CN113234819A

CN113234819A - Application method of long-chain non-coding RNA LOC107987064 and detection and treatment preparation

Info

Publication number: CN113234819A
Application number: CN202110439801.8A
Authority: CN
Inventors: 彭淑平; 帅词俊; 钟雁城; 刘映; 贺甜甜; 莫雨晴; 邹姿
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2021-08-10
Anticipated expiration: 2041-04-23
Also published as: CN113234819B

Abstract

The invention discloses an application method of long-chain non-coding RNA LOC107987064 and a detection and treatment preparation. The kit is a detection reagent for detecting the platinum chemotherapy tolerance of ovarian cancer, and particularly prepares a kit for detecting whether ovarian cancer is tolerant to platinum drugs by a real-time fluorescence quantitative analysis method and a platinum tolerance treatment preparation for human ovarian cancer. The research proves that LncRNA LOC107987064 is highly expressed in platinum-resistant ovarian cancer tissues, and LOC107987064 is a platinum-resistant marker of ovarian cancer, so that the expression of LOC107987064 is applied to the detection of platinum chemotherapy resistance of ovarian cancer, and the platinum chemotherapy sensitivity of human ovarian cancer is enhanced by inhibition, and the method has profound clinical significance and popularization.

Description

Application method of long-chain non-coding RNA LOC107987064 and detection and treatment preparation

Technical Field

The invention relates to the field of molecular biology, in particular to an application method of long-chain non-coding RNA LOC107987064 and a detection and treatment preparation.

Background

Ovarian cancer is a highly malignant disease, which is hidden from disease and lacks specific clinical symptoms at an early stage, and most of the ovarian cancer is found in clinical stages III and IV. Ovarian cancer cytoreductive surgery and platinum-paclitaxel combination chemotherapy are the standard treatment regimens for advanced ovarian cancer patients, but more than 25% of patients in the early stages experience resistance to platinum chemotherapy, i.e., relapse within 6 months after cessation of chemotherapy, and some drug resistant patients have less than 30% responsiveness to subsequent platinum chemotherapy regimens. Research on novel biomarkers related to platinum resistance of ovarian cancer is continuously carried out, but the research on the series of molecular changes such as gene, transcription, protein, metabolism and the like has no major breakthrough.

Disclosure of Invention

The invention aims to provide an application method of a platinum tolerance detection molecular marker for human ovarian cancer, in particular to application of the platinum tolerance detection molecular marker for human ovarian cancer in preparation of a platinum tolerance detection preparation for human ovarian cancer.

An application method of long-chain non-coding RNA LOC107987064 in preparing a platinum tolerance detection preparation for human ovarian cancer, wherein the sequence of the long-chain non-coding RNA LOC107987064 is shown in SEQ NO: 1.

the platinum tolerance detection preparation for human ovarian cancer comprises a reagent for detecting the expression level of long-chain non-coding RNA LOC107987064 in human ovarian cancer tissues.

The reagent for detecting the expression quantity of the long-chain non-coding RNA LOC107987064 in the human ovarian cancer tissue comprises a real-time fluorescent quantitative PCR detection reagent; preferably, the real-time fluorescent quantitative PCR detection reagent comprises a specific primer of real-time fluorescent quantitative PCR:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3', see SEQ NO: 2;

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3' is shown in SEQ NO: 3.

the real-time fluorescent quantitative PCR detection reagent comprises:

specific primers of internal reference beta-actin:

5'-CCTATCGAGCATGGAGTGGT-3', respectively; see SEQ NO: 4;

5'-CTGAGGCATAGAGGGACAGC-3', respectively; see SEQ NO: 5;

specific primers for internal control GAPDH:

5'-AACGGATTTGGTCGTATTGG-3', respectively; see SEQ NO: 6

5'-TTGATTTTGGAGGGATCTCG-3', respectively; see SEQ NO: 7.

the invention provides a platinum tolerance detection kit for human ovarian cancer, which comprises a reagent for detecting the expression quantity of long-chain non-coding RNA LOC107987064 in human ovarian cancer tissues by real-time fluorescent quantitative PCR, preferably, the reagent for detecting the expression quantity of long-chain non-coding RNA LOC107987064 in human ovarian cancer tissues by real-time fluorescent quantitative PCR comprises a specific primer for real-time fluorescent quantitative PCR:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3', respectively;

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3' are provided.

The kit further comprises: (1) extracting total RNA from the induced ovarian cancer drug-resistant cells and ovarian cancer tissues by using a reagent comprising Trizol reagent, trichloromethane, isopropanol and enzyme-free water; (2) the reagent for reverse transcription of lncRNA LOC107987064 into cDNA by taking total RNA as a template comprises a reverse transcription buffer solution, dNTP, an RNase inhibitor, MMLV reverse transcriptase and a random primer; (3) reagents for real-time quantitative PCR of cDNA, including primers specific to lncRNA LOC107987064, real-time fluorescent quantitative SYBR dye, enzyme-free water:

specific primers for fluorescent quantitative PCR:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3', respectively;

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3' are provided.

Specific primers of internal reference beta-actin:

5'-CCTATCGAGCATGGAGTGGT-3'；

5'-CTGAGGCATAGAGGGACAGC-3'；

specific primers for internal control GAPDH:

5'-AACGGATTTGGTCGTATTGG-3'；

5'-TTGATTTTGGAGGGATCTCG-3'。

the use method of the detection preparation comprises the following steps: (1) collecting tumor tissues from patients with ovarian cancer, and extracting total RNA; (2) reverse transcribing LncRNA LOC107987064 into cDNA with total RNA as template; (3) the relative expression quantity is obtained by performing real-time fluorescent quantitative PCR amplification by using an LncRNA LOC107987064 specific primer and an internal reference primer, and when the fold is higher, the platinum curative effect of the ovarian cancer patient is more tolerant.

The third purpose of the invention is to provide a human ovarian cancer platinum tolerance detection preparation, which comprises the product expression quantity obtained by real-time fluorescence quantitative PCR amplification by using the following specific primers, and when the Cq (LOC107987064-Actin) is less than or equal to 11, the preparation is judged to be human ovarian cancer platinum tolerance:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3', respectively;

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3' are provided.

The fourth purpose of the invention is to provide an application method of long non-coding RNA LOC107987064 in preparing a platinum-resistant therapeutic preparation for human ovarian cancer, wherein the sequence of the long non-coding RNA LOC107987064 is shown in SEQ NO: 1.

the human ovarian cancer platinum tolerance treatment preparation comprises a preparation for enhancing the chemotherapy sensitivity of the human ovarian cancer platinum drugs.

The platinum-resistant therapeutic preparation for human ovarian cancer comprises an agent for inhibiting the expression of long-chain non-coding RNA LOC 107987064;

the preferred agent for inhibiting the expression of long non-coding RNA LOC107987064 is a short hairpin RNA expression vector designed for RNA interference against this LOC 107987064.

Further preferably, the 2 interference target sequences required for preparing the short hairpin RNA expression vector for RNA interference are as follows:

shLOC107987064#1 5'-GGAACATCAAAGGTAGAAA-3'；

shLOC107987064#2 5'-CCAGCTTGAAAGAAGCATT-3'。

most preferably: aiming at 2 interference target point sequences and vectors, designing oligonucleotide single chains capable of forming hairpin structures and reverse complementary sequences thereof, and annealing to form DNA double chains with two ends respectively provided with cohesive ends of restriction enzyme sites, wherein the specific sequences are as follows:

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCT TTGATGTTCCTTTTTG-3’；

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTC TACCTTTGATGTTCCA-3’；

or

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTC TTTCAAGCTGGTTTTTG-3’；

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAAT GCTTCTTTCAAGCTGGA-3'。

It is a fifth object of the invention to provide a platinum-resistant therapeutic agent for human ovarian cancer comprising an agent that inhibits the expression of long non-coding RNA LOC 107987064.

The preparation for inhibiting the expression of the long-chain non-coding RNA LOC107987064 is a short hairpin RNA expression vector for RNA interference designed aiming at the LOC 107987064;

preferably, the 2 interference target sequences required by the short hairpin RNA expression vector for preparing RNA interference are as follows:

shLOC107987064#1 5'-GGAACATCAAAGGTAGAAA-3'

shLOC107987064#2 5'-CCAGCTTGAAAGAAGCATT-3'；

further preferably: aiming at 2 interference target point sequences and vectors, designing oligonucleotide single chains capable of forming hairpin structures and reverse complementary sequences thereof, and annealing to form DNA double chains with two ends respectively provided with cohesive ends of restriction enzyme sites, wherein the specific sequences are as follows:

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCT TTGATGTTCCTTTTTG-3’

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTC TACCTTTGATGTTCCA-3’

or

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTC TTTCAAGCTGGTTTTTG-3’

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAAT GCTTCTTTCAAGCTGGA-3'。

The sixth purpose of the invention is to provide the interfering target sequence of the vector and the reverse complementary sequence thereof, wherein the sequence is as follows:

shLOC107987064#1

5'-GGAACATCAAAGGTAGAAA-3', see SEQ NO: 8;

5'-TTTCTACCTTTGATGTTCC-3', see SEQ NO: 9;

shLOC107987064#2

5'-CCAGCTTGAAAGAAGCATT-3', see SEQ NO: 10;

5'-AATGCTTCTTTCAAGCTGG-3', see SEQ NO: 11.

the seventh purpose of the invention is to provide a single oligonucleotide strand of the hairpin structure and the reverse complementary sequence thereof for preparing the carrier, the sequence is as follows:

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCT TTGATGTTCCTTTTTG-3', see SEQ NO: 12;

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTC TACCTTTGATGTTCCA-3', see SEQ NO: 13;

or

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTC TTTCAAGCTGGTTTTTG-3' is shown in SEQ NO: 14;

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAAT GCTTCTTTCAAGCTGGA-3', see SEQ NO: 15.

the blank vector for inhibiting the expression of LncRNA LOC107987064 includes, but is not limited to, pLKO.1puro (available from Addgene) vector.

The earlier stage research shows that the applicant firstly discovers that the expression of LncRNA LOC107987064 is analyzed by collecting tumor tissue samples of ovarian cancer patients with platinum resistance and platinum sensitivity, extracting total RNA of each tumor tissue sample, carrying out real-time fluorescence quantitative PCR after reverse transcription, and then: expression of LncRNA LOC107987064 was higher in tumor tissues of platinum-resistant patients than in platinum-sensitive patients with statistical differences, which is also consistent with the trend of earlier high-throughput sequencing, and accordingly, the applicant proposed the use of LncRNA LOC107987064 for the preparation of marker detection reagents resistant to platinum treatment for human ovarian cancer. Further provides a kit for evaluating the curative effect of platinum chemotherapy of ovarian cancer, which has high cost performance and is easy to popularize and apply.

In addition, the invention also discovers that the platinum-based drug chemotherapy sensitivity of the platinum-resistant cells infected with the ovarian cancer can be enhanced by inhibiting the expression of LncRNA LOC 107987064. Further provides a preparation for enhancing the chemotherapy sensitivity of the platinum-based drug for human ovarian cancer.

The detection reagent can be used for detecting the expression level of LncRNA LOC107987064 in a human ovarian cancer tissue sample, provides a lncRNA molecular marker for predicting the platinum curative effect for a human ovarian cancer patient, and simultaneously can also provide a preparation for enhancing the chemotherapy sensitivity of platinum drugs for human ovarian cancer, and has profound practical significance and popularization.

Drawings

FIG. 1 is a high throughput sequencing heatmap analysis;

FIG. 2 shows the PCR primers designed according to the present invention are specifically aligned by Primer-BLAST;

FIG. 3 is a graph showing the difference in expression of LncRNA LOC107987064 in the tissues of human ovarian cancer platinum-sensitive and platinum-resistant patients analyzed by real-time fluorescent quantitative PCR;

in FIG. 3, R-1, R-2, R-3, R-4 and R-5 are tissues of a patient with drug resistance to platinum for human ovarian cancer, S-1, S-2 and S-3 are tissues of a patient with platinum sensitivity to human ovarian cancer, and Actin is an internal reference;

FIG. 4 is a real-time fluorescent quantitative PCR analysis of the difference in expression of LncRNA LOC107987064 in human ovarian cancer cells and induced ovarian cancer platinum-resistant cells;

FIG. 5 is the RNA expression level of LOC107987064 after infection of ovarian cancer SKOV3 and OVCAR3 cells by LOC107987064 inhibitor;

FIG. 6 is a graph of cell viability of SKOV3 and OVCAR3 treated with varying concentrations of carboplatin and cisplatin following infection of SKOV3 and OVCAR3 cells with LOC107987064 inhibitor;

FIG. 7 is the apoptosis rate of OVCAR3 treated with carboplatin and cisplatin following OVCAR3 cell infection with LOC107987064 inhibitor.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

High throughput sequencing was used in the early stages and the results of the thermographic analysis are shown in fig. 1, where LncRNA, including LncRNA LOC107987064, expressed significantly up-regulated by the first 8 in platinum resistant ovarian cancer tissues.

Example 1

Detection kit for LncRNA LOC107987064 in tumor tissues derived from human ovarian cancer patients

1. Isopropanol 100ml

Trizol reagent 100ml

3. Chloroform 50ml

4.1 μ M random reverse transcription primer 50 μ l

5. 2ml of enzyme-free water

6. 10mM dNTP 100μl

7.200U/. mu.l RNA reverse transcriptase 50. mu.l

8.5 Xreverse transcription buffer 1ml

9.40U/. mu.l RNase inhibitor 500. mu.l

10.Premix Ex Taq 50μl

11.10. mu.M IncRNA LOC107987064 specific primer 50. mu.l

Specific primers for fluorescent quantitative PCR:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3'

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3'

12.10 μ M of each 50 μ l of internal reference control primer

Specific primers of internal reference beta-actin:

5'-CCTATCGAGCATGGAGTGGT-3'

5'-CTGAGGCATAGAGGGACAGC-3'

specific primers for internal control GAPDH:

5'-AACGGATTTGGTCGTATTGG-3'

5'-TTGATTTTGGAGGGATCTCG-3'。

the designed PCR primers were specifically aligned by Primer-BLAST, and the alignment result shows that the PCR Primer of LOC107987064 only specifically detects LOC107987064 as shown in FIG. 2.

Example 2

In the previous research work, the applicant collected 8 tumor tissue samples of ovarian cancer platinum-resistant and platinum-sensitive patients, extracted the total RNA of each tumor tissue sample, and analyzed the expression of LncRNA LOC107987064 by real-time fluorescence quantitative PCR after reverse transcription, and found that: expression of LncRNA LOC107987064 was higher in tumor tissues of platinum-resistant patients than in platinum-sensitive patients (see fig. 3) and was statistically different, which is also consistent with the trend of earlier high-throughput sequencing, and accordingly, the applicant proposed the use of LncRNA LOC107987064 for the preparation of a marker detection reagent for platinum resistance of human ovarian cancer. Repeated detection of LOC107987064 expression in platinum-resistant and platinum-sensitive patient tissues by qPCR experiments revealed that LOC107987064 has a cycle number Cq in platinum-resistant tissues of < 26 when the cycle number Cq of internal control Actin is 15, i.e.that ovarian cancer tissues are platinum-resistant when the cycle number Cq (LOC107987064-Actin) is < 11.

Example 3

Detection of human ovarian cancer cells and induced ovarian cancer platinum resistant cells LncRNA LOC107987064

(1) Collecting human ovarian cancer parent cells and induced platinum-resistant ovarian cancer cells, extracting total RNA: removing the culture medium in the culture plate, adding 1ml of Trizol, horizontally placing for 5min at room temperature to uniformly distribute the lysate on the cell surface, and blowing the cells by using a pipette to make the cells fall off; transferring the cell lysate to a centrifuge tube, adding 200 ul of chloroform, tightly covering the tube cap of the centrifuge tube, shaking up and down for 15 seconds, standing on ice for 5 minutes, 12000g, and centrifuging at 4 ℃ for 15 minutes. Taking out the centrifuge tube, and dividing the sample into three layers: a pale supernatant phase, a middle white layer and a pink lower organic phase. Carefully sucking the light supernatant, phase-shifting to another centrifuge tube, adding isopropanol with the same volume, gently mixing, standing at-20 deg.C for 30min, then 12000g, centrifuging at 4 deg.C for 15 min, and observing white RNA precipitate at the bottom of the tube. The supernatant was carefully removed and 1mL of 75% ethanol was slowly added along the tube wall and gently mixed. 750g, centrifuge for 5 minutes at 4 ℃ and carefully pipette off the supernatant. Drying the precipitate at room temperature for 2-5 minutes, adding 30-50 mu L of non-enzyme water to dissolve the RNA precipitate, detecting the concentration and the quality of the RNA by a spectrophotometer, and storing at-80 ℃ with the OD260/280 ratio of 1.8-2.0.

(2) Reverse transcribing LncRNA LOC107987064 into cDNA with total RNA as template;

Oligo(dT)18primer(100μM) 1μl

Total RNA 1μg

total 12. mu.l of enzyme-free water

First step procedure of reverse transcription 5min at 65 ℃

Reverse transcription second step procedure 60 min at 42 ℃ and 5min at 72 ℃.

(3) Real-time fluorescent quantitative PCR was performed using LncRNA LOC107987064 specific primers and internal reference primers: LOC107987064 specific primer DNA sequences were synthesized by Invitrogen.

Firstly, the reverse transcription product is diluted by 5 times and mixed evenly, and a 20 mu l reaction system comprises the following components:

real-time fluorescent quantitative PCR reaction at 95 ℃ for 30 seconds, 40 cycles of 95 ℃ for 5 seconds, 65 ℃ for 30 seconds, 72 ℃ for 30 seconds, 65-95 ℃ with 0.5 ℃ increase every 0.05 seconds.

Specific primers for fluorescent quantitative PCR:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3'

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3'

Specific primers of internal reference beta-actin:

5'-CCTATCGAGCATGGAGTGGT-3'

5'-CTGAGGCATAGAGGGACAGC-3'

specific primers for internal control GAPDH:

5'-AACGGATTTGGTCGTATTGG-3'

5'-TTGATTTTGGAGGGATCTCG-3'

(4) determination of expression: the experimental data were analyzed by using GraphPad Prism using a relatively quantitative analysis method with β -actin and GAPDH as reference genes. lncRNA LOC107987064 was significantly upregulated in ovarian cancer cells that successfully induced platinum resistance, the results are shown in fig. 4, and the differences were statistically significant (p < 0.05).

Example 4

The gene is prepared for interfering the expression of the gene and promoting the enhancement of the sensitivity of ovarian cancer cells to platinum chemotherapeutic drugs.

The preparation is a short hairpin RNA expression vector designed aiming at LncRNA LOC107987064 and used for RNA interference.

The 2 interference targets required by the short hairpin RNA expression vector for preparing RNA interference are as follows:

shLOC107987064#1 5'-GGAACATCAAAGGTAGAAA-3'

shLOC107987064#2 5'-CCAGCTTGAAAGAAGCATT-3'

aiming at a target sequence and a vector, designing an oligonucleotide single strand capable of forming a hairpin structure and a reverse complementary sequence thereof, and annealing the oligonucleotide single strand and the reverse complementary sequence to form a DNA double strand with two ends respectively provided with a restriction enzyme cutting site cohesive end, wherein the specific sequence is as follows:

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCT TTGATGTTCCTTTTTG-3’

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTC TACCTTTGATGTTCCA-3’

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTC TTTCAAGCTGGTTTTTG-3’

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAAT GCTTCTTTCAAGCTGGA-3'

the preparation method of the short hairpin RNA expression vector for RNA interference comprises the following steps:

1) design of shRNA

Firstly, according to the sequence of LncRNA LOC107987064, searching shRNA target point and target point

The sequence is as follows:

shLOC107987064#1 5'-GGAACATCAAAGGTAGAAA-3'

shLOC107987064#2 5'-CCAGCTTGAAAGAAGCATT-3'

the widely used scramble sequence without any target in the human genome was used as a negative control and the sequence was as follows:

scramble RNA 5'-GCTGCAGTGTCTACTAAGT-3', see SEQ NO: 16;

2) aiming at a target sequence and a scramblel sequence, and a pLKO.1puro vector (purchased from Addgene), an oligonucleotide single chain capable of forming a hairpin structure and a reverse complementary sequence thereof are designed, and after annealing, a DNA double chain with two sticky ends with restriction enzyme cutting sites at two ends can be formed, wherein the specific sequences are as follows:

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCT TTGATGTTCCTTTTTG-3’

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTC TACCTTTGATGTTCCA-3’

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTC TTTCAAGCTGGTTTTTG-3’

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAAT GCTTCTTTCAAGCTGGA-3'

scramble RNA

5'-CCGGTGCTGCAGTGTCTACTAAGTTTCAAGAGAACTTAGTAG ACACTGCAGCTTTTTG-3', see SEQ NO: 17;

5'-AATTCAAAAAGCTGCAGTGTCTACTAAGTTCTCTTGAAACTT AGTAGACACTGCAGCA-3', see SEQ NO: 18.

3) construction of shRNA vectors

Chemically synthesizing 6 single-stranded Oligo sequences of the shRNA and the control, dissolving the synthesized Oligo into 20 mu l by using an Oligo annealing buffer, taking 10 mu l of complementary single strands respectively, heating the Oligo mixture in a PCR instrument at 95 ℃ for 5 minutes, and naturally cooling to room temperature to form double-stranded Oligo fragments:

the plasmid pLKO.1puro was double digested with AgeI and EcoRI, the 7.0kb vector fragment was recovered, and the annealed DNA at the cohesive end and the digested recovered vector were digested according to the following 3: 1, using T4 ligase, ligating at 25 ℃ for 1 hour, transforming Ecoli competence, selecting monoclone, shaking bacteria, sequencing and identifying, and judging whether the target fragment is accurately inserted.

The invention provides an RNA interference inhibitor with ovarian cancer gene treatment potential and prospect for the first time, and has profound significance and popularization.

Example 5

Construction of ovarian cancer cell line stably knocking-down LOC107987064

OVCAR3 and SKOV3 cells were purchased from the cell resource center of the institute of basic medicine of Chinese academy of medical sciences, and 1640 culture medium used for cell culture, fetal bovine serum and trypsin used for digesting cells were all products of Gibco of America.

The ratio of cells in good condition is 2X 10⁵The cells/well were seeded in 6-well plates, and the 6-well plates were placed at 37 ℃ with 5% CO₂In a cell culture box, the transfection of the shRNA expression vector can be started when the cell grows to 70-80% in density, and the transfection process is as follows:

(1) add 4. mu.l lipofectamine2000 to 100. mu.l serum-free medium in sterile EP tube and mix for 5 min;

(2) adding the constructed shRNA expression vector into 100 mul of serum-free culture medium, then uniformly mixing the shRNA expression vector with 100 mul of serum-free culture medium containing lipofectamine2000 in the step (1), and standing for 30min at room temperature to enable the DNA and the liposome to form a complex;

(3) washing the cells with D-hank's solution 3 times;

(4) adding the mixture into 800 μ l serum-free culture medium (without antibiotic), mixing, adding into corresponding cells in 6-well plate, placing 6-well plate at 37 deg.C with 5% CO₂After 6 hours of culture in an incubator, the supernatant was aspirated,adding complete culture medium and continuing culturing for 48 hours;

(5) and detecting the effect of shRNA interference on LOC107987064 expression by real-time quantitative PCR.

OVCAR3 and SKOV3 cells transfected by various shRNA vectors extract total RNA, 2 mu g of RNA is subjected to reverse transcription to form cDNA, and then fluorescence real-time quantitative PCR is carried out, wherein the primer sequence of LOC107987064 is as follows:

5'-TCAGTGAGGGCCCCAGTTAG-3'

5'-CTTTCAAGCTGGACAGAGCAT-3'

primer sequences for control β -actin and GAPDH are:

β-actin

5'-CCTATCGAGCATGGAGTGGT-3'

5'-CTGAGGCATAGAGGGACAGC-3'

GAPDH

5'-AACGGATTTGGTCGTATTGG-3'

5'-TTGATTTTGGAGGGATCTCG-3'

a fluorescent real-time quantitative PCR reaction system:

a fluorescent real-time quantitative PCR reaction step:

and after the reaction is finished, confirming an amplification curve and a melting curve of the fluorescent real-time quantitative PCR, and calculating a P value by adopting a group t-test after the expression intensity of each gene is normalized according to a CT value (threshold cycle values) and an internal reference gene.

2. Results

1) The effect of shRNA interfering with LOC107987064 expression.

Both OVCAR3 and SKOV3 cells were infected with the shRNA vector, which significantly down-regulated the expression level of LOC107987064 (fig. 5).

2) shRNA interference with LOC107987064 increased ovarian cancer OVCAR3 and SKOV3 cells sensitivity to platinum-based drug chemotherapy.

According to the invention, the OVCAR3 and SKOV3 cells are stably infected by the shRNA expression vector, and are treated by different concentrations of carboplatin and cisplatin with scramble as a control, so that after the gene expression is interfered by the shRNA technology, the cell survival activity of OVCAR3 and SKOV3 is reduced (figure 6), the apoptosis rate is increased (figure 7), and the sensitivity of ovarian cancer cells to platinum-based drug chemotherapy is enhanced after the LOC107987064 expression is inhibited. The shRNA expression vector and the derivative reagent thereof constructed by the invention have potential gene therapy effects.

Sequence listing

<110> university of south-middle school

Application method of <120> long-chain non-coding RNA LOC107987064 and detection and treatment preparation

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<170> SIPOSequenceListing 1.0

<210> 1

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<213> Intelligent (Homo sapiens)

<400> 1

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agaggttccc aagtggaagc tgcacccggc atctcatcag tgagggcccc agttagcagg 180

aacatcaaag gtagaaatgc tggaacctga aaaaaagaat gctctgtcca gcttgaaaga 240

agcattctac tgaagtgtgg tgataagatc cggattagaa tcagcattga tggatcatag 300

aaggattaaa gtgacagggt cccaaa 326

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcagtgaggg ccccagttag 20

<210> 3

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctttcaagct ggacagagca t 21

<210> 4

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<213> Artificial Sequence (Artificial Sequence)

<400> 4

cctatcgagc atggagtggt 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctgaggcata gagggacagc 20

<210> 6

<211> 20

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<213> Artificial Sequence (Artificial Sequence)

<400> 6

aacggatttg gtcgtattgg 20

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttgattttgg agggatctcg 20

<210> 8

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggaacatcaa aggtagaaa 19

<210> 9

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<213> Artificial Sequence (Artificial Sequence)

<400> 9

tttctacctt tgatgttcc 19

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccagcttgaa agaagcatt 19

<210> 11

<211> 19

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<213> Artificial Sequence (Artificial Sequence)

<400> 11

aatgcttctt tcaagctgg 19

<210> 12

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccggtggaac atcaaaggta gaaattcaag agatttctac ctttgatgtt cctttttg 58

<210> 13

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aattcaaaaa ggaacatcaa aggtagaaat ctcttgaatt tctacctttg atgttcca 58

<210> 14

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccggtccagc ttgaaagaag cattttcaag agaaatgctt ctttcaagct ggtttttg 58

<210> 15

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aattcaaaaa ccagcttgaa agaagcattt ctcttgaaaa tgcttctttc aagctgga 58

<210> 16

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gctgcagtgt ctactaagt 19

<210> 17

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ccggtgctgc agtgtctact aagtttcaag agaacttagt agacactgca gctttttg 58

<210> 18

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

aattcaaaaa gctgcagtgt ctactaagtt ctcttgaaac ttagtagaca ctgcagca 58

Claims

1. An application method of long-chain non-coding RNA LOC107987064 in preparing a platinum tolerance detection preparation for human ovarian cancer, wherein the sequence of the long-chain non-coding RNA LOC107987064 is shown in SEQ NO: 1.

2. the use of the method as claimed in claim 1, wherein the platinum tolerance detection preparation for human ovarian cancer comprises a reagent for detecting the expression level of long-chain non-coding RNA LOC107987064 in human ovarian cancer tissues.

3. The use method as claimed in claim 2, wherein the reagent for detecting the expression level of long non-coding RNA LOC107987064 in human ovarian cancer tissue comprises real-time fluorescent quantitative PCR detection reagent; preferably, the real-time fluorescent quantitative PCR detection reagent comprises a specific primer of real-time fluorescent quantitative PCR:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3', respectively;

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3' are provided.

4. The platinum tolerance detection kit for the human ovarian cancer is characterized by comprising a reagent for detecting the expression quantity of long-chain non-coding RNA LOC107987064 in a human ovarian cancer tissue by real-time fluorescent quantitative PCR (polymerase chain reaction), preferably the reagent for detecting the expression quantity of long-chain non-coding RNA LOC107987064 in the human ovarian cancer tissue by the real-time fluorescent quantitative PCR comprises a specific primer of the real-time fluorescent quantitative PCR:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3', respectively;

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3' are provided.

5. A platinum tolerance detection preparation for human ovarian cancer is characterized by comprising a preparation for judging the platinum tolerance of human ovarian cancer by using the following specific primers to carry out real-time fluorescent quantitative PCR amplification on the expression level of a product:

lncRNA LOC107987064 forward primer: 5'-TCAGTGAGGGCCCCAGTTAG-3', respectively;

lncRNA LOC107987064 reverse primer: 5'-CTTTCAAGCTGGACAGAGCAT-3' are provided.

6. An application method of long-chain non-coding RNA LOC107987064 in preparing a platinum tolerance treatment preparation for human ovarian cancer, wherein the sequence of the long-chain non-coding RNA LOC107987064 is shown in SEQ NO: 1.

7. the method of use of claim 6, wherein said human ovarian cancer platinum-resistant therapeutic agent comprises an agent that enhances the sensitivity of human ovarian cancer platinum-based chemotherapy.

8. The use of claim 6 or 7, wherein said human ovarian cancer platinum-resistant therapeutic agent comprises an agent that inhibits the expression of long-chain non-coding RNA LOC 107987064;

the preferred agent for inhibiting the expression of long non-coding RNA LOC107987064 is a short hairpin RNA expression vector designed for RNA interference against said LOC 107987064;

shLOC107987064#1 5'-GGAACATCAAAGGTAGAAA-3'

shLOC107987064#2 5'-CCAGCTTGAAAGAAGCATT-3'；

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCTTTGATGTTCCTTTTTG-3’

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTCTACCTTTGATGTTCCA-3’

or

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTCTTTCAAGCTGGTTTTTG-3’

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAATGCTTCTTTCAAGCTGGA-3'。

9. A platinum-resistant therapeutic agent for human ovarian cancer, which comprises an agent that inhibits the expression of long non-coding RNA LOC 107987064.

10. The platinum-resistant therapeutic preparation for human ovarian cancer according to claim 9, wherein said preparation inhibiting the expression of long non-coding RNA LOC107987064 is a short hairpin RNA expression vector designed for RNA interference against said LOC 107987064;

shLOC107987064#1 5'-GGAACATCAAAGGTAGAAA-3'

shLOC107987064#2 5'-CCAGCTTGAAAGAAGCATT-3'；

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCTTTGATGTTCCTTTTTG-3’

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTCTACCTTTGATGTTCCA-3’

or

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTCTTTCAAGCTGGTTTTTG-3’

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAATGCTTCTTTCAAGCTGGA-3'。

11. An interfering target sequence and its reverse complement for the preparation of the vector of claim 10, wherein the sequence is as follows:

shLOC107987064#1

5'-GGAACATCAAAGGTAGAAA-3'

5'-TTTCTACCTTTGATGTTCC-3'

shLOC107987064#2

5'-CCAGCTTGAAAGAAGCATT-3'

5'-AATGCTTCTTTCAAGCTGG-3'。

12. a single oligonucleotide strand with a hairpin structure and its reverse complement for preparing the vector of claim 10, wherein the sequence is as follows:

ShLOC107987064#1

5'-CCGGTGGAACATCAAAGGTAGAAATTCAAGAGATTTCTACCTTTGATGTTCCTTTTTG-3’

5'-AATTCAAAAAGGAACATCAAAGGTAGAAATCTCTTGAATTTCTACCTTTGATGTTCCA-3’

or

ShLOC107987064#2

5'-CCGGTCCAGCTTGAAAGAAGCATTTTCAAGAGAAATGCTTCTTTCAAGCTGGTTTTTG-3’

5'-AATTCAAAAACCAGCTTGAAAGAAGCATTTCTCTTGAAAATGCTTCTTTCAAGCTGGA-3'。