CN103233007A

CN103233007A - Hsa-miR-545miRNA and use thereof

Info

Publication number: CN103233007A
Application number: CN2013100472474A
Authority: CN
Inventors: 张必良; 杜博文
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2013-02-05
Filing date: 2013-02-05
Publication date: 2013-08-07

Abstract

The invention discloses hsa-miR-545miRNA and a use thereof. The hsa-miR-545miRNA has base composition shown in the formula of SEQ ID NO: 1. The hsa-miR-545miRNA can effectively reduce cancer cell proliferation, can induce cancer cell apoptosis and animal tumorigenicity capability, can be used as an anticancer drug and can especially be used for lung cancer treatment.

Description

Hsa-miR-545miRNA and application thereof

Technical field

The invention belongs to technical field of biomedical materials, specifically relate to a kind of hsa-miR-545miRNA relevant with the cancer generation and the application in pharmacy thereof.

Background technology

Cancer also claims malignant tumour, the not normal and disease that causes of the control growth and proliferation of cell mechanism of serving as reasons.Cancer cells except grow out of control, healthy tissues even transfer to other parts of health via body-internal-circulation system or lymphsystem arround also can local invading.Cancer is one of the highest disease of world today's mortality ratio.Serious threat human health.

MircoRNA is called for short miRNA, a normal length 19-25 Nucleotide, pure with various physiological clock, be evolve to go up conservative small molecules single stranded RNA.

The miRNA gene is normally transcribed by rna plymerase ii (polII) in nuclear, initial product pri-miRNA.Pri-miRNA is processed into the pre-miRNA that 70 Nucleotide are formed under the effect of Drosha RNase and its cofactor Pasha.RNA – GTP and exportin5 are transported to pre-miRNA in the tenuigenin.Subsequently, another Dicer RNase shears it and produces the miRNA:miRNA* two strands that is about 22 length of nucleotides.This two strands is directed in silencing complex (RISC) complex body very soon, and wherein the strand miRNA of a maturation is retained in this complex body.Ripe miRNA is attached to the site of the mRNA complementary with it by the base pairing regulate gene expression.

MiRNA is by regulating target gene expression functionating in cell at post-transcriptional level.In cell proliferation, growth, differentiation plays an important role in the important physiological process of apoptosis.Therefore, the abnormal expression of miRNA often with the generation of tumour, shift etc. relevant.MiRNA more than 50% is positioned at tumor-related gene group zone, comprises chromosome amplification district and fragile site.These all show, we can disclose the tumour mechanism from the angle of miRNA regulation and control, and provide new gene target spot and molecule marker for diagnosing tumor and treatment.

Still have nothing to do at present and be used for the report of pulmonary cancer diagnosis and treatment in hsa-miR-545.

Summary of the invention

One of purpose of the present invention provides a kind of new hsa-miR-545miRNA, and this hsa-miR-545miRNA can be used for the treatment of tumour.

The technical scheme that realizes above-mentioned purpose is as follows.

A kind of hsa-miR-545miRNA, its based composition is SEQ ID NO:1.

Another object of the present invention provides the application of above-mentioned hsa-miR-545miRNA.

Concrete technical scheme is as follows.

The application of hsa-miR-545miRNA in the preparation antitumor drug.

Another object of the present invention provides a kind of pharmaceutical composition for the treatment of tumour.

Concrete technical scheme is as follows:

A kind of pharmaceutical composition for the treatment of tumour, its activeconstituents includes aforesaid hsa-miR-545miRNA.

Among embodiment, described tumour is lung cancer therein.

The present invention carries out the exploitation of anti-tumor medicine by the research to miRNA, and a kind of new hsa-miR-545miRNA and the purposes in the medicine for treating tumor thing thereof are provided.Hsa-miR-545 of the present invention can effectively reduce the propagation of cancer cells, cancer cell specific induction of apoptosis and animal become the knurl ability, therefore can be used as a kind of cancer therapy drug, particularly the application in treatment lung cancer has very important application prospects in its cancer therapy.

Description of drawings

Fig. 1 is that real-time quantitative PCR detects hsa-miR-545 at paired lung cancer and the expression level in the cancer beside organism, does stdn with the expression level of 5S, data from 3 independent experiments (± SD); *, P＜0.05; *, P＜0.01; * *, P＜0.001.

Fig. 2 is the proliferation function that detects the cancer cells of hsa-miR-545 with the celltiter test kit.A, the influence of the A549 cell proliferation of hsa-miR-545; B, the NIH-H460(H460 of hsa-miR-545) influence of cell proliferation; *, P＜0.05; *, P＜0.01; * *, P＜0.001.

Fig. 3 detects the cancer cell-apoptosis effect of hsa-miR-545.A, the apoptotic influence of the A549 of hsa-miR-545; B, the apoptotic influence of the H460 of hsa-miR-545; *, P＜0.05; *, P＜0.01; * *, P＜0.001.

Fig. 4 is the effect that animal becomes the subcutaneous one-tenth knurl of the knurl experiment detection lung cell A549 of hsa-miR-545 ability, A, tumour picture; B, gross tumor volume; C, tumor quality; *, P＜0.05; *, P＜0.01; * *, P＜0.001.

Embodiment

In order more to be expressly understood technology contents of the present invention, be described with reference to the accompanying drawings especially exemplified by following examples.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Used various chemical reagent commonly used are the commercially available prod among the embodiment.

The miR-545mimics sequence:

Mature sequence: 5 '-UCAGCAAACAUUUAUUGUGUGC-3 ' (hsa-miR-545miRNA, SEQ ID NO.1)

Complementary sequence: 3 '-AGUCGUUUGUAAAUAACACACG-5 ' (SEQ ID NO.2).

The miR-545inhibitor sequence:

5’-mGmCmAmCmAmCmAmAmUmAmAmAmUmGmUmUmUmGmCmUmGmA-3’（SEQ ID NO.3)。Wherein, m is the modification that methylates.

Medicine by hsa-miR-545miRNA preparation of the present invention can be Nanoparticulate compositions, lipid composition, by biocompatiblity molecules and/or biodegradable molecular composition.

Medicine by hsa-miR-545miRNA preparation of the present invention can be in intestines or parenteral admin.Administration is that the oral administration parenteral admin is administration in administration in administration in administration in intravascular administration, encephalic administration, the pleura, the tumour, intraperitoneal administration, intramuscular administration, lymph administration, the gland, subcutaneous administration, topical, segmental bronchus administration, the tracheae, intranasal administration, inhalation or dropleting medicine-feeding in the intestines.

During the medicinal application by hsa-miR-545miRNA preparation of the present invention, to the 10mg/kg body weight, miR-506miRNA obtains by chemical synthesis process is synthetic concentration in the 0.01mg/kg body weight; Perhaps expressing the back purifying by carrier construction, is 1X10 with every dosage then ⁵To 1X10 ¹⁴The DNA plasmid vector of individual virion or 100mg to 4000mg.

Embodiment 1: real-time quantitative PCR is analyzed hsa-miR-545 and is expressed:

1. sample is prepared: collect 15 routine lung cancer patient excision samples ,-80 ℃ frozen, the liquid nitrogen transportation.

2. the extraction of total RNA:

1) extracting tissue adds 1ml Trizol reagent tissue is carried out cracking 10min.

2) in above-mentioned EP pipe, add the 0.2ml chloroform, cover EP pipe lid, in hand, firmly shook 15 seconds, at room temperature place 5 minutes after, 12000r/min, 4 ℃ of centrifugal 15min;

3) get the upper strata water and place new EP pipe, add the 0.5ml Virahol, ice bath was placed 10 minutes, 12000r/min, 4 ℃, centrifugal 15min;

4) abandon supernatant, add 1ml75% ethanol and wash, vortex mixed, 7500r/min, 4 ℃ of centrifugal 5min abandon supernatant;

5) allow the precipitation RNA seasoning at room temperature;

6) with RNase-free water dissolving RNA precipitation.

3. reverse transcription: use the Promega Reverse Transcription System of company, Random Primer buys the company in Tiangen

System is as follows:

RNA	2μg
		miR-545RT Primer（10μM)	0.2μl
Random Primer（25μM）	1μl
		The RNA enzyme inhibitors	0.5μl
dNTP（2.5mM each）	3μl
		Reversed transcriptive enzyme	0.5μl
5X Reaction Buffer	4μl
		DEPC H ₂O	Polishing to 20 μ l

MiR-545RT Primer is synthetic by Shanghai JaRa company

MiR-545RT Primer sequence

5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCACACAA-3′（SEQ ID NO.4)

Reaction process is as follows

16℃	30min
		37℃	30min
42℃	30min
		85℃	5min

4. real-time quantitative PCR: use the Tiangen RealMasterMix of company test kit

Reaction system

H ₂O	7μl
		cDNA	2μl
Forward Primer	1μl
		Reverse Primer	1μl

2.5×RealMasterMix

9μl

The miR-545 primer, confidential reference items 5S rRNA primer is by Shanghai JaRa company and synthetic

miR-545Forward Primer5′-CCATCAGTCAGCAAACATT-3′（（SEQ ID NO.5)；

miR-545Reverse Primer5′-AGTGCAGGGTCCGAGGTAT-3′（SEQ ID NO.6)；

5S rRNA Forward Primer5′-ACGGCCATACCACCCTGAAC-3′（SEQ ID NO.7)；

5S rRNA Reverse Primer5′-GGCGGTCTCCCATCCAAGTA-3′（SEQ ID NO.8)。

Reaction system

95℃ 30s

95℃ 5s

55℃ 30s

40 circulations

95℃ 5min。

The real-time quantitative PCR result as shown in Figure 1, hsa-miR-545 expresses in cancerous lung tissue and is lower than normal lung tissue.

The influence of the proliferation of lung cancer cells of embodiment 2:hsa-miR-545

Adopt cell counting-8kit(CCK-8) detect ability of cell proliferation, (A549 is H460) with normal lung inoblast (HFL1) to select two strain lung cancer cell lines.Lung carcinoma cell transfection miR-545mimics is experimental group, transfection NC(Negative Control, buy the sharp rich biotech firm in Guangzhou) be control group, normal lung cell transfecting miR-545inhibitor is experimental group, and transfection Inhibitor Negative Control(buys the sharp rich biotech firm in Guangzhou) be control group.

1) inoculating cell: the above-mentioned cell of logarithmic phase is made single cell suspension, every hole 5000 cell inoculations, 96 porocyte culture plates with the RPMI1640 or the F12-K nutrient solution that contain 10% foetal calf serum.

2) cell transfecting: cell inoculation carried out transfection with lipofectamine2000 (Invitrogen company) according to its specification sheets after 1 day, renewed bright culture medium culturing behind the 6h.

3) detect: respectively at cell cultures 24h, 48h, 72h the time standby celltiter test kit (Promega company) detect cell proliferation.

Celltiter result shows, as shown in Figure 2, compares with control group, and the A549 of transfection miR-545mimics and H460 ability of cell proliferation reduce.Compare with control group, transfection the HFL1 ability of cell proliferation of miR-545inhibitor strengthen, this prompting hsa-miR-545 suppresses tumor growth.

Embodiment 3: the apoptosis of tumor cells experiment

Adopt apoptosis test regent box (BD company) to detect apoptosis, select two strain lung cancer cell lines (A549, H460).Lung carcinoma cell transfection miR-545mimics is experimental group.NC(negative control) is control group.

1) inoculating cell: the above-mentioned cell of logarithmic phase is made single cell suspension with the RPMI1640 or the F12-K nutrient solution that contain 10% foetal calf serum, inoculate 6 porocyte culture plates.

2) cell transfecting:

Behind the cell inoculation 1 day, carry out transfection miR-545mimics and NC with lipofectamine2000 (Invitrogen company) respectively according to its specification sheets, renew bright culture medium culturing behind the 6h.

3) detect: respectively cell cultures 72h the time standby apoptosis test regent box (BD company) detect apoptosis.

The apoptosis detected result shows that as shown in Figure 3, compare with control group, the A549 of transfection miR-545mimics and H460 apoptosis obviously increase.This prompting hsa-miR-545 can inducing apoptosis of tumour cell.

Embodiment 4: nude mice becomes the knurl experiment to detect hsa-miR-545 in vivo to the influence of lung carcinoma cell

Use male BALB/c nude mice to be model, become the knurl ability in the detection bodies.

With the A549 cell of logarithmic phase, with aforesaid method transfection miR-545mimics, peptic cell is made suspension behind the 24h, calculates cell count, with PBS with cell dilution to 1 * 10 ⁷Individual cell/ml.Select 6 4-6 male BALB/c nude mice in all ages, on its hind leg oxter subcutaneous injection cell suspension (left side: NC, left and right sides; Right: miR-545mimics) since the 16th day, gross tumor volume of measurement in per 4 days.Gross tumor volume V calculation formula is as follows: V=L * W ²/ 2, L is maximum diameter, and W is minimum diameter).After 48 days, mouse is put to death and goes tumour to weigh.

Become the knurl experimental result to show in the nude mouse, as shown in Figure 4, cross and express the interior one-tenth of the body knurl ability that hsa-miR-545 can significantly suppress tumour cell, prompting hsa-miR-545 has the gene activity that similar tumour presses down the cancer factor.

This shows that the hsa-miR-545 among the present invention is the effective propagation of anticancer in experiment in vitro not only, and also effectively anticancer tumour formation in the experiment in vivo.Therefore hsa-miR-545 can be used as the potential drug of following oncotherapy.

The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims

1. hsa-miR-545miRNA that based composition is SEQ ID NO:1.

2. the application of the described hsa-miR-545miRNA of claim 1 in the preparation antitumor drug.

3. application according to claim 1 is characterized in that, described tumour is lung cancer.

4. a pharmaceutical composition for the treatment of tumour is characterized in that, its activeconstituents includes the described hsa-miR-545miRNA of claim 1.

5. pharmaceutical composition according to claim 4 is characterized in that, described tumour is lung cancer.