CN109666741B

CN109666741B - Application of novel gastric cancer marker gene circPTPDC1

Info

Publication number: CN109666741B
Application number: CN201910094141.7A
Authority: CN
Inventors: 康宁; 朱桂芳; 曹秀峰
Original assignee: Jiangsu Wancheng Biomedical Research Institute Co ltd
Current assignee: Jiangsu Wancheng Biomedical Research Institute Co ltd
Priority date: 2019-01-30
Filing date: 2019-01-30
Publication date: 2022-06-10
Anticipated expiration: 2039-01-30
Also published as: CN109666741A

Abstract

The invention discloses application of a novel gastric cancer marker gene circPTPDC1, relates to discovery, detection and application of a circPTPDC1 molecular marker, and designs and synthesizes a detection primer specifically used for real-time quantitative PCR. Aiming at the current situations that target groups have poor compliance on the screening of the gastric cancer endoscope and most of the clinically confirmed gastric cancer patients are in middle and late stages, an ideal molecular early warning signal is found in the process of the canceration of the gastric cancer, and endoscopic examination and pathological confirmation are performed in a targeted manner, so that the pain of the patients is relieved, over-medical treatment is avoided, and medical resources are saved. The invention finds that the blood circPTPDC1 can be used as a marker for lymph node metastasis of a gastric cancer patient and monitoring prognosis.

Description

Application of novel gastric cancer marker gene circPTPDC1

Technical Field

The invention relates to the field of tumor molecular biology, in particular to application of a novel gastric cancer marker gene circPTPDC 1.

Background

Currently, gastric cancer is still ranked third in cancer-related deaths worldwide and is one of the major health burdens of society. In china, gastric cancer is the second most prevalent cancer and the second leading cause of cancer death, with 679,100 new cases and 498,000 deaths estimated in 2015. Despite advances in surgical techniques and combination chemotherapy regimens, the 5-year overall survival of gastric cancer is still less than 30% in most countries due to the lack of early diagnostic markers for gastric cancer. Therefore, it is necessary to carefully explore the molecular characteristics associated with gastric cancer and develop a reasonable method for early detection of gastric cancer.

Circular RNA (circRNA) is thought to result from non-classical splicing of linear pre-mRNA and is widely found in virus-like infectious particles such as viroids, circovirus and hepatitis delta virus. The circular RNAs appear to be the result of erroneous RNA splicing, and its critical role and function have not been discovered until recently and have permanently altered our view of cancer, particularly carcinogenesis and cancer progression. To date, some endogenous circrnas have been found in mammalian cells, and they can be present in high abundance and evolutionarily conserved in tumor progression. In addition, numerous studies have demonstrated that circular RNAs may regulate transcription and manipulate pathways of micrornas. Several circrnas are thought to be responsible for the malignant biological behavior of cancer cells. However, since our understanding of circrnas is limited, many circrnas that can function as micrornas may not have been discovered. There is increasing evidence that circRNA may play an important role in the development and progression of cancer, including gastric cancer, and may be a neogenetic marker of cancer. Zhang J, team found that circLARP4 was down-regulated in gastric cancer tissues and targeted to LATS1 gene by amplifying miR-424, inhibiting biological behavior of gastric cancer cells, which is an independent prognostic factor for overall survival of gastric cancer patients.

Currently, in the prior art, the disease condition of a patient is evaluated by detecting tumor indexes such as CEA/CA199/CA153 in the blood of the patient, and the indexes have poor specificity and sensitivity.

Disclosure of Invention

In order to solve the problems, the invention discloses application of a novel gastric cancer marker gene circPTPDC1, which aims at the current situations that target people have poor compliance on gastric cancer endoscopy screening and most of clinically diagnosed gastric cancer patients are in middle and late stages, an ideal molecular early warning signal is found in the gastric cancer canceration process, and endoscopic examination and pathological diagnosis are performed in a targeted manner to relieve pain of the patients, avoid over-treatment and save medical resources. The invention discovers that the blood circPTPDC1 can be used as a lymph node metastasis of a gastric cancer patient and a marker for monitoring prognosis.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention discloses application of a novel gastric cancer marker gene circPTPDC1, wherein the nucleotide sequence of the circPTPDC1 is SEQ ID NO.1, and the circPTPDC1 is used as a marker for diagnosis and prognosis of gastric cancer.

In the present invention, the reagent for gastric cancer diagnosis is a real-time quantitative PCR kit.

In the invention, 2 pairs of primers are designed according to circPTPDC1 with the nucleotide sequence of SEQ ID NO.1 for detecting the sequence, and the nucleotide sequences of the primers are shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.

GAPDH was used as internal control, GAPDH (F); 5; "GCATCCTGGGGCTACACATG-; GAPDH (R) < 5 > ACTTCAGGAGCATCTTGAATAGGT-3 < 3 > and the nucleotide sequence is SEQ ID NO. 3.

A primer pair, an upstream primer: 5'-CTTTCATGGAGGCTGGCATT-3', having the nucleotide sequence of SEQ ID NO.4 was used. The downstream primer, nucleotide sequence 5'-TGCAGCCATGGTAGTCTGTT-3' is SEQ ID NO. 5.

The invention also discloses a detection method of circPTPDC1, which comprises RNA extraction, reverse transcription and real-time quantitative PCR. The method comprises the following specific steps: extracting total RNA in tissue by using TRIzol reagent and total RNA in plasma by tiamamp Virus RNA kit; reverse transcription of RNA into cDNA TM using Prime-Script one-step RT-PCR kit, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal control; the following primer pairs were used: 5'-CTTTCATGGAGGCTGGCATT-3' (Forward, or F) and 5'-TGCAGCCATGGTAGTCTGTT-3' (reverse, R), circPTPDC1 expression levels were determined by qRT-PCR; GAPDH was used as an internal control, using primer pair 5'-GCATCCTGGGCTACACTG-3' (F) and 5'-ACTTCAGGAGCATCTGAAATAGGT-3' (R). All qRT-PCR reactions were performed using ABI7500 system and SYBR Green PCR Master Mix. To accurately verify the fold change in circPTPDC1 expression in GC tissues, the calculated Ct values were normalized to GAPDH (Ct ═ ctted-CtGAPDH) amplified from the same samples, and the- Δ Ct method was used to estimate the value of the change. Each sample was replicated three times and all reactions were independently replicated three times to ensure reproducibility of all data. Curtoff value of circPTPDC1 was-12.55, when detected ^-When the Δ Ct is larger than this value, the cancer is gastric cancer, and the specificity is 66.67% and the sensitivity is 69.44%.

The invention also discloses a real-time quantitative PCR detection kit for gastric cancer diagnosis, which comprises a detection primer which is designed and synthesized according to the circPTPDC1 molecular marker with the nucleotide sequence of SEQ ID NO.1 and is specifically used for real-time quantitative PCR.

In the invention, further, the specific primers comprise an upstream primer shown in SEQ ID NO.4 and a downstream primer shown in SEQ ID NO. 5.

The invention also discloses a molecular marker for gastric cancer diagnosis, which is a circPTPDC1 molecular marker with the nucleotide sequence of SEQ ID NO. 1.

Specific primers for detecting the circPTPDC1 molecular marker, wherein the specific primers comprise an upstream primer shown in SEQ ID NO.4 and a downstream primer shown in SEQ ID NO. 5.

The invention has the following advantages:

(1) the use of circPTPDC1 is proposed. (2) Circular RNA has the characteristics of mechanism stability, abundance degree, tissue-specific expression and the like. And the expression quantity of the circPTPDC1 in the gastric cancer and the tissue beside the gastric cancer is different and is related to stages, so the circPTPDC1 has an application prospect of becoming a gastric cancer biomarker.

The invention is provided aiming at the current situations that target people have poor compliance on the screening of the gastric cancer endoscope and most of gastric cancer patients diagnosed clinically are in middle and late stages, and hopes that an ideal molecular early warning signal can be found in the canceration process of the gastric cancer, and endoscopic examination and pathological confirmation are carried out in a targeted manner, so that the pain of the patients is relieved, over-medical treatment is avoided, and medical resources are saved. The patent finds that blood circPTPDC1 can be used as a marker for diagnosis of gastric cancer patients.

Drawings

FIG. 1, showing that circPTPDC1 is derived from PTPDC1 exons 2-6;

FIG. 2, qRT-PCR of the abundance of circPTPDC1 and PTPDC1 mRNA in GC cells treated with RNase R;

FIG. 3, expression of circPTPDC1 expression level was significantly higher than that of adjacent non-cancerous tissue of GC patients;

FIG. 4, ROC curve;

FIG. 5, upregulation of circPTPDC1 levels was positively correlated with TNM staging;

FIG. 6, the expression level of circPTPDC1 in the blood of GC patients was significantly higher than that of normal persons.

In the figure: p <0.05, P < 0.01.

Detailed Description

The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.

The invention discloses application of a novel gastric cancer marker gene circPTPDC1, wherein the nucleotide sequence of a circPTPDC1 molecular marker is SEQ ID NO.1, and the circPTPDC1 is used as a marker for diagnosis and prognosis of gastric cancer. The following experimental methods are specific examples, and the experimental results are shown in fig. 1 to 5.

The experimental steps are as follows:

RNA Sanger sequencing

(1) The amplification products were inserted into a T-vector for Sanger (Sanger) sequencing to determine their full length. Different primers were designed to confirm the loop-back splice point of circPTPDC 1. Primers were synthesized by Invitrogen (shanghai, china) and sanger sequencing was performed by Realgene, inc.

RNase R digestion

2mg of total RNA were incubated at 37 ℃ for 20 minutes with or without 3U/mg RNase R, followed by the RNeasy MinElute kit.

3. Patient and case screening

A total of 36 GC tissues and corresponding adjacent non-tumor tissue samples were collected from GC patients, all from the department of general surgery of the nanjing hospital affiliated to the university of medical, nanjing (between 1 month in 2013 and 12 months in 2017). All patients in the group underwent radiotherapy and chemotherapy prior to surgery, and their tissue samples were immediately stored in RNA fixative reagents and immediately stored in a-80 ℃ freezer. The paired adjacent non-tumor tissues were pathologically free of tumor cells and were located 5cm from the tumor body border. All tumors were staged accurately according to the tumor-lymph node metastasis (TNM) staging system of the international anticancer consortium. Each patient signed a written informed consent and the study protocol was approved by the local medical ethics committee.

In this study, there were 21 blood samples from GC cancer patients and 17 normal human blood samples.

4. Clinical tissue sample sampling

(1) Preparation items:

liquid nitrogen tank (checking liquid nitrogen volume, filling liquid nitrogen in advance in time), ice box, ice physiological saline, sterile enzyme-free freezing tube (subpackaging RNA protective solution and numbering for later use), sterilized gloves (without talcum powder), mask, cap, tweezers, scissors, low-temperature marker, etc.

(2) The operation process comprises the following steps:

A. determining that a blood sample is left before a patient operation, processing according to a blood sample extraction process, and extracting

Transferring the serum into Greiner freezing tube, marking with red low-temperature marker pen, and storing at low temperature

Refrigerator, and record in case data table and its electronic version.

B. Filling in the case data sheet of the tissue specimen in advance (recording effective contact way for follow-up visit), paying attention to filling

The integrity of the content. The number of the freezing tube for reserving the cancer and the tissue beside the cancer is predetermined and recorded

Recorded in the case data table.

C. Immediately placing the tissue in a bent disc after the tissue is separated from the body, and quickly showering the tissue by using ice brine (the RNA enzyme activity can be temporarily inhibited at low temperature)

Striving for sampling time; washing mixed blood in the tissue sample to reduce pollution; maintaining tissue samples

So as to facilitate rapid penetration of the RNA protection solution and to exert an enzyme inhibiting effect).

D. Selecting gastric mucosa tissue, decomposing the tissue into small blocks with the size of about soybean, and subpackaging, wherein the sampling sequence is as follows: get

2-4 paracancer normal tissues and 2-4 tumor tissues with the thickness not more than 5mm, taking the normal tissues first, and then

Taking tumor tissue, a freezing tube is suitable for storing a sample only, so that the tumor and normal tissue beside the tumor are not mixed

The number of the frozen tube is stored.

E. Screwing the freezing tube after sampling (if liquid nitrogen penetrates into the freezing tube without screwing the bottle cap, taking out the freezing tube at normal temperature

The bursting of the freezing tube is easily caused when the tube is frozen), the tube is turned upside down gently and shaken to uniformly mix the tissue and the protective solution,

then quickly placing the mixture in a liquid nitrogen tank.

F. Checking whether the tissue specimen case data sheet is completely filled, and determining in advance in' refrigerator specimen positioning sheet

Recording the storage position of the sample in the deep low temperature refrigerator, and timely transferring the sample in the liquid nitrogen tank into the deep low temperature refrigerator

A refrigerator. The phenomenon that the sample is wasted and unusable after liquid nitrogen is volatilized and depleted due to forgetfulness is avoided.

G. Tracking the pathological report result one week after operation, and timely recording the result into a case data sheet to determine whether the sample can be used

Study in group.

RNA extraction, reverse transcription and real-time quantitative PCR

Total RNA in tissues was extracted by using TRIzol reagent, and total RNA in plasma was extracted by tiamamp Virus RNA kit. RNA was reverse transcribed into cDNA TM using Prime-Script one-step RT-PCR kit, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. The following primer pairs were used: 5'-CTTTCATGGAGGCTGGCATT-3' (Forward, or F) and 5'-TGCAGCCATGGTAGTCTGTT-3' (reverse, R.) the level of circPTPDC1 expression was determined by qRT-PCR. GAPDH was used as an internal control, using primer pair 5'-GCATCCTGGGCTACACTG-3' (F) and 5'-ACTTCAGGAGCATCTGAAATAGGT-3' (R). All qRT-PCR reactions were performed using ABI7500 system and SYBR Green PCR Master Mix. To accurately verify the fold change in circPTPDC1 expression in GC tissues, the calculated Ct values were normalized to GAPDH amplified from the same sample (Ct ═ ctted-CtGAPDH) and the value of the change was estimated using the- Δ Ct method. Each sample was replicated three times and all reactions were independently replicated three times to ensure reproducibility of all data. The Cutoff value of circPTPDC1 was-12.55, and when a- Δ Ct larger than this value was detected, gastric cancer was detected, and the specificity was 66.67% and the sensitivity was 69.44%.

The experimental operation steps are as follows:

(1) trizol tissue RNA extraction:

A. wearing a mask and gloves, opening the 4 ℃ low-temperature centrifuge, and setting the temperature for later use. The number of samples to be extracted was counted and a 1.5ml sterile enzyme-free EP tube was prepared in duplicate and placed on an EP tube holder for future use.

B. Pouring a proper amount of liquid nitrogen into the grinding vessel for precooling, putting 50-100mg of tissues into the liquid nitrogen, and grinding the tissues into powder.

C. 1ml Trizol was added to the milling vessel and the mixture of coagulated tissue and Trizol continued to be milled.

D. Standing for liquefaction, and transferring to an RNase free 1.5ml EP tube.

E. The mixture was allowed to stand at room temperature for 5min, and 200. mu.l of chloroform was added to 1ml of Trizol.

F. Shaking vigorously for 15s, and standing at room temperature for 2-3 min.

G.4 ℃, 12000g-14000g, and centrifuging for 15 min.

H. The aqueous phase was transferred to a fresh RNase free 1.5ml EP, 500. mu.l of isopropanol was added per 1ml of Trizol, and the mixture was left at room temperature for 10 min.

I.4 ℃, 12000g and centrifuging for 10 min. 75% ethanol (prepared with DEPC water) was used to prepare the RNA for washing.

J. The supernatant was carefully discarded and the RNA pellet was washed by adding at least 1ml 75% ethanol per tube and vortexing.

K.4 ℃, 7500 and 10000g, and centrifuging for 5-10 min. The supernatant was carefully discarded.

L, air-drying the RNA precipitate (the RNA is colorless after air-drying), dissolving the precipitate with DEPC-water (about 10-80ul is added according to the size of the precipitate), and sucking, beating and blowing uniformly by using a gun head.

(2) Trizol extraction of cellular RNA:

A. the cells to be extracted were washed twice with PBS.

B. The appropriate amount of Trizol was added to the culture vessel (1 ml per well of vial/six well plate, 3ml per dish, 5ml per vial).

C. The pipetting was repeated several times and transferred to RNase free 1.5ml EP tube.

D. The mixture was allowed to stand at room temperature for 5min, and 200. mu.l of chloroform was added to 1ml of Trizol.

E. Shaking vigorously for 15s, and standing at room temperature for 2-3 min.

F.4 ℃, 12000-14000g, and centrifuging for 15 min.

G. The aqueous phase was transferred to a fresh RNase free 1.5ml EP, 500. mu.l of isopropanol was added per 1ml of Trizol, and the mixture was left at room temperature for 10 min.

H.4 ℃, 12000g, and centrifuging for 10 min.

I. The supernatant was discarded and the RNA pellet was washed by adding at least 1ml of 75% ethanol and vortexing.

10000g of J.4 ℃, 7500 and centrifugation for 5-10 min.

K. The RNA pellet was air-dried and the pellet was dissolved in DEPC-water (about 10-80ul was added depending on the pellet size).

(3) Detecting the concentration of RNA in the extracted sample:

A. each sample to be tested (1 ul +99ul DEPC water per sample to be tested) was placed in a new RNase free200ul EP tube at a concentration of 1%.

B. The UV spectrophotometer was turned on, the cuvette was cleaned, and 100ul DEPC water was taken to set a blank control.

C. And injecting samples to be detected in sequence, detecting and recording the concentration of the samples and the OD260/280 ratio.

(4) Reverse transcription of RNA:

A. The reverse transcription kit was removed from the-20 ℃ freezer, 2 enzymes were inserted into ice-box ice, the other reagents were thawed on ice, and RNase Free water was thawed at room temperature for use.

B. Based on the measured RNA concentration, the volume of RNA required for reverse transcription per tube was calculated, and the DEPC water volume was also calculated.

C. Placing the PCR tube frame on ice, placing the PCR tube frame into a sterile 200ul enzyme-free PCR tube according to the requirement, and marking according to the number. After sucking a proper amount of RNA by a liquid transfer device, vertically inserting the gun head into the bottom of the PCR tube, and gently beating out liquid at a constant speed.

D. A pipette sucks a proper amount of DEPC water, and the pipette tip is stuck to the rear wall and is driven into the PCR tube.

E. Reverse transcription Step 1: add gDNA Eraser 1ul and 5x gDNA Eraser Buffer 2ul per tube.

F. Placing on an eight-tube centrifuge, slightly bouncing off bubbles at the bottom or on the wall of the tube, and centrifuging for a short time.

RT-PCR instrument at 42 ℃ for 2 minutes, and the reaction product is placed on ice for later use.

H. Reverse transcription Step 2: 1.5ml of EP tube was used, and each tube of reaction Mix was prepared according to the following formulation

Composition (I)	Volume (uL)
		5x PrimerScript Buffer2	4
Rnase Free dH2O	4
		PrimerScript RT Enzyme Mix I	1
RT Primer Mix	1

Setting reaction conditions: 15min at 37 ℃; 85 ℃ for 5 sec; maintaining the temperature at 4 ℃.

I. And fastening the pipe cover, taking the pipe cover off the pipe frame, placing the pipe cover on the centrifuge tube frame, slightly bouncing the bottom of the pipe for a person with bubbles, centrifuging for a short time, confirming that the liquid level of each pipe is consistent and no bubble exists in the pipe, and then placing the pipe cover on ice again.

J. Setting reaction conditions: 15min at 37 ℃; 85 ℃ for 5 sec; maintaining the temperature at 4 ℃. Operating on the machine, and storing the cDNA at-20 ℃ after 15-20 min.

(5) qRT-PCR step:

A. preparing an ice box, taking the cDNA, the primers and the PCR reagent of the sample to be detected out of a refrigerator at the temperature of-20 ℃, and unfreezing on ice.

B. The eight-tube connector, the tube cover, the eight-tube connector frame, the tweezers, the sterile 1.5ml EP tube, the EP tube frame, the pipettor with the appropriate measuring range and the sterile film gloves are prepared.

C. And (3) sequentially carrying out short-time oscillation centrifugation on the unfrozen cDNA, the primers and the PCR reagent, and standing on ice for later use.

D. And dividing different target gene sample adding areas according to the number of the detection samples and the indexes. The eight-joint pipe frame is placed on ice, and the eight-joint pipe is clamped by tweezers and laid on the eight-joint pipe frame.

E. Take 1.5ml EP tube, prepare eight-tube PCR Mix according to the following 19ul per well, and shake and centrifuge.

F. According to the preset position of each sample and the position of the multiple holes in the eight-connecting pipe, 1ul of cDNA and 19ul of PCR Mix of the samples are sequentially added.

G. Centrifuging to remove bubbles, operating on a machine, and setting reaction conditions.

(6) qRT-PCR reaction System:

composition (I)	Volume (ul)
		SYBR Premix Ex Taq TM II	10
Forward Primer	0.5
		Reverse Primer	0.5
ROC Reference Dye/II	0.4
		dH2O	7.6
cDNA	1

qRT-PCR reaction conditions:

6. and (3) data analysis:

the results of the experiments are shown in FIGS. 1-6, FIG. 1, showing that circPTPDC1 is derived from exons 2-6 of PTPDC 1. The amplified products were inserted into a T-vector for Sanger sequencing to determine their full length. Different primers were designed to confirm that circPTPDC 1: 5'-AGGGCTTGGTCGAACAG-3' (sense) and 5'-ACTACCATGGCTGCAGG-3' (antisense) splice junctions; FIG. 2, qRT-PCR of the abundance of circPTPDC1 and PTPDC1 mRNA in GC cells treated with RNase R; FIG. 3, expression of circPTPDC1 expression level was significantly higher than that of adjacent non-cancerous tissue of GC patients. The correlation of-delta Ct values of the two is determined; FIG. 4, the ROC curve has been used to evaluate the potential diagnostic value of circPTPDC1, with an area under the ROC curve (AUC) totaling 0.758. The expression AUC of circPTPDC1 in the GC late (IIIA-IV) TNM phase was 0.809, which is higher than that in the early (IA-IIB) TNM phase; FIG. 5, upregulation of circPTPDC1 levels was positively correlated with TNM staging; FIG. 6, the expression level of circPTPDC1 in the blood of GC patients was significantly higher than that of normal persons. In the figure: p <0.05, P < 0.01.

The correlation of circPTPDC1 with clinical staging data and prognosis of gastric cancer patients was performed using t-test and chi-square test and survival analysis. Receiver Operating Characteristic (ROC) curves were performed to evaluate their diagnostic value. All statistical analyses were performed using SPSS for Windows v.17.0. For all results, P <0.05 was considered statistically significant.

The technical means disclosed in the invention scheme are not limited to the technical means disclosed in the above embodiments, but also include the technical scheme formed by any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.

Sequence listing

<110> Jiangsu Wancheng biomedical research institute Co Ltd

<120> application of novel gastric cancer marker gene circPTPDC1

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aaagtagcta tccattgtca tgcagggctt ggtcgaacag actaccatgg ctgcaggagt 120

cttgcctcag aatgaacaac catattctac cttggtgaat aacagcgagt gtgttgcaaa 180

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Claims

1. The application of a reagent for detecting the expression level of a circPTPDC1 marker in blood in preparing a gastric cancer diagnosis reagent is disclosed, wherein the nucleotide sequence of the circPTPDC1 marker is SEQ ID NO.1, and the circPTPDC1 is used as the marker for diagnosing gastric cancer.

2. The use of claim 1, wherein: the reagent for detecting the expression level of the circPTPDC1 marker in blood is a real-time quantitative PCR reagent.

3. Use according to claim 1 or 2, characterized in that: 1 pair of primers is designed according to circPTPDC1 with the nucleotide sequence of SEQ ID NO.1 for detecting the sequence, and the nucleotide sequences of the primers are shown as SEQ ID NO.4 and SEQ ID NO. 5.

4. The use of claim 1, wherein: the detection method for detecting the expression level of the circPTPDC1 marker in blood comprises the steps of RNA extraction, reverse transcription and real-time quantitative PCR.

5. The use of claim 4, wherein: the detection method comprises the following specific steps: extracting total RNA in tissue by using TRIzol reagent and total RNA in plasma by tiamamp Virus RNA kit; reverse transcribing the RNA to cDNA TM using Prime-Script one-step RT-PCR kit; glyceraldehyde 3-phosphate dehydrogenase was used as an internal control, the circPTPDC1 expression level was determined by qRT-PCR using the primer pair shown in SEQ ID No.4 and SEQ ID No.5, and all qRT-PCR reactions were performed using ABI7500 system and SYBR Green PCR Master Mix.