO ₂ The cell culture was continued in the incubator.

2. Construction of stable over-expression cell line for target cell transfected by lentivirus

(1) Collection of viruses: 36 hours after transfection, cell supernatants were collected with a pipette in 15ml centrifuge tubes and centrifuged at 1300g for 15 minutes; the supernatant was collected and filtered using a 0.45 μm filter to remove 293T cells and their debris and collect the virus suspension.

(2) Infection with virus: the day before infection, MG63 cells in logarithmic growth phase are digested to single cells by trypsin, a proper amount of cells are inoculated to a 6-hole plate, and the cell fusion degree is about 30% when the cells are infected; on the day of infection, the virus suspension was mixed with fresh culture medium at a ratio of 1:1, and polybrene was added at a final concentration of 8ug/ml, and thoroughly mixed to prepare a virus infection solution. After removal of the culture medium to be infected with MG63 cells, the aforementioned virus-infected liquid was added and incubated overnight.

(3) Screening of stable cell strains: the next day of infection, the virus-infected fluid was removed and fresh cell culture fluid was added. And adding puromycin after 3 days of cell recovery to screen the over-expressed positive cells.

(4) Identification of stable cell lines: the positive cells grown in the culture medium containing puromycin were MG63-MYOD1 cells and MG63-V cells, respectively, and the overexpression of the MYOD1 gene was identified by RT-qPCR as described above.

FIG. 3A control cell line MG63-V of MYOD1 overexpressing cell line MG63-MYOD1 and transfected empty vector pCDH-puro was established in human osteosarcoma cell line MG63 by stabilizing the vector pCDH-MYOD-puro overexpressing the human MYOD1 gene (the overexpressing vector was prepared by Hunan Feng Hui Biotechnology Co., ltd.). Cells were treated with TPA 0.1uM added to the cell growth medium for 48 hours, and then the cell growth medium was changed to an induced differentiation medium, and TPA 0.1uM was maintained therein. After 96 hours, the morphology of MG63-V cells was not significantly changed under the microscope, and the cells were still uniformly sized epithelial-like cells; but MG63-MYOD1 cell morphology was significantly different, cell size was very different, and a significant portion of cell morphology was significantly larger, exhibiting myofibrillar changes, indicating that cells were myodifferentiated.

Example 4: immunofluorescence was performed to examine MG63-MYOD1 cell lines induced to differentiate in example 3 and MG63-V as a control cell line.

Immunofluorescence assay

1.1 preparation of cell samples

(1) Cover slips were coated with polyethylenimine and left to stand at room temperature for 1 hour.

(2) The coverslips were washed 3 times with sterile water for 1 hour each.

(3) The coverslip was sterilized by exposure to ultraviolet light in a room temperature drying environment for 4 hours, and the coverslip was sufficiently dried.

(4) Cells were cultured on glass coverslips.

1.2 cell immobilization

(1) The cell culture solution was aspirated and the cell samples on the coverslips were briefly rinsed with phosphate buffer.

(2) Adding 100% methanol precooled at-20 ℃ into a sample, and incubating for 5 minutes at room temperature;

1.3 permeabilization

(1) The samples were incubated with phosphate buffer containing 0.1-0.25% Triton X-100 for 10 minutes.

(2) Cells were washed 3 times for 5 minutes each with phosphate buffer.

1.4 blocking and immunostaining

(1) Samples were incubated with phosphate buffer containing 1% bovine serum albumin, 0.1% tween 20 for 30 minutes at room temperature.

(2) The primary antibody anti-MHC (Santa Cruz, sc-376157) was diluted with 1% bovine serum albumin, 0.1% Tween 20 in phosphate buffer and incubated with the sample for 1 hour at normal temperature.

(3) The primary antibody incubation was removed and the cells were washed 3 times for 5 minutes with phosphate buffer.

(4) The secondary antibody was diluted with phosphate buffer of 1% bovine serum albumin, 0.1% tween 20: fluorescent-labeled mouse antibody (ThermoFisher, A-31571) was incubated with the sample in the dark at room temperature for 1h.

(5) The secondary antibody incubation was removed and washed 3 times for 5 minutes with phosphate buffer in a dark environment.

1.5 counterstaining

(1) Cells were incubated with 0.1-1. Mu.g/mL 4', 6-diamidino-2-phenylindole for 1 min at ambient temperature.

(2) Cells were washed 3 times for 5 minutes each with phosphate buffer.

1.6 sealing plate

(1) The coverslip was blocked on the slide with a blocking medium.

(2) Observed under a fluorescence microscope and photographed.

Fig. 4: to further verify that the stable cell line MG63-MYOD1 overexpressed by MYOD1 has myogenic differentiation ability and can differentiate into myofibers, we detected the MG63-MYOD1 cell line induced to differentiate by the above method and its control cell line MG63-V by immunofluorescence using the molecular marker MHC specific for myofibers. The nuclear fluorescent dye DAPI also dyes the nucleus along with MHC, facilitating cell counting. The results showed that when DAPI staining showed a comparable number of cells, MG63-MYOD1 cells were induced to differentiate and reacted positively with antibodies to the muscle fiber specific molecular marker MHC (Santa Cruz, sc-376157); whereas the control cell line MG63-V was negative. From this, it was further confirmed that MG63-MYOD1 has the capacity to differentiate myoblastly and can be induced to differentiate into myofibers.

Sequence listing

<110> Xiangya two hospitals at university of south China

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cgccatccgc tatatcgagg 20

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ctgtagtcca tcatgccgtc g 21

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acaactttgg tatcgtggaa gg 22

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Claims

Application of an over-expression vector of MYOD1 gene in preparation of a preparation for enabling MG63 osteosarcoma cells to have myogenic differentiation capacity, wherein CDS sequence of MYOD1 gene is SEQ ID NO.1.