CN112877437B - Application of lncRNA in diagnosis and treatment of oral squamous cell carcinoma - Google Patents
Application of lncRNA in diagnosis and treatment of oral squamous cell carcinoma Download PDFInfo
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Abstract
The invention discloses an application of lncRNA in diagnosing and treating oral squamous cell carcinoma, wherein the lncRNA is IGF2BP2-AS 1. In the specific embodiment of the invention, through detecting oral squamous carcinoma tissue and paracarcinoma tissue samples, IGF2BP2-AS1 is found to be significantly highly expressed in the oral squamous carcinoma tissue. The invention also discloses a method for screening the candidate drugs for oral squamous cell carcinoma.
Description
Technical Field
The invention belongs to the field of biomedicine, relates to a biomarker, and particularly relates to application of LncRNA in diagnosis and treatment of oral squamous cell carcinoma.
Background
Oral Squamous Cell Carcinoma (OSCC), abbreviated as Oral squamous cell carcinoma, is a malignant tumor with the highest incidence rate of Oral maxillofacial region, and the proportion of the Oral squamous cell carcinoma is as high as about 90% in Oral malignant diseases. Oral squamous carcinoma can appear in different parts of the oral cavity, mostly close to important tissues and organs, such as cranium, upper respiratory tract and important neurovascular vessels of the neck, so that the range of the operation is severely restricted, and because the vessels of the head, neck and maxillofacial tissues and the lymphatic vessels are rich and the tongue is frequently mechanically moved, early cervical lymph node metastasis is often generated, the prognosis is poor, and the life health of people is seriously threatened. With the development of comprehensive treatment of tumors, the treatment of oral squamous cell carcinoma is advanced to a certain extent, but in recent years, the 5-year survival rate of patients is not improved obviously. The current clinical treatment methods for oral squamous cell carcinoma comprise surgical treatment, preoperative or postoperative chemoradiotherapy and the like, but the treatment methods are far from meeting the survival needs of patients, and some patients still have postoperative recurrence and metastasis possibility even after the operation and the auxiliary chemoradiotherapy are carried out, so that the overall survival time of the patients is seriously influenced. The reason is that the existing pathological diagnosis technology can not accurately judge the biological characteristics of oral squamous cell carcinoma, and lacks specific and efficient treatment medicines and schemes aiming at the oral squamous cell carcinoma.
Biomarkers are indicative of the physiological state and changes of cells during the course of a disease, have unique advantages in accurately and sensitively evaluating early and low levels of damage, and can be used for early diagnosis, prognosis prediction and clinical treatment of cancer. lncrnas are a class of non-coding RNAs that are over 200 nucleotides in length, and although lncrnas do not encode proteins, they can be involved in the development and progression of disease by altering chromatin conformation, mRNA transcription, post-transcriptional RNA splicing, protein degradation, and trafficking. The research shows that lncRNA can regulate the growth, proliferation, invasion, metastasis, angiogenesis and the like of cancer cells, can be used as an oncogene or a tumor suppressor to regulate a cancer-related signal pathway in a direct or indirect mode, and influences the development of tumors. The method has important significance for finding lncRNA with accurate diagnosis, prediction prognosis and clinical treatment capacity for oral squamous cell carcinoma.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a biomarker related to oral squamous cell carcinoma, thereby providing a molecular means for diagnosing and treating the oral squamous cell carcinoma.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect, the invention provides a product for diagnosing oral squamous cell carcinoma or predicting prognosis of oral squamous cell carcinoma, wherein the product comprises a reagent for detecting the expression level of IGF2BP2-AS 1.
The IGF2BP2-AS1 refers to the Gene which is located on chromosome 3 and has the Gene ID of 646600.
The term "prognosis" as used herein refers to the prediction of the likely course and outcome of a clinical condition or disease. The prognosis of a patient is usually determined by evaluating factors or symptoms indicative of a favorable or unfavorable disease process or outcome. The term "prognosis" does not refer to a process or outcome that is capable of 100% accurately predicting a condition. Rather, the term "prognosis" refers to an increased probability that a particular process or outcome will occur, as will be understood by those skilled in the art; that is, the process or result is more likely to occur in a patient exhibiting a given condition than in an individual not exhibiting the condition. Prognosis may be expressed as the amount of time a patient is expected to survive. Alternatively, prognosis may refer to the likelihood of remission of the disease or the amount of time the disease is expected to remain in remission. Prognosis can be expressed in different ways; for example, prognosis can be expressed as a percentage probability that a patient survives one year later, five years later, ten years later, etc. Optionally, prognosis can be expressed as the average number of months that a patient can expect to survive due to a disorder or disease. The prognosis of a patient can be considered a relative manifestation, with many factors affecting the final outcome. For example, for a patient with a particular condition, prognosis may be suitably expressed as the likelihood that the condition is treatable or curable, or the likelihood that the disease will be alleviated, while the prognosis for a patient with a more severe condition may be more suitably expressed as the likelihood of survival for a particular time.
Further, the product comprises a reagent for detecting the expression level of IGF2BP2-AS1 by PCR, in situ hybridization or a high-throughput sequencing platform.
The high-throughput sequencing platform is a special tool, and with the continuous development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the knowledge that the abnormality of IGF2BP2-AS1 gene is related to the occurrence or prognosis of oral squamous cell carcinoma in high-throughput sequencing also belongs to the novel application of the IGF2BP2-AS1, and is also within the protection scope of the invention.
Further, the reagent comprises a probe or a primer.
Furthermore, the sequence of the primer is shown as SEQ ID NO. 1-2.
In the present invention, the term "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.
The term "primer" means 7 to 50 nucleic acid sequences capable of forming a base pair (base pair) complementary to a template strand and serving as a starting point for replication of the template strand. The primers are generally synthesized, but naturally occurring nucleic acids may also be used. The sequence of the primer does not necessarily need to be completely identical to the sequence of the template, and may be sufficiently complementary to hybridize with the template. Additional features that do not alter the basic properties of the primer may be incorporated. Examples of additional features that may be incorporated include, but are not limited to, methylation, capping, substitution of more than one nucleic acid with a homolog, and modification between nucleic acids.
The second aspect of the invention provides an application of lncRNA in preparing a product for diagnosing oral squamous cell carcinoma or predicting oral squamous cell carcinoma prognosis, wherein the lncRNA is IGF2BP2-AS 1.
Furthermore, the product is used for detecting the expression level of IGF2BP2-AS1 in a sample of a subject, and compared with a normal person, the expression level of IGF2BP2-AS1 in the sample of the subject is up-regulated, so that the subject is diagnosed AS an oral squamous cell carcinoma patient or the prognosis of the oral squamous cell carcinoma is poor.
The term "subject" as used in the terms means any animal, also human and non-human animals. The term "non-human animal" includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic or pet animal; and non-mammals, such as chickens, amphibians, reptiles, and the like.
In a preferred embodiment, the subject is a human.
The terms "sample" and "sample" are used interchangeably herein to refer to a composition obtained or derived from a subject (e.g., an individual of interest) that comprises cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cells and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture fluids, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.
In a preferred embodiment, the sample is selected from the group consisting of tissues.
The most reliable results are possible when processing samples in a laboratory environment. For example, a sample may be taken from a subject in a doctor's office and then sent to a hospital or commercial medical laboratory for further testing. However, in many cases, it may be desirable to provide immediate results at the clinician's office or to allow the subject to perform the test at home. In some cases, the need for testing that is portable, prepackaged, disposable, ready to use by the subject without assistance or guidance, etc., is more important than a high degree of accuracy. In many cases, especially in the case of physician visits, it may be sufficient to perform a preliminary test, even a test with reduced sensitivity and/or specificity. Thus, an assay provided in product form can involve detecting and measuring relatively small amounts of lncRNA to reduce the complexity and cost of the assay.
Further, the product comprises a chip or a kit. Wherein, the chip comprises a gene chip; the kit comprises a gene detection kit. The gene chip comprises a solid phase carrier and oligonucleotide probes fixed on the solid phase carrier, wherein the oligonucleotide probes comprise oligonucleotide probes for detecting gene expression level and aiming at IGF2BP2-AS1 genes; the gene detection kit comprises a primer or a chip for detecting the expression level of IGF2BP2-AS1 gene.
In a third aspect, the invention provides a pharmaceutical composition for treating oral squamous cell carcinoma, wherein the pharmaceutical composition comprises an inhibitor of functional expression of IGF2BP2-AS 1. The inhibitor is selected from: an interfering molecule which uses IGF2BP2-AS1 or its transcript AS a target sequence and can inhibit IGF2BP2-AS1 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.
The term "treatment" in the present invention refers to the medical management of a patient for the purpose of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term includes active therapy, i.e., treatment aimed specifically at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at removing the cause of the associated disease, pathological condition, or disorder. Moreover, the term also includes palliative treatments, i.e., treatments designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, i.e. treatment for supplementing another specific therapy with the purpose of improving the associated disease, pathological state or condition.
Further, the inhibitor is siRNA.
Furthermore, the sequence of the siRNA is shown as SEQ ID NO. 5.
The siRNA of the present invention may include partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may include adding non-nucleotide material, e.g., to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that render the siRNA resistant to nuclease digestion.
The fourth aspect of the invention provides an application of IGF2BP2-AS1 in preparing a pharmaceutical composition for treating oral squamous cell carcinoma, wherein the pharmaceutical composition can also comprise an effective amount of a medicament for treating oral squamous cell carcinoma and a pharmaceutically acceptable carrier and/or an auxiliary material.
The term "effective amount" as used herein, refers to an amount that has a therapeutic effect or is required to produce a therapeutic effect in a subject being treated. For example, a therapeutically or pharmaceutically effective amount of a drug refers to the amount of drug required to produce the desired therapeutic effect, which can be reflected in the results of clinical trials, model animal studies, and/or in vitro studies. The pharmaceutically effective amount will depend on several factors including, but not limited to, the characteristics of the subject (e.g., height, weight, sex, age, and medical history), and the severity of the disease.
The pharmaceutical composition and the medicament for treating oral squamous cell carcinoma can be prepared into separate preparations for combined application, and the two can also be prepared into one preparation for application in the form of a composition.
The carrier and/or adjuvant includes pharmaceutically acceptable carrier, diluent, filler, binder and other excipients, which depend on the administration mode and the designed dosage form. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols (e.g., polyethylene glycol, water, sucrose, ethanol, glycerol), various preservatives, lubricants, dispersants, flavoring agents, wetting agents, sweeteners, flavorants, emulsifiers, suspending agents, preservatives, antioxidants, colorants, stabilizers, salts, buffers, and the like, which may also be added.
Suitable pharmaceutically acceptable carriers and/or adjuvants are well described in Remington's Pharmaceutical Sciences, for assisting the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odour in the case of oral administration, as required, and the formulations which may be used in such Pharmaceutical compositions may be in the form of their original compounds per se, or optionally in the form of their pharmaceutically acceptable salts. The pharmaceutical composition thus formulated may be administered by any suitable means known to those skilled in the art, as desired, by administering a safe and effective amount of the drug of the present invention to a human.
The appropriate dose of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the method of preparation, the mode of administration, the age, body weight, sex, disease state, diet, time of administration, route of administration, excretion rate and reaction sensitivity of the patient, and a skilled physician can easily determine the prescription and the dose of administration effective for the desired treatment or prevention.
Further, the pharmaceutical composition comprises an inhibitor of functional expression of IGF2BP2-AS 1. The inhibitor is selected from: an interfering molecule which uses IGF2BP2-AS1 or its transcript AS a target sequence and can inhibit IGF2BP2-AS1 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.
Further, the inhibitor is siRNA.
Furthermore, the sequence of the siRNA is shown as SEQ ID NO. 5.
In a fifth aspect, the present invention provides a method for screening a candidate drug for treating oral squamous cell carcinoma, the method comprising:
(1) treating a system expressing or containing IGF2BP2-AS1 with a test agent;
(2) detecting the expression of IGF2BP2-AS1 in said system;
(3) selecting a test agent that reduces the expression level of IGF2BP2-AS1 AS a drug candidate.
Further, the method further comprises the following steps: the candidate drug obtained in the above step is further tested for its effect of inhibiting oral squamous cell carcinoma, and if the test substance has a significant inhibitory effect on oral squamous cell carcinoma, the substance is a candidate substance for preventing or treating oral squamous cell carcinoma.
Further, the system includes (but is not limited to): cell, subcellular, solution, tissue, organ or animal systems (e.g., animal models, preferably non-human mammalian animal models such as mice, rabbits, sheep, monkeys, etc.).
Further, when the drug candidate selected by the screening method of the present invention is administered to a human or other mammal, including but not limited to mouse, rat, guinea pig, rabbit, cat, dog, sheep, pig, cow, monkey, baboon, chimpanzee, the drug candidate may be directly administered, or it may be formulated into various dosage forms using known drug preparation methods. For example, the drug may be orally administered as sugar-coated tablets, capsules, elixirs and microcapsules, as desired; or as a sterile solution or suspension in water or any other pharmaceutically acceptable liquid for parenteral administration in the form of an injection. For example, the drug candidate may be mixed together in unit dosage form as required for generally accepted modes of drug administration, with pharmaceutically acceptable carriers or media including, but not limited to, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders and the like. Depending on the content of the active ingredient in these preparations, an appropriate amount to be administered within a prescribed range can be obtained.
The invention has the following advantages and beneficial effects:
the invention provides a biomarker of oral squamous cell carcinoma, and the biomarker is IGF2BP2-AS 1.
The IGF2BP2-AS1 can be applied to diagnosis of oral squamous cell carcinoma or prediction of oral squamous cell carcinoma prognosis.
The invention also provides application of IGF2BP2-AS1 in treating oral squamous cell carcinoma.
Drawings
Fig. 1 is a differential expression profile and survival plot of IGF2BP2-AS1 in a dataset, wherein (a) is a differential expression profile of IGF2BP2-AS1 in OSCC tissue samples and normal tissue samples of the dataset, and (b) is a survival curve of IGF2BP2-AS 1.
FIG. 2 is a graph showing differential expression of IGF2BP2-AS1 and localization of IGF2BP2-AS1 in cells in a sample, wherein (a) is a graph showing the results of experiments for verifying differential expression of IGF2BP2-AS1 by RT-qPCR, and (b) is a graph showing the results of experiments for detecting the localization of IGF2BP2-AS1 in cells by FISH.
FIG. 3 is a graph showing the results of an experiment for measuring cell proliferation by a cell colony formation method.
Fig. 4 is a graph showing the results of the cell scratch test.
FIG. 5 is a graph showing the results of the Transwell experiment.
FIG. 6 is a graph showing the results of an OSCC cell cycle distribution experiment after detecting the down-regulation of IGF2BP2-AS1 gene expression by flow cytometry.
FIG. 7 is a graph showing the results of an OSCC apoptosis experiment after detecting the down-regulation of IGF2BP2-AS1 gene expression by flow cytometry.
FIG. 8 is a graph showing the results of Western Blot analysis of the expression of Bax, Cyclin D1, MMP2 and beta-catenin in OSCC cells.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.
Experimental Material
1. Reagent:
TRIzol reagent, HiFi-Script cDNA first chain synthesis kit, UltraSYBR mix purchased from Beijing kang, century Biotechnology Co., Ltd; ECL chemiluminescence detection kit was purchased from Proteintech, USA; the transfection reagent Lipofectamine 2000 was purchased from Invitrogen Life technologies, Inc., USA; the apoptosis detection kit and the cell cycle detection kit are purchased from Changchun praying health biological products, Inc.; RIPA lysate was purchased from Biyuntian Biotechnology Ltd; the BCA protein quantitative kit is purchased from Shaanxi Prodoad Biotech development Co., Ltd.
2. Cell lines: the OSCC cell strain (Cal27, Tca8113) is from the cell bank of Chinese academy of sciences.
3. Antibody: the cyclin D1 murine antibody, the beta-actin murine antibody, the beta-catenin rabbit antibody and the Bax rabbit antibody are purchased from Proteintech company in the United states; MMP2 rabbit derived antibodies were purchased from wuhanseville biotechnology ltd; anti-rabbit IgG and anti-mouse IgG were purchased from Affinity Biosciences, USA.
4. The instrument comprises the following steps:
Example 1 biomarkers associated with oral squamous carcinoma
A data set containing 372 samples (including 339 OSCC patient tissue samples and 33 normal tissue samples) was downloaded from the TCGA database and analyzed for differential expression of IGF2BP2-AS1 using the edgeR package. Based on the expression level of IGF2BP2AS1, patients were divided into two groups at optimal cut-off values, and survival curves were constructed using the Kaplan-Meier method. A p-value <0.05 is considered statistically significant.
As a result: as shown in FIG. 1(a), the expression level of IGF2BP2-AS1 was significantly increased in the tissues of OSCC patients AS compared with the normal tissues. Furthermore, AS shown in fig. 1(b), the expression level of IGC2BP2-AS1 was negatively correlated with the survival of OSCC patients (P ═ 0.031). The results of this experiment suggest that IGF2BP2-AS1 can be used for diagnosis of OSCC and is associated with poor prognosis in OSCC patients.
Example 2 expression of IGF2BP2-AS1 in OSCC tissues and cells.
1. Sample acquisition
30 OSCC tissues and 20 tissues beside the cancer (more than 5cm away from the primary tumor) are collected from Shandong provincial hospital, and the cut samples are all quickly frozen at-80 ℃ and confirmed by pathological diagnosis. All the above samples were obtained with the consent of the ethical committee of the provincial hospital, Shandong.
2. mRNA extraction and RT-qPCR
Firstly, extracting total RNA of a sample by using a TRIzol reagent, then carrying out RNA reverse transcription by using a HiFi-Script cDNA first strand synthesis kit to obtain cDNA, and finally carrying out qPCR amplification by using an UltraSYBR mix premix system, wherein specific primer sequences are as follows:
IGF2BP2-AS1：
the forward primer is 5'-ATGGTGGTGCATGGAGGAAG-3' (SEQ ID NO. 1);
the reverse primer was 5'-CTCTTCAGCAAAGCAGACTTC-3' (SEQ ID NO. 2).
β-actin：
The forward primer is 5'-AGACCTGTACGCCAACACAG-3' (SEQ ID NO. 3);
the reverse primer was 5'-CGGACTCGTCATACTCCTGC-3' (SEQ ID NO. 4).
3. Statistical analysis
Statistical analysis was performed using the statistical software Graphpad Prism 8.0, and the data were expressed as mean. + -. standard deviation (mean. + -. SD). Values and percent differences between the two groups were compared using the Student's t test, with differences of P <0.05 being statistically significant.
4. FISH technology is adopted to detect the location of IGF2BP2-AS1 in OSCC cells (Cal27, Tca 8113).
5. Results
(1) AS shown in fig. 2(a), the expression level of IGF2BP2-AS1 was significantly increased in OSCC tissues compared to the para-carcinoma tissues, with statistical significance (. about.p < 0.05).
(2) As shown in FIG. 2(b), IGF2BP2-AS1 is mainly localized in the cytoplasm of OSCC cells.
Example 3 silencing assay and functional validation of IGF2BP2-AS1
1. Cell culture
At 5% CO2Cells (Cal27, Tca8113) were cultured in a 37 ℃ incubator in DMEM medium containing 10% FBS.
2. Cell transfection
siRNA against IGF2BP2-AS1 was synthesized by Beijing Oligobio Co., Ltd (Beijing, China) and had the following sequence:
si-IGF2BP2-AS1 is GGTCATGCCTGGTTAACAT (SEQ ID NO. 5).
The sequences of sirnas as controls were as follows:
si-NC TTCTCCGAACGTGTCACGT (SEQ ID NO. 6).
Transfection was performed using the Lipofectamine 2000 kit, and the specific procedures were performed according to the instructions.
3. Cell colony formation assay
Cells (Cal27, Tca8113) were seeded into 35mm diameter petri dishes (1000 cells/well) and cultured for 14 days. Cells were fixed with 4% paraformaldehyde solution for 15min and stained with 0.1% crystal violet for 10 min. Finally, the cell colonies were counted under a microscope, each cell colony consisting of at least 50 cells.
4. Cell scratch test
Cells (Cal27, Tca8113) were seeded into 6-well plates and cultured for 24 hours, after transfection of si-IGF2BP2-AS1 or si-NC, the cell layer was streaked using a 10. mu.L pipette tip; cells were washed with Phosphate Buffered Saline (PBS), serum-free medium was added, cells were placed in an incubator for 48 hours, samples were taken at 0 and 48 hours, photographed under an inverted microscope, and the scratch closure area was measured using ImageJ software.
5. Transwell experiment
Cells (Cal27, Tca8113) transfected for 24h were trypsinized to obtain 6X 10 cells4The cells were seeded in 100. mu.L of serum-free medium in the upper chamber of a Transwell chamber, and medium containing 30% FBS was added to the lower chamber, and the culture was continued in an incubator for 48 hours; the cells remaining on the upper surface were removed, and the cells migrated to the lower side of the membrane were washed with PBS, fixed with 4% paraformaldehyde solution for 15min, and then stained with 0.1% crystal violet for 10 min. Finally, under a microscopeCell counts were performed.
6. Flow cytometry detection of cell cycle distribution
After cells (Cal27, Tca8113) are transfected for 72h, an apoptosis detection kit is adopted to detect apoptosis by combining with flow cytometry. Cell cycle was analyzed using a cell cycle assay kit. The specific operation is carried out according to the instruction.
7. Western Blot analysis (Western Blot)
Proteins were released by lysis of cells (Cal27, Tca8113) using RIPA lysis buffer and protein concentration was quantified using BCA protein quantification kit. Total protein samples (20. mu.g/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membrane. The membrane was blocked in 5% skim milk at room temperature for 2h, and separately added with cyclin D1 murine antibody (1: 5,000 dilution), β -actin antibody (1: 5000 dilution), β -catenin rabbit antibody (1: 5000 dilution), Bax antibody (1: 2000 dilution) and MMP2 antibody (1: 1000 dilution), and incubated overnight at 4 ℃. After washing the membrane 3 times with tris buffer containing 0.05% Tween-20(TBST), a secondary anti-rabbit IgG (1: 5000 dilution) or anti-mouse IgG (1: 5000 dilution) was added to the membrane, allowed to stand at room temperature for 1 hour, and washed 3 times with TBST buffer. And (3) carrying out substrate detection by using an ECL chemiluminescence detection kit, analyzing by using an Amersham Imager 600 chemiluminescence imaging system, and finally quantifying the band of the target protein by using Image J software.
8. Statistical analysis
Statistical analysis was performed using the statistical software Graphpad Prism 8.0, and the data were expressed as mean. + -. standard deviation (mean. + -. SD). Values and percent differences between the two groups were compared using the Student's t test, with differences of P <0.05 being statistically significant.
9. As a result:
(1) AS shown in figure 3, down-regulation of IGF2BP2-AS1 expression resulted in a significant reduction in the number of cell colonies (. P <0.05), which data indicate that down-regulation of IGF2BP2-AS1 expression can inhibit OSCC cell proliferation.
(2) Cell scoring experiments showed that the migration of OSCC cells was significantly reduced in the experimental group transfected with si-IGF2BP2-AS1 compared to the control group transfected with si-NC (AS shown in fig. 4), with a statistical difference P < 0.05.
(3) Results of Transwell experiments showed that upon down-regulation of IGF2BP2-AS1, the number of migrating cells in the experimental group was significantly reduced (AS shown in fig. 5), with statistical significance of the difference (. about.p < 0.05).
(4) Flow cytometry detection cell cycle distribution experimental results show that the cell cycle progression is delayed by blocking the G2/M phase after down-regulation of IGF2BP2-AS1 gene expression compared with the transfected si-NC group and the control group (AS shown in FIG. 6).
(5) The apoptosis test result of flow cytometry shows that the apoptosis rate of the IGF2BP2-AS1 siRNA transfected group is obviously higher than that of the si-NC transfected control group and the siRNA untransfected control group (AS shown in FIG. 7). This data suggests that down-regulation of IGF2BP2-AS1 expression may increase OSCC apoptosis.
(6) The Western Blot experiment result shows that the expression levels of beta-catenin, Cyclin D1 and MMP2 proteins in the cells with down-regulated IGF2BP2-AS1 expression are lower, the bax protein level is higher, and the beta-actin is the internal reference (AS shown in FIG. 8) compared with the control group.
The experimental results prove that IGF2BP2-AS1 regulates the proliferation, apoptosis and migration of OSCC cells through a Wnt/beta-catenin pathway, and the IGF2BP2-AS1 can be applied to OSCC treatment.
The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.
In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.
Sequence listing
<110> Shandong first medical university affiliated provincial Hospital (Shandong provincial Hospital)
<120> application of IncRNA in diagnosis and treatment of oral squamous cell carcinoma
<141> 2021-03-07
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Claims (11)
1. The application of the reagent for detecting the expression level of lncRNA in preparing products for diagnosing oral squamous cell carcinoma or predicting the prognosis of oral squamous cell carcinoma is characterized in that the lncRNA is IGF2BP2-AS 1.
2. The use of claim 1, wherein the product comprises reagents for detecting the expression level of IGF2BP2-AS1 by PCR, in situ hybridization, or high throughput sequencing platform.
3. The use of claim 1, wherein the reagent comprises a probe or a primer.
4. The use of claim 3, wherein the primer has the sequence shown in SEQ ID No. 1-2.
5. The use of claim 1, wherein the product is used to detect the expression level of IGF2BP2-AS1 in a subject sample, and wherein an upregulation of the expression level of IGF2BP2-AS1 in the subject sample, AS compared to a normal human, diagnoses the subject AS a squamous cell carcinoma patient or AS having a poor prognosis of squamous cell carcinoma.
6. The use of claim 5, wherein the sample is tissue.
7. The use according to any one of claims 1 to 6, wherein the product is a chip, or a kit.
Use of an inhibitor of the functional expression of IGF2BP2-AS1 for the preparation of a pharmaceutical composition for the treatment of oral squamous cell carcinoma.
9. The use of claim 8, wherein the inhibitor is an siRNA.
10. The use of claim 9, wherein the siRNA has the sequence shown in SEQ ID No. 5.
11. A method of screening for a candidate agent for treating oral squamous cell carcinoma, said method comprising:
(1) treating a system expressing or containing IGF2BP2-AS1 with a test agent;
(2) detecting the expression of IGF2BP2-AS1 in said system;
(3) selecting a test agent that reduces the expression level of IGF2BP2-AS1 AS a drug candidate.
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