CN110616265B

CN110616265B - Molecular marker for preventing, diagnosing and treating tongue squamous carcinoma and application thereof

Info

Publication number: CN110616265B
Application number: CN201911116648.4A
Authority: CN
Inventors: 郑颖; 刘奕; 郑博文; 安娜; 刘帆; 时逸; 贡雨嘉; 刘崇良
Original assignee: HOSPITAL OF STOMATOLOGY CHINA MEDICAL UNIVERSITY
Current assignee: HOSPITAL OF STOMATOLOGY CHINA MEDICAL UNIVERSITY
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2022-11-04
Anticipated expiration: 2039-11-15
Also published as: CN110616265A

Abstract

The invention relates to the technical field of molecular biology and biomedicine, in particular to a molecular marker for preventing, diagnosing and treating tongue squamous carcinoma and application thereof. The invention discloses a molecular marker for preventing, diagnosing and treating tongue squamous carcinoma, which is LncRNA DANCR. The application of the molecular marker in screening or preparing a reagent for diagnosing the tongue squamous carcinoma and the application of the molecular marker in screening or preparing a medicine for treating the tongue squamous carcinoma. The LncRNA DANCR is found to be highly expressed in the tongue squamous cell carcinoma tissue and is an independent index for diagnosing the tongue squamous cell carcinoma, judging the progression of the tongue squamous cell carcinoma and judging the survival prognosis; and the silencing LncRNA DANCR can inhibit the proliferation, cell migration and invasion of tongue squamous carcinoma cells in vitro. The application proves that LncRNA DANCR is an important carcinogenic factor of tongue squamous cell carcinoma for the first time, and can be used as a molecular marker for tongue squamous cell carcinoma diagnosis and a novel target for treatment.

Description

Molecular marker for preventing, diagnosing and treating tongue squamous carcinoma and application

Technical Field

The invention relates to the technical field of molecular biology and biomedicine, in particular to a molecular marker for preventing, diagnosing and treating tongue squamous carcinoma and application thereof.

Background

Squamous cell carcinoma of the Tongue (TSCC) is the major type of Head and Neck Squamous Cell Carcinoma (HNSCC), with high recurrence rate, high metastasis rate, and poor prognosis. Despite major advances in prevention and treatment, the survival rates of TSCC patients remain low. The results indicate that tumor invasion and migration are the main causes of tumorigenesis and development. Therefore, by exploring the underlying molecular mechanisms of TSCCs, the development of new therapeutic strategies has been imminent.

Long non-coding RNAs (incrnas) are a group of long non-coding RNAs with a length of more than 200 nucleotides. A number of reports have shown that LncRNAs play important roles in a variety of biological processes such as cell proliferation, differentiation, apoptosis, migration and invasion. In particular, lncRNAs were found to be closely associated with the development of various tumors and TSCCs. For example, high expression of lncRNA AFAP1-AS1 in TSCC tumor tissue via activation of the β -catenin Wnt signaling pathway promotes tumor progression. NKILA is a key determinant of TSCC metastasis, reducing cell migration and invasion by inhibiting the epithelial-mesenchymal transition (EMT) process. The literature reports that lncRNA DANCR (differentiation antagonistic non-protein coding RNA) can inhibit the differentiation of epidermal cells and promote the proliferation of liver cancer cells. DANCR is also known as an oncogenic lncRNA for a variety of cancers, such as prostate, gastric, and colorectal cancers. However, a related art of the mechanism by which DANCR occurs in TSCC has not been elucidated.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have been shown to regulate the expression of a target gene. Recent researches show that miR-135a-5p is a main regulatory factor for tumor invasion and metastasis. In non-small cell lung cancer (NSCLC), miR-135a-5p inhibits cell migration and invasion by targeting Kruppel-like factor 8 (KLF 8). As is well known to those skilled in the art, KLF8 has been extensively demonstrated to be involved in the regulation of cell cycle proliferation, transformation, EMT and invasion. By bioinformatics analysis, it is predicted that DANCR may have a specific binding site with miR-135a-5p, so the inventors speculate that DANCR may influence the occurrence and development of TSCC by regulating the miR-135a-5p/KLF8 axis.

To further investigate the effect of DANCR on TSCC malignancies, the inventors constructed CAL-27 and TCa-8113 cells with DANCR silencing, as well as SCC9 and TSCA cells with DANCR overexpression. The effect of DANCR on TSCC cell proliferation, migration, and invasion was studied. Further, it was demonstrated that the miR-135a-5p/KLF8 axis is an important pathway for the DANCR to promote TSCC progression. The prior art does not report about the expression condition of DANCR in tongue squamous cell carcinoma tissues, and an effective early onset biomarker is found, and a related molecular mechanism has important significance for guiding the clinical diagnosis, treatment and prognosis of tongue squamous cell carcinoma. In general, further study of the role of LncRNA DANCR in the development of squamous cell carcinoma of tongue is very important for the prevention, diagnosis, treatment and prognosis of squamous cell carcinoma of tongue.

Disclosure of Invention

In view of the problems in the prior art, the invention aims to provide a molecular marker for preventing, diagnosing and treating tongue squamous carcinoma and application thereof, and aims to provide a novel diagnosis marker for tongue squamous carcinoma, which can clearly and clearly represent the occurrence and development of tongue squamous carcinoma and has a specific high expression phenomenon in human clinical tongue squamous carcinoma tissues compared with corresponding normal tongue squamous carcinoma tissues; and also shows specific high expression phenomenon in human tongue squamous carcinoma cell line. LncRNADARNR is used as a diagnosis marker of tongue squamous cell carcinoma, provides a molecular target for tongue squamous cell carcinoma treatment, and can be applied to preparation of anti-tumor drugs.

In order to achieve the purpose, the invention adopts the following technical scheme.

A molecular marker for preventing, diagnosing and treating tongue squamous carcinoma is LncRNA DANCR, and the nucleotide sequence of the marker is shown as SEQ ID No. 1.

Use of LncRNA DANCR gene or its expression product as molecular marker in screening or preparing reagent or chip for diagnosing tongue squamous carcinoma.

Use of a reagent for detecting an expression product of LncRNA DANCR gene in the preparation of a kit for diagnosing tongue squamous cell carcinoma.

The LncRNA DANCR gene or the expression product thereof is used as a target for screening or preparing a medicament for treating tongue squamous carcinoma.

Use of an inhibitor of LncRNA DANCR gene expression, which is siRNA that inhibits LncRNA DANCR gene expression, in the preparation of a medicament for treating squamous cell carcinoma of the tongue.

A medicament for treating squamous cell carcinoma of tongue, comprising an inhibitor for inhibiting expression of LncRNA DANCR gene, which is siRNA for inhibiting expression of LncRNADANCR gene, and a pharmaceutically acceptable carrier.

Compared with the prior art, the invention has the following beneficial effects.

The invention discovers that LncRNA DANCR can be used as a tongue squamous cell carcinoma diagnosis biomarker for the first time, and provides a new treatment target for prevention, diagnosis and treatment of TSCC. And the TSCC tumor progression is verified to be regulated by the DANCR/miR-135a-5p/KLF8 axis, the expression of MMP-9 and MMP-2 is changed through the DANCR/miR-135a-5p/KLF8 axis, and a regulation network of the TSCC tumor on malignant tumors is further verified from a molecular level. DANCR, as a "sponge" for miR-135a-5p, activates the KLF8/MMP-2/9 signaling pathway, thereby stimulating the development and progression of TSCC.

Drawings

FIG. 1 shows the relative expression of DANCR in different TSCC cell lines measured by qRT-PCR.

FIG. 2 shows the transfection efficiency of CAL-27 and TCa-8113 cells transfected with si-DANCR, measured by qRT-PCR.

FIG. 3 shows MTT assay to examine the proliferation potency of CAL-27 and TCa-8113 cells after transfection of si-DANCR.

FIG. 4 is a scratch test to examine the migration ability of CAL-27 and TCa-8113 cells after transfection of si-DANCR.

FIG. 5 is a Transwell experiment to examine the invasive ability of CAL-27 and TCa-8113 cells after transfection of si-DANCR.

FIG. 6 shows the qRT-PCR detection of the transfection efficiency of pcDNA3.1 vector overexpressing DANCR transfected SCC9 and TSCCA cells.

FIG. 7 shows the MTT assay for detecting the proliferation capacity of SCC9 and TSCCA cells after overexpression of DANCR.

FIG. 8 is a scratch test to examine the migration ability of SCC9 and TSCCA cells after overexpression of DANCR.

FIG. 9 shows the invasion capacity of SCC9 and TSCCA cells after overexpression of DANCR in the Transwell assay.

FIG. 10 is a sequence alignment of DANCR and potential targeting sites of miR-135a-5 p.

FIG. 11 is a luciferase reporter assay demonstrating the binding of DANCR to miR-135a-5 p.

FIG. 12 shows the change in relative expression of miR-135a-5p after si-DANCR transfection in CAL-27 cells measured by qRT-PCR.

FIG. 13 shows the change of relative expression of miR-135a-5p after si-DANCR transfection in TCa-8113 cells by qRT-PCR.

FIG. 14 is a graph showing the change in relative expression of miR-135a-5p after si-DANCR transfection in SCC9 cells by qRT-PCR.

FIG. 15 shows the change in relative expression of miR-135a-5p after transfection of si-DANCR in TSCCA cells by qRT-PCR.

FIG. 16 shows the change in the relative expression of KLF8 after transfection of si-DANCR in CAL-27 cells measured by qRT-PCR.

FIG. 17 shows the change in the relative expression of KLF8 after transfection of si-DANCR in TCa-8113 cells by qRT-PCR.

FIG. 18 is a graph showing the change in the relative expression of KLF8 after transfection of si-DANCR in SCC9 cells by qRT-PCR.

FIG. 19 shows the variation of the relative expression of KLF8 after transfection of si-DANCR in TSCCA cells by RT-PCR.

FIG. 20 shows that relative expression of miR-135a-5p in different TSCC cell lines is detected by qRT-PCR.

FIG. 21 shows the transfection efficiency of miR-135a-5p plasmid overexpression in CAL-27 and TCa-8113 cells tested by qRT-PCR.

FIG. 22 shows that MTT test detects the change of proliferation capacity of CAL-27 and TCa-8113 cells after over-expressing miR-135a-5 p.

FIG. 23 shows the change of migration ability of CAL-27 and TCa-8113 cells after miR-135a-5p is over-expressed by scratch test.

FIG. 24 shows that MTT assay detects the invasive ability change of CAL-27 and TCa-8113 cells after miR-135a-5p is over-expressed.

FIG. 25 shows the relative expression of KLF8 mRNA after miR-135a-5p overexpression by cells CAL-27 and TCa-8113 detected by qRT-PCR.

FIG. 26 shows KLF8 expression levels after detection of CAL-27 and TCa-8113 cells over-expressing miR-135a-5p by western blot.

FIG. 27 is a graph of miR-135a-5p inhibitor partially reversing the inhibition of transfected si-DANCR-mediated tumor cell proliferation in CAL-27 and TCa-8113 cells.

FIG. 28 is a graph of miR-135a-5p inhibitor partially reversing the inhibition of transfected si-DANCR-mediated tumor cell migration in CAL-27 and TCa-8113 cells.

FIG. 29 is a graph of miR-135a-5p inhibitor partially reversing the inhibition of transfected si-DANCR-mediated tumor cell invasion in CAL-27 and TCa-8113 cells.

FIG. 30 shows the change in MMP-2 and MMP-9 expression after the addition of miR-135a-5p inhibitor in CAL-27 and TCa-8113 cells transfected with si-DANCR by Western Blot detection.

FIG. 31 shows the expression change of KLF8 after the addition of miR-135a-5p inhibitor to CAL-27 and TCa-8113 cells transfected with si-DANCR by Western Blot detection.

FIG. 32 shows the expression level of KLF8 protein after transfection of si-KLF8 in SCC9 cells silencing miR-135a-5p by Western Blot assay.

FIG. 33 shows the MTT assay to detect changes in tumor cell proliferation after transfection of si-KLF8 in miR-135a-5 p-silenced SCC9 cells.

FIG. 34 is a scoring experiment to detect changes in tumor cell migration following transfection of si-KLF8 in SCC9 cells silencing miR-135a-5 p.

FIG. 35 is a drawing. The Transwell experiment detects the change of tumor cell invasion after siKLF 8 transfection in SCC9 cells silencing miR-135a-5 p.

FIG. 36 shows the expression changes of MMP-2 and MMP-9 in SCC9 cells with miR-135a-5p silencing by Western Blot assay after transfection of si-KLF 8.

FIG. 37 is a comparison of tumor tissue volume for downregulation of DANCR with normal tumor tissue volume.

FIG. 38 is a comparison of tumor body tissue weight downregulated by DANCR with normal tumor body tissue weight.

FIG. 39 is a Western Blot detecting the change of MMP-2 and MMP-9 protein expression in tumor tissues at 25 days in the tumor transplantation experiment.

FIG. 40 shows that Western Blot detects the expression change of KLF8 protein in tumor tissues at 25 days in the experimental transplantation of tumors.

FIG. 41 shows KLF8 changes at 25 days of the experiment for immunofluorescence staining of transplantable tumors.

Detailed Description

The following detailed description of the present invention is provided in connection with the accompanying drawings and examples. The following examples are provided to illustrate the present invention, but these examples are only for illustrating the present invention and the present invention is not limited to these. The operation methods not specifically described in the examples are all the conventional operation methods in the art.

Examples are given.

1. Cell culture and reagents.

4 human TSCC cell lines SCC9, TSCCA, TCa-8113, and CAL-27 cells were used in this experiment. SCC9 cells (Cellcook, guangzhou) were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (SH 30084.03, hyclone, south Logan, UT, USA); TSCCA cells (Procell, wuhan) were cultured in DMEM medium (12100-46, gibco) containing 10% fetal bovine serum; the TCa-8113 and CAL-27 cell lines (Procell, wuhan) were cultured in RPMI-1640 medium (31800-014, gibco, gauthersbueg, md., USA) containing 10% fetal bovine serum. All these cell lines were cultured at 37 ℃ in a standard environment of 5% CO 2. MiR-135a-5p mimetics/inhibitors and the corresponding negative control mimetics/inhibitors were purchased from JTS scientific (Beijing).

1. Construction of siRNAs and shRNAs.

The sequences of human DANCR-targeted siRNAs (5 '-3') were designed as follows.

si-DANCR-1 sense strand (SEQ ID No. 2): GUUGACAACUACAGGCACATT.

si-DANCR-1 antisense strand (SEQ ID No. 3): UGUGUGCCUGUAGUUGUCAACTT.

si-DANCR-2 sense strand (SEQ ID No. 4): cuagagcaggugacaaugcutt.

si-DANCR-2 antisense strand (SEQ ID No. 5): AGCAUUGUCUGCUCUCUAGTT.

NC siRNA sequence (5 '-3') is.

Sense strand (SEQ ID No. 6): UUCCCGAACGUGUCACGUTT.

Antisense strand (SEQ ID No. 7): ACGUGACACGUUCGGAGAATT.

The shRNAs against DANCR and the corresponding negative control sh-NC were constructed using pRNAH1.1 plasmid vector (Genscript, nanjing). The human KLF8 interference sequence (5 '-3') was designed as follows.

si-KLF8 sense strand (SEQ ID No. 8): CGAUAUGGAUAAACUCAUATT.

si-KLF8 antisense strand (SEQ ID No. 9): uaugaguuuauccauucgac.

2. And (3) constructing an overexpression plasmid.

Human DANCR (NR _ 024031.2) was amplified using a pair of specific primers (sense strand: 5. The amplified product was inserted into pcDNA3.1 plasmid (V790-20, invitrogen, carlsbad, CA, USA) between BamHI and XhoI cleavage sites to induce overexpression of DANCR. The empty pcDNA3.1 plasmid was used as a control.

3. And (4) cell transfection.

When the cells reached 70% confluence, DANCR-targeted siRNAs or shRNAs were transfected into CAL-27 and TCa-8113 cells, as directed by Lipofectamine 2000 reagent (11668-019, invitrogen) according to the manufacturer's instructions. All experiments were performed 48h after transfection. Moreover, miR-135a-5p mimetics or NC mimetics are transfected into CAL-27 or TCa-8113 cells, and inhibitors or NC inhibitors thereof are transfected into SCC9 cells as described above, to overexpress or silence miR-135a-5 p. In addition, the co-transfection of miR-135a-5p inhibitors with si-DANCR or si-KLF8 was also mediated by Lipofectamine 2000 reagents.

4. Real-time quantitative polymerase chain reaction (qRT-PCR).

Total RNA in TSCC cell lines was extracted using an RNAscope Total RNA kit (DP 419, TIANGEN, beijing) and reverse transcribed to cDNA using M-MLV reverse transcriptase (NG 212, TIANGEN)In the template. The designed specific primer sequence was synthesized by sangon bioth. Subsequently, the cDNA template, specific primers, SYBR Green reagent (SY 1020, solarbio, beijing) and Taq PCR MasterMix (KT 201, TIANGEN) were mixed to amplify the gene of interest, and qRT-PCR analysis was performed in Exicycler 96 PCR system (Bioneer, daejeon, korea). DANCR and KLF8 expression GAPDH, and miR-135a-5p expression U6. Use 2 ^-ΔΔCT The method calculates a relative expression.

5. MTT assay.

TSCC cells were cultured at 4X 10 ³ The density of individual cells/well was plated on 96-well plates for 0, 24, 48 and 72 hours, respectively. Then incubated in complete medium (KGA 311, keyGEN, nanjing) containing 0.5 mg/ml MTT for 4 hours. After dissolution in DMSO (ST 038, beyotime), viable cells were determined at an optical density of 570 nm using a microplate reader (ELX-800, BIOTEK, winooski, VT, USA).

6. And (5) scratching test.

Scratch test was used to assess cell migration ability. Cells were treated with mitomycin C (M0503, sigma) in serum-free medium for 1 hour. Scratches were formed by a 200 μ l pipette tip and recorded by phase contrast microscopy (IX 53, olympus, tokyo). After 24 hours, the cell migration ability was calculated by measuring the cell migration distance using Image Pro Plus software (Media Cybernetics, silver Springs, MD, USA).

7. Transwell assay.

The invasion capacity of the cells was evaluated by the Transwell method. Cell suspensions (2 × 104 cells/well) were seeded in Transwell (3422, corning, USA, new york) upper chamber and the matrix gel was pre-coated with serum-free medium (356234, bd Biosciences, san Jose, CA, USA). The lower chamber was filled with medium containing 30% fetal bovine serum, and after 48 hours of incubation, the upper chamber cells were taken and washed 2 times with PBS. Cells were then fixed with 4% paraformaldehyde and stained with 0.4% crystal violet (0528, amresco, solon, OH, USA). The number of the cells in the lower chamber is observed by using a phase contrast microscope with 200 times magnification, and 5 fields are randomly selected from each image for counting.

8. Luciferase reporter experiments.

Bioinformatics analysis predicts that lncRNA DANCR is likely to bind to miR-135a-5 p. Wild type (wt) or mutant (mut) luciferase reporter vectors were constructed using the pmirGLO vector (E133A, promega, madison, wis., USA) containing NheI and SalI cleavage sites. Site-directed mutagenesis of DANCR was used to verify its target effect with miR-135a-5 p. 293T cells (New China Biotech, inc., shanghai, china) were then seeded in 12-well plates and wt-DANCR or mut-DANCR was co-transfected with miR-135a-5p or NC mimic using Lipofectamine 2000. 48 hours after transfection, the binding activity was measured by calculating luciferase activity/renal luciferase activity using a dual luciferase reporter kit (E1910, promega).

9. Immunoblotting.

Total protein (P0100, solarbio) of TSCC cell lines or tumor tissues was isolated using RIPA lysate (R0010, solarbio) containing PMSF and quantified using BCA assay kit (PC 0020, solarbio). Equal amounts of protein were then loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF membranes (IPVH 00010, millipore, billerica, MA, USA). After washing in TBST, the membranes were incubated overnight at 4 ℃ with one of the following specific antibodies: MMP-2 antibody (1. Subsequently, HRP-conjugated goat anti-rabbit antibodies (1.

10. Xenograft tumor model analysis.

The study was approved by the ethical committee of the oral medical college of medical university of china (No. G2018007). All animal experimental procedures were performed according to the laboratory guidelines for animal care and use. Balb/c nude mice (4-5 weeks, 1)8-20 g) purchased from Beijing Fukang bioscience, inc. of China, and placed in a standard environment. sh-DANCR or sh-NC cells were stably transfected with G418 antibiotic (A1720, sigma, st. Louis, MO, USA). Then, sh-DANCR or sh-NC stably transfected CAL-27 cells or TCa-8113 cells were injected subcutaneously into the right side of the axilla at a density of 1X 106 cells per animal. According to tumor volume (mm) ³ ) = (length x width) ² ) Equation of/2 tumor volume was measured every 4 days with calipers. Mice were sacrificed 25 days later and tumor weights were measured.

11. Immunofluorescence.

Tumor tissues collected by immunofluorescence staining were fixed with paraformaldehyde, and 5 μm-thick sections were prepared. Paraffin sections were incubated overnight at 4 ℃ with KLF8 antibody (NBP 2-57740, NOVUS, centennaal, CO, USA) and FITC-labeled goat-anti-rabbit secondary antibody (A0562 Beyotime) at room temperature for 60 minutes. After counterstaining with DAPI, the immunopositive material was observed using an optical microscope (BX 53, olympus) at × 400 magnification and photographed with a digital camera (DP 73, olympus).

12. And (5) performing statistical analysis.

Data are expressed as mean ± standard deviation and analyzed using GraphPad Prism software (san diego, california, usa). P is <0.05 using t-test or one-way anova after Bonferroni test.

13. And (6) obtaining the result.

1) The DANCR knockout inhibited proliferation, migration and invasion of TSCC cell lines.

The expression profile of DANCR was first detected in 4 different TSCC cell lines, as shown in FIG. 1. As can be seen from the figure, the expression of DANCR was significantly higher in CAL-27 and TCa-8113 cells than in SCC9 and TSCCA cells. Thus, in further experiments, the use of CAL-27 and TCa-8113 cells inhibited DANCR, while SCC9 and TSCCA cells were forced to express DANCR. As expected, targeting of DANCR with specific siRNAs significantly reduced levels in CAL-27 and TCa-8113 cells, as shown in FIG. 2.

The effect of si-DANCRs on TSCC cell proliferation, migration, and invasion was then assessed. The MTT method was considered as a marker of cell proliferation, and the results showed that the number of cells surviving CAL-27 and TCa-8113 decreased when DANCR was knocked down, as shown in FIG. 3. Inhibition of DANCR significantly reduced the migratory and invasive capacity of TSCC cells by scratch and transwell invasion experiments, as shown in FIGS. 4 and 5, indicating that in vitro DANCR knockdown can attenuate the biological properties of TSCC malignancies.

2) DANCR overexpression promotes proliferation, migration, and invasion of TSCC cell lines.

Overexpression of DANCR was used to study its biological function in SCC9 and TSCCA cells. It was observed that the overexpression plasmid significantly increased the expression of DANCR in SCC9 and TSCCA cells, as shown in FIG. 6. Functional analysis of SCC9 and TSCCA cells showed that ectopic expression of DANCR induced an increase in cell viability, migration distance and number of invasive cells, as shown in fig. 7-9. Experimental data indicate that DANCR can promote proliferation, migration and invasion of TSCC cells in vitro.

3) DANCR targeting miR-135a-5p regulates the expression of KLF8 in TSCC cell lines.

As shown in FIG. 10, bioinformatics predicted the presence of complementary sequences for DANCR and miR-135a-5p, which has been confirmed by a dual-luciferase reporter experiment. The results show that the miR-135a-5p mimic can obviously inhibit the luciferase activity of wt-DANCR, but has no influence on mut-DANCR, as shown in FIG. 11. A significant increase in the levels of miR-135a-5p observed with SI-DANCR transfected by CAL-27 and TCa-8113 cells, as shown in FIGS. 12 and 13, and a significant decrease in the levels of miR-135a-5p observed with pcDNA3.1-DANCR transfected by SCC9 and TSCCA cells, as shown in FIGS. 14 and 15. In addition, CAL-27 (FIG. 16) and TCa-8113 cells (FIG. 17) were transfected with si-DANCR, with KLF8 mRNA down-regulated, but KLF8 mRNA levels were up-regulated following DANCR overexpression in SCC9 cells (FIG. 18) and TSCCA cells (FIG. 19). These data indicate that miR-135a-5p is a direct target for DANCR, whereas KLF8 is involved in the regulation of the TSCC malignant phenotype by DANCR.

4) MiR-135a-5p overexpression inhibited tumor cell progression and KLF8 expression in TSCC cell lines.

miR-135a-5p is expressed in SCC9 and TSCCA cells more than TCa-8113 and CAL-27 cells, as shown in FIG. 20. To further study the role of miR-135a-5p, the inventors further studied its specific mimetics. It was demonstrated that miR-135a-5p expression was increased by mimetics in CAL-27 and TCa-8113 cells, as shown in FIG. 21. FIGS. 22-24 show that the living cell number of CAL-27 cells and TCa-8113 cells can be reduced, the migration distance is shortened, and the invasiveness is reduced by over-expressing miR-135a-5 p. In addition, miR-135a-5p can also inhibit the expression of KLF8 mRNA and protein, as shown in FIGS. 25 and 26. All results indicate that miR-135a-5p is involved in inhibiting KLF8, and thus in inhibiting the progression of tumor cells in TSCC cell lines.

5) Tumor cell progression and KLF8 expression were inhibited by targeting miR-135a-5p DANCR knockdown in TSCC cell lines.

Although miR-135a-5p has been shown to target DANCR and promote TSCC progression, it is unclear whether miR-135a-5p can reverse the effect of DANCR on malignancy. As shown in FIG. 27, miR-135a-5p inhibitors can enhance the inhibition of live cells by DANCR knockdown. In addition, inhibition of miR-135a-5p reversed si-DANCR-mediated cell migration and invasion inhibition, as shown in fig. 28 and 29. It is well known that Matrix Metalloprotease (MMP) family proteins are the main biomarkers of tumor invasion and metastasis. As shown in FIG. 30, miR-135a-5p inhibitors can partially increase the reduction in MMP-2 and MMP-9 protein levels induced by DANCR silencing. In addition, it was found that miR-135a-5p inhibitors could reverse si-DANCR-mediated reduction of KLF8 expression, as shown in fig. 31. These results further indicate that DANCR/miR-135a-5p regulates the progression of TSCC by modulating KLF 8.

6) Progression of tumor cells was exacerbated by activation of inhibitors of KLF8MiR-135a-5p in TSCC cell lines.

Using siRNA specific for KLF8, it was further demonstrated whether KLF8 acts on the regulation of DANCR/miR-135a-5p in SCC9 cells. As expected, the miR-135a-5p inhibitor induced KLF8 elevation was inhibited by siRNA from KLF8 itself, as shown in figure 32. Downregulation of KLF8 attenuated the effect of miR-135a-5p inhibitors on SCC9 cell proliferation, migration, and invasion, as shown in fig. 33-35. Similarly, KLF8 silencing also inhibited the development of tumors, such as MMP-2 and MMP-9, as shown in FIG. 36, confirming that tumor malignancy was altered at the molecular level. Taken together, these findings indicate that KLF8 affects progression in TSCC by modulating DANCR/miR-135a-5 p.

7) Activated in vivo by KLF8, DANCR knocks down the formation of disrupted tumors.

To test the effect of DANCR on tumor growth in vivo, CAL-27 or TCa-8113 cells were stably transfected with shRNAs and injected subcutaneously into the right axilla of nude mice. As shown in fig. 37 and 38, tumor size and weight can be inhibited by downregulating DANCR. At the molecular level, by inhibiting DANCR, the expression of MMP-2 and MMP-9 was also reduced in the tumor tissues, as shown in FIG. 39. As shown in fig. 40 and 41, both immunoblot and immunofluorescence staining results showed significant down-regulation of KLF8 expression in stable tumor tissues transfected with DANCR shRNA. Taken together, these in vivo results indicate that DANCR activates the expression of KLF8 and MMPs, affecting the growth of TSCC tumors.

Sequence listing

< 110 > oral Hospital affiliated to Chinese medical university

Less than 120 molecular marker for preventing, diagnosing and treating tongue squamous carcinoma and application thereof

＜160＞9

＜210＞1

＜211＞915

＜212＞RNA

＜213＞Homo sapiens

＜400＞1

gcccttgccc agagtcttcc cgggattggc tgcgggcctc gcgaccctcc tgcttccctc 60

cccgccccgc gccgcctctc tggtttgtgc gcccgtcgca ggtcgcaggc ctctttgtca 120

gctggagttg cgcgggctga cgcgccacta tgtagcgggt ttcgggcggg ccacgcgtgc 180

gggacaggaa cccaacccca gccgaccttg agctccagga gttcgtctct tacgtctgcg 240

gaagtgcagc tgcctcagtt cttagcgcag gttgacaact acaggcacaa gccattgaag 300

ctggaatgtc ctgttgctgg tatttcaatt gacttaagcc aactatccct tcagttacaa 360

taggaaagtg cctctaataa ggccaaatat gcgtactaac ttgtagcaac cacgtgtccg 420

tgcagtgcca caggagctag agcagtgaca atgctggtgg caacagggca gtgtagcagg 480

tgcttcatgt tcaccttttc aaccttttca tttaattgtc acaactcgga ggtggattct 540

gttagggaca ggctgcccca ggaccactcc gcccccgcta actcaatgca gctgaccctt 600

accctgaata ctctgcagct gcattcctga accgttatct aggcgctata gcaaggtcac 660

cagacttgct acaccgaagc cctctgggtg gcacggggga ggtcatgaga aacgtggatt 720

acaccccctt gtaaattcct attttcacaa gataatatat tgtaagccgg tcatgagatt 780

atatgtggta aagttaattg actaacaacc ccagggtctc tctcccccat ataaacccct 840

cattttgtaa gctcagggct gccacctccg actggtggag aagcctggca ggttaataaa 900

cttacttggc ctgac 915

＜210＞2

＜211＞21

＜212＞DNA

< 213 > is artificially synthesized

＜400＞2

guugacaacu acaggcacat t 21

＜210＞3

＜211＞19

＜212＞DNA

< 213 > is artificially synthesized

＜400＞3

ugugccugua guugucaact t 21

＜210＞4

＜211＞21

＜212＞DNA

< 213 > Artificial Synthesis

＜400＞4

cuagagcagu gacaaugcut t 21

＜210＞5

＜211＞21

＜212＞DNA

< 213 > is artificially synthesized

＜400＞5

agcauuguca cugcucuagt t 21

＜210＞6

＜211＞21

＜212＞DNA

< 213 > Artificial Synthesis

＜400＞6

uucuccgaac gugucacgut t 21

＜210＞7

＜211＞21

＜212＞DNA

< 213 > is artificially synthesized

＜400＞7

acgugacacg uucggagaat t 21

＜210＞8

＜211＞21

＜212＞DNA

< 213 > is artificially synthesized

＜400＞8

cgauauggau aaacucauat t 21

＜210＞9

＜211＞21

＜212＞DNA

< 213 > Artificial Synthesis

＜400＞9

uaugaguuua uccauaucga c 21

Claims

1. Use of a reagent for detecting an expression product of LncRNA DANCR gene in the preparation of a kit for diagnosing tongue squamous cell carcinoma, wherein the nucleotide sequence of the marker LncRNA DANCR is shown as SEQ ID No. 1.

Use of an inhibitor of LncRNA DANCR gene expression in the preparation of a medicament for treating tongue squamous carcinoma, wherein the inhibitor is siRNA inhibiting the LncRNA DANCR gene expression, and the nucleotide sequence of the marker LncRNA DANCR is shown in SEQ ID No. 1.