CN1730658A - Recombinant plasmid for curing prostate gland cancer and melanoma - Google Patents

Abstract

The invention relates to a recombinant plasmid for treating prostatic csarcinoma and melanoma, wherein the GRIM19 expression sequences are located under the CMV promotor of the vector PcDNA3.1(+) and linked by Kpn I and EcoR cleavages, the U6 promotor-siRNA-stat3 specific sequence is located in front of the PcDNA3.1(+)CMV promotor, and linked by Bgl II and NruI cleavages.

Description

A kind of prostate cancer and melanomatous recombinant plasmid of being used for the treatment of

Technical field

The invention belongs to biological technical field, be specially a kind of recombinant plasmid, relate to and use the overexpression of RNAi technology inhibition Stat3 in prostate cancer and melanoma to reach the purpose of treatment tumour, GRIM19 can obviously strengthen its restraining effect to tumour.

Background technology

The excessive activation of signal transducer and transcription activator Stat3 and expression can cause cell indeterminate growth, and canceration takes place the opposing apoptosis.At present, in many kinds of malignant tumours, found to have the overexpression of Stat3, comprised prostate cancer and melanoma,, can bring new hope to tumor treatment therefore with the target spot of Stat3 as oncotherapy.

GRIM19 is a kind of gene in normal tissue expression, discovers, its expression is closed during oncogenesis, its expression plasmid is imported cancer cells can induce this apoptosis.

The RNA interference is the gene silencing after a kind of the transcribing.It can trigger certain and transcribe the back watchdog routine, causes the degraded of specific mRNA strand.The main mechanism of its effect is that mRNA is the small segment of 21-23bp through nuclease degradation, and is template with it, specific site, the specific interval corresponding mRNA of sequence with it that degrades.

At present STAT3siRNA expression vector and the GRIM19 expression vector method by subclone is configured to a kind of new recombinant plasmid jointly, is used for prostate cancer and melanomatous treatment, no relevant report.

Summary of the invention

The invention provides a kind of prostate cancer and melanomatous recombinant plasmid of being used for the treatment of, to solve the problem that does not have local injection treatment prostate cancer and melanomatous recombinant plasmid at present.The technical scheme that the present invention takes is:

The GRIM19 expressed sequence is positioned under the CMV promotor of carrier PcDNA3.1 (+), link to each other by Kpn I and EcoR I restriction enzyme site, before U6 promotor-siRNA-stat3 distinguished sequence is positioned at PcDNA3.1 (+) CMV promotor, link to each other by BglII and NruI restriction enzyme site.

The siRNA-stat3 distinguished sequence is as described in the SEQ ID NO.1.

The GRIM19 expressed sequence is as described in the SEQ ID NO.2.

Construction of recombinant plasmid process of the present invention:

(1), the structure of pSilencer U6-Stat3 siRNA carrier: pSilencer U6 plasmid is purchased the ambion company in the U.S., according to known person Stat3mRNA sequence NM003150 among the gene BANK, seek suitable target site (2143-2162), the dna profiling of composite coding siRNA, after pSilencer U6 usefulness BamH I and Hind III linearizing, under the effect of T4 ligase enzyme, be connected with it;

(2), the structure of GRIM19 expression vector: angle the encoding sequence of getting GRIM19 with the RT-PCR method, with expression vector pCXN2mycA, U.S. invitrogen company after KpnI and EcoR I linearizing, connects both with the T4 ligase enzyme;

(3), be template with PCR method with (one), angle the encoding sequence of getting encode U6 promotor and Stat3siRNA; With PcDNA3.1 (+) expression vector, purchase invitrogen company in the U.S., with BglII and NruI linearizing, the method by subclone connects both under the effect of T4 ligase enzyme;

(4), be template with the RT-PCR method with (two), angle the encoding sequence of getting GRIM19, by KpnI and EcoR I restriction enzyme site it is cloned under the CMV promotor of PcDNA3.1 (+)-U6-Stat3 siRNA that (three) obtain; By said process will encode U6-Stat3 siRNA sequence and the coding GRIM19 sequence be building up to jointly on PcDNA3.1 (+) expression vector.

Beneficial effect of the present invention:

1 pSilencerU6-Stat3 siRNA recombinant plasmid local injection prostate cancer and melanoma can obviously suppress its growth, induce its apoptosis.

2 GRIM19 expression plasmids can obviously strengthen the restraining effect of pSilencerU6-Stat3 siRNA to prostate cancer and melanoma local injection.

3 pSilencerU6-Stat3 siRNA and GRIM19 co-expression plasmid PcDNA3.1 (+)-U6stat3 siRNA-GRIM19 local injection can obviously suppress prostate cancer and melanomatous growth, induce its apoptosis.Its cancer suppressing action is higher than 1 and 2.

Topical be can adopt when the present invention uses, local injection or osmosis comprised.

Description of drawings

Behind Fig. 1, the nude mice prostate cancer local injection recombinant plasmid (20 μ g/mouse), gross tumor volume obviously dwindles;

Behind Fig. 2, the nude mice prostate cancer local injection recombinant plasmid (20 μ g/mouse), apoptosis appears in tumour cell.

Behind Fig. 3, the nude mice prostate cancer local injection recombinant plasmid (20 μ g/mouse), stat3mRNA expresses obviously and reduces in the knurl body; A, Northern blot analyze, and the 1st road blank group is played on the left side, the 2nd road negative control group, the 3rd road stat3siRNA group, the 4th road stat3 siRNA-GRIM19 group; B, RT-PCR analyze, and the 1st road blank group is played on the left side, the 2nd road negative control group, the 3rd road stat3siRNA group, the 4th road stat3siRNA-GRIM19 group.

Behind Fig. 4, the nude mice prostate cancer local injection recombinant plasmid (20 μ g/mouse), stat3 and p-stat3 protein expression obviously reduce in the knurl body; A, Western blot analyze stat3 and express, and the 1st road blank group is played on the left side, the 2nd road negative control group, the 3rd road stat3siRNA group, the 4th road stat3 siRNA-GRIM19 group; B, Western blot analysis-p-stat3 express, and the 1st road blank group is played on the left side, the 2nd road negative control group, the 3rd road stat3siRNA group, the 4th road stat3 siRNA-GRIM19 group.

Behind Fig. 5, the nude mice prostate cancer local injection recombinant plasmid (20 μ g/mouse), cyclin D1 in the knurl body, C-Myc, BcL2 albumen obviously reduces; A, Western blot analyze the BcL2 protein expression, and the 1st road blank group is played on the left side, the 2nd road negative control group, the 3rd road stat3 siRNA-GRIM19 group; B, Western blot analyze Cyclin D1 and express, and the 1st road the 1st road blank group is played on the left side, the 2nd road negative control group, the 3rd road stat3siRNA group, the 4th road stat3 siRNA-GRIM19 group; C, RT-PCR analyze the C-myc protein expression, and the 1st road blank group is played on the left side, the 2nd road negative control group, the 3rd road stat3siRNA group, the 4th road stat3siRNA-GRIM19 group.

Fig. 6, A C57 murine melanoma local injection recombinant plasmid (20ug/mouse), local tumor obviously diminishes, B C57 murine melanoma local injection recombinant plasmid (20ug/mouse), tumor growth curve.

Embodiment

Experimental example recombinant plasmid PcDNA3.1 of the present invention (+)-U6stat3 siRNA-GRIM19 is to nude mice prostate cancer and the effect of C57 murine melanoma growth-inhibiting

Inhibition test in the 1 tumor bearing nude mice body

15 of nude mices, every 2 * 10 ⁶/ 100ulPC3 cell is seeded to left veutro wall, and measure with vernier callipers every day, and volume is m by formula ₁ ²* m ₂* 0.5236 calculates, wherein m ₁For short wheelbase from, m ₂Be the major axis distance, when treating that gross tumor volume grows to a certain size, animal be divided into four groups, 1, blank saline control group, 2 feminine genders (19 with human body in all sequences siRNA expression vector of making up of homologous base sequence not) control group; The 3pSilencerU6-stat3siRNA group; 4PcDNA3.1-siRNAstat3-GRIM19 recombinant plasmid group.The plasmid injected dose is 20 μ g/ only (7 days repeat administrations at interval), finishes the back in injection and carries out electrotransfection to improve plasmid at the intravital transfection efficiency of knurl in injection site.Observation tumour size is grown to enough when the control group tumour after the inoculation and is put to death animal when big, gets tumour, measures size, paraffin embedding partly, section, H﹠amp; E dyeing, groupization; Another partly extracts albumen.Found that PcDNA3.1-U6stat3 siRNA-GRIM 19 locally injected into tumor obviously suppress tumor growth (Fig. 1), and induce it that apoptosis (Fig. 2) takes place; PcDNA3.1-U6stat3 siRNA-GRIM 19 locally injected into tumor (dosage 20 μ g/ only) obviously suppress stat3 expression in the tumour, and cause Bcl-2, Cyclin D1 and C-Myc expression level downward modulation (Fig. 3, Fig. 4, Fig. 5);

2 PcDNA3.1-U6stat3 siRNA-GRIM19 press down the knurl effect to C57 murine melanoma model

21 of C57 mouse, every with 2 * 10 ⁶/ 100ul B16 cell was seeded to right Toe pad, all went out knurl in postvaccinal the 9th day, and measuring the tumour mean size is (4.12mm ³), it is divided into three groups at random, the saline control group, negative (19 with human body in all sequences siRNA expression vector of making up of homologous base sequence not) control group, the experimental group injected dose is 20-30ug/, the injection site electrotransfection, in back 30 days of inoculation, the control group tumour was long to 12.1mm ³, the empty plasmid control group is 9.89mm ³, and PcDNA3.1-U6stat3 siRNA-GRIM19 transfection group is 5.25mm ³(Figure29), compare significant difference (P＜0.01) with control group (Fig. 6).

Embodiment: invention construction of recombinant plasmid process

(1), the structure of pSilencer U6-Stat3 siRNA carrier: pSilencer U6 plasmid is purchased the ambion company in the U.S., according to known person Stat3mRNA sequence NM003150 among the gene BANK, seek suitable target site (2143-2162), the dna profiling of composite coding siRNA, after pSilencerU6 usefulness BamH I and Hind III linearizing, under the effect of T4 ligase enzyme, be connected with it;

(2), the structure of pCXN2mycA-GRIM19 expression vector: angle the encoding sequence of getting GRIM19 with the RT-PCR method,, purchase invitrogen company, after KpnI and EcoRI linearizing, both are connected with the T4 ligase enzyme in the U.S. with expression vector pCXN2mycA;

(3), be template with PCR method with (one), angle the encoding sequence of getting encode U6 promotor and Stat3 siRNA; With PcDNA3.1 (+) expression vector, purchase invitrogen company in the U.S., with BglII and NruI linearizing, the method by subclone connects both under the effect of T4 ligase enzyme;

(4), be template with the RT-PCR method with (two), angle the encoding sequence of getting GRIM19, by KpnI and EcoR I restriction enzyme site it is cloned under the CMV promotor of PcDNA3.1 (+)-U6-Stat3 siRNA that (three) obtain; By said process will encode U6-Stat3 siRNA sequence and the coding GRIM19 sequence be building up to jointly on PcDNA3.1 (+) expression vector.

Sequence table

＜110〉Jilin University

＜120〉be used for the treatment of prostate cancer and melanomatous recombinant plasmid

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Claims (4)

1, a kind of prostate cancer and melanomatous recombinant plasmid of being used for the treatment of, it is characterized in that: the GRIM19 expressed sequence is positioned under the CMV promotor of carrier PcDNA3.1 (+), link to each other by Kpn I and EcoR I restriction enzyme site, before U6 promotor-siRNA-stat3 distinguished sequence is positioned at PcDNA3.1 (+) CMV promotor, link to each other by Bgl II and NruI restriction enzyme site.

2, prostate cancer and the melanomatous recombinant plasmid of being used for the treatment of according to claim 1 is characterized in that: the siRNA-stat3 distinguished sequence is as described in the SEQ ID NO.1.

3, prostate cancer and the melanomatous recombinant plasmid of being used for the treatment of according to claim 1 is characterized in that: the GRIM19 expressed sequence is as described in the SEQ ID NO.2.

4, prostate cancer and the melanomatous construction of recombinant plasmid process of being used for the treatment of as claimed in claim 1: comprise the following steps:

(1), the structure of pSilencer U6-Stat3 siRNA carrier: according to known person Stat3mRNA sequence NM003150 among the gene BANK, seek suitable target site (2143-2162), the dna profiling of composite coding siRNA, after pSilencer U6 usefulness BamH I and Hind III linearizing, under the effect of T4 ligase enzyme, be connected with it;

(2), the structure of GRIM 19 expression vectors: angle the encoding sequence of getting GRIM19 with the RT-PCR method, with expression vector pCXN2mycA with KpnI and EcoR I linearizing after, with the T4 ligase enzyme both are connected;

(3), be template with PCR method with (one), angle the encoding sequence of getting encode U6 promotor and Stat3 siRNA; With BglII and NruI linearizing, the method by subclone connects both under the effect of T4 ligase enzyme with PcDNA3.1 (+) expression vector;

(4), be template with the RT-PCR method with (two), angle the encoding sequence of getting GRIM19, by KpnI and EcoR I restriction enzyme site it is cloned under the CMV promotor of PcDNA3.1 (+)-U6-Stat3 siRNA that (three) obtain; By said process will encode U6-Stat3 siRNA sequence and the coding GRIM19 sequence be building up to jointly on PcDNA3.1 (+) expression vector.

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