CN118166098B - Application of reagent for detecting MAGEA4-AS1 in preparation of kit for diagnosing oral squamous cell carcinoma - Google Patents

Abstract

The present application discloses the use of a reagent for detecting MAGEA4-AS1 in the preparation of a kit for diagnosing oral squamous cell carcinoma. According to the application of the embodiment of the present application, at least the following beneficial effects are achieved: the expression of MAGEA4-AS1 is significantly upregulated in tumor tissues and OSCC cells of OSCC patients, and the subject characteristic curve shows that the AUC value is above 0.7; at the same time, MAGEA4-AS1 has a regulatory effect on the proliferation, migration and invasion of OSCC cells. Therefore, MAGEA4-AS1 plays an important regulatory role in the occurrence and development of OSCC and can be used as an effective clinical diagnostic marker for OSCC.

Description

Application of reagents for detecting MAGEA4-AS1 in the preparation of a kit for diagnosing oral squamous cell carcinoma

Technical Field

The present application relates to the technical field of oral squamous cell carcinoma, and in particular to the use of a reagent for detecting MAGEA4-AS1 in the preparation of a kit for diagnosing oral squamous cell carcinoma.

Background Art

Oral squamous cell carcinoma (OSCC) is one of the most common oral malignancies in the oral and maxillofacial region. Although there has been an increasing number of studies on OSCC in recent years and some improvements have been made in clinical treatment, the prognosis of most OSCC patients is still poor, with a 5-year overall survival rate of 55% to 65%. Therefore, it is necessary to screen for OSCC, but the potential biomarkers that have been discovered and are closely related to the occurrence and development of OSCC are mostly protein molecules and miRNAs. The specificity and sensitivity of traditional markers are not high. The same marker may indicate the risk of multiple cancers, and there is also a high chance of missed diagnosis and misdiagnosis.

Long non-coding RNA (lncRNA) is an RNA with a length of more than 200 nucleotides and is not translated into functional proteins. More and more studies have shown that lncRNA plays an important role in the regulation of gene expression. Compared with other RNA molecules, the expression of lncRNA has obvious tissue specificity and spatiotemporal specificity. The expression of lncRNA varies between different tissues, and the expression of lncRNA in the same tissue or organ at different growth stages also changes. In addition, some lncRNAs can be directly detected in body fluids such as blood and urine, which are suitable for non-invasive examinations. Therefore, it is necessary to explore lncRNA diagnostic markers for OSSC.

Summary of the invention

The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes the use of a reagent for detecting MAGEA4-AS1 in the preparation of a kit for diagnosing oral squamous cell carcinoma.

In a first aspect of the present application, there is provided use of a reagent for detecting MAGEA4-AS1 in preparing a kit for diagnosing oral squamous cell carcinoma.

According to the application of the embodiment of the present application, there are at least the following beneficial effects:

The expression of MAGEA4-AS1 is tissue-specific, and its expression is significantly upregulated in tumor tissues and OSCC cells of OSCC patients. The receiver operating characteristic curve shows that the AUC value is above 0.7. At the same time, MAGEA4-AS1 has a regulatory effect on the proliferation, migration and invasion of OSCC cells. Therefore, MAGEA4-AS1 plays an important regulatory role in the occurrence and development of OSCC and can be used as an effective clinical diagnostic marker for OSCC.

Among them, MAGEA4-AS1 is the antisense lncRNA of the coding gene melanoma-associated antigen 4 (MAGEA4), located at chrX: 151904431-chrX: 151911455. Antisense lncRNA is an RNA transcribed from the antisense strand of the coding gene as a template. Its name is based on the location of the adjacent coding gene, but there are many different possibilities for antisense lncRNA to be complementary to the coding region sequence or non-coding region sequence of the adjacent coding gene. Although some research results show that some antisense lncRNAs that have complementary pairing with the coding region of the coding gene can improve the mRNA stability of the source gene, the transcription template of MAGEA4-AS1 is located upstream of the MAGE-A4 coding region and is not complementary to the MAGE-A4 coding region, and the expression of the two is not related. In addition, studies have found that antisense lncRNA has more complex functions than the proteins encoded by adjacent coding genes, and can cause changes in upstream signaling pathways at multiple levels such as epigenetic level, transcriptional level and post-transcriptional level, thereby exerting expression regulation function and affecting the occurrence and development of tumors. Therefore, although MAGEA4 is widely present in a variety of cancer cells, the research results of this application show that MAGEA4-AS1 and MAGEA4 may have different functions in the occurrence and development of OSCC.

The nucleotide sequence of MAGEA4-AS1 is as follows, with a length of 432 bp:

AGTTGATTTGGGAACCAGAGAACAGCAGCCTAAGTGTGACTGTAATTTTTGGCAGCTACAGATTCCCAAGACAACTAGGAAGGGAAAAGGAACCCTTCAAGGAAGTTCCAGGAAAAACGTCTCTCCCAAAAAGACCACCGCTTCAGGAAAACCTGAGGAAGGGAGTGGGCCAACAGTATCCTGCCACTGTGACCAGCTGAACGAGGAACTCCATCAAAATGAGCCACAGCTGGACCAAAACAGTGAGGAGACCTCCACCTT CTTCAGTGTCTCCCTGAGCAGTCAGTAATTTCAGGGCAAGCTGAGAGGTCTCCAGAGGTCTAACCCCAGATAAATAGCCAACAGGGTCTAGAGTACATTTTATACCCAACAGGGTATACCCCATGGTGATCAAAATAAAATAAACCTGTTGTAAAATGAAAAAAAAAAAAA (SEQ ID NO. 5).

In some embodiments, oral squamous cell carcinoma refers to a cancer with squamous differentiation that originates from the oral mucosal epithelium, including at least one of the buccal mucosa, gingival mucosa, retromolar trigone mucosa, tongue (anterior 2/3 of the terminal sulcus) mucosa, floor of mouth mucosa, hard palate mucosa and lip mucosa.

In some embodiments, the reagent for detecting MAGEA4-AS1 is a reagent for detecting the expression level of MAGEA4-AS1 in a sample.

In some embodiments, the sample is at least one of a cell, a tissue, or a body fluid.

In some embodiments, the sample is at least one of oral mucosal tissue, blood, urine, and saliva.

In some embodiments, for the kit for diagnosing oral squamous cell carcinoma, when the expression level of MAGEA4-AS1 in the sample of the subject is higher than a threshold value, the subject is diagnosed with oral squamous cell carcinoma.

In some embodiments, the threshold value is a cutoff value when a ROC curve constructed by the OSCC patient group and the control group meets a certain specificity and/or sensitivity, for example, the cutoff value when the sum of the specificity and the sensitivity is the maximum.

In some embodiments, the OSCC patient group and the control group are cancer tissues and paracancerous tissues of OSCC patients, respectively. In other embodiments, the OSCC patient group and the control group can also be cancer tissues of OSCC patients and sample tissues of normal persons, respectively.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene, for example, it can be any one of β-Actin, GAPDH, tubulin, etc.

In some embodiments, the internal reference gene used to calculate the relative expression level is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene calculated according to the 2 ^−ΔΔCt formula, for example, the internal reference gene is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to the internal reference gene GAPDH calculated according to the 2 ^-ΔΔCt formula. When the expression level of MAGEA4-AS1 in the subject's sample is higher than a threshold, the subject is diagnosed with oral squamous cell carcinoma, and the threshold is 6.265.

In some embodiments, the reagent for detecting MAGEA4-AS1 includes a primer pair for amplifying MAGEA4-AS1. The specific primer pair can be designed based on the nucleotide sequence of MAGEA4-AS1 according to the lncRNA primer design method combined with primer design software commonly used in the art, such as Prime 5.

In some embodiments, the primer pair comprises:

an upstream primer having a nucleotide sequence as shown in TGGCAGCTACAGATTCCCAAG (SEQ ID NO. 1), and

The nucleotide sequence of the downstream primer is shown as GAGTTCCTCGTTCAGCTGGT (SEQ ID NO. 2).

In some embodiments, the kit further comprises an RNA extraction reagent and a reverse transcription reagent.

In some embodiments, the RNA extraction reagent includes TRIzol total RNA extraction reagent.

In some embodiments, the reverse transcription reagents include reverse transcription polymerase, dNTPs, and RT primers.

In some embodiments, the reverse transcriptase polymerase comprises Evo M-MLV RTase.

In some embodiments, the RT primer comprises at least one of a random primer and oligo dT.

In some embodiments, the reverse transcription reagent further comprises an RNase inhibitor.

In some embodiments, the reverse transcription reagents further comprise a gDNA removal reagent.

In some embodiments, the reverse transcription reagent further comprises RNase-free water.

In some embodiments, the kit further comprises an internal reference primer pair.

In some embodiments, the internal reference primer pair is a primer pair for GAPDH.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in GAAGGTGAAGGTCGGAGTC (SEQ ID NO. 3), and

The nucleotide sequence of the downstream primer is shown as GAAGATGGTGATGGGATTTC (SEQ ID NO. 4).

In a second aspect of the present application, a computer-readable storage medium is provided, wherein the computer-readable storage medium stores computer-executable instructions, and the computer-executable instructions are used to cause a computer to perform the following operations:

Step 1: Obtaining information on the expression level of MAGEA4-AS1 in the subject's sample:

Step 2: Compare the expression level with a threshold value, and indicate whether the subject has oral squamous cell carcinoma based on the comparison result.

In some embodiments, the information on the expression level of MAGEA4-AS1 in a sample from a subject is obtained by detecting the sample using a reagent for detecting MAGEA4-AS1.

In some embodiments, the sample is at least one of a cell, a tissue, or a body fluid.

In some embodiments, the sample is at least one of oral mucosal tissue, blood, urine, and saliva.

In some embodiments, when the expression level of MAGEA4-AS1 in the sample of the subject is higher than a threshold value, it indicates that the subject has oral squamous cell carcinoma.

In some embodiments, the threshold value is a cutoff value when a ROC curve constructed by the OSCC patient group and the control group meets a certain specificity and/or sensitivity, for example, the cutoff value when the sum of the specificity and the sensitivity is the maximum.

In some embodiments, the OSCC patient group and the control group are cancer tissues and paracancerous tissues of OSCC patients, respectively, or cancer tissues of OSCC patients and sample tissues of normal persons, respectively.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene, such as any one of β-Actin, GAPDH, tubulin, etc. In some embodiments, the internal reference gene used to calculate the relative expression level is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene calculated according to the 2 ^−ΔΔCt formula, for example, the internal reference gene is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to the internal reference gene GAPDH calculated according to the 2 ^-ΔΔCt formula. When the expression level of MAGEA4-AS1 in the subject's sample is higher than a threshold, it indicates that the subject has oral squamous cell carcinoma, and the threshold is 6.265.

In some embodiments, the reagents for detecting MAGEA4-AS1 include a primer pair for amplifying MAGEA4-AS1.

In some embodiments, the primer pair comprises:

an upstream primer having a nucleotide sequence as shown in TGGCAGCTACAGATTCCCAAG (SEQ ID NO. 1), and

The nucleotide sequence of the downstream primer is shown as GAGTTCCTCGTTCAGCTGGT (SEQ ID NO. 2).

In some embodiments, the kit further comprises an RNA extraction reagent and a reverse transcription reagent.

In some embodiments, the RNA extraction reagent includes TRIzol total RNA extraction reagent.

In some embodiments, the reverse transcription reagents include reverse transcription polymerase, dNTPs, and RT primers.

In some embodiments, the reverse transcriptase polymerase comprises Evo M-MLV RTase.

In some embodiments, the RT primer comprises at least one of a random primer and oligo dT.

In some embodiments, the reverse transcription reagent further comprises an RNase inhibitor.

In some embodiments, the reverse transcription reagents further comprise a gDNA removal reagent.

In some embodiments, the reverse transcription reagent further comprises RNase-free water.

In some embodiments, the kit further comprises an internal reference primer pair.

In some embodiments, the internal reference primer pair is a primer pair for GAPDH.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in GAAGGTGAAGGTCGGAGTC (SEQ ID NO. 3), and

The nucleotide sequence of the downstream primer is shown as GAAGATGGTGATGGGATTTC (SEQ ID NO. 4).

According to a third aspect of the present application, a device is provided, including a processor and a memory, wherein a computer program executable on the processor is stored in the memory, and the processor implements the following operations when executing the computer program:

Step 1: Obtaining information on the expression level of MAGEA4-AS1 in the subject's sample:

Step 2: Compare the expression level with a threshold value, and indicate whether the subject has oral squamous cell carcinoma based on the comparison result.

In some embodiments, the information on the expression level of MAGEA4-AS1 in a sample from a subject is obtained by detecting the sample using a reagent for detecting MAGEA4-AS1.

In some embodiments, the sample is at least one of a cell, a tissue, or a body fluid.

In some embodiments, the sample is at least one of oral mucosal tissue, blood, urine, and saliva.

In some embodiments, when the expression level of MAGEA4-AS1 in the sample of the subject is higher than a threshold value, it indicates that the subject has oral squamous cell carcinoma.

In some embodiments, the threshold value is a cutoff value when a ROC curve constructed by the OSCC patient group and the control group meets a certain specificity and/or sensitivity, for example, the cutoff value when the sum of the specificity and the sensitivity is the maximum.

In some embodiments, the OSCC patient group and the control group are cancer tissues and paracancerous tissues of OSCC patients, respectively, or cancer tissues of OSCC patients and sample tissues of normal persons, respectively.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene, such as any one of β-Actin, GAPDH, tubulin, etc. In some embodiments, the internal reference gene used to calculate the relative expression level is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene calculated according to the 2 ^−ΔΔCt formula, for example, the internal reference gene is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to the internal reference gene GAPDH calculated according to the 2 ^-ΔΔCt formula. When the expression level of MAGEA4-AS1 in the subject's sample is higher than a threshold, it indicates that the subject has oral squamous cell carcinoma, and the threshold is 6.265.

In some embodiments, the reagents for detecting MAGEA4-AS1 include a primer pair for amplifying MAGEA4-AS1.

In some embodiments, the primer pair comprises:

an upstream primer having a nucleotide sequence as shown in TGGCAGCTACAGATTCCCAAG (SEQ ID NO. 1), and

The nucleotide sequence of the downstream primer is shown as GAGTTCCTCGTTCAGCTGGT (SEQ ID NO. 2).

In some embodiments, the kit further comprises an RNA extraction reagent and a reverse transcription reagent.

In some embodiments, the RNA extraction reagent includes TRIzol total RNA extraction reagent.

In some embodiments, the reverse transcription reagents include reverse transcription polymerase, dNTPs, and RT primers.

In some embodiments, the reverse transcriptase polymerase comprises Evo M-MLV RTase.

In some embodiments, the RT primer comprises at least one of a random primer and oligo dT.

In some embodiments, the reverse transcription reagent further comprises an RNase inhibitor.

In some embodiments, the reverse transcription reagents further comprise a gDNA removal reagent.

In some embodiments, the reverse transcription reagent further comprises RNase-free water.

In some embodiments, the kit further comprises an internal reference primer pair.

In some embodiments, the internal reference primer pair is a primer pair for GAPDH.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in GAAGGTGAAGGTCGGAGTC (SEQ ID NO. 3), and

The nucleotide sequence of the downstream primer is shown as GAAGATGGTGATGGGATTTC (SEQ ID NO. 4).

In a fourth aspect of the present application, a risk assessment system for oral squamous cell carcinoma is provided, comprising:

The acquisition module is used to obtain the information of the expression level of MAGEA4-AS1 in the sample of the subject:

The evaluation module is used for comparing the expression level with a threshold value, and indicating whether the subject has oral squamous cell carcinoma according to the comparison result.

In some embodiments, the method further comprises a detection module for detecting the expression level of MAGEA4-AS1 based on a sample from a subject.

It is understandable that the acquisition module can obtain the information on the expression level of MAGEA4-AS1 in the sample of the subject according to the detection result of MAGEA4-AS1 in a direct or indirect manner.

In some embodiments, the sample is at least one of a cell, a tissue, or a body fluid.

In some embodiments, the sample is at least one of oral mucosal tissue, blood, urine, and saliva.

In some embodiments, the detection module includes a reagent for detecting MAGEA4-AS1.

In some embodiments, the reagents for detecting MAGEA4-AS1 include a primer pair for amplifying MAGEA4-AS1.

In some embodiments, the primer pair comprises:

an upstream primer having a nucleotide sequence as shown in TGGCAGCTACAGATTCCCAAG (SEQ ID NO. 1), and

The nucleotide sequence of the downstream primer is shown as GAGTTCCTCGTTCAGCTGGT (SEQ ID NO. 2).

In some embodiments, the kit further comprises an RNA extraction reagent and a reverse transcription reagent.

In some embodiments, the RNA extraction reagent includes TRIzol total RNA extraction reagent.

In some embodiments, the reverse transcription reagents include reverse transcription polymerase, dNTPs, and RT primers.

In some embodiments, the reverse transcriptase polymerase comprises Evo M-MLV RTase.

In some embodiments, the RT primer comprises at least one of a random primer and oligo dT.

In some embodiments, the reverse transcription reagent further comprises an RNase inhibitor.

In some embodiments, the reverse transcription reagents further comprise a gDNA removal reagent.

In some embodiments, the reverse transcription reagent further comprises RNase-free water.

In some embodiments, the kit further comprises an internal reference primer pair.

In some embodiments, the internal reference primer pair is a primer pair for GAPDH.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in GAAGGTGAAGGTCGGAGTC (SEQ ID NO. 3), and

The nucleotide sequence of the downstream primer is shown as GAAGATGGTGATGGGATTTC (SEQ ID NO. 4).

In some embodiments, in the evaluation module, when the expression level of MAGEA4-AS1 in the sample of the subject is higher than a threshold, it indicates that the subject has oral squamous cell carcinoma.

In some embodiments, the threshold value is a cutoff value when a ROC curve constructed by the OSCC patient group and the control group meets a certain specificity and/or sensitivity, for example, the cutoff value when the sum of the specificity and the sensitivity is the maximum.

In some embodiments, the OSCC patient group and the control group are cancer tissues and paracancerous tissues of OSCC patients, respectively, or cancer tissues of OSCC patients and sample tissues of normal persons, respectively.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene, such as any one of β-Actin, GAPDH, tubulin, etc. In some embodiments, the internal reference gene used to calculate the relative expression level is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to an internal reference gene calculated according to the 2 ^−ΔΔCt formula, for example, the internal reference gene is GAPDH.

In some embodiments, the expression level of MAGEA4-AS1 is the relative expression level of MAGEA4-AS1 relative to the internal reference gene GAPDH calculated according to the 2 ^-ΔΔCt formula. When the expression level of MAGEA4-AS1 in the subject's sample is higher than a threshold, it indicates that the subject has oral squamous cell carcinoma, and the threshold is 6.265.

The fifth aspect of the present application provides the use of a substance that specifically inhibits the expression of MAGEA4-AS1 in the preparation of a1 to a2: a1. A drug for treating oral squamous cell carcinoma; a2. A drug for inhibiting the proliferation, migration or invasion of oral squamous cell carcinoma cells.

In some embodiments, the substance includes any one of the antisense oligonucleotide (ASO), small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), clustered regularly interspaced short palindromic repeats (CRSIPR) system, transcription activator-like effector nuclease (TALEN) system, and zinc finger nuclease (ZFN) system of MAGEA4-AS1. Among them, ASO can be, for example, GapmeR, and the CRSIPR system can be, for example, a CRISPR-Cas13 system.

In some embodiments, the nucleotide sequences of the Top strand and the Bottom strand of the shRNA are shown as SEQ ID NO.6 and SEQ ID NO.7, respectively;

in:

GATCCGAGAGGTCTCCAGAGGTCTACTCGAGTAGACCTCTGGAGACCTCTTTTTT G(SEQ IDNO.6);

AATTCAAAAAAAGAGGTCTCCAGAGGTCTACTCGAGTAGACCTCTGGAGACCTCT CG (SEQ ID NO. 7).

In some embodiments, the substance also includes a carrier loaded with the above-mentioned ASO, siRNA, shRNA, miRNA, CRS IPR system, TALEN system, and ZFN system.

In some embodiments, the carrier is any one of an inorganic carrier, an organic carrier, and an inorganic-organic composite carrier.

In some embodiments, the vector comprises any one of a plasmid, a lentivirus, an adenovirus, and an adeno-associated virus.

In some embodiments, the oral squamous cell carcinoma cell is a tongue squamous cell carcinoma cell.

In some embodiments, the oral squamous carcinoma cells include at least one of HSC-3, HN6, and CAL-27.

According to the above application, the embodiments of the present application also relate to a drug combination, including a substance that specifically inhibits the expression of MAGEA4-AS1.

In some embodiments, the drug combination further includes a combined drug, such as at least one of a chemotherapy drug, a photosensitizer, a photothermal agent, and an immunotherapy drug for oral squamous cell carcinoma. Among them, the chemotherapy drug can be, for example, at least one of paclitaxel, camptothecin, 5-fluorouracil, cisplatin, doxorubicin, mitomycin, and epirubicin; the photosensitizer can be, for example, at least one of boron dipyrrole, dihydrochlorin, and Bengal red; the photothermal agent can be, for example, at least one of precious metal nanoparticles, organic polymers, carbon-based nanomaterials, magnetic nanomaterials, and semiconductor nanomaterials; the immunotherapy drug can be, for example, at least one of an immune cell therapy drug and an immune checkpoint inhibitor. It is understandable that the combined drug and MAGEA4-AS1 or a substance that increases the expression of MAGEA4-AS1 can be used simultaneously or successively.

In some embodiments, the pharmaceutical combination further comprises a targeted delivery system.

The embodiments of the present application also relate to the use of a substance that specifically inhibits the expression of MAGEA4-AS1 in treating oral squamous cell carcinoma or inhibiting the proliferation, migration or invasion of oral squamous cell carcinoma cells.

The present application also relates to a method for treating oral squamous cell carcinoma, comprising administering to a patient an effective dose of a substance that specifically inhibits the expression of MAGEA4-AS1.

The embodiments of the present application also relate to a method for inhibiting the proliferation, migration or invasion of oral squamous cell carcinoma cells, comprising mixing an effective dose of a substance that specifically inhibits the expression of MAGEA4-AS1 with the oral squamous cell carcinoma cells.

Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the differential expression analysis result of lncRNA in tumor tissues of OSCC patients in the TCGA database according to an embodiment of the present application.

Figure 2 shows the relative expression levels of MAGEA4-AS1 in cancerous tissues and adjacent tissues of OSCC patients in the examples of the present application, wherein the Wilcoxon test was used, *p<0.05.

Figure 3 shows the relative expression levels of MAGEA4-AS1 in different OSCC cell lines in the examples of the present application, wherein multiple t-tests were used, and the p-values were corrected by the FDR method, **p _-corrected < 0.01, ***p _-corrected < 0.001.

Figure 4 is a ROC curve of the diagnostic efficacy of MAGEA4-AS1 for OSCC using cancer tissue and paracancerous tissue in the present application, wherein AUC is the area under the curve, the specificity is 88.24%, the sensitivity is 52.94%, the cutoff value is 6.265, and p=0.0201.

Figure 5 shows the results of CCK-8 experiments in HN6 stably transfected cell lines, where independent sample t-test was used, *p<0.05, **p<0.01.

Figure 6 shows the results of CCK-8 experiments in CAL-27 stably transfected cell lines, where independent sample t-test was used, *p<0.05.

Figure 7 shows the results of cell migration experiments of HN6 and CAL-27 stable cell lines, where A is a staining photo under a microscope, with a scale of 200 μm; B is the number of migrated cells calculated based on the photo, using an independent sample t-test, *p<0.05, **p<0.01.

Figure 8 shows the results of cell invasion experiments of HN6 and CAL-27 stably transfected cell lines, where A is a staining photo under a microscope, with a scale of 200 μm; B is the number of invasive cells calculated based on the photo, using an independent sample t-test, *p<0.05.

DETAILED DESCRIPTION

The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

In the description of this application, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, "above", "below", "within", etc. are understood to include the number itself, and "about" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the number itself. If there is a description of "first" or "second", it is only used to distinguish the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

The sources of the main reagents and instruments involved in the following examples of this application are as follows:

TRIzol reagent (Dalian TaKaRa Company);

Chloroform (Shanghai Lingfeng Chemical Reagent Co., Ltd.);

Isopropyl alcohol, anhydrous ethanol (Guangdong Huaguang Technology Co., Ltd.);

High-fidelity PCR polymerase 2×Vazyme Lamp Master Mix (Dye Plus), Cat.#P312-01 (Nanjing Novozyme Biotechnology Co., Ltd.);

Reverse transcription kit Evo M-MLV RT Mix Kit with gDNA Clean for qPCR, Cat.#AG11728, fluorescent quantitative PCR reagent SYBR Green Premix Pro Taq HS qPCR Kit II, Cat.#AG11702 (Hunan Aikerui Bioengineering Co., Ltd.);

RNase-free water (Biosharp, China);

Primers (Shanghai Sangon Biotechnology Co., Ltd.);

human OSCC cell lines (HSC-3, HN6, and CAL-27) human oral keratinocytes (HOK);

DMEM (Dulbecco's Modified Eagle Medium), streptomycin-penicillin double antibody mixture (Thermo Fisher Scientific, USA);

Fetal bovine serum (PAN-Biotech, Germany);

Lentivirus, polybrene (Hanheng Biotechnology Shanghai Co., Ltd.);

CCK-8 (Cell Counting Kit-8) cell proliferation detection kit (Shenzhen Aipno Biomedical Technology Co., Ltd.);

Matrigel (Corning, USA);

10% paraformaldehyde (Guangzhou Weiges Biotechnology Co., Ltd.);

PCR instrument (Applied Biosystems, USA);

NanoDrop One Nucleic Acid Analyzer (Thermo Fisher Scientific, USA);

Lightcycler480Ⅱ real-time fluorescence quantitative PCR instrument (Roche, Switzerland);

Multiskan GO fully automatic microplate reader (Thermo Fisher Scientific, USA);

DMi8 inverted fluorescence microscope (LEICA, Germany).

Example 1: TCGA database analysis

Transcriptome data of head and neck squamous cell carcinoma patient tissues were downloaded from the TCGA (The cancer genome atlas) database, and 447 OSCC patients were screened out. The transcriptome data of tumor tissues and adjacent tissues of OSCC patients were analyzed for lncRNA differential expression, and the top 20 differential lncRNAs were screened from high to low according to the differential expression level, and MAGEA4-AS1 was selected as the target of subsequent research. Referring to Figure 1, the sample data of the above patients showed that the log ₂ (Fold change) of MAGEA4-AS1 was >5, and -log ₁₀ (FDR corrected p value) was >2.

MAGEA4-AS1 is the antisense lncRNA of the gene encoding MAGE family member A4 (MAGEA4), which is also known as CT1.4, MAGE4, MAGE4A, MAGE4B, MAGE-41 and MAGE-X2. MAGEA4-AS1 is located at chrX:151904431-chrX:151911455, with a length of 432bp and the nucleotide sequence is as follows:

>NR_136578.1Homo sapiens MAGEA4 antisense RNA 1(MAGEA4-AS1), long non-coding RNA

AGTTGATTTGGGAACCAGAGAACAGCAGCCTAAGTGTGACTGTAATTTTTGGCAGCTACAGATTCCCAAGACAACTAGGAAGGGAAAAGGAACCCTTCAAGGAAGTTCCAGGAAAAACGTCTCTCCCAAAAAGACCACCGCTTCAGGAAAACCTGAGGAAGGGAGTGGGCCAACAGTATCCTGCCACTGTGACCAGCTGAACGAGGAACTCCATCAAAATGAGCCACAGCTGGACCAAAACAGTGAGGAGACCTCCACCTT CTTCAGTGTCTCCCTGAGCAGTCAGTAATTTCAGGGCAAGCTGAGAGGTCTCCAGAGGTCTAACCCCAGATAAATAGCCAACAGGGTCTAGAGTACATTTTATACCCAACAGGGTATACCCCATGGTGATCAAAATAAAATAAACCTGTTGTAAAATGAAAAAAAAAAAAA (SEQ ID NO. 5).

Example 2: Detection of relative expression of lncRNA in OSCC patient tissues and different OSCC cell lines

According to the diagnostic criteria for OSCC of the World Health Organization, approved by the Ethics Committee of Peking University Shenzhen Hospital, and after the patients themselves or their families agreed and signed the informed consent form, cancer tissues and adjacent mucosal tissues (no tumor cells) of 17 patients treated in the Department of Oral and Maxillofacial Surgery of Peking University Shenzhen Hospital were collected. The selection criteria for patients were as follows: ① All patients were diagnosed based on histopathological results; ② All patients had complete follow-up data after surgery; ③ All patients underwent thorough primary lesion cleaning surgery and selective neck lymph node dissection. All patients had not received preoperative radiotherapy or chemotherapy, and the patients themselves had no history of other systemic diseases such as rheumatoid arthritis, cardiovascular disease, diabetes, hyperthyroidism, or other tumor diseases. The pathological data of the patients are shown in Table 1.

Table 1. Clinical information of patient tissues

(1) OSCC tissues and adjacent paracancerous tissues from 17 patients were ground and total RNA was extracted from each tissue using the TRIzol method. Total RNA from HOK, HSC-3, HN6, and CAL-27 cells was also extracted.

(2) After the total RNA concentration was measured by a nucleic acid analyzer, cDNA was obtained using the reverse transcription kit Evo M-MLV RT Mix Kitwith gDNA Clean for qPCR.

(3) The fluorescence quantitative PCR reagent SYBR Green Premix Pro Taq HS qPCR Kit II and Lightcycler480Ⅱ real-time fluorescence quantitative PCR instrument were used to detect the cycle threshold (Ct) of the internal reference gene (GAPDH) and MAGEA4-AS1 in each sample. Finally, the 2 ^-ΔΔCt formula was used to calculate the relative expression level of MAGEA4-AS1 in each cancer tissue, adjacent tissue and different OSCC cell lines. The primer sequences used for qRT-PCR are as follows:

MAGEA4-AS1 upstream primer: TGGCAGCTACAGATTCCCAAG (SEQ ID NO. 1);

MAGEA4-AS1 downstream primer: GAGTTCCTCGTTCAGCTGGT (SEQ ID NO. 2);

GAPDH upstream primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO. 3);

GAPDH downstream primer: GAAGATGGTGATGGGATTTC (SEQ ID NO. 4).

The results are shown in Figures 2 and 3. Compared with adjacent adjacent tissues, the expression level of MAGEA4-AS1 in OSCC cancer tissues was significantly upregulated; and compared with HOK cells, the expression levels of MAGEA4-AS1 in the three OSCC cell lines, HSC-3, CAL-27 and HN6, were significantly increased.

In addition, the ROC curve was established based on the relative expression levels of MAGEA4-AS1 in cancer tissues and adjacent paracancerous tissues of 17 patients to verify its diagnostic efficacy. The results are shown in Figure 4. As can be seen from the figure, the area under the ROC curve can reach 0.7336, the specificity is 88.24%, the sensitivity is 52.94%, the cutoff value is 6.265, and p = 0.0201, indicating that circular RNA MAGEA4-AS1 has a certain diagnostic significance for OSCC.

Example 3: Cell experiment

HN6 and CAL-27 cells were inoculated in cell culture well plates and cultured at 37°C and 5% _CO2 . The culture medium consisted of 90% DMEM and 10% FBS. Then, the experimental group cells (Si-lnc) were infected with the lentivirus carrying the knockdown MAGEA4-AS1 plasmid using lentivirus suspension and polybrene, and the negative control group cells (Si-vector) were infected with the lentivirus carrying the control plasmid, respectively, to construct two stable cell lines of OSCC cells, and the expression levels were detected by qRT-PCR according to the primers in Example 2. As shown in the results, the expression levels of MAGEA4-AS1 were significantly knocked down in both experimental group cells (Si-lnc), indicating that the stable cell lines of two OSCC cells were successfully constructed.

Among them, the shRNA sequences used to knock down MAGEA4-AS1 are as follows:

Top strand:

GATCCGAGAGGTCTCCAGAGGTCTACTCGAGTAGACCTCTGGAGACCTCTTTTTTT G(SEQ IDNO.6);

Bottom strand:

AATTCAAAAAAAGAGGTCTCCAGAGGTCTACTCGAGTAGACCTCTGGAGACCTCT CG (SEQ ID NO. 7).

1.CCK-8 Experiment

In the cell culture well plate, the control group (Si-vector) and the experimental group (Si-lnc) cells were inoculated, and 1:10 CCK-8 reagent was added at 24h, 48h, 72h and 96h, respectively. After incubation for 1h, the absorbance at 450nm was detected by a microplate reader. The cell proliferation curve was drawn using Graphpad prism software, and statistical analysis was completed. The results are shown in Figures 5 and 6, indicating that the knockdown of MAGEA4-AS1 inhibited the proliferation rate of the two OSCC cells.

2. Cell Migration Assay

The control group (Si-vector) and experimental group (Si-lnc) cells suspended in serum-free DMEM were inoculated in the permeable cell culture chamber, and DMEM containing 10% serum was added under the chamber. After the cells migrated in the incubator (37°C, 5% CO ₂ ), they were washed 2-3 times with PBS, fixed with 10% paraformaldehyde, and stained with 1% crystal violet solution. Finally, the dye was washed off and the residual dye in the chamber was wiped clean with a cotton swab. The number of migrated cells was calculated using Image J software after microscopic photography. Graphpad prism software was used to draw statistical graphs and perform statistical analysis. The results are shown in Figure 7, indicating that the knockdown of MAGEA4-AS1 inhibited the migration ability of the two OSCC cells.

3. Cell invasion assay

Matrigel was added to the bottom of the permeable cell culture chamber. After drying the matrigel, cells of the control group (Si-vector) and the experimental group (Si-lnc) suspended in serum-free DMEM were inoculated respectively. DMEM containing 10% serum was added to the bottom of the chamber. The cells were cultured in an incubator (37°C, 5% CO ₂ ) until the cells invaded. Then, the cells were washed 2 to 3 times with PBS, fixed with 10% paraformaldehyde, and stained with 1% crystal violet solution. Finally, the dye was washed off and the residual dye in the chamber was wiped clean with a cotton swab. The number of invaded cells was calculated by using Image J software after being photographed under a microscope. Graphpad prism software was used to draw statistical graphs and perform statistical analysis. The results are shown in Figure 8, indicating that the knockdown of MAGEA4-AS1 inhibited the invasion ability of the two OSCC cells.

Based on the above results, it can be seen that the expression of MAGEA4-AS1 is tissue-specific, and the expression of MAGEA4-AS1 is significantly upregulated in tumor tissues and OSCC cells of OSCC patients; at the same time, MAGEA4-AS1 has a regulatory effect on the proliferation, migration and invasion of OSCC cells. Therefore, MAGEA4-AS1 plays an important regulatory role in the occurrence and development of OSCC and is expected to become an effective clinical diagnostic marker and targeted therapeutic drug for OSCC.

Example 4: Diagnostic Assay

According to the method and selection criteria of Example 2, multiple OSCC patients and normal subjects were enrolled, and cancer tissues of patients and oral mucosal tissue samples of normal subjects were collected to detect the relative expression level of MAGEA4-AS1. The results showed that the relative expression level of the patient group was significantly higher than that of the control group of normal subjects, and the difference was statistically significant (p<0.05); the ROC curve showed that the AUC value was greater than 0.7. These results further demonstrated the diagnostic ability of MAGEA4-AS1 for OSCC.

The present application is described in detail above in conjunction with the embodiments, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims (3)

1. Use of a reagent for detecting MAGEA4-AS1 in the preparation of a kit for diagnosing oral squamous cell carcinoma, said reagent comprising a primer pair for amplifying MAGEA4-AS 1; the primer pair comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown as SEQ ID NO. 2.

2. The use of claim 1, wherein the kit further comprises RNA extraction reagents and reverse transcription reagents.

3. The application of a substance for specifically inhibiting MAGEA4-AS1 expression in preparing a medicament for inhibiting proliferation, migration or invasion of oral squamous carcinoma cells is disclosed, wherein the substance is shRNA of MAGEA4-AS1, and the nucleotide sequences of Top strand and Bottom strand of the shRNA are respectively shown AS SEQ ID NO.6 and SEQ ID NO. 7.