CN106974929A - Purposes of the miR 647 in the medicine for preparing treatment stomach cancer - Google Patents
Purposes of the miR 647 in the medicine for preparing treatment stomach cancer Download PDFInfo
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Abstract
Suppressing the potential medical value of Metastasis of Gastric Cancer the invention discloses the chemical synthesis analog agomir 647 of miR 647.The present invention confirms that miR 647 expresses downward and related to patient's lymphatic metastasis and prognosis in stomach cancer by stomach organization qPCR and in situ hybridization etc.；Tested by bioinformatics target prediction, metastasis related gene screening verification and the functional verifications of stomach cancer cell line miR 647 etc., it is found that miR 647/SRF/MYH9 axles play crucial inhibitory action in Metastasis of Gastric Cancer.
Description
Technical field
The present invention relates to a kind of miR-647, and in particular to purposes of the miR-647 in the medicine for preparing treatment stomach cancer.
Background technology
MiRNA agomir are the miRNA analogs modified by special chemical, are entered by simulating miRNA
Enter miRISC compounds and played a role to adjust the expression of said target mrna.Compared with common miRNA mimics, agomir exists
There is higher stability and miRNA facilitation effects in animal body, and the obstacles such as internal cell membrane, tissue can be overcome to be enriched in
Target cell.It can be administered in zoopery with the method such as whole body or local injection, suction, medicine feed, action effect is lasting
Time is 6 weeks.Existing many miRNA agomir correlative study achievements are delivered.This is studied for zoopery
Agomir-647 is purchased from Guangzhou Ribo Bio Co., Ltd.'s (production code member：miR40003317-1-10).It there is no at present
The experimental research achievements report of the agomir-647.
The content of the invention
A kind of medicine for treating stomach cancer is provided it is an object of the invention to still undesirable for stomach cancer clinical treatment curative effect
Compositions, the invention provides purposes of the miR-647 in the medicine for preparing treatment stomach cancer, the invention provides agomir-
647 purposes in the medicine for preparing treatment stomach cancer.
To achieve the above object, the technical scheme taken：A kind of pharmaceutical composition for treating stomach cancer, the drug regimen
Thing includes miR-647 analogs agomir-647.The agomir-647 is the miR-647 classes through chemical modification synthesized in vitro
Like thing.
The invention provides purposes of the miR-647 in the medicine for preparing treatment stomach cancer.
The invention provides purposes of the agomir-647 in the medicine for preparing treatment stomach cancer.Specifically it there is provided
Purposes of the agomir-647 in the medicine for suppressing Metastasis of Gastric Cancer is prepared.
The present invention is pressed down by infrastest after cell and animal level first confirm that transcriptions of the miR-647 to SRF mRNA
Make and use, and find that it can suppress the related important skelemin of transfer, SRF downstream molecules II type myosins A indirectly
(MYH9) expression.
The beneficial effects of the present invention are：The present invention confirms that miR-647 exists by stomach organization qPCR and in situ hybridization etc.
Expression is lowered and related to patient's lymphatic metastasis and prognosis in stomach cancer；By bioinformatics target prediction, metastasis related
Because of the experiment such as screening verification and stomach cancer cell line miR-647 functional verifications, find miR-647/SRF/MYH9 axles in Metastasis of Gastric Cancer
In play crucial inhibitory action.
Brief description of the drawings
Fig. 1 expresses downward for miR-647 in the embodiment of the present invention 1 in stomach cancer, and related to patient's lymphatic metastasis
Picture；
Fig. 2 is miR-647 influence metastasis related gene SRF and the MYH9 tables in gastric carcinoma cell lines in the embodiment of the present invention 2
The picture reached；
Fig. 3 significantly inhibits the picture of stomach cancer cell migration for miR-647/SRF/MYH9 signal shafts in the embodiment of the present invention 3；
Fig. 4 influences patient's prognosis, and the figure related to SRF horizontal expressions for miR-647 expression in the embodiment of the present invention 4
Piece；
It is the picture by influenceing SRF expression to realize that Fig. 5 plays cancer suppressing action for miR-647 in the embodiment of the present invention 5.
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.MiR-647 analogs of the present invention refer to agomir-647.
Embodiment 1：MiR-647 expresses downward in stomach cancer, and related to patient's lymphatic metastasis
Experimental method and result are following (result is as shown in Figure 1)：
A in Fig. 1：109 Human Stomach Tissues (T) and corresponding normal mucosa tissues (N) are detected by qRT-PCR
Middle miR-647 expressions, wherein being used as internal reference using U6RNA.***p<.001
B in Fig. 1：With pie chart and Waterfall plot displaying miR-647 Human Stomach Tissues and corresponding normal mucosa tissues
Middle mrR-647 relative expression levels.The change multiple (T/N) of miR-647 relative levels>2 or<Think there is statistics meaning for 1/2
Justice.Waterfall plot：Change multiple (the log of miR-647 relative levels2[T/N])>1 or<- 1 is thought statistically significant.
As shown in figure 1, A and B illustrates that miR-647 is delivered up to downward in stomach cancer intermediate frequency by different statistical methods in Fig. 1.
C in Fig. 1：MiR-647 expressions and the phase of patients with gastric cancer clinical stages, lymphatic metastasis or local infiltration situation
Close relationship analysis.*P<.05；**P<.01.As shown in C in figure Fig. 1, miR-647 expression is related to T, N and TNM clinical stages.
D in Fig. 1：QRT-PCR detects miR-647 expression results in 5 kinds of stomach cancer cell lines.MGC 80-3 as shown in the figure
Expression is minimum, AGS and SNU-5 expression highests.
E, F in Fig. 1：MGC 80-3 after qRT-PCR detection transfections miR-647 and miR-647 tables in MKN45 stomach cancer cells
Up to level.***P<.001.As shown in E, F in figure Fig. 1, two kinds of cells have preferable transfection efficiency.
G in Fig. 1：3D cell migration Matrigel * * * P<.001
H in Fig. 1：Cell monolayer scratch test is used for 2D cell migrations.***P<.001.
As shown in figure 1, G and H observe the stable overexpressing cell strains of miR-647 and turned again in wink after being overexpressed respectively in Fig. 1
Suppress migration and the invasion and attack situation of its expression cell strain, as a result find after miR-647 overexpressions under cell migration and invasive ability
Drop, the ability after miR-647 that suppresses has to be recovered to a certain degree, illustrates the migration of miR-647 expression inhibitings stomach cancer cell and invasion and attack energy
Power.
Embodiment 2：MiR-647 influence metastasis related gene SRF and MYH9 expression in gastric carcinoma cell lines
Experimental method and result are following (result is as shown in Figure 2)：
A in Fig. 2：Bioinformatics Prediction mrR-647 potential target gene is carried out by 4 conventional miRNA databases.
Which show part gene related to cell motility and related with Rho paths (labeled as red).
B in Fig. 2：With miRBase Targets databases (http://microrna.sanger.ac.uk/targets)
Carry out the enrichment analysis of mrR-647 target genes path.Use 4 databases and miRGator (Nam, et al., Nucleic
Acids Research.36:D159-64；http://genome.ewha.ac.kr/miRGator).Cell movement function is related
Gene as shown in the chart.
C in Fig. 2：The expression of Rho passageway related genes in MGC 80-3 and AGS is detected by qRT-PCR.Wherein by
It is low in MGC 80-3 stomach cancer cell miR-647 expressions, therefore give MGC 80-3 stomach cancer cells to transfect miR-647 analogs；
And miR-647 expressions are high in AGS stomach cancer cells, therefore AGS stomach cancer cells are given to transfect miR-647 inhibitor.U6 is as interior
Ginseng.***P<.001.As shown in C in Fig. 2, SRF and MYH9 expression are substantially influenceed in two kinds of cells by miR-647 expression.
D-E in Fig. 2：The expression water of SRF and MYH9 in 5 plants of stomach cancer cell lines is detected with western blot and qRT-PCR
It is flat.GADPH is used as internal reference.As illustrated, AGS and SNU-5 SRF and MYH9 expression is relatively fewer；MGC 80-3 and MKN45 is thin
SRF and the MYH9 expression of born of the same parents is of a relatively high.
F in Fig. 2：Western blot detect that MGC 80-3 (infect what miR-647 was overexpressed with MKN45 stomach cancer cells
Slow virus or control group slow virus) in SRF and MYH9 protein expression levels.GADPH is used as internal reference.As shown in F in Fig. 2, plus
Enter miR-647 to be overexpressed after slow virus, cell SRF and MYH9 expression are lowered.
G in Fig. 2：The MGC 80-3- of western blot detections infection miR-647 inhibitor or its negative control (INC)
LV-miR-647 and SRF and MYH9 in MKN45-LV-miR-647 stomach cancer cells protein expression level.GADPH is used as internal reference.
As shown in G in Fig. 2, adding original two kinds of cells SRF and the MYH9 expression for being overexpressed miR-647 after miR-647 inhibitor has
Recovered.
Embodiment 3：MiR-647/SRF/MYH9 signal shafts significantly inhibit stomach cancer cell migration
Experimental method and result are following (result is as shown in Figure 3)：
A in Fig. 3：Predict miR-647 at SRF and MYH9mRNA 3 ' ends by TargetScan and miRanda databases
The binding sequence of noncoding region.
B, C in Fig. 3：To stable expression LV-miR-647 or LV-NC MGC 80-3 and AGS (MGC80-3-LV-miR-
647 and AGS-LV-miR-647) it is transferred to 3 ' end noncoding region reporter plasmids (pGV306-WT1-3), site mutation reporter plasmid
(pGV306-MUT1-4) the plain enzyme activity assay of fluorescence associated or after control plasmid (pGV306 is unloaded).With renilla luciferase
Activity is used as reference.***P<.001.As shown in B, C in Fig. 3, SRF 3 ' end noncoding regions the 1st and the 3rd position prediction binding site
It is miR-647 binding sequence；MYH9 prediction binding site is not detected by miR-647 binding activity.Point out miR-647 targetings
Suppress SRF rather than MYH9mRNA 3 ' end noncoding regions.
D, E in Fig. 3：With Relative luciferase activity assay checking determine SRF by target combination MYH9 promoters come
Adjust MYH9 expression.Predicted and turned in MYH9 promoters by Bioinformatics (Proscan and JASPAR) analyzing web site
Record the potential sequence that the factor is combined.(D) this research has synthesized MYH9 promoters full length sequence (luciferase plasmids structure) pGL3-
MYH9-1300 (wild type) and 5 sections block fragment (pGL3-MYH9-1000, pGL3-MYH9-903, pGL3-MYH9-753,
pGL3-MYH9-528and pGL3-MYH9-328).The sequence of SRF binding sites is contained in -589 to -439 sites, in fluorescein
Find that correspondence uciferase activity is significantly increased (D) in enzyme Reporter Gene Experiments.It is real by further luciferase reporter gene
Checking is real, SRF can directly in conjunction with MYH9 promoter wild types sequence (CCATATATGG is in -589 to -439 sites), and
Its correspondence mutant sequences (AACGACCAGAG) (E) can not be combined.***P<.001.Therefore, SRF is by combining MYH9 promoters
Area CCATATATGG promotes MYH9 transcriptions.
F in Fig. 3：Combined with the analysis checking of ChIP-qPCR experimental methods in MGC 80-3 with SRF in AGS stomach cancer cells
MYH9 starts subcase.Rna plymerase ii (RNAII) can be attached in GADPH promoters, therefore selects it right as the positive
According to.Displaying (mean ± SD is used as using the IgG enrichment times relative to control；*p<.05；**p<.01；***p<.001).Such as Fig. 3
Shown in middle F, the MYH9 promoter region DNA small fragments that SRF and MRTF antibody can leave behind using CCATATATGG as core, further
Illustrate that both SRF and MRTF are incorporated into CCATATATGG regional controls MYH9 expression.
G in Fig. 3：CarG box's in Protein S RF target combination MYH9 promoters illustrates.
H in Fig. 3：The MGC80-3-LV-miR- for being transferred to SRF plasmids or control group plasmid is tested and analyzed with Western blot
647 change with SRF in AGS-LV-miR-647 stomach cancer cells with MYH9 expressions.GADPH is used as internal reference.Meanwhile, Western
Blot is also used for testing and analyzing the MGC 80-3-LV-miR-647 and AGS-LV-miR- that are transferred to MYH9 plasmids or control group plasmid
SRF changes with MYH9 expressions in 647 stomach cancer cells.GADPH is used as internal reference.As shown in H in Fig. 3, it is overexpressed under miR-647
Adjust SRF and MYH9 expression；It is overexpressed SRF and recovers SRF and MYH9 protein expressions；And be overexpressed MYH9 and only recover MYH9 albumen tables
Reach, SRF expression has no significant change.This explanation miR-647 suppresses MYH9 and SRF expression, and SRF promotes MYH9 expression.With reference to glimmering
Light element enzyme reporter gene testing result prompting, miR-647 targeted inhibitions SRF expression is so as to lower the MYH9 of SRF dependent transcriptions
Expression.
I in Fig. 3：3D cell migration Matrigels：Respectively：Transfect the AGS-LV-miR-647 of SRF, MYH9 and empty carrier
Stomach cancer cell.Each bar represents the mean±SD.***p<.001.As shown in I in Fig. 3, miR-647 crosses table
Reduced up to caused cell migration invasion and attack and obtain a certain degree of recovery after SRF or MYH9 expression is recovered.With reference to testing before
As a result, it is to influence stomach cancer cell migration invasion and attack by SRF/MYH9 axles to illustrate miR-647.
Embodiment 4：MiR-647 expression influence patient's prognosis, and it is related to SRF horizontal expressions
Experimental method and result are following (result is as shown in Figure 4)：
A in Fig. 4：SRF albumen in 109 stomach organizations and correspondence normal mucosa carries out SABC.Original magnification,
100 × multiplication factor.
B in Fig. 4：Scoring is carried out according to the ImmunohistochemistryResults Results of SRF expressions to take statistics analysis (Chi-square Test, * * * p
<.001).As shown in B in Fig. 4, the SRF expression scorings of normal structure and cancerous tissue, which exist, scores high in significant difference, cancerous tissue
Occupy the majority that (scoring 3 accounts for 44.96%, and 32.11%) scoring 2 accounts for
C in Fig. 4：SRF expressions and the dependency relation of miR-647 levels are analyzed in 109 stomach organization samples, * * * p
<.001.As shown in C in Fig. 4, miR-647 and SRF qPCR detection of expression results show that expressions of both is negatively correlated.
D in Fig. 4：Hybridization in situ experiment detects miR-647 in stomach organization and the expression feelings in neighbouring normal mucosa tissue
Condition.Original magnification, 100 ×, 200 ×.(DIG labeled LNAprobes for U6and scramble were used
As positive and negative controls, respectively.) DIG label L NA probes.It is right respectively as the positive
According to (U6) and negative control (scramble).As shown in D in Fig. 4, miR-647 is expressed in cancer substantially to be lowered compared with normal epithelial.
E, F in Fig. 4：Dependency relation between miR-647 and SRF and MYH9 expressions in 109 gastric cancer cases.It show two
Representative case shows (E).Original magnification, 200 ×.MiR-647 low expressions or height are expressed as a percentage in tissue specimen,
Analysis and the dependency relation of SRF and MYH9 expressions.*p<.05.MiR-647 expression and MYH9, SRF as shown in E, F in Fig. 4
It is negatively correlated.
G in Fig. 4：With the corresponding SRF expression of Kaplan-Meier survival analysises method statistic analysis patients with gastric cancer existence
Horizontal dependency relation (log-rank test).As shown in G in Fig. 4, miR-647 expression height, miR- related to survival of patients
647 low expressions point out prognosis mala.
Embodiment 5
A-C is overexpressed plasmid with miR-647 overexpressions slow virus, SRF in Fig. 5 and the two corresponding control structure stomach cancer is thin
(LV-NC/vector is control group to the stable strain of born of the same parents' gene unconventionality expression；LV-miR-647/vector is that miR-647 is overexpressed
Stomach cancer cell；LV-miR-647/SRF is the cell line that the expression of SRF plasmid stabilisations is added after miR-647 is overexpressed, for function
Reply experiment, it was demonstrated that miR-647 function is by suppressing SRF expression realization).With three kinds of cell nude mice by subcutaneous into after knurl again
Original position plantation is observed to coat of the stomach, every group each 8,1 anesthetic death of control group.Anatomic observation after two months is planted in original position,
Primary tumo(u)r size and metastasis site and quantity are recorded, it is obvious by two sample t inspection statistics group differences.Illustrate miR-647
The stomach cancer cell one-tenth knurl ability and transfer ability of overexpression are significantly lowered, and the phenomenon obtains certain journey after SRF levels are overexpressed
The reverse of degree, it is by influenceing SRF expression realizations to further illustrate that miR-647 plays cancer suppressing action in vivo.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (3)
1. a kind of pharmaceutical composition for treating stomach cancer, it is characterised in that described pharmaceutical composition includes miR-647 analogs
agomir-647。
Purposes of the 2.miR-647 in the medicine for preparing treatment stomach cancer.
3.agomir-647 the purposes in the medicine for preparing treatment stomach cancer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097473A (en) * | 2018-08-24 | 2018-12-28 | 南京求臻基因科技有限公司 | A kind of non-small cell lung cancer auxiliary diagnostic box |
| JP2021031420A (en) * | 2019-08-21 | 2021-03-01 | 日東電工株式会社 | Cancer metastasis inhibition using myh9 inhibitory substance |
-
2017
- 2017-03-24 CN CN201710186450.8A patent/CN106974929A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| WENLONG CAO等: "Role of miR-647 in human gastric cancer suppression", 《ONCOLOGY REPORTS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097473A (en) * | 2018-08-24 | 2018-12-28 | 南京求臻基因科技有限公司 | A kind of non-small cell lung cancer auxiliary diagnostic box |
| JP2021031420A (en) * | 2019-08-21 | 2021-03-01 | 日東電工株式会社 | Cancer metastasis inhibition using myh9 inhibitory substance |
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