CN118166099B - Application of reagents for detecting hsa_circ_0014088 in the preparation of a kit for diagnosing oral squamous cell carcinoma - Google Patents

Abstract

The application discloses application of a reagent for detecting hsa_circ_0014088 in preparation of a kit for diagnosing oral squamous cell carcinoma. The application according to the embodiment of the application has at least the following beneficial effects: the expression of hsa_circ_0014088 has tissue specificity, and the expression quantity thereof is obviously down-regulated in the tumor tissue and OSCC cells of an OSCC patient; meanwhile, hsa_circ_0014088 has a regulatory effect on proliferation, migration and invasion of OSCC cells. Therefore, hsa_circ_0014088 plays an important role in regulating the occurrence and development of OSCC, and is expected to become an effective OSCC clinical diagnosis marker.

Description

Application of reagents for detecting hsa_circ_0014088 in the preparation of a diagnostic kit for 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 hsa_circ_0014088 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%. Part of the reason why it is difficult to significantly improve the survival rate of OSCC is that its pathogenic mechanism and cell biological characteristics are not fully understood. Therefore, it is necessary to find potential biomarkers for OSCC and find suitable breakthroughs for early diagnosis and other aspects.

Circular RNA (circRNA) is a special type of abundant non-coding RNA, whose expression level can be about 10 times higher than that of linear isomers, and often shows the characteristics of specific expression at different developmental stages. In addition, the closed ring structure of circular RNA makes it less susceptible to RNase degradation, so it can exist more stably in the organism. Multiple evidences show that circRNA has abnormal expression in many types of cancer and may be a potential clinical diagnostic marker. However, the potential biomarkers that have been discovered and are closely related to the occurrence and development of OSCC are mostly protein molecules and miRNAs, and there are relatively few circRNA markers.

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 hsa_circ_0014088 in the preparation of a kit for diagnosing oral squamous cell carcinoma.

In a first aspect of the present application, a reagent for detecting hsa_circ_0014088 is provided for use 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:

In the examples of this application, it was found that the expression of hsa_circ_0014088 was tissue-specific, and its expression was significantly downregulated in tumor tissues and OSCC cells of OSCC patients; at the same time, hsa_circ_0014088 had a regulatory effect on the proliferation, migration and invasion of OSCC cells. Therefore, hsa_circ_0014088 plays an important regulatory role in the occurrence and development of OSCC and is expected to become an effective clinical diagnostic marker for OSCC.

Among them, hsa_circ_0014088 is located at chr1:150999708-chr1:151001420, and is formed by the cyclization of exons 6 and 7. The maternal gene is the prune exopolyphosphatase (PRUNE) gene, which is also known as DRES17, HTCD37, NMIHBA, DRES-17 or H-PRUNE. The nucleotide sequence of the circRNA is as follows, with a length of 254bp:

GACTGACCACTGAGCAGATGCTGAGAAAAGACCAGAAGACTATCTATAGACAAGGCGTCAAGGTGGCCATTAGTGCAATATATATGGATTTGGAGGCCTTTCTGCAGAGGTCTAACCTCCTTGCAGATCTCCATGCTTTCTGCCAGGCTCACAGCTATGATGTCCTGGTTGCCATGACTATCTTTTTCAACACTCACAATGAGCCAGTGCGGCAGTTGGCTATTTTCTGTCCCCATGTGGCACTCCAAACAACG(SEQ ID NO.7).

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 hsa_circ_0014088 is a reagent for detecting the expression level of hsa_circ_0014088 in a sample.

In some embodiments, the sample is a sample of oral mucosal tissue.

In some embodiments, for a kit for diagnosing oral squamous cell carcinoma, when the expression level of hsa_circ_0014088 in a sample of the subject is lower 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 hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene, for example, any one of β-Actin, GAPDH, tubulin, etc.

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

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene calculated according to the 2 ^-ΔΔCt formula, for example, the internal reference gene is β-Actin.

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to the internal reference gene β-Actin calculated according to the 2 ^-ΔΔCt formula. When the expression level of hsa_circ_0014088 in the subject's sample is lower than a threshold, the subject is diagnosed with oral squamous cell carcinoma, and the threshold is 2.122.

In some embodiments, the reagent for detecting hsa_circ_0014088 includes a primer pair for amplifying hsa_circ_0014088. The specific primer pair can be designed based on the nucleotide sequence of hsa_circ_0014088 according to the circRNA primer design method combined with primer design software commonly used in the art, such as Prime 5.

In some embodiments, the primer pair comprises:

The nucleotide sequence is shown as an upstream primer of GGCTCACAGCTATGATGTCCTG (SEQ ID NO. 1), and,

A downstream primer having a nucleotide sequence as shown in GGCAGAAAGCATGGAGATCTG (SEQ ID NO. 2);

or,

The nucleotide sequence is shown as an upstream primer of CACTCACAATGAGCCAGTGC (SEQ ID NO. 3), and,

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

The above two primer pairs amplify the reverse splicing site of circRNA to obtain products of different lengths, which are suitable for sequencing and qPCR respectively. Among them, sequencing has requirements on the length of PCR products, so longer amplification products are required, while the products of qPCR are generally no more than 300bp.

In some embodiments, the kit further comprises RNA extraction reagents and reverse transcription reagents.

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 include 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 β-Actin.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in AAACTGGAACGGTGAAGGTG (SEQ ID NO.5), and

The nucleotide sequence of the downstream primer is shown as AGTGGGGTGGCTTTTAGGAT (SEQ ID NO. 6).

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: Obtain information on the expression level of hsa_circ_0014088 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, information on the expression level of hsa_circ_0014088 in a sample of a subject is obtained by detecting the sample with a reagent for detecting hsa_circ_0014088.

In some embodiments, the sample is a sample of oral mucosal tissue.

In some embodiments, when the expression level of hsa_circ_0014088 in the sample of the subject is lower 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 hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene. In some embodiments, the internal reference gene is β-Actin.

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene calculated according to the 2 ^-ΔΔCt formula, for example, the internal reference gene is β-Actin.

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to the internal reference gene β-Actin calculated according to the 2 ^-ΔΔCt formula. When the expression level of hsa_circ_0014088 in the subject's sample is lower than a threshold, it indicates that the subject has oral squamous cell carcinoma, and the threshold is 2.122.

In some embodiments, the reagent for detecting hsa_circ_0014088 includes a primer pair for amplifying hsa_circ_0014088. In some embodiments, the primer pair can be at least one of a divergent primer and a convergent primer, which are used to amplify a circular transcript and a linear transcript of hsa_circ_0014088, respectively.

In some embodiments, the primer pair comprises:

The nucleotide sequence is shown as an upstream primer of GGCTCACAGCTATGATGTCCTG (SEQ ID NO. 1), and,

A downstream primer having a nucleotide sequence as shown in GGCAGAAAGCATGGAGATCTG (SEQ ID NO. 2);

or,

The nucleotide sequence is shown as an upstream primer of CACTCACAATGAGCCAGTGC (SEQ ID NO. 3), and,

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

In some embodiments, the kit further comprises RNA extraction reagents and reverse transcription reagents.

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 include 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 β-Actin.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in AAACTGGAACGGTGAAGGTG (SEQ ID NO.5), and

The nucleotide sequence of the downstream primer is shown as AGTGGGGTGGCTTTTAGGAT (SEQ ID NO. 6).

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 on the memory, and the processor implements the following operations when executing the computer program:

Step 1: Obtain information on the expression level of hsa_circ_0014088 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, information on the expression level of hsa_circ_0014088 in a sample of a subject is obtained by detecting the sample with a reagent for detecting hsa_circ_0014088.

In some embodiments, the sample is a sample of oral mucosal tissue.

In some embodiments, when the expression level of hsa_circ_0014088 in the sample of the subject is lower 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 hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene. In some embodiments, the internal reference gene is β-Actin.

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene calculated according to the 2 ^-ΔΔCt formula, for example, the internal reference gene is β-Actin.

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to the internal reference gene β-Actin calculated according to the 2 ^-ΔΔCt formula. When the expression level of hsa_circ_0014088 in the subject's sample is lower than a threshold, it indicates that the subject has oral squamous cell carcinoma, and the threshold is 2.122.

In some embodiments, the reagent for detecting hsa_circ_0014088 includes a primer pair for amplifying hsa_circ_0014088. In some embodiments, the primer pair can be at least one of a divergent primer and a convergent primer, which are used to amplify a circular transcript and a linear transcript of hsa_circ_0014088, respectively.

In some embodiments, the primer pair comprises:

The nucleotide sequence is shown as an upstream primer of GGCTCACAGCTATGATGTCCTG (SEQ ID NO. 1), and,

A downstream primer having a nucleotide sequence as shown in GGCAGAAAGCATGGAGATCTG (SEQ ID NO. 2);

or,

The nucleotide sequence is shown as an upstream primer of CACTCACAATGAGCCAGTGC (SEQ ID NO. 3), and,

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

In some embodiments, the kit further comprises RNA extraction reagents and reverse transcription reagents.

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 include 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 β-Actin.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in AAACTGGAACGGTGAAGGTG (SEQ ID NO.5), and

The nucleotide sequence of the downstream primer is shown as AGTGGGGTGGCTTTTAGGAT (SEQ ID NO. 6).

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 hsa_circ_0014088 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, a detection module is further included for detecting the expression level of hsa_circ_0014088 based on a sample from a subject.

It can be understood that the acquisition module can obtain information on the expression level of hsa_circ_0014088 in the subject's sample based on the detection results of hsa_circ_0014088 in a direct or indirect manner.

In some embodiments, the sample is a sample of oral mucosal tissue.

In some embodiments, the detection module includes a reagent for detecting hsa_circ_0014088.

In some embodiments, the reagent for detecting hsa_circ_0014088 includes a primer pair for amplifying hsa_circ_0014088. In some embodiments, the primer pair can be at least one of a divergent primer and a convergent primer, which are used to amplify a circular transcript and a linear transcript of hsa_circ_0014088, respectively.

In some embodiments, the primer pair comprises:

The nucleotide sequence is shown as an upstream primer of GGCTCACAGCTATGATGTCCTG (SEQ ID NO. 1), and,

A downstream primer having a nucleotide sequence as shown in GGCAGAAAGCATGGAGATCTG (SEQ ID NO. 2);

or,

The nucleotide sequence is shown as an upstream primer of CACTCACAATGAGCCAGTGC (SEQ ID NO. 3), and,

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

In some embodiments, the kit further comprises RNA extraction reagents and reverse transcription reagents.

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 include 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 β-Actin.

In some embodiments, the internal reference primer pair comprises:

an upstream primer having a nucleotide sequence as shown in AAACTGGAACGGTGAAGGTG (SEQ ID NO.5), and

The nucleotide sequence of the downstream primer is shown as AGTGGGGTGGCTTTTAGGAT (SEQ ID NO. 6).

In some embodiments, in the evaluation module, when the expression level of hsa_circ_0014088 in the sample of the subject is lower 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 hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene. In some embodiments, the internal reference gene is β-Actin.

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to an internal reference gene calculated according to the 2 ^-ΔΔCt formula, for example, the internal reference gene is β-Actin.

In some embodiments, the expression level of hsa_circ_0014088 is the relative expression level of hsa_circ_0014088 relative to the internal reference gene β-Actin calculated according to the 2 ^-ΔΔCt formula. When the expression level of hsa_circ_0014088 in the subject's sample is lower than a threshold, it indicates that the subject has oral squamous cell carcinoma, and the threshold is 2.122.

In the fifth aspect of the present application, there is provided an application of hsa_circ_0014088 or a substance that increases the expression level of hsa_circ_0014088 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 that increases the expression level of hsa_circ_0014088 includes an overexpression vector of hsa_circ_0014088.

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

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

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

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 hsa_circ_0014088 or a substance that increases the expression level of hsa_circ_0014088.

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 noble 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 hsa_circ_0014088 or a substance that increases the expression of hsa_circ_0014088 can be used simultaneously or successively.

The embodiments of the present application also relate to the use of hsa_circ_0014088 or a substance that increases the expression of hsa_circ_0014088 in the treatment of oral squamous cell carcinoma or the inhibition of the proliferation, migration or invasion of oral squamous cell carcinoma cells.

The present application embodiment also relates to a method for treating oral squamous cell carcinoma, comprising administering to a patient an effective dose of hsa_circ_0014088 or a substance that increases the expression level of hsa_circ_0014088.

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 hsa_circ_0014088 or a substance that increases the expression of hsa_circ_0014088 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

Figure 1 is a schematic diagram of the structure of hsa_circ_0014088 in an embodiment of the present application.

Figure 2 is the relative expression level of hsa_circ_0014088 in cancerous tissues and adjacent tissues of OSCC patients in the examples of the present application. Among them, the Wilcoxon test was used, **p<0.01.

Figure 3 shows the relative expression levels of hsa_circ_0014088 in different OSCC cell lines in the examples of the present application. Multiple t-tests were used, and the p-values were corrected by the FDR method, ***p _correction < 0.001.

Figure 4 is a ROC curve for verifying the diagnostic efficacy of hsa_circ_0014088 for OSCC using cancer tissue and paracancerous tissue in the embodiment of the present application, wherein AUC is the area under the curve, the specificity is 65%, the sensitivity is 75%, the cutoff value is 2.122, and p=0.0074.

Figure 5 shows the relative expression of hsa_circ_0014088 in the experimental group cells (OE-circ) and the negative control group cells (OE-vector) of the stably transfected cell line constructed with HN6 cells. The independent sample t test was used, *p<0.05.

Figure 6 shows the relative expression of hsa_circ_0014088 in the experimental group cells (OE-circ) and the negative control group cells (OE-vector) of the stable cell line constructed by CAL-27 cells. Among them, the independent sample t test was used, **p<0.01.

Figure 7 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, ***p<0.001.

Figure 8 shows the results of CCK-8 experiments in CAL-27 stably transfected cell lines, where independent sample t-test was used, **p<0.01, ***p<0.001.

Figure 9 shows the scratch test results of HN6 stable cell line, where A is a scratch photo under a microscope, with a scale of 50 μm; B is the cell migration rate result calculated based on the photo, using independent sample t test, ***p<0.001.

Figure 10 shows the scratch test results of CAL-27 stable cell line, where A is a scratch photo under a microscope, with a scale of 50 μm; B is the cell migration rate result calculated from the photo, using independent sample t test, ***p<0.001.

Figure 11 is the result of cell migration experiment of HN6 stable cell line, wherein A is the staining photo under microscope, the scale is 100 μm; B is the number of migrated cells calculated based on the photo, using independent sample t test, ***p<0.001.

Figure 12 shows the results of the cell migration experiment of the CAL-27 stable cell line, where A is a staining photo under a microscope, with a scale of 100 μm; B is the number of migrated cells calculated based on the photo, using an independent sample t-test, ***p<0.001.

Figure 13 shows the results of the cell invasion experiment of the HN6 stable cell line, where A is a staining photo under a microscope, with a scale of 100 μm; B is the number of migrated cells calculated based on the photo, using an independent sample t-test, **p<0.01.

Figure 14 shows the results of the cell invasion experiment of the CAL-27 stable cell line, where A is a staining photo under a microscope, with a scale of 100 μm; B is the number of migrated cells calculated based on the photo, using an independent sample t-test, ***p<0.001.

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 for the purpose of distinguishing technical features, and cannot be understood as indicating or implying 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.

In the preliminary experiment, in accordance with the Declaration of Helsinki and the NIH regulations on the use of clinical specimens, approved by the Ethics Committee of Peking University Shenzhen Hospital, and after the patients fully informed and signed the informed consent form, cancer and paracancerous mucosal tissues were collected from 42 patients. None of the patient samples received other treatments before surgery, all oral squamous cell carcinoma tissues were confirmed by strict pathological examination, and there were no cancer cell components in the paracancerous mucosal tissues. The clinical and pathological characteristics of the patients were based on the WHO classification and UICC TNM classification. Among them, the cancer tissues and paracancerous mucosal tissues of 8 patients were extracted with total RNA by TRIzol method, enriched and purified, and the library was constructed and high-throughput deep sequencing was performed. The sequencing data were analyzed to identify multiple differentially expressed circRNAs. Reverse transcription quantitative PCR (RT-qPCR) was performed to verify these differentially expressed circRNAs based on all 42 samples, confirming that hsa_circ_0014088 was differentially expressed (|Fold change|≥2, p≤0.05), refer to Front Oncol.2018Oct8:8:398. For confidentiality, hsa_circ_0014088 and its related results were not disclosed in the article.

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 gDNAClean 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: Identification of hsa_circ_0014088

The total RNA of HOK cells was extracted by TRIzol method, and cDNA was synthesized by reverse transcription kit Evo M-MLV RT Mix Kitwith gDNAClean for qPCR. The hsa_circ_0014088 in HOK cells was amplified by high-fidelity PCR polymerase 2×Vazyme LampMaster Mix (Dye Plus) and PCR instrument. The reaction solution was then sent to Shanghai Biotech Co., Ltd. for sequencing. The primer sequences used for amplification are as follows:

Upstream primer: GGCTCACAGCTATGATGTCCTG (SEQ ID NO. 1);

Downstream primer: GGCAGAAAGCATGGAGATCTG (SEQ ID NO. 2).

Referring to Figure 1, hsa_circ_0014088 is located at chr1:150999708-chr1:151001420, is formed by the cyclization of exon 6 and exon 7, is 254 bp in length, and has a nucleotide sequence as shown in SEQ ID NO. 7. Its parent gene is the prune exopolyphosphatase (PRUNE) gene, which is also known as DRES17, HTCD37, NMIHBA, DRES-17 or H-PRUNE.

GACTGACCACTGAGCAGATGCTGAGAAAAGACCAGAAGACTATCTATAGACAAGGCGTCAAGGTGGCCATTAGTGCAATATATATGGATTTGGAGGCCTTTCTGCAGAGGTCTAACCTCCTTGCAGATCTCCATGCTTTCTGCCAGGCTCACAGCTATGATGTCCTGGTTGCCATGACTATCTTTTTCAACACTCACAATGAGCCAGTGCGGCAGTTGGCTATTTTCTGTCCCCATGTGGCACTCCAAACAACG(SEQ ID NO.7).

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

In this embodiment, according to the diagnostic criteria for OSCC of the World Health Organization, approved by the Ethics Committee of Peking University Shenzhen Hospital, the patients themselves or their families agreed and signed the informed consent form, and collected cancer tissue and adjacent mucosal tissue (no tumor cells) from 20 patients treated in the Department of Oral and Maxillofacial Surgery of Peking University Shenzhen Hospital. The selection criteria for patients were: ① All patients were diagnosed based on histopathological results; ② All patients had complete follow-up data after surgery; ③ All patients underwent a thorough primary lesion cleaning operation and selective neck lymph node dissection. All patients had not undergone preoperative radiotherapy and 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 20 patients were ground and total RNA was extracted from each tissue using the TRIzol method. Total RNA from 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 (β-Actin) and hsa_circ_0014088 in each sample. Finally, the 2 ^-ΔΔCt formula was used to calculate the relative expression level of hsa_circ_0014088 in each cancer tissue, adjacent tissue and different OSCC cell lines. The primer sequences used for qRT-PCR are as follows:

hsa_circ_0014088 upstream primer: CACTCACAATGAGCCAGTGC (SEQ ID NO. 3);

hsa_circ_0014088 downstream primer: GCACTAATGGCCACCTTGAC (SEQ ID NO. 4);

β-Actin upstream primer: AAACTGGAACGGTGAAGGTG (SEQ ID NO. 5);

β-Actin downstream primer: AGTGGGGTGGCTTTTAGGAT (SEQ ID NO. 6).

The results are shown in Figures 2 and 3. Compared with adjacent adjacent tissues, the expression level of hsa_circ_0014088 in OSCC cancer tissues was significantly downregulated; and compared with HOK cells, the expression levels of hsa_circ_0014088 were significantly reduced in three OSCC cell lines, HSC-3, CAL-27 and HN6 cells.

In addition, the ROC curve was established based on the relative expression levels of hsa_circ_0014088 in cancer tissues and adjacent paracancerous tissues of 20 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 0.7475, indicating that hsa_circ_0014088 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 (OE-circ) were infected with the lentivirus carrying the overexpression hsa_circ_0014088 plasmid using lentivirus suspension and polybrene, and the negative control group cells (OE-vector) were infected with the lentivirus carrying the control plasmid. Two stable cell lines of OSCC cells were constructed, and the overexpression efficiency was detected by qRT-PCR according to the primers in Example 2.

The results are shown in Figures 5 and 6. In the two experimental groups of cells (OE-circ), the expression levels of hsa_circ_0014088 were significantly increased, indicating that the stable cell lines of two OSCC cells were successfully constructed.

1.CCK-8 Experiment

In the cell culture well plate, the control group (OE-vector) and the experimental group (OE-circ) 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 7 and 8, indicating that the overexpression of hsa_circ_0014088 inhibited the proliferation rate of the two OSCC cells.

2. Scratch test

In the cell culture well plate, the control group (OE-vector) and the experimental group (OE-circ) cells were inoculated respectively. When the cell coverage was 100%, the gun tip was perpendicular to the bottom of the well plate to scrape a uniform width of scratch. After culturing for a period of time, the migration of cells at the scratch was observed under a microscope and photographed. The scratch area was calculated using Image J, and the cell migration rate (%) = (initial scratch area-scratch area at time t)/initial scratch area × 100%. Graphpad prism software was used to draw statistical graphs and perform statistical analysis. The results are shown in Figures 9 and 10, indicating that the overexpression of hsa_circ_0014088 inhibited the migration ability of the two OSCC cells.

3. Cell Migration Assay

The control group (OE-vector) and experimental group (OE-circ) cells suspended in serum-free DMEM were inoculated in the permeable cell culture chamber, and DMEM containing 10% serum was added to the bottom of 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 then 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 Figures 11 and 12, which also show that the overexpression of hsa_circ_0014088 inhibited the migration ability of the two OSCC cells.

4. Cell invasion assay

Matrigel was added to the bottom of the permeable cell culture chamber. After drying the matrigel, cells of the control group (OE-vector) and the experimental group (OE-circ) suspended in serum-free DMEM were inoculated respectively. DMEM containing 10% serum was added to the bottom of the chamber. After the cells were cultured in an incubator (37°C, 5% CO ₂ ) until invasion occurred, they were washed 2 to 3 times with PBS, fixed with 10% paraformaldehyde, and then 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 Figures 13 and 14, indicating that the overexpression of hsa_circ_0014088 inhibited the invasion ability of the two OSCC cells.

Based on the above results, it can be seen that the expression of hsa_circ_0014088 is tissue-specific, and the expression of hsa_circ_0014088 is significantly downregulated in tumor tissues and OSCC cells of OSCC patients; at the same time, hsa_circ_0014088 has a regulatory effect on the proliferation, migration and invasion of OSCC cells. Therefore, hsa_circ_0014088 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, several OSCC patients and normal subjects were enrolled, and the cancer tissues of the patients and the oral mucosal tissue samples of the normal subjects were collected to detect the relative expression level of hsa_circ_0014088. The results showed that the relative expression level of the patient group was significantly lower 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 hsa_circ_0014088 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 (5)

1. Use of a reagent for detecting hsa_circ_0014088 in the preparation of a kit for diagnosing oral squamous cell carcinoma, the reagent comprising a primer pair for amplifying hsa_circ_0014088, the primer pair comprising: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; or, The nucleotide sequence of the upstream primer is shown as SEQ ID NO.3 and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.4. 2. The use according to claim 1, characterized in that the kit further comprises an RNA extraction reagent and a reverse transcription reagent. 3. A computer-readable storage medium, 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: Obtain information on the expression level of hsa_circ_0014088 in the subject's sample: Step 2: comparing the expression level with a threshold value, and indicating whether the subject suffers from oral squamous cell carcinoma according to the comparison result; The information on the expression level of hsa_circ_0014088 in the sample of the subject is obtained by detecting the sample with a reagent for detecting hsa_circ_0014088; the reagent includes a primer pair for amplifying hsa_circ_0014088, and the primer pair includes: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; or, The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4. 4. A device, comprising a processor and a memory, wherein the memory stores a computer program that can be run on the processor, and the processor implements the following operations when running the computer program: Step 1: Obtain information on the expression level of hsa_circ_0014088 in the subject's sample: Step 2: comparing the expression level with a threshold value, and indicating whether the subject suffers from oral squamous cell carcinoma according to the comparison result; The information on the expression level of hsa_circ_0014088 in the sample of the subject is obtained by detecting the sample with a reagent for detecting hsa_circ_0014088; the reagent includes a primer pair for amplifying hsa_circ_0014088, and the primer pair includes: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; or, The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4. 5. An oral squamous cell carcinoma risk assessment system, characterized in that it comprises: An acquisition module, the acquisition module is used to obtain information on the expression level of hsa_circ_0014088 in a sample of a subject: an evaluation module, the evaluation module being used to compare the expression level with a threshold value, and indicating whether the subject suffers from oral squamous cell carcinoma according to the comparison result; The information on the expression level of hsa_circ_0014088 in the sample of the subject is obtained by detecting the sample with a reagent for detecting hsa_circ_0014088; the reagent includes a primer pair for amplifying hsa_circ_0014088, and the primer pair includes: An upstream primer having a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.2; or, The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3 and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.