CN107236817A - The application of LncRNA ENST00000424523.1 and its gene in diagnosis and medicine - Google Patents
The application of LncRNA ENST00000424523.1 and its gene in diagnosis and medicine Download PDFInfo
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Abstract
The invention discloses the application of LncRNA ENST00000424523.1 and its gene in diagnosis and medicine, belong to biomedicine technical field, the application of LncRNA ENST00000424523.1 and its gene in screening or preparing chip, preparation or the kit of stomach cancer clinical diagnosis or prognosis, the application of LncRNA ENST00000424523.1 and its gene in screening or preparing curing gastric cancer medicine, LncRNA ENST00000424523.1 gene order number is LOC101927497.Preparation of the present invention to the clinical diagnosis, prognosis and medicine of stomach cancer has facilitation.
Description
Technical field
Exist the invention belongs to biomedicine technical field, more particularly to LncRNA ENST00000424523.1 and its gene
Application in diagnosis and medicine.
Background technology
Stomach cancer is one of most common alimentary system malignant tumour in the world, and the patients with gastric cancer of statistics display 70% belongs to hair
It is national in exhibition；Wherein, the patients with gastric cancer of nearly half is belonged into the sub- country in East Asia, particularly China.Pathogeny of gastric cancer is complicated, and it is related to
Factor have helicobacter pylori infections, diet, environmental carcinogen exposure and genetic predisposition etc.；Environmental carcinogen is considered as to lead
Cause one of most important factor of stomach carcinogenesis.
N- nitroso compounds (NOCs) are the class strong carcinogens related to stomach carcinogenesis, are mainly derived from life
It is all kinds of to pickle thing, sootiness thing or formed by endogenous.N- methyl-N '-nitro-N '-nitrosoguanidine (MNNG) is used as N- nitrosos
One kind of compound, is to generally acknowledge the chemical mutagen and carcinogen being widely present in environment.Although current people have built MNNG
The animal model and cell model of stomach cancer are induced, the research of stomach cancer is obtained certain progress, but the stomach cancer mechanism that MNNG is related to
Still need to further clearly.
Past, chemical carcinogen induces the Mechanism Study of stomach cancer mainly for encoding gene, in recent years with high-flux sequence
Etc. the development of biotechnology, non-coding RNA induces associating for stomach cancer with chemical carcinogen and is gradually valued by people.Long-chain is non-
Coding RNA (Long noncoding RNA, LncRNA) is the non-coding RNA that a class length is more than 200 nucleotides.LncRNA is
The important gene expression regulation element of one class, can be in a variety of levels (after epigenetic, the transcription and transcription etc.) table of controlling gene
Reach, and, generation, development, apoptosis, migration and the adhesion of such as tumour closely related with a variety of diseases.According to LncRNA in tumour
Effect in generation, is divided into the LncRNA of carcinogenesis and the LncRNA of cancer suppressing action at present.
Stomach cancer turns into the disease problem of today's society, and the clinical diagnosis and prognosis of early stage are conducive to the treatment of stomach cancer, together
When stomach cancer medicine await further research and development.It is examining for stomach cancer therefore, it is necessary to which LncRNA is combined with stomach cancer
Disconnected, prognosis and treatment provide new treatment thoughts.
The content of the invention
Present invention aims to overcome that prior art exist deficiency, and provide LncRNA ENST00000424523.1 and
Application of its gene in diagnosis and medicine.To achieve the above object, the technical scheme taken of the present invention is：LncRNA
ENST00000424523.1 and its gene are in screening or preparing chip, preparation or the kit of stomach cancer clinical diagnosis or prognosis
Application, LncRNA ENST00000424523.1 gene order number is LOC101927497.
In addition, the present invention also provides LncRNA ENST00000424523.1 and its gene is screening or prepared curing gastric cancer
Application in medicine, LncRNA ENST00000424523.1 gene order number is LOC101927497.
In addition, the reagent that the present invention also provides detection LncRNA ENST00000424523.1 faces in screening or preparation stomach cancer
Application in chip, preparation or the kit of bed diagnosis or prognosis.
In addition, the present invention also provides chip, preparation or the kit of a kind of stomach cancer clinical diagnosis or prognosis, including determine
The specific primer of LncRNA ENST00000424523.1 transcription amounts, the specific primer include such as Seq ID NO.1 and
DNA sequence dna shown in Seq ID NO.2.
The present invention confirms LncRNA ENST00000424523.1 gene by high-flux sequence RNA-Seq first
(LOC101927497) transcription in stomach cancer cell and negative cells has differences, and is prepared for detecting LncRNA
ENST00000424523.1 specific primer, can be used as the label of clinical diagnosis and the prognosis of stomach cancer.
In addition, the present invention, which also provides LncRNA ENST00000424523.1 accelerator, is screening or is preparing curing gastric cancer medicine
Application in thing.
As the improvement of above-mentioned technical proposal, the accelerator of LncRNA ENST0000042452 3.1 includes promotion base
Because of the plasmid that LOC101927497 is overexpressed, the plasmid contains gene LOC101927497 DNA sequence dna.
The present invention builds the plasmid that gene LOC101927497 is overexpressed, and design synthesizes gene LOC101927497
SiRNA, experiment find：In GES-1-T (people's gastric mucosal cell of vicious transformation) and two kinds of cells of MKN-28 (stomach cancer cell)
In, up-regulated expression LncRNA ENST00000424523.1 can suppress proliferation of human gastric cancer cell, lower expression LncRNA
ENST00000424523.1 can promote proliferation of human gastric cancer cell.
In addition, the present invention also provides a kind of medicine of stomach cancer, the medicine includes LncRNA
ENST00000424523.1 accelerator.
As the improvement of above-mentioned technical proposal, the LncRNA ENST00000424523.1 accelerator includes promotion gene
The plasmid that LOC101927497 is overexpressed, the plasmid contains gene LOC101927497 DNA sequence dna.
Further improved as above-mentioned technical proposal, the medicine is also comprising acceptable carrier on Students Majoring in Pharmacy.
In the above-mentioned technical solutions, the solvent of " pharmaceutically acceptable carrier " including any and all physical compatibility,
Decentralized medium, be coated material, antibacterium and antifungal agent, etc. blend absorption delayer etc..The example of pharmaceutically acceptable carrier
Including one or more of water, salt solution, phosphate buffered saline, dextran, glycerine, ethanol etc. and combinations thereof.
In many cases, preferably isotonic agent such as sugar, polyalcohol or sodium chloride are included in the composition.It is pharmaceutically acceptable
Carrier can also can improve the shelf life or the auxiliary substance of validity of antibody or antibody moiety, such as wetting agent or breast comprising a small amount of
Agent, preservative or buffer solution.
In addition, the present invention also provides a kind of gene LOC101927497 siRNA, the siRNA be siRNA-1,
SiRNA-2 or siRNA-3, the siRNA-1 are made up of the RNA sequence as shown in Seq ID NO.3 and Seq ID NO.4, institute
State siRNA-2 to be made up of the RNA sequence as shown in Seq ID NO.5 and Seq ID NO.6, the siRNA-3 is by such as Seq ID
RNA sequence composition shown in NO.7 and Seq ID NO.8.
The beneficial effects of the present invention are：The present invention provides LncRNA ENST00000424523.1 and its gene in diagnosis
With the application in medicine, the present invention confirms LncRNA by high-flux sequence RNA-Seq first
Transcription of the ENST00000424523.1 gene (LOC101927497) in stomach cancer cell and negative cells has differences；And
It is prepared for detecting LncRNA ENST00000424523.1 specific primer, can apply to the clinical diagnosis of stomach cancer and pre-
Afterwards；RNA-Seq data and qRT-PCR data are all shown：LncRNA ENST00000 424523.1 glue in the stomach of vicious transformation
Downward, up-regulated expression LncRNA are expressed in theca cell GES-1-T and stomach cancer cell (MKN-28, MGC-803)
ENST00000424523.1 can suppress proliferation of human gastric cancer cell, and stomach can be promoted by lowering expression LncRNA ENST00000424523.1
Cancer cell multiplication；As can be seen here, LncRNA ENST00000424523.1 and gene can be applied to the clinical diagnosis of stomach cancer, prognosis
And treatment.
Brief description of the drawings
Fig. 1 is the high flux RNA-Seq sequencing result figures of GES-1-T cells and GES-1-N cells；Figure 1A shows two kinds
The LncRNA of Cell differentials expression, Figure 1B show the mRNA of two kinds of Cell differentials expression；In figure, differential expression LncRNA and mRNA
Marked with circle；
Fig. 2 is the statistical chart of checking LncRNA differential expression；Fig. 2A is shown in GES-1-T cells and GES-1-N cells
LncRNA expression quantity, EN~452050.1 are ENST00000452050.1 abbreviation, and EN~435695.1 are
ENST00000435695.1 abbreviation, EN~424523.1 are ENST00000424523.1 abbreviation；Fig. 2 B show that GES-1 is thin
LncRNA ENST00000424523.1 relative levels in born of the same parents, MKN-28 cells and MGC-803 cells；In figure, * represents p<
0.05, * * represents p<0.01；
Influence of Fig. 3 display up-regulation LncRNA ENST00000424523.1 expression to proliferation of human gastric cancer cell；Fig. 3 A show
Show LncRNA in the GES-1-T cells and MKN-28 cells for import over-express vector pcDNA-LOC101927497
ENST00000424523.1 relative expression quantity；Fig. 3 B displays import over-express vector pcDNA-LOC101927497 GES-
The propagation of 1-T cells and MKN-28 cells is inhibited；In figure, pcDNA-LncRNA is quiding gene LOC101927497's
Over-express vector, pcDNA3.1 is negative control；
Influence of the LncRNA ENST00000424523.1 expression to proliferation of human gastric cancer cell is lowered in Fig. 4 display displays；Figure
LncRNA ENS T00000424523.1 relative expression in 4A display transfections siRNA GES-1-T cells and MKN-28 cells
Amount；Fig. 4 B display transfections siRNA GES-1-T cells and the propagation of MKN-28 cells are promoted；In figure, siRNA is feminine gender
Control.
Embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with specific embodiment, subordinate list and attached
The invention will be further described for figure.
The high flux RNA-Seq sequencing results of embodiment 1 are analyzed, and the GES-1-T cells of MNNG processing can produce differential expression
LncRNA
Method：The vicious transformation people gastric mucosal cell GES-1-T (T induced with MNNG：Conversion) and DMSO processing feminine gender
Control cell GES-1-N (N：Normally) it is experimental subjects, by high flux RNA-Seq sequencing technologies, analyzes in two kinds of cells
LncRNA and mRNA differential expression.The LncRNA expression quantity in GES-1-T and GES-1-N cells is analyzed, with differential expression times
Count more than 2 times or less than 0.5 times, p<0.05 is the LncRNA that standard determines differential expression, and difference is determined with same standard
The mRNA of expression.
As a result：Found by analysis, detect to examine in LncRNA 17626, GES-1-N cells in GES-1-T cells
Measure LncRNA 17916.There are 253 compared with the LncRNA with differential expression in GES-1-N cells in GES-1-T cells, its
The LncRNA of middle up-regulated expression has 94, and the LncRNA that expression is lowered has 159 (as shown in Figure 1A)；Simultaneously, it was also found that two kinds
There are the mRNA totally 751 of differential expression, the mRNA of wherein up-regulated expression there are 244 in cell, the mRNA that expression is lowered has 507
Individual (as shown in Figure 1B).As shown in table 1, list and most obvious 3 LncRNA are lowered in GES-1-T cells.
Table 1
Embodiment 2LncRNA ENST00000424523.1 low expressions in GES-1-T cells and stomach cancer cell
Method：In order to further find the LncRNA that may be played a role in MNNG induces GES-1 cells evil to turn, I
More significant 3 LncRNA of variation are chosen from the LncRNA with different expression, existed with qRT-PCR method
GES-1-T cells in GES-1-N cells with detecting its expression.In addition, we also have detected stomach cancer cell by qRT-PCR
LncRNA ENST00000424523.1 expression in MKN-28 and MGC-803.LncRNA used in qRT-PCR and internal reference
As shown in table 2, Forward is forward primer to GAPDH primer sequences in table 2, and Reverse is reverse primer；LncRNA
ENST00000424523.1 cDNA primer sequences Forward-AATGCTTGGAATGTGGGAGC is (such as Seq ID NO.1 institutes
Show), Reverse-GGCAGCTTTCAGGGGTTTTA (as shown in Seq ID NO.2).
As a result：3 more significant LncRNA of variation are analyzed, we have found that LncRNA from testing result
ENST00000424523.1, its NCBI gene number is LOC101927497, is a not studied LncRNA.With GES-
1-N cells are compared, and (fold change=are substantially lowered in expression of the LncRNA ENST00000424523.1 in GES-1-T
0.3974799 ± 0.26, p<0.05) (as shown in Figure 2 A)；LncRNA ENST00000424523.1 are in MKN-28, MGC-803
Low expression level (as shown in Figure 2 B) is equally shown as in both stomach cancer cells.These results indicate that LncRNA
ENST00000424523.1 in the MNNG evils induced turn cell GES-1-T and stomach cancer cell line lower by expression, gene
LOC101927497 plays a significant role possible as tumor suppressor gene during MNNG induces stomach cancer.
Table 2
The propagation of the up-regulation LncRNA of embodiment 3 ENST00000424523.1 expression inhibiting stomach cancer cell
Method：In order to inquire into functions of the gene LOC101927497 during MNNG induces stomach cancer, by building table
Up to carrier pcDNA-LOC101927497, gene LOC101927497 is in vicious transformation stomach cancer cell GES-1-T and stomach cancer for research
Effect in cell line MKN28.Transfect GES-1-T and MKN-28 cells, 48 hours respectively with pcDNA-LOC101927497
Afterwards, LncRNA ENST00000424523.1 overexpression effect is detected with qRT-PCR methods, and was detected with CCK-8 kits
Influences of the LncRNA ENST00000424523.1 of table to proliferation of human gastric cancer cell.
As a result：Transfect after pcDNA-LOC101927497, LncRNA ENST00000424523.1 expression exists
GES-1-T and MKN-28 cells have substantially up-regulation (as shown in Figure 3A)；Secondly, the LncRNA of overexpression
ENST00000424523.1 substantially weakens the propagation (as shown in Figure 3 B) of GES-1-T cells and MKN-28 cells.The result shows
Up-regulation LncRNA ENST00000424523.1 expression has obvious inhibiting effect to the multiplication capacity of stomach cancer cell.
Embodiment 4 lowers the propagation of LncRNA ENST00000424523.1 expression promotion stomach cancer cell
Method：Disturb LncRNA ENST00000424523.1 expression with siRNA, detection to GES-1-T cells and
The situation of MKN-28 cells propagation.3 specific siRNAs are synthesized：SiRNA-1, siRNA-2, siRNA-3 (as shown in table 3),
GES-1-T cells and MKN-28 cells are transfected respectively with 3 siRNA, and after 48 hours, LncRNA is detected with qRT-PCR
ENST00000424523.1 expression is further used under the detection of CCK-8 kits with verifying siRNA interference effect
Influences of the LncRNA ENST00000424523.1 that mileometer adjustment reaches to proliferation of human gastric cancer cell.
As a result：Transfect after 3 siRNA, LncRNA ENST00000424523.1 expression is in GES-1-T cells
With (as shown in Figure 4 A) is decreased obviously in MKN-28 cells；After the expression for striking low LncRNA ENST00000424523.1,
GES-1-T and MKN-28 ability of cell proliferation is significantly increased, and MKN-28 cells are more substantially (as shown in Figure 4 B).
Table 3
Finally, it should be noted that above example is to illustrate technical scheme rather than to present invention protection
The limitation of scope, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should manage
Solution, technical scheme can be modified or replaced on an equal basis, without departing from technical solution of the present invention essence and
Scope.
Sequence table
<110>Attached 5th hospital of Guangzhou medical university
<120>The application of LncRNA ENST00000424523.1 and its gene in diagnosis and medicine
<130> 2017.7.3
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Claims (10)
1.LncRNA ENST00000424523.1 and its gene are screening or prepared chip, the system of stomach cancer clinical diagnosis or prognosis
Application in agent or kit, LncRNA ENST00000424523.1 gene order number is LOC101927497.
The application of 2.LncRNA ENST00000424523.1 and its gene in screening or preparing curing gastric cancer medicine, LncRNA
ENST00000424523.1 gene order number is LOC101927497.
3. detect LncRNA ENST00000424523.1 reagent screen or prepare stomach cancer clinical diagnosis or prognosis chip,
Application in preparation or kit.
4. a kind of stomach cancer clinical diagnosis or chip, preparation or the kit of prognosis, it is characterised in that：Including determining LncRNA
The specific primer of ENST00000424523.1 transcription amounts, the specific primer includes such as Seq ID NO.1 and Seq ID
DNA sequence dna shown in NO.2.
Application of the 5.LncRNA ENST00000424523.1 accelerator in screening or preparing curing gastric cancer medicine.
6. application as claimed in claim 5, it is characterised in that：The LncRNA ENST00000424523.1 accelerator includes
The plasmid for promoting gene LOC101927497 to be overexpressed, the plasmid contains gene LOC101927497 DNA sequence dna.
7. a kind of medicine of stomach cancer, it is characterised in that the medicine promotees comprising LncRNAENST00000424523.1
Enter agent.
8. medicine as claimed in claim 7, it is characterised in that：The LncRNA ENST00000424523.1 accelerator
Including the plasmid for promoting gene LOC101927497 to be overexpressed, the plasmid contains gene LOC101927497 DNA sequence dna.
9. medicine as claimed in claim 7 or 8, it is characterised in that：The medicine is also included and can connect on Students Majoring in Pharmacy
The carrier received.
10. a kind of gene LOC101927497 siRNA, it is characterised in that：The siRNA be siRNA-1, siRNA-2 or
SiRNA-3, the siRNA-1 are made up of the RNA sequence as shown in Seq ID NO.3 and Seq ID NO.4, the siRNA-2
It is made up of the RNA sequence as shown in Seq ID NO.5 and Seq ID NO.6, the siRNA-3 is by such as Seq ID NO.7 and Seq
RNA sequence composition shown in ID NO.8.
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