CN110699458A - Mechanism for regulating miRNA-196a by using long-chain non-coding RNA Gas-5 and application thereof - Google Patents
Mechanism for regulating miRNA-196a by using long-chain non-coding RNA Gas-5 and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of medical diagnosis, discloses a mechanism for regulating miRNA-196a by long-chain non-coding RNA Gas-5, and also discloses application of the mechanism for regulating miRNA-196a by long-chain non-coding RNA Gas-5 in a kit and a chip for diagnosing and treating esophageal cancer. The invention takes esophageal squamous cell carcinoma as a model, develops a specific mechanism for regulating miRNA-196a by long-chain non-coding RNA Gas-5, defines a binding site on pri-miRNA-196a and long-chain non-coding RNA Gas-5 and a mechanism for interfering the generation of miRNA-196a by the binding site, and also defines a plurality of binding sites on long-chain non-coding RNA Gas-5 and DGCR8 protein and a mechanism for interfering the generation of miRNA-196a by the binding sites.
Description
Technical Field
The invention relates to the technical field of medical diagnosis, in particular to a mechanism for regulating miRNA-196a by long-chain non-coding RNA Gas-5 and application thereof.
Background
Esophageal cancer is a common tumor of the digestive tract, and about 30 million people die of esophageal cancer every year worldwide. The morbidity and mortality varies greatly from country to country. China is one of the high-incidence areas of esophageal cancer in the world, and the average death rate of people is about 15 ten thousand every year. More men than women, the onset age is usually over 40 years. Esophageal cancer typically has progressive dysphagia, which is characterized by difficulty swallowing dry food, followed by semifluid food, and finally, water and saliva.
Long non-coding RNAs (lncrnas) are a class of RNA molecules with transcripts longer than 200nt, which do not encode proteins but regulate gene expression in RNA form at various levels. Although research on lncRNA has been advanced rapidly in recent years, the functions of most lncRNA are still unclear, and the excavation of new functions of lncRNA is one of the hot spots of non-coding RNA research nowadays. Long-chain non-coding RNAgas-5 was discovered to be highly expressed in mouse NIH3T3 fibroblasts with growth inhibition in 1998, and in human, the long-chain non-coding RNAgas-5 is located in a non-coding chromosome region 1q25.1 and is a sheared, polyadenylated non-protein coding RNA, and the long-chain non-coding RNAgas-5 is abnormally expressed in various tumors, but the expression and the effect of the long-chain non-coding RNAgas-5 in esophageal cancer are not reported yet.
miRNA is a small molecule non-coding RNA with the length of about 22nt, miRNA genes are generally generated by transcription of RNA polymerase II (polII) in a cell nucleus, the initial product is large pri-miRNA with a cap structure (7MGpppG) and a poly A tail, the pri-miRNA is cut into pre-miRNA consisting of about 70 nucleotides under the action of a nuclease Drosha enzyme, then the pre-miRNA is delivered into cytoplasm by ran-GTP and Exportin-5, and then the pre-miRNA is cut into miRNA double strands with the length of about 22 nucleotides by another nuclease Dicer enzyme. This double strand is quickly directed into the silencing complex (RISC), where a single mature single-stranded miRNA is retained. miRNA-196a is located on chromosome 12, is an upstream gene of Hox family, can regulate and control the expression of genes such as HOXA9, HOXB7, HOXC8 and the like, and plays a very important role in the development process of vertebrates; recent studies indicate that the abnormal expression of miRNA-196a gene is related to various diseases, and the expression of miRNA-196a shows a gradually increased trend in the process of the development from normal esophageal mucosa to esophageal adenocarcinoma.
Current studies show that pre-miRNAs are generated mainly by cleavage of pri-miRNAs by proteins called "Microprocessor complex" (Drosha, DGCR8, p68, p72, etc.) in the nucleus, and then transported out of the nucleus by Exportin-5 and ran-GTP, and are prevented from being degraded in the nucleus. The Drosha protein is a nuclear protein consisting of a proline-rich region and the N-terminus consisting of an arginine-and serine-rich region, two RNaseIII domains and a dsRBD domain, and must be assisted by DGCR8 to specifically cleave the pri-miRNA molecule. The DGCR8 protein is able to bind to bases in the pri-miRNA molecule that form a hairpin structure, helping the Drosha protein locate at the cleavage site, i.e. the pri-miRNA molecule stem is 11bp from the junction between the double-stranded stem and the flanking ssRNA sequence.
The inventor finds that the long-chain non-coding RNA Gas-5 inhibits the growth of esophageal cancer cells in vivo and in vitro, and further finds that the long-chain non-coding RNA Gas-5 can inhibit the expression of miRNA-196a at the post-transcriptional level, which indicates that lncRNAGAs-5 participates in the maturation process of miRNA. Therefore, the invention aims to research the mechanism of long-chain non-coding RNA Gas-5 participating in esophageal cancer generation, research the specific mechanism of Gas-5 participating in the generation process of miRNA-196a, and determine the specific mechanism of lncRNA Gas-5 participating in the inhibition of the growth of esophageal cancer cells by inhibiting the generation of miRNA-196a, thereby laying a preliminary foundation for developing and developing a kit and a chip for diagnosing and treating esophageal cancer.
Disclosure of Invention
Based on the problems, the invention provides the application of a mechanism for regulating and controlling miRNA-196a by long-chain non-coding RNA Gas-5 in a kit and a chip for diagnosing and treating esophageal cancer, the invention takes esophageal squamous cell carcinoma as a model, researches a mechanism for exerting the effect of an anti-cancer gene by the long-chain non-coding RNA Gas-5 through influencing the generation process of miRNA-196a, analyzes a new regulation and control mode of lncRNA Gas-5 on miRNA-196a through the invention, and lays a preliminary foundation for developing and developing the kit and the chip for diagnosing and treating esophageal cancer.
In order to solve the technical problems, the invention provides a mechanism for regulating and controlling miRNA-196a by long-chain non-coding RNA Gas-5, which comprises the following two mechanisms:
mechanism 1: a binding site I with long-chain non-coding RNAGAs-5 exists on the pri-miRNA-196a, the binding site I is a cleavage site of Drosha enzyme and Dicer enzyme, and the long-chain non-coding RNAGAs-5 prevents the Drosha enzyme and the Dicer enzyme from cleaving the pri-miRNA-196a by directly binding with the pri-miRNA-196a, so that the generation of pre-miRNA-196a is inhibited, and the generation of miRNA-196a is further inhibited;
mechanism 2: the long-chain non-coding RNAGAs-5 has a binding site II with DGCR8 protein, and the long-chain non-coding RNAGAs-5 and pri-miRNA-196a competitively bind with DGCR8 protein, so that the generation of pre-miRNA-196a by assisting Drosha enzyme to cut pri-miRNA-196a by the DGCR8 protein and the generation of miRNA-196a are inhibited.
Furthermore, the sequence of the binding site I is 5'-UAGGUAGUUUCAUGUUGUUGGG-3'.
Further, the sequence of the binding site II is: 5'-GUGGAGUCCAACUUGCCU-3' are provided.
In order to solve the technical problems, the invention also provides application of a mechanism of regulating miRNA-196a by using long-chain non-coding RNA Gas-5 in a kit and a chip for diagnosing and treating esophageal cancer.
Compared with the prior art, the invention has the beneficial effects that: the invention takes esophageal squamous cell carcinoma as a model, develops a specific mechanism for regulating miRNA-196a by long-chain non-coding RNA Gas-5, defines a binding site on pri-miRNA-196a and long-chain non-coding RNA Gas-5 and a mechanism for interfering the generation of miRNA-196a by the binding site, and also defines a plurality of binding sites on long-chain non-coding RNAGAs-5 and DGCR8 protein and a mechanism for interfering the generation of miRNA-196a by the binding sites.
Drawings
FIG. 1 is a primer set of miRNA-196a in an embodiment of the invention;
fig. 2 is a technical roadmap of a research process of an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example (b):
the mechanism of long-chain non-coding RNAGAs-5 for regulating miRNA-196a comprises the following two mechanisms:
mechanism 1: a binding site I for long-chain non-coding RNAGAs-5 exists on pri-miRNA-196a, and the sequence of the binding site I is 5'-UAGGUAGUUUCAUGUUGUUGGG-3'; the binding site I is a cleavage site of Drosha enzyme and Dicer enzyme, and the long-chain non-coding RNA Gas-5 is directly combined with pri-miRNA-196a to prevent Drosha enzyme and Dicer enzyme from cleaving pri-miRNA-196a, so that the generation of pre-miRNA-196a is inhibited, and the generation of miRNA-196a is further inhibited;
mechanism 2: the long-chain non-coding RNAGAs-5 has a binding site II with DGCR8 protein, and the sequence of the binding site II is as follows: 5'-GUGGAGUCCAACUUGCCU-3', respectively; the long-chain non-coding RNAGAs-5 and pri-miRNA-196a competitively bind to DGCR8 protein, so that DGCR8 protein is inhibited to assist Drosha enzyme to shear pri-miRNA-196a to generate pre-miRNA-196a, and further generation of miRNA-196a is inhibited.
The application of the mechanism of long-chain non-coding RNA Gas-5 regulation miRNA-196a in a kit and a chip for diagnosing and treating esophageal cancer.
The specific mechanism of the long-chain non-coding RNA Gas-5 participating in the miRNA-196a generation process is researched by the technologies of RT-PCR, RIP, RNA antisense purification, RNA affinity, in vitro processing assay and the like, the research of the embodiment explains the new function of the long-chain non-coding RNA Gas-5 participating in the miRNA-196a generation, partially explains the recognition mechanism of miRNA gene regulated after transcription, and improves the understanding of the interaction between non-coding RNAs.
The specific study procedure is as follows:
(1) MiRNA chip detection
Detection of miRNA chip is completed by Shanghai Bohao Biotechnology Co., Ltd, the chip is Affymetrix4.0 chip, and experimental sample total RNA (containing miRNA) adopts Affymetrix miRNA chip matched kit FlashtagTMBiotin HSR RNA Labeling Kit (P/N901911, Affymetrix) and Standard protocolsThe process labels miRNA in the sample total RNA. According to the hybridization standard flow and the matched kit which are provided by matching the Affymetrix miRNA chip,
hybridization, Wash and Stain Kit (P/N900720, Affymetrix Santa Clara CA, US), GeneChip Eukaryotic Hybridization Control Kit (P/N900454, Affymetrix Santa Clara CA, US) performed 48 ℃ and 16 hours rolling Hybridization in a rolling Hybridization Oven (P/N00-0331 (220V), Affymetrix Santa Clara CA, US), after which the washing of the chips was performed in a washing workstation Fluidics Station 450 (P/N00-0079, Affymetrix Santa Clara CA, US) according to the standard protocol provided by Affymetrix. Chip result adoption
Scanner 7G (Affymetrix, Santa Clara, Calif., US) was scanned and the raw data read using Command Console Software 3.2(Affymetrix, Santa Clara, Calif., US); the data qualified in quality control were normalized by expression Console (Affymetrix, Santa Clara, Calif., US) using the algorithm RMA + DABG.
(2) Detection of qRT-PCRpri-miRNA, pre-miRNA and mature miRNA expression
The miRNA gene is generally transcribed by RNApolII, as is the common mRNA gene, so that different primers are designed according to pri and pre to detect the expression of pri-miRNA and pre-miRNA. F1R: pri-miRNA; F2R: the primers of pri-miRNA + pre-miRNA and miRNA-196a are shown in figure 1, and the expression of mature miRNA is detected by using a method of a Taq-Man probe.
(3) In vitro transcription of long-chain non-coding RNAGAs-5
T7 RNA polymerase is adopted for in vitro transcription, the constructed long-chain non-coding RNAgas-5 expression vector pcDNA3.1-Gas-5 and mutant pcDNA3.1-Gas-5 (sequencing and identification) are utilized, the plasmid is subjected to single enzyme digestion linearization treatment by XhoI enzyme, the template DNA is removed after in vitro transcription by T7 enzyme, and the RNA is purified by RNA ethanol and is frozen at minus 80 ℃ for standby;
the pcDNA3.1-Gas-5 vector primers were constructed as follows:
CTAGCTAGCTGTGAGGTATGGTGCTGG Nhe1
CGGGATCCAAATTGGAGACACTGTTT BamH1
the pcDNA3.1-antisense-Gas-5 vector primer is:
GAS5-as-F：CGGGATCCTGTGAGGTATGGTGCTGG
GAS5-as-R：CTAGCTAGCAAATTGGAGACACTGTTT
(4)RNAantisense purification(RAP)
after long-chain non-coding RNAGAs-5antisense RNA probes are transcribed in vitro, biotin labeling is carried out, and the antisense RNA probes have miRNA-196a binding sites and RT-PCR amplification regions and also cover the full length of Gas-5. Esophageal cancer cells were cross-linked with 2m MDSG at room temperature for 45min and fixed with 3% formaldehyde at 37 ℃ for 10 min. For each purification reaction, 100ng of biotin-labeled probe was added to the cell lysate, mixed and incubated at 45 ℃, followed by capture of the probe by streptavidin magnetic beads, elution of the relevant RNA and DNA, and detection of RNA and DNA expression by RT-qPCR.
(5) Luciferase reporter gene
All vectors take sea cucumber luciferase as an indicator gene, a target sequence is inserted into the downstream of a luciferin translation termination site, a target sequence or an unrelated sequence of 3 xmiRNA-196 a is constructed into a multiple cloning site, cells are collected for double-luciferase activity detection 48 hours after transfection, and 3 repeating groups are set in each group of experiments. The Dual Luciferase activity was detected using the Dual-Luciferase Reporter Assay System from Promega with the following primers:
GAS5-Rep-F：CTAGCTAGCTGTGAGGTATGGTGCTGG
GAS5-Rep-R：CGGGATCCAAATTGGAGACACTGTTT。
(6) RNA chromatin co-immunoprecipitation (RIP)
The binding condition of the DGCR8 protein and the long-chain non-coding RNA Gas-5 is verified by using the RIP technology. Capturing endogenous RNA binding protein in nucleus or cytoplasm by using DGCR8 antibody, binding the protein on magnetic beads, then carrying out immunoprecipitation to separate the RNA binding protein and RNA bound by the RNA binding protein, detecting the abundance of long-chain non-coding RNA Gas-5 (control IgG antibody) by using RT-PCR technology, extracting total RNA by TRIzol, carrying out reverse transcription to obtain cDNA, and detecting the expression condition of Gas-5 by using SYBGreen method; RT-PCR primers were as follows:
upstream: 5'-AACTACACTGTGTGGAGCAAG-3'
Downstream: 5'-TTGTGCCATGAGACTCCATCAG-3'
(7)RNApull-down
Taking 5ul of RNA, adding 1ul of DMSO, heating at 85 ℃ for 5min, removing an RNA secondary structure, incubating 30% PEG for 10min under a water bath condition at 37 ℃, sequentially adding an RNA connection reaction reagent according to the specification of RNA 3' -End Dethiomethylation kit (Pierce, USA), incubating overnight at 16 ℃, and extracting the RNA by a phenol chloroform method; marking RNA by Biotin, and detecting marking efficiency by using a dot hybridization experiment; adding 50ul1 XRNACAPture Buffer to resuspend streptavidin-treated magnetic beads, adding 50pmol RNA probe to the magnetic beads, mixing, incubating at room temperature for 30min to allow RNA to bind to streptavidin magnetic beads; 100ul of 1 Xprotein-RNABinding Buffer balance-bound RNA beads were added, mixed, placed in a Magnetic stand to aspirate the liquid, 100ul of Protein-RNABinding Reaction Master Mix was prepared according to the Magnetic RNA-Protein Pull-Down Kit (Pierce, USA) instructions, and the beads were resuspended. Incubate at 4 ℃ for 60min, and elute RNA-Bindingproteins. Westernblot detects the expression of the protein.
(8)Electrophoretic mobility shift assay(EMSA)
Wild-type and mutant miRNA-196a vectors (pcDNA3.1(+) _ prem-196 a, pcDNA3.1(+) _ prem-196 a mut) were constructed, labeled with biotin after in vitro transcription, combined in 1x binding buffer (20mM Tris-HCl, pH8.0, 1mM DTT, 1mM MgCl 2, 20mM KCl,10mM Na2HPO4-NaH2 PO4, pH8.0) reaction solution, biotin-labeled RNAs were added during the reaction, then unlabeled Gas-5 (wild-type and mutant) were added gradually at different concentrations (0.5-6.25pmol), each reaction was pre-warmed at 70 ℃ for 5min, then cooled to 30 ℃ for 20min, subjected to 5% native polyacrylamide gel electrophoresis, transferred to nylon membrane, and detected with Ambion's BioBright BioDennisotopic Detection kit.
(9) RNA affinity purification (RNAaffinitypurification)
Researching the combination of DGCR8 and pri-miRNA-196a in the presence and absence of long-chain non-coding RNAGAs-5 by using an RNA affinity purification method; pri-miRNA-196a generated by in vitro transcription and mutant pri-miRNA-196a are covalently bound to activated agarose beads, the activated agarose beads are incubated with protein extracts of 293T cells, wild type and mutant long-chain non-coding RNA Gas-5 with different concentrations are added in the incubation process, and the expression condition of DGCR8 protein is detected by using western blot after pull-down.
(10)In vitro pri-miRNAprocessing assay
In vitro production of the substrate for pri-miRNA-196a required for the experiment, [ alpha-32P ] was labeled with ATP (PerkinElmer) after in vitro transcription. Adding different concentrations (0.21,0.42and 0.83uM) of long-chain non-coding RNAGAs-5(wt or mutant) into each pri-miRNA-196a, or adding 100mM, 200mM and 500mM of 12-base 2' -O-methyl-oligonucleotide matched with the pri-miRNA-196a, preheating the RNA mixture at 65 ℃ for 2min, and cooling to 30 ℃; HEK293T cells & extracts were added to the reaction mixture and incubated at 30 ℃ for 90min, RNA extracted and subjected to 8% denaturing polyacrylamide gel electrophoresis.
The experimental results obtained in the above studies are as follows:
1. expression of long-chain non-coding RNAGAs-5 in esophageal cancer cells and tissues
The expression quantity of the long-chain non-coding RNAGAs-5 in the esophageal cancer cell strain is lower than that of normal esophageal epithelial cells, and the expression of miRNA-196a in the esophageal cancer cell strain is increased; the expression level of long-chain non-coding RNA Gas-5 in esophageal cancer tissues is reduced relative to distant cancer tissues, the internal parameter is GAPDH, and N is 50; the expression level of long-chain non-coding RNA Gas-5 is reduced in most esophageal cancer tissues relative to paracarcinoma tissues (36/50), and the internal reference is GAPDH; in addition, the expression of the long-chain non-coding RNAGAs-5 is related to the stage of esophageal squamous cell carcinoma, and the expression quantity of the long-chain non-coding RNAGAs-5 in the esophageal carcinoma of the stage I and the stage II is higher than that in the esophageal carcinoma of the stage III and the stage IV; compared with a para-carcinoma tissue, the expression level of miRNA-196a in an esophageal cancer tissue is increased, the internal parameter is U6, and N is 50; the long-chain non-coding RNAGAs-5 is in negative correlation with the expression of miRNA-196a, and N is 50.
2. Long-chain non-coding RNAGAs-5 inhibits growth of esophageal squamous cell carcinoma cells in vivo and in vitro
Transfecting long-chain non-coding RNA Gas-5siRNA and an over-expression vector to an EC109 cell, wherein Edu experiments show that long-chain non-coding RNAGAs-5 obviously inhibits esophagus cancer cell proliferation (P <0.05), and nude mice experiments after over-expressing long-chain non-coding RNAGAs-5 show that the tumor formation of esophagus cancer cells is inhibited after over-expressing long-chain non-coding RNAGAs-5.
3. Long-chain non-coding RNAGAs-5 influences the post-transcriptional regulation of miRNA-196a
After long-chain non-coding RNAGAs-5 is over-expressed, the expression of miRNA is detected by using a miRNA chip, wherein fold change is more than 3.0, and 18 miRNA with reduced pre-miRNA expression are obtained; after long-chain non-coding RNA Gas-5 is over-expressed, RT-RCR is used for detecting the expression of pre-miRNA-196a and mature miRNA-196a, and the expression of pre-miRNA-196a and mature miRNA-196a is found to be remarkably reduced, and the expression of pri-miRNA-196a is not changed.
4. Specific mechanism for regulating miRNA-196a by long-chain non-coding RNAGAs-5
Mechanism 1: a binding site I for long-chain non-coding RNAGAs-5 exists on pri-miRNA-196a, and the sequence of the binding site I is 5'-UAGGUAGUUUCAUGUUGUUGGG-3'; the binding site I is a cleavage site of Drosha enzyme and Dicer enzyme, and the long-chain non-coding RNA Gas-5 prevents Drosha enzyme and Dicer enzyme from cleaving pri-miRNA-196a by directly binding with pri-miRNA-196a, so that the generation of pre-miRNA-196a is inhibited, and the generation of miRNA-196a is further inhibited.
Mechanism 2: the long-chain non-coding RNAGAs-5 has a binding site II with DGCR8 protein, and the sequence of the binding site II is as follows: 5'-GUGGAGUCCAACUUGCCU-3', respectively; the long-chain non-coding RNAGAs-5 and pri-miRNA-196a competitively bind to DGCR8 protein, so that DGCR8 protein is inhibited to assist Drosha enzyme to shear pri-miRNA-196a to generate pre-miRNA-196a, and further generation of miRNA-196a is inhibited.
The DGCR8 and IgG antibody pull-down RNA are used for RT-PCR detection, the enrichment of pre-miRNA-196a and long-chain non-coding RNAGAs-5 is obviously improved compared with that of an IgG antibody, and the DGCR8 can be combined with the pre-miRNA-196a and can be combined with the long-chain non-coding RNAGAs-5.
The binding site, the binding relationship and the competitive relationship in the specific mechanism of regulating miRNA-196a by the long-chain non-coding RNA Gas-5 provided by the invention can be used for preparing a kit and a chip for diagnosing and treating esophageal cancer, and a new way is opened for diagnosing and treating esophageal cancer.
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.
Sequence listing
<110> China people liberation army, military and medical university
Mechanism for regulating miRNA-196a by long-chain non-coding RNA Gas-5 and application thereof
<130>20191010
<160>2
<170>SIPOSequenceListing 1.0
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<211>22
<212>RNA
<213>Human esophageal cancer cell
<400>1
uagguaguuu cauguuguug gg 22
<210>2
<211>18
<212>RNA
<213>Human esophageal cancer cell
<400>2
guggagucca acuugccu 18
Claims (4)
1. The mechanism for regulating miRNA-196a by long-chain non-coding RNAGAs-5 is characterized by comprising the following two mechanisms:
mechanism 1: a binding site I with long-chain non-coding RNAGAs-5 exists on the pri-miRNA-196a, the binding site I is a cleavage site of Drosha enzyme and Dicer enzyme, and the long-chain non-coding RNAGAs-5 prevents the Drosha enzyme and the Dicer enzyme from cleaving the pri-miRNA-196a by directly binding with the pri-miRNA-196a, so that the generation of pre-miRNA-196a is inhibited, and the generation of miRNA-196a is further inhibited;
mechanism 2: the long-chain non-coding RNAGAs-5 has a binding site II with DGCR8 protein, and the long-chain non-coding RNAGAs-5 and pri-miRNA-196a competitively bind with DGCR8 protein, so that the generation of pre-miRNA-196a by assisting Drosha enzyme to cut pri-miRNA-196a by the DGCR8 protein and the generation of miRNA-196a are inhibited.
2. The mechanism of claim 1, wherein the binding site I has the sequence 5'-UAGGUAGUUUCAUGUUGUUGGG-3' as long chain non-coding RNAGAs-5 regulating miRNA-196 a.
3. The mechanism of claim 1, wherein the long chain non-coding RNAGas-5 regulates miRNA-196a, wherein the sequence of binding site ii is: 5'-GUGGAGUCCAACUUGCCU-3' are provided.
4. The use of the mechanism of long non-coding RNA Gas-5 regulating miRNA-196a of any one of claims 1-3 in kits and chips for diagnosis and treatment of esophageal cancer.
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