CN113652483A - Long-chain non-coding RNA and application of siRNA for specifically interfering expression of long-chain non-coding RNA - Google Patents
Long-chain non-coding RNA and application of siRNA for specifically interfering expression of long-chain non-coding RNA Download PDFInfo
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Abstract
The invention relates to the technical field of medical examination, in particular to application of long-chain non-coding RNA and siRNA for specifically interfering expression of the long-chain non-coding RNA, wherein the long-chain non-coding RNA is LINC 00601. Patients with liver cancer tissues high-expression long-chain non-coding RNA (ribonucleic acid) -LINC 00601 resist to oxaliplatin treatment, and the prognosis is poor; according to the invention, LINC00601 gene detection is carried out on liver cancer tissues before treatment, the treatment effect of oxaliplatin is predicted so as to fill up the clinical blank, and other systemic treatment schemes such as targeting or immunotherapy are suggested for patients with potential chemotherapy resistance, so that the prognosis of patients with late-stage liver cancer is better improved.
Description
Technical Field
The invention relates to the technical field of medical examination, in particular to application of long-chain non-coding RNA and siRNA for specifically interfering expression of the long-chain non-coding RNA.
Background
Since early symptoms of liver cancer are not typical, most patients are diagnosed in middle and late stages, and hepatic artery chemoembolization (TACE, interventional therapy) and systemic chemotherapy are the main treatment methods adopted by middle and late stage patients. Oxaliplatin is the most frequently used chemotherapeutic drug for interventional therapy and systemic chemotherapy, and in clinic, 80% of middle and late stage liver cancer patients receive the oxaliplatin treatment; because the tumor heterogeneity of the middle and late stage liver cancer patients is strong, the individual difference of the curative effect of oxaliplatin chemotherapy is large, and the survival benefit difference of the patients is very large. However, no effective prediction index exists in clinic so far, and the treatment effect can be predicted before patients receive oxaliplatin chemotherapy.
Disclosure of Invention
In view of the above problems, the present invention provides the use of long non-coding RNA and siRNA that specifically interferes with the expression of long non-coding RNA, which is LINC 00601. Patients with liver cancer tissues high-expression long-chain non-coding RNA (ribonucleic acid) -LINC 00601 resist to oxaliplatin treatment, and the prognosis is poor; according to the invention, LINC00601 gene detection is carried out on liver cancer tissues before treatment, the treatment effect of oxaliplatin is predicted so as to fill up the clinical blank, and other systemic treatment schemes such as targeting or immunotherapy are suggested for patients with potential chemotherapy resistance, so that the prognosis of patients with late-stage liver cancer is better improved.
The purpose of the invention can be realized by the following technical scheme:
the invention aims to provide application of a long-chain non-coding RNA in preparation of a reagent for evaluating liver cancer prognosis, wherein the long-chain non-coding RNA is LINC00601, and a gene sequence is shown as SEQ ID No. 1.
In one embodiment of the invention, the long-chain non-coding RNA is applied to preparing a reagent for evaluating the treatment efficacy of the hepatoma oxaliplatin.
The second purpose of the invention is to provide an application of a long non-coding RNA in the preparation of a kit for evaluating the prognosis of liver cancer, wherein the long non-coding RNA is LINC00601, and the gene sequence is shown as SEQ ID No. 1.
In one embodiment of the invention, the long-chain non-coding RNA is applied to preparing a kit for evaluating the treatment efficacy of oxaliplatin for liver cancer.
In one embodiment of the present invention, the kit further comprises instructions that describe the method for evaluating the prognosis of liver cancer by using long non-coding RNA, wherein the method comprises:
(1) collecting a sample and extracting RNA: collecting a sample and extracting total RNA in the sample by using a purification kit,
(2) reverse transcription into cDNA: configuring reverse transcription reaction system, reverse transcription reaction to obtain cDNA,
(3) q-PCR quantitative detection of LINC 00601: and (3) preparing a q-PCR reaction system, and carrying out fluorescent quantitative detection by using a fluorescent quantitative PCR instrument.
In one embodiment of the present invention, in step (1), the sample comprises a tissue sample, a cell sample;
in one embodiment of the present invention, in the step (2), the reverse transcription reaction system is: 5 μ L of 4 XEZscript RT Mix II, 1 μ g of total RNA, and make up the reaction to 20 μ L with DEPC water;
the reaction process is as follows: 42 ℃ for 15min, 85 ℃ for 10 sec.
In one embodiment of the present invention, in step (3), the q-PCR reaction system is:
| 2×SYBR Green qPCR Master Mix(ROX2 plus) | 10μL |
| positive and negative PCR primer (10. mu.M) | 0.4. mu.L each |
| Reverse transcription of cDNA | 4μL |
| ddH2O | 5.2μL |
| Total reaction system | 20μL |
The reaction process is as follows:
activation of hot start enzyme: multiplying at 95 ℃ for 5 min;
and (3) PCR amplification: 95 ℃ X10 sec → 60 ℃ X30 sec: a total of 40 cycles;
and (3) detection of a dissolution curve: 95 ℃ X15 sec → 60 ℃ X1 min → 95 ℃ X30 sec → 60 ℃ X15 sec.
The third purpose of the invention is to provide an application of long non-coding RNA in preparing a liver cancer treatment drug, wherein the long non-coding RNA is LINC00601, and the gene sequence is shown as SEQ ID No. 1.
In one embodiment of the invention, the long-chain non-coding RNA is applied to preparing a drug for treating oxaliplatin-resistant liver cancer cells.
The fourth purpose of the invention is to provide siRNA for specifically interfering the expression of long-chain non-coding RNA, wherein the siRNA is selected from siRNA1 with a gene sequence shown as SEQ ID No.4, siRNA2 with a gene sequence shown as SEQ ID No.5 or siRNA3 with a gene sequence shown as SEQ ID No. 6.
The fifth purpose of the invention is the application of siRNA for specifically interfering the expression of long-chain non-coding RNA in the preparation of liver cancer treatment drugs, wherein the siRNA is selected from siRNA1 with a gene sequence shown as SEQ ID No.4, siRNA2 with a gene sequence shown as SEQ ID No.5 or siRNA3 with a gene sequence shown as SEQ ID No. 6.
In one embodiment of the invention, the siRNA is applied to preparing a drug for treating oxaliplatin-resistant liver cancer cells.
In one embodiment of the invention, the expression level of LINC00601 is remarkably improved after the chemotherapeutic drug oxaliplatin acts on or in oxaliplatin-resistant hepatoma cells, and the sensitivity of oxaliplatin-resistant hepatoma cells to oxaliplatin chemotherapy can be remarkably improved after the expression of LINC00601 is interfered.
In one embodiment of the invention, the expression of LINC00601 is much higher in oxaliplatin-tolerant liver cancer cells than control liver cancer cells. Furthermore, the invention uses RNA interference technology to research gene function, uses LINC00601 specific siRNA to interfere its expression, and uses CCK8 cell activity experiment to research the function of the gene in oxaliplatin-resistant liver cancer cells.
Compared with the prior art, the invention has the following advantages:
(1) LINC00601 has different degrees of high expression in liver cancer tissue than in the tissue beside the cancer, and the expression in oxaliplatin-resistant liver cancer cells is obviously improved compared with control liver cancer cells; it can be seen that LINC00601 can be used as a prediction marker of hepatocellular carcinoma chemotherapy effect.
(2) Specific interference on the expression of LINC00601 can obviously improve the sensitivity of oxaliplatin-resistant hepatoma carcinoma cells to oxaliplatin chemotherapy.
Drawings
FIG. 1 is a schematic diagram of the expression difference of LINC00601 between liver cancer tissue and paracancerous tissue.
FIG. 2 is a schematic diagram showing the expression difference of LINC00601 in hepatoma carcinoma cells and oxaliplatin-resistant hepatoma carcinoma cells.
FIG. 3 is a diagram showing the expression efficiency of siRNA-specific interference LINC 00601.
FIG. 4 is a schematic diagram showing the effect of siRNA-specific interference on the expression of LINC00601 on oxaliplatin sensitivity of hepatoma cells.
Detailed Description
The invention aims to provide application of a long-chain non-coding RNA in preparation of a reagent for evaluating liver cancer prognosis, wherein the long-chain non-coding RNA is LINC00601, and a gene sequence is shown as SEQ ID No. 1.
In one embodiment of the invention, the long-chain non-coding RNA is applied to preparing a reagent for evaluating the treatment efficacy of the hepatoma oxaliplatin.
The second purpose of the invention is to provide an application of a long non-coding RNA in the preparation of a kit for evaluating the prognosis of liver cancer, wherein the long non-coding RNA is LINC00601, and the gene sequence is shown as SEQ ID No. 1.
In one embodiment of the invention, the long-chain non-coding RNA is applied to preparing a kit for evaluating the treatment efficacy of oxaliplatin for liver cancer.
In one embodiment of the present invention, the kit further comprises instructions that describe the method for evaluating the prognosis of liver cancer by using long non-coding RNA, wherein the method comprises:
(1) collecting a sample and extracting RNA: collecting a sample and extracting total RNA in the sample by using a purification kit,
(2) reverse transcription into cDNA: configuring reverse transcription reaction system, reverse transcription reaction to obtain cDNA,
(3) q-PCR quantitative detection of LINC 00601: and (3) preparing a q-PCR reaction system, and carrying out fluorescent quantitative detection by using a fluorescent quantitative PCR instrument.
In one embodiment of the present invention, in step (1), the sample comprises a tissue sample, a cell sample;
in one embodiment of the present invention, in the step (2), the reverse transcription reaction system is: 5 μ L of 4 XEZscript RT Mix II, 1 μ g of total RNA, and make up the reaction to 20 μ L with DEPC water;
the reaction process is as follows: 42 ℃ for 15min, 85 ℃ for 10 sec.
In one embodiment of the present invention, in step (3), the q-PCR reaction system is:
| 2×SYBR Green qPCR Master Mix(ROX2 plus) | 10μL |
| positive and negative PCR primer (10. mu.M) | 0.4. mu.L each |
| Reverse transcription of cDNA | 4μL |
| ddH2O | 5.2μL |
| Total reaction system | 20μL |
The reaction process is as follows:
activation of hot start enzyme: multiplying at 95 ℃ for 5 min;
and (3) PCR amplification: 95 ℃ X10 sec → 60 ℃ X30 sec: a total of 40 cycles;
and (3) detection of a dissolution curve: 95 ℃ X15 sec → 60 ℃ X1 min → 95 ℃ X30 sec → 60 ℃ X15 sec.
The third purpose of the invention is to provide an application of long non-coding RNA in preparing a liver cancer treatment drug, wherein the long non-coding RNA is LINC00601, and the gene sequence is shown as SEQ ID No. 1.
In one embodiment of the invention, the long-chain non-coding RNA is applied to preparing a drug for treating oxaliplatin-resistant liver cancer cells.
The fourth purpose of the invention is to provide siRNA for specifically interfering the expression of long-chain non-coding RNA, wherein the siRNA is selected from siRNA1 with a gene sequence shown as SEQ ID No.4, siRNA2 with a gene sequence shown as SEQ ID No.5 or siRNA3 with a gene sequence shown as SEQ ID No. 6.
The fifth purpose of the invention is the application of siRNA for specifically interfering the expression of long-chain non-coding RNA in the preparation of liver cancer treatment drugs, wherein the siRNA is selected from siRNA1 with a gene sequence shown as SEQ ID No.4, siRNA2 with a gene sequence shown as SEQ ID No.5 or siRNA3 with a gene sequence shown as SEQ ID No. 6.
In one embodiment of the invention, the siRNA is applied to preparing a drug for treating oxaliplatin-resistant liver cancer cells.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
And (3) detecting the expression quantity of LINC00601 in liver cancer tissues, paracarcinoma tissues and oxaliplatin-resistant liver cancer cells.
The invention collects the liver cancer cell lines 97H, 3B and oxaliplatin resistant cell lines 97H-OXR, 3B-OXR thereof, and a certain amount of hepatocellular carcinoma and paracarcinoma tissue samples, extracts RNA and carries out reverse transcription to obtain cDNA, and analyzes the expression level of LINC00601 by adopting a fluorescence quantitative PCR method, wherein the specific method comprises the following steps:
1. collecting a sample and extracting RNA:
tissue samples were collected at the Zhongshan Hospital repository affiliated at the university of Compound denier, and total RNA in the Tissue samples was extracted using the Tissue RNA Purification Kit PLUS Kit from EZBioscience, Inc. according to the instruction procedures.
The cell samples were stored at the liver cancer research institute of Zhongshan Hospital affiliated to the university of Compound Dan, and total RNA in the cell samples was extracted using the EZ-press RNA Purification Kit of EZBioscience, Inc. according to the instruction procedures.
2. Reverse transcription into cDNA:
about 1. mu.g of RNA was collected from each sample, and Reverse Transcription reaction was carried out in a 20. mu.L system using 4 XEZscript Reverse Transcription Mix II, a Reverse Transcription reagent from EZBioscience. The reaction system is specifically as follows: 5 μ L of 4 XEZscript RT Mix II, 1 μ g of total RNA, and make up the reaction to 20 μ L with DEPC water; the reaction process is as follows: 42 ℃ for 15min, 85 ℃ for 10 sec.
3. q-PCR relative quantification of LINC00601
Using 2 XSSYBR Green qPCR Master Mix (ROX2 plus) reagent from EZBioscience and a fluorescent quantitative PCR instrument from ABI, Forward and Reverse PCR primers were a Forward primer shown in SEQ ID No.2 and a Reverse primer shown in SEQ ID No.2, respectively, and the reaction system was configured as follows:
| 2×SYBR Green qPCR Master Mix(ROX2 plus) | 10μL |
| positive and negative PCR primer (10. mu.M) | 0.4. mu.L each |
| Reverse transcription of cDNA | 4μL |
| ddH2O | 5.2μL |
| Total reaction system | 20μL |
The q-PCR reaction was carried out as follows:
activation of hot start enzyme: multiplying at 95 ℃ for 5 min;
and (3) PCR amplification: 95 ℃ X10 sec → 60 ℃ X30 sec: a total of 40 cycles;
and (3) detection of a dissolution curve: 95 ℃ X15 sec → 60 ℃ X1 min → 95 ℃ X30 sec → 60 ℃ X15 sec.
The experimental result shows that: the expression level of LINC00601 in the liver cancer tissue is increased in different degrees (in figure 1, different straight lines respectively represent the comparison of the expression level of LINC00601 in the tumor tissue/the tissue beside the cancer of a liver cancer patient by 10,****，P<0.0001), and the expression level is obviously increased in oxaliplatin-resistant hepatoma cells (fig. 2, fig. 2 expresses the change of LINC00601 expression level in oxaliplatin-resistant cells and control cells, and in fig. 2, 97H: MHCC-97H cell line; 97H-OXR: MHCC-97H oxaliplatin-resistant cell line; 3B: hep-3B cell line; 3B-OXR: hep-3B oxaliplatin-resistant cell line; ***: p<0.001；****：P<0.0001)。
Example 2
The siRNA specifically interferes with the expression efficiency of LINC 00601.
Three siRNAs aiming at LINC00601 are designed, and the gene sequences are as follows:
| siRNA1(SEQ ID No.4) | GAGCAGAAATTGCTGCTACAT |
| siRNA2(SEQ ID No.5) | CCATCCATTCCACCACATA |
| siRNA3(SEQ ID No.6) | GCAGCCTTGCAAATAATTT |
target cells were transfected with lipofectamine 3000, a transfection reagent from ThermoFisher, and the efficiency of siRNA interference with LINC00601 expression was examined using the q-PCR method (FIG. 3, FIG. 3 expresses the change in the expression level of LINC00601 in cells after siRNA transfection; NC: control; siRNA: small interfering RNA; P < 0.0001).
The experimental result shows that: the detection result of the q-PCR method shows that the expression quantity of LINC00601 of the three targeted LINC00601 siRNA sequences (SEQ ID No.4-6) after transfecting cells is remarkably reduced compared with that of a control transfection group, and the fact that the expression efficiency of LINC00601 in oxaliplatin resistant cells can be remarkably interfered by the use of the siRNAs is proved.
Example 3
The siRNA specifically interferes with the influence of LINC00601 on the sensitivity of oxaliplatin-resistant hepatoma cells to oxaliplatin.
Cells transfected with siRNA and control cells were seeded in 96-well plates at 5000 cells per well. After 24h the cells were treated with oxaliplatin at a concentration gradient of 0, 1, 2, 3, 5, 10, 15, 20, 30, 40 μ M for 48h, after which each well was changed to 110 μ L of medium containing 10 μ L of CCK8 solution, incubated at 37 ℃ for 1.5h and the absorbance at 450nm was measured.
The experimental result shows that: the CCK8 experimental result shows that the cell activity curve of the oxaliplatin-resistant cells transfected with siRNA is obviously shifted downwards, the activity of the cells is obviously reduced under the action of the same oxaliplatin concentration, and the sensitivity of the cells to oxaliplatin is improved. Therefore, the result indicates that the sensitivity of the oxaliplatin-resistant hepatoma cells to oxaliplatin can be obviously improved after the expression of the LINC00601 is specifically interfered (FIG. 4, the change of a cell activity curve after the oxaliplatin-resistant cells are transfected with the siRNA targeting the LINC00601 is expressed in FIG. 4, 97H-OXR: MHCC-97H oxaliplatin-resistant cell line, 3B-OXR: hep-3B oxaliplatin-resistant cell line, sinC: a control transfection group and siLNC: a transfection-targeting LINC00601-siRNA group).
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
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Claims (10)
1. The application of the long-chain non-coding RNA in the preparation of the reagent for evaluating the prognosis of the liver cancer is characterized in that the long-chain non-coding RNA is LINC00601, and the gene sequence is shown as SEQ ID No. 1.
2. The use of a long non-coding RNA (ribonucleic acid) in the preparation of a reagent for evaluating the prognosis of liver cancer according to claim 1, wherein the long non-coding RNA is used in the preparation of a reagent for evaluating the treatment effect of oxaliplatin on liver cancer.
3. The application of the long-chain non-coding RNA in the preparation of the liver cancer prognosis evaluation kit is characterized in that the long-chain non-coding RNA is LINC00601, and the gene sequence is shown as SEQ ID No. 1.
4. The use of a long non-coding RNA in the preparation of a kit for assessing the prognosis of liver cancer according to claim 3, wherein the long non-coding RNA is used in the preparation of a kit for assessing the treatment efficacy of oxaliplatin for liver cancer.
5. The use of the long non-coding RNA of claim 3 in the preparation of a kit for the assessment of the prognosis of liver cancer, wherein the kit further comprises instructions, the instructions carrying: the method for the prognosis evaluation of the long-chain non-coding RNA on the liver cancer comprises the following steps:
(1) collecting a sample and extracting RNA: collecting a sample and extracting total RNA in the sample by using a purification kit,
(2) reverse transcription into cDNA: configuring reverse transcription reaction system, reverse transcription reaction to obtain cDNA,
(3) q-PCR quantitative detection of LINC 00601: preparing a q-PCR reaction system, performing fluorescent quantitative detection by using a fluorescent quantitative PCR instrument,
in the step (1), the samples comprise tissue samples and cell samples;
in the step (2), the reverse transcription reaction system is as follows: 5 μ L of 4 XEZscript RT Mix II, 1 μ g of total RNA, and make up the reaction to 20 μ L with DEPC water; the reaction process is as follows: 15min at 42 ℃ and 10sec at 85 ℃;
in the step (3), the q-PCR reaction system is as follows:
2×SYBR Green qPCR Master Mix(ROX2 plus) 10μL
positive and negative PCR primers 10. mu.M each 0.4. mu.L
Reverse transcription of cDNA 4. mu.L
ddH2O 5.2μL
The total reaction system is 20 mu L;
the reaction process is as follows:
activation of hot start enzyme: multiplying at 95 ℃ for 5 min;
and (3) PCR amplification: 95 ℃ X10 sec → 60 ℃ X30 sec: a total of 40 cycles;
and (3) detection of a dissolution curve: 95 ℃ X15 sec → 60 ℃ X1 min → 95 ℃ X30 sec → 60 ℃ X15 sec.
6. The application of the long-chain non-coding RNA in preparing the liver cancer treatment drug is characterized in that the long-chain non-coding RNA is LINC00601, and the gene sequence is shown as SEQ ID No. 1.
7. The use of a long non-coding RNA according to claim 6 in the preparation of a medicament for treating liver cancer, wherein the long non-coding RNA is used in the preparation of a medicament for treating oxaliplatin-resistant liver cancer cells.
8. An siRNA for specifically interfering the expression of long-chain non-coding RNA is characterized in that the siRNA is selected from siRNA1 with a gene sequence shown as SEQ ID No.4, siRNA2 with a gene sequence shown as SEQ ID No.5 or siRNA3 with a gene sequence shown as SEQ ID No. 6.
9. Use of siRNA for specifically interfering with the expression of long non-coding RNA in the preparation of a medicament for treating liver cancer, wherein the siRNA is according to claim 8.
10. The use of siRNA of specific interfering long-chain non-coding RNA expression of claim 9 in the preparation of a medicament for treating liver cancer, wherein said siRNA is used in the preparation of a medicament for treating oxaliplatin-resistant liver cancer cells.
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| US20190161730A1 (en) * | 2016-07-07 | 2019-05-30 | Rubius Therapeutics, Inc. | Compositions and methods related to therapeutic cell systems expressing exogenous rna |
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| CN112458168A (en) * | 2020-11-20 | 2021-03-09 | 中国人民解放军海军军医大学 | Application of long-chain non-coding RNA in preparation of hepatocellular carcinoma diagnostic product or therapeutic drug |
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| US20170306406A1 (en) * | 2014-10-22 | 2017-10-26 | The Hospital For Sick Children | Dna methylation markers for neurodevelopmental syndromes |
| US20190161730A1 (en) * | 2016-07-07 | 2019-05-30 | Rubius Therapeutics, Inc. | Compositions and methods related to therapeutic cell systems expressing exogenous rna |
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