CN112941183B - Application of non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment - Google Patents

Application of non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment Download PDF

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CN112941183B
CN112941183B CN202110279486.7A CN202110279486A CN112941183B CN 112941183 B CN112941183 B CN 112941183B CN 202110279486 A CN202110279486 A CN 202110279486A CN 112941183 B CN112941183 B CN 112941183B
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liver cancer
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primary liver
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周俊
孙丹丹
王腊
邓洋
李海涛
毛昌霞
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Wuhan University of Science and Engineering WUSE
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Abstract

The invention provides application of a non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment, and particularly relates to a kit for diagnosing primary liver cancer by using the non-coding gene miR-187-5 p. The low expression of the miR-187-5p gene can obviously promote the proliferation of the human primary liver cancer cell Huh7, and the overexpression inhibits the proliferation of the primary liver cancer cell Huh7, so that the miR-187-5p gene can be used as an early diagnosis index for detecting primary liver cancer and can also be used as a targeted medicine for preparing the human primary liver cancer cell Huh 7.

Description

Application of non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment
Technical Field
The invention relates to the field of biological medicines, in particular to application of a non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment.
Background
Primary liver cancer (HCC) is a malignant tumor with high morbidity and mortality in China, and about 60 million new cases occur each year. Although the current tumor diagnosis and treatment level is continuously improved, the conventional treatment means still has no revolutionary breakthrough, and the morbidity and mortality are still high. Liver transplantation and liver cancer resection are the most effective treatment methods at present, but the application is limited, the total survival rate in 5 years is less than 5%, and the prognosis is very poor. Therefore, the development of new therapeutic targets and new therapeutic approaches by improving the existing therapeutic protocols is the main direction of research.
microRNA (miRNA) is a non-coding small segment RNA which is widely present in eukaryotes and can be widely involved in various physiological and pathological processes of organisms by regulating the expression of target genes. During the growth and development process of the human primary liver cancer, miRNA induces normal cells to transform to abnormal cells by down regulating the activity of cancer suppressor genes, or reduces cell differentiation and suppresses apoptosis genes so as to enable abnormal cells to grow without control; some miRNAs also have the effects of inhibiting oncogenes, inhibiting proto-oncogenes, transferring proliferation related genes and the like, and further inhibiting the proliferation and migration processes of liver cancer cells. At present, the action principle of the microRNA targeted drug in the primary liver cancer is only limited on the surface curative effect, and the deep mechanism still needs to be further researched. Therefore, the mechanism of the microRNA targeted drug in treating the primary liver cancer is deeply researched, a basis is provided for safer and more reasonable medication, and a foundation is laid for further optimizing the drug.
Disclosure of Invention
In view of the above, the invention provides a non-coding gene miR-187-5p for inhibiting cancer cell proliferation and application thereof in primary liver cancer diagnosis and treatment.
The technical scheme of the invention is realized as follows: the invention provides application of a non-coding gene miR-187-5p in primary liver cancer diagnosis and treatment.
The application of the non-coding gene miR-187-5p in diagnosis and treatment of the primary liver cancer, and the non-coding gene miR-187-5p is used as a kit for diagnosing the primary liver cancer.
On the basis of the technical scheme, preferably, the nucleotide sequence of the non-coding gene miR-187-5p is shown in SEQ ID NO. 1.
On the basis of the technical scheme, preferably, the expression of the non-coding gene miR-187-5p in the primary liver cancer cell is obviously reduced.
On the basis of the technical scheme, preferably, the non-coding gene miR-187-5p is from a miR-187 precursor sequence, and the miR-187-5p mature sequence is obtained by cutting the miR-187 precursor sequence.
On the basis of the technical scheme, preferably, the miR-187 precursor sequence is shown in SEQ ID NO. 2.
On the basis of the technical scheme, preferably, the kit comprises a primer capable of specifically amplifying the non-coding gene miR-187-5p or a probe capable of specifically recognizing the non-coding gene miR-187-5 p.
On the basis of the technical scheme, preferably, the kit further comprises a reverse transcription primer of the non-coding gene miR-187-5 p.
On the basis of the technical scheme, preferably, the reverse transcription primer is shown as SEQ ID NO. 3.
On the basis of the technical scheme, preferably, the kit further comprises a qRT-PCR primer pair of a non-coding gene miR-187-5 p:
miR-187-5p F：5'-CGCAGTGGCTACAACACAGG-3'
miR-187-5p R：5'-AGTGCAGGGTCCGAGGTATTC-3'。
on the basis of the technical scheme, preferably, the kit further comprises a positive control substance, a negative control substance, a buffering agent, an auxiliary agent and a solvent.
Based on the above technical solution, preferably, the sample to be tested is from cells, serum or plasma.
On the basis of the technical scheme, preferably, the low expression of the non-coding miR-187-5p gene remarkably promotes the proliferation of the human primary liver cancer cell Huh7, and the over-expression inhibits the proliferation of the primary liver cancer cell Huh 7.
Further preferably, the application of the non-coding gene miR-187-5p in preparation of the anti-human primary liver cancer Huh7 cell drug is also included.
Compared with the prior art, the application of the non-coding gene in primary liver cancer diagnosis and treatment has the following beneficial effects:
(1) The low expression of the non-coding gene miR-187-5p promotes the proliferation of the primary liver cancer cell, and the overexpression can inhibit the proliferation of the primary liver cancer cell Huh7, so that the gene can be used as an early diagnosis index for detecting the primary liver cancer, and can also be used as a targeted medicine for preparing the anti-human primary liver cancer cell Huh 7.
(2) The invention discloses that the down regulation of miR-187-5p gene in cells is an index for predicting primary liver cancer diagnosis.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a box diagram of miR-187-5p of the present invention;
FIG. 2 is a graph of miR-187-5p cell levels of the present invention;
FIG. 3 is a miR-187-5p qRT-PCR assay of the present invention;
FIG. 4 is a graph of the experimental results of miR-187-5p cck-8 of the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
miR-187 is located at chromosome 18q12.2, and a miR-187 precursor sequence is shown in SEQ ID No. 2. The miR-187 precursor sequence is transcribed, and the 5' end is cut to obtain a miR-187-5p mature body sequence, namely a miR-187-5p sequence, which is shown in SEQ ID NO. 1.
Example one analysis of miR-187-5p expression in TCGA database
1. Data collection
Collecting miRNA expression profile data of 369 primary liver cancer tissues and 49 para-cancer tissues from a TCGA database, and analyzing the expression level of miR-187-5p in the primary liver cancer tissues and the para-cancer tissues; box plots are drawn.
By TCGA database analysis, miR-187-5p has down-regulation in primary liver cancer tissue and normal tissue in clinical samples (figure 1).
Example expression of the DimiR-187-5 p Gene in liver cancer cell lines
1. Cell resuscitation
Wearing a protective mask and gloves, taking out LO2 cells, hepG2 cells and Huh-7 cell freezing tubes from a liquid nitrogen container, rapidly shaking the frozen cells in a water bath at 42 ℃ for 30sec, rapidly thawing the frozen cells in a water bath kettle at 37 ℃ by shaking, and centrifuging at 200 Xg room temperature for 4min. Discarding the frozen stock solution in a sterile environment between cell cultures, adding 1mL of complete medium, pipetting the suspended cells, transferring into a 6cm cell culture dish, adding complete medium, placing at 37 deg.C, and 5% CO 2 Cultured in an incubator for subsequent experiments.
2. Subculturing of cells
When the cell closure in the dish reached about 95%, the medium was aspirated and discarded, 1mL of PBS solution was added, the dish was tilted so that the PBS solution covered and washed all the cells, 1mL of trypsin solution was added after discarding the PBS, and digestion was performed at room temperature for 1-2min. After a large piece of cells fell off the surface of the dish, 2mL of complete medium was added. Blowing the cell suspension with a pipette 4-8 times until it is uniform, adding 1mL of cell suspension into a new culture medium dish, adding complete culture medium to 6mL, adding 37 deg.C, and 5% CO 2 Cultured in an incubator for subsequent experiments.
3. Extraction of miRNA
Using RNeasy Mini KitAnd (6) rows. The sample was lysed with guanidinium thiocyanate in buffer (which rapidly inactivates rnases), ethanol was added to provide the appropriate binding conditions, the sample was transferred to an RNeasy Mini spin column to allow total RNA to bind to the membrane, and contaminants were effectively washed away. High quality RNA was then eluted in 30-100. Mu.L of water. The kit selectively excludes most<200 nucleotides of RNA (e.g., 5.8S rRNA,5S rRNA, and tRNA). The size distribution of the purified RNA was comparable to the size obtained by CsCl buffer centrifugation. The method comprises the following specific steps: digesting the cell with pancreatin to obtain 3-5 × 10 6 Centrifuging the cells at 4 ℃ at 250 Xg for 3min, removing the cells collected from the supernatant, or removing the tumor tissue of 20-40 mg, adding 1mL Buffer RLT reagent, mixing by gentle blowing with a pipette or grinding with a glass grinder, standing on ice for 10min, adding 200 μ L chloroform, covering a tube cover, performing vortex oscillation for 15sec, centrifuging at 14000 Xg at 4 ℃ for 8min, taking the supernatant, adding 75% ethanol with the same volume, reversing, mixing uniformly, and adding the mixture into an adsorption column Spin Columns RM filled in a collecting tube. 14000 Xg was centrifuged at 4 ℃ for 30sec, and the waste liquid was discarded, and the waste liquid in the collection tube was discarded. mu.L of Buffer RW1, 14000 Xg was added and centrifuged at 4 ℃ for 30sec, and then the waste liquid was discarded. 600. Mu.L of Buffer RPE,14000 Xg was added, and after centrifugation at 4 ℃ for 30sec, the waste liquid was discarded. 14000 Xg is centrifuged at 4 ℃ for 2min, and the column is put into a new EP tube and dried at 25 ℃ for 30 min. 40 μ L of deionized water was added to the middle part of the adsorption column, and the column was left at 25 ℃ for 2min, and then centrifuged at 14000 Xg for 1min at room temperature to collect an RNA solution. The content is measured by a ultramicro spectrophotometer and then is directly used for subsequent reverse transcription or stored at-80 ℃.
4. Reverse Transcription (RT)
The extracted miRNA is firstly digested by DNase to remove DNA, and the digestion system is shown in Table 1. The prepared mixed solution is incubated for 2min in water bath at 42 ℃. The digested mixture was used to prepare a mixture for first strand synthesis of RNA and cDNA, and the system for first strand synthesis of cDNA is shown in Table 2. The prepared mixture was subjected to reverse transcription as shown in Table 3. The primer sequences are shown in Table 4.
TABLE 1 DNA digestion System in RNA
RNase-free ddH 2 O To10μL
5×g DNA Wiper Mix 2μL
Total RNA 10pg-1μg
TABLE 2 cDNA Synthesis System Table
RNase-free ddH 2 O To20μL
The mixed liquid of the last step 10μL
RT primer 1μL
10×RT Mix 2μL
HiScript Ⅱ Enzyme Mix 2μL
TABLE 3 first Strand cDNA Synthesis reaction
Temperature of Time
25℃ 5min
50℃ 15min
85℃ 5min
TABLE 4 primer sequences
Figure BDA0002978154610000071
5. Real-time fluorescent quantitative PCR (qRT-PCR)
Adding excessive SYBR fluorescent dye molecules which do not emit fluorescent signals into an RT-PCR reaction system, specifically doping the SYBR fluorescent dye molecules into a DNA double strand to emit the fluorescent signals in a DNA amplification reaction, synchronizing the increase of the fluorescent signals with PCR products due to the linear correlation between the Ct value of the template and the initial copy number of the template in the exponential period of the PCR amplification, and quantitatively analyzing a specific DNA sequence in a sample to be detected by a method for measuring the total amount of products after each PCR cycle. And after the reaction cycle is finished, transferring the reaction product into a PCR product melting curve graph to verify the specificity of the qRT-PCR primer and the PCR amplification product. The intergroup data was normalized by detection of the internal parameters.
And carrying out qRT-PCR analysis by using qRT-PCR premix liquid by adopting an SYBR Green method. And (3) taking a cDNA sample obtained by reverse transcription or miRNA first strand cDNA as a template, adding a primer corresponding to a target gene to prepare a qRT-PCR system, and detecting U6 as an internal reference. Each set of three replicate wells. The reaction system is shown in Table 5. The prepared qRT-PCR mixed solution is added into an eight-connected tube and put into a qRT-PCR instrument for reaction, and the reaction program is shown in Table 6. All the used qRT-PCR primer sequence information is shown in Table 7.
TABLE 5 qRT-PCR systems Table
Figure BDA0002978154610000081
TABLE 6 reaction schedule
Figure BDA0002978154610000082
TABLE 7 primer sequences
Figure BDA0002978154610000083
The qRT-PCR experiment result shows that: compared with the expression levels of normal cells LO2, liver cancer cells Huh-7 and HepG2, miR-187-5p, the expression levels of the normal cells LO2, the liver cancer cells Huh-7 and HepG2 are respectively 1.00000, 0.00016 and 0.44302, and Huh-7 is most obvious (figure 2). Further detecting the Huh-7 cells, transfecting miR-187-5p mimics and a control thereof, detecting the transfection efficiency through qRT-PCR, and indicating that: the minic expression level was 2627.62927, the minic NC expression level was 1.00000, and miR-187-5p was overexpressed (FIG. 3).
EXAMPLE three CCK-8 assay for cell proliferation
1. Cells were diluted to 1-2X10 6 One cell/mL (density is determined according to the subsequent treatment mode and detection time) is subcultured into a 96-well cell culture plate according to 100 mu L/well, and each group has 6 duplicate wells.
2. After cells adhere, the cells were transfected using lip2000 transfection reagent at 1:3 ratio to carry out miR-187-5p transfection and a mimic and a control thereof;
3. by means of thin websComplete medium for cell-specific assay CCK-8 solution was diluted according to the manufacturer's instructions, the original cell culture medium was replaced by 110. Mu.L/well, returned to 37 ℃ C., 5% CO 2 The incubator continues to culture.
4. 1-3h after CCK-8 addition, the incubation was completed and the absorbance (OD) was measured at 450nm in a full wavelength microplate reader.
The CCK8 detection result is shown in figure 4, compared with a negative control NC group and a blank control group, the cell proliferation of an experimental group transfected with miR-187-5P is obviously inhibited (P is less than 0.05), and the miR-187-5P overexpression has an obvious inhibition effect on the cell proliferation capacity of Huh 7.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Sequence listing
<110> Wuhan science and technology university
Application of <120> non-coding gene miR-187-5p in diagnosis and treatment of primary liver cancer related to hepatitis B virus
<130> 2021
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggcuacaaca caggacccgg gc 22
<210> 2
<211> 109
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ggucgggcuc accaugacac agugugagac cucgggcuac aacacaggac ccgggcgcug 60
cucugacccc ucgugucuug uguugcagcc ggagggacgc agguccgca 109
<210> 3
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgcccgg gt 52

Claims (1)

1. The application of the non-coding gene miR-187-5p in the preparation of the medicine for inhibiting the human primary liver cancer Huh7 cell proliferation is characterized in that: the nucleotide sequence of the non-coding gene miR-187-5p is shown in SEQ ID NO. 1.
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CN106459867A (en) * 2014-06-18 2017-02-22 东丽株式会社 Liver cancer detection kit or device, and detection method

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Publication number Priority date Publication date Assignee Title
CN106459867A (en) * 2014-06-18 2017-02-22 东丽株式会社 Liver cancer detection kit or device, and detection method

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