CN109182515A

CN109182515A - The new application of molecular marker miR-99a

Info

Publication number: CN109182515A
Application number: CN201811040405.2A
Authority: CN
Inventors: 张晓娜
Original assignee: Sixth Affiliated Hospital of Sun Yat Sen University
Current assignee: Sixth Affiliated Hospital of Sun Yat Sen University
Priority date: 2018-09-06
Filing date: 2018-09-06
Publication date: 2019-01-11

Abstract

The invention discloses purposes of the colorectal cancer early molecule marker miR-99a in the diagnostic kit of the relevant colorectal cancer hypotype of preparation diabetes.The invention demonstrates that miR-99a expresses downward in colorectal cancer cell, and the downward in the colorectal cancer cell of the clinical diabetes that occur together in the more simple colorectal cancer of expression of miR-99a is more significant, the expression of its target gene mTOR then increases, and shows that miR-99a has direct negative regulation to act on mTOR；The present invention shows that miR-99a can inhibit the proliferation of colorectal cancer cell HCT-116, transfer ability simultaneously, further demonstrates the cancer suppressing action of miR-99a.

Description

The new application of molecular marker miR-99a

Technical field

The present invention relates to diagnosis of colorectal carcinoma and treatment technology field, the especially new application of molecular marker miR-99a.

Background technique

Colorectal cancer is one of (colorectal cancer, CRC) most common tumor in digestive tract, disease incidence and dead The world Wang Shuaijunju malignant tumour third position.In China, annual new cases are up to ten thousand people of 13-16, and disease incidence is just with 4.2% Speed be gradually incremented by, far surpass 2% world level.Diabetes (diabetes mellitus, DM) are to seriously endanger in the world One of big non-communicable diseases of the three of human health, will be up to more than 3.6 hundred million people to whole world diabetes patient in 2025.China is sugar Niao Bing big country, according to the data that the World Health Organization in 2016 issues, the disease incidence of diabetes mellitus in China is 9.4%.Clinical and stream Row disease data shows that there are common risk factor, such as obesity, lacks movement at high caloric diet for diabetes and colorectal cancer Deng in recent years, it is close that more and more researches show that both sides relations.Domestic and international many studies have shown that diabetics and non-saccharide Urine patient compares, and other than the risk for suffering from colorectal cancer obviously increases, the recurrence rate and the death rate of cancer are also higher for the former, sugar Urine disease is the independent hazard factor of colorectal cancer illness.

With the colorectal cancer molecule parting development in recent years based on gene expression, different colorectal cancer classification is emerged Method, different subtype colorectal cancer has unique biological characteristics, Clinical symptoms and the targeting intervention based on hypotype, with sugar The colorectal cancer of urine disease may be one of special hypotype, and molecular mechanism is still not clear, and urgently to be resolved.

MicroRNAs (miRNAs) is the single stranded RNA of non-coding, about 21-23 nucleotide of length, by with said target mrna The interaction in complementary site inhibits the protein expression after transcribing in mode.MiRNAs cell grow, proliferation, differentiation, movement, It plays an important role in autophagy and apoptosis.Current study show that the unconventionality expression of some miRNAs and many diseases, including it is swollen Tumor is related with the generation of diabetes, but its effect in the colorectal cancer hypotype occurrence and development with diabetes not yet appears in the newspapers Road.

Summary of the invention

Based on the above issues, providing it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art a kind of can have The molecular marker of the relevant colorectal cancer hypotype of effect diagnosis diabetes, can also effectively inhibit cancer using the molecular marker Progress.

To achieve the above object, the technical solution that the present invention takes includes the following aspects:

In the first aspect, the present invention provides molecular marker miR-99a in the relevant colorectal cancer of preparation diabetes Purposes in the diagnostic kit of hypotype.

Preferably, the sequence of the molecular marker miR-99a is as shown in SEQ ID NO:1.

Preferably, the diagnostic kit by detection subject's Colorectal Carcinoma in miR-99a content, and with list The Average expression level of miR-99a compares in the Colorectal Carcinoma of pure colorectal cancer patients, to judge that the subject is The no colorectal cancer hypotype with diabetes.

Preferably, the diagnostic kit also passes through the target gene of miR-99a in detection subject's Colorectal Carcinoma The expression of mTOR, and compared with the Average expression level of mTOR in the Colorectal Carcinoma of simple colorectal cancer patients, To judge whether the subject suffers from the colorectal cancer hypotype with diabetes.It should be noted that present inventor It is found by test of many times, the more simple Colon and rectum of expression of the miR-99a of the colorectal cancer subgroups for the diabetes that occur together Much lower in cancer patient, the more simple colorectal cancer patients of the expression of target gene mTOR then have raising.

In the second aspect, the present invention provides a kind of diagnostic kit of the colorectal cancer hypotype of diabetes that occur together, institutes Stating kit includes Colorectal Carcinoma total RNA extraction reagent, reverse transcription PCR reagent and quantitative PCR reagent.Wherein, quantitative PCR Reagent is preferably used for the specific forward primer and general reverse primer of quantitative miR-99a；Include in reverse transcription PCR reagent The reverse transcriptase primer of miR-99a.

Preferably, the kit further includes the extraction reagent of mTOR albumen and determining for mTOR albumen in Colorectal Carcinoma Measure reagent.

In the third aspect, the present invention provides colorectal cancer early molecule marker miR-99a occurs together in preparation treatment Purposes in the drug of the colorectal cancer hypotype of diabetes.Present invention applicant has found that miR-99a can inhibit by test of many times The proliferation of HCT-116, migration, invasive ability further confirm the cancer suppressing action of miR-99a；On the other hand, miR-99a can lead to Targeting and negative regulation mTOR gene are crossed, cancer suppressing action is played.

In the fourth aspect, the present invention provides a kind of drug for the treatment of cancer, miR-99a or energy are contained in the drug The drug of miR-99a expression quantity is promoted in cancerous tissue.

Preferably, also containing the neutralization that can reduce the drug or mTOR of the expression quantity of mTOR in cancerous tissue in the drug Agent.It should be noted that neutralizer is preferably the antibody of mTOR, but be not restricted to that the antibody of mTOR, as long as cancer can be reduced The drug of the expression of mTOR or reagent can be used as the neutralizer in tissue.

Preferably, the cancer is the colorectal cancer hypotype of diabetes of occurring together.

In conclusion the invention has the benefit that

The inventor of the present application discovered that miR-99a expresses downward in colorectal cancer cell, and the knot for the diabetes that occur together is straight Downward in the more simple colorectal cancer of the expression of miR-99a in intestinal cancer hypotype tissue and cell is more significant, target gene The expression of mTOR then increases, and shows that miR-99a has direct negative regulation to act on mTOR；Present inventor also sends out Existing, miR-99a can inhibit the proliferation of HCT-116 cell, migration, invasive ability, and the suppression cancer for further demonstrating miR-99a is made With.

Detailed description of the invention

Fig. 1 be occur together diabetes colorectal cancer hypotype tissue in miR-99a expression testing result result figure；Its Middle A figure be extract normal Colon and rectum mucous membrane, colorectal cancer, the diabetes that occur together colorectal cancer hypotype tissue in total RNA, into The detection of row Agilent miRNA chip of expression spectrum, then carries out clustering to the miRNA of difference；B figure is colorectal cancer and companion There is the VN figure of the colorectal cancer hypotype histological difference expression miRNAs of diabetes；C figure is that the normal Colon and rectum of qPCR experimental verification is viscous MiR-99a level (n=20) in film (n=20) and Colorectal Carcinoma；D figure is colorectal cancer (n=20) and with diabetes Colorectal cancer hypotype (n=20) tissue in miR-99a expression qPCR analysis；E figure be normal enterocyte NCM460 and MiR-99a expression (p < 0.05, * p < in the colorectal cancer cells such as HCT-15, HCT-116, HCT-8, SW480, LoVo 0.01, * * * p < 0.001)；

Fig. 2 is the testing result of colorectal cancer cell HCT-116 proliferation and invasive ability that miR-99a inhibits in vitro culture Figure；MiR-99a mimics transiently transfects HCT-116 cell for 24 hours, and MTT measures cells survival rate and lowers (A figure)；transwell (B and C figure) (p < 0.05, * p < 0.01, * * * p < is lowered with the measuring cell migration of the cell boyden and invasive ability 0.001)；

Fig. 3 be turn HCT-116 cell 48h miR-99a mimics wink after target gene mTOR in colorectal cancer cell HCT- Detection of expression result figure in 116；Wherein, A figure is qPCR measurement mTOR expression；B figure is Western blot detection MTOR protein expression level；C speculates miR-99a binding site in 3 '-UTR of mTOR, and mTOR is shown in seed sequence The change of 3 '-UTR mutant；Dual-Luciferase test confirms that miR-99a directly adjusts 3 '-UTR of mTOR；D figure is miR- 99a microRNA target prediction (p < 0.05, * p < 0.01, * * * p < 0.001)；

Fig. 4 is the testing result figure of the expression of miR-99a；Wherein, A figure is HCT-116 cell through 100mg/L AGE After saccharification processing 48h, qPCR measures miR-99a expression；QPCR experiment analyzes (B figure) to mTOR expression；western Blot is analyzed (C figure) to mTOR protein expression level；D figure is colorectal cancer of the IHC method detection with diabetes, Colon and rectum MTOR expresses (p < 0.05, * p < 0.01, * * * p < 0.001) in cancer and normal Colon and rectum mucosal tissue (n=20).

Specific embodiment

MiRNA plays a significant role during tumor development, is the forward position studied at present.It from now on, if can be just Ordinary person, with the colorectal cancer precancerous lesion patient and different phase of diabetes with being established in the colorectal cancer patients of diabetes The miRNA differential expression of system perfecting is composed, it will help is illustrated the Colorectal Cancer mechanism with diabetes, is colorectal cancer Diagnoses and treatment bring new research direction and thinking.How the activator of miRNA and inhibitor specifically to be introduced into tumour Cell, and how using the express spectra of miRNA for early stage provide diagnosis basis with the colorectal cancer of diabetes, be all future Research direction.MiRNA perhaps can become with the colorectal cancer early diagnosis of diabetes, specific treatment and assessment prognosis Small molecule marker.Therefore, miRNA is of great significance in colorectal cancer diagnosis and treatment.

Present inventor carried out it is corresponding experiment to explore expression of the miRNA in colorectal cancer patients, Including the following aspects:

1) Agilent Human miRNA chip detects: choosing normal person's Colon and rectum mucous membrane, simple colorectal cancer and companion Colorectal cancer hypotype every group of clinical tissue sample each 1 for sending out diabetes, and to above-mentioned three groups of sample tissue extractions total RNA, chip detection, and clustering is carried out to the miRNA of difference；In the miRNA of differential expression, expression multiple is filtered out most High difference miRNA is candidate miRNA.

2) filtering out and lowering most apparent miR-99a is purpose miRNA, expands the verifying of clinical tissue sample and target gene is pre- Survey: qPCR experiment detection miR-99a is not accompanied by diabetic, normal person's Colon and rectum with diabetic, intestinal cancer in intestinal cancer It is expressed in mucosal tissue each 20, verifies said chip result.

3) miR-99a functional analysis in the cell: qPCR first is verified in normal intestinal epithelial cell NCM460 and Colon and rectum MiR-99a expression in cancerous cell line (HCT-116, HCT-15, HCT-8, SW480, LoVo), and pick out suitable cell Strain is studied for difference miRNA cell function；Chemical synthesis difference miR-99a mimics (is mentioned by GenePharma company The synthetics of the miR-99a of confession transfect the expression that can raise miR-99a after entering cell, enhance the function of miR-99a), And the lower colon-cancer cell system HCT-116 of relative expression is transfected, detection miR-99a mimics grows cell and migrates, invades Attack the influence of ability.

4) the target gene verifying of miR-99a: in conjunction with three microRNA target prediction method (PicTar, TargetScan and MiRanda), the potential target gene mTOR of miR-99a is obtained；MiR-99a pairs is analyzed using luciferase Reporter System Articles are made in the direct regulation and control of 3 ' UTR of mTOR gene；Intracellular miR-99a mimics is detected to the mRNA and protein level of mTOR The influence of expression.

5) the intracellular function of miR-99a is verified after saccharification colorectal cancer cell in vitro: being to determine that AGEs saccharification is handled HCT-116 cell inoculation is trained base 48h in 100mg/L AGEs by the no expression for influencing miR-99a, and then qPCR detects miR- MTOR mRNA and protein expression level change in the cell of expression and AGEs the saccharification processing of 99a.

6) expression of the target gene mTOR of miR-99a in colorectal cancer hypotype tissue of the clinic with diabetes is assessed: Normal Colorectal mucosa is detected with immunohistochemical experiment, straight with the colorectal cancer hypotype of diabetes and without the knot of diabetes MTOR protein expression level in each 20, intestinal cancer tissue.

In the course of the research, the inventor of the present application discovered that in the colorectal cancer hypotype of the clinical diabetes that occur together, miR-99a The more simple colorectal cancer of expression in it is lower, the expression of target gene mTOR then has raising；It is overexpressed miR-99a energy Proliferation, the migration, invasive ability for inhibiting HCT-116 cell, lower the expression of target gene mTOR；And AGE-BSA saccharification is handled HCT-116 cell can lower miR-99a expression and improve the expression of its target gene mTOR；Clinic occur together diabetes knot it is straight It is higher in the more simple colorectal cancer of the expression of mTOR in intestinal cancer hypotype.The above experiment confirms miR-99a with diabetes Colorectal cancer hypotype in cancer suppressing action.MiR-99a can become with the colorectal cancer hypotype diagnosis of diabetes, spy as a result, The small molecule marker of opposite sex treatment and assessment prognosis.

Experimental material involved in the present invention is briefly introduced with experimental method below.Unless otherwise instructed, this hair Material and reagent in bright can from the market or other public's channels obtain.Unless otherwise instructed, the reagent in the present invention Concentration is mass concentration.In this application, the relevant colorectal cancer hypotype of diabetes be occur together diabetes colorectal cancer it is sub- Type；Primer involved in the application (including reverse transcriptase primer) can be designed according to known target sequence, belong to this field Routine techniques, do not need carry out creative work can be obtained.

(1) clinical samples

The colorectal cancer patients that ZhongShan University attached No.6 Hospital is hospitalized during flesh tissue sample is derived from 2017-2018 And healthy people.Colorectal cancer patients are preoperative not to carry out radiotherapy or chemotherapy, and patient does not merge other malignant tumours such as oesophagus Cancer, gastric cancer etc..Tumor tissues are collected after the excision of sample underwent operative is in vitro.It is gland cancer that tumor tissues, which pass through proved by pathology,.Health Examinee's row enteroscopy takes Colon and rectum mucous membrane as normal control tissue.Each group tissue specimen carries out number and registration.It collects Tissue specimen be put into -80 DEG C of refrigerators and save backup in vitro 30min.Corresponding number is normal control sample respectively This: 177005, simple colorectal cancer sample number: 176906, occur together diabetes B colorectal cancer sample number: 178373.

(2) tumour cell

Colorectal cancer tumor cell line, including HCT-116 (Guangzhou forever promise biology Co., Ltd), using containing 10% tire ox blood Clearly, the DMEM culture medium of 1% dual anti-, 1% nonessential amino acid, 1% glutaminol and 1 ‰ Ciprofloxacins is placed in 37 DEG C, 5% CO₂Incubator in cultivate.

(3) key instrument equipment

(4) main agents

(5)

The main experimental methods being related to are as follows:

1) tissue, cell total RNA extracting；

2) tissue, cell protein extracting；

3)Agilent Human miRNA(8*60K)array；

4) real time fluorescent quantitative qRT-PCR；

5) luciferase Reporter System detects；

6)Western blot；

7) cell culture；

8) cell transfecting；

9) mtt assay detection cell viability variation；

10) cell migration of the cell Transwell, boyden and Matrigel；

(6) statistical analysis

This experimental data measurement data is carried out using 7 software of GraphPad Prism.P value is examined using sided t.P value < 0.05 is considered to have statistical significance, and value < 0.01 P is considered to have obvious statistical significance.

To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair The present invention is described further.

1 Transwell Cell migration assay of embodiment

Experimental method:

1, after the enzymic digestion of HCT-116 cell tryptase, appropriate cell suspension is taken, 1000rpm is centrifuged 5 minutes.

2, supernatant is sucked, after cell is resuspended with serum-free supernatant, cell count simultaneously adjusts cell concentration with basal medium To 1 × 10⁵/ ml takes 100 μ l to be added to the cell Transwell upper chamber, and 600 μ l complete mediums are added in lower room.

3, it is put into after being cultivated 24~48 hours in incubator, takes out cell, the cell of upper chamber, 4% poly are wiped with cotton swab Formaldehyde fixes 15 minutes, and PBS washed once, and 1% violet staining 10 minutes, PBS washed once, and microscopically observation cell is It is no to pass through aperture.If any passing through, terminate other experimental groups, take pictures and count across cell number.

The experiment of 2 Transwell cell invasion of embodiment

Experimental method:

1, Matrigel is placed in 4 DEG C and is dissolved overnight, press Matrigel: culture medium=1 with the basal medium of pre-cooling: 3 dilution proportion takes 40 μ l to be added in the cell Transwell of pre-cooling, and movement is slow, avoids generating bubble.

2,37 DEG C of incubations solidify Matrigel in 2 hours.

3,100 μ l and 600 μ l basal mediums, 37 DEG C of hydrated overnights are added in upper and lower room respectively.Next day sucks culture medium.

4, after the digestion of experimental cell pancreatin, appropriate cell suspension is taken, 800rpm is centrifuged 5 minutes.

5, supernatant is sucked, after cell is resuspended with basal medium, cell count simultaneously adjusts cell concentration with basal medium To 1 × 10⁶/ ml takes 100 μ l to be added to the cell Transwell upper chamber, and 600 μ l complete mediums are added in lower room.

6, it is put into after being cultivated 24~48 hours in incubator, takes out cell, the cell of upper chamber, 4% poly are wiped with cotton swab Formaldehyde fixes 15 minutes, and PBS washed once, and 1% violet staining 10 minutes, PBS washed once, and microscopically observation cell is It is no to pass through aperture.If any passing through, terminate other experimental groups, take pictures and count across cell number.

3 immunohistochemical experiment of embodiment

Experimental method:

1. fixed: 4%PFA room temperature fixed 10min, PBS wash each 5min twice.

2. punching: being washed 2 times, 5min/ times with TBST (0.1%Triton X-100in TBS).

3. configuring fresh 3%H with distilled water or PBS₂O₂, room temperature closes 5~10min, and PBS washes 3 times, each 2min.

4. confining liquid is added dropwise, room temperature ten minutes.Surplus liquid is got rid of, is not washed.

5. FAS protein primary antibody is added dropwise, 4 DEG C overnight (4 DEG C overnight after in room temperature rewarming 45min).

6.PBS washes 3 each 2min.

7. polymerization HRP, which is added dropwise, marks anti-rabbit/mouse IgG secondary antibody, it is incubated at room temperature 1h.

8.PBS is washed 3 times, 2min/ times.

9.DAB colour developing: DAB colour reagent box or autogamy chromogenic reagent (colour developing degree is grasped under mirror), observation to mesh Signal depth background color it is shallow when terminated immediately with distilled water.

10. distillation washing, haematoxylin redye 15s.

11. dehydration: slice is put into 50%, 70%, 80%, 90%, 95%, 100% ethyl alcohol and is dehydrated, each 2min.

12. transparent: it is transparent that slice being put into 100% dimethylbenzene 10min.

13. resinene 50ul mounting, room temperature preservation.

4 protein immunoblot experiment of embodiment

Experimental method:

(1) albumen is extracted

Experimental procedure:

1) match cell pyrolysis liquid: 120 holes μ l RIPA/ (hole 35mm).

2) it has marked the EP of buckle to manage, has prepared ice chest, cell scraper, beaker, vacuum extractor etc..

3) cell plates inclined-plane is placed on to operate on ice.Thorough Aspirate medium (inhaling 2 times), 120 μ l RIPA are added in every hole (PBS that the cell containing serum is first pre-chilled with 4 DEG C is washed 2 times).With cell scraper cell scraping, it is collected into corresponding EP pipe that (EP pipe is set In dedicated ice chest).It is careful not to ice to be splashed into cell hole when operation.

4) ultrasound (interrupts genomic DNA, avoids the too sticky influence loading of sample.) ultrasound intensity 37%, 0.8s/ times.With Distilled water washes 3 probes, dries.The sample of each EP pipe effectively ultrasound 3 times.Ultrasonic albumen should be Western in time Blotting, especially detection phosphorylated protein.Without special circumstances, Western must be on the same day by surveying phosphorylated protein Blotting, remaining sample are stored in -20 DEG C.

(2) protein quantification (BCA method)

Experimental procedure:

1) standard items are diluted to 0.5ug/ul with 1 × PBS.

2) BCA reagent A liquid and B liquid are configured by 50:1 by appropriate working solution according to sample size, mixed well.BCA work Liquid is placed at room temperature for 24 hours interior stabilization.

3) standard items are added in 96 orifice plates by 0,1,2,4,8,12,16,20ul, 1 × PBS polishing is added to every hole 20ul.Sample to be tested dilutes by a certain percentage, and 20ul is added into 96 orifice plates.

4) 200ul BCA working solution is added in every hole.

5) it reacts 30 minutes for 60 DEG C.Microplate reader 562nm measures absorbance.

6) concentration of specimens is calculated according to standard curve, by all Sample Dilutions at minimum concentration.

(3) Western Blot detects protein expression

Experimental procedure:

Extract cell protein it is quantitative after, using the polyacrylamide gel testing goal albumen of 12%, 8%, 10% concentration, Albumen is gone on pvdf membrane after SDS-PAGE electrophoresis, 5% skim milk closing after, by corresponding primary antibody (FAS, mTOR, WNT1), 4 DEG C be incubated overnight after, after being washed twice within 7 minutes every time with TBST, with accordingly dilute secondary antibody (HRP label secondary antibody, Forevergen after) being incubated for 1~2h at room temperature, after being washed three times with TBST each minute, progress chemiluminescence development (ECL, Forevergen).It is used as internal reference with GAPDH { (HC301), 1:5000 }, compares the difference of above-mentioned protein expression after different disposal It is different.

The experiment of 5 luciferase gene reporting system of embodiment

(1) it transfects

Experimental procedure:

1) mentioning the previous day seeds cells into 24 orifice plates, and density is about 70% when transfection.

2) cell is taken out, culture medium is changed into serum free medium, puts back to incubator culture.

3) EP of sterilizing is taken to manage, as required mixed plasmid (hole about 0.8ug/, wherein pmirGLO-3 ' UTR carrier 0.2ug, MiR-99a or NC 0.6ug, that is, 100nM), every pipe adds 50ul Opti-MEM, mixes.

4) separately EP is taken to manage, lipofectamine 2000 (hole 2ul/) and Opti-MEM (hole 50ul/) is added, mixes gently After be stored at room temperature 5min.

5) mixed liquor in 4 is added in 3 EP pipe, is mixed.

6) it is stored at room temperature 20min, lipofectamine 2000- plasmid mixed liquor is dripped in 24 orifice plates, the hole 100ul/.

7) cell is put back into incubator culture, the complete medium containing serum is gained after 4h.

(2) Luciferase Activity determination

Experimental procedure:

8) match lysate: 5 × passive lysis buffer is thawed from -20 DEG C of taking-ups, 1 is made into MiniQ ×.

9) cell is taken out, sucks culture medium at room temperature, 1xPBS is washed one time, and 1 × passive of 100ul is added in every hole lysis buffer。

10) 24 orifice plates are placed on shaking table and shake 15min, be transferred in EP pipe for subsequent experimental or storage.

11) substrate is got out, LAR II is taken out to defrosting from -80 DEG C；Stop and Glo: 50 × Stop and Glo substrate is made into 1 with Stop and Glo buffer ×；Avoid light place.

12) cell pyrolysis liquid is transferred to and has been marked in EP pipe, brief centrifugation.

13) measurement pipe has been marked, 50ul cell pyrolysis liquid is added in every pipe.

14) program is arranged in by specification.

15) 100ul LAR II solution is added in every measurement pipe, quickly mixes, measurement.

16) 100ul Stop and Glo solution is added in every measurement pipe, quickly mixes, measurement.

17) data are arranged, it is for statistical analysis with GraphPad Prism software.

6 quantitative PCR experiment step of embodiment

(1) RNA is extracted

Experimental procedure:

1) preparation: inspection proposes the dedicated pipette tips of RNA, EP pipe and PBS, Trizol, chloroform, isopropanol, 75% second Alcohol, DEPC water (0.1%), ice chest, homogenizer.

2) restore room temperature from 4 DEG C of taking-up Trizol, centrifuge is pre-chilled to 4 DEG C.

3) cell is handled: 4 DEG C of PBS are washed once, cell are collected into the EP pipe of pre-cooling and 1mlTrizol is added, with 1ml rifle Head is blown and beaten 10 times repeatedly；Centrifugation 15 minutes, 12000g takes supernatant.

4) 200ul chloroform is added into supernatant, is firmly mixed by inversion half a minute up and down, stands 3 minutes.

5) 4 DEG C, 12000g is centrifuged 15 minutes, and visible lysate divides three layers at this time: upper layer is the RNA of water phase；Middle layer is DNA, lipid etc.；Lower layer is cell residue, albumen, polysaccharide etc..

6) it takes in supernatant 500ul to new EP pipe, 167ul inhales three times；Isometric isopropanol is added, mixes, stands 10 After minute, 4 DEG C, 12000g is centrifuged 10 minutes.

7) carefully remove supernatant, be careful not to lose RNA precipitate, 75% ethyl alcohol of 1ml is added, turns upside down, make to precipitate block It is resuspended.

8) 4 DEG C, 12000g is centrifuged 10 minutes, is carefully removed supernatant, is blotted the liquid of tube wall as far as possible, is careful not to lose RNA precipitate, if precipitating, which loosens, to be centrifuged again.It dries about 15 minutes, until tube wall no liquid.

9) be added appropriate volume (20-30ul) DEPC water dissolve RNA, 58 DEG C water-bath 10 minutes.

10) it is quantitative to take out 2ul, measures buffer:10mM TrisCl (pH7.8), reverse transcription is carried out according to quantitative result. (1_A260=40 μ g/ml, A260/A280=1.8~2.1).

11) reverse transcription.

(2) cDNA reverse transcription (wherein the primer of miR-99a is using primer sequence disclosed in CN201210306109)

Experimental system:

(3) QPCR amplification experiment

1) experimental system:

2) reaction condition: 95 DEG C of thermal denaturation, 120s, 1cycle；Then 95 DEG C are denaturalized, 15sec, annealing extend 60 DEG C, 60~95 DEG C of 30sec, solubility curve, 40cycle.

3) design of primers and synthesis

According to the gene order of mTOR, GAPDH in genebank, using 5.0 software Design primers of Primer, related sequence Column are as shown in table 1 below.

1 base sequence of table

Experimental result:

The experimental result of Examples 1 to 6 includes the following aspects:

1, in the Colorectal Carcinoma for the diabetes that occur together miR-99a level than being lowered more in simple Colorectal Carcinoma Significantly (result is referring to Fig. 1).

In order to identify differential expression of the miRNA in the colorectal cancer with diabetes, inventor has chosen normal person's knot The colorectal cancer hypotype (178373) of mucous membrane of rectum (177005), simple Colorectal Carcinoma (176906) and the diabetes that occur together Clinical sample each 1, with Agilent Human miRNA (8*60K) array chip analysis expression water of 2549 miRNA It is flat, and clustering (Figure 1A) is carried out to the miRNA of difference；Then intersection is taken, there are 17 with the colorectal cancer group of diabetes MiRNAs differential expression, wherein there is 15 to be overexpressed and 2 low expression miRNAs (Figure 1B).Compared with normal colorectal carcinoma, MiR-99a is highest with fold differences in simple Colorectal Carcinoma in the colorectal cancer hypotype tissue with diabetes miRNA.In order to further proofing chip as a result, in normal Colon and rectum mucous membrane, with the colorectal cancer hypotype, simple of diabetes The expression of miR-99a is verified in each 20 tissues of colorectal cancer by qPCR, as shown in Figure 1 C, miR-99a is in Colon and rectum It is significantly lowered in cancerous tissue, is significantly lower than simple Colon and rectum with miR-99a expression in the colorectal cancer hypotype tissue of diabetes Expression (Fig. 1 D) in cancerous tissue.Normal person Colorectal mucosa epithelial cell NCM460 and colorectal cancer cell system HCT-15, The expression of miR-99a, as referring to figure 1E, qPCR are detected in HCT-116, HCT-8, SW480 and LoVo the results show that all Colorectal cancer cell miR-99a expression lower.It may be between diabetes and colorectal cancer that result above, which prompts miR-99a, Potential association, and adjust the development of the special hypotype of colorectal cancer with diabetes.

2, miR-99a can inhibit colorectal cancer cell growth, migration, invasion (referring to fig. 2) in vitro

The effect for developing and shifting to study miR-99a to colorectal cancer, inventor use miR-99a mimics mistake Express the level of miR-99a in HCT-116.MTT measurement display miR-99a is overexpressed proliferation (Fig. 2A) capable of inhibiting cell. Colorectal cancer cell migration and invasive ability decline (figure after the cell transwell and boyden experiment display miR-99a is overexpressed 2B and C).These discovery prompts miR-99a is the negative regulatory factor of colorectal cancer growth and transfer.

3, miR-99a can inhibit mTOR signal path by binding directly the 3 '-UTR of mTOR (referring to Fig. 3)

Mature miRNAs is combined by the 3 '-UTR with target gene and is blocked following protein expression to adjust the table of gene It reaches.In conjunction with three microRNA target prediction methods (PicTar, TargetScan and miRanda), inventor obtains the 13 of miR-99a A potential target gene (Fig. 3 D).In these target genes, mTOR plays an important role in network interaction.Next, hair Bright people assesses effect of the miR-99a targeting mTOR access to colorectal cancer cell.As shown in figs 3 a andb, miR-99a mimics The significant mRNA and protein level for lowering mTOR.In addition, luciferase reporting analysis result confirm miR-99a by with Colon and rectum The 3 '-UTR of mTOR bind directly and lower mTOR expression in cancer cell.The 3 '-UTR of mTOR and saltant type of 3 '-UTR of wild type (having 3 base mutations in seed region sequence) is inserted into pmiRGL- luciferase reporting plasmid (Fig. 3 C) respectively.miR- The overexpression of 99a inhibits relative luciferase activity in HCT-116 cell, and does not observe the suppression in the cell of saltant type Production is used.These results suggest that miR-99a is targeted in colorectal cancer and negative regulation mTOR signal path.

4, saccharification colorectal cancer cell can promote the expression (referring to fig. 4) of mTOR by lowering miR-99a

Glycosylation end products (AGEs) is formed by protein and the non-glycosylation of lipid and reduced sugar.For determination Whether AGEs influences the expression of miR-99a, colorectal cancer HCT-116 cell inoculation is trained base 48h in 100mg/L AGEs, then The expression of qPCR detection miR-99a.The results show that compared with the control group, under the cell miR-99a through saccharification processing is obvious It adjusts (Fig. 4 A).In addition, AGEs saccharification processing HCT-116 cell in mTOR mRNA and protein expression level increase (Fig. 4 B and C).Effect of the miR-99a in the colorectal cancer with diabetes for further evaluation, inventor are had detected normally with IHC Colorectal mucosa, CRC+DM and CRC+NONDM organize the protein expression level of mTOR in each 20, as shown in Figure 4 D, and simple Colorectal Carcinoma is compared, and is significantly increased with mTOR expression in the colorectal cancer hypotype tissue of diabetes.These results The expression of high Glyco inhabiting miR-99a is prompted to activate mTOR access, promotes to be in progress with the colorectal cancer hypotype of diabetes.

Interpretation of result:

In many researchs prompt diabetes on epidemiological level and the substantial connection between colorectal cancer^[1~4], but only There is a small number of research to attempt to connect diabetes and colorectal cancer from molecular level.MiRNAs widely with many pathology mistakes The development of journey is related, and the expression of miRNAs widely changes in cancer and diabetes^[5~7].Alam KJ has found that miR-375 is logical Cross the regulation colorectal cancer growth of inhibition CTGF-EGFR signal path and proliferation^[8].Laudato S research discovery miR-30e-5p is logical Cross the invasion transfer that targeting ITGA6 and ITGB1 inhibits colorectal cancer^[9].The growth of miR-760 inhibition colorectal cancer^[10]。Roux M has found that miR-152-3p is related to the diabetic nephropathy of type 2 diabetic patient in serum^[11].Liang YZ is to multiple about sugar The meta analysis of the relevant miRNA regulation of urine disease finds that miR-223, miR-130a, miR-19a, miR-26b and miR-27b are 2 The potential cycling markers of patients with type Ⅰ DM, and miR-146a and miR-21 can be used as tissue marker object^[12].MiR-99a can be targeted MTOR/p-4E-BPI/p-S6K1 inhibits breast cancer tumor growth, promotes apoptosis^[13].In smooth muscle cell, miR-99a is logical Targeting IGF-1R and mTOR is crossed, regulation hyperinsulinemia promotes smooth muscle cell growth, invasion^[14].In addition, miR-99a It is a variety of swollen in lung cancer, liver cancer, carcinoma of endometrium, head and neck scale carcinoma, carcinoma of mouth, Patients with Urinary System Tumors and esophageal squamous cell carcinoma etc. Expression quantity is remarkably decreased in tumor, and the prognosis of the lower patient of miR-99a expression is poorer^[15-21].MiR-99a is contaminated at No. 21 The tumor suppressor gene of a variety of human cancers it has been reported as on colour solid, the overexpression of exogenous miR-99a significantly inhibits carcinoma of mouth, preceding The transfer of column gland cancer, non-small cell lung cancer and colorectal cancer cell^[22-25]。

MiRNAs may be the crosstalk signal between diabetes and colorectal cancer? inventor's supposition, specific miRNA's Change may be the biological factor fallen ill with the colorectal cancer hypotype of diabetes.

Present inventor is first with the detection of Agilent Human miRNA chip and screening goes out with diabetes Colorectal cancer, colorectal cancer, and the normally highest miRNAs of differential expression multiple in Colon and rectum mucous membrane, wherein miR-99a is companion Have and lowers most apparent miRNA in the colorectal cancer of diabetes；Then, by fluorescent quantitative PCR experiment with diabetes MiR-99a is demonstrated with sugar in colorectal cancer, colorectal cancer and normal Colon and rectum mucous membrane each 20 clinical tissue samples It is expressed in the colorectal cancer of urine disease minimum.In next experiment in vitro after verifying saccharification processing colorectal cancer HCT-116 cell, MiR-99a expression is lowered, and cell Proliferation, migration, invasive ability enhancing, illustrates that miR-99a is straight to tying in high saccharide ring border Intestinal cancer growth, migration, invasion have negative regulation effect.

MiRNA is combined with 3 '-UTR of said target mrna and is interfered translation^[26].Previous studies are it has been reported that miR-99a can be with Inhibit the expression of mTOR, including the cancer of the esophagus and cervical carcinoma by being directly targeted the 3 '-UTR of mTOR^[27,28]。Yan-Jie Zhang etc. is studies have shown that the activity of mTOR signal path is more much higher than normal Colon and rectum mucous membrane in colorectal cancer, and mTOR Excessive activation be used as earliest events in tumour generating process, and with from normal cell to hyperplasia to tumor phenotypes Progress and express raising^[29]。

MTOR is serine threonine protein kinase, belongs to phosphatidylinositol-3-kinase (PI3K) associated kinase family. Regulation of the mTOR activity by tumor suppression compound TSC1/2^[30].Growth factor and nutrient, such as glucose, insulin and ammonia Base acid activates PI3K-PKB and reduces TSC1/2 activity, mTOR is caused to activate.The mTOR of activation by adjust S6 kinase and EIF4E Binding Protein 1 (4EBP1) phosphorylation adjusts translation initiation mechanism, so as to cause protein synthesis and cell growth Increase^[31,32].MTOR signal path be regulating cell growth with proliferation a critical path, the access will from nutrient molecule, The signal integration that energy state and growth factor transmit together, regulates and controls a large amount of life process, including autophagy, ribose The life assemblage of body and metabolism etc..The imbalance of the access is related to a variety of human diseases, and the imbalance of the access will will lead to a system Generation of column disease, including tumour, obesity, diabetes B etc.^[33].MTOR can to extracellularly include growth factor, insulin, A variety of stimulations such as nutrient, amino acid, glucose generate response.It is mainly realized by PI3K/Akt/mTOR approach to thin The regulating and controlling effect of the different physiological roles such as intracellular growth, cell cycle^[34].Hyperglycemia, hyperinsulinemia/IGF-1 axis and this is logical Road is closely related^[35]。

Inventor extends these researchs, incorporates the phase of prediction and each target gene based on bioinformatics Interaction network, in the target gene that 13 are predicted, discovery mTOR is the most important potential target gene of miR-99a.Pass through Luciferase experimental verification miR-99a can combine 3 '-UTR of mTOR gene.Phase can be significantly reduced in miR-99a mimics To the activity of luciferase.In the cell that 3 '-UTR of mTOR gene mutation are transfected, miR-99a mimics couple is not observed The inhibiting effect of relative luciferase activity.These are the result shows that miR-99a can be by tying with the 3 '-UTRs of mTOR mRNA Close and directly adjust the expression of mTOR gene.After saccharification processing colorectal cancer HCT-116 cell, miR-99a is lowered, and by upper MTOR expression is adjusted, inhibits colorectal cancer cell growth, migration, invasion, illustrates that miR-99a can be targeted and be born in high saccharide ring border Regulate and control mTOR signal path.By immunohistochemistry detection discovery in the colorectal cancer group with diabetes in clinical tissue sample MTOR expression is knitted compared with lowering in Colorectal Carcinoma, normal Colon and rectum mucous membrane.These results suggest that miR-99a is by targeting simultaneously Negative regulation mTOR signal path inhibits colorectal cancer growth, the invasion with diabetes, has cancer suppressing action.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention And range.

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Claims

1. Use of miR-99a, an early molecular marker of colorectal cancer, in the preparation of a diagnostic kit for diabetes-related colorectal cancer subtypes.

2 . The use according to claim 1 , wherein the sequence of the molecular marker miR-99a is shown in SEQ ID NO: 1. 3 .

3. purposes according to claim 1 or 2, is characterized in that, described diagnostic kit detects the content of miR-99a in subject's colorectal cancer tissue, and compares with colorectal cancer tissue of simple colorectal cancer patients. The mean expression levels of miR-99a in the test subjects were compared to determine whether the subjects had diabetes-related colorectal cancer subtypes.

4. purposes according to claim 3, it is characterized in that, described diagnostic kit also detects the expression level of the target gene mTOR of miR-99a in subject's colorectal cancer tissue, and compares with simple colorectal cancer patient's expression level. The mean expression levels of mTOR in colorectal cancer tissues were compared to determine whether the subjects had diabetes-related colorectal cancer subtypes.

5. A diagnostic kit for diabetes-related colorectal cancer subtypes, characterized in that the kit comprises a colorectal cancer tissue total RNA extraction reagent, a reverse transcription PCR reagent and a quantitative PCR reagent.

6 . The kit according to claim 5 , wherein the kit further comprises an extraction reagent for mTOR protein in colorectal cancer tissue and a quantitative reagent for mTOR protein. 7 .

7. Use of miR-99a, a molecular marker for early diagnosis of colorectal cancer, in the preparation of a medicament for treating diabetes-related colorectal cancer subtypes.

8. A drug for the treatment of cancer, characterized in that the drug contains miR-99a or a drug capable of increasing the expression of miR-99a in cancer tissue.

9 . The medicine according to claim 8 , wherein the medicine further contains a medicine that can reduce the expression of mTOR in cancer tissue or a neutralizing agent for mTOR. 10 .

10. The medicine according to claim 8 or 9, wherein the cancer is a diabetes-related colorectal cancer subtype.