CN108245526A - A kind of liver cancer cell growth inhibitor and its application in prognosis in hcc judgement - Google Patents
A kind of liver cancer cell growth inhibitor and its application in prognosis in hcc judgement Download PDFInfo
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Abstract
A kind of application the invention discloses liver cancer cell growth inhibitor and its in prognosis in hcc judgement, belongs to biomedicine field.Liver cancer cell growth inhibitor miR 378a 3p provided by the invention can significantly inhibit proliferation, growth, the self-renewing of stem cell and the external migration of liver cancer cells, invasion in vitro, and can inhibit the distal end Lung metastases of subcutaneous Liver Cancer Bearing Nude Mice, the growth of liver orthotopic transplantation tumor and liver cancer cells in vivo.Functional verification the result shows that, miR 378a 3p take on the role of tumor-inhibitory microRNA in liver cancer.
Description
Technical field
A kind of application the present invention relates to liver cancer cell growth inhibitor and its in prognosis in hcc judgement, belongs to biological doctor
Medicine field.
Background technology
Liver cancer is one of higher malignant tumour of global incidence and the death rate, the annual whole world new hair of more than half
China is happened at dead liver cancer patient.Wherein, most liver cancer belongs to hepatocellular carcinoma.Find suitable biomarker
It is of great significance for the diagnose and treat of liver cancer.MicroRNA is the non-coding that a kind of length is about 22-24 nucleotide
RNA, they can the level up regulation gene expression after transcription.Due to the particularity of function itself, microRNA has preferable
Expression specificity can be stable in the presence of in tissue and blood, can be as the biological marker of medical diagnosis on disease, prognosis and treatment
Object.
Invention content
Verification and liver cancer tissue the present invention is based on miR-378a-3p differential expressions in liver cancer clinical samples/cell line
MiR-378a-3p expressions and the analysis of clinical pathological factors correlation, further determined that miR-378-3p is thin to liver cancer
Intracellular growth, migration, invasion, the influence for shifting biological behaviour, and 7 potential target genes of candidate miR-378a-3p.
Based on the above results, the present invention provides miR-378a-3p (SEQ ID NO.1) in the drug for preparing treatment liver cancer
In application.
Specifically, refer to miR-378a-3p prepare inhibit liver cancer cells self-renewing drug in application, it is described from
I, which updates, includes proliferation, differentiation, growth, clone.
Specifically, further include miR-378a-3p prepare inhibit the migrations of liver cancer cells, transfer, invasive ability drug
In application.
The present invention the result shows that, miR-378a-3p can significantly inhibit the proliferation of liver cancer cells, growth, stem cell in vitro
Self-renewing and external migration, invasion, and Liver Cancer Bearing Nude Mice subcutaneous, the growth of liver orthotopic transplantation tumor and liver cancer can be inhibited in vivo
The distal end Lung metastases of cell.Functional verification the result shows that, miR-378a-3p takes on tumor-inhibitory microRNA's in liver cancer
Role.
Description of the drawings
Fig. 1 CCK methods detect influences of the miR-378a-3p to liver cancer cells growth in vitro.(a) miR-378a-3p pairs
The influence of MHCC97H growths；(b) influence that antagonism miR-378a-3p grows SMMC-7721.MHCC97H：With
AgomiRcontrol compares, the 1st day after transfection, * p<0.05, difference the 2nd, 3,4 day after transfection, * * p<0.01；SMMC-
7721：Compared with antagomiRcontrol, difference the 1st, 2,3 day after transfection, * p<0.05, the 4th day after transfection, * * p<
0.01。
Fig. 2 plates of cells colony formation detects influences of the miR-378a-3p to liver cancer cells clonality.
(a) tumor colonies form representative photo；(b) opposite clonality column diagram.MHCC97H：With agomiRcontrol ratios
Compared with * * p<0.01；SMMC-7721：Compared with antagomiRcontrol, * * p<0.01.
Influences of Fig. 3 tumor stem cells microballoon experiment detection miR-378a-3p to liver-cancer stem cell self-renewal capacity.
(a) tumor stem cell microballoon representative photo；(b) tumour microballoon number column diagram.MHCC97H：With agomiRcontrol ratios
Compared with * * p<0.01；SMMC-7721：Compared with antagomiRcontrol, * * p<0.01.
Fig. 4 scratch experiments detect influences of the miR-378a-3p to fucosylation ability.(a) scratch experiment is representative
Photo；(b) tumour cell relative migration ability column diagram.MHCC97H：Compared with agomiRcontrol, * * p<0.01；SMMC-
7721：Compared with antagomiRcontrol, * p<0.05.
Fig. 5 Transwell Matrigels detect influences of the miR-378a-3p to Invasive Ability of Hepatocellular Carcinoma.(a)
Transwell Matrigel representative photos；(b) tumour cell is with respect to invasive ability column diagram.MHCC97H：With
AgomiRcontrol compares, * * p<0.01；SMMC-7721：Compared with antagomiRcontrol, * * p<0.01.
Influence of Fig. 6 liver cancer subcutaneous transplantation knurls into knurl scale-model investigation miR-378a-3p to liver cancer cells nude mice tumor growth.
(a) into the knurl time；(b) gross tumor volume；(c) small animal living body imaging representative diagram；(d) subcutaneous knurl photo；(e) knurl weight.Into
The knurl time：Compared with 97H-agomiRcontrol, * * p<0.01；Gross tumor volume：Compared with 97H-agomiRcontrol, swollen
The 2nd week after oncocyte inoculation, * p<0.05, difference the 3rd, 4 week after inoculation, * * p<0.01；Knurl weight：With 97H-
AgomiRcontrol compares, * * p<0.01.
Fig. 7 liver cancer subcutaneous transplantations knurl treats influences of the scale-model investigation miR-378a-3p to liver cancer cells nude mice tumor growth.
(a) gross tumor volume；(b) small animal living body imaging representative diagram；(c) subcutaneous knurl photo；(d) knurl weight.Gross tumor volume：With
AgomiRcontrol treatment groups compare, the 2nd week after transplantable tumor treatment, * p<0.05, respectively the 3rd, 4 after transplantable tumor treatment
Week, * * p<0.01；Knurl weight：Compared with agomiRcontrol treatment groups, * * p<0.01.
Fig. 8 liver cancer orthotopic transplantation tumor treats influences of the scale-model investigation miR-378a-3p to liver cancer cells nude mice tumor growth.
(a) small animal living body imaging representative diagram；(b) liver cancer original position knurl photo；(c) knurl weight.Knurl weight：It is controlled with agomiRcontrol
Treatment group compares, * p<0.05.
Influences of Fig. 9 miR-378a-3p to liver cancer cells tail vein injection Lung metastases ability.(a) nude mice lung tissue is substantially
Sample；(b) lung tissue HE dyes representative photo；(c) neoplasm lung metastasis tubercle column diagram.With agomiRcontrol treatment groups
Compare, * * p<0.01.
Influences of Figure 10 miR-378a-3p to liver cancer orthotopic transplantation tumor Lung metastases ability.(a) nude mice lung tissue is substantially marked
This；(b) lung tissue HE dyes representative photo；(c) neoplasm lung metastasis tubercle column diagram.With agomiRcontrol treatment groups ratio
Compared with * * p<0.01.
Figure 11 miR-378a-3p compare schematic diagram with 7 candidate target-gene sequence complementations.
The uciferase activity of 293T cells under Figure 12 difference transfection conditions.
Specific embodiment
1st, cell proliferation experiment
A. it takes the logarithm MHCC97H, SMMC-7721 liver cancer cells in growth period, with 0.25% trypsin digestion and blows and beats
Into individual cells, and cell is suspended in the DMEM culture solutions of 10% fetal calf serum, 1X10 is made5The cell suspension of/ml, 96
Inoculating cell suspension (100 μ l/ holes) in orifice plate；
B.24 agomiRcontrol, agomiR-378a-3p are used after hour；pantagomiRcontrol、antagomiR-
378a-3p transfects above-mentioned cell, final concentration of 200nM, and cell is grouped into：
First group：MHCC97H/agomiRcontrol vs MHCC97H/agomiR-378a-3p
Second group：SMMC-7721/antagomiRcontrol vs SMMC-7721/antagomiR-378a-3p
Every group is all provided with 6 multiple holes；
Wherein, agomiRcontrol, agomiR-378a-3p；antagomiRcontrol、antagomiR-378a-3p
Base sequence be respectively：
agomiR-378a-3p：
5’-ACUGGACUUGGAGUCAGAAGGC-3’
3’-UGACCUGAACCUCAGUCUUCCG-5’
Double-strand, and No. 2 position carbon atom methoxyl group modifications, No. 5 position carbon atom cholesterol modifications；agomiRcontrol、
AgomiR negative control are synthesized by Guangzhou Ribo Bio Co., Ltd.；
antagomiR-378a-3p：5 '-ACUGGACUUGGAGUCAGAAGGC-3 ', it is single-stranded, and No. 2 position carbon atom methoxies
Base modification, No. 5 position carbon atom cholesterol modifications；AntagomiRcontrol, antagomiR negative control are by wide
Rui Bo bio tech ltd of state city synthesizes.
C. culture plate is placed on incubator culture (37 DEG C, 5%CO2), it is cultivated 4 days after transfection；
D. respectively after transfection for 24 hours, 48h, 72h, 96h each time point per hole add in 10 μ l CCK-8 solution, after will training
It supports plate to be incubated in incubator 4 hours, the absorbance at 450nm is measured with microplate reader；
E. in triplicate, m- growth curve analysis result during drafting.
2nd, plates of cells colony formation
A. take the logarithm growth period MHCC97H, SMMC-7721 liver cancer cells count after adjustment cell concentration for 0.5 ×
105/ ml is inoculated in 24 well culture plates per hole 1ml；
B. secondary daily agomiRcontrol, agomiR-378a-3p；antagomiRcontrol、antagomiR-378a-
3p transfects above-mentioned cell, final concentration of 200nM, and cell is grouped into：
First group：MHCC97H/agomiRcontrol vs MHCC97H/agomiR-378a-3p
Second group：SMMC-7721/antagomiRcontrol vs SMMC-7721/antagomiR-378a-3p
C. it is suspended in respectively with 0.25% trypsin digestion and piping and druming into individual cells, and cell after transfecting 48 hours
It is spare in the DMEM culture solutions of 10% fetal calf serum.Cell suspension is made into the dilution of gradient multiple, every group of cell is respectively with every hole
400th, the Graded Density of 800,1600 cells is inoculated with respectively in 6 orifice plates of 37 DEG C of pre-temperature culture solutions containing 5ml, and is gently rotated,
Cell is made to be uniformly dispersed；
D. 37 DEG C, 5%CO are put2And it is cultivated 2~3 weeks in the cell incubator of saturated humidity；
E. observation daily when there is macroscopic clone in 6 orifice plates, terminates culture；
F. it moves and abandons supernatant, carefully embathed with PBS 2 times, 4% paraformaldehyde for adding in 5ml fixes cell 15 minutes, then
Fixer is removed, appropriate violet staining liquid is added to contaminate 10~30 minutes, then dyeing liquor is slowly washed away with flowing water, is air-dried；
G. 6 orifice plates are inverted and are superimposed a transparent film with grid, counted in microscope (low power lens) and be more than 10
Clone's number of cell calculates cloning efficiency, every group of setting by cloning efficiency=(clone's number/inoculating cell number) × 100%
Three multiple holes, in triplicate, statistic analysis result simultaneously calculate opposite clonality.
3rd, microballoon is tested
A. it takes the logarithm MHCC97H, SMMC-7721 liver cancer cells in growth period, with 0.25% trypsin digestion and blows and beats
Into individual cells, and cell is suspended in the DMEM culture solutions of 10% fetal calf serum, adjusted after counting cell concentration for 0.5 ×
105/ ml is inoculated in 24 well culture plates, secondary daily agomiRcontrol, agomiR-378a-3p per hole 1ml；
AntagomiRcontrol, antagomiR-378a-3p transfect above-mentioned cell, final concentration of 200nM, and cell is grouped into
MHCC97H/agomiRcontrol vs MHCC97H/agomiR-378a-3p；SMMC-7721/antagomiRcontrol vs
SMMC-7721/antagomiR-378a-3p；
B. transfect 48 hours after respectively with 0.25% trypsin digestion and piping and druming into individual cells, with 2ml's
MammoCultTMComplete medium (MammoCultTM basal medium+MammoCultTM proliferation
Supplement it) suspends, it is 2 × 10 that cell concentration is adjusted after counting3Cell/ml；
C.4×103/ 2ml is seeded on 6 well culture plates of ultralow adherency, puts 37 DEG C, 5%CO2Cell incubator culture；
D. it cultivates 1 week, observation experiment group and the difference of control group microballoon, three multiple holes of every group of setting, in triplicate, statistics
Analysis result.
4th, scratch experiment
A. it is marked behind with 6 porocyte culture plates before marking pen cell inoculation, it then will be in exponential phase
MHCC97H, SMMC-7721 liver cancer cells are suspended after the digestion of 0.25% pancreatin with the DMEM complete mediums of 10%FBS；
B. adjustment cell concentration is 5 × 10 after counting5/ ml is inoculated in 6 well culture plate (each of above-mentioned label per hole 1ml
6 holes of each inoculation of cell difference), 37 DEG C, 5%CO2Overnight incubation makes its 70% fusion remittance piece；
C. secondary daily agomiRcontrol, agomiR-378a-3p；antagomiRcontrol、antagomiR-378a-
3p transfects above-mentioned cell, final concentration of 200nM, and cell is grouped into：
First group：MHCC97H/agomiRcontrol vs MHCC97H/agomiR-378a-3p
Second group：SMMC-7721/antagomiRcontrol vs SMMC-7721/antagomiR-378a-3p
Every group is all provided with 3 multiple holes；
D. after transfecting for 24 hours, pipette tips clean 2 times with PBS and remove the cell that scrapes in mark cut；
E. cut for 24 hours after, take pictures under the common low power X100 light microscopics visual field, with ImageJ softwares analysis cell migration area take
Its average value, and calculated respectively using MHCC97H/agomiRcontrol, SMMC-7721/antagomiRcontrol as control
Tumour cell relative migration ability is tested in triplicate, influences of the statistical analysis miR-378a-3p to liver cancer transfer ability.
5th, Transwell Matrigels
A. by 50 μ l matrigels (Matrigel) and mixed liquor (50mg/l matrigels and the DMEM of plasma-free DMEM medium
Serum free medium is than 1:7, matrigel suitable 1:8 dilutions) equably it is layered on 24 hole Transwell cells (8 μm of aperture) substrate
The upper chamber face of film, 37 DEG C or 4 DEG C of air-dried plastics；
B. before refinement born of the same parents, 37 DEG C of effect 30min aquation bases of DMEM serum free mediums of the 100 μ l containing 0.5%BSA are added in
Counterdie；
C.200nM the two groups of cells of final concentration transfection for 24 hours：
First group：MHCC97H/agomiRcontrol vs MHCC97H/agomiR-378a-3p
Second group：SMMC-7721/antagomiRcontrol vs SMMC-7721/antagomiR-378a-3p
0.25% pancreatin digests and prepares single cell suspension, cell with the DMEM serum free medium suspensions containing 0.5%BSA
Cell concentration is adjusted after counting to 1 × 106/ml；
D. 100 μ l cell suspensions (1 × 10 are taken5Cell) add in the 24 of 8 μm of aperture for being covered with matrigel (Matrigel)
Hole Transwell cells (Transwell chamber), Transwell cells lower floor are complete with the DMEM of 500 μ l 10%FBS
Culture medium covers, and tests every group and sets 3 multiple holes respectively；
E.24h Transwell cells are taken out afterwards, and Transwell cells upper cell, 4% poly first are wiped with cotton swab head
Aldehyde fixes 0.1% violet staining 20min after 20min；
F. the cell that 5 visuals field counting invasion are randomly selected under the X200 high power light microscopics visual field takes its average value, and respectively
Using MHCC97H/agomiRcontrol, SMMC-7721/antagomiRcontrol as control, the opposite invasion of tumour cell are calculated
Ability is tested in triplicate, influences of the statistical analysis miR-378a-3p to liver cancer invasive ability.
6 transplanted human hepatocellular carcinoma animal models
(1) microRNA incubated in vitro is subcutaneously tested into knurl
A. the MHCC97H liver cancer marked using agomiRcontrol, agomiR-378a-3p incubated in vitro luciferase is thin
Born of the same parents transfect final concentration of 200nM；
B. after transfecting 3 days, serum-free is suspended in into individual cells, and cell with 0.25% trypsin digestion and piping and druming
DMEM culture solutions, after counting adjust cell concentration be 2 × 107/ ml is noted with 29G insulin syringes syringe needle in nude mice by subcutaneous
Penetrating the 100 μ l of single cell suspension that have been suspended, (cell number is about 2 × 106), every group of 6 nude mices；
C. mouse is observed after tumor cell inoculation into knurl situation, record experimental group and control group into the knurl time, with swollen
The a diameter of 0.5cm of knurl is into knurl standard；
D. every 1 week after tumor cell inoculation, luciferase is injected intraperitoneally by 150 μ g/g weight concentration are pressed after mouse anesthesia
Substrate carries out miR-378a-3p pairs of fluorescein image supervisory control using Caliper IVIS Lumina II living imaging systems
The influence of MHCC97H liver cancer cells tumor growths；
E. it is measured after tumor cell inoculation and records the major diameter A of hepatocellular carcinoma in nude mice subcutaneous transplantation knurl and minor axis B, tumor volume meter
Calculation formula is V=A × B2/ 2, the change curve of m- volume relationship when drawing tumour；
F. tumor cell inoculation puts to death the above-mentioned nude mice into knurl using de- cervical approach, weighs after removing tumour, calculate after 4 weeks
The tumour inhibiting rate of each group, tumour inhibiting rate (%)=[1- experimental groups average quality/control group average quality] × 100%；
G. it is for use the tumor tissues of stripping to be placed in fixation, paraffin embedding in 10% neutral formalin.
(2) subcutaneous transplantation knurl microRNA Experiment on therapy
A. MHCC97H liver cancer cells luciferase marked with 0.25% trypsin digestion and are blown and beaten into single thin
Born of the same parents, and cell is suspended in the DMEM culture solutions of serum-free, it is 2 × 10 that cell concentration is adjusted after counting7/ ml, with 29G insulin
(cell number is about 2 × 10 to the 100 μ l of single cell suspension that syringe needle has been suspended in mouse bare subcutaneous injection6)；
B. after into knurl, two groups (every group 6) are randomly divided into knurl mouse by above-mentioned, multiple spot in knurl is carried out to tumour and is noted
It penetrates miR-378a-3p control agent and agonist agomiRcontrol, agomiR-378a-3p is treated, therapeutic dose
It 2nmol/ times, treats within every 3 days 1 time, treats 4 times altogether；
C. treatment before and treatment after 4 weeks (every 1 week), using Caliper IVIS Lumina II living imaging systems into
Influences of the row fluorescein image supervisory control miR-378a-3p to MHCC97H Growth of Transplanted Hepatocarcinoma in Mice；
D. measure and record treatment before and treatment after the major diameter A of hepatocellular carcinoma in nude mice subcutaneous transplantation knurl and minor axis B, tumor volume meter
Calculation formula is V=A × B2/ 2, the change curve of m- volume relationship when drawing tumour；
E. 4 weeks after treating, the nude mice into knurl is put to death using de- cervical approach, weighs after removing tumour, calculates the tumour inhibiting rate of each group,
Tumour inhibiting rate (%)=[1- experimental groups average quality/control group average quality] × 100%；
F. it is for use the tumor tissues of stripping to be placed in fixation, paraffin embedding in 10% neutral formalin.
(3) liver orthotopic transplantation tumor microRNA Experiment on therapy
A. the MHCC97H liver cancer cells of luciferase label with 0.25% trypsin digestion and are blown and beaten into individual cells,
And cell being suspended in the DMEM culture solutions of serum-free, it is 2 × 10 that cell concentration is adjusted after counting7/ ml is noted with 29G insulin
(cell number is about 2 × 10 to the 100 μ l of single cell suspension that emitter syringe needle has been suspended in mouse bare subcutaneous injection6)；
B. observation mouse is into knurl situation after injecting, and the excised tumor when subcutaneous transplantation knurl grows to diameter about 1cm is immersed in
In PBS, 1-2mm3 fritters are cut into after stripping removal slough；
C. after experiment nude mice carries out intraperitoneal anesthesia using 1% yellow Jackets (60mg/kg), in upper left abdomen row transverse incision,
Exposure liver；
D. above-mentioned tumor tissue fritter is taken, is implanted into the essence of nude mice right lobe of liver deep with thick needle in vitro 40min, and complete
Layer closes abdomen；
E. the latter week left and right of orthotopic transplantation will press 150 μ g/g weight concentration intraperitoneal injection luciferase bottom after mouse anesthesia
Object carries out the growth feelings of fluorescein image supervisory control in situ tumor using Caliper IVIS Lumina II living imaging systems
Condition；
F. living imaging monitoring is found have the mouse in situ into knurl to be grouped at random, end of line intravenous injection miR-378a-3p
Control agent and agonist agomiRcontrol, agomiR-378a-3p are treated, therapeutic dose 5nmol/ times, are treated within every 3 days
It 1 time, treats 4 times altogether；
G. before tail vein injection treatment and after treatment 4 weeks (every 1 week), fluorescein imaging is carried out using living imaging system
Monitor influences of the miR-378a-3p to the growth of MHCC97H livers orthotopic transplantation tumor, transfer；
H. after treating 4 weeks, the nude mice into knurl is put to death using de- cervical approach, stripping liver in situ tumor tissue is weighed, and analyzes miR-
The influence that 378a-3p grows MHCC97H livers orthotopic transplantation tumor, and liver orthotopic transplantation tumor tissue is placed in 10% neutral formal
Fixation, paraffin embedding are for use in woods；Lung is taken to check metastases tubercle, parallel 10% neutral formalin is fixed, paraffin embedding
After be sliced, HE dyeing detection tumour lung micrometastasis situation, analysis miR-378a-3p to liver orthotopic transplantation tumor DISTANT METASTASES IN ability
It influences.
(4) tail vein injection Lung metastases are tested
A. the MHCC97H liver cancer marked using agomiRcontrol, agomiR-378a-3p incubated in vitro luciferase is thin
Born of the same parents transfect final concentration of 200nM；
B. after transfecting 3 days, single cell suspension is prepared into PBS suspensions after being digested using 0.25% pancreatin, after cell count
Cell concentration is adjusted to 1 × 107/ml；
C. the 200 μ l (cell numbers of single cell suspension being suspended with 29G insulin syringes syringe needle in nude mice tail vein injection
About 2 × 106), every group of 6 nude mices；
D. the 4th week after injecting, solution takes lung and checks metastases tubercle, and parallel 10% neutral formalin is fixed, paraffin
It is sliced after embedding, HE dyeing detection tumour lung micrometastasis situations, miR-378a-3p is to transfer ability in liver cancer cells body for analysis
It influences.7th, the statistical test of experimental result
This paper experimental sections carry out Pearson ' s χ 2test, Student's t test, Dan Yin using SPSS13.0 softwares
Element/two-way ANOVA analytical statistics meaning, p<0.05 variant, the p that is considered as statistics<0.01 to be considered as statistics significant
Difference.
The In vitro cell model that 1 miR-378a-3p of embodiment influences liver cancer cell growth, migration, invasion is analyzed
MHCC97H liver cancer cells is selected to carry out the exploration that miR-378a-3p obtains sexual function；Select SMMC-7721 liver cancer
Cell carries out miR-378a-3p and loses sexual function exploration.
(1) influences of the miR-378a-3p to liver cancer cells growth in vitro
The MHCC97H of opposite low expression miR-378a-3p is transfected into agomiR-378a-3p, agomiRcontrol respectively
(control group)；Relatively it is high expression miR-378a-3p SMMC-7721 transfect respectively antagomiR-378a-3p,
AntagomiRcontrol (control group).In the same terms culture 4 days after transfection, CCK-8 cell proliferation experiments were utilized every 1 day
Influence and miR-378a-3p antagonism of the detection miR-378a-3p overexpressions to MHCC97H liver cancer cell growths are to SMMC- respectively
The influence of 7721 liver cancer cell growths.
Using OD450nm as ordinate, the time (Day) draws growth of tumour cell curve (Fig. 1) for abscissa.It can by Fig. 1
See, miR-378a-3p can significantly inhibit the growth (p of MHCC97H liver cancer cells<0.05or p<0.01), antagonism endogenous
MiR-378a-3p can promote the growth (p of SMMC-7721 liver cancer cells<0.05orp<0.01), and miR-378a-3p inhibits
MHCC97H or antagonism miR-378a-3p promote the effects of SMMC-7721 liver cancer cell growths with after transfection incubation time prolong
Long enhancing, is presented regular hour dependence.CCK-8 results are prompted, and miR-378a-3p has apparent inhibition liver cancer cell growth
Effect.
Plates of cells colony formation (Fig. 2) also confirms that, using agomiRcontrol as control, miR-378a-3p crosses table
Less (the p of clone's smaller, number formed up to rear MHCC97H liver cancer cells<0.01), it is with respect to clonality
The 42% of agomiRcontrol control groups, and the clone that SMMC-7721 liver cancer cells are formed after miR-378a-3p antagonisms compared with
AntagomiRcontrol control groups clone becomes larger, number becomes more (p<0.01), it is with respect to clonality
1.70 times of antagomiRcontrol control groups.As a result it prompts, miR-378a-3p has the clone for significantly inhibiting liver cancer cells
Proliferative capacity.
Tumor stem cell microballoon experiment (Fig. 3) further confirmation, MHCC97H liver cancer cells after miR-378a-3p is overexpressed
The tumor stem cell microballoon formed the also less (p of smaller, number<0.01) SMMC-7721 livers, and after miR-378a-3p antagonisms
The tumor stem cell microballoon that cancer cell is formed becomes larger, number becomes more (p<0.01), prompting miR-378a-3p, which also has, inhibits liver
The activity of tumor stem cells self-renewal.Our result show that miR-378a-3p can inhibit liver cancer cells proliferation,
Growth and the self-renewing of liver-cancer stem cell, miR-378a-3p reduces the runaway growth for promoting liver cancer cells in liver cancer cells.
(2) influence of miR-378a-3p external transfer abilities to liver cancer cells
MHCC97H liver cancer cells are transfected into agomiR-378a-3p, agomiRcontrol (control group) respectively；SMMC-
7721 liver cancer cells transfect antagomiR-378a-3p, antagomiRcontrol (control group) respectively, and pass through scratch experiment
The wound healing ability of comparative analysis liver cancer cells.From fig. 4, it can be seen that miR-378a-3p can significantly inhibit MHCC97H liver cancer cells
Migration and wound healing, using agomiRcontrol as control group, miR-378a-3p group relative migrations ability be 57% (p<
0.01)；Antagonism miR-378a-3p can promote the transfer ability of SMMC-7721 liver cancer cells, and miR-378a-3p antagonism groups are opposite
Transfer ability is 1.48 times of (p of antagomiRcontrol control groups<0.05).
(3) influences of the miR-378a-3p to liver cancer cells vitro invasion ability
AgomiR-378a-3p, agomiRcontrol (control group) transfect MHCC97H liver cancer cells；antagomiR-
After 378a-3p, antagomiRcontrol (control group) transfection SMMC-7721 liver cancer cells, by adding matrigel
Transwell Matrigel comparative analyses MHCC97H agomiR-378a-3p groups, MHCC97H agomiRcontrol groups；
The vitro invasion ability of SMMC-7721antagomiR-378a-3p groups, antagomiRcontrol group liver cancer cells.It can by Fig. 5
See, compared with agomiRcontrol, the cell number of agomiR-378a-3p groups MHCC97H invasion significantly reduces, opposite to invade energy
Power is the 32% of agomiRcontrol control groups；And the invasion of antagomiR-378a-3p group SMMC-7721 liver cancer cells is thin
Born of the same parents' digital display work increases, with respect to 2.75 times of (p that invasive ability is antagomiRcontrol control groups<0.01).The above results carry
Show, miR-378a-3p has the function of to inhibit fucosylation, invasion.
The animal model that 2 miR-378a-3p of embodiment influences liver cancer cell growth, transfer is analyzed
(1) whether this ability for inhibiting liver cancer cells growth in vitro for clear and definite miR-378a-3p may occur in vivo,
After we mark MHCC97H liver cancer cells with luciferase, nude mice MHCC97H liver cancer subcutaneous transplantation knurls are established into knurl model,
By measuring into knurl time, gross tumor volume, weight and living imaging (Fig. 6), MHCC97H-agomiR- is detected and compared
378a-3p, MHCC97H-agomiRcontrol (control group) are in the subendothelial speed of growth of BALB/c nude mouses.
By Fig. 6 (a) as it can be seen that compared with MHCC97H-agomiRcontrol control groups, MHCC97H-agomiR-378a-3p compositions
The knurl time is obviously prolonged, and MHCC97H-agomiRcontrol groups, MHCC97H-agomiR-378a-3p groups are average into the knurl time point
It Wei not 6.33,10.83 days (p<0.01).With gross tumor volume (mm3) for ordinate, inoculation time (Week) is drawn for abscissa
Growth curve (Fig. 6 (b)) of the MHCC97H liver cancer cells in nude mouse, and pass through small animal living body imaging (Fig. 6 (c)) monitoring
Growth.By Fig. 6 (b), Fig. 6 (c) as it can be seen that above-mentioned MHCC97H-agomiR-378a-3p, pMHCC97H-agomiRcontrol
After liver cancer cells nude mice by subcutaneous is inoculated with the 2nd, 3,4 week, MHCC97H-agomiR-378a-3p groups are compared with MHCC97H-
Growth of the agomiRcontrol control groups in nude mouse significantly slowly (p<0.05or p<0.01).Tumor cell inoculation the 4th
Zhou Hou, all mouse put to death, subcutaneous transplantation knurl knurl are taken to take pictures (Fig. 6 (d)) and weighs (Fig. 6 (e)).MHCC97H-
AgomiRcontrol groups, MHCC97H-agomiR-378a-3p group transplantable tumor knurl weights are respectively 1.10,0.48g, more in system
Meter learns difference (p<0.01).
To further determine that miR-378a-3p inhibits the effect of liver cancer cells nude mice tumor growth, we are by luciferase
The MHCC97H of label first establishes MHCC97H liver cancer subcutaneous transplantations knurl, orthotopic transplantation tumor respectively, and then MHCC97H liver cancer is subcutaneously moved
It plants knurl and is divided into 2 groups：1 group of row intracellular injection agomiR-378a-3p treatment, the injection agomiRcontrol (treatments of another 1 group of row intracellular
Control group)；MHCC97H liver cancer orthotopic transplantation tumors are also divided into 2 groups：1 group of end of line intravenous injection agomiR-378a-3p treatments, another 1
Group end of line intravenous injection agomiRcontrol (treatment control group).MHCC97H liver cancer subcutaneous transplantation knurls agomiR-378a-3p is controlled
Forward and backward measurement gross tumor volume, weight and living imaging are treated, tumor growth curve (Fig. 7) is drawn, has detected miR-378a-3p pairs
The therapeutic effect of MHCC97H liver cancer subcutaneous transplantation knurls.By Fig. 7 (a), Fig. 7 (b) as it can be seen that using agomiRcontrol treatment groups as pair
According to MHCC97H liver cancer subcutaneous transplantation knurls are grown after agomiR-378a-3p is treated significantly slows down (p<0.05 or p<0.01).
After subcutaneous transplantation knurl is treated the 4th week, all mouse are put to death, and subcutaneous transplantation knurl knurl is taken to take pictures (Fig. 7 (c)) and (Fig. 7 that weighs
(d)).Compared with agomiRcontrol treatment control groups (knurl weight 1.65g), the transplantable tumor knurl of agomiR-378a-3p treatment groups
There was only 0.93g (p again<0.01).MHCC97H liver cancer orthotopic transplantation tumor passes through small animal living body image supervisory control agomiR-378a-3p
Treat the growth (Fig. 8 (a)) of forward and backward liver cancer orthotopic transplantation tumor, and after treating the 4th week, all mouse are put to death, and liver cancer is taken to move in situ
Knurl knurl is planted to take pictures (Fig. 8 (b)), and (Fig. 8 (c)) (p that weighs<0.05).As a result show that miR-378a-3p can also inhibit liver cancer former
The growth of position transplantable tumor.Above-mentioned 3 kinds of liver cancer xenograft models confirm that miR-378a-3p can significantly inhibit in nude mouse
The growth of MHCC97H transplanted human hepatocellular carcinomas, it is consistent with our cell model result.
(2) to inquire into influences of the miR-378a-3p to being shifted in liver cancer cells body, we pass through MHCC97H liver cancer cells
It is established respectively by tail vein injection in BALB/c nude mices after agomiR-378a-3p, agomiRcontrol (control group) transfection
Tumour tail vein injection Lung metastases model.After tail vein injection 4 weeks, mouse is put to death, lung tissue (Fig. 9 (a)) is taken to find,
There is neoplasm lung metastasis tubercle in the lung tissue of 6 mouse of MHCC97H-agomiRcontrol control group tail vein injections
(6/6), MHCC97H-agomiR-378a-3p groups only have 2/6 Lung metastases occur；Naked eyes gross examination of skeletal muscle, MHCC97H-agomiR-
378a-3p Lung metastases tubercle is significantly reduced compared with MHCC97H-agomiRcontrol control groups.Lung tissue is fixed, paraffin embedding
Neoplasm lung metastasis situation (Fig. 9 (b), Fig. 9 (c)) is further checked in row HE dyeing after slice.Also, it was found that MHCC97H-
AgomiR-378a-3p inhibits the ability (p of MHCC97H liver cancer cells distant place Lung metastases in nude mouse<0.01).Moreover,
Mouse is put to death after agomiR-378a-3p is treated the 4th week and takes lung tissue, fixation, paraffin by MHCC97H liver cancer orthotopic transplantation tumor
Embedded section, HE dyeing.It has been found that (Figure 10), MHCC97H liver cancer orthotopic transplantation tumors agomiRcontrol treatment control groups
5/6 mouse there are distant place Lung metastases, and agomiR-378a-3p treatment groups only have 3/6 Lung metastases occur.Naked eyes are substantially seen
Examine confirms that miR-378a-3p can also significantly inhibit the distant place Lung metastases ability (p of liver cancer primary tumor with HE dyeing<0.01).
The target gene screening of 3 miR-378a-3p of embodiment
The potential target gene of miR-378a-3p includes：PLAG1、SULF1、SLC7A6、NPAT、HDAC4、PLAGL2、
Gli3.The sequence complementation of miR-378a-3p and PLAG1, SULF1, SLC7A6, NPAT, HDAC4, PLAGL2, Gli3 compare signal
Figure is as indicated at 11.
By microRNAorg, PITA, TargetScan database filter out PLAG1, SULF1, SLC7A6, NPAT,
Potential target gene of HDAC4, PLAGL2, GLI37 genes as miR-378a-3p, the lucky triumphant chemical gene technology in commission Shanghai
3 ' the UTR Dual-Luciferases report base of Co., Ltd structure PLAG1, SULF1, SLC7A6, NPAT, HDAC4, PLAGL2, GLI3
Because of plasmid.By miR-378a-3p mimics (200nM) respectively with PLAG1-3 ' UTR, SULF1-3 ' UTR, SLC7A6-3 ' UTR,
NPAT-3 ' UTR, HDAC4-3 ' UTR, PLAGL2-3 ' UTR, GLI3-3 ' UTR luciferase reporter genes plasmid are used
2000 cotransfection 293T cells of Lipofectamine, and set miR-378a-3p mimics negative control (NC)
As control.After transfecting 48h, uciferase activity is detected, preliminary analysis miR-378a-3p is to above-mentioned 7 kinds of candidate targets
Effect.It is bis- glimmering that the most strong PLAGL2 of selection miR-378a-3p targeted inhibition effects further builds PLAGL2-3 ' UTR mutant
After light element enzyme reporter plasmid, by various concentration (200,100,50,25nM) miR-378a-3p mimics, miR-
378a-3p mimics NC (control) (compare) cotransfection 293T with PLAGL2-3 ' UTR, PLAGL2-3 ' UTR mutant respectively
Uciferase activity is detected after cell 48h, specifies the target gene that PLAGL2 is miR-378a-3p.
Wherein, miR-378a-3p mimics：
5’-ACUGGACUUGGAGUCAGAAGGC-3’
3’-UGACCUGAACCUCAGUCUUCCG-5’
Double-strand；
MiR-378a-3p mimics negative control are synthesized by Guangzhou Ribo Bio Co., Ltd..
As a result as shown in figure 12：
It is utilized respectively 3 ' the UTR Dual-Luciferase reports of PLAG1, SULF1, SLC7A6, NPAT, HDAC4, PLAGL2, GLI3
It accuses genic system and (compares) cotransfection 293T with the miR-378a-3p mimics or miR-378a-3p mimics NC of 200nM
Uciferase activity is detected after cell to find, compared with miR-378a-3p mimics NC control groups, miR-378a-3p
PLAG1-3 ' UTR, SLC7A6-3 ' UTR, PLAGL2-3 ' the UTR uciferase activities of mimics transfection groups decline (p<
0.05orp<0.01).Wherein, PLAGL2-3 ' the UTR uciferase activity downward trends of miR-378a-3p mimics transfection groups
Significantly, relative luciferase activity only has the 30.8% of miR-378a-3p mimics NC control groups.The above results carry
Show, in our 7 candidate potential target genes of miR-378a-3p, PLAGL2 may be potential target base the most closely related
Cause.And it was found that miR-378a-3p mimics differences transfection concentrations (200,100,50,25nM) inhibit PLAGL2-3 '
Uciferase activity (the p of UTR<0.05or p<0.01), different transfection concentrations are to the fluorescence of PLAGL2-3 ' UTR mutant
The plain equal unrestraint effect of enzymatic activity.It is noted that miR-378a-3p mimics inhibit the fluorescein of PLAGL2-3 ' UTR
Apparent dosage effect is presented in enzymatic activity, and with the increase of concentration, rejection ability also gradually enhances.It further confirms, PLAGL2
It is the potential target gene of miR-378a-3p.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>University Of Suzhou
<120>A kind of liver cancer cell growth inhibitor and its application in prognosis in hcc judgement
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> RNA
<213>People
<400> 1
acuggacuug gagucagaag gc 22
<210> 2
<211> 22
<212> RNA
<213>People
<400> 2
acuggacuug gagucagaag gc 22
<210> 3
<211> 22
<212> RNA
<213>People
<400> 3
ugaccugaac cucagucuuc cg 22
<210> 4
<211> 22
<212> RNA
<213>People
<400> 4
acuggacuug gagucagaag gc 22
<210> 5
<211> 22
<212> RNA
<213>People
<400> 5
acuggacuug gagucagaag gc 22
<210> 6
<211> 22
<212> RNA
<213>People
<400> 6
ugaccugaac cucagucuuc cg 22
Claims (8)
1. a kind of liver cancer cells self-renewing inhibitor, which is characterized in that containing miR-378a-3p, the miR-378a-3p's
Shown in base sequence such as 5 '-ACUGGACUUGGAGUCAGAAGGC-3 '.
A kind of 2. liver cancer cells self-renewing inhibitor according to claim 1, which is characterized in that the self-renewing packet
Include proliferation, differentiation, growth, clone.
3. a kind of fucosylation, transfer, invasive ability inhibitor, which is characterized in that it is described containing miR-378a-3p
Shown in the base sequence of miR-378a-3p such as 5 '-ACUGGACUUGGAGUCAGAAGGC-3 '.
Applications of the 4.miR-378a-3p in the drug for preparing treatment liver cancer.
5. application according to claim 4, which is characterized in that be included in the drug for preparing and inhibiting liver cancer cells self-renewing
In application.
6. application according to claim 5, which is characterized in that the self-renewing includes proliferation, differentiation, growth, clone.
7. application according to claim 4, which is characterized in that preparing inhibition liver cancer cells including miR-378a-3p
Migration is shifted, the application in the drug of invasive ability.
8. a kind of drug for treating liver cancer, which is characterized in that containing miR-378a-3p, the base sequence of the miR-378a-3p
As shown in 5 '-ACUGGACUUGGAGUCAGAAGGC-3 '.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111150848A (en) * | 2020-01-21 | 2020-05-15 | 中国药科大学 | PLAGL2 and application thereof in liver cancer |
| CN111518886A (en) * | 2020-04-23 | 2020-08-11 | 浙江大学 | MicroRNA related to sorafenib drug resistance of tumor cells and application thereof |
| CN111534491A (en) * | 2020-05-09 | 2020-08-14 | 胡宗强 | Experimental method for inhibiting growth of liver cancer cells by miR-9 inhibitor |
| CN113604569A (en) * | 2021-08-04 | 2021-11-05 | 广州医科大学 | Application of miR-6883-3p in preparation of anti-liver cancer or liver cancer prognosis evaluation product |
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| CN104388561A (en) * | 2014-11-14 | 2015-03-04 | 浙江理工大学 | Liver cancer biomarkers and application thereof |
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| CN104388561A (en) * | 2014-11-14 | 2015-03-04 | 浙江理工大学 | Liver cancer biomarkers and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111150848A (en) * | 2020-01-21 | 2020-05-15 | 中国药科大学 | PLAGL2 and application thereof in liver cancer |
| CN111150848B (en) * | 2020-01-21 | 2022-02-15 | 中国药科大学 | PLAGL2 and application thereof in liver cancer |
| CN111518886A (en) * | 2020-04-23 | 2020-08-11 | 浙江大学 | MicroRNA related to sorafenib drug resistance of tumor cells and application thereof |
| CN111518886B (en) * | 2020-04-23 | 2021-08-17 | 浙江大学 | MicroRNA related to sorafenib drug resistance of tumor cells and application thereof |
| CN111534491A (en) * | 2020-05-09 | 2020-08-14 | 胡宗强 | Experimental method for inhibiting growth of liver cancer cells by miR-9 inhibitor |
| CN113604569A (en) * | 2021-08-04 | 2021-11-05 | 广州医科大学 | Application of miR-6883-3p in preparation of anti-liver cancer or liver cancer prognosis evaluation product |
| CN113604569B (en) * | 2021-08-04 | 2023-11-17 | 广州医科大学 | Application of miR-6883-3p in preparation of anti-liver cancer or liver cancer prognosis evaluation product |
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Application publication date: 20180706 |