CN113774170A

CN113774170A - Application of miRNA 21 in preparation of reagent for judging survival rate of HPV-infected head and neck squamous cell carcinoma patient after radiotherapy

Info

Publication number: CN113774170A
Application number: CN202111259259.4A
Authority: CN
Inventors: 郭兰伟; 杨福娜; 殷玉林
Original assignee: Cancer Hospital and Institute of CAMS and PUMC; Henan Cancer Hospital
Current assignee: Cancer Hospital and Institute of CAMS and PUMC; Henan Cancer Hospital
Priority date: 2021-10-28
Filing date: 2021-10-28
Publication date: 2021-12-10
Anticipated expiration: 2041-10-28
Also published as: CN113774170B

Abstract

The invention belongs to the technical field of medical treatment, and discloses application of miRNA 21 in preparation of a reagent for judging the survival rate of head and neck squamous cell carcinoma patients infected by HPV after radiotherapy. The reagent is used for quantitatively detecting the expression quantity of the miRNA 21. The invention obtains the co-expression of HPV E6/E7mRNA and host miRNA 21 as independent prognostic factors influencing the head and neck tumor by detecting the expression conditions of HPV E6/E7mRNA and candidate ray sensitivity related miRNA in the HPV positive head and neck tumor tissues before radiotherapy and performing single-factor and multi-factor regression analysis, can be used for radiotherapy prognosis judgment of head and neck tumor patients with positive HPV infection in China, provides reference for the formulation of individualized treatment schemes of the head and neck tumor, and finally achieves the purpose of reducing the death rate of the head and neck tumor.

Description

Application of miRNA 21 in preparation of reagent for judging survival rate of HPV-infected head and neck squamous cell carcinoma patient after radiotherapy

Technical Field

The invention belongs to the technical field of medical treatment, and relates to application of miRNA 21 in preparation of a reagent for judging the survival rate of head and neck squamous cell carcinoma infected by HPV after radiotherapy, a kit and a screening method.

Background

Head and Neck Cancer (HNC) refers to a malignant tumor located in the oral cavity, pharynx (oropharynx, nasopharynx, and hypopharynx) and larynx, and is the sixth most common malignant tumor worldwide, accounting for about 5% of the total number of cancers worldwide. Worldwide, about 88 million new cases of outbreaks in 2018 (including 377713 oral cancer cases, 316020 pharyngeal cancer cases and 184615 laryngeal cancer cases) and 40 million deaths are attributed to head and neck tumors. The five-year survival rate of the head and neck tumor is only 40% -50%, and the functions of human vision, hearing, smell, taste, sound production and the like are managed by the hands of various facial amplifiers, and the occurrence of the tumors at the parts can directly influence the daily life and the psychological state of a patient. Radiotherapy (radiotherapy) is currently the main treatment modality for head and neck tumors, and most patients after early tumor surgery need preventive radiotherapy. Because the total dose of the radiotherapy of the head and neck tumors generally reaches 70Gy, patients often have serious stomatitis, dry mouth, speech and swallowing dysfunction and the like, and the quality of life is greatly influenced. Therefore, finding out a suitable prognostic influence factor, developing prognostic risk assessment according to the prognostic influence factor, and adopting a more individualized treatment scheme to reduce excessive treatment and improve the quality of life are the hot and difficult problems of the existing head and neck tumor treatment.

Disclosure of Invention

The invention aims to provide an application of miRNA 21 in preparing a reagent for evaluating the survival rate of head and neck squamous cell carcinoma patients infected by HPV (human papilloma Virus) prognosis.

The invention also aims to provide a kit for judging the survival rate of the head and neck squamous cell carcinoma patients infected by HPV after radiotherapy.

The invention also aims to provide a screening method for judging the influence factors of the survival rate of the head and neck squamous cell carcinoma patients infected by HPV after radiotherapy.

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

the invention provides an application of miRNA 21 in preparing a reagent for evaluating the survival rate of head and neck squamous cell carcinoma patients infected by HPV (human papilloma virus), wherein the reagent is used for quantitatively detecting the expression quantity of the miRNA 21.

Preferably, the reagent comprises a primer designed based on the miRNA 21 and used for reverse transcription.

Preferably, the primer sequence is shown as SEQ ID NO. 1.

The invention also provides a kit for judging the survival rate of the head and neck squamous cell carcinoma patient infected by HPV after radiotherapy, and the kit comprises a reagent for quantitatively detecting the miRNA 21 expression quantity.

Preferably, the primer sequence is shown as SEQ ID NO. 1.

The invention also provides a screening method for judging the influence factors of the survival rate of the head and neck squamous carcinoma patients infected by HPV after radiotherapy, which comprises the following steps:

step 1, collecting a plurality of fresh tumor tissue specimens of new head and neck squamous carcinoma cases meeting the standard, storing the specimens in a refrigerator at the temperature of-80 ℃, and carrying out HPV DNA detection; the HPV virus comprises 7 low-risk subtypes and 17 high-risk subtypes, wherein the 7 low-risk subtypes are HPV6, HPV 11, HPV 34, HPV 42, HPV 67, HPV 70 and HPV 81, and the 17 high-risk subtypes are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV 82;

step 2, carrying out HPV E6/E7mRNA and 7 kinds of ray-sensitive miRNA detection on the positive cases of HPV DNA detection, and analyzing the relation between HPV infection and miRNA high expression rate; the 7 radiation-sensitive miRNAs are miRNA 21, miRNA 34a, miRNA 101, miRNA 155, miRNA 106b, miRNA 221 and miRNA 222;

step 3, calculating the positive rates of HPV DNA, HPV E6/E7mRNA and miRNA of head and neck squamous cell carcinoma and sub-parts thereof in the case, and judging the influence of HPV infection on miRNA expression;

step 4, calculating clinical characteristics of cases, HPV DNA negative and positive groups, HPV E6/E7mRNA negative and positive groups and median survival time of different miRNA expression levels by using a single-factor Kaplan-Meier method to obtain candidate influencing factors of the radiotherapy prognosis survival rate of the head and neck squamous cell carcinoma; the clinical characteristics of the cases included gender, age, location of cancer and clinical stage;

and 5, carrying out multi-factor regression analysis according to candidate influence factors of the radiotherapy prognosis survival rate obtained by single-factor analysis, and finally determining the influence factors of the radiotherapy prognosis survival rate of the head and neck squamous cell carcinoma.

Preferably, the HPV DNA detection employs HPV genotyping techniques.

Compared with the prior art, the invention has the beneficial effects that:

the invention adopts a molecular epidemiology method, in head and neck patients who are positive for HPV infection and receive radiotherapy based on hospitals, by detecting the expression conditions of HPV E6/E7mRNA and candidate ray sensitivity related miRNA in HPV positive head and neck tumor tissues before radiotherapy, and by single-factor and multi-factor regression analysis, the co-expression of HPV E6/E7mRNA and host miRNA 21 is obtained as an independent prognostic factor influencing head and neck tumors, and the molecular epidemiology method can be used for radiotherapy prognosis judgment of head and neck tumor patients positive for HPV infection in China, provides reference for the formulation of individual treatment schemes of head and neck tumor patients in China, and finally achieves the purpose of reducing the death rate of the head and neck tumors.

Drawings

FIG. 1 is a survival curve for HPV E6/E7mRNA in combination with 7 miRNAs.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

Epidemiological and clinical medical studies have found that about 95% of head and neck tumors are squamous carcinomas and are tumors associated with viral infections. About 70% of squamous cell carcinomas are caused by infection with high-risk Human Papilloma Virus (HPV) except that nasopharyngeal carcinoma is closely related to EB (epstein-barr virus, EBv) virus infection, and the onset of these subtypes is on a growing trend year by year. More importantly, studies have shown that HPV infection is not only associated with the risk of developing head and neck tumors, but also greatly affects the susceptibility of head and neck tumor patients to radiation therapy, and that both progression-free survival and overall survival for HPV infection-positive patients are superior to HPV-negative patients (Marur S, DSouza G, Westra WH, et al. Hpv-associated head and novel cancer: A virus-related cancer epidemic [ J ]. Lancet Oncol,2010,11(8):781-789.DOI:10.1016/S1470-2045(10) 70017-6). However, clinical studies have also found that HPV-positive head and neck tumor patients have a five-year survival rate of only 60%, suggesting that nearly 40% of cases remain insensitive to radiation therapy. Therefore, if the patients with positive HPV infection head and neck tumors can be further subjected to radiotherapy sensitivity detection and prognosis risk assessment, and the prognosis of HPV positive individual radiotherapy is judged, more individualized treatment schemes can be favorably formulated, the over-treatment proportion is reduced, and the physiological, psychological and economic burdens of the patients are reduced.

The main mechanisms for a better prognosis of patients with HPV-positive head and neck tumors are as follows: inactivating p53 gene against HPV negative tumor cell p53 gene, which is inhibited by HPV E6 protein, but may also express a small amount of wild-type p53(Gillison ML, Shah KV. human papillomavir us-associated head and negative square cell Cancer: Mount elevation role for an ocular roll for a human papillomavir in a subset of head and negative Cancer [ J ]. Current Optin Oncol,2001,13(3):183-188.) such that TP 53-mediated tumor cell cycle arrest and cell apoptosis are critical for HPV positive tumor patients (Kimple RJ, Smith MA, Blittzer GC, et al. enhanced radiation in a pv-cell Cancer strain [ 20100 ], 4800 ] Cancer antigen J.3; ② because the inhibition of HPV E7 to Rb leads to P16 overexpression, reduces the homologous recombination mediated DNA damage repair bypass, thereby inhibits the double-stranded DNA damage repair ability to cause tumor cell apoptosis (Dok R, Kalev P, Van Limbergen EJ, et al.16ink4a antigens and homologus recombination-mediated DNA repair in human papillomavir-positive head and negtechnologies [ J ]. cer Res,2014,74(6):1739-1751.), and leads to higher sensitivity of HPV positive tumor cells to radiotherapy. Although the mechanism has not been clearly studied, it can be seen that the HPV E6/E7 oncoprotein plays a major role therein. The main biological basis for the HPV E6/E7 oncoprotein to exert the efficacy is that HPV infects head and neck cells, integrates the DNA of the HPV into the host cell genome in a non-homologous recombination mode, realizes the expression of the oncoprotein after mRNA is transcribed, and then inhibits the functions of host oncostatin p53 and pRb respectively, thereby causing the canceration or the change of the radiation sensitivity of the host cells.

At present, the conventional HPV detection means is mainly detection of HPV DNA, and positive HPV DNA can only indicate that a host is infected by HPV and cannot indicate whether HPV viral genes are integrated with host genes and exert the effects of the HPV viral genes, so that the HPV E6/E7 gene product is used as positive evidence of HPV infection of head and neck tumors and can judge the sensitivity of tumor individuals to radiotherapy, and the detection is more specific than the detection of HPV DNA. However, since the existing protein detection technology is difficult to purify the corresponding antibody of recombinant non-denatured E6/E7 oncoprotein, polypeptide fragments or denatured protein are often used, which leads to low sensitivity (Tungteakhun SS, Duerksen-Hughes PJ. cellular binding partners of the human papillomavir E6 protein [ J ]. Arch Virol,2008,153(3): 397. 408.), limiting the application effect of HPV E6/E7 oncoprotein detection as HPV infection in head and neck tumors. With the progress of biological technology, recent studies have found that the integration of HPV with host genome and virus activity can be better reflected by detecting the mRNA expression activity of HPV E6/E7 oncogene, i.e., positive detection of HPV E6/E7mRNA indicates the presence of biologically active HPV (Schwartz S. Papilomavir transports and posttranscriptional regulation [ J ]. Virology,2013,445(1-2): 187-196.). Therefore, the transcriptional activity of the viral oncogenes can be more accurately reflected by measuring the expression level of HPVE6/E7 mRNA, thereby more accurately predicting the sensitivity of tumor individuals to radiation therapy.

Besides the influence of HPV infection on the radiotherapy prognosis of tumor individuals, the influence of HPV infection on the activity and the integration capability of HPV E6/E7 oncogene, the response of host genome to the regulation and control of HPV E6/E7 oncogene is also one of important reasons for determining the sensitivity of host cells to radiation and further influencing the radiotherapy prognosis. Recent studies have shown that high-risk HPV E6/E7 oncogenes can regulate the expression of common microRNAs (microRNAs/miRNAs) in host cells by controlling transcription factors c-Myc, p53 and E2F (Liu X, Dakic A, Chen R, et Al, cell-regulated expression by human papillomalus ligands with expression and expression of the htert promoter by Myc [ J ]. J Virol,2008,82(23):11568 and 11576; Al Moustafla AE, Foulkeys WD, Benlimamame N, et Al, E6/E7 proteins of HPV type 16 and erbb-2 gene expression vector of human genes [ biological J ]. 23, molecular J.: coding J.23. biological J.,350. molecular J.,358, 2015,15(3):213-218.), including the down-regulation of miRNA-21, 155, 221, etc. (Wald AI, Hoskins EE, Wells SI, et al., alteration of microrna profiles in squarosus cells of the Head and in cell lines by human papillomanavirus [ J ]. Head New, 2011,33(4): 504-512.; lajer CB, von Buchwald C, the role of human papillomavir in head and the like cancer [ J ]. APMIS,2010,118(6-7): 510-. Meanwhile, another study finds that p53 can up-regulate miRNA-34a so as to inhibit the expression of Bcl-2, influence the resistance of tissues to radiotherapy and increase the radiotherapy sensitivity of tissues (Liu C, Zhou C, Gao F, et al, mir-34a in age and tissue related radiation-sensitivity and serum mir-34a as a novel indicator of radiation in J. Int J Biol Sci,2011,7(2): 221-; miRNA-101 can down-regulate the expression level of DNA repair gene proteins such as ATM and DNA-PKcs and the like to increase the sensitivity of tumors to radiation (Wang Y, Wang J, Huang Y. Micrornas new biomakers for human papilloma virus related head and new burners [ J ]. Cancer Biomark,2015,15(3): 213-218.); miRNA-155 increases tumor resistance to radiotherapy by mediating the effects of hypoxia prevalent in solid tumors (Babar IA, Czochor J, Steinmetz A, et al. inhibition of hypoxia-induced mir-155 radiosensing hypoxic lung Cancer cells [ J ]. Cancer Biol Ther,2011,12(10): 908-914.); the change of expression of miRNA-106b can be observed after partial tumor cell radiotherapy, and the survival ability of tumor cells is increased through the mediation of angiogenesis effect by TGF-beta and other factors (Smith AL, Iwanaga R, Drasin DJ, et AL. the mir-106b-25cluster target smad7, activators TGF-beta signaling, and indenes emt and tumor inducing cell characterization down stream of six1 in human breast cancer [ J ]. Oncogene,2012,31(50): 5162-; MiRNA-21, 17-92, 221 and 222, etc., modulate radiation resistance of tumor cells by changes in autophagy-related proteins such as PTEN, pAKT, PI3K, etc. (Santekadur PK, Das SK, Gredler R, et al. Multifunctino protein biochemical binding 1(snd1) proteins tumor tissue peptides in human hepatocellular nuclear protein vaccine and mir-221[ J ] Biol Chem,2012,287(17): 13952-. Therefore, we further scientifically hypothesized that the HPV E6/E7 oncoprotein may up-regulate the sensitivity of tumor cells to radiotherapy by regulating the expression of certain radiotherapy-sensitive miRNAs in the host, such as miRNAs 17-92, 21, 34a, 101, 155, 106b, 221 and 222, thereby increasing the sensitivity of patients to radiotherapy and obtaining a better prognosis. Therefore, the study on the expression of host ray sensitivity related miRNA is helpful for judging the prognosis of HPV positive person radiotherapy.

Example one

1.1 study selection

New head and neck squamous carcinoma cases (including tumors of oral cavity, oropharynx and laryngopharynx) are selected for head and neck surgery in tumor hospitals of 12-month Henan province and tumor hospitals of Chinese medical academy of sciences in 2016 to 2018.

The inclusion criteria are as follows: 1) age above 18 years; 2) there is a definitive histological or cytological diagnosis, the morphology being squamous carcinoma. The following primary head and neck squamous carcinomas will be included in this study (encoded by international disease classification oncology): c00 lip; c01 tongue base; other parts of the C02 tongue; c03 gingival; c04 mouth bottom; c05 jaw; other parts of the C06 oral cavity; c09 tonsil; c10 oropharynx; c12 pear-shaped; c13 hypopharynx; c14 other parts of the lips, mouth, pharynx; c32 (including all subcategories); 3) positive HPV DNA detection; 4) the diagnosis time is no more than 6 months, and the patient is not treated by radiotherapy, chemotherapy or operation; 5) signing an informed consent, agreeing to questionnaire survey, collecting tumor tissues for the survey and receiving follow-up visits within the study time. Exclusion criteria: 1) one not meeting any of the above-mentioned conditions; 2) the following sites are not included: salivary glands (C07-08), nasopharynx (C11), thyroid (C73) and oesophagus (C15); 3) patients with cervical cancer, HIV or other sexually transmitted diseases and other systemic diseases, and patients after organ transplantation or using immunosuppressants for a long time.

The questionnaire survey mainly includes demographic information (age, sex, nationality, residence, education condition, etc.), smoking history, environmental smoking exposure history, drinking history, eating habits, history of various diseases, oral hygiene, sexual life history, etc.

A total of 121 subjects were collected and had a mean age of 58.53 ± 10.35 years. 103 cases in male (85.12%), 41 cases in overweight people (33.88%), 11 cases in obese people (9.09%), 58 cases in smokers (47.93%), 60 cases in drinker (49.59%), 45 cases in drinker (37.19%), 3 cases with brushing frequency <7 times (2.48%), 36 cases in denture wearer (29.75%), 22 cases with family history of tumor (18.18%).

Of 121 patients with head and neck tumors, oral cancer 27 (22.31%), laryngeal cancer 76 (62.81%), oropharyngeal cancer 18 (14.88%), and stage I to IV 34 (28.10%), 19 (15.70%), 30 (24.79%) and 34 (28.10%), respectively. The vast majority of surgeries (99.17%) were performed without chemotherapy (84.30%) and targeted therapy (98.35%). See table 1 for details.

TABLE 1 general condition of the study and clinical case data

(Note: BMI, body Mass index)

1.2 detection assay

Fresh tumor tissue was collected for cases meeting inclusion criteria and used for detection of HPV DNA. In each case, fresh pathological or surgical specimens are collected and frozen in a-80 ℃ freezer within 20 minutes of removal. Whether the tissue specimens were frozen immediately or not should be recorded on a recording sheet. And histological and/or immunohistochemical reports of the cases were taken from the pathologist. All cases were followed by telephone each year and their survival was recorded as well as further diagnosis, treatment and recurrence of metastases.

1.2.1 HPV DNA detection

By using

HPV genotyping technology is used for HPV DNA detection.

HPV genotyping technology amplifies characteristic sequences containing different HPV subtypes by PCR, then amplifies the corresponding mutation sites by specific single-base extension primers (extension primers), and in a ddNTP reaction system, the extension primers only amplify bases complementary to the specific mutation sites of the HPV to be detected, namely termination. And analyzing the final extension product by a Time of Flight (TOF) mass spectrometry system, and typing the characteristic HPV according to the molecular weight difference of different bases. The technology can simultaneously detect 24 HPV subtypes, including 7 low-risk subtypes (HPV 6, HPV 11, HPV 34, HPV 42, HPV 67, HPV 70 and HPV 81) and 17 high-risk subtypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV 82).

1.2.1.1 HPV Virus DNA extraction and quality control

HPV viral DNA in tissues was extracted using a commercial DNA extraction kit. Detecting OD value of all DNA samples by using a NanoDrop2000 instrument, detecting by using 1.25% agarose gel electrophoresis, and performing quality inspection to evaluate whether the DNA samples meet the requirements

The HPV typing DNA quality requirements and quality inspection standards are shown in Table 2.

TABLE 2 HPV Virus DNA extraction quality control standards

Quality control project	Standard of merit
		DNA concentration	More than 20ng/ul
OD260/280 value	2.2～1.6
		OD260/230 value	>0.6
OD230nm	No absorption peak
		Results of DNA electrophoresis	gDNA integrity without severe degradation

And then transferring the qualified sample to a 96-well plate as working solution, and storing at-20 ℃ for later use.

1.2.1.1 PCR amplification reactions

Specific sequences of different HPV subtypes are amplified through PCR, the PCR amplification sequences are shown in Table 3, and ACGTTGGATG labels are added to the 5' ends of the primers in order to ensure that the molecular weights of the primers are similar and the Tm value and the CG% content are in the optimal range.

TABLE 3 PCR amplification sequences

1) A1.5 ml EP tube was used to prepare a PCR master mix, and the tube was centrifuged at low speed with shaking. The reaction components are shown in table 4.

TABLE 4 PCR master mix reaction-related reagent formulation components

2) Using an 8-channel or 12-channel pipette, add 4. mu.l of PCR master mix to each well of 384-well plate, add 1. mu.l of template DNA (20 ng/. mu.l), mix well, cover the 384-well plate carefully, and press each well tightly to prevent evaporation during the PCR procedure. Centrifuge at 1000rpm for 1 min.

3) The PCR reaction plate was placed on a PCR machine according to the following PCR amplification reaction procedure (Table 5), and the procedure was started.

TABLE 5 PCR amplification reaction procedure

94℃	5min
			94℃	20sec
56℃	30sec	45cycles
			72℃	1min
72℃	3min
			4℃	∞

1.2.1.2 alkaline phosphatase (SAP) reaction

In this step, dNTPs in the PCR reaction system are phosphorylated by alkaline phosphatase treatment.

1) After the PCR reaction was completed, the PCR product was treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs from the system.

2) Alkaline phosphatase treatment reaction solutions were prepared in new 1.5ml EP tubes, and SAP Mix reaction components are shown in Table 6.

TABLE 6 SAP reaction Components

SAP mix of Reagent	Concentration	Volume(1rxn)
			Water(HPLC grade)	NA	1.53μl
SAP Buffer	10x	0.17μl
			SAP Enzyme	1U/μl	0.30μl
Total volume	-	2.00μl

3) SAP mix was added to 384-well PCR reaction plates in a total reaction volume of 7. mu.l, 5. mu.l of PCR product and 2. mu.l of SAP mix per alkaline phosphatase treated reaction well.

4) After the completion of pipetting, 384-well sealing plates were carefully covered and each well was firmly pressed to prevent evaporation and the like during the PCR procedure, and the following reaction procedure was carried out after centrifugation.

5) Setting up SAP reaction program: 20min at 37 ℃; 5min at 85 ℃; infinity at 4 ℃. And a 384 well reaction plate was placed on the PCR instrument and the procedure was started.

1.2.1.3 Single base extension reactions

Single base extension reactions were performed in ddNTP systems by UEP primers (see Table 7) to form single base extension products complementary to the specific mutation sites of the specific HPV viruses to be detected.

1) After the alkaline phosphatase treatment, the single-base extension reaction was carried out in a total volume of 9. mu.l.

2) Single base extension reaction solution was prepared in a new 1.5ml EP tube, and the EXTEND Mix reaction solution components are shown in Table 8.

TABLE 7 UEP primers

TABLE 8 Single-base extension reaction solution Components

3) EXTEND Mix was added to 384 well reaction plates. For each reaction well, the single base extension reaction system is shown in table 9.

TABLE 9 Single-base extension reaction System

Reagents	Volume(μl)
		EXTEND Mix	2
SAP+PCR reaction	7
		Total Volume[μl]	9

5) The set-up extension reaction procedure is shown in table 10.

TABLE 10 elongation reaction procedure

1.2.1.4 resin purification

1) The resin was uniformly filled in an 384/6MG Dimple plate and left to dry for 10 minutes.

2) Add 16. mu.L of water to each well of 384 sample plates.

3) The 384 sample plate was gently inverted and snapped onto the sample plate and then tapped to drop the resin into each well of the sample plate.

4) The 384 sample plates were placed in a tumbling centrifuge and spun for 30 minutes at room temperature.

1.2.1.5 chip spotting

Starting up

The Nanodiproperser RS1000 spotter transfers the resin purified extension product to 384-well SpectroCHIP bioarray.

1.2.1.6 Mass Spectrometry detection and data output

Analyzing the spotted SpectroCHIP chip by using a MALDI-TOF mass spectrometer, acquiring original data and a genotyping chart by using TYPER4.0 software according to a detection result, checking the integrity and correctness of a data file, storing the result into a corresponding storage medium and submitting the result to a biological information room for analysis.

1.2.2 HPV E6/E7mRNA detection

The HPV E6/E7mRNA is detected by an RT-qPCR method.

The method mainly comprises three steps of mRNA extraction, reverse transcription and fluorescence real-time quantitative detection:firstly, Trizol is added in the process of sample cracking or homogenizing, chloroform is added for extraction after the sample is fully cracked to release RNA, and the extracted RNA exists in an aqueous phase layer. After collecting the aqueous layer, RNA was precipitated using isopropanol to obtain Total RNA. The isolated mRNA was then reverse transcribed into cDNA (primers are shown in Table 11). The method adds poly (T) and random primer into reverse transcription system, uses MMLV reverse transcriptase to reverse transcribe all RNA into cDNA to maximum extent for subsequent fluorescent quantitative detection. And finally, quantitatively detecting the reverse transcription product by using a SYBR Green I RT-qPCR method. SYBRGreen I is a double stranded DNA binding dye that binds in the minor groove. After binding to double-stranded DNA, its fluorescence is greatly enhanced. With the continuous accumulation of PCR reaction products, the fluorescence signal intensity is proportionally increased, and a fluorescence amplification curve is finally obtained by monitoring the fluorescence intensity in real time in the PCR reaction process. Comparison of Ct values of target mRNA and reference RNA (. beta. -actin) by use of 2^-ΔΔCtThe relative expression quantity of the target mRNA is calculated by the method.

TABLE 11 HPV E6/E7mRNA reverse transcription primers

1.2.3 MiRNA detection

The miRNA is also detected by an RT-qPCR method.

The method mainly comprises three steps of miRNA extraction, miRNA tailing and reverse transcription and fluorescence real-time quantitative detection: firstly, Total RNA is extracted from a sample, the obtained Total RNA passes through a miRNA enrichment column, miRNA is adsorbed on a membrane under the action of the adsorption column, and RNase Free H is used₂And O, eluting the miRNA on the adsorption column to obtain the enriched miRNA. Then, miRNA tailing and reverse transcription. Since miRNAs are short (18-22nt) and do not have a poly (A) tail, a tailing reaction is required before reverse transcription. The method comprises polymerizing E.coli poly (A)The synthase adds poly-A tail poly (A) at the 3' end of miRNA, then uses oligo (dT) -univarial tag universal reverse transcription primer (see table 12) to carry out reverse transcription reaction (see table 13), finally generates cDNA chain corresponding to miRNA. Finally, the reverse transcription products were quantitatively determined by SYBR Green I RT-qPCR (see tables 14-15). SYBR Green I is a double stranded DNA binding dye that binds in the minor groove. After binding to double-stranded DNA, its fluorescence is greatly enhanced. With the continuous accumulation of PCR reaction products, the fluorescence signal intensity is proportionally increased, and a fluorescence amplification curve is finally obtained by monitoring the fluorescence intensity in real time in the PCR reaction process. Comparison of Ct values of target miRNA and internal reference RNA (U6) by using 2^-△△CtThe relative expression quantity of the target miRNA is calculated by the method.

Reverse transcription primer of table 127 radiation-sensitive miRNAs

TABLE 13 reverse transcription System and procedure

TABLE 14 SYBR Green I PCR System

TABLE 15 PCR reaction procedure

1.3 data management and statistical analysis

All data were checked and statistically analyzed using SAS 9.4 software. The positive rates of HPV DNA, HPVE6/E7 mRNA and host miRNA of the head and neck squamous carcinoma and sub-parts thereof are respectively calculated, and the results are shown in tables 16-18.

TABLE 16 HPV DNA Positive detection in the population of different tumor sites

As can be seen from Table 16, among 121 subjects, the HPV infection rate was 66.94%, and the highest HPV infection rate was HPV52 (65.29%). As representative of high-risk HPV, the infection rate of HPV16 was 4.13% each, which was much lower than the analysis result of 28 previous studies on head and neck tumors (24.7%) (Guo L, Yang F, Yin Y, et al. Presence of human cervical type-16in head and neck tumors and neck cancer amplification: A meta-analysis [ J ]. Front Oncol,2018,8 (619.); infection rate of HPV18 was 6.61% each, which was slightly higher than the analysis result of 1881 head and neck tumors in 19 previous studies (6.0%) (Guo L, Yang F, Yin Y, et al. prediction of human cervical type-16in head and neck tumors [ A-amplification ] A-8. Frontol, 619.) (Guo L, Yang F, Yin Y, et al. prediction: 2018).

TABLE 17 Positive rates for HPV E6/E7mRNA in a population of different tumor sites

Recent studies have found that the integration of HPV with the host genome and viral activity can be better reflected by detecting the mRNA expression activity of HPV E6/E7 oncogene, i.e., positive detection of HPV E6/E7mRNA indicates the presence of biologically active HPV (Schwartz S. Papillomavir transcripts and posttranscriptional regulation [ J ]. Virology,2013,445(1-2): 187-196.). Thus, HPV E6/E7mRNA as evidence of positive HPV infection of head and neck tumors should theoretically be more specific than the detection of HPV DNA. As can be seen from Table 17, the HPV E6/E7mRNA is detected by the RT-qPCR method, and the positive rate of the HPV E6/E7mRNA in the research object is 28.93 percent and is far lower than that of HPV DNA (66.94 percent).

Dividing the patients with head and neck tumor into high expression group and low expression group by taking the average value of the miRNA expression ratio in cancer and cancer adjacent tissue as a demarcation point. The results are shown in Table 18. The invention adopts an RT-qPCR method to detect 7 radiotherapy sensitive miRNAs (miRNA 21, 34a, 101, 105, 106b, 221 and 222), and as can be seen from Table 18, the patient is divided into a high expression group and a low expression group by taking the average value of the miRNA expression ratio in the cancer and the paracancer tissues as a dividing point, and the high expression rate range of the 7 miRNAs is 23.97-40.50%.

TABLE 18 high expression rates of miRNA in populations with different tumor sites

TABLE 19 detection of high expression of miRNA in populations with different HPV infections

Besides the influence of HPV infection on the radiotherapy prognosis of tumor individuals, the influence of the activity and the integration capability of HPV E6/E7mRNA, the reaction of the expression of common miRNA in host cells on the regulation of HPV E6/E7mRNA is also one of important reasons for determining the sensitivity of the host cells to rays so as to influence the radiotherapy prognosis. As can be seen from Table 19, the high expression rates of miRNA in HPV positive persons are all higher than those of HPV negative persons, and the differences are all statistically significant (P < 0.001); the high expression rate of miRNA in HPV E6/E7mRNA positive persons is higher than that of HPV E6/E7mRNA negative persons, but the difference is not statistically significant (P >0.05), which indicates that the miRNA expression is influenced by HPV infection.

Clinical characteristics of cases, HPV DNA negative and positive groups, HPV E6/E7mRNA negative and positive groups and median survival time of different miRNA expression levels are calculated by using a one-factor Kaplan-Meier method, and results are shown in Table 20, so that candidate influencing factors of the radiotherapy prognosis survival rate of the head and neck squamous cell carcinoma are obtained.

TABLE 20 Single factor analysis results

From table 20, it can be seen that age, clinical stage, high risk type HPV infection (including HPV35, HPV52, HPV 59), HPV E6/E7mRNA, seven radiation sensitive mirnas (miRNA 21, miRNA 34a, miRNA 101, miRNA 105, miRNA 106b, miRNA 221, and miRNA 222) are candidate contributors to the prognosis survival rate for radiotherapy of head and neck squamous carcinoma (P < 0.05).

And (4) incorporating the candidate variables of the result of the single-factor analysis into the multi-factor regression analysis. Two variables, age and clinical stage, were adjusted and the results are shown in table 21. Overall Survival (OS) is the time from the first radiation treatment to the time of death (death case) or the last follow-up (non-death or missed-visit, by 8/31 days 2020). The significance level was taken as bilateral α ═ 0.05, with P <0.05 being statistically significant. As seen from table 21, compared to the HPV E6/E7mRNA negative and miRNA 21 low expression group, the HPV E6/E7mRNA positive or miRNA 21 high expression group had better prognosis (HR 0.28, 95% CI 0.11-0.70), and the HPV E6/E7mRNA positive and miRNA 21 high expression group had the best prognosis (HR values could not be calculated due to no death cases), thereby co-expressing HPV E6/E7mRNA and miRNA 21 as the independent prognostic survival influencing factor for head and neck tumors.

TABLE 21 multifactor analysis results

Finally, HPV E6/E7mRNA was combined with seven radiation-sensitive miRNA expression results and then subjected to regression analysis, with the results shown in Table 22. As can be seen from Table 22, the HPV E6/E7mRNA is positive and the miRNA high expression group has the best prognosis, and then the HPV E6/E7mRNA positive or miRNA high expression group has the worst prognosis and is HPV E6/E7mRNA negative and miRNA low expression group, and the survival curve of 7 groups is shown in figure 1, and the HPV E6/E7mRNA positive and miRNA 21 high expression group has the best survival (P < 0.001).

TABLE 22 HPV E6/E7mRNA and seven radiation-sensitive miRNAs combined analysis results

The above-mentioned embodiments are merely preferred embodiments of the present invention, which are merely illustrative and not restrictive, and it should be understood that other embodiments may be easily made by those skilled in the art by replacing or changing the technical contents disclosed in the specification, and therefore, all changes and modifications that are made on the principle of the present invention should be included in the scope of the claims of the present invention.

SEQUENCE LISTING

<110> Ohwian Oncology academy of Chinese medicine tumor Hospital

Application of <120> miRNA 21 in preparation of reagent for judging survival rate of HPV-infected head and neck squamous cell carcinoma patient after radiotherapy

<130> 2021

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 22

<212> DNA

<213> Artificial sequence

<400> 1

cggtcgtagc ttatcagact ga 22

Claims

The application of miRNA 21 in preparing a reagent for judging the survival rate of head and neck squamous cell carcinoma patients infected by HPV after radiotherapy, which is characterized in that the reagent is used for quantitatively detecting the expression quantity of miRNA 21.
2. The use of claim 1, wherein the reagent comprises a primer designed based on the miRNA 21 and used for reverse transcription.
3. The use of claim 1, wherein the primer sequence is as shown in SEQ ID No. 1.
4. A kit for judging the survival rate of a head and neck squamous carcinoma patient infected by HPV after radiotherapy is characterized by comprising a reagent for quantitatively detecting the miRNA 21 expression quantity.
5. The kit of claim 4, wherein the reagents comprise primers designed based on the miRNA 21 and used for reverse transcription.
6. The kit according to claim 4, wherein the primer sequence is shown as SEQ ID No. 1.
7. A screening method for judging influencing factors of the survival rate of patients suffering from HPV-infected head and neck squamous cell carcinoma after radiotherapy is characterized by comprising the following steps:

step 1, collecting a plurality of fresh tumor tissue specimens of new head and neck squamous carcinoma cases meeting the standard, storing the specimens in a refrigerator at the temperature of-80 ℃, and carrying out HPV DNA detection; the HPV virus comprises 7 low-risk subtypes and 17 high-risk subtypes, wherein the 7 low-risk subtypes are HPV6, HPV 11, HPV 34, HPV 42, HPV 67, HPV 70 and HPV 81, and the 17 high-risk subtypes are HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 and HPV 82;

step 2, carrying out HPV E6/E7mRNA and 7 kinds of ray-sensitive miRNA detection on the positive cases of HPV DNA detection, and analyzing the relation between HPV infection and miRNA high expression rate; the 7 radiation-sensitive miRNAs are miRNA 21, miRNA 34a, miRNA 101, miRNA 155, miRNA 106b, miRNA 221 and miRNA 222;

step 3, calculating the positive rates of HPV DNA, HPV E6/E7mRNA and miRNA of head and neck squamous cell carcinoma and sub-parts thereof in the case, and judging the influence of HPV infection on miRNA expression;

step 4, calculating clinical characteristics of cases, HPV DNA negative and positive groups, HPV E6/E7mRNA negative and positive groups and median survival time of different miRNA expression levels by using a single-factor Kaplan-Meier method to obtain candidate influencing factors of the radiotherapy prognosis survival rate of the head and neck squamous cell carcinoma; the clinical characteristics of the cases included gender, age, location of cancer and clinical stage;

and 5, carrying out multi-factor regression analysis according to candidate influence factors of the radiotherapy prognosis survival rate obtained by single-factor analysis, and finally determining the influence factors of the radiotherapy prognosis survival rate of the head and neck squamous cell carcinoma.
8. The screening method according to claim 7, wherein the HPV DNA detection is by HPV genotyping.