CN115354078A - Osteosarcoma detection marker and application thereof - Google Patents

Abstract

The invention provides a biomarker for detecting osteosarcoma, which is a LACTB gene and has missense mutation of one and/or more site mutations of p.Met5Leu/c.13A > C, p.Lys255Gln/c.763A > C, p.Arg469Lys/c.1406G > A and/or synonymous mutation of p.Ser475Ser/c.1425G > A, p.Asp355Asp/c.1065T > C. Also provides application of the biomarker and a corresponding detection kit. Unlike other tumors, the LACTB protein in osteosarcoma exerts oncogenic effects and undergoes five-site mutations. The marker can be used for early prevention, diagnosis and treatment, and provides a new scheme for the diagnosis and treatment of osteosarcoma.

Description

Osteosarcoma detection marker and application thereof

Technical Field

The invention relates to the fields of biotechnology and clinical medicine, in particular to an osteosarcoma detection marker and application thereof.

Background

Osteosarcoma (OS) is a highly-developed tumor in children and adolescents, poses a great threat to the family of solitary children and women, and brings great economic loss to the society. Through the efforts of clinical medical workers, the five-year survival rate of newly-assisted chemotherapy and extensive excision is close to 60% under the current regulation. Osteosarcoma is polymorphic, so that it is clinically difficult to diagnose in early stages, and death rate is high. The establishment of an osteosarcoma early diagnosis system and the search of a corresponding new therapeutic target point are urgently needed.

An enhancer is a DNA sequence on the genome that can cooperate with a transcription factor to enhance gene expression. For example, an enhancer encoding the upstream of the beta globin gene in HeLa cells of cervical cancer cells can enhance the expression of the beta globin gene by 200 times. However, recent studies have shown that an enhanced version of the enhancer, super enhancer (super enhancer), is present in cells. The control of super enhancers covers a variety of cancer lineages, where cancer cells, by assembling their own super enhancers, overproduce malignant oncogenes, display proliferative, invasive and metastatic cancer marker features. Therefore, the research on the osteosarcoma super enhancer can find the oncogene highly expressed in the tumor cell, and is expected to play an important role in the aspect of disease diagnosis and treatment.

LACTB is a mitochondrial transmembrane protein from the Penicillin Binding Proteins (PBPs)/β -lactamase protein family, is present in all vertebrates and is involved in peptidoglycan synthesis. Research shows that the beta-lactamase serine-like protein LACTB forms fibrous fibroid mitochondrial membrane in the inter-membrane space to directly or indirectly regulate mitochondrial phospholipid metabolism, and the mitochondrial phospholipid metabolism is closely related to cell proliferation and tumor formation, which indicates that the expression of the beta-lactamase serine-like protein LACTB participates in the proliferation and differentiation of tumors. The higher the clinical pathological stage in most tumors, the lower the intramitochondrial expression of the beta-lactamase serine-like protein LACTB. For example, in breast cancer patients, only 34-42% of people detect the beta-lactamase serine-like protein LACTB in their breast tissue, whereas that of normal breast is 100%.

In untransformed cells, LACTB silencing is insufficient to cause transformation, but when bound to the expression of the oncogenes RAS or MYC, it leads to tumor formation in vivo. The research shows that in the solid tumor tissue, the expression of LACTB reduces the growth rate of tumor cells, promotes the apoptosis of the tumor cells and sometimes even can completely eliminate and return the tumor cells of the initial cancer focus to normal.

However, according to years of clinical and research experience of the inventor, LACTB presents opposite results to the existing research in osteosarcoma, and an early diagnosis system of osteosarcoma can be established around the LACTB.

Disclosure of Invention

The invention aims to overcome the defect that the existing biomarker is difficult to diagnose early osteosarcoma, and provides a biomarker for detecting osteosarcoma and application thereof.

In a first aspect of the invention, a biomarker for detecting osteosarcoma is provided.

A biomarker for detecting osteosarcoma is a LACTB gene, and one or more site mutations of missense mutations p.Met5Leu/c.13A > C, p.Lys255Gln/c.763A > C, p.Arg469Lys/c.1406G > A, synonymous mutations p.Ser475Ser/c.1425G > A and p.Asp355Asp/c.1065T > C exist.

In some of these embodiments, p.Met5Leu/c.13A > C and/or p.Arg469Lys/c.1406G > A in missense mutations are preferred. And still further preferably further comprises at least one of p.Lys255Gln/c.763A > C and synonymous mutations p.Ser475Ser/c.1425G > A, p.Asp355Asp/c.1065T > C.

In some of these examples, the biomarker for osteosarcoma detection is the LACTB gene and there are missense mutations p.Met5Leu/c.13A > C, p.Lys255Gln/c.763A > C, p.Arg469Lys/c.1406G > A, the synonymous mutations p.Ser475Ser/c.1425G > A, and p.Asp355Asp/c.1065T > C.

A biomarker for detecting osteosarcoma, wherein LACTB protein expressed by LACTB gene with site mutation.

The second aspect of the invention provides the application of detecting the expression level of the LACTB gene or the content of the LACTB protein in preparing an osteosarcoma detection kit.

In a third aspect of the invention, a kit for detecting osteosarcoma is provided.

A kit for detecting osteosarcoma comprises a reagent for detecting LACTB gene and having missense mutation of one and/or more of p.Met5Leu/c.13A > C, p.Lys255Gln/c.763A > C, p.Arg469Lys/c.1406G > A and/or synonymous mutation of p.Ser475Ser/c.1425G > A and p.Asp355Asp/c.1065T > C.

A kit for detecting osteosarcoma comprises a kit for detecting LACTB gene expression or LACTB protein level in a sample to be detected.

The LACTB gene has one and/or more site mutations of missense mutation p.Met5Leu/c.13A > C, p.Lys255Gln/c.763A > C, p.Arg469Lys/c.1406G > A and/or synonymy mutation p.Ser475Ser/c.1425G > A and p.Asp355Asp/c.1065T > C, and is used as a biomarker in the preparation of a kit for detecting osteosarcoma.

The detection method may be any one of PCR, fluorescent quantitative PCR, RT-PCR, sequencing method, and liquid chip method. The kit comprises reagents for detection by the above methods.

In one embodiment, the method further comprises detecting at least one of CEP55, CDK4, EEF1A2, gli2, SCD1, SREBF1, TCF7L2, SRSF3, further preferably LACTB, CEP55, CDK4, EEF1A2, gli2, SCD1 in combination.

1. In long-term clinical practice and exploration, the inventor of the invention unexpectedly finds that the LACTB has a different effect in osteosarcoma from the low expression mode of other clinically common tumors and has high expression in osteosarcoma patients, so that the LACTB gene or LACTB can be used as a detection index of osteosarcoma to diagnose the osteosarcoma timely and accurately. Furthermore, it was found that there are five site mutations in the LACTB gene of osteosarcoma patients, in particular wherein the mutations of p.Met5Leu/c.13A > C and/or p.Arg469Lys/c.1406G > A are highly correlated with the development of osteosarcoma.

2. In the treatment process, according to the discovery at the 1 st point, the change of the LACTB and the combined marker thereof in the serum of the patient can be detected, so that the treatment scheme can be timely adjusted, the health of the patient can be better recovered, and the damage of tumor medicaments to the patient can be reduced.

3. The mutation detection of LACTB can be added into physical examination items of teenagers to prevent osteosarcoma from occurring early.

4. By detecting the change of the LACTB level, a judgment basis can be provided for the prognosis of the patient, and the early detection of the recurrence of the tumor and other poor prognosis is facilitated.

Drawings

FIG. 1 is a diagram showing the Western Blot detection results of osteosarcoma cell line and hFOB cell.

FIG. 2 is a diagram showing the Western Blot detection results after knocking down LACTB in osteosarcoma cell line and hFOB cell.

FIG. 3 is a graph showing the results of cell proliferation potency after knocking out LACTB in osteosarcoma cell line and hFOB cell.

FIG. 4 is a graph showing the results of the scoring experiments after knockdown of LACTB in osteosarcoma cell lines and hFOB cells.

FIG. 5 is a schematic representation of the results of the Transwell experiment after knockdown of LACTB in osteosarcoma cell line and hFOB cells.

FIG. 6 is a schematic diagram showing the result of LACTB mutation in 8 osteosarcoma cell line gene sequencing.

FIG. 7A is the information for 15 osteosarcoma patients.

FIG. 7B is a schematic diagram showing the result of LACTB mutation in gene sequencing of 15 osteosarcoma patient specimens.

FIG. 8 is a schematic diagram showing the results of detecting tissue proteins in 7 cases of osteosarcoma patients.

FIG. 9 is a graph showing the results of HE staining and immunohistochemical staining of osteosarcoma tissue.

FIG. 10 is a graph showing the results of Western Blot analysis of the expression levels of LACTB, CEP55, CDK4, EEF1A2, gli2, SCD1, SREBF1, TCF7L2, and SRSF3 proteins in osteosarcoma patients.

Detailed Description

In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention is further illustrated by the following specific examples, which are not intended to limit the scope of the invention.

Example 1

1.1 the medicine THZ2 (a CDK7 inhibitor which can affect the function of a super enhancer after CDK7 is inhibited) is used for interfering the transcription of the super enhancer in osteosarcoma cells U2-OS and SJSA-1, and related genes with the maximum downstream change multiple of the super enhancer in osteosarcoma are screened and enriched through transcriptome sequencing, gene chips and other high-throughput screening means to obtain the LACTB gene.

The test process is as follows: planting U2OS and SJSA-1 osteosarcoma cells in large dishes at a rate of 3X 10/dish ⁶ 4 dishes each. 37 ℃ C. 5% CO ₂ The cell culture box is kept still for culture, after the cells grow to be full of 70 percent, the culture solution is discarded, the cells are washed by PBS, and a complete culture medium (high-sugar DMEM culture medium, 10 percent fetal calf serum and 1 percent penicillin-streptomycin) with the THZ2 drug concentration of 25, 100 and 400nmol/L is added. The cells were cultured in an incubator for 6 hours. Discarding the culture solution, washing with 4 ℃ precooled PBS solution, adding 4 ℃ precooled Trizol, scraping cells by using a cell scraper, transferring into an EP tube, adding into a dry ice box, and sending to Huada gene sequencing.

1.2 Western Blot assay was performed on osteosarcoma cell lines and hFOB cells (human osteoblast line, non-tumor cells, purchased from Shanghai cell Collection). Total cellular protein was collected and expression of LACTB was detected by Western Blot. The LACTB primary antibody used was purchased from proteintech, cat #: 18195-1-AP, diluted with 5% BSA solution at a dilution ratio of 1:4000.

the results of the experiments demonstrated that LACTB is highly expressed ubiquitously in osteosarcoma cell lines and less expressed in hFOB (see FIG. 1). This result, in contrast to the traditional clinical pathology of LACTB, suggests that LACTB plays a different role in osteosarcoma. In traditional cognition, LACTB is low in expression in tumors, and high in expression in normal cells, so that the LACTB has the effect of inhibiting the tumors. In the experiment, the expression level of LACTB in an osteosarcoma cell line is higher than that of a normal cell line h-Fob, which shows that LACTB plays a role in promoting the development of osteosarcoma.

1.3cck-8, 1000 ZOSL-1 cells are respectively taken, planted in a 96-well plate, added with 100 microliter of culture solution (complete culture medium (high-glucose DMEM culture medium, 10% fetal bovine serum and 1% penicillin-streptomycin), added with 10 microliter of cck-8 reagent (purchased from the same kernel of Japan) the next day, incubated for 1h in the dark, detected in the light absorption value at 450nm, replaced with fresh culture solution, continuously detected for 7 days, cell growth curve is drawn, and multiplication time is calculated.

We knocked down LACTB in both cells by shRNA, and Western Blot assay showed that knocking down effect was significant (fig. 2). Then, the proliferation capacity of the knocked-out LACTB cells was detected by a CCK-8 kit, and the results showed that the proliferation capacity of U2-OS cells and SJSA-1 cells was reduced after knocking-out LACTB (FIG. 3).

The experimental process comprises the following steps: respectively taking 2X10 ⁵ U2OS and SJSA-1 cells of (b), were seeded in six-well plates. After the cell density reached about 60%, the culture medium was removed, rinsed with PBS, and 1mL of 10% FBS-free medium (high-glucose DMEM medium, 10% fetal bovine serum), 1mL of LACTB shRNA virus solution (Guangzhou Eryside Biotech Co., ltd.), 8-10ug/mL Polybrene, cultured at 37 ℃ for 10 hours. And replacing the complete culture solution, and carrying out passage after the cells grow full. Puromycin was added at a concentration of 1ug/ml to screen out untransformed virus cells. And collecting the cell protein.

1.4 migration function experiment: 5 x10 ⁴ Planting cells in 6-hole plateAfter the cells are fully grown, scratching is carried out along the symmetry line of the culture dish, after PBS is cleaned, serum-free culture solution is added, and the scratching healing conditions are observed for 0h, 24h and 48 h.

Transwell experiment: 20 by 10 ⁴ The cells were suspended in 200. Mu.L of serum-free medium, added to the chamber, plated in 24-well plates, and 500. Mu.L of complete medium was added to the bottom. 37 ℃ 5% of CO ₂ After the incubator is kept still for 24 hours, the liquid inside and outside the small chamber is sucked off, the cells which are not penetrated on the inner surface of the bottom membrane of the small chamber are lightly wiped off by a small cotton swab, the inner part of the small chamber is washed by distilled water, and the cells are wiped off by the small cotton swab again. The cell was fixed in methanol for 30min; after the cell was aspirated, 500uL of crystal violet solution was added and the staining was performed at room temperature for 30min. Washing with ultrapure water, air drying, and performing microscopic examination.

The results of the scratch test showed that the migration function of U2-OS cells and SJSA-1 cells was significantly inhibited after knocking down LACTB (FIG. 4).

The migration and invasion ability of cells after knocking down LACTB was examined by Transwell experiment, and the results showed that the migration and invasion functions of U2-OS cells and SJSA-1 cells were significantly inhibited after knocking down LACTB (FIG. 5).

Thus, it was found that LACTB plays a significant role in the development of osteosarcoma, and that it plays a pro-cancerous, rather than a suppressive, role in osteosarcoma.

Example 2

Collecting 6 osteosarcoma cell lines (SaoS-2, MG63, HOS, G292, SJSA-1 and U2-OS) and 2 primary osteosarcoma cell lines (ZOS-1 and ZOSL-1), amplifying cells, collecting cell RNA, and sending to Huada gene for whole exon sequencing. The results (FIG. 6) show that the p.Met5Leu/c.13A > C missense mutation and the p.Arg469Lys/c.1406G > A missense mutation were present in 8 cells; p.lys255gln/c.763a > C missense mutation was detected in G292 cells; the synonymous mutation Ser475Ser/c.1425G > A was detected in ZOS-1, YQOS, MG63, HOS while the synonymous mutation p.Asp355Asp/c.1065T > C was detected only in U2-OS.

Example 3

15 specimens of osteosarcoma patients (the specimens were approved by the ethical committee of experimental animals and clinical research in the first hospital affiliated to Zhongshan university (lot No.: lun review [2019 ]) 060), and the patients signed informed consent forms), surgery information and pathological results of the patients were collected as shown in FIG. 7A. All specimens were harvested on the same day of surgery, and the non-necrotic area under the tumor envelope was excised and washed with PBS shaking to remove blood from the tissue. And cutting the specimen into pieces, preserving the pieces by liquid nitrogen, and after collection, sending the pieces together to the Huada gene for sequencing the whole exon. The results show (FIG. 7B) that the missense mutations p.Met5Leu/c.13A > C and p.Arg469Lys/c.1406G > A were shown in 15 specimens, while the synonymous mutation p.Ser475Ser/c.1425G > A was shown in 5 specimens.

Example 4

The osteosarcoma tissue and peripheral normal muscle tissue specimens of the 7 osteosarcoma patients after amputation surgery were collected, the tissues were ground using a tissue grinder, and the supernatant was collected after centrifugation. The protein concentration is detected by using a BCA method, and the protein loading amount is adjusted. Western Blot was used to detect LACTB protein status.

As shown in FIG. 8, the LACTB content in normal muscle tissue (M) was significantly lower than that in tumor tissue (T) at the same protein level (GAPDH).

Example 5 pathological examination of osteosarcoma patients

Collecting osteosarcoma tissue and peripheral normal muscle tissue specimens after the amputation operation of an osteosarcoma patient, and respectively carrying out HE staining and immunohistochemical staining.

As shown in FIG. 9, HE staining of muscle tissue showed marked muscle texture, while tumor tissue showed marked nuclear fission and larger nuclei. Immunohistochemistry showed (immunohistochemistry with primary LACTB antibody purchased from proteintech, cat # 18195-1-AP diluted to 1:200 with PBS solution) that there was no expression of LACTB in muscle tissue, but significant expression of LACTB in tumor tissue.

Example 7 detection of marker combinations in serum of patients with bone tumors

Sera were collected from 2 osteosarcoma patients at different time points. The serum collection time of one patient is after diagnosis, chemotherapy and amputation; another patient was diagnosed, treated with chemotherapy once, treated with chemotherapy three times, and subjected to amputation for 12 days and one month later. After centrifugation, the proteins were collected, and Western Blot was used to detect the expression levels of TCF7L2, SRSF3, SCD1, SAA, LACTB, gli2, EEF1A2, CEP55, CDK7, CDK4, SCD1, SREBF1, and other proteins (LACTB and super enhancer pathway-related proteins).

The results are shown in FIG. 10, and the expression levels of LACTB, CEP55, CDK4, EEF1A2, gli2 and SCD1 were highest at the time of diagnosis under the same protein content, and decreased with chemotherapy and surgery, indicating the correlation between these markers and the course of osteosarcoma.

All possible combinations of the technical features of the above embodiments may not be described for the sake of brevity, but should be considered as within the scope of the present disclosure as long as there is no contradiction between the combinations of the technical features.

Claims (10)

1. A biomarker for detecting osteosarcoma, which is LACTB gene and has at least one site mutation of missense mutation p.Met5Leu/c.13A > C, p.Lys255Gln/c.763A > C, p.Arg469Lys/c.1406G > A synonymous mutation p.Ser475Ser/c.1425G > A and p.Asp355Asp/c.1065T > C.

2. The osteosarcoma detection biomarker of claim 1, wherein the marker is a LACTB gene and is p.Met5Leu/c.13A > C and/or p.Arg469Lys/c.1406G > A with missense mutation; further preferred are the presence of missense mutations p.Met5Leu/c.13A > C, p.Lys255Gln/c.763A > C, p.Arg469Lys/c.1406G > A, the synonymous mutations p.Ser475Ser/c.1425G > A, and p.Asp355Asp/c.1065T > C.

3. A biomarker for detecting osteosarcoma, wherein the LACTB protein expressed by the site-mutated LACTB gene of claim 1 or 2.

4. Use of a reagent for detecting the expression level of the LACTB gene of claim 1 or 2 or the LACTB protein content of claim 3 in the preparation of an osteosarcoma detection kit.

5. A kit for detecting osteosarcoma, comprising a reagent for detecting the mutation or expression level of the LACTB gene according to claim 1 or 2 in a sample to be detected.

6. A kit for detecting osteosarcoma, comprising a reagent for detecting the LACTB protein of claim 3 in a sample to be detected.

7. The kit for detecting osteosarcoma according to claim 5 or 6, wherein the kit comprises reagents for detecting PCR, fluorescent quantitative PCR, RT-PCR, sequencing method, and liquid chip method.

8. The kit for detecting osteosarcoma according to claim 5 or 6, wherein the kit comprises reagents for enzyme linked immunosorbent assay and protein chip.

9. Use of the LACTB gene having a site mutation as claimed in claim 1 or 2 as a biomarker for the preparation of a kit for detecting osteosarcoma, wherein the method comprises detecting whether the expression of the LACTB gene as claimed in claim 1 or 2 is up-regulated or the level of LACTB protein as claimed in claim 3 is present in the sample to be tested.

10. The use of claim 9, further comprising detecting at least one of CEP55, CDK4, EEF1A2, gli2, SCD1, SREBF1, TCF7L2, SRSF3 protein levels.

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