CN116622703A - siRNA and shRNA for interfering LINC02518 expression and application thereof - Google Patents
siRNA and shRNA for interfering LINC02518 expression and application thereof Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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Abstract
The invention belongs to the technical field of oncology medicaments, and discloses siRNA, shRNA and application for interfering LINC02518 expression, wherein the sequence of LINC02518 is SEQ ID NO:1, the sequence of the siRNA that interferes with LINC02518 expression is SEQ ID NO:2, the sequence of shRNA interfering with LINC02518 expression is SEQ ID NO:3. the invention discloses LINC02518 which is a novel long-chain non-coding RNA for the first time, has the functions of promoting tumor growth, invasion and metastasis in liver cancer, and can reduce the expression of LINC02518 by interfering in vitro experiments, thereby inhibiting the tumor growth, invasion and metastasis. The siRNA and the shRNA thereof developed by the invention can play a role in inhibiting cancer by intervening the expression of LINC02518, and provide a new direction for treating liver cancer.
Description
Technical Field
The invention belongs to the technical field of oncology medicines, and particularly relates to siRNA, shRNA and application of interfering LINC02518 expression.
Background
Currently, invasive growth of tumor cells is a multi-step complex process in which various biochemical factors are involved. Tumor cells must adhere to the extracellular matrix, affecting cell adhesion, movement and migration by promoting extracellular matrix signaling that is dependent on PTK kinase activity.
RNA interference (RNAi) refers to the phenomenon of highly conserved, double-stranded RNA (dsRNA) -induced, highly efficient and specific degradation of homologous mRNA during evolution. Since the expression of a specific gene can be specifically knocked out or shut down using RNAi technology, the technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors.
Chinese patent CN201310084396.8 discloses an siRNA for anti-tumor comprising one selected from the following nucleotide sequences: the RNA sequence shown as SEQ ID NO. 7-12 or SEQ ID NO. 15-126 has more than 90% homology with the RNA sequence limited by SEQ ID NO. 7-12 or SEQ ID NO. 15-126 and has the same function.
However, the patent is to inhibit the expression of FAK gene, and the related technical scheme for interfering LINC02518 expression has not been reported in the prior art. Therefore, the RNA interference technology is utilized, and the function of LINC02518 is combined, so that good guiding significance is provided for the treatment of tumors.
Through the above analysis, the problems and defects existing in the prior art are as follows: related technical schemes for interfering LINC02518 expression have not been reported in the prior art.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides siRNA, shRNA and application for interfering LINC02518 expression, in particular to a sequence of LINC02518, siRNA, shRNA and application for interfering LINC02518 gene expression.
The invention is realized in such a way that LINC02518 is a novel long-chain non-coding RNA, and the sequence of LINC02518 is SEQ ID NO:1.
further, the LINC02518 has the effect of promoting tumor growth, invasion and metastasis in liver cancer.
Another object of the present invention is to provide an siRNA that interferes with LINC02518 expression using said LINC02518, wherein the sequence of the siRNA that interferes with LINC02518 expression is SEQ ID NO:2.
another object of the present invention is to provide a shRNA expressed by LINC02518 using the LINC02518, where the sequence of the shRNA expressed by LINC02518 is SEQ ID NO:3.
another object of the present invention is to provide an application of the siRNA in preparing a tumor suppressor.
The invention also aims to provide an application of the shRNA in preparing a tumor inhibitor.
Another object of the present invention is to provide a medicament for treating tumor, which comprises an effective amount of the siRNA and a pharmaceutically acceptable carrier.
Another object of the present invention is to provide a medicament for treating tumor, which comprises an effective amount of shRNA and a pharmaceutically acceptable carrier.
Further, the tumor includes liver cancer.
Further, the siRNA and shRNA exert a cancer suppressing effect by interfering with expression of LINC02518.
In combination with the above technical solution and the technical problems to be solved, please analyze the following aspects to provide the following advantages and positive effects:
first, aiming at the technical problems in the prior art and the difficulty in solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
the invention discloses a sequence of LINC02518, siRNA interfering LINC02518 gene expression, shRNA and application thereof, wherein the sequence of LINC02518 is shown as SEQ ID No.1; the sequence of siRNA1 is shown as SEQ ID No.2, and shRNA2 is shown as SEQ ID No. 3.
The invention discloses LINC02518 which is a novel long-chain non-coding RNA for the first time, has the functions of promoting tumor growth, invasion and metastasis in liver cancer, and can reduce the expression of LINC02518 by interfering in vitro experiments, thereby inhibiting the tumor growth, invasion and metastasis.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
the siRNA and the shRNA thereof developed by the invention can play a role in inhibiting cancer by intervening the expression of LINC02518, and provide a new direction for treating liver cancer.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of screening of LINC02518, diagnostic value in HCC, and clinical significance provided by an embodiment of the present invention;
FIG. 2 is a schematic diagram of chromosome localization, secondary structure and sequencing of LINC02518 provided in an embodiment of the invention;
FIG. 3 is a schematic diagram showing the expression, cell sub-localization, cell strain stabilization and cancer promotion function of LINC02518 preliminarily proved by animal experiments in different liver cancer cell lines of LINC02518 provided by the embodiment of the invention;
fig. 4 is a schematic diagram showing the effect of LINC02518 on proliferation, invasion and migration formation ability of liver cancer cells according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides siRNA, shRNA and application for interfering LINC02518 expression, and the invention is described in detail below with reference to the accompanying drawings.
1. The embodiments are explained. In order to fully understand how the invention may be embodied by those skilled in the art, this section is an illustrative embodiment in which the claims are presented for purposes of illustration.
The embodiment of the invention provides a LINC02518 sequence, siRNA interfering LINC02518 gene expression, shRNA thereof and application.
The LINC02518 provided by the embodiment of the invention has a sequence shown in SEQ ID No.1;
SEQ ID No.1：
atttttttttaacaacaaggggagtctctgaggagctatcaaaaactgcttgccatctttccaaatcatctctgtctgcagtgaccatgtcttaatcctgaaggaaaacaaggtggaaattgatttttacttctctgcagctaagtatgtggcagtcttggccacatggtgactcagcagaggagcctatgtactggctgtcctgatgctcttcctgattcaactgattctctacacttgatcttagccaaaaggccgagaagcaatgattcaactgattctcaaaaggacaccgctgcaggaataaacccacttctaaatatggtacacttggaaagttgtccagctcgcaatgtcagagtcacagacttcatgcacactcatctcaagtcaatgttatctaacacatttccactaattattggatgacactagcaagaaactctgatatttttcacctaacctttgctaatttaaatgtggccagcccaccctcatactgcattgtgatggggatataaaagccaatgaaagataccaaaaaaaaaaaaaaaaaaaaaaaaaaaa。
the sequence of the siRNA1 provided by the embodiment of the invention is shown as SEQ ID No. 2;
SEQ ID No.2：
sense(5'-3')ggacaccgcugcaggaauatt；
antisense(5'-3')uauuccugcagcggugucctt。
the shRNA2 provided by the embodiment of the invention is shown as SEQ ID No. 3;
SEQ ID No.3：
sense(5'-3')gcucgcaaugucagagucatt；
antisense(5'-3')ugacucugacauugcgagctt。
according to the invention, LINC02518 is a novel long-chain non-coding RNA, has the effects of promoting tumor growth, invasion and metastasis in liver cancer, and in vitro experiments show that the interference of LINC02518 can cause the reduction of the expression of LINC02518, so that the tumor growth, invasion and metastasis are inhibited, and the developed siRNA and shRNA thereof can play the role of inhibiting cancer by interfering the expression of LINC02518, so that a new direction is provided for the treatment of liver cancer.
2. Application example. In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.
The invention discloses LINC02518 which is a novel long-chain non-coding RNA for the first time, has the functions of promoting tumor growth, invasion and metastasis in liver cancer, and can reduce the expression of LINC02518 by interfering in vitro experiments, thereby inhibiting the tumor growth, invasion and metastasis. The siRNA and shRNA thereof can play a role in inhibiting cancer by intervening the expression of LINC02518, and provide a new direction for treating liver cancer.
3. Evidence of the effect of the examples. The embodiment of the invention has a great advantage in the research and development or use process, and has the following description in combination with data, charts and the like of the test process.
(1) 218 lncRNAs with differential expression beside liver cancer and cancer are successfully screened out by using a self-made lncRNA chip, and a novel intergenic lncRNA LINC02518 closely related to prognosis of survival of HCC patients and the like is further screened out.
In the invention, malignant tumor related LncRNA is screened from an LncRNA database (Noncode, genecode, lncRNAdb, refseq), 2412 LncRNA transcripts are screened out totally, an oligonucleotide probe specific to each transcript is designed by using Array design 4.4 software, and the oligonucleotide probe is synthesized by Invitrogen company, and then chips are prepared. And detecting 125 LncRNA expression profiles (shown in figure 1A) of liver cancer tissues and paired cancer tissues by using a self-made LncRNA chip, and finding that 218 LncRNAs with different expression multiples exceeding 1.25 times are shared in the liver cancer tissues and the cancer tissues by SAM analysis [ False Discovery Rate (FDR) is set to 0], wherein 85 LncRNAs are up-regulated in the liver cancer tissues and 133 LncRNAs are down-regulated in the liver cancer tissues.
Analysis of the relationship between differentially expressed LncRNA and HCC patient prognosis by single factor Cox regression revealed that 17 LncRNA are closely related to prognosis such as survival of HCC patient; one of the differential expression top 5 was chosen from among them and named LINC02518. The fluorescence levels of LINC02518 in liver cancer and paracancestral tissues are shown in fig. 1B, and further HCC diagnostic assays were performed by ROC curves to find LINC02518 to be distinguishable (auc=0.66) (see fig. 1C). Prognosis analysis suggests: high expression levels of LINC02518 were inversely correlated with HCC patient survival (survivin), disease-free survival (DFS), relapse-free survival (RFS), metastasis-free survival (MFS) (see fig. 1D-G).
FIG. 1A is a diagram showing the result of hybridization of LncRNA chip of RNA of liver cancer and other tissues; total RNA hybridization results labeled with Cy5-dUTP (left), total RNA hybridization results labeled with Cy3-dUTP (right); FIG. 1B is a graph showing the differential expression of LINC02518 in liver cancer and paracancestral tissues; FIG. 1C is a ROC curve of LINC02518 in the diagnosis of HCC; FIGS. 1D-G are expression of LINC02518 and various clinical prognostic related DFS: disease-free survival time; RFS: no recurrence life time; MFS: no transfer lifetime.
(2) The chromosomal location, secondary structure, full length and non-coding characteristics of LINC02518 were successfully verified.
LINC02518 is found in the antisense strand by UCSC, LNCipedia et al database, belongs to intergenic lncRNA, is located on chromosome 6 (chr6: 113428540-113433421), has two exons, and has two transcripts (see FIG. 2A), and the secondary results are shown in FIG. 2B. The full length of the LINC02518 sequence was successfully obtained by further designing specific primers for 3'RACE and 5' RACE (see FIG. 2C). Analysis and prediction by PRIDE, phyloCSF, ORF, lee translation initiation sites, etc., found that INC02518 does not have the function of encoding a protein (see table 1).
In FIG. 2, FIG. 2A is a schematic representation of LINC02518 and its site exons and transcripts on human chromosome 6q21 in UCSC genome browser (GRCh 37/hg 38); FIG. 2B is a secondary structure of LINC02518 in the LNCipedia database (http:// www.lncipedia.org); FIG. 2C is the result of obtaining the full length sequence of LINC02518 by 3 'and 5' RACE sequencing.
TABLE 1 identification of LINC02518 non-coding proteins
(3) Detecting expression conditions and cell subdocalization in different liver cancer cell lines of LINC02518 by adopting RT-qPCR, and successfully constructing a sh-LINC02518 stable expression cell line capable of expressing Green Fluorescent Protein (GFP) knockdown LINC 02518; the preliminary discovery of Western blot and animal experiments shows that LINC02518 plays a role in promoting cancer in liver cancer.
The expression of LINC02518 in liver cancer cell lines SMMC-7721, bel-7402, hepG2, hep3B, MHCC-97H and Huh7 is detected by RT-qPCR, and the relative expression situation of LINC02518 in the liver cancer cell lines and human normal liver cell line LO2 is compared. The results show that LINC02518 has a certain difference in expression level in different cell lines, and LINC02518 has high expression in Bel-7402 and Hep3B relative to human normal liver cell line LO2 and relatively low expression in HepG2, SMMC-7721 and M97H (see figure 3A). After separating RNA of cytoplasm and nucleus in Hep3B, hepG and liver cancer cell, qRT-PCR is adopted to detect expression condition of LINC02518 in cytoplasm and nucleus, and simultaneously, beta-actin is mainly expressed in cytoplasm and U6 is mainly expressed in nucleus, so that beta-actin and U6 are used as markers for successfully separating cytoplasmic RNA and reference for LncRNA expression level. The results showed that LINC02518 was detected mainly in cytoplasmic RNA, with a small distribution of nuclei as well (see fig. 3B).
Four instantaneous knockdown siRNA interference sequences are synthesized according to LINC02518 sequence design, and interference efficiency is verified through RT-qPCR, and the results show that si-LINC02518-2 and si-LINC02518-3 knockdown efficiency is higher in Bel-7402 and Hep3B liver cancer cell lines (see figure 3C). Then constructing low-expression LINC02518 lentivirus with GFP fluorescent marker according to the instantaneous knockdown siRNA sequence, infecting Bel-7402, identifying puromycin medicine sieve by RT-qPCR, and successfully obtaining Bel-7402 cell strain with stable low-expression LINC02518 (see figure 3D).
WesternBlot detection shows that the overexpression of LINC02518 can obviously raise cell cycle proliferation related proteins CDK2 and Cyclin D1, obviously lower expression of cancer suppressor genes PTEN and P53, and that the overexpression of LINC02518 can down regulate E-cadherein and raise expression of N-cadherin, vimentin, SNAIL protein when detecting metastasis related proteins (see figure 3E). The nude mice subcutaneous tumor experiments are carried out by adopting the Hep3B cell strain knocked down LINC02518 and the negative control Hep3B cell strain, two groups of nude mice are injected, each group of 5 nude mice is observed for 6 weeks, and experimental results show that the growth of the nude mice subcutaneous tumor formed by the Hep3B cell strain knocked down LINC02518 is obviously slower than that of the negative control group, and the volume of the finally formed nude mice subcutaneous tumor is smaller (see figure 3F). Immunohistochemical staining of Ki-67 suggested that tap-down LINC02518 had weaker proliferation activity of Hep3B cells, demonstrating that tap-down LINC02518 could also inhibit proliferation of tumor cells in vivo (see fig. 3G). The results suggest that LINC02518 plays a role in promoting cancer in the development of liver cancer.
In fig. 3, fig. 3A shows the expression of LINC02518 in different liver cancer cell lines; FIG. 3B is a cell subdocalization of LINC02518 in HepG2 and Hep3B hepatoma cells; FIG. 3C is knockdown of LINC02518 by four independent siRNAs in Bel-7402 and Hep 3B; FIG. 3D is a Bel-7402 cell line stably expressing LINC02518 with GFP fluorescent markers; FIG. 3E is the effect of over-expression LINC02518 on proliferation and metastasis associated protein expression; FIGS. 3F and G are knockdown LINC02518 inhibiting tumorigenesis of hepatoma cells in vivo; transfecting sh-LINC02518-NC (control group) or sh-LINC02518-3 (experimental group) into Hep3B cells, and respectively injecting into nude mice Hep3B cells; compared with the control group, the tumor of the experimental group is obviously smaller (F); immunohistochemical staining of Ki-67 suggested that Hep3B cells knockdown LINC02518 had weaker proliferative activity (G).
(4) LINC02518 was found to promote hepatoma cell proliferation, invasion and migration formation by CCK8 experiments, transwell invasion and migration experiments.
Firstly, the influence of LINC02518 on the proliferation capacity of liver cancer cells is studied through CCK8 experiments at an in vitro level, and the result shows that the knockdown LINC02518 can remarkably inhibit the proliferation capacity of liver cancer cells Bel-7402 and Hep3B in vitro (see figures 4A and 4B), and the overexpression of LINC02518 promotes the proliferation of liver cancer cells HepG2 and M97H (see figures 4C and 4D). Subsequently, the effect of the overexpressed LINC02518 on the migration and invasion capacities of Bel-7402 and HepG2 hepatoma cells was examined by using a Transwell migration and invasion (matrigel cell) experiment, and the result shows that the knockdown LINC02518 significantly inhibits the migration and invasion capacities of Bel-7402 hepatoma cells (see FIG. 4E), while the overexpressed LINC02518 significantly promotes the migration and invasion of HepG2 hepatoma cells (see FIG. 4F).
In FIG. 4, FIGS. 4A and B are graphs showing that knockdown LINC02518 inhibits proliferation of hepatoma cell lines Bel-7402 and Hep 3B; FIGS. 4C and D are graphs showing that LINC02518 is overexpressed to promote proliferation of hepatoma cell lines HepG2 and M97H; FIG. 4E is the ability of knockdown LINC02518 to inhibit migration and invasion of Bel-7402 hepatoma cells; FIG. 4F is the ability of overexpressing LINC02518 to promote migration and invasion of HepG2 liver cancer cells.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (10)
1. LINC02518, wherein LINC02518 is a novel long-chain non-coding RNA, and wherein LINC02518 has the sequence of SEQ ID NO:1.
2. the LINC02518 of claim 1, wherein said LINC02518 has tumor growth, invasion and metastasis promoting effects in liver cancer.
3. An siRNA that interferes with LINC02518 expression using LINC02518 according to any one of claims 1 to 2, wherein the sequence of the siRNA that interferes with LINC02518 expression is SEQ ID NO:2.
4. a shRNA that interferes with LINC02518 expression using LINC02518 according to any one of claims 1-2, wherein the sequence of the shRNA that interferes with LINC02518 expression is SEQ ID NO:3.
5. use of the siRNA of claim 3 for the preparation of a tumor suppressor.
6. Use of the shRNA of claim 4 in the preparation of a tumor suppressor.
7. A medicament for treating tumors, which comprises an effective amount of the siRNA of claim 3 and a pharmaceutically acceptable carrier.
8. A medicament for treating tumors, comprising an effective amount of the shRNA of claim 4 and a pharmaceutically acceptable carrier.
9. A medicament for the treatment of tumors according to any of claims 7 to 8, characterized in that said tumors comprise liver cancer.
10. The agent for treating tumors according to any one of claims 7 to 8, wherein the siRNA and shRNA exert a cancer suppressing effect by interfering with the expression of LINC02518.
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