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

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

Use of miRNA in the preparation of a kit for detecting latent infection of KSHV

technical field

The invention belongs to the field of medical biotechnology, in particular to the use of miRNA in the preparation of a kit for detecting latent infection of KSHV.

Background technique

Kaposi's Sarcoma (KS) is a malignant tumor mainly originating from endothelial cells, which mostly occurs in soft tissues. According to epidemiological and clinical characteristics, KS can be divided into classic type, AIDS-related type (AIDS-KS), immunosuppressive type and African endemic type. In my country, classic KS mainly occurs in Xinjiang Uygur and Kazakh.

The etiological basis of the pathogenesis of KS is Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8) infection. found in. Subsequently, high expression of KSHV was found in all types of KS. The study found that KSHV is an essential and main factor in the occurrence of Kaposi's sarcoma, and is closely related to the occurrence of KS.

After KSHV infects cells, it exists in two ways, namely persistent latent infection and transient lytic infection. For latent infection, accurate detection is often difficult.

miRNAs are a class of small non-coding RNAs about 23 nucleotides in length. It is widespread in multicellular organisms. miRNAs are complementary to the 3'UTR of the target mRNA after gene transcription to degrade the target mRNA or inhibit 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 still a lack of research and application of using miRNA to detect latent infection of KSHV.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the above-mentioned technical problems, and to provide the use of miRNA in the preparation of KSHV latent infection detection kit. In the present invention, the microRNA chip was screened by using the tumor tissues and normal tissues adjacent to the tumor of 4 cases of classic KS patients, and further verified by RT-qPCR, and it was unexpectedly found that miR-485-5p and miR-155 can be used as the detection of latent infection of KSHV. markers, thereby completing the present invention.

One aspect of the present invention provides use of a miRNA selected from at least one of miR-155 and miR-485-5p in the preparation of a kit for detecting latent infection of KSHV.

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

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

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

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

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

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

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

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

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

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

The beneficial effects of the present invention

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

1. The present invention can be detected in the latent infection stage of KSHV, so that the experiment can be intervened early, and it has important clinical application value.

2. The present invention can be detected by using the peripheral blood of patients, which is non-invasive, convenient and fast.

3. The present invention provides a new marker for detecting latent infection of KSHV, which is suitable for clinical use and can provide better assistance for clinical treatment of KSHV.

Description of drawings

Figure 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 stands for iSLK-BAC cells, and 219 stands for iSLK-219 cells.

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

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

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

Figure 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).

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

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

Figure 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 ways

In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

Example

The following examples are used herein to demonstrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention, and thus can be regarded as preferred solutions for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein and still obtain the same or similar results, without departing from the spirit or scope of the present invention.

Unless otherwise defined, 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, the public references herein and the materials to which they refer are incorporated by reference .

Those skilled in the art will recognize, or be aware of through routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are to be included in the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory equipment unless otherwise specified; the test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

Example 1 miRNA chip analysis

1 Tissue sample collection

The tumor tissue and normal tissue adjacent to the tumor were collected from 14 patients with Kaposi's sarcoma (KS patients) who were treated in the Sixth People's Hospital of Xinjiang Uygur Autonomous Region. They were stored in liquid nitrogen immediately after surgical resection. All specimens were confirmed by histological examination. For Boehner's sarcoma, all specimens were subjected to immunohistochemical experiments with mouse monoclonal antibody HHV-8[LN35] (1:50; Abcam, Cambridge, UK). At the same time, peripheral blood samples were drawn from KS patients and KSHV latently infected patients (KSHV positive), and another 10 healthy individuals (KSHV negative) were taken as controls. Among them, patients with latent infection were determined by detecting KSHV latent protein LANA and lytic phase protein RTA. In the present invention, a total of 17 patients were determined to be KSHV latent infection by detecting the expression of LANA protein but not RTA protein.

Kaposi's sarcoma tumors and peritumoral tissues (a total of 28 samples) were used for miRNA chip analysis, and peripheral blood samples (14 patients with KS, 17 patients with latent infection with KSHV, and 10 healthy individuals) were used for RT-qPCR analysis.

2 Tissue specimen collection

(1) After obtaining the consent and signature of the patients, all patients took photos of the skin lesion area, obtained detailed disease information, and performed skin biopsy in the biopsy room.

(2) Routinely disinfect the skin, and take the material a few minutes after local anesthesia.

(3) Use a sharp knife to make a prismatic incision. The knife should be perpendicular to the skin, and the direction of the incision should be consistent with the skin texture.

(4) The incision should be as deep as the subcutaneous tissue. The tumor tissue should be 2cm long and 0.5cm wide, and the normal tissue adjacent to the tumor should be cut. The tumor tissue with a size of 0.5cm×0.5cm was placed in 10% formaldehyde fixative solution for pathological diagnosis. Tumor tissue and paratumor tissue were placed in cold storage tubes and stored in liquid nitrogen. and marked accordingly.

(5) Try not to damage the tissue so as not to affect the quality of the specimen.

(6) Suture the incision.

(7) 5-7 days to remove sutures.

3miRNA chip analysis

The seventh generation miRCURY LNA ^™ Array (v.18.0) (Exiqon, Copenhagen, Denmark) includes 3100 capture probes covering all human, murine and all viral miRNAs, in addition to 25 miRPlus ^™ human miRNAs.

3.1 RNA extraction

1) Tissue sample homogenate

The tissue samples were first frozen and crushed using a Biopulverizer, and then the crushed tissue samples were homogenized with a Mini-Bead-Beater-16.

2) Two-phase separation: First, the homogenized tissue sample was incubated at room temperature for 5 minutes, then 0.2 mL of chloroform was added to 1 mL of the sample, vigorously shaken for 15 sec, and incubated at room temperature for 3 minutes. Centrifuge at 12,000 × g for 15 min at 4°C.

3) Precipitate RNA: transfer the upper aqueous phase to a new centrifuge tube, then add 1.5 times the volume of 100% alcohol to the new centrifuge tube, and invert the centrifuge tube upside down to mix thoroughly.

4) Pipette 700 μL of sample, add it to a 2mL Rneasy Mini spin column, centrifuge at room temperature ≥8000×g for 15sec, and discard the supernatant. If the liquid is greater than 700 μL, aspirate the remaining samples and repeat step 4, and discard the supernatant.

5) Add 700μL of Buffer RWT to the 2mL RNeasy Mini spin column, centrifuge at room temperature ≥8000×g for 15sec, and discard the supernatant.

6) Add 500ul RPEBuffer to the Rneasy Minis pin collection column, centrifuge at room temperature ≥8000×g for 15sec, and discard the supernatant.

7) Transfer the Rneasy Mini spin collection column to a new 2mL centrifuge tube and centrifuge at full speed for 1min.

8) Transfer the Rneasy Mini spin collection column to a new 1.5mL centrifuge tube, then directly add 20 μL of RNase-free water to the membrane of the Rneasy Mini spin collection column, and centrifuge at room temperature ≥8000×g for 1 min. .

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

3.2 RNA quality detection

ND-1000测定RNA浓度和纯度1) Ultraviolet absorption measurement method: use

ND-1000 Determination of RNA Concentration and Purity

Concentration determination: A reading of 1 at 260nm represents 40ng RNA/μL. The calculation formula of sample RNA concentration is: A260×40ng/μL. If RNA is dissolved in 20 μL of DEPC water, 1 μL is taken for determination, and A260=65.003 is measured, then RNA concentration=65.003×40ng/μL=2600.12ng/μL. The remaining sample RNA was 19 μL, and the total amount of remaining RNA was: 19 μL×2600.12ng/μL=49.4 μg.

Purity test: The A260/A280 ratio ranges from 1.8 to 2.1, indicating pure purity.

The total RNA extraction and quality inspection results of the 4 pairs of KS tumor and paratumor tissue were satisfactory.

2) Denaturing agarose gel electrophoresis

First prepare a denaturing agarose gel, take 3 μg of RNA sample and mix it with 3 times the volume of the loading dye solution, and add EB to the loading dye solution to a final concentration of 10 μg/mL. Heat the mixed sample dye solution to 70°C, incubate for 15 min, spot the sample on an agarose gel, and run electrophoresis at a voltage of 5–6 V/cm. When the bromophenol blue indicator enters the gel for 2–3 cm, stop the electrophoresis and remove it. The gel was observed under UV transmitted light and photographed.

The electrophoresis bands 18S and 28S of total RNA were clear, indicating that the nucleic acid composition was complete, without degradation, and without protein and organic solvent contamination.

3.3 Labeling miRNA

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

1) Thaw all reagents except enzymes on ice for 15-20min, shake and mix well and then centrifuge slightly;

2) Prepare the reaction solution according to the following table, mix well, and all operations are performed on ice;

3) Incubate the reaction solution at 37°C for 30min;

4) Immediately after terminating the reaction at 95°C, place the reaction tube on ice for 2-15min and centrifuge briefly;

5) Prepare the CIP reaction solution according to the table below, add fluorescent labeling reagents to the reaction tube, and mix thoroughly:

6) Incubate the reaction tube at 16°C for 1h in the dark;

7) The reaction tube was incubated at 65°C for 15 minutes to terminate the labeling reaction, and the reaction tube was placed in a 4°C refrigerator after slight centrifugation.

3.4 miRNA microarray hybridization

1) Prepare the chip hybridization solution according to the following table:

2) Incubate the hybridization solution at 95°C in the dark for 2min;

3) After incubation, place on ice for 2-15min and mix well;

4) Take out the cover glass;

5) Place the miRCURY ^TM chip on the cover glass, leaving a gap in the middle to make a hybridization chamber;

6) Put the hybridization chamber composed of the chip and the lid fragment into a heat-shrinkable hybridization bag of 3.1 cm×9 cm.

7) The opening of the heat-shrinkable hybridization bag is clamped with a metal clip, and it is quickly immersed in hot water at 95°C, and the hybridization bag shrinks and tightly wraps the hybridization chamber.

8) Take out the heat-shrinkable hybridization bag and dry the water outside the bag, cut off the excess part of the top of the hybridization bag with scissors, and then put it into a drying box at 50°C for more than 10 minutes.

9) Add 180ul of hybridization mixture from the sample addition hole, and supplement the liquid with 1× hybridization buffer, and add it to a position about 0.5cm from the top of the liquid surface.

10) Cut the second heat shrink hybridization bag in half and fit it vertically over the first heat shrink hybridization bag containing the hybridization chamber.

11) Hold the top of the hybridization chamber with tweezers, and immerse the whole in 95°C hot water vertically.

12) Put the hybridization chamber into a drying oven at 56°C and place it on a shaker overnight.

3.5 Chip washing

After hybridization, take the chip out of the hybridization bag, first wash the chip with washing solution A at 56°C for 2 min, then gently wash the chip with washing solution B at room temperature, then wash the chip with washing solution B at room temperature for 2 min, and then wash the chip with washing solution B at room temperature for 2 min. The chip was washed in liquid C at room temperature for 2 min, then washed with water, and finally the chip was dried by centrifugation at 1000 rpm for 5 min, and the chip was scanned immediately after drying.

3.6 miRNA microarray scanning and analysis

The chip was scanned with Axon GenePix 4000B chip scanner, and the original data analysis of chip results was carried out with Gene Pixpro V6.0.

Methods as below:

The green signal intensity of each probe on the chip was de-background, and the average value of 4 replicate probes was taken. The data is standardized by the median standardization method, and then the standardized data is obtained, and the correction value (foreground value-background value) on each chip of the non-control probes >=50 is selected for standardization. The probe median is used as a normalization factor to normalize the points of the entire chip, that is, each miRNA correction value/median value=standard value. After normalization, the differentially expressed miRNAs were calculated using the T test. Finally, cluster analysis and correlation analysis were performed on the differentially expressed miRNA microarray data.

3.7 Statistical analysis

Scanned images were imported into GenePix Pro 6.0 software for grid calibration and data extraction. Repeated miRNAs were averaged, and samples with miRNAs intensity ≥30 across all samples were selected for normalization factor calculation. Data were normalized using medians. miRNAs with a fold difference greater than 2 and p < 0.05 were considered statistically different in the two groups.

The results showed that among the 3100 miRNA probes, there were 185 differentially expressed miRNAs, including 74 up-regulated and 111 down-regulated miRNAs, and the most significant up-regulated miRNAs were hsa-miR-485-5p, hsa-miR-155 and hsa-miR-374a-5p (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 fluorescence quantitative PCR (RT-qPCR) verification

Cells iSLK-BAC and iSLK-219 were infected with KSHV, and the expressions of miR-485-5p, miR-374a-5p and miR-155 in cells after infection were detected, and iSLK-PURO cells without KSHV infection were used as control.

At the same time, the expressions of miR-485-5p, miR-374a-5p and miR-155 in the serum of KS patients and KSHV-positive patients (KSHV latent infection) were detected respectively, and KSHV-negative healthy individuals were used as controls.

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 PCR primers:

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

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

miR-374a-5p real-time PCR primers:

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

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

miR-155 real-time PCR primers:

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

Downstream: 5'-CCAGGTGCAGGGTCCGAGGT-3' (SEQ ID No. 10).

Internal reference U6 real-time quantitative PCR primers:

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

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

1 Extraction of miRNA from the sample

1) Add lysate MZ to the sample, take 200 μL of each of the sample and lysate MZ, and mix and shake well on a shaker for 30 sec.

2) The mixed solution is allowed to stand for 5 minutes. The purpose of this step is to fully disperse the nucleic acid protein complex.

3) The following steps are performed at room temperature. The supernatant is centrifuged and placed in a RNase-free centrifuge tube. The speed is set to 12,000 rpm and centrifuged for 10 min.

4) 200 μL of chloroform, mix well, shake vigorously for 15 sec, and let stand at room temperature for 5 min.

5) Set the rotation speed to 12,000 rpm and centrifuge for 15 min. The RNA is in the uppermost layer of the three layers. Transfer the upper layer of RNA to an RNase-free centrifuge tube and save the liquid for later use.

6) Add 66 μL of absolute ethanol to the stored liquid. The absolute ethanol added at this time is about 1/4 of the total volume. Before transferring to the adsorption column miRspin, ensure that the solution is fully mixed. Sometimes the solution will precipitate. Set it for 2 min, set the rotation speed to 12,000 rpm, and centrifuge for 15 sec. After centrifugation, the adsorption column miRspin is discarded, and the liquid is saved for later use.

7) Add 132 μL of absolute ethanol to the stored liquid. The absolute ethanol added at this time is about 1/2 of the total volume. Before transferring to the adsorption column miRelute, ensure that the solution is fully mixed. Sometimes the solution will precipitate. Set for 2 min, the speed is set to 12,000 rpm, the centrifugation time is 30 sec, the effluent is discarded after centrifugation, and the adsorption column miRspin is retained.

8) Add 500 μL of MRD deproteinized solution to the adsorption column miRelute, let it stand for 2 minutes, and then centrifuge. The speed is set to 12,000 rpm, and the centrifugation time is 30 sec.

9) 600 μL of rinsing solution RW was added to the adsorption column miRelute, left for 2 min, the rotation speed was set to 12,000 rpm, the centrifugation time was 30 sec, and the waste liquid was discarded.

10) Repeat step 9).

11) Put the adsorption column miRelute into a 2ml collection tube, set the rotation speed to 12,000rpm, and centrifuge for 1min to remove the residual liquid. Place the adsorption column miRelute on the ultra-clean workbench and let it sit for a while to dry. The purpose of this step is to remove the rinse solution from the adsorption column.

12) Take a new RNase-free 1.5ml centrifuge tube, put the adsorption column miRelute in it, add 20 μL of RNase-free ddH ₂ O, let it stand for 2 minutes, and then centrifuge. The speed is set to 12,000 rpm and the centrifugation time is 2 minutes.

13) Extract the total sRNA in the sample, and obtain the total sRNA with good quality and purity, and the fragment size is less than 200bp. Finally, the concentration and purity of total sRNA were measured with a nucleic acid analyzer, and the sRNA was stored at -80°C for later use.

2RNA reverse transcription

The reverse transcription process is mainly divided into two steps. This process is carried out using Tiangen's first-strand synthesis kit (miRcutemiRNA cDNA):

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

1) Cool the RNase-free reaction tube on ice, and the total reverse transcription system is 20 μL.

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

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

4) After the reaction tube is balanced and centrifuged, let it stand at 37°C in the incubator for 60 minutes, and the reaction solution can be directly used for subsequent experiments, or it can be stored at -20°C or at -80°C. The storage time at -80°C is longer.

2.2Poly(A) modified miRNA for reverse transcription reaction:

1) Oligo(dT)-Universal Tag was used in the reverse transcription reaction. The universal reverse transcription primer was used in this method. The RNase-free reaction tube was cooled on ice, and the total reverse transcription system was 20 μL.

2) Cool for 10 minutes, and add the reagents shown in the table below in sequence.

3) Mix the reaction with a pipette, briefly centrifuge at 37°C for 60 minutes and let it stand for reaction to generate the first strand of cDNA, and store the reaction solution at -20°C for later use.

4) Use agarose gel electrophoresis to detect the integrity of the miRNA.

3 Fluorescence quantitative detection

1) The fluorescence quantitative detection kit (FP401) of Tiangen Company was used in the fluorescence quantitative experiment. The materials to be used are Reverse Primer, 2×miRcute miRNA Premix.

2) Gently mix and invert 2×miRcute miRNA Premix up and down to prevent the generation of air bubbles, otherwise it will affect the experiment. It is used after a brief centrifugation.

3) The ABI eight-link reaction tube was cooled on ice, and the total reverse transcription system was 20 μL.

4) After pre-cooling for 10min, proceed according to the following parameters.

5) Use ABI's 7500Fast Fluorescence Quantitative Detector to perform fluorescence quantitative detection, and perform three repeated determinations on the sample.

6) After thorough mixing, centrifuge for 30 s to avoid foaming of the reaction solution and make the sample gather to the bottom of the tube. Put the eight-connected reaction tube into the 7500Fast fluorescence quantitative detector for fluorescence quantitative detection.

7) Fluorescence quantification was performed according to the following parameters. Melting curve analysis: 95°C for 15sec, 60°C for 1min, 95°C for 15sec (the fluorescence signal was collected at this time), and 60°C for 15min.

8) U6 RNA was used as the internal reference. U6 RNA was purchased from Tiangen Company. The copy number of U6 RNA was used as the calibration base to obtain the Ct value of each sample. The Ct value of RNA in the same sample was subtracted from the Ct value of U6 to obtain the △Ct value. . The ΔΔCt value in the serum of healthy controls was used as the calibration base to obtain the ΔΔCt value, and the miRNA expression in each sample was calculated using the 2 ^-ΔΔCt formula.

4 Statistical analysis

SPSS 20.0 software was used for statistical analysis of data, and measurement data were expressed as mean ± standard deviation. One-way analysis of variance was used to compare the differences between groups of miRNAs, and P<0.05 was considered statistically significant. Statistical software was used to plot the test results, and the receiver operating characteristic curve (ROC curve) analysis was performed on the results. The abscissa was 1-specificity, and the ordinate was sensitivity. Under the curve, AUC), AUC>0.9 indicates high diagnostic accuracy, AUC=0.7-0.9 indicates good diagnostic accuracy, AUC=0.5-0.7 indicates low diagnostic accuracy, and AUC<0.5 indicates no diagnostic value.

The amplification graph of the cycle number threshold and the fluorescence signal threshold of the sample was detected by real-time quantitative PCR. The curve amplification showed that the target sample was well amplified, and the sample was repeated three times and showed good repeatability.

Fluorescence quantitative PCR detection results are as follows:

(1) miRNA expression level after KSHV infection of cells

The results showed (as shown in Figure 1): Compared with cells without KSHV infection, the expression of the three miRNAs in KSHV-infected cells increased to varying degrees. Among them, relative to different cell lines, the expression of three miRNAs in iSLK-219 cells infected with KSHV was significantly higher than that in iSLK-BAC cells infected with KSHV. In the same cell line, the relative expression of miR-155 was the highest, while the relative expression of miR-347a-5p was not significantly increased.

(2) miRNA expression levels in KS patients and KSHV-positive patients

The results showed (Figure 2): Compared with KSHV-negative serum, (1) miR-155 and miR-485-5p were highly expressed in serum of KS patients and KSHV-positive patients, among which miR-155 had the highest relative expression, while The relative expression of miR-347a-5p was not significantly different from the control. (2) In the serum of KS patients, the relative expression levels of miR-155 and miR-485-5p were higher than that in KSHV-positive serum.

The ROC curve analysis results are as follows:

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

The ROC curve analysis results of miR-155 and miR-485-5p between KS patients and healthy controls (KSHV negative) are shown in Figure 3 and Figure 4, respectively. The AUC of miR-155 reached 0.991, and the sensitivity and specificity were 96.7% and 93.3%, indicating that miR-155 can distinguish KS patients and healthy controls well, so as to confirm the diagnosis of KS; ROC curve analysis of miR-485-5p in KSHV-positive serum and KSHV-negative serum controls showed that AUC was 0.980, and the sensitivity and specificity were 90.0% and 100%, respectively, indicating that miR-485-5p also had a certain diagnostic accuracy between KS patients and healthy controls.

The ROC curve analysis results of the combination of miR-155 and miR-485-5p between KS patients and healthy controls (KSHV-negative) are shown in Figure 5. The AUC reached 0.996, and the sensitivity and specificity were 100% and 93.3%, respectively, indicating that The combined detection of miR-155 and miR-485-5p can better distinguish KS patients from healthy controls, thereby confirming the diagnosis of KS.

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

The ROC curve analysis results of miR-155 and miR-485-5p between KSHV-positive individuals and healthy controls (KSHV-negative) are shown in Figure 6 and Figure 7, respectively. The AUC of miR-155 reached 0.969, and the sensitivity and specificity were respectively The ROC curve analysis of miR-485-5p in KSHV positive serum and KSHV negative serum control showed that the AUC was 0.931, the sensitivity and specificity were 93.3% and 86.7%. The sexes were 86.7% and 86.7%, respectively, indicating that miR-485-5p can also be used as a detection marker for latent infection of KSHV.

The ROC curve analysis results of the combined detection of miR-155 and miR-485-5p between KSHV-positive individuals and healthy controls (KSHV-negative) are shown in Figure 8, respectively. The AUC reached 0.984, and the sensitivity and specificity were 96.7% and 93.3%, respectively, indicating that miR-155 and miR-485-5p were both used as detection markers for latent infection of KSHV, and the detection effect was better.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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<210> 9

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 9

acactccagc tgggttaatg ctaatcgtga t 31

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 10

ccagtgcagg gtccgaggt 19

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 11

gcttcggcag cacatatact aaaat 25

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 12

cgcttcacga atttgcgtgt cat 23

Claims (6)

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

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