CN102978278B - Endogenic non-coding tiny RNA s and application thereof - Google Patents

Abstract

The present invention provides the i.e. miR of a kind of Microrna 644 and precursor sequence thereof diagnosing, prevent, treat and/or purposes in prognosis evaluation malignant tumor.The Microrna that the present invention provides is the nucleic acid or its bioactive functions fragment or variant comprising the nucleotide sequence shown in SEQ ID NO:1: 5 ' AGUGUGGCUUUCUUAGAGC 3 ', the precursor of described Microrna is to comprise the nucleotide of nucleotide sequence shown in SEQ ID NO:3 or the fragment of its bioactive functions or variant.This Microrna can be used for prevention and/or the treatment of malignant tumor as a kind of novel pharmaceutical composition, additionally, this Microrna is also used as a kind of new biomarker is applied to diagnosis and/or the prognosis evaluation of malignant tumor.

Description

Endogenic non-coding tiny RNA s and application thereof

Technical field

The invention belongs to biological medicine engineering field, relate to a kind of endogenic non-coding tiny RNA s and application thereof, specifically Relate to a kind of Microrna (microRNA, miRNA) and precursor sequence thereof and they are in preparation prevention and/or treatment malignant tumor Application in the test kit of disease.

Background technology

Current malignant tumor is the disease that whole world mortality rate is higher, and the sickness rate of malignant tumor increases, seriously year by year Threaten the health of the mankind.Third National coroner's inquest result shows, urban and rural residents of China mortality of malignant tumors belongs to generation Boundary's higher level, and in lasting growth trend, malignant tumor has become China urbanite first place cause of the death (25%).In recent years The next research acquirement remarkable progress about tumor pathogenesis and treatment and prevention of tumour, but in the world, the preventing and treating of malignant tumor It is still a difficult point in current life science field.

Apoptosis plays a significant role in Tumorigenesis, and the suppression of apoptosis of tumor cells is sent out with tumor Exhibition has close relationship.The gene of many apoptosis involvement regulation and control plays a significant role in tumor develops, these bases Cause can be as diagnosing tumor and the action target spot for the treatment of.Therefore, find apoptosis involvement new in tumor cell protein factor, MiRNA etc. and illustrate its mechanism of action in apoptosis of tumor cells there is extremely important using value.

MicroRNA (miRNA) is the class length endogenous non-coding tiny RNA at about 22nt, be widely present in animal, In the multiple organism such as plant, virus.It mainly by with 3 ' UTR of target gene completely or incomplete pairing, target of degrading Gene mRNA or suppress it to translate, thus participate in body development, apoptosis, breed and the vital movement such as differentiation, it was predicted that Human gene more than 1/3 is conservative miRNA target gene.Experimental evidence shows, miRNA can be by regulating and controlling its target gene The signal path participated in, affects generation and the development of tumor, plays the function being similar to oncogene or antioncogene.miRNA The research being found to be Tumorigenesis provide new thinking, provide new strategy for tumor diagnosis and therapy.

Along with the development of science and technology and deepening continuously, based on bio-target molecule of tumor cell signal transduction pathways research Tumor biotherapy become the focus of Effect of Anti tumour medicine.The biotherapy of tumor is with molecular biology, cytobiology Based on analyzing immunology, routine treatment effect big with Radiotherapy chemotherapy three is complementary to one another, and the treatment of tumor is greatly improved Curative effect.Biotechnology is combined with radiotherapy chemotherapy, introduces and promote the drug resistance that the molecule of apoptosis can produce with reversing tumor cell Property, strengthen the tumor cell sensitivity to antitumor drug, thus avoid the drug resistance that traditional radiotherapy chemotherapy method causes With the shortcoming of toxic and side effects, reach to treat the effect of tumor.This area in the urgent need to understand promote apoptosis of tumor cells relevant because of Son, is applied in the treatment of clinic as the target molecule of Drug therapy, therefore finds to promote apoptosis of tumor cells relevant The factor and radiotherapy chemotherapy combined treatment tumor disease, have broad prospects.

Summary of the invention

It is an object of the invention to provide a kind of new Microrna i.e. miR-644 with rush apoptosis of tumor cells function, should MiRNA is one section of endogenous miRNA in organism, has the function promoting apoptosis of tumor cells, in tumor models and naked The experiment of Mus lotus tumor model all enhances the amycin therapeutic effect to tumor cell.

For above-mentioned technical purpose, technical scheme is as follows:

On the one hand, the present invention provides a kind of Microrna in preparation for preventing, treat and/or prognosis evaluation malignant tumor Medicine or test kit in purposes, wherein said Microrna is miR-644 and precursor sequence thereof.

Preferably, the sequence of described miRNA-644 is to comprise the nucleoside of the nucleotide sequence shown in following SEQ ID NO:1 Acid or its bioactive functions fragment or variant:

5′-AGUGUGGCUUUCUUAGAGC-3′。

Preferably, the sequence of described miRNA-644 precursor is to comprise the nucleotide sequence shown in following SEQ ID NO:3 Nucleotide or the fragment of its bioactive functions or variant:

5′-UUUUUUUUUAGUAUUUUUCCAUCAGUGUUCAUAAGGAA

UGUUGCUCUGUAGUUUUCUUAUAGUGUGGCUUUCUUAGAGCAAA

GAUGGUUCCCUA-3′。

Preferably, the sequence of described miRNA-644 has carried out thio-modification, methoxy modifies or cholesterol is modified.

Preferably, the sequence of described miRNA-644 or its precursor contains miRNA-by proceeding to known expression vector establishment 644 or the expression vector of its precursor sequence, and the expression vector containing miRNA-644 or its precursor sequence built is proceeded to disease Poison is expressed and is obtained.

Preferably, described known expression vector is pSilencer Adeno CMV1.0 shuttle vector (pSilencer Adeno CMV1.0shuttle vector), described virus is adenovirus.

On the other hand, the invention provides a kind of pharmaceutical composition for preventing and/or treat malignant tumor, it comprises The above-mentioned miR-644 of therapeutically effective amount and precursor sequence thereof and pharmaceutically acceptable virus, carrier or adjuvant, wherein said micro- Tiny RNA is nucleic acid or its bioactive functions fragment or the variant comprising following SEQ ID NO:1 sequence: 5 '- AGUGUGGCUUUCUUAGAGC-3′。

Preferably, described pharmaceutically acceptable carrier or adjuvant are solid selected from plasmid expression vector, virus, chitosan, gallbladder Alcohol, liposome, nano-particle etc..

Preferably, the administering mode of described pharmaceutical composition is oral administration or drug administration by injection；It is highly preferred that described injection Administering mode is selected from intravenous injection, intramuscular injection, direct tumor intratumor injection etc..

Another aspect, present invention also offers a kind of test kit for prognosis evaluation malignant tumor, described test kit bag Contain the probe for the above-mentioned miR-644 of specific detection or primer.

Preferably, the probe comprised in described test kit or primer have the nucleotides sequence shown in following SEQ ID NO:4 Row:

5′-GCTCTAAGAAAGCCACACT-3′。

In sum, the present inventor by substantial amounts of it is demonstrated experimentally that miRNA-644 (5 '-AGUGUGGCUUUCUUAGAGC- 3 ') improve the tumor cell sensitivity to chemotherapeutics by inducing apoptosis of tumour cell, comment in tumor prevention, treatment and prognosis There is in estimating important using value.

Processing through chemotherapeutic drugs Doxorubicin, in tumor cell, miRNA-644 expresses notable rise, the rise of miRNA-644 The generation of apoptosis of tumor cells may be take part in.In tumor cell, process LAN miRNA-644 can be with inducing apoptosis of tumour cell Occur.MiRNA-644 is combined with suitable carrier formation medicine, imports tumor locus or internal, will to malignant tumor patient There is prevention and therapeutical effect.Inventor can adenovirus infection adenocarcinoma of stomach SGC-of process LAN miRNA-644 by building 7901 cell lines and cancer Hela cells, find that miRNA-644 can induce two kinds of tumor cell line apoptosis, in cellular water The flat miRNA-644 that demonstrates reaches to treat the latent effect of tumor by inducing apoptosis of tumour cell.Anti-by miRNA-644 The expression of MODN suppression miRNA-644, can suppress the apoptosis of tumor cells that amycin is induced.Nude mice by subcutaneous inoculation is swollen Oncocyte, inoculation position is injected miRNA-644 analogies simultaneously, can be significantly inhibited nude mice by subcutaneous tumor growth.In whole animal Level also demonstrates the potential prevention of miRNA-644, therapeutical effect.

Process LAN miRNA-644 in tumor cell, uses the amycin of relatively low-dose to get final product inducing apoptosis of tumour cell, carries The high tumor cell sensitivity to medicine, can overcome the toxic and side effects of chemotherapeutics and the shape of drug resistance in oncotherapy Become.

Therefore, the invention provides miRNA-644 and precursor thereof and suitable carrier or adjuvant such as eukaryotic gene expression load The packaging such as body, adenovirus, chitosan, cholesterol, liposome, nano-particle forms medicine, by oral, intravenous injection, muscle Injection or the mode of directly tumor locus injection, be used for preventing and/or treating malignant tumor.

Accompanying drawing explanation

Hereinafter, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Fig. 1 is the experiment knot that amycin processes the change of miRNA-644 expression during inducing apoptosis of tumour cell Really schematic diagram；MiRNA-644 expression experimental result schematic diagram during wherein Figure 1A is SGC-7901 tumor cell；Figure 1B is MiRNA-644 expression experimental result schematic diagram in Hela tumor cell；

Fig. 2 be proceed to virus after miRNA-644 process LAN adenovirus construction carrier after the experimental result expressed of miRNA-644 Schematic diagram；Wherein Fig. 2 A is the schematic diagram of the pSilencer Adeno CMV1.0 shuttle vector containing miRNA-644；Fig. 2 B is PCR checking obtains the electrophoretogram of the positive colony of the pSilencer Adeno CMV1.0 shuttle vector containing miRNA-644, figure In 1 be DNA marker (DNA Marker), 2 is the miRNA-644 fragment that amplification obtains 610bp；Fig. 2 C is miRNA-644 adenopathy Poison infects SGC-7901 tumor cell miRNA-644 expression test experience result schematic diagram；Fig. 2 D is miRNA-644 adenopathy Poison infects Hela tumor cell miRNA-644 expression test experience result schematic diagram；

Fig. 3 is can be with the experimental result schematic diagram of inducing apoptosis of tumour cell by adenovirus process LAN miRNA-644；Its Middle Fig. 3 A is the experimental result schematic diagram of SGC-7901 apoptosis of tumor cells；Fig. 3 B is the experiment knot of apoptosis in Hela tumor cell Really schematic diagram；

Fig. 4 is that the miRNA-644 analogies process LAN miRNA-644 by transfection synthetic can be with inducing tumor cell The experimental result schematic diagram of apoptosis；Wherein Fig. 4 A, 4B is SGC-7901 tumor cell and Hela tumor cell transfection miRNA-644 After analogies 24 hours, the result schematic diagram of real time fluorescence quantifying PCR method detection miR-644 expression；Fig. 4 C, 4D are The experimental result schematic diagram of apoptosis of tumor cells after SGC-7901 and Hela tumor cell transfection miRNA-644 analogies；

Fig. 5 is the growth curve that nude mice by subcutaneous inoculated tumour injection location miRNA analogies lump is formed；

Fig. 6 is to be expressed by miRNA-644 in miRNA-644 antisense oligonucleotide suppression tumor cell, tumor cell pair The experimental result schematic diagram that the apoptosis susceptibility of amycin induction strengthens, wherein Fig. 6 A is that Hela cell transfecting miRNA-644 is anti- After MODN, the experimental result schematic diagram of the expression of miRNA-644；After Fig. 6 B is for suppression endogenous miRNA-644, The experimental result schematic diagram of the apoptosis situation of Hela cell under low dosage amycin effect.

Detailed description of the invention

Referring to specific embodiment, the present invention is described.Only it will be appreciated by those skilled in the art that these embodiments For the present invention is described, it limits the scope of the present invention never in any form.

The change of miRNA-644 expression during embodiment 1 amycin process inducing apoptosis of tumour cell

This experiment processes SGC-7901 and Hela tumor cell with 2uM amycin respectively and (is purchased from Chinese Academy of Sciences Shanghai thin Born of the same parents storehouse) 3h, collects cell, extracts total serum IgE with Trizol, use the method pair of real-time fluorescence quantitative PCR after 6h, 12h, 24h The expression of miR-644 detects, and observes the expression change of miR-644 during amycin processes tumor cell. As it is shown in figure 1, along with amycin processes the increase of SGC-7901 and Hela tumor cell time, the expression of miR-644 exists Two kinds of tumor cells all present the trend of substantially rising.

The structure of embodiment 2miRNA-644 adenovirus

The pSilencer Adeno 1.0-CMV carrier system using Ambion company builds miRNA-644 process LAN and carries Body or adenovirus.Respective carrier and adenovirus is built according to company's description.

With rat gene group as template, PCR amplification miRNA-644 and the sequence of each nearly 300 bp in upstream and downstream two ends thereof, Amplification obtains segment about 600bp (SEQ ID NO:2, as follows), and amplimer is as follows:

F:5 '-CTAGTGTTTCCTATCCCTGTTGTG-3 ' (SEQ ID NO:7)；

R:5 '-GGTAATGATGTGGTGTAAGTCCTG-3 ' (SEQ ID NO:8).

Amplified fragments sequence is as follows:

CTAGTGTTTCCTATCCCTGTTGTGGAGTGTTTTATCATGAAAGTGTATTAAATTTTG

TAAAATCCTTTTTTGGTATCAATTGAGATGATCATGTGATTTTTTTCCCCCCCTCAT

TCTGTTAATGTGGTATATTATGTTGATTTTTCATAGGTTGAACCTTTTTTGCATTCC

AGGAATAAATCCTACTTAGTCAAAAGGATTAAAGTGTGCTTTTAATATGCTGCTGA

GATTTTTTTGCTGATTTTTTTTTAGTATTTTTCCATCAGTGTTCATAAGGAATGTTGC

TCTGTAGTTTTCTTATAGTGTGGCTTTCTTAGAGCAAAGATGGTTCCCTATTACTTT

CTAATTTTATACTTCTACACATTAACAACTTTTATATTTAAAGCAGAAACTGGAAA

ATCAGGCCAATTTGGTATTAATGAAATTACAGAGGTAATTTAGATATGGGAATAA

ATTACCGATAATATTATTCTATCATTCATTTTAGTTACAATAAGTTTGGGTGCATAT

AATAGAAAACACTAAAATAATAGTGGTTTATGTAAGATAGAAGTGTATTTGTCCCC

ATTATTTAAAAATAGTCCAGCAGGACTTACACCACATCATTACC

The segment that amplification obtains is cloned into carrier T, is subcloned into pSilencer Adeno CMV1.0 shuttle vector (pSilencer Adeno CMV1.0shuttle vector), it is thus achieved that the carrier (such as Fig. 2 A) containing miRNA-644.Pass through PCR And sequence verification obtains the clone containing miRNA-644 carrier, PCR result is shown in Fig. 2 B.Pac this carrier of I linearization for enzyme restriction and adenopathy Poison skeleton (Adenovirus LacZ Backbone), cotransfection HEK-293 cell (purchased from U.S. ATCC), pack adenopathy Poison, amplification is collected virus, is measured virus titer.Real-time quantitative PCR (Real-time PCR) identifies the expression of miRNA-644.

MiRNA-644 mutant virus builds: miR-644pSilencer Adeno CMV1.0 shuttle vector is passed through base Because of rite-directed mutagenesis method suddenly change miRNA-644 sequence four bases (by QuikChange site-directed mutagenesis kit (Stratagene company) carries out site-directed point mutation, the explanation operation of concrete grammar reference reagent box), mutant primer is as follows, prominent The base become is drawn horizontal line and is marked:

5′-GCTCTGTAGTTTTCTTATAGTTGACCTTTCTTAGAGCAAAGATGG-3 ' (SEQ ID NO:5)

5′-CCATCTTTGCTCTAAGAAAGGTCAACTATAAGAAAACTACAGAGC-3 ' (SEQ ID NO:6).

MiR-644pSilencer Adeno CMV1.0 shuttle vector after being suddenlyd change is according to above-mentioned identical method structure Build miRNA-644 mutant adenovirus.Virus is all dissolved in serum-free medium, and-80 DEG C store for future use.

By miRNA-644 adenovirus infection SGC-7901 and Hela tumor cell, after 24 hours, collect cell, extract RNA, Real-time PCR detects miRNA-644 expression, and the cell of miRNA-644 adenovirus is infected in result display MiRNA-644 expression is significantly raised, and the cell miRNA-644 infecting miRNA-644 mutant virus expresses and is not changed in, (such as Fig. 2 C, 2D).

Embodiment 3 adenovirus process LAN miRNA-644 can be with inducing apoptosis of tumour cell

By miRNA-644 adenovirus infection SGC-7901 and Hela tumor cell, adenovirus infection 12,24,36,48 hours After, detect apoptosis situation by original position end gap labelling method (TUNEL).As it is shown on figure 3, after process LAN miRNA-644 The prolongation over time of SGC-7901 and Hela tumor cell all has apoptosis to occur.

Embodiment 4miRNA-644 analogies process LAN miRNA-644 can be with inducing apoptosis of tumour cell

By the miRNA-644 analogies of SGC-7901 and Hela tumor cell transfection synthetic, (analogies are in Shang Haiji Agate company synthesizes, and is the oligonucleotide sequence of 2 '-methoxy modification, and its sequence is SEQ ID NO:1), after transfection, 12,24,48 is little Time, detect apoptosis situation by original position end gap labelling method (TUNEL).As shown in Figure 4, after process LAN miRNA-644 The prolongation over time of SGC-7901 and Hela tumor cell all has apoptosis to occur.

Embodiment 5 nude mice by subcutaneous inoculated tumour injection location miRNA analogies lump forms growth curve

Cultivate Hela tumor cell, collected by trypsinisation cell, inoculate 1 × 10⁷Cell is in athymic BALB/c nude mice (purchased from animal experiment company limited of Beijing dimension tonneau China) dorsal sc.After inoculating cell 3 days, inject in inoculation position MiRNA-644 analogies (same with the miRNA-644 analogies in embodiment 4) (analogies synthesize in Shanghai Ji Ma company), note Penetrate miRNA analogies negative control as a control group.Often 6 BALB/c nude mices of group are tested, every 3 after inoculated tumour cell It measures the size of tumor mass, and Continuous Observation 4 weeks, by formula: long × wide 2/2 calculates lump volume, draws tumor growth curve, sees Examine miR-644 in nude mice level to swollen neoplastic impact.As it is shown in figure 5, the nude mouse tumor of injection miRNA-644 grows relatively Matched group is slow, becomes tumor small volume.

Process LAN miRNA-644 in embodiment 6 tumor cell, the apoptosis susceptibility that amycin is induced by tumor cell strengthens

SGC-7901 and Hela tumor cell infects miRNA-644 adenovirus and mutant virus thereof, after infecting 24 hours, The amycin (0.2 μM) using low dosage processes cell, thin by end gap labelling method (TUNEL) detection in situ after 24 hours Born of the same parents' apoptosis situation.As shown in Figure 6, the amycin (0.2 μM) using low dosage processes SGC-7901 and Hela tumor cell, tumor Apoptosis is the most obvious.After process LAN miRNA-644, then can be obviously promoted apoptosis of tumor cells with the amycin of this dosage Generation, namely process LAN miRNA-644 can increase the sensitivity of apoptosis of tumor cells of amycin induction.

Claims (6)

1. a human miRNAs-644 and precursor thereof are in preparing pharmaceutical composition or the test kit for treating malignant tumor Purposes,

Wherein, described malignant tumor is adenocarcinoma of stomach and cervical cancer.

Purposes the most according to claim 1, it is characterised in that described miRNA-644 is for comprising following SEQ ID NO:1 institute The nucleotide of the nucleotide sequence shown or its bioactive functions fragment or variant:

5′-AGUGUGGCUUUCUUAGAGC-3′。

Purposes the most according to claim 1, it is characterised in that the sequence of described miRNA-644 precursor is for comprising following SEQ The nucleotide of the nucleotide sequence shown in ID NO:3 or the fragment of its bioactive functions or variant:

5′-UUUUUUUUUAGUAUUUUUCCAUCAGUGUUCAUAAGGAAUGUUGCUCUGUAGUUUUCUUAUAGUGUGGCUU UCUUAGAGCAAAGAUGGUUCCCUA-3′。

4. according to the purposes described in any one of claims 1 to 3, it is characterised in that the sequence of described miRNA-644 has carried out sulfur In generation, modifies, methoxy modifies or cholesterol is modified.

5. according to the purposes described in any one of Claims 1-4, it is characterised in that described miRNA-644 or its precursor are by turning Enter known expression vector establishment and contain the expression vector of miRNA-644 or its precursor, and by build containing miRNA-644 or its The expression vector of precursor proceeds to expressing viral and obtains.

Purposes the most according to claim 5, it is characterised in that described known expression vector is p Silencer Adeno CMV1.0 shuttle vector, described virus is adenovirus.