CN111057790B

CN111057790B - Application of miRNA in preparation of kit for detecting KSHV latent infection

Info

Publication number: CN111057790B
Application number: CN201911266332.3A
Authority: CN
Inventors: 曾妍; 郑军; 徐江; 张铁军; 赵娟; 周政; 袁武梅; 刘振球; 方绮雯
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2022-08-30
Anticipated expiration: 2039-12-11
Also published as: CN111057790A

Abstract

The invention discloses application of miRNA in preparation of a kit for detecting KSHV latent infection, and belongs to the technical field of medical biology. Wherein the miRNA is selected from at least one of miR-155 and miR-485-5 p. The invention also discloses application of a reagent for quantitatively detecting the miRNA expression quantity in preparing a kit for detecting KSHV latent infection, and the kit for detecting KSHV latent infection comprises the reagent for quantitatively detecting the miRNA expression quantity. The invention can be used for detecting by using peripheral blood of an object, is noninvasive, convenient and quick, and has important clinical application value.

Description

Application of miRNA in preparation of kit for detecting KSHV latent infection

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to application of miRNA in preparation of a kit for detecting KSHV latent infection.

Background

Kaposi's Sarcoma (KS) is a malignant tumor with lesions mostly occurring in soft tissues, with endothelial cells as the major source. KS can be classified into classic, AIDS-related form (AIDS-KS), immunosuppressive form and African preformance form according to epidemiological and clinical characteristics. Typical KS mainly occurs in Uygur and Kazak in Xinjiang in China.

The etiological basis for the onset of KS was the infection with Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpes Virus type 8 (HHV-8), first discovered by Chang et al from the sarcoma tissue of AIDS-KS patients. Subsequently, high expression of KSHV was found in each type of KS. KSHV is found to be an essential factor and a main factor of the occurrence of Kaposi's sarcoma and is closely related to the occurrence of KS.

KSHV infects cells followed by two ways, persistent latent infection and transient lytic infection. For latent infections, accurate detection is often difficult.

mirnas are a class of small non-coding RNAs of about 23 nucleotides in length. It is widely found in multicellular organisms. miRNA complementarily binds to target mRNA 3' UTR after gene transcription, degrading the target mRNA or inhibiting protein translation. They are involved in signal transduction, proliferation, apoptosis, invasion or tumor angiogenesis. There are many mature studies in the fields of oncology, virology, neurobiology, embryonic development, etc.

However, there is currently a lack of research and application for the detection of latent infection with KSHV using miRNA.

Disclosure of Invention

The invention aims to solve the technical problems and provides the application of miRNA in preparing a KSHV latent infection detection kit. The invention utilizes 4 cases of tumor tissues and paraneoplastic normal tissues of typical KS patients to carry out screening of microRNA chips, further through RT-qPCR verification, miR-485-5p and miR-155 are unexpectedly found to be used as markers for detecting KSHV latent infection, and thus the invention is completed.

The invention provides application of miRNA in preparing a kit for detecting KSHV latent infection, wherein the miRNA is selected from at least one of miR-155 and miR-485-5 p.

In some embodiments of the invention, the miRNA is selected from two of miR-155 and miR-485-5 p.

In some embodiments of the invention, the miRNA is derived from peripheral blood.

The second aspect of the invention provides application of a reagent for quantitatively detecting the expression quantity of miRNA in preparing a kit for detecting KSHV latent infection, wherein the miRNA is selected from at least one of miR-155 and miR-485-5 p.

In some embodiments of the present invention, the expression amount refers to an expression amount of the miRNA in peripheral blood.

In some embodiments of the present invention, the reagent for quantitatively detecting the expression amount of miRNA is a real-time fluorescent quantitative PCR related reagent. In other embodiments of the present invention, the reagent for quantitatively detecting the expression amount of miRNA is a miRNA sequencing reagent.

In a third aspect, the invention provides a kit for detecting KSHV latent infection, which comprises a reagent for quantitatively detecting the expression amount of miRNA, wherein the miRNA is selected from at least one of miR-155 and miR-485-5 p.

In some embodiments of the present invention, the reagent for quantitatively detecting the expression amount of miRNA is a real-time fluorescent quantitative PCR related reagent. In some embodiments of the invention, the real-time fluorescent quantitative PCR-related reagents comprise a primer combination capable of specifically amplifying a reverse transcription product of the miRNA. In some preferred embodiments of the invention, the primer combination capable of specifically amplifying miR-485-5p comprises an upstream primer having a nucleotide sequence shown in SEQ ID No.5 and a downstream primer having a nucleotide sequence shown in SEQ ID No. 6; the primer combination capable of specifically amplifying miR-155 comprises an upstream primer with a nucleotide sequence shown in SEQ ID No.9 and a downstream primer with a nucleotide sequence shown in SEQ ID No. 10.

In some embodiments of the invention, the real-time fluorescent quantitative PCR related reagent further comprises a fluorescent dye. In other embodiments of the present invention, the real-time fluorescent quantitative PCR related reagent further comprises a probe capable of specifically hybridizing to a reverse transcription product of the miRNA.

In other embodiments of the present invention, the reagent for quantitatively detecting the expression amount of miRNA is a miRNA sequencing reagent.

The invention has the advantages of

Compared with the prior art, the invention at least has the following technical effects:

1. the invention can be detected in the KSHV latent infection stage, thereby intervening in the early stage of the experiment and having important clinical application value.

2. The invention can detect by using the peripheral blood of the patient, and is noninvasive, convenient and quick.

3. The invention provides a novel marker for detecting KSHV latent infection, which is suitable for clinical application and can provide better assistance for clinically treating KSHV.

Drawings

FIG. 1 shows the expression levels of miR-485-5p, miR-374a-5p and miR-155 in cells infected with KSHV relative to cells not infected with KSHV. BAC represents iSLK-BAC cells, and 219 represents iSLK-219 cells.

FIG. 2 shows expression levels of miR-485-5p, miR-374a-5p and miR-155 in KS patients, KSHV positive patients relative to healthy controls.

FIG. 3 shows the results of ROC curve analysis of miR-155 between KS patients and healthy controls.

FIG. 4 shows the results of ROC curve analysis of miR-485-5p between KS patients and healthy controls.

FIG. 5 shows the results of ROC curve analysis of the combination of miR-155 and miR-485-5p between KS patients and healthy controls (KSHV negative).

FIG. 6 shows the results of ROC curve analysis of miR-155 between KSHV positive patients and healthy controls.

FIG. 7 shows the results of ROC curve analysis of miR-485-5p between KSHV positive patients and healthy controls.

FIG. 8 shows the results of ROC curve analysis of the combined detection of miR-155 and miR-485-5p between KSHV positive individuals and healthy controls (KSHV negative).

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.

Examples

The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples, unless otherwise specified, were all conventional laboratory instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1miRNA chip analysis

1 tissue sample Collection

Tumor tissues and paratumoral normal tissues of 14 Kaposi sarcoma patients (KS patients) who were examined in the sixth national hospital of Uygur autonomous area of Xinjiang were collected, and immediately stored in liquid nitrogen after surgical excision, Kaposi sarcoma was confirmed by histological examination of all specimens, and immunohistochemical experiments were performed on all specimens using murine monoclonal antibody HHV-8[ LN35] (1: 50; Abcam, Cambridge, UK). Meanwhile, peripheral blood samples of KS patients and KSHV latently infected patients (KSHV positive) were drawn, and another 10 healthy individuals (KSHV negative) were drawn as controls. Wherein, the latently infected patients are determined by detecting KSHV latenti protein LANA and cleavage phase protein RTA, and in the invention, 17 patients are determined to be KSHV latenti-infection by detecting the expression of the LANA protein and the non-expression of the RTA protein.

Kaposi's sarcoma tumor and paraneoplastic tissue (28 samples in total) were used for miRNA chip analysis, and peripheral blood samples (14 patients with KS, 17 patients with KSHV latent infection, 10 healthy individuals) were used for RT-qPCR analysis.

2 taking tissue specimens

(1) After patient consent and signature were obtained, all patients were photographed in skin lesion areas and disease information was retained in detail and then skin biopsies were performed in biopsy rooms.

(2) The skin is disinfected conventionally, and the materials are taken after local anesthesia for several minutes.

(3) The knife is used as a prismatic incision, the knife is perpendicular to the skin, and the incision direction is consistent with the dermatoglyph.

(4) The incision should reach the subcutaneous tissue, 2cm long tumor tissue and 0.5cm wide tumor tissue are taken, normal tissue around the tumor is cut, 0.5cm x 0.5cm tumor tissue is reserved and put into 10% formaldehyde fixing solution for pathological diagnosis, and the rest tumor tissue and tissue around the tumor are respectively put into a cold storage tube and stored in liquid nitrogen. And marked accordingly.

(5) The tissue is not damaged as much as possible so as not to influence the quality of the specimen.

(6) The incision is sutured.

(7) And (5) disconnecting the lines after 7 days.

3miRNA chip analysis

Using a seventh generation miRCURY LNA ^TM Array (v.18.0) (Exiqon, Copenhagen, Denmark) contains 3100 capture probes covering all human, murine and all viral miRNAs, and in addition, 25 miR Plus ^TM Human miRNA.

3.1RNA extraction

1) Tissue sample homogenate

The tissue samples were first crushed by freezing using a Biopulverizer crusher and then homogenized by Mini-Bead-Beater-16.

2) Two-phase separation: the homogenized tissue samples were incubated for 5min at room temperature, then 0.2mL of chloroform was added to 1mL of samples, shaken vigorously for 15sec, and incubated for 3min at room temperature. Centrifuge at 12,000 Xg for 15min at 4 ℃.

3) And (3) RNA precipitation: the upper aqueous phase was transferred to a new centrifuge tube, then 100% alcohol was added to the new tube in an amount of 1.5 times the volume of the tube, and the tube was inverted upside down to mix thoroughly.

4) A700. mu.L sample was aspirated, added to a 2mL Rneasy Mini spin collection column, centrifuged at room temperature ≥ 8000 Xg for 15sec, and the supernatant discarded. If the liquid is larger than 700 mu L, sucking the rest sample to repeat the fourth step, and discarding the supernatant.

5) To a 2mL Rneasy Mini spin collection column was added 700. mu.L of Buffer RWT, centrifuged at room temperature ≥ 8000 Xg for 15sec, and the supernatant discarded.

6) 500ul RPEBuffer was added to the Rneasy Mini pin collection column, centrifuged at room temperature ≥ 8000 Xg for 15sec, and the supernatant discarded.

7) The Rneasy Mini spin collection column was transferred to a new 2mL centrifuge tube and centrifuged at full speed for 1 min.

8) The Rneasy Mini spin collection column was transferred to a new 1.5mL centrifuge tube, 20. mu.L of RNase-free water was added directly to the membrane of the Rneasy Mini spin collection column, and centrifuged at room temperature ≥ 8000 Xg for 1 min. .

9) The eluted RNA is subjected to RNA quality detection.

3.2RNA quality detection

1) Ultraviolet absorption measurement method: use of

ND-1000 determination of RNA concentration and purity

And (3) concentration determination: a reading at 260nm of 1 indicates 40ng RNA/. mu.L. The formula for calculating the RNA concentration of the sample is as follows: A260X 40 ng/. mu.L. If RNA is dissolved in 20 μ L DEPC water and 1 μ L is taken for the assay, the concentration of RNA is 65.003 × 40ng/μ L-2600.12 ng/μ L, measured as a 260-65.003. The remaining sample RNA was 19. mu.L, and the total amount of remaining RNA was: 19 μ L × 2600.12ng/μ L49.4 μ g.

And (3) purity detection: the A260/A280 ratio ranged from 1.8 to 2.1, indicating purity.

4, the total RNA extraction and quality inspection results of KS tumor and paraneoplastic tissues are ideal, the A260/A280 value of the total RNA is between 1.8 and 2.1, and the A260/A230 is more than 1.8.

2) Denaturing agarose gel electrophoresis

Firstly, preparing denatured agarose gel, taking 3 mu g of RNA sample and mixing with a sample dye solution with 3 times volume, and adding EB into the sample dye solution until the final concentration is 10 mu g/mL. Heating the mixed sample dye liquor to 70 ℃, incubating for 15min, then spotting on agarose gel, carrying out electrophoresis at a voltage of 5-6V/cm, stopping electrophoresis when the bromophenol blue indicator enters 2-3 cm, taking out the gel, observing under ultraviolet transmitted light, and taking a picture.

The electrophoresis bands 18S and 28S of the total RNA are clear, which shows that the nucleic acid components are complete, and the nucleic acid components are not degraded and polluted by protein, organic solvent and the like.

3.3 labeling of miRNA

Reagents and kits: mircurY TM Array Power laboratory kit (Cat #208032-A, Exiqon)

1) Thawing all reagents except enzyme on ice for 15-20min, shaking, mixing, and centrifuging;

2) preparing reaction liquid according to the following table, fully and uniformly mixing, and operating all operations on ice;

3) incubating the reaction solution for 30min at 37 ℃;

4) after the reaction is stopped at 95 ℃, immediately placing the reaction tube on ice for 2-15min, and centrifuging for a short time;

5) preparing CIP reaction liquid according to the following table, adding a fluorescence labeling reagent into a reaction tube, and fully and uniformly mixing:

6) incubating the reaction tube at 16 ℃ in a dark place for 1 h;

7) the reaction tube was incubated at 65 ℃ for 15min and the labeling reaction was stopped, after gentle centrifugation, the reaction tube was placed in a 4 ℃ freezer.

3.4 hybridization of miRNA chips

1) Chip hybridization solutions were prepared according to the following table:

2) incubating the hybridization solution at 95 ℃ in the dark for 2 min;

3) incubating, standing on ice for 2-15min, and mixing;

4) taking out the cover glass;

5) mixing miRCURY ^TM Placing the chip on a cover glass, and leaving a gap in the middle to form a hybridization chamber;

6) the hybridization chamber consisting of the chip and the cover fragment was filled in a heat-shrinkable hybridization bag of 3.1cm × 9 cm.

7) The opening of the heat-shrinkable hybridization bag was clamped with a metal clamp, and the bag was rapidly immersed in hot water at 95 ℃ to shrink and tightly wrap the hybridization chamber.

8) Taking out the heat-shrinkable hybridization bag, wiping off water outside the bag, shearing off the excess part at the top end of the hybridization bag by using scissors, and then drying the hybridization bag in a drying oven at 50 ℃ for more than 10 min.

9) 180ul of hybridization mixture was added from the well and the solution was supplemented with 1 Xhybridization buffer to a level of about 0.5cm from the top.

10) The second heat-shrinkable hybridization bag is cut to half and vertically sleeved on the first heat-shrinkable hybridization bag provided with the hybridization chamber.

11) The top of the hybridization chamber was held with tweezers and the whole was immersed vertically in hot water at 95 ℃.

12) The hybridization chamber was placed in a 56 ℃ oven and placed on a shaker overnight.

3.5 chip washing

After hybridization, the chip was removed from the hybridization bag, washed first with washing solution A at 56 ℃ for 2min, then with washing solution B at room temperature gently to wash the chip, then with washing solution B at room temperature for 2min, then with washing solution C at room temperature to wash the chip for 2mim, then with water to wash the chip, finally spin-dry the chip at 1000rpm for 5min, and immediately scan the chip after drying.

3.6MiRNA chip scanning and analysis

The chip was image scanned using the Axon GenePix 4000B chip scanner and the chip results were analyzed for raw data using Gene Pixpro V6.0.

The method comprises the following steps:

the green signal intensity of each probe on the chip was back-set and averaged over 4 replicate probes. And (3) carrying out standardization processing on the data by adopting a median standardization method to further obtain the standardized data, selecting a non-control probe with the corrected value (foreground value-background value) of 50 on each chip for standardization, and carrying out standardization processing on the points of the whole chip by taking the median of the part of probes as a standardization factor, namely, taking each miRNA corrected value/median value as a standard value. After normalization, differentially expressed mirnas were calculated using T-test. And finally, carrying out clustering analysis and correlation analysis on the miRNA chip data of the differential expression.

3.7 statistical analysis

The scanned images were input into GenePix Pro 6.0 software to achieve grid calibration and data extraction. The miRNAs were averaged and samples with miRNAs intensity > 30 in all samples were selected for calculation of normalization factor. Data were normalized using median. Mirnas that differed by a fold greater than 2 and p <0.05 in the 2 group samples were considered statistically different.

The results show that: of the 3100 miRNA probes, 185 differentially expressed miRNAs, including 74 up-and 111 down-regulated miRNAs, the most significant up-regulated miRNAs were hsa-miR-485-5p, hsa-miR-155, and hsa-miR-374a-5p (as shown in the table below).

miRNAs	Differential expression fold (n ═ 4)
		hsa-miR-485-5p	6.73±0.24
hsa-miR-155	3.77±4.05
		hsa-miR-374a-5p	2.34±0.89

Example 2 real-time fluorescent quantitative PCR (RT-qPCR) validation

Cells iSLK-BAC and iSLK-219 are infected by KSHV respectively, the expression of miR-485-5p, miR-374a-5p and miR-155 in the infected cells is detected, and the iSLK-PURO of uninfected KSHV cells is used as a control.

Meanwhile, the expressions of miR-485-5p, miR-374a-5p and miR-155 in the serum of a KS patient and the serum of a KSHV positive patient (KSHV latent infection) are respectively detected, and a KSHV negative healthy individual is used as a control.

miR-485-5p stem loop primer sequence (SEQ ID No. 1):

5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAATTC-3'。

miR-374a-5p stem-loop primer sequence (SEQ ID No. 2):

5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACTTA-3'。

miR-155 stem-loop primer sequence (SEQ ID No. 3):

5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACCCCTAT-3'。

internal reference U6 stem loop primer sequence (SEQ ID No. 4):

5'-CGCTTCACGAATTTGCGTGTCAT-3'。

miR-485-5p real-time fluorescent quantitative PCR primer:

upstream: 5'-CGAGAGGCTGGCCGTGAT-3' (SEQ ID No.5),

downstream: 5'-AGTGCAGGGTCCGAGGTATT-3' (SEQ ID No. 6).

miR-374a-5p real-time fluorescent quantitative PCR primer:

upstream: 5'-CGCGCGTTATAATACAACCTGA-3' (SEQ ID No.7),

downstream: 5'-AGTGCAGGGTCCGAGGTATT-3' (SEQ ID No. 8).

miR-155 real-time fluorescent quantitative PCR primer:

upstream: 5'-ACACTCCAGCTGGGTTAATGCTAATCGTGAT-3' (SEQ ID No.9),

downstream: 5'-CCAGTGCAGGGTCCGAGGT-3' (SEQ ID No. 10).

Internal reference U6 real-time fluorescent quantitative PCR primer:

upstream: 5'-GCTTCGGCAGCACATATACTAAAAT-3' (SEQ ID No.11),

downstream: 5'-CGCTTCACGAATTTGCGTGTCAT-3' (SEQ ID No. 12).

1 extraction of MiRNA from samples

1) The sample was added with the lysate MZ, and 200. mu.L of each of the sample and the lysate MZ was mixed and shaken well on a shaker for 30 sec.

2) The mixed solution was allowed to stand for 5min, which was intended to disperse the nucleic acid protein complex sufficiently.

3) The following procedure was performed at room temperature, the supernatant was centrifuged and placed in RNase-free centrifuge tubes at 12,000rpm for 10 min.

4) Chloroform 200. mu.L, mixed well, shaken vigorously for 15sec, and left to stand at room temperature for 5 min.

5) Setting the rotating speed at 12,000rpm, centrifuging for 15min, enabling RNA to be on the uppermost layer of the three layers, transferring the RNA on the upper layer into a centrifuge tube without RNase, and preserving liquid for later use.

6) Adding 66 mu L of absolute ethyl alcohol into the preserved liquid, wherein the added absolute ethyl alcohol is about 1/4 of the total volume, ensuring that the solution is fully mixed before being transferred into the adsorption column miRspin, sometimes leading the solution to generate precipitate, standing at room temperature for 2min, setting the rotating speed at 12,000rpm, centrifuging for 15sec, discarding the adsorption column miRspin after centrifugation, and preserving the liquid for later use.

7) Adding 132 μ L of anhydrous ethanol into the stored liquid, wherein the added anhydrous ethanol is about 1/2 of the total volume, ensuring that the solution is fully mixed before transferring into an adsorption column mirplus, sometimes leading the solution to precipitate, standing at room temperature for 2min, setting the rotating speed at 12,000rpm, centrifuging for 30sec, discarding the effluent after centrifuging, and keeping the adsorption column miRspin.

8) Adding 500 μ L deproteinized solution MRD into adsorption column miRelute, standing for 2min, centrifuging at 12,000rpm for 30sec, and discarding the liquid.

9) The rinse solution RW (600. mu.L) was added to the adsorption column miRelute, and the mixture was allowed to stand for 2min at a rotation speed of 12,000rpm for 30sec, and the waste solution was discarded.

10) Operation step 9) is repeated.

11) The adsorption column miRelute was placed in a 2ml collection tube at 12,000rpm for 1min to remove residual liquid. And (3) placing the adsorption column miRelute on an ultra-clean workbench, standing and airing for a moment. The purpose of this step is to remove the rinse liquid from the adsorption column.

12) Taking a new No-RNase 1.5ml centrifuge tube, putting an adsorption column MiRelute into the centrifuge tube, and adding No-RNase ddH ₂ O20 mu L, standing for 2min, and centrifuging at 12,000rpm for 2 min.

13) And extracting the total sRNA in the sample to obtain the total sRNA with better quality and purity and the fragment size of less than 200 bp. Finally, the concentration and purity of total sRNA were determined using a nucleic acid analyzer, and sRNA was stored at-80 ℃ for future use.

2RNA reverse transcription

The reverse transcription process is mainly divided into two steps, and the process is carried out by using a first strand synthesis kit (miRcute miRNA cDNA) of the tiangen company:

2.1miRNA 3' reverse transcription plus poly (A) treatment:

1) the RNase-free reaction tube was cooled on ice and the total reverse transcription volume was 20. mu.L.

2) Cool for 10 minutes and add the reagents in the following table in order.

3) Finally, E.coli Poly (A) Polymerase is added, and the prepared reaction solution is gently mixed by a pipette.

4) After the balance and centrifugation of the reaction tube, the reaction tube is kept still and reacts for 60min at 37 ℃ in an incubator, and the reaction solution is directly used for subsequent experiments and can also be stored at-20 ℃ or-80 ℃ for a longer time at-80 ℃.

2.2Poly (A) modified miRNA is subjected to reverse transcription reaction:

1) the reverse transcription reaction used oligo (dT) -Universal Tag, which used Universal reverse transcription primer, and no RNase reaction tube was cooled on ice, and the total reverse transcription was 20. mu.L.

2) Cooled for 10min and the reagents shown in the table below were added in order.

3) And (3) fully mixing and reacting by a pipettor, standing and reacting at 37 ℃ for 60min after short-time centrifugation to generate a first chain of cDNA, and storing reaction liquid at-20 ℃ for later use.

4) Agarose gel electrophoresis was used to detect the integrity of the mirnas.

3 fluorescent quantitative detection

1) When the fluorescent quantitative experiment is carried out, a fluorescent quantitative detection kit (FP401) of Tiangen company is selected. The materials to be used are Reverse Primer, 2 × miRcute miRNA Premix.

2) The 2 × miRcute miRNA Premix is mixed and inverted slightly and uniformly from top to bottom to prevent bubbles from being generated, otherwise, the experiment is influenced, and the miRNA Premix is used after short-time slight centrifugation.

3) The ABI eight-linked reaction tube was cooled on ice, and the reverse transcription system was 20. mu.L in total.

4) After precooling for 10min, the following parameters were followed.

5) The fluorescence quantitative determination was performed using 7500Fast fluorescence quantitative determination apparatus of ABI, and 3 repeated determinations were performed on the sample.

6) After mixing well, the mixture was centrifuged for 30s to avoid foaming of the reaction solution and to allow the sample to accumulate at the bottom of the tube. Placing the eight-link reaction tube into a 7500Fast fluorescent quantitative detector for fluorescent quantitative detection.

7) Fluorescence quantification was performed according to the following parameters. Dissolution curve analysis: 95 ℃ for 15sec, 60 ℃ for 1min, 95 ℃ for 15sec (at which time the fluorescence signal was collected), 60 ℃ for 15 min.

8) U6 RNA was used as an internal reference, U6 RNA was purchased from Tiangen corporation, Ct values of each sample were obtained using the copy number of U6 RNA as the base number of correction, and the Ct value of U6 was subtracted from the Ct value of RNA in the sample to obtain the Δ Ct value. The delta-delta Ct value was obtained using the value of delta Ct in serum of healthy control as the correction base, and 2 was used ^-△△Ct And calculating the expression quantity of miRNA in each sample by a formula.

4 statistical analysis

Statistical analysis is carried out on the data by adopting SPSS 20.0 software, the mean value plus or minus standard deviation is used for representing measured data, the difference between groups of miRNA is compared by single-factor variance analysis, and the difference has statistical significance when P is less than 0.05. And (2) drawing the detection result by using statistical software, and carrying out receiver operating characteristic curve (ROC) analysis on the result, wherein the abscissa is 1-specificity, the ordinate is sensitivity, and the area under the curve (AUC) is calculated, wherein the AUC >0.9 represents higher diagnostic certainty, the AUC (0.7-0.9) represents better diagnostic certainty, the AUC (0.5-0.7) represents lower diagnostic certainty, and the AUC <0.5 represents no diagnostic value.

And (3) detecting an amplification graph of the cycle number threshold and the fluorescence signal threshold of the sample by using fluorescence quantitative PCR, wherein the curve amplification shows that the amplification of the target sample is good, and the three-time repetition of the sample shows that the repeatability is good.

The results of the fluorescent quantitative PCR detection are as follows:

(1) MiRNA expression levels following KSHV infection of cells

The results show (as in fig. 1): all three mirnas were expressed to varying degrees in KSHV-infected cells relative to KSHV-infected cells. Wherein expression of the three miRNAs is significantly higher in KSHV-infected iSLK-219 cells than in KSHV-infected iSLK-BAC cells, relative to different cell lines. In the same cell line, the relative expression level of miR-155 is highest, and the relative expression level of miR-347a-5p is not obviously increased.

(2) MiRNA expression levels in KS patients, KSHV-positive patients

The results show (as in fig. 2): compared with KSHV negative serum, the miR-155 and miR-485-5p in the serum of (1) KS patients and KSHV positive patients have high expression phenomena, wherein the relative expression of miR-155 is the highest, and the relative expression of miR-347a-5p has no significant difference from that of a control. (2) In KS patients, relative expression levels of miR-155 and miR-485-5p are higher in serum compared with KSHV positive serum.

ROC curve analysis results are as follows:

(1) ROC curve analysis of KS patients and healthy controls

The results of ROC curve analysis of miR-155 and miR-485-5p between KS patients and healthy controls (KSHV negative) are respectively shown in FIG. 3 and FIG. 4, the AUC of miR-155 reaches 0.991, and the sensitivity and specificity are respectively 96.7% and 93.3%, which indicates that miR-155 can well distinguish KS patients from healthy controls, so that KS can be diagnosed; ROC curve analysis of miR-485-5p in KSHV positive serum and KSHV negative serum controls showed that AUC was 0.980, and sensitivity and specificity were 90.0% and 100%, respectively, indicating that miR-485-5p also has some diagnostic certainty between KS patients and healthy controls.

The ROC curve analysis result of the combination of miR-155 and miR-485-5p between KS patients and healthy controls (KSHV negative) is shown in figure 5, the AUC reaches 0.996, and the sensitivity and specificity are respectively 100% and 93.3%, which indicates that the combined detection of miR-155 and miR-485-5p can better distinguish the KS patients from the healthy controls, so that KS can be diagnosed assuredly.

(2) ROC curve analysis for KSHV positive patients and healthy controls

The results of ROC curve analysis of miR-155 and miR-485-5p between a KSHV positive individual and a healthy control (KSHV negative) are respectively shown in FIG. 6 and FIG. 7, the AUC of miR-155 reaches 0.969, and the sensitivity and specificity are respectively 93.3% and 86.7%, which indicates that miR-155 can be used as a detection marker for KSHV latent infection; ROC curve analysis of miR-485-5p in KSHV positive serum and KSHV negative serum controls shows that the AUC is 0.931, and the sensitivity and the specificity are 86.7 percent and 86.7 percent respectively, which indicates that miR-485-5p can also be used as a detection marker of KSHV latent infection.

The results of ROC curve analysis of the combined miR-155 and miR-485-5p assays between KSHV positive individuals and healthy controls (KSHV negative) are shown in FIG. 8, respectively. AUC reaches 0.984, sensitivity and specificity are 96.7% and 93.3% respectively, which shows that miR-155 and miR-485-5p are simultaneously used as detection markers of KSHV latent infection, and the detection effect is better.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> river university

Use of <120> miRNAs for the preparation of a kit for the detection of KSHV latent infection

<130> XY-2019-1-W-088

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgaattc 50

<210> 2

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccactta 50

<210> 3

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctcaactggt gtcgtggagt cggcaattca gttgagaccc ctat 44

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cgcttcacga atttgcgtgt cat 23

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgagaggctg gccgtgat 18

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agtgcagggt ccgaggtatt 20

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgcgcgttat aatacaacct ga 22

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agtgcagggt ccgaggtatt 20

<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acactccagc tgggttaatg ctaatcgtga t 31

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccagtgcagg gtccgaggt 19

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcttcggcag cacatatact aaaat 25

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgcttcacga atttgcgtgt cat 23

Claims

Use of a miRNA in the preparation of a kit for detecting a latent infection of human KSHV, wherein the miRNA is selected from at least one of hsa-miR-155 and hsa-miR-485-5 p.
2. The use according to claim 1, wherein the miRNA is derived from peripheral blood.
3. Use of a reagent for quantitatively detecting the expression quantity of miRNA in the preparation of a kit for detecting human KSHV latent infection, wherein the miRNA is selected from at least one of hsa-miR-155 and hsa-miR-485-5 p.
4. The use according to claim 3, wherein the expression level is an expression level of the miRNA in peripheral blood.
5. The use according to claim 3 or 4, wherein the reagent for quantitatively detecting the expression amount of miRNA is a real-time fluorescent quantitative PCR related reagent.
6. The use according to claim 3 or 4, wherein the reagent for quantitatively detecting the expression amount of miRNA is miRNA sequencing reagent.