CN112899371A

CN112899371A - Application of hsa _ circ _0000231 in treatment of tongue squamous cell carcinoma

Info

Publication number: CN112899371A
Application number: CN202110348026.5A
Authority: CN
Inventors: 吴昊; 韩靓; 汤明明
Original assignee: Affiliated Hospital of Nantong University
Current assignee: Affiliated Hospital of Nantong University
Priority date: 2021-03-31
Filing date: 2021-03-31
Publication date: 2021-06-04
Anticipated expiration: 2041-03-31
Also published as: CN112899371B

Abstract

The invention belongs to the technical field of biological medical treatment, and discloses a clinical diagnosis marker for tongue squamous cell carcinoma, which is characterized in that the diagnosis marker is hsa _ circ _ 0000231. The invention also discloses application of knocking down hsa _ circ _0000231 expression in treating tongue squamous cell carcinoma. The invention also discloses application of the small interfering RNA in preparing a medicament for treating tongue squamous cell carcinoma, which is characterized in that the small interfering RNA targets the hsa _ circ _0000231 connecting region, and the sequence of the target point is 5'-CTGAACAGATAA GGGTTTAAA-3'. A kit for detecting tongue squamous cell carcinoma is characterized by comprising Trizol reagent for extracting total RNA, reverse transcription reagent, hsa _ circ _0000231 upstream primer, hsa _ circ _0000231 upstream primer, GAPDH upstream primer sequence and GAPDH downstream primer.

Description

Application of hsa _ circ _0000231 in treatment of tongue squamous cell carcinoma

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of hsa _ circ _0000231 expression in treatment of tongue squamous cell carcinoma, in particular to a clinical diagnosis marker of tongue squamous cell carcinoma, application of reducing hsa _ circ _0000231 expression in treatment of tongue squamous cell carcinoma, application of small interfering RNA in preparation of a medicine for treating tongue squamous cell carcinoma, a kit for detecting tongue squamous cell carcinoma and a using method thereof.

Background

Tongue cancer is the most common oral cancer, more men than women. Most tongue cancer is squamous carcinoma, which mostly occurs in the tongue margin, and then in the tongue tip, tongue back and tongue root, which are often ulcerated or infiltrative. Generally, the degree of malignancy is high, the growth is fast, the infiltration is strong, and the tongue movement is limited due to the constant wave and the tongue muscle, so that the speaking, eating and swallowing are difficult. The development of tumors is a complex process with multiple gene involvement, multiple steps and multiple stages. Circular RNA (circRNA) is a class of noncoding transcriptional regulators widely found in mammals, which are highly conserved, most of which may exert regulatory effects at the transcriptional or post-transcriptional level.

At present, the correlation between the TSCC and the micro rna and the lncRNA are researched more, and the circRNA is not reported yet. In our earlier studies, paired fresh TSCC tissue samples were subjected to circRNA detection and identification by second generation High throughput sequencing technology, the identified circRNA was annotated using the circBase database and the circ2 trap disease database and the sequencing results were uploaded to the GEO public database (High-throughput sequencing data area accessible using GEO ID: GSE 118750). 322 differentially expressed circRNAs were selected with Fold Change (FC) ≥ 2.0 and P-value <0.05 as the threshold for differential circRNAs. The host gene of the circRNA is subjected to GO function and KEGG pathway analysis, and hsa _ circ _0000231 is selected as a research object.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems or the defects in the prior art, the invention provides a clinical diagnosis marker of tongue squamous cell carcinoma, application of knocking down hsa _ circ _0000231 expression in treating tongue squamous cell carcinoma, application of small interfering RNA in preparing a medicament for treating tongue squamous cell carcinoma, a kit for detecting tongue squamous cell carcinoma and a using method thereof.

In order to achieve the above object, an embodiment of the present invention provides a clinical diagnostic marker for squamous cell carcinoma of tongue, wherein the diagnostic marker is hsa _ circ _ 0000231.

Further, the hsa _ circ _0000231 promotes the proliferation, migration and invasion of tongue squamous cell carcinoma cells by activating the Wnt/beta-catenin signaling pathway.

Embodiments of the invention also provide for the use of knocking down hsa _ circ _0000231 expression in the treatment of squamous cell carcinoma of the tongue.

Specifically, the application of knocking down hsa _ circ _0000231 expression in treating tongue squamous cell carcinoma is characterized by comprising the following steps: (1) constructing hsa _ circ _0000231 gene interference and lentivirus transfection to realize the reduction of hsa _ circ _0000231 in TSCC cell lines CAL-27 and Tca-8113; (2) qRT-PCR detection of hsa _ circ _0000231 interference and control cells in hsa _ circ _0000231 expression; (3) and selecting the cells with the highest knocking efficiency to perform a CCK-8 experiment, a clone formation experiment, a Transwell invasion experiment and a scratch experiment, detecting the expression of EMT related proteins E-cadherin, Snail protein and N-cadherin vimentin in the TSCC cells of the interference group and the control group by a Western blot method, and comparing the proliferation, invasion and transfer capacities of the TSCC cells of the interference group and the control group.

The invention also provides application of the small interfering RNA in preparing a medicine for treating tongue squamous cell carcinoma, which is characterized in that the small interfering RNA targets the hsa _ circ _0000231 connecting region, and the sequence of the target point is 5'-CTGAACAGATAA GGGTTTAAA-3'.

The invention also provides a kit for detecting tongue squamous cell carcinoma, which is characterized by comprising Trizol reagent for extracting total RNA, a reverse transcription reagent, an hsa _ circ _0000231 upstream primer, an hsa _ circ _0000231 upstream primer, a GAPDH upstream primer sequence and a GAPDH downstream primer.

Specifically, the sequence of the hsa _ circ _0000231 upstream primer is as follows: 5'-GACACGCTCATTTGGTCAT-3' the flow of the air in the air conditioner,

hsa _ circ _0000231 downstream primer sequence: 5'-CCCACTTCTGTCAGCCATT-3', respectively;

GAPDH upstream primer sequence: 5'-TGACTTCAACAGCGACACCCA-3' the flow of the air in the air conditioner,

GAPDH downstream primer sequence: 5'-CACCCTGTTGCTGTAGCCAAA-3' are provided.

The invention also provides a use method of the kit for detecting the tongue squamous cell carcinoma, which is characterized by comprising the following steps: extracting total RNA by using a Trizol reagent, detecting the concentration and purity of the RNA by using a Nanodrop 2000/2000C spectrophotometer, carrying out reverse transcription on the total RNA into cDNA by using a reverse transcription kit, and carrying out qRT-PCR detection by using specific primers hsa _ circ _0000231 and GAPDH respectively, wherein the upstream primer sequence of hsa _ circ _0000231 is 5'-GACACGCTCATTTGGTCAT-3', and the downstream primer sequence is 5'-CCCACTTCTGTCAGCCATT-3'; the GAPDH upstream primer sequence is 5'-TGACTTCAACAGCGACACCCA-3', and the downstream primer sequence is 5'-CACCCTGTTGCTGTAGCCAAA-3'; by using 2^-△△CTThe method calculates the expression of hsa _ circ _0000231 and GAPDH gene as data normalization reference.

The technical scheme of the invention has the following beneficial effects:

(1) experiments verify that hsa _ circ _0000231 is up-regulated in TSCC tissues, saliva samples and TSCC cell lines and is an independent prognostic factor of TSCC; the highly expressed hsa _ circ _0000231 promotes the proliferation, invasion and metastasis of TSCC cells, the biological behavior may be due to the fact that the biological behavior activates a Wnt/beta-catenin signaling pathway, and hsa _ circ _0000231 is expected to become a TSCC non-invasive clinical diagnosis marker and a clinical treatment target.

(2) The invention provides a kit and a detection method for detecting tongue squamous cell carcinoma, which provide a new possibility for detecting TSCC; meanwhile, the invention also provides application of the small interfering RNA in preparing the drug for treating the tongue squamous cell carcinoma, and provides a therapeutic drug possibility for treating the tongue squamous cell carcinoma.

Drawings

FIG. 1 is a graph of differential expression of hsa _ circ _0000231 verified by qRT-PCR in an embodiment of the invention. Wherein, FIG. 1A is a relative expression diagram of TSCC tissue hsa _ circ _0000231 detected by qRT-PCR; FIG. 1B is a graph of relative expression of hsa _ circ _0000231 in para-cancerous tissue detected by qRT-PCR; FIG. 1C is a graph of the expression levels of hsa _ circ _0000231 in saliva of 10 TSCC patients and 10 healthy controls tested by qRT-PCR; FIG. 1D is a graph of the relative expression of hsa _ circ _0000231 in TSCC cell lines and HOK as detected by qRT-PCR. In the figure, TSCC: squamous cell carcinoma of the tongue; para: tissue adjacent to the cancer; HC: healthy control. P < 0.05.

FIG. 2 is a graph of the expression level of hsa _ circ _0000231 as a function of prognosis of TSCC patient survival in an embodiment of the invention; FIG. 2A is a graph of the relationship between expression of hsa _ circ _0000231 and overall survival of TSCC patients; FIG. 2B is a graph plotting the expression of hsa _ circ _0000231 versus disease-free survival for TSCC patients.

FIG. 3 is a graph of hsa _ circ _0000231 regulating cell proliferation, metastasis, invasion and EMT transformation in an example of the invention; in FIG. 3A, the upper two graphs are the transfection efficiency graphs of qRT-PCR and Tca-8113 detected by an electron fluorescence microscope, respectively, and the lower two graphs are the transfection efficiency graphs of CAL-27 cells detected by qRT-PCR and an electron fluorescence microscope, respectively; FIG. 3B is a three-panel diagram of CCK-8 assay blank, negative control and interfering group cell proliferation potential in sequence; FIG. 3C is a graph of the effect of hsa _ circ _0000231 on the proliferative capacity of Tca-8113 and CAL-27 cells as determined by colony formation experiments; FIG. 3D is a graph of the effect of scratch assay on cell migration of hsa _ circ _ 0000231; FIG. 3E is a graph of the effect of the Transwell assay on the ability of hsa _ circ _0000231 to invade Tca-8113 and CAL-27 cells; FIG. 3F is a diagram showing the effect of Western blot detection of expression of Snail, E-cadherin, N-13 cadherin and vimentin in Tca-8113 and CAL-27 cells by hsa _ circ _ 0000231; in the figure, MOCK is blank control; sh-NC is negative control; sh-circ hsa _ circ _0000231 interference group. P < 0.05.

FIG. 4 is a graph of the inhibition of the growth of subcutaneous nude mouse graft tumors by knocking down the expression of hsa _ circ _0000231 in accordance with an embodiment of the present invention; FIG. 4A is a comparison graph of the gross appearance of the transplanted tumor in nude mice in the interference group and the negative control group; fig. 4B is a graph comparing the volumes of subcutaneous transplantable tumors of the interference group and the negative control group. P < 0.05.

FIG. 5 is a diagram showing the effect of hsa _ circ _0000231 on Wnt/beta-catenin signaling pathway-related proteins in Tca-8113 and CAL-27 cells as verified by Western blot assay in the examples of the present invention; FIG. 5A is the expression diagram of Wnt/beta-catenin related protein of Tca-8113 and CAL-27 cells in the interference group and the negative control group; FIG. 5B is a graph showing the expression of Wnt/beta-catenin-related proteins in Tca-8113 and CAL-27 cells of LiCl-treated and untreated interference groups. In the figure, Control: interfering with the cells of the group; LiCl interference group cells after LiCl treatment.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

Example I, Experimental materials

1. Patient and specimen: 60 pairs of fresh TSCC tissue specimens, corresponding paracancerous tissue specimens, and 10 TSCC saliva specimens were obtained from patients admitted to the southern university hospital affiliated and the southern city tumor hospital from 12 months 2014 to 12 months 2017, and the control group saliva specimens were obtained from healthy volunteers. All tissue specimens were diagnosed by two skilled pathologists, had no history of other tumors, and were not treated with chemotherapy or radiotherapy prior to surgery.

Samples were taken and frozen immediately and stored in liquid nitrogen until total RNA extraction. The study was approved and approved by ethical review committees of affiliated hospitals of southern university and tumor hospitals of southern city, and patients and their families signed informed consent. All animal studies were approved by the university of south china animal experimental ethics committee.

Example II cell lines and Primary reagents

1. HOK was purchased from bio-technologies ltd, unp (shanghai);

2. CAL-27 was purchased from the Kjeldahl gene;

3. tca-8113 and HN-4 are presented by the ninth people hospital in Shanghai;

4. fetal bovine serum, MEM, DMEM and RPMI-1640(Gibco, Waltham, MA);

5. TRIzol reagent (Invitrogen, usa), reverse transcription kit (Thermo scientific), ECL kit (Santa Cruz Biotechnology);

6. small interfering RNA (jecqie, shanghai, china);

7. e-cadherin, GAPDH, Snail, N-cadherin and vimentin (Santa Cruz Biotechnology), Bcl-2 and β -Catenin (CST), MMP-9 and C-myc (abcam), CyclinD1 (Proteintetech).

Example III, Experimental methods

1. Collection of saliva samples and extraction of total RNA: saliva samples were collected from the morning, fasted, alcohol and smoke were removed before collection, and no teeth were brushed, gargled or flossed for teeth within 2 hours. Saliva samples were centrifuged at 3000 Xg for 15 minutes at 4 ℃ and the supernatant collected, the supernatant was centrifuged at 12000 Xg for 10 minutes at 4 ℃ and the supernatant was again stored at-80 ℃ until total RNA was extracted. Total RNA was extracted using TRIzol reagent according to the instructions.

2. Cell culture: HOK cells are subcultured in MEM containing 10% fetal calf serum, CAL-27 and HN-4 are cultured in DMEM medium containing 10% fetal calf serum, Tca-8113 is subcultured in RPMI-1640 medium containing 10% fetal calf serum, and the cells are routinely cultured in an incubator at 37 ℃ and 5% CO2 saturated humidity, and all the cell strains are not subcultured for more than 6 months.

qRT-PCR to detect hsa _ circ _0000231 expression, and analyze its relation to TSCC clinical pathological parameters and prognosis: extracting total RNA by using Trizol reagent, detecting RNA concentration and purity by using a Nanodrop 2000/2000C spectrophotometer, carrying out reverse transcription on the total RNA into cDNA by using a reverse transcription kit, and carrying out qRT-PCR by using specific primers of hsa _ circ _0000231 and GAPDH respectively. hsa _ circ _0000231 upstream primer sequence: 5'-GACACGCTCATTTGGTCAT-3', hsa _ circ _0000231 downstream primer sequence: 5'-CCCACTTCTGTCAGCCATT-3', respectively;

GAPDH downstream primer sequence: 5'-CACCCTGTTGCTGTAGCCAAA-3' are provided.

By using 2^-△△CTThe method calculates the expression of hsa _ circ _0000231 and GAPDH gene as data normalization reference. According to the qRT-PCR result, the average value of the relative expression amount of hsa _ circ _0000231 is used as a boundary, the average value which is equal to or more than the relative expression amount of hsa _ circ _0000231 is defined as 'high expression', the average value which is lower than the relative expression amount of hsa _ circ _0000231 is defined as 'low expression', and the average value is combined with follow-up resourcesMaterial analysis hsa _ circ _0000231 expression correlates with clinical pathology and prognosis of TSCC patients.

4. Cell transfection: small interfering RNA (si-RNA) targeted to the hsa _ circ _0000231 junction with a 5'-CTGAACAGATAA GGGTTTAAA-3' target sequence. The density of the preparation prepared by adding complete medium is 4X 10⁴2ml of cell suspension per ml in 6-well plates. Culturing at 37 deg.C for 24 hr until cell confluency is 20-30%, adding virus and HlitransG P infection enhancing solution (25x), mixing, culturing for 12 hr, and replacing complete culture medium.

And selecting a group of CAL-27 and Tca-8113 cells with the highest transfection efficiency for functional deletion research through qRT-PCR detection and electron fluorescence microscope observation.

CCK-8 experiment: at 2X 10³Each cell/well was inoculated into a 96-well plate, and after 1 day, 2 days, 3 days, 4 days and 5 days of conventional culture, 10. mu.l of CCK-8 reagent was added to each well. After incubation at 37 ℃ for 2 hours at 5% CO2, absorbance values at 450nm were measured using an Infinite M200 microplate reader (Tecan Infinite, Switzerland).

6. Clone formation experiments: the six-well plate was covered with a layer of 0.6% agar, and 0.3% agar containing 103 cells was added to the six-well plate, and cultured at 37 ℃ under 5% CO2 for 2 weeks. The number of colonies per well was fixed, stained with crystal violet, imaged and counted.

7. Scratch test: CAL-27 and Tca-8113 cells at 5X 10⁴Cell concentrations per ml were seeded in 6-well plates. When the cell fusion degree reaches 80%, drawing a straight line by using a 10-microliter pipette tip, and taking a picture after 8 hours.

Transwell invasion assay: pre-cooled serum-free medium was used at a rate of 1: 8, 100. mu.L of matrigel was added to a precooled Transwell chamber and incubated at 37 ℃ for 2h to coagulate the matrigel. The treated cells were resuspended in 200. mu.L serum-free medium in the upper chamber, 600. mu.L complete medium was added to the lower chamber, and after 24 hours of incubation, the cells on the lower side of the membrane were fixed, stained, photographed and counted.

Detecting EMT and Wnt/beta-catenin signal channel related proteins by Western blot: total protein was extracted using protein lysis buffer, 10% SDS-PAGE, gel electrophoresed, transferred to PVDF membrane, blocked with monoclonal antibody overnight at 4 ℃ and the membrane incubated with secondary antibody at room temperature for 2 h. Visualization was performed using enhanced chemiluminescence. E-cadherin, Snail protein, N-cadherin, vimentin (dilution ratio 1: 1000), GAPDH (dilution ratio 1: 1000), beta-catenin (dilution ratio 1: 1000), C-myc (dilution ratio 1: 1000), Bcl-2 (dilution ratio 1: 1000), MMP-9 (dilution ratio 1: 1000), and CyclinD1 (dilution ratio 1: 5000).

10. Nude mice subcutaneous tumor formation experiment: female BALB/C nude mice at 4 weeks of age were sourced from the southern university animal testing center. Two cell lines of CAL-27 and Tca-8113 are selected for experiments, and the subcutaneous injection concentration of the nude mice is 2 multiplied by 10⁷mu.L of si-NC or si-circ cancer cell suspension per ml (6 mice per group), the long diameter (a) and short diameter (b) of the tumor mass were measured once every 4 days, the nude mice were sacrificed after 28 days by terminating the observation, and the tumor volume was calculated by the following formula: volume ═ a × b²)/2。

Example four statistical analysis

Performing data analysis by using SPSS 25.0 statistical analysis software, and performing comparison between groups by using a t test and a c2 test; Kaplan-Meier survival analysis plots Total survival (OS) and Disease Free Survival (DFS) curves; analyzing single clinical pathological parameter and TSCC patient survival time by Log-rank single factor; the multi-factor analysis among clinical pathological parameters adopts a Cox proportional risk regression model, and Pearson correlation test is used for analyzing the pairwise expression correlation. P <0.05 indicates that the difference is statistically significant.

The experimental results are as follows:

one, hsa _ circ _0000231 expression in TSCC and paracarcinoma tissues, saliva samples and cell lines

The qRT-PCR results showed that expression of hsa _ circ _0000231 was significantly higher in TSCC tissue than paired paracancer tissue. Hsa _ circ _0000231 appeared to be highly expressed in TSCC patient saliva relative to healthy controls.

Hsa _ circ _0000231 expression was higher in CAL-27, Tca-8113 and HN-4 cells than in HOK (FIG. 1). In FIG. 1, the data for qRT-PCR is given by 2^-△△CTMethods were performed and the expression of the GAPDH gene was used as a reference for data normalization, data are expressed as mean ± SEM.

Second, the relation between the expression of hsa _ circ _0000231 in TSCC patients and the clinical pathological parameters and prognosis

The qRT-PCR result shows that the number of samples with the relative expression of hsa _ circ _0000231 in TSCC tissue is 36 samples which are higher than the mean value of 1.37, and 24 samples which are lower than the mean value; hsa _ circ _0000231 expression levels were positively correlated with tumor size (p ═ 0.032), T grade (p ═ 0.018), lymph node metastasis (p ═ 0.02) and degree of differentiation (p ═ 0.01), regardless of age and gender (table 1). Among 60 TSCC patients, the 3-year overall survival rate of the hsa _ circ _0000231 low-expression group is 83.34%, and the disease-free survival rate of 77.92% is remarkably higher than the overall survival rate of 36.11% and the disease-free survival rate of 33.33% of the hsa _ circ _0000231 high-expression group. The survival function plotted by Kaplan-Meier survival analysis showed that TSCC patients with high expression of hsa _ circ _0000231 had a poor prognosis (FIG. 2). Log rank one-way analysis showed that the expression of differentiation grade (p ═ 0.008), T grade (p <0.001) and hsa _ circ _0000231 (p <0.001) correlated with TSCC patient prognosis; multivariate analysis showed that hsa _ circ _0000231 expression upregulation (p ═ 0.016) and T-score (p ═ 0.029) were independent prognostic factors for poor prognosis in patients with TSCC (table 2).

TABLE 1 relationship between Hsa _ circ _0000231 expression and clinical pathological index in tongue squamous cell carcinoma tissue (example)

TABLE 2 has _ circ _0000231 and the relationship between the clinical pathological parameters and the prognosis of survival

Third, the effect of hsa _ circ _0000231 expression on TSCC cell proliferation, invasion and epithelial-mesenchymal transition (EMT).

Constructing CAL-27 and Tca-8113 cell models expressed by hsa _ circ _0000231 by siRNA knockdown, detecting the transfection efficiency by qRT-PCR and a fluorescence electron microscope, and selecting the sh-circ-2 line of subsequent experiments with the highest transfection efficiency. CCK8 and clonogenic experiments showed that knocking down hsa _ circ _0000231 expression significantly reduced CAL-27 and Tca-8113 cell proliferation and colony formation ability. The scratch test and the Transwell invasion test show that knocking down hsa _ circ _0000231 expression inhibits the migration and invasion capacity of CAL-27 and Tca-8113 cells. The results of western blot detection of EMT-associated proteins show that knocking down hsa _ circ _0000231 expression can significantly increase the expression of E-cadherin in CAL-27 and Tca-8113 cells and reduce the expression of Snail, N-cadherin and vimentin (FIG. 3).

Fourthly, the expression of hsa _ circ _0000231 is knocked down to obviously inhibit the growth of subcutaneous transplanted tumor of nude mouse

The nude mouse tumor formation experiment shows that the tumor formation rate is 100%, the tumor volume of the subcutaneous transplanted tumor of the hsa _ circ _0000231 interference group is obviously smaller than that of the negative control group, and the growth speed is slower than that of the negative control group (figure 4).

Fifth, the influence of the expression of hsa _ circ _0000231 on the expression of Wnt/beta-catenin signaling pathway related protein in TSCC cells

The expression of CAL-27 and Tca-8113 cells is reduced after the expression of hsa _ circ _0000231 is knocked down, and the expression of beta-catenin, C myc, Bcl-2, MMP-9 and CyclinD1 is reduced. After the cells of the interfering group are treated by 20mM LiCl which is an activator of Wnt/beta-catenin signaling pathway for 24 hours, western blot detects the proteins again, and the results show that the expression of the beta-catenin, C-myc, Bcl-2, MMP-9 and CyclinD1 proteins of the cells after the LiCI treatment is increased compared with the cells of the non-treated interfering group. LiCl reversed the tendency to knock down the expression of Wnt/β -catenin related proteins caused by expression of hsa _ circ _0000231 in TSCC cells (FIG. 5).

The invention adopts real-time fluorescence quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect the expression level of hsa _ circ _0000231 in 60 pairs of fresh paired TSCC tissues, 10 pairs of saliva samples and 3 TSCC cell lines. And analyzing the relation between the relative expression of hsa _ circ _0000231 and the clinical pathological characteristics and prognosis of the patient by combining with follow-up data. Constructing hsa _ circ _0000231 gene interference and lentivirus transfection, realizing the reduction of hsa _ circ _0000231 in TSCC cell lines CAL-27 and Tca-8113, detecting the expression of hsa _ circ _0000231 interference group (si-circ) and idle slow virus group (Negative control group Negative control, NC) cells by qRT-PCR, selecting the cell line CCK-8 experiment with the highest knocking efficiency, clone forming experiment, Transwell invasion experiment and scratch experiment, detecting EMT related protein E-cadherin, Snail protein, N-cadherin and vimentin expression in the TSCC cells of the interference group and the control group by Western blot method, and comparing the proliferation, invasion and transfer capacities of the TSCC cells of the interference group and the control group. The nude mice were injected subcutaneously with TSCC cells from the interfering and control groups and the effect of knockdown of hsa _ circ _0000231 on the growth of subcutaneous transplanted tumors in nude mice was compared. And detecting the expression of Wnt/beta-catenin signal pathway related proteins beta-catenin, C-myc, Bcl-2, MMP-9 and CyclinD1 in an interference group and a control group by using Western blot, and detecting the expression change of the proteins again by using the Western blot method after co-culturing the Wnt/beta-catenin signal pathway activator LiCl and the TSCC cells of the interference group. Results hsa _ circ _0000231 was highly expressed in TSCC patient tissues, saliva samples and cell lines CAL-27, Tca-8113 and HN-4, and was less expressed in paired paracancerous tissues, healthy human saliva samples and normal human oral mucosal cells (HOK). Log-rank one-way analysis showed that the expression level of hsa _ circ _0000231 (p <0.001), degree of tumor differentiation (p ═ 0.008) and T-score (p <0.001) correlated with the prognosis of survival in TSCC patients. Multifactorial Cox risk regression model analysis showed that hsa _ circ _0000231 expression level (p ═ 0.016) and T-score (p ═ 0.029) were independent impact factors for poor prognosis in TSCC patients. Knock-down of hsa _ circ _0000231 reduced the proliferation, invasion and metastatic capacity of CAL-27 and Tca-8113 cells. The growth rate of the subcutaneous transplanted tumor of the interference group is slower than that of the negative control group, and the tumor volume is obviously smaller than that of the negative control group. Western blot experiments show that the expression of proteins, namely beta-catenin, C-myc, Bcl-2, MMP-9 and cyclinD1, related to Wnt/beta-catenin signal pathway of the interfering group cells is reduced compared with that of a control group, and the protein expression reduction trend of the interfering group cells is reversed after the Wnt/beta-catenin pathway activator LiCl is co-cultured with the interfering group TSCC cells. Conclusion hsa _ circ _0000231 was up-regulated in TSCC tissues, saliva samples, and TSCC cell lines, and was an independent prognostic factor for TSCC; the highly expressed hsa _ circ _0000231 promotes the proliferation, invasion and metastasis of TSCC cells, the biological behavior may be due to the fact that the biological behavior activates a Wnt/beta-catenin signaling pathway, and hsa _ circ _0000231 is expected to become a TSCC non-invasive clinical diagnosis marker and a clinical treatment target.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

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Claims

1. A clinical diagnostic marker for squamous cell carcinoma of the tongue, wherein said diagnostic marker is hsa _ circ _ 0000231.

2. The clinical diagnostic marker for tongue squamous cell carcinoma according to claim 1, wherein hsa _ circ _0000231 promotes the proliferation, migration and invasion of tongue squamous cell carcinoma cells by activating Wnt/beta-catenin signaling pathway.

3. Use of knocking down hsa _ circ _0000231 expression for the treatment of squamous cell carcinoma of the tongue.

4. The use of knocking down hsa _ circ _0000231 expression according to claim 3 for the treatment of squamous cell carcinoma of the tongue, comprising the steps of: (1) constructing hsa _ circ _0000231 gene interference and lentivirus transfection to realize the reduction of hsa _ circ _0000231 in TSCC cell lines CAL-27 and Tca-8113; (2) qRT-PCR detection of hsa _ circ _0000231 interference and control cells in hsa _ circ _0000231 expression; (3) and selecting the cells with the highest knocking efficiency to perform a CCK-8 experiment, a clone formation experiment, a Transwell invasion experiment and a scratch experiment, detecting the expression of EMT related proteins E-cadherin, Snail protein and N-cadherin vimentin in the TSCC cells of the interference group and the control group by a Western blot method, and comparing the proliferation, invasion and transfer capacities of the TSCC cells of the interference group and the control group.

5. Use of a small interfering RNA for the manufacture of a medicament for the treatment of squamous cell carcinoma of the tongue, wherein said small interfering RNA targets the hsa _ circ _0000231 junction region and has the target sequence 5'-CTGAACAGATAA GGGTTTAAA-3'.

6. A kit for detecting tongue squamous cell carcinoma is characterized by comprising Trizol reagent for extracting total RNA, reverse transcription reagent, hsa _ circ _0000231 upstream primer, hsa _ circ _0000231 upstream primer, GAPDH upstream primer sequence and GAPDH downstream primer.

7. The kit for detecting squamous cell carcinoma of tongue according to claim 6, wherein the primer sequence upstream of hsa _ circ _ 0000231: 5'-GACACGCTCATTTGGTCAT-3', hsa _ circ _0000231 downstream primer sequence: 5'-CCCACTTCTGTCAGCCATT-3', respectively; GAPDH upstream primer sequence: 5'-TGACTTCAACAGCGACACCCA-3', GAPDH downstream primer sequence: 5'-CACCCTGTTGCTGTAGCCAAA-3' are provided.

8. Use of a kit for detecting squamous cell carcinoma of tongue according to claim 7, characterized in that it comprises the following steps: extracting total RNA by using a Trizol reagent, detecting the concentration and purity of the RNA by using a Nanodrop 2000/2000C spectrophotometer, carrying out reverse transcription on the total RNA into cDNA by using a reverse transcription kit, and carrying out qRT-PCR detection by using specific primers hsa _ circ _0000231 and GAPDH respectively, wherein the upstream primer sequence of hsa _ circ _0000231 is 5'-GACACGCTCATTTGGTCAT-3', and the downstream primer sequence is 5'-CCCACTTCTGTCAGCCATT-3'; the GAPDH upstream primer sequence is 5'-TGACTTCAACAGCGACACCCA-3', and the downstream primer sequence is 5'-CACCCTGTTGCTGTAGCCAAA-3'; by using 2^-△△CTThe method calculates the expression of hsa _ circ _0000231 and GAPDH gene as data normalization reference.