CN111269985A

CN111269985A - Application of hsa _ circRNA6448-14 in diagnosis and prognosis prediction of esophageal squamous cell carcinoma

Info

Publication number: CN111269985A
Application number: CN202010203383.8A
Authority: CN
Inventors: 周福有; 张耀文; 樊锐太; 王立东; 杨海军; 李军扩; 代宁涛; 原翔
Original assignee: Anyang Tumor Hospital
Current assignee: Anyang Tumor Hospital
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-06-12
Anticipated expiration: 2040-03-20
Also published as: CN111269985B

Abstract

The invention belongs to the technical field of medical biology, and particularly discloses a marker for diagnosing and prognostically predicting esophageal squamous cell carcinoma, namely hsa _ circRNA 6448-14. hsa _ circRNA6448-14 is highly expressed in esophageal squamous carcinoma tissues and expressed in paracancerous normal tissues. By detecting the expression level of hsa _ circRNA6448-14 in the tissues, the diagnosis and prognosis prediction of esophageal squamous carcinoma can be carried out. The invention provides a new way for diagnosis and prognosis prediction of esophageal squamous cell carcinoma, and provides a reference basis for diagnosis and disease analysis of esophageal squamous cell carcinoma by clinicians.

Description

Application of hsa _ circRNA6448-14 in diagnosis and prognosis prediction of esophageal squamous cell carcinoma

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to application of hsa _ circRNA6448-14 in diagnosis and prognosis prediction of esophageal squamous cell carcinoma.

Background

Esophageal squamous carcinoma (ESCC), also known as Esophageal squamous cell carcinoma, is a malignant tumor occurring in human Esophageal epithelial tissue, is one of six malignant tumors worldwide, about 50 ten thousand Esophageal cancer patients are newly added worldwide each year, more than half of them occur in China, and the incidence rate is 100 times higher than that in western countries. The national cancer center 2018 shows the latest national cancer report that in 2014, the number of new cases of esophageal cancer in China is 18.85/10 ten thousand, the number of new cases of esophageal cancer is the sixth in malignant tumor, and the number of cases of esophageal cancer death is 14.11/10 ten thousand, the number of new cases of esophageal cancer is the fourth in malignant tumor. At present, diagnosis and prognosis prediction of esophageal cancer face three major challenges: late clinical symptoms, uncomfortable and costly endoscopy, insensitivity and non-specificity of biomarkers. Therefore, diagnosis and prognosis of ESCC urgently requires new highly sensitive, cost-effective biomarkers.

The circRNA (circular RNA) is an endogenous non-coding RNA which has good conservative property and high specificity and can play a role in regulating and controlling a target gene. Compared with other RNAs such as miRNA and lncRNA, the circRNA is more suitable as a potential cancer biomarker. First, circRNA has a covalently closed loop structure, lacking a free end that is resistant to RNase R, and therefore has greater stability than mRNA or linear ncRNA; second, they are usually expressed by specific cell types or in specific pathological conditions, with tissue and developmental stage specificity; third, their abundant presence in various tissues and body fluids, including blood, plasma, serum, and even in exosomes, makes them potential candidates for fluid biopsy biomarkers. At present, various circRNAs are proved to be biomarkers for diagnosis and prognosis of malignant tumors, including prostate cancer, liver cancer, gastric cancer, lung cancer and the like. However, the mechanism of action of circRNA in esophageal squamous carcinoma is not clear, and the use of circRNA as a biomarker for ESCC has been poorly explored. Furthermore, the expression profiles of circRNA for ESCC were not identical due to the non-uniform detection platforms. Therefore, the construction of the circRNA expression profile of ESCC in high incidence areas of China and the discussion of the functions and mechanisms of key circRNA in ESCC have important significance for the diagnosis and clinic of esophageal cancer. In the research, a micro-array analysis is adopted to obtain a circRNA expression profile of esophageal cancer in a high-incidence area in China, and then key circRNA related to the occurrence and development of esophageal cancer is found from the circRNA expression profile so as to discover a potential circRNA marker of esophageal cancer in the high-incidence area.

Disclosure of Invention

The invention aims to provide circRNA which can be used for diagnosing and prognostically predicting esophageal squamous cell carcinoma.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention firstly provides the application of the quantitative detection reagent of hsa _ circRNA6448-14 in the preparation of products for predicting esophageal squamous cell carcinoma prognosis. Wherein the nucleotide sequence of the hsa _ circRNA6448-14 is shown as SEQ ID NO. 3.

According to the above applications, preferably, the product detects the expression level of hsa _ circRNA6448-14 in a sample by real-time quantitative PCR, in situ hybridization, a chip or a high-throughput sequencing platform.

Further, the product contains a probe that hybridizes to hsa _ circRNA6448-14 or a primer that specifically amplifies hsa _ circRNA 6448-14.

Further, the product for detecting the expression level of hsa _ circRNA6448-14 in a sample by real-time quantitative PCR comprises a pair of primers for specifically amplifying hsa _ circRNA 6448-14.

Further, a product for detecting the expression level of hsa _ circRNA6448-14 in a sample by in situ hybridization comprises a probe that hybridizes to the nucleotide sequence of hsa _ circRNA 6448-14.

Further, a product for detecting the expression level of hsa _ circRNA6448-14 in a sample by a chip comprises a probe that hybridizes to the nucleotide sequence of hsa _ circRNA 6448-14.

Further, the primer sequences for specifically amplifying hsa _ circRNA6448-14 are shown as SEQ ID NO.1 and SEQ ID NO. 2.

Further, the product includes (but is not limited to) a chip, a preparation or a kit. Further, the chip includes 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 the expression level of hsa _ circRNA 6448-14. The gene detection kit comprises a specific primer for detecting the expression level of hsa _ circRNA 6448-14.

The invention also provides a product for predicting the prognosis of esophageal squamous cell carcinoma, which comprises a reagent for detecting the expression level of hsa _ circRNA6448-14 in a sample.

Further, the reagents include reagents for detecting the expression level of hsa _ circRNA6448-14 by real-time quantitative PCR, in situ hybridization, a chip, or a high throughput sequencing platform.

The invention also provides application of the quantitative detection reagent of hsa _ circRNA6448-14 in preparing an esophageal squamous cell carcinoma diagnosis product.

Further, the product detects the expression level of hsa _ circRNA6448-14 in a sample by real-time quantitative PCR, in situ hybridization, a chip or a high-throughput sequencing platform.

In the present invention, 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. In the present invention, the term "primer" refers to a nucleic acid sequence having a free 3' hydroxyl group which is capable of binding complementarily to a template and enabling reverse transcriptase or DNA polymerase to initiate template replication. Primers are nucleotides having a sequence complementary to a nucleic acid sequence of a specific gene. In the present invention, the term "prognosis" refers to the prediction of the course and outcome of disease progression, such as the likelihood of long-term survival, disease-free survival, etc.

Compared with the prior art, the invention has the following positive beneficial effects:

(1) the invention discovers for the first time that hsa _ circRNA6448-14 is differentially expressed in cancer tissues and paracancer normal tissues of esophageal squamous carcinoma patients, and the expression level of hsa _ circRNA6448-14 in ESCC cancer tissues is remarkably increased (p is 0.003) compared with that of paracancer normal tissues, and ROC curve analysis shows that hsa _ circRNA6448-14 can distinguish esophageal squamous carcinoma tissues from paracancer normal tissues by the product 0.906 under the ROC curve (95% CI:0.899-0.903, p is 0.021), the detection sensitivity is 82.9%, and the specificity is 85.5%; therefore, the hsa _ circRNA6448-14 can be used as a biological marker for diagnosing the esophageal squamous cell carcinoma patients and is used for assisting the clinical diagnosis of the esophageal squamous cell carcinoma.

(2) The invention discovers that the expression of hsa _ circRNA6448-14 is related to the prognosis of an esophageal squamous cell carcinoma patient, the Disease-free Survival (DFS) and the Overall Survival (OS) of the esophageal squamous cell carcinoma patient with high expression of hsa _ circRNA6448-14 are poorer, and the high expression of hsa _ circRNA6448-14 is related to the poor prognosis of the esophageal squamous cell carcinoma, so that the prognosis of the esophageal squamous cell carcinoma patient can be predicted by detecting the expression level of hsa _ circRNA6448-14, thereby providing a new way for judging the prognosis prediction of the esophageal squamous cell carcinoma and providing a reference basis for a clinician to analyze the condition of the esophageal squamous cell carcinoma.

Drawings

FIG. 1 is a diagram showing the results of circRNA expression analysis of a whole sample of esophageal squamous carcinoma; wherein, A: cluster analysis graph, B: correlation analysis graph, C: boxline graph, D: principal component analysis chart.

FIG. 2 is a diagram showing the results of differential expression analysis of cirRNA of esophageal squamous cell carcinoma; wherein, A: cluster analysis graph, B: scatter plot, C: volcanic chart, D: circle diagram, E: circRNA chromosome profile, F: circular RNA source map.

FIG. 3 is a scatter plot of the results of the differential expression of 5 differentially expressed circRNAs in esophageal squamous carcinoma tissue and paracarcinoma normal tissue by qRT-PCR (normal in the figure, cancer is paracarcinoma normal tissue, cancer is ESCC tissue;. represents p <0.05, and. represents p < 0.01).

FIG. 4 is a graph showing the results of ROC curve analysis of 26 pairs of hsa _ circRNA6448-14 and hsa _ circRNA6448-14 in a sample.

FIG. 5 is a graph showing the results of differential expression of hsa _ circRNA6448-14 in 50 pairs of esophageal squamous carcinoma tissues and paracarcinoma tissues and ROC curve analysis.

FIG. 6 is a graph of ROC curve analysis of differential expression of hsa _ circRNA6448-14 in 76 versus esophageal squamous carcinoma tissue and paracancerous tissue.

FIG. 7 is a Kaplan-meier plot of 76 cases of ESCC post-operative OS; wherein, A: family history of esophageal cancer, B: differentiation, C: pT stage, D: pN staging, E: pTNM staging, F: hsa _ circRNA6448-14 expression.

FIG. 8 is a Kaplan-meier plot of post-operative DFS for 76 ESCC patients, where A: family history of esophageal cancer, B: differentiation, C: pT stage, D: pN staging, E: pTNM staging, F: hsa _ circRNA6448-14 expression.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.

Example 1: construction of circRNA expression profiles and screening for differentially expressed circRNAs

1. Sample source and sample processing

6 patients who receive esophageal cancer radical treatment in thoracic surgery of tumor hospital in Anyang city from 2015 6/month 4 to 2016 4/month 5 are selected and diagnosed as esophageal squamous carcinoma through histopathology. Wherein 3 cases of men and 3 cases of women are aged 45-77 years, and the median age is 61 years. All patients did not receive any radiation, chemotherapy, targeting or other anti-tumor treatment prior to surgery. Collecting ESCC tissue and paired paracancer normal tissue specimens respectively during operation, and storing in an ultra-low temperature refrigerator at-80 deg.C for later use. The matched paracancer normal tissues are taken from the position which is more than 5cm away from the edge of the esophageal cancer tissue, no obvious abnormality is seen by naked eyes, and the paracancer normal tissues are determined to be normal tissues through pathological detection and have no atypical hyperplasia and cancer cell infiltration. Grouping experiments: cancer (ESCC tissue), Non-Ca (paired paracancerous normal tissue).

The study was approved by ethical committee of tumor hospital in Anyang to obtain informed consent of patients and family members, and signed written informed consent.

2. Experimental methods

2.1 extraction and quality control of Total RNA

(1) Extracting total RNA:

the tissue pieces were minced with ophthalmic scissors and ground with a small amount of liquid nitrogen, according to the Invitrogen trizol kit instructions, until the samples were ground to a uniform white powder. Adding Trizol lysis solution into 50-100mg tissue/ml Trizol, and homogenizing for about 1-2 min. 200 μ L of chloroform was added thereto, followed by shaking rapidly for 15 seconds and standing at room temperature for 5 min. Centrifuging at 12000rpm for 15min at 4 deg.C, wherein the liquid in the centrifuge tube is divided into three layers, the RNA is mainly in the upper colorless solution (water phase), and transferring the upper colorless solution to a new 1.5ml EP tube (note: this step is critical, and ten million cannot be absorbed into the middle layer to avoid RNA contamination). Adding equal volume of isopropanol, mixing, and standing at room temperature for 10 min. Centrifuging at 12000rpm for 15min at 4 deg.C, discarding supernatant, and precipitating at the bottom of the tube to obtain RNA. 1ml of 75% ethanol was added, gently shaken and the precipitate was suspended. Centrifuge at 7500rpm for 5min at 4 deg.C, discard the supernatant and repeat one wash (note: after centrifugation, ethanol was sucked as clean as possible, but not to the bottom white precipitate). The precipitate is dried in the air at room temperature for 5-10 min (note: RNA is not dried too much, otherwise it is difficult to dissolve). Dissolving the RNA precipitate with proper amount of pure DEPC water, and storing at-80 deg.C.

(2) And (3) controlling the total RNA quality:

the RNA purity and concentration were measured by a NanoDrop ND-1000 spectrophotometer, the RNA concentration and Optical Density (OD) values at 260nm to 280nm were automatically generated, OD260/280 between 1.80 to 2.00 indicates high RNA purity, and the RNA concentration was recorded. The integrity of the RNA samples was tested using the Agilent 2100 bioanalyzer system RNArano 6000Assay Kit.

2.2 construction of circRNA expression profiles

(1) Reverse transcription to synthesize First Strand cDNA:

the total RNA was taken at 200 ng and 500ng, and the corresponding volume of Spike-in was added according to the following table. A single reaction system reverse transcription Master Mix was prepared as follows: mu.L of First Strand Enzyme Mix 1. mu.L, First Strand Buffer Mix 4. mu.L, total 5. mu.L, were mixed well and centrifuged and then ice-washed. mu.L Master Mix was transferred to a 0.2ml EP tube containing the Total RNA sample, mixed and centrifuged, 2h at 42 ℃ and ice-washed.

(2) Synthesis of Second Strand cDNA:

a single reaction system, Second Strand Master Mix, was formulated as follows: mu.L of nucleic-free Water 13. mu.L, Second Strand Enzyme Mix 2. mu.L, Second Strand Buffer Mix 5. mu.L, 20. mu.L in total, were mixed and centrifuged and then iced. Add 20. mu.L of Second Strand Master Mix to each sample tube in (1) and Mix well at 16 ℃ for 1h, 65 ℃ for 10min, ice bath.

(3) In vitro transcription synthesis of cRNA:

in vitro transcription Master Mix was prepared (Nuclear-free Water 4. mu.L, Nuclear-free Water 20. mu.L, T7 Enzyme Mix 6. mu.L, 30. mu.L total), mixed and centrifuged. And (3) adding 30 mu L of in-vitro transcription MasterMix into each sample tube in the step (2), uniformly mixing, standing at 40 ℃ for 8-14 h, and purifying cRNA through a column.

(4) Reverse transcription of cRNA:

taking 5 mu g of a cRNA purified product, adjusting the volume to 7.5 mu L, adding 4 mu L of Random Primer, mixing uniformly, reacting at 65 ℃ for 5min, and carrying out ice bath for 5 min. The cRNA was reverse transcribed to Master Mix (4. mu.L of CbcScript II Buffer, 5. mu.L of CbcScript II, 1.5. mu.L of CbcScript II, 2. mu.L of 0.1M DTT, total 8.5. mu.L), mixed and centrifuged. Adding cRNA reverse transcription Master Mix 8.5. mu.L, mixing, 25 deg.C 10min, 37 deg.C 1.5 h. Adding 5 μ L of terminator Solution, mixing, and standing at 65 deg.C for 10min and room temperature for 5 min. Add 1. mu.L of the Solution and mix well. Push button

Extract II kit (MACHEREY-NAGEL Germany) was purified and quantified by UV spectrophotometer.

(5) Fluorescence labeling:

and (3) concentrating the reverse transcription product of the cRNA prepared in the step (4) to 14 mu L, adding 4 mu L Random Primer, mixing uniformly, centrifuging, carrying out 3min at 95 ℃, and carrying out ice bath for 5 min. Adding 4 XKlenow Buffer 5 μ L, Cy3-dCTP1 μ L, Cy3-dCTP 1.2 μ L sequentially, beating for 2-3 times, mixing, centrifuging for a short time, reacting at 37 deg.C for 1.5h, and reacting at 70 deg.C for 5 min. The labeled product is purified and quantified, and the DNA with the fluorescent group can be used for chip hybridization.

(6) Chip hybridization:

hybridization mix (hybridization mix) was prepared in the following volumes, reacted at 95 ℃ for 3min, and ice-cooled for 2 min. Add 45. mu.L (or 105. mu.L) of hybridization solution to the hybridization cap, place the circRNA chip, screw the hybridization device, place in the hybridization oven overnight for hybridization, approximately 16 h.

(7) Chip cleaning and scanning:

after the hybridization of the chip, the chip was washed with 0.2% SDS-2 XSSC in wash solution I at about 42 ℃ for 5min and then with 0.2 XSSC in wash solution II at room temperature for 5 min. Scanning by an Agilent chip scanner to obtain tiff format hybridization pictures.

(8) Pretreatment and analysis of a whole sample:

scanning the tiff picture by adopting an Agilent Feature Extraction (v10.7) software processing chip to obtain an original data file (.txt), importing the original data file into GeneSpring software to perform data normalization and annotation analysis, performing Cluster analysis by Cluster 3.0 software, and performing visual graphic output by treeview software.

(9) Differential comparison analysis and screening of circrnas:

the screening standard of the circRNA with obvious difference is that the difference multiple FC (abs) is more than or equal to 2.0, and p is less than 0.05. A larger FC value indicates a larger difference between the two groups; a smaller p indicates a higher reliability of the differential gene.

3. Results of the experiment

3.1 Whole sample cirRNA expression analysis

And 6, carrying out microarray analysis on the ESCC tissues and paracancerous normal tissues to detect 149789 circRNAs in a total way and recording the specific expression condition of the CircRNAs. The visualized graphs show the expression of the ESCC full-sample cirRNA, wherein Ca-973 to Ca-917 represent 6 ESCC tissue specimens, and Con-973 to Con-917 represent 6 matched paracancer normal tissues.

Clustering analysis showed that there was a large amount of circRNAs expressed between samples in Ca-group and Non-Ca group, consistent with the expected grouping (FIG. 1A). Correlation analysis showed high correlation between the tissue samples selected in this experiment, with expression similarity (FIG. 1B). The boxplot shows that the intensity of the fluorescence signal is basically consistent after the circRNA expression profile is normalized, which indicates that the expression level of the overall genes of the two groups of samples tends to be uniform and comparable (FIG. 1C). PCA 3D showed relative clustering of the corresponding spots for both sets of samples, indicating better sample similarity within each set (fig. 1D).

3.2 CirRNA differential expression analysis

(1) Screening for differentially expressed circRNA: the FC is more than or equal to 2.0, p is less than 0.05, as the screening condition of the gene for differentially expressing the circRNA, the result shows that the expression difference of the circRNA of an ESCC tissue group and a paracancer normal tissue group is obvious, 15908 circRNAs are detected to be abnormally expressed in the ESCC tissue, wherein the total number of the up-regulated circRNAs is 7161 (45.01%), and the total number of the down-regulated circRNAs is 8747 (54.98%).

(2) And (3) visual graphic display: FIG. 2 shows the significant difference in the expression of circRNA between ESCC tissue and paracancer normal tissue in different graphs, Ca-for ESCC tissue and Con-for paracancer normal tissue.

① hierarchical clustering analysis chart (FIG. 2A) in which red is up-regulated differential circRNA and green is down-regulated differential circRNA, each column represents the circRNA expression profile of a tissue sample, and each row corresponds to a circRNA, the results show that there are significant differences in the circRNA expression profiles of different tissue samples.

② scatter diagram (FIG. 2B) shows that the abscissa is mean expression value of Con-group sample of para-carcinoma tissue, the ordinate is mean expression value of ESCC group sample, both are values after log2 conversion, black represents circRNA without significant difference, scatter diagram outside black shows that the circRNA expression level FC in two groups is more than or equal to 2.0, p is less than 0.05, red is significantly up-regulated circRNA, green is significantly down-regulated circRNA, and the result shows that the circRNA expression difference in ESCC tissue is significant.

③ volcano plot (FIG. 2C) with abscissa of-log 10(p value) and ordinate of log2(FC value), black indicating no significant difference in circRNA, black outside indicating FC > 2.0, p <0.05 difference in circRNA, red indicating significant up-regulation of circRNA and green indicating significant down-regulation of circRNA, the closer the points are to the top left and right, the more significant the difference, the results show significant deregulation of circRNA in ESCC tissue.

④ circled graph (FIG. 2D) showing that red is up-regulated differential circRNA and green is down-regulated differential circRNA, the length of the column indicates the fold of the differential circRNA gene, the longer the column indicates the greater the fold of the circRNA difference, and the results indicate that the circRNA difference in ESCC tissues is significant.

⑤ the distribution of circRNA expressed differentially on human chromosome shows that most of the circRNA is distributed on chr1, chr2, chr3, chr5, chr7 and chr17, the circRNA expressed differentially on chromosome 1 is most, and the circRNA is rarely distributed on chr21, chr22, chrX or chrY, and the detail is shown in FIG. 2E.

⑥ statistics on the source classes revealed that most of the differentially expressed circRNAs were exon-derived, 13366 of the differentially total circRNAs were exon-derived, accounting for 84.02% (13366/15908), 88.41% (6331/7161) for exon-origin up-regulated circRNAs, and 80.43% (7035/8747) for exon-origin down-regulated circRNAs, as shown in FIG. 2F.

Example 2: screening 5 kinds of differential expression circRNAs and finding out key circRNAs

From the 15908 circrnas significantly differentially expressed in example 1, 5 circrnas were selected based on fold difference, p-value and signal value, and were: hsa _ circRNA6448-14, hsa _ circ _0110255, hsa _ circRNA15930-8, hsa _ circ _0064369 and hsa _ circ _0024108, 3 up-regulated and 2 down-regulated, all of which are exon-derived, and the detailed biological information is given in Table 1. The expression of 5 circRNAs in ESCC tissues and para-cancer tissues is verified by 26 qRT-PCR experiments in the first step, and key circRNAs are found out; and then the expression of key circRNAs in ESCC tissues and para-cancer tissues is verified by 50 steps of qRT-PCR experiments.

Table 1 screening of 5 differentially expressed circRNAs in ESCC

Name (R)

P value

FC value

Trend of the

Chr

Origin of origin

Gene abbreviation

hsa-circRNA6448-14

0.002686278

23.9508689

up

chr5

exon

TGFBI

hsa_circ_0110255

7.52115E-05

195.346854

up

chr1

exon

COL11A1

hsa-circRNA15930-8

8.81852E-05

16.81513982

down

chr9

exon

SH2D3C

hsa_circ_0064369

1.63138E-05

8.445969538

down

chr3

exon

TMEM40

hsa_circ_0024108

3.25051E-05

330.2744199

up

chr11

exon

MMP1

1. Sample source and sample processing

76 patients who receive esophageal cancer radical surgery in thoracic surgery of tumor hospital of Anyang city from 2015 6/month 4 to 2016 4/month 5 are selected, wherein 46 men and 30 women are selected, and the patients are aged 55-74 years (median age 61), and general conditions and clinical data of 76 patients are collected. Referring to International Cancer Control (UICC) of 2017 and American Joint Committee on Cancer (AJCC) 8 th edition esophageal Cancer TNM staging standard, 6 cases in the upper chest, 53 cases in the middle chest and 17 cases in the lower chest of 76 patients; stage pT1, stage pT2, stage pT3 and stage pT 43; 40 cases in the pN0 stage, 26 cases in the pN1 stage and 10 cases in the pN3 stage; pathological stages I-II and III-IV 33 cases. Of the 76 patient samples, 26 were used for the first qRT-PCR validation and 50 were used for the second qRT-PCR validation.

None of the 76 patients received any radiation, chemotherapy, targeting or other anti-tumor treatment prior to surgery. Collecting ESCC tissue and paired paracancer normal tissue specimens respectively during operation, and storing in an ultra-low temperature refrigerator at-80 deg.C for later use. The matched paracancer normal tissues are taken from the position which is more than 5cm away from the edge of the esophageal cancer tissue, no obvious abnormality is seen by naked eyes, and the paracancer normal tissues are determined to be normal tissues through pathological detection and have no atypical hyperplasia and cancer cell infiltration. Grouping experiments: cancer (ESCC tissue), Non-Ca (paired paracancerous normal tissue).

2. qRT-PCR experiment verifies 26 expression of 5 circRNAs in ESCC tissue and para-cancer tissue

2.1 Experimental methods

2.1.1 Total RNA extraction

The specific operation of total RNA extraction is the same as that of 2.1 in example 1, and is not described herein again. The extracted total RNA is then reverse transcribed.

2.1.2 reverse transcription

A12. mu.L system of Mix I (Primer (10. mu.M) 1.0. mu.L, dNTP Mix (10mM) 1.0. mu.L, DEPC-treated water 10. mu.L) was prepared. Mix I was incubated at 65 ℃ for 5min, ice-washed for 1min, and then 5 XFirst-Strand buffer 4.0. mu.L, 0.1M dTT 2.0. mu.L, RNaseout 40U/. mu.L 1.0. mu.L, SuperScript III RT (200U/. mu.L) 1.0. mu.L were added in this order to prepare Mix II, which amounted to 20. mu.L. Treating Mix II at 25 deg.C for 5min, 42 deg.C for 60min, and 70 deg.C for 15min to inactivate enzyme, and immediately placing on ice for use or storing in a refrigerator at-20 deg.C.

2.1.3 qRT-PCR

① A reaction solution was prepared in the following manner.

② qRT-PCR reaction conditions:

③ amplification primers were as follows:

④ calculating the relative expression quantity of circRNA, repeating all quantitative PCR for 3 times, observing the amplification curve, analyzing whether the melting curve is a single peak, determining whether the primer is specific, extracting the Ct value of the sample, and calculating the expression level of the circRNA in each tissue sample by using a 2-delta Ct (delta Ct ═ Ct target gene-Ct internal reference gene, delta Ct ═ delta Ct cancer tissue-delta Ct cancer tissue) formula.

2.2 results of the experiment

Differential analysis of 2.2.15 circRNAs at 26 ESCC samples

The results of the qRT-PCR validation of the 26 pairs of samples are shown in table 2 and figure 3. As can be seen from Table 2 and FIG. 3, compared to paracancerous normal tissue, hsa-circRNA6448-14 expression was significantly up-regulated in esophageal squamous carcinoma tissue (p <0.01), hsa-circRNA15930-8 expression was significantly down-regulated in esophageal squamous carcinoma tissue (p <0.05), and the trends of change were not significant for hsa _ circ _0110255, hsa _ circ _0064369, and hsa _ circ _0024108 (p > 0.05).

Expression of 25 circRNAs in 26 pairs of esophageal squamous carcinoma samples

2.2.2 ROC Curve analysis of 5 circRNAs

ROC curves were prepared from the qRT-PCR quantitative results of hsa-circRNA6448-14 and hsa-circRNA15930-8 in the esophageal squamous cell carcinoma tissues and paracarcinoma normal tissues of 26 patients with esophageal squamous cell carcinoma, and the sensitivity and specificity were evaluated by the ROC curves, and the results are shown in Table 3 and FIG. 4. As can be seen from Table 3 and FIG. 4, the AUC of hsa _ circRNA6448-14 and hsa _ circRNA15930-8 were 0.846 (95% CI: 0.738-0.954, p ═ 0.045) and 0.673 (95% CI: 0.519-0.828, p ═ 0.032), respectively, indicating that hsa _ circRNA6448-14 and hsa _ circRNA15930-8 can distinguish esophageal squamous cell carcinoma tissue from paracarcinoma normal tissue. The sensitivity and specificity of Hsa _ circRNA6448-14 and Hsa _ circRNA15930-8 were 80.8% and 77%, respectively; 73.1% and 50%; compared with hsa _ circRNA15930-8, hsa _ circRNA6448-14 has stronger specificity and higher sensitivity for diagnosing esophageal squamous cell carcinoma, so hsa _ circRNA6448-14 is selected for the next step of qRT-PCR verification of a larger sample.

Table 326 sensitivity and specificity for hsa _ circRNA6448-14 and hsa _ circRNA15930-8 in samples

Name of circRNA	AUC	95％CI	P value	Sensitivity of the composition	Specificity of
						hsa_circRNA6448-14	0.846	0.738～0.954	0.045	80.8％	77％
hsa_circRNA15930-8	0.673	0.519～0.828	0.032	73.1％	50％

3. qRT-PCR validation 50 pairs of expression of hsa _ circRNA6448-14 in ESCC and para-carcinoma tissues

3.1 Experimental methods

The qRT-PCR verification method is the same as that of 2.1 in the embodiment, and is not described herein again.

3.2 results of the experiment

3.2.1 differential analysis of hsa _ circRNA6448-14 on ESCC samples at 50

The results of the qRT-PCR validation of 50 pairs of samples are shown as a in figure 5. As can be seen from a in fig. 5, expression of hsa _ circRNA6448-14 in ESCC tissue was significantly higher than that in paracancerous normal tissue, with a significant up-regulation trend (p ═ 0.003); the results were consistent with the chip analysis and the verification results of 26 samples.

3.2.2 ROC Curve analysis of hsa-circRNA6448-14

Based on the results of qRT-PCR quantification of hsa-circRNA6448-14 in the esophageal squamous cell carcinoma tissue and paracarcinoma normal tissue of 50 patients with esophageal squamous cell carcinoma, ROC curves were prepared, and sensitivity and specificity were evaluated by the ROC curves, as shown in B in FIG. 5. As shown in FIG. 5B, the AUC of hsa _ circRNA6448-14 was 0.941 (95% CI: 0.893-0.990, p ═ 0.025), and sensitivity and specificity were 84.6% and 86.0%, respectively.

Based on the results of qRT-PCR quantification of hsa-circRNA6448-14 in the esophageal squamous cell carcinoma tissue and paracarcinoma normal tissue of 76 cases of esophageal squamous cell carcinoma patients, ROC curves were prepared, and sensitivity and specificity were evaluated by the ROC curves, as shown in FIG. 6. As can be seen in FIG. 6, the AUC of hsa _ circRNA6448-14 was 0.906 (95% CI:0.899-0.903, p ═ 0.021), and the sensitivity and specificity were 82.9% and 85.5%, respectively.

The results show that the sensitivity and specificity of hsa _ circRNA6448-14 for distinguishing esophageal squamous cell carcinoma tissues from paracancerous normal tissues are high, and the hsa _ circRNA6448-14 is expected to be a potential target for diagnosis and treatment of esophageal squamous cell carcinoma patients.

Example 3: application of hsa _ circRNA6448-14 in prognosis prediction of esophageal squamous cell carcinoma

To clarify the clinical significance of hsa _ circRNA6448-14 expression in esophageal squamous carcinoma, the 76 patient samples collected in example 2 were subjected to clinical data statistics and postoperative follow-up statistics, and the 76 patient samples were analyzed for the relationship between hsa _ circRNA6448-14 expression and ESCC clinical pathological characteristics and post-operative DFS (Disease-free Survival) and OS (Overall Survival) according to the statistical results.

1. Follow-up and statistical analysis

Performing neck, chest and abdomen CT and upper abdomen and double neck color ultrasound for 1 time every 3 months in 2 years after operation, and performing gastroscope and PET-CT examination if necessary; rechecking every half year for 1 time within 2 to 5 years; after 5 years, the patient is reviewed every 1 year, and if bone metastasis is suspected, the patient is examined by ECT or MRI, and if brain metastasis is suspected, the patient is examined by MRI or CT for the head. Follow-up visits are carried out by means of outpatient reexamination, telephone follow-up visits, home visits, letters, public security information and the like, and the follow-up visits are carried out by 12 months and 25 days in 2019. OS is defined as the time from the beginning of the surgery to the last follow-up or death. The disease-free survival DFS is defined as the time from the beginning of surgery to the appearance of metastasis or recurrence. Death was used as the endpoint event, loss of visit and survival as tail-biting data. The clinical data statistics and postoperative follow-up information statistics of 76 patient samples are shown in table 4.

Analyzing the relation between hsa _ circRNA6448-1 expression and clinical pathological characteristics in ESCC tissues by using a chi-square test or an exact probability method (Fisher test); calculating OS and DFS by a Kaplan-Meier method, testing by a Log-rank method and drawing a survival curve by single factor analysis; the multi-factor analysis affecting post-operative OS and DFS employs a Cox regression model. Analysis was performed using SPSS 26.0(spssinc., Chicago, IL, USA) and Graphpad Prism v8.0(Graphpad Software inc., San Diego, CA, USA) statistical Software, with p <0.05 as the difference being statistically significant.

Clinical data statistics for Table 476 patients with esophageal squamous carcinoma

2. Analysis of the relationship between hsa _ circRNA6448-14 expression and clinical pathological characteristics in cancer tissues of 76 patients with esophageal squamous carcinoma

The correlation between hsa _ circRNA6448-14 expression and clinical pathology in cancer tissues from 76 patients with esophageal squamous carcinoma was analyzed using the chi-square test, and the results are shown in Table 5.

TABLE 576 relation between Hsa _ circRNA6448-14 expression and clinical pathological characteristics of patients with esophageal squamous carcinoma

As can be seen from Table 5, expression of hsa _ circRNA6448-14 was positively correlated with differentiation and pTNM staging (both p <0.05) in 76 ESCC patients, i.e., high expression of hsa _ circRNA6448-14 was observed in patients with low differentiation and late pTNM staging.

3. Single factor analysis of prognosis after operation in 76 ESCC patients

Single factor analysis of OS after surgery in 3.176 ESCC patients

The results of single factor analysis of the factors related to OS after surgery on 76 patients with esophageal squamous cell carcinoma using Kaplan-Meier method and Log-rank test are shown in FIG. 7. As can be seen from fig. 7, OS after ESCC surgery is related to esophageal cancer family history, degree of differentiation, pT stage, pN stage, pTNM stage, and hsa _ circRNA6448-14 expression, and the differences have statistical significance (p ═ 0.036, p ═ 0.023, p ═ 0.001, p ═ 0.026, p < 0.001); that is, patients with family history of esophageal cancer, low differentiation degree, deep infiltration depth, late pathological stage and high hsa _ circRNA6448-14 expression have short OS and poor survival prognosis.

3.276 patients with ESCC postoperative DFS one-factor analysis

The results of single-factor analysis of the factors related to DFS after surgery on 76 patients with esophageal squamous cell carcinoma by Kaplan-Meier method and Log-rank test are shown in FIG. 8. As can be seen in fig. 8, post-ESCC DFS is associated with pT staging and hsa _ circRNA6448-14 expression, with statistical differences (p ═ 0.024, p ═ 0.006); i.e., patients with deep tumor infiltration and high expression of hsa _ circRNA6448-14, have short DFS and are prone to local recurrence or distant metastasis.

4. Multi-factor analysis of prognosis after surgery in 76 ESCC patients

Multi-factor analysis of OS after surgery in 4.176 ESCC patients

One-way analysis of indicators associated with prognosis of postoperative OS survival: the 6 esophageal cancer family history, degree of differentiation, pT stage, pN stage, pTNM stage and hsa _ circRNA6448-14 expression were analyzed in a Cox proportional risk model, and the possibility of using the esophageal cancer family history, degree of differentiation, pT stage, pN stage, pTNM stage and hsa _ circRNA6448-14 expression as independent postoperative OS prognosis prediction indicators for patients with esophageal squamous cell carcinoma was evaluated, with the results shown in Table 6 (B in Table 6 is a covariate coefficient, and exp (B) is relative risk).

TABLE 676 results of Cox multifactorial analysis of OS after surgery in ESCC patients

As can be seen from table 6, esophageal cancer family history, pT staging and hsa _ circRNA6448-14 were expressed as independent prognostic factors for ESCC postoperative OS (p 0.033, p 0.007, p 0.003), and HR values were 2.149 (95% CI 1.062. 347), 2.516 (95% CI 1.280. 4.947) and 0.199 (95% CI 0.069. 0.573), respectively. The mortality risk of patients with family history of esophageal cancer is 2.149 times higher than that of patients without family history of esophageal cancer, suggesting that family history of esophageal cancer is an adverse factor. With each grade increase in pT staging, the patient's risk of mortality increased 2.516-fold, suggesting that late pT staging is an adverse factor. The mortality risk of patients with low expression of Hsa _ circRNA6448-14 was 0.199 times higher than that of patients with high expression of Hsa _ circRNA6448-14, suggesting that low expression of Hsa _ circRNA6448-14 is a favorable factor.

Further, we found that the mortality risk increased 1.223 times (95% CI: 0.677 to 2.210), 1.056 times (95% CI: 0.471 to 2.369), and 1.547 times (95% CI: 0.506 to 4.729) in the differentiation, pN and pTNM stages, respectively, at one stage of increase, but the differences were not statistically significant (p: 0.504, p: 0.895, and p: 0.444).

Thus, without a family history of esophageal cancer, low expression of pT 1-stage 2 and hsa _ circRNA6448-14 is a protective factor in the postoperative OS prognosis in patients with esophageal cancer.

4.276 patients with ESCC postoperative DFS multifactorial analysis

Single factor analysis is an indicator related to DFS: the 2 terms pT stage and hsa _ circRNA6448-14 expression were included in the Cox proportional hazards model for analysis, and the possibility of expression of pT stage, hsa _ circRNA6448-14 as a prognostic predictor of post-operative independent DFS in patients with esophageal squamous cell carcinoma was evaluated, with the results shown in Table 7 (B in Table 7 is a covariate coefficient, exp (B) is relative risk).

TABLE 776 results of Cox multifactorial analysis of postoperative DFS in ESCC patients

As can be seen from table 7, the pT fraction and hsa _ circRNA6448-14 were expressed as independent DFS prognostic factors (p 0.031 and p 0.014) after esophageal squamous cell carcinoma surgery, and HR values were 1.755 (95% CI 1.054 to 2.921) and 0.376 (95% CI 0.173 to 0.819), respectively. In the pT stage, the risk of relapse or metastasis increases by 1.755 times for each increase of one stage, suggesting that a high pT stage is a negative factor; the risk of relapse or metastasis in patients with low hsa _ circRNA6448-14 expression is 0.376 times higher than in patients with high hsa _ circRNA6448-14 expression. Therefore, low pT staging and low expression of hsa _ circRNA6448-14 are protective factors for post-operative DFS prognosis in patients with esophageal cancer.

Example 4: diagnostic kit for esophageal squamous carcinoma

A kit for diagnosing esophageal squamous carcinoma comprises a primer sequence for specifically amplifying hsa _ circRNA6448-14, wherein the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2. The kit adopts a qRT-PCR method to carry out quantitative detection on hsa _ circRNA6448-14 in an esophageal squamous carcinoma endoscopic biopsy tissue sample, determines the expression level of hsa _ circRNA6448-14 in the esophageal squamous carcinoma endoscopic biopsy tissue sample, and realizes early diagnosis of esophageal carcinoma according to the expression level of hsa _ circRNA 6448-14. Further, the kit also contains reagents commonly used in a qRT-PCR reaction system (see example 2 for the qRT-PCR reaction system and the commonly used reagents).

Example 5: kit for predicting esophageal squamous carcinoma prognosis

A kit for predicting esophageal squamous cell carcinoma prognosis comprises a primer sequence of specific amplification hsa _ circRNA6448-14, wherein the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2. The kit adopts a qRT-PCR method to carry out quantitative detection on hsa _ circRNA6448-14 of an esophageal squamous cell carcinoma surgical excision tissue sample, determines the expression level of hsa _ circRNA6448-14 in the esophageal squamous cell carcinoma surgical excision tissue sample, and distinguishes the life cycle of an esophageal squamous cell carcinoma patient according to the expression level of hsa _ circRNA 6448-14. Further, the kit also contains reagents commonly used in a qRT-PCR reaction system (see example 2 for the qRT-PCR reaction system and the commonly used reagents).

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 present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.

Sequence listing

<110> Anyang city tumor hospital

<120> application of hsa _ circRNA6448-14 in diagnosis and prognosis prediction of esophageal squamous cell carcinoma

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tcatgccgtg tttcagctca 20

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ctccttgcac gggccggccc agcttccccg cccctggcgt ccgctccctc ccgctcgcag 60

cttacttaac ctggcccggg cggcggaggc gctctcactt ccctggagcc gcccgcttgc 120

ccgtcggtcg ctagctcgct cggtgcgcgt cgtcccgctc catggcgctc ttcgtgcggc 180

tgctggctct cgccctggct ctggccctgg gccccgccgc gaccctggcg ggtcccgcca 240

agtcgcccta ccagctggtg ctgcagcaca gcaggctccg gggccgccag cacggcccca 300

acgtgtgtgc tgtgcagaag gttattggca ctaataggaa gtacttcacc aactgcaagc 360

agtggtacca aaggaaaatc tgtggcaaat caacagtcat cagctacgag tgctgtcctg 420

gatatgaaaa ggtccctggg gagaagggct gtccagcagc cctaccactc tcaaaccttt 480

acgagaccct gggagtcgtt ggatccacca ccactcagct gtacacggac cgcacggaga 540

agctgaggcc tgagatggag gggcccggca gcttcaccat cttcgcccct agcaacgagg 600

cctgggcctc cttgccagct gaagtgctgg actccctggt cagcaatgtc aacattgagc 660

tgctcaatgc cctccgctac catatggtgg gcaggcgagt cctgactgat gagctgaaac 720

acggcatgac cctcacctct atgtaccaga attccaacat ccagatccac cactatccta 780

atgggattgt aactgtgaac tgtgcccggc tgctgaaagc cgaccaccat gcaaccaacg 840

gggtggtgca cctcatcgat aaggtcatct ccaccatcac caacaacatc cagcagatca 900

ttgagatcga ggacaccttt gagacccttc gggctgctgt ggctgcatca gggctcaaca 960

cgatgcttga aggtaacggc cagtacacgc ttttggcccc gaccaatgag gccttcgaga 1020

agatccctag tgagactttg aaccgtatcc tgggcgaccc agaagccctg agagacctgc 1080

tgaacaacca catcttgaag tcagctatgt gtgctgaagc catcgttgcg gggctgtctg 1140

tagagaccct ggagggcacg acactggagg tgggctgcag cggggacatg ctcactatca 1200

acgggaaggc gatcatctcc aataaagaca tcctagccac caacggggtg atccactaca 1260

ttgatgagct actcatccca gactcagcca agacactatt tgaattggct gcagagtctg 1320

atgtgtccac agccattgac cttttcagac aagccggcct cggcaatcat ctctctggaa 1380

gtgagcggtt gaccctcctg gctcccctga attctgtatt caaagatgga acccctccaa 1440

ttgatgccca tacaaggaat ttgcttcgga accacataat taaagaccag ctggcctcta 1500

agtatctgta ccatggacag accctggaaa ctctgggcgg caaaaaactg agagtttttg 1560

tttatcgtaa tagcctctgc attgagaaca gctgcatcgc ggcccacgac aagaggggga 1620

ggtacgggac cctgttcacg atggaccggg tgctgacccc cccaatgggg actgtcatgg 1680

atgtcctgaa gggagacaat cgctttagca tgctggtagc tgccatccag tctgcaggac 1740

tgacggagac cctcaaccgg gaaggagtct acacagtctt tgctcccaca aatgaagcct 1800

tccgagccct gccaccaaga gaacggagca gactcttggg agatgccaag gaacttgcca 1860

acatcctgaa ataccacatt ggtgatgaaa tcctggttag cggaggcatc ggggccctgg 1920

tgcggctaaa gtctctccaa ggtgacaagc tggaagtcag cttgaaaaac aatgtggtga 1980

gtgtcaacaa ggagcctgtt gccgagcctg acatcatggc cacaaatggc gtggtccatg 2040

tcatcaccaa tgttctgcag cctccagcca acagacctca ggaaagaggg gatgaacttg 2100

cagactctgc gcttgagatc ttcaaacaag catcagcgtt ttccagggct tcccagaggt 2160

ctgtgcgact agcccctgtc tatcaaaagt tattagagag gatgaagcat tagcttgaag 2220

cactacagga ggaatgcacc acggcagctc tccgccaatt tctctcagat ttccacagag 2280

actgtttgaa tgttttcaaa accaagtatc acactttaat gtacatgggc cgcaccataa 2340

tgagatgtga gccttgtgca tgtgggggag gagggagaga gatgtacttt ttaaatcatg 2400

ttccccctaa acatggctgt taacccactg catgcagaaa cttggatgtc actgcctgac 2460

attcacttcc agagaggacc tatcccaaat gtggaattga ctgcctatgc caagtccctg 2520

gaaaaggagc ttcagtattg tggggctcat aaaacatgaa tcaagcaatc cagcctcatg 2580

ggaagtcctg gcacagtttt tgtaaagccc ttgcacagct ggagaaatgg catcattata 2640

agctatgagt tgaaatgttc tgtcaaatgt gtctcacatc tacacgtggc ttggaggctt 2700

ttatggggcc ctgtccaggt agaaaagaaa tggtatgtag agcttagatt tccctattgt 2760

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Claims

The application of the quantitative detection reagent of hsa _ circRNA6448-14 in preparing a product for predicting esophageal squamous cell carcinoma prognosis.
2. The use of claim 1, wherein the product is used to detect the expression level of hsa _ circRNA6448-14 in a sample by real-time quantitative PCR, in situ hybridization, a chip, or a high throughput sequencing platform.
3. The use according to claim 1, wherein the product comprises a probe that hybridizes to hsa _ circRNA6448-14 or a primer specific for amplification of hsa _ circRNA 6448-14.
4. The use according to claim 3, wherein the specific primer sequences for amplification of hsa _ circRNA6448-14 are shown in SEQ ID NO.1 and SEQ ID NO. 2.
5. The use according to claim 1, wherein the product is a chip, a preparation or a kit.
6. A product for predicting prognosis of esophageal squamous carcinoma, the product comprising an agent for detecting the expression level of hsa _ circRNA6448-14 in a sample.
7. The product of claim 6, wherein the reagents comprise reagents for detecting the expression level of hsa _ circRNA6448-14 by real-time quantitative PCR, in situ hybridization, a chip, or a high throughput sequencing platform.
The application of the quantitative detection reagent of hsa _ circRNA6448-14 in preparing the diagnosis product of esophageal squamous cell carcinoma.
9. The use of claim 8, wherein the product is used to detect the expression level of hsa _ circRNA6448-14 in a sample by real-time quantitative PCR, in situ hybridization, a chip, or a high throughput sequencing platform.
10. The use of claim 8 wherein the product comprises a probe that hybridizes to hsa _ circRNA6448-14 or a primer that specifically amplifies hsa _ circRNA 6448-14.