CN113151456A - Diagnostic marker for head and neck cell carcinoma and application thereof - Google Patents
Diagnostic marker for head and neck cell carcinoma and application thereof Download PDFInfo
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Abstract
The invention discloses a diagnostic marker for head and neck cell carcinoma, which is LncRNA-RP11-215P8.4, and has a nucleotide sequence shown as SEQ ID NO. 1 and a length of 669 bp. Also relates to the application of LncRNA-RP11-215P8.4 in the preparation of a kit for diagnosing and/or prognostically evaluating the squamous cell carcinoma of head and neck; also relates to an agent for preventing and/or treating head and neck squamous cell carcinoma, comprising an effective active ingredient in an amount that inhibits the expression of LncRNA-RP11-215P8.4 in cancer cells. For a patient suspected of suffering from head and neck squamous cell carcinoma, the detection reagent provided by the invention can be used for detection so as to facilitate early discovery and early treatment.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a diagnostic marker for head and neck cell carcinoma and application thereof.
Background
Squamous Cell Carcinoma of Head and Neck (SCCHN) refers to a squamous cell carcinoma that occurs in the oral cavity, pharynx, larynx, nasal cavity, sinuses, salivary glands, cervical airways and esophagus, and is one of the most common types of head and neck malignancies, which have a high degree of malignancy and a poor prognosis. It is one of the leading causes of morbidity and mortality worldwide, accounting for approximately four percent of all cancers. In 2018, it is expected that about 51540 new patients with oral and throat cancer will die of 10030 patients with the disease. Alcohol and tobacco abuse is a common causative factor in oral, oropharyngeal, hypopharyngeal and laryngeal cancers, with a 5-year survival rate of only 63% in SCCHN patients, primarily because about 80-90% of advanced SCCHN patients develop local recurrence or distant metastasis. Therefore, the identification of novel biomarkers for early diagnosis of primary head and neck squamous cell carcinoma plays an important role in improving outcome.
LncRNA is an RNA that is more than 200 nucleotides in length and has no protein coding function. Studies have shown that high throughput chip and RNA sequencing technologies have identified large numbers of lncrnas that are aberrantly expressed in various cancers. With the advent of a continuously updated big data era, there has been a great progress in the research on head and neck squamous cell carcinoma, in which LncRNAs are becoming new biomarkers in tumor diagnosis.
Disclosure of Invention
The invention aims to provide a diagnostic marker for head and neck cell carcinoma and application thereof, and provides a means for diagnosis or treatment scheme selection or prognosis evaluation of head and neck squamous cell carcinoma so as to find early treatment.
The technical scheme adopted by the invention is as follows:
a diagnostic marker for head and neck cell cancer is LncRNA-RP11-215P8.4, the nucleotide sequence of the LncRNA-RP11-215P8.4 is shown as SEQ ID NO. 1, and the length is 669 bp.
Use of LncRNA-RP11-215P8.4 for the preparation of a kit for the diagnostic and/or prognostic evaluation of head and neck squamous cell carcinoma, wherein the nucleotide sequence of LncRNA-RP11-215P8.4 is shown in SEQ ID NO:1 and is 669bp in length.
Further, primers for amplifying the nucleotide sequence of LncRNA-RP11-215P8.4 are shown in SEQ ID NO. 2 and SEQ ID NO. 3.
A reagent kit for detecting the expression quantity of LncRNA-RP11-215P8.4 is an RT-PCR reagent and an RNA detection probe for detecting the expression quantity of LncRNA-RP11-215P 8.4.
Further, the RT-PCR reagent comprises an RNA extraction reagent, a reverse transcription reagent, a PCR quantitative reagent, a forward amplification primer such as SEQ ID NO. 2, a reverse amplification primer such as SEQ ID NO. 3, a cDNA sample used as a negative control and a cDNA sample used as a positive control and highly expressing RP11-215P 8.4.
An agent for preventing and/or treating head and neck squamous cell carcinoma, which comprises an effective active ingredient in an amount that inhibits the expression of LncRNA-RP11-215P8.4 in cancer cells.
Further, the active ingredient for inhibiting the expression amount of LncRNA-RP11-215P8.4 in cancer cells is siRNA for inhibiting the expression of LncRNA-RP11-215P8.4, and the sequence of the siRNA is shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.
A group of interfering RNAs aiming at RP11-215P8.4 gene targets, wherein the sequence of the RP11-215P8.4 gene targets is shown as SEQ ID NO:1, and the interfering RNAs comprise at least two of siRNA-1, siRNA-2 or siRNA-3, wherein:
the sequence of the siRNA-1 is shown as SEQ ID NO. 4;
the sequence of the siRNA-2 is shown as SEQ ID NO. 5;
the sequence of the siRNA-3 is shown as SEQ ID NO. 6.
The present inventors have found that the expression of LncRNA RP11-215P8.4 in tumors is different from that in normal tissues by analyzing RNA data from SCCHN patients and cancer-free controls. Then, further experiments are carried out, the influence on the migration and invasion of tumor cells is observed by knocking down the expression of the lncRNA, the influence shows inhibition, and the lncRNA has potential as a biomarker in the tumor cells based on the experimental data, so the invention provides the application of the LncRNA RP11-215P8.4 in preparing the diagnosis agent for the squamous cell carcinoma of head and neck.
The present invention relates to an LncRNA marker for detecting squamous cell carcinoma of head and neck. Also relates to a kit for diagnosing the head and neck squamous cell carcinoma, which comprises a reagent for detecting the expression level of the marker; and an agent for treating head and neck squamous cell carcinoma comprising an active ingredient which inhibits the expression amount of LncRNA in cancer cells. For a patient suspected of suffering from head and neck squamous cell carcinoma, the detection reagent provided by the invention can be used for detection so as to facilitate early discovery and early treatment
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a statistical graph showing the expression levels of RP11-215P8.4 in 11 normal tissues and 22 SCCHN tissue samples;
FIG. 2 is a scatter plot showing the distribution of the expression levels of RP11-215P8.4 in SCCHN tissues and control normal tissue samples;
FIG. 3 is a statistical graph of the expression levels of RP11-215P8.4 in normal tissue cells (NC) and tumor cell lines (UMSCC, 10B, Tca8113, OSCC-15, HSC-4);
FIG. 4 is a statistical graph of the expression levels of RP11-215P8.4 after transfer of 3 siRNAs into Tca8113, respectively, wherein the data show that: 0.01 < P < 0.05; 0.001 < P < 0.01; p < 0.001;
FIG. 5 is a statistical graph of the expression level of RP11-215P8.4 after transferring 3 siRNAs into HSC-4, respectively, 0.01 < P < 0.05; 0.001 < P < 0.01; p < 0.001;
FIG. 6 is a statistical chart of the expression levels of RP11-215P8.4 after transfer of 3 siRNAs into OSCC-15, respectively, 0.01 < P < 0.05; 0.001 < P < 0.01; p < 0.001;
FIG. 7 is a photograph of a scratch test for Tca 8113;
FIG. 8 is a relative migratory capacity of Tca8113 calculated from the experimental photograph of FIG. 7;
FIG. 9 is a photograph showing the scratch test of HSC-4;
FIG. 10 is a graph of the relative migratory capacity of HSC-4 as calculated from the experimental photograph of FIG. 9;
FIG. 11 is a photograph showing the scratch test of OSCC-15;
FIG. 12 is a relative migration capability of OSCC-15 calculated from the experimental photograph of FIG. 11;
FIG. 13 is a photograph of a transwell experiment of Tca8113, HSC-4, OSCC-15;
FIG. 14 is the relative migratory capacity of Tca8113 calculated from the experimental photograph of FIG. 13;
FIG. 15 is a graph of the relative migratory capacity of HSC-4 as calculated from the experimental photograph of FIG. 13;
FIG. 16 is a relative migration capability of OSCC-15 calculated from the experimental photograph of FIG. 13;
FIG. 17 is a plot of experimental data for the detection of CCK8 based on Tca 8113;
FIG. 18 is a graph plotting experimental data for the detection of CCK8 based on HSC-4;
FIG. 19 is a graph plotting the experimental data for the detection of CCK8 based on OSCC-15.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
Detection of expression level of LncRNA RP11-215P8.4 in 1.22 SCCHN tissue samples 1.1RNA extraction
(1) Grinding a tissue sample by using liquid nitrogen, adding 1mL of Trizol for cracking, washing a cell sample twice by using PBS (phosphate buffer solution), adding 1mL of Trizol for cracking, transferring the ground tissue or cell into a 1.5mL centrifuge tube without RNase, and standing at room temperature for 7 min;
(2) chloroform was added to 1mL of Trizol in a ratio of 200. mu.L of chloroform, vigorously shaken vigorously, and allowed to stand at room temperature for 3 min. Centrifuging at 12000g at 4 deg.C for 15 min;
(3) taking out the centrifuge tube, dividing the liquid into three layers, taking the upper layer as the target RNA extract, sucking 500 mu L of supernatant liquid by a liquid-moving machine, and carefully transferring the supernatant liquid into a new centrifuge tube;
(4) adding isopropanol into a new centrifuge tube according to the proportion of 1:1, reversing, mixing uniformly, and standing for 10min at room temperature. Centrifuging at high speed for 10min at 4 ℃ and 12000 g;
(5) the supernatant was discarded, and the precipitate was discarded, and washed by adding 75% ethanol/mL (prepared in 750. mu.L absolute ethanol + 250. mu.L DEPC water). Centrifuging at 8000g for 5min at 4 deg.C;
(6) repeating the step (5) once or twice;
(7) pouring out supernatant, centrifuging instantly, sucking out residual liquid at the bottom of the centrifuge tube by using a pipettor, drying and precipitating in a fume hood for 3min, adding 20 mu L of NF water to dissolve RNA precipitate after the precipitate is dried, and carefully blowing, beating and mixing uniformly;
(7) mu.L of the RNA solution was taken and the RNA concentration and purity were measured with NanoDrop.
1.2 reverse transcription of RNA
Kang is HiFiScript cDNA Synthesis Kit:
total reaction 20 μ L: 5 × RT Buffer 4 μ L, dNTP Mix 4 μ L, PrimerMix 2 μ L, DTT 2 μ L, HiFiScript 1 μ L, RNA template 1 μ g, NF water to 20 μ L;
1.3qPCR
naoza ChamQ SYBR qPCR Master Mix:
total reaction 20 μ L: 2 × SYBR Green PCR Master mix 10 μ L, upstream primer 0.5 μ L, downstream primer 0.5 μ L, cDNA 1 μ L, NF water 8 μ L;
the primer sequence is as follows:
RP11-215P8.4-F: 5'-ACAGCCCAGACAAACTTCTTCCC-3' as shown in SEQ ID NO:2,
RP11-215P8.4-R: 5'-AATGAATGGACCCAGAAGGCAGAC-3', shown as SEQ ID NO: 3.
The amplification reaction was performed using an FTC3000 fluorescent quantitative PCR instrument, programmed as follows: 95-30 s; circulating for 40 seconds at 95-10 seconds and 60-30 seconds; 95-30 s, 60-90 s and 94-10 s.
Wherein the fluorescent signal is collected in 60-30 s.
As shown in FIG. 1, 11 normal tissues and 22 samples diagnosed as SCCHN tissues showed that LncRNA RP11-215P8.4 was highly expressed in tumor tissues to different extents, and FIG. 2 showed that LncRNA RP11-215P8.4 was significantly more highly expressed than normal bladder tissues (P < 0.05), and thus LncRNA RP11-215P8.4 was considered as one of diagnostic markers of SCCHN.
Detection of expression level of LncRNA RP11-215P8.4 in SCCHN cell line
The expression level of LncRNA RP11-215P8.4 in five SCCHN cell lines, namely UMSCC, 10B, Tca8113, OSCC-15 and HSC-4, was determined by the PCR method and compared with that in normal tissue cells NC.
As a result, as shown in FIG. 3, the expression level of LncRNA RP11-215P8.4 in the five cells was higher than that of normal tissue cell NC, especially the differences among Tca8113, OSCC-15 and HSC-4 were more obvious.
Knockdown of LncRNA RP11-215P8.4
According to the experimental results, Tca8113, OSCC-15 and HSC-4 cell lines are selected to be used for carrying out the knockdown experiment of LncRNA RP11-215P8.4, and the knocked down cells are subjected to the detection of related gene expression and cell migration capacity.
The knockdown of LncRNA RP11-215P8.4 is realized by using a method of siRNA, which comprises the following steps: spreading cells in a six-well plate, and starving for 30min by using Opti-MEM when the culture plate is full of 50% in the next day;
2.5nmol siRNA/siNC was dissolved in 125. mu.L DEPC water, 10. mu.L per well. Taking two tubes as reference numbers 1 and 2, and adding 100 mu L of Opti-MEM into each tube; add 10. mu.L lipofectamine 3000 to tube 2, add siRNA or siNC 10. mu.L to tube 2, mix well and let stand for 5min at room temperature. And (3) fully and uniformly mixing the tube 1 and the tube 2, standing for 15min, adding the mixture into a six-hole plate, and transfecting for 6-8 h.
Three siRNAs were used in the study of the present invention, the sequences are as follows:
siRNA-1：5’-AUUGAAGAACAAAAUCCUCCU-3’；
siRNA-2：5’-UGGUUAAUGAAAUUUCAGGGA-3’；
siRNA-3：5’-AGACUUUAUCUUGUGAAUGAU-3’；
FIGS. 4, 5 and 6 show that the expression level of RP11-215P8.4 was decreased in the three cell lines after transferring the above three siRNAs into Tca8113, HSC-4 and OSCC-15 cell lines, respectively.
3.2 migration Capacity test of tumor cells
Tumor cells were subjected to scarification experiments (fig. 6-12) and transwell experiments (fig. 13-16).
The scratch test was carried out as follows:
after transfection, when the cell density reaches about 70%, the cell is quickly scribed in the cell culture plate perpendicular to the pore plate (2 lines are scribed in the horizontal and vertical directions), and the width of each scratch is ensured to be consistent as much as possible. The cell culture medium was discarded and the cells were washed three times with PBS buffer. The medium without serum was added and photographed with an inverted microscope and the photographing time was recorded as 0 h. The cells were cultured in a box, photographed at 24h and 48h under an inverted microscope at the same field of view, and observed for migration of the cells in the control group and the experimental group.
transwell experiment:
after transfection, cells were digested and centrifuged, cells were resuspended in serum-free medium, and after cell counting, cells were resuspended in serum-free medium at 2X 10 per cell4The cell number was prepared as a cell suspension. 500 μ L of complete medium containing 10% FBS was added under a Transwell chamber (i.e., the bottom of a 24-well plate), 200 μ L of the formulated cell suspension was added to the chamber, and cultured at 37 ℃ for 36-48h in an incubator containing 5% CO 2. The Transwell chamber was removed and the medium was discarded. Fixing with 4% paraformaldehyde at room temperature for 30 min. Adding 500 mul of 0.1% crystal violet staining solution into each hole, completely immersing the bottom membrane of the transwell cell in the crystal violet staining solution, and staining for 30min at normal temperature. The number of cells passing through the chamber at the bottom of the chamber was observed by taking a picture under an inverted microscope.
As shown in the figure, compared with the SCCHN cells which are not knocked down, the Tca8113, HSC-4 and OSCC-15 cells transferred with siRNA have reduced migration capability.
3.3 viability assay of tumor cells
Digesting transfected cells, centrifuging and resuspending cells, counting the number of cells by using a cell counting plate, setting the number of cells in a control group and an experimental group to be 0h, 24h and 48h respectively, and setting the number of cells in three groups, rotating a 96-well plate to 37 ℃, and carrying out cell separation on the cells containing 5% CO2In the cell culture box of (1), 0h after cell transfection,and taking out the culture medium after 24h and 48h, adding a CCK-8 reagent accounting for 10 percent of the total volume of the culture medium into each hole under the condition of keeping out of the light, continuing culturing for 50min, and changing the color of the solution in the 96-hole plate from colorless to yellow. The absorbance (OD value) of each well in the 96-well plate was measured at a wavelength of 450nm using a microplate reader.
The results are shown in FIGS. 17-19, where the viability of the tumor cells was significantly reduced.
The results show that the expression inhibitor of LncRNA-RP11-215P8.4 can effectively inhibit the migration of SCCHN cells highly expressing RP11-215P8.4, and thus can be used as a therapeutic agent or an adjuvant therapeutic agent for such SCCHN.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Although the embodiments of the present invention have been described in conjunction with the accompanying drawings, those skilled in the art may make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations fall within the scope defined by the appended claims.
Claims (8)
1. The diagnostic marker for the head and neck cell carcinoma is LncRNA-RP11-215P8.4, the nucleotide sequence of the LncRNA-RP11-215P8.4 is shown as SEQ ID NO. 1, and the length is 669 bp.
Use of LncRNA-RP11-215P8.4 for the preparation of a kit for the diagnostic and/or prognostic evaluation of head and neck squamous cell carcinoma, wherein the nucleotide sequence of LncRNA-RP11-215P8.4 is as shown in SEQ ID NO:1 and has a length of 669 bp.
3. The use according to claim 2, wherein the primers for amplifying the nucleotide sequence of LncRNA-RP11-215P8.4 are shown as SEQ ID NOs 2 and 3.
4. A kit for detecting the expression quantity of LncRNA-RP11-215P8.4 is characterized in that a reagent for detecting the expression quantity of LncRNA-RP11-215P8.4 is an RT-PCR reagent for detecting the expression quantity of LncRNA-RP11-215P8.4 and an RNA detection probe.
5. The kit of claim 4, wherein the RT-PCR reagent comprises an RNA extraction reagent, a reverse transcription reagent, a PCR quantification reagent, a forward amplification primer such as SEQ ID NO:2, a reverse amplification primer such as SEQ ID NO:3, a cDNA sample as a negative control, and a cDNA sample as a positive control with high expression of RP11-215P 8.4.
6. An agent for preventing and/or treating head and neck squamous cell carcinoma, characterized in that the agent comprises an effective active ingredient in an amount to inhibit the expression of LncRNA-RP11-215P8.4 in cancer cells.
7. The agent according to claim 6, wherein the active ingredient for suppressing the expression level of LncRNA-RP11-215P8.4 in cancer cells is siRNA having a sequence as shown in SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6, which suppresses the expression level of LncRNA-RP11-215P 8.4.
8. A group of interfering RNAs aiming at RP11-215P8.4 gene targets is characterized in that the sequence of the RP11-215P8.4 gene targets is shown as SEQ ID NO:1, and the interfering RNAs comprise at least two of siRNA-1, siRNA-2 or siRNA-3, wherein:
the sequence of the siRNA-1 is shown as SEQ ID NO. 4;
the sequence of the siRNA-2 is shown as SEQ ID NO. 5;
the sequence of the siRNA-3 is shown as SEQ ID NO. 6.
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| US20140329704A1 (en) * | 2013-03-28 | 2014-11-06 | President And Fellows Of Harvard College | Markers for mature beta-cells and methods of using the same |
| KR20190048019A (en) * | 2017-10-30 | 2019-05-09 | 이화여자대학교 산학협력단 | Compositions comprising lncRNA for diagnosis and prognosis of epithelial-mesenchymal transition in lung cancer |
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