CN102229928B - Small-interfering RNA (Ribonucleic Acid) of human RBBP6 (Retinoblastoma-binding Proteingene) and application thereof - Google Patents

Abstract

The invention relates to a group of small-interfering RNA (siRNA) aiming at the human RBBP6 gene, nucleic acid constructs thereof, a slow virus carrier for expressing the siRNA and a slow virus, and application of the siRNA, the nucleic acid constructs, the slow virus carrier and the slow virus. Sequences on a coding area of the human RBBP6 gene are used as target sites of the siRNA and the siRNA is designed according to continuous 10-30 (preferably 15-27, more preferably 19-23) base sequences in the target sites. The nucleic acid constructs and the slow virus for expressing the siRNA are obtained through utilizing gene cloning. Cell experiments prove that the siRNA can reduce the expression of the human RBBP6 gene by the sequence specificity and can effectively inhibit the proliferation of tumor cells.

Description

The siRNA of people RBBP6 gene and application thereof

Technical field

The present invention relates to nucleic acid field, relate in particular to RNA perturbation technique, relate more specifically to have the small molecule interference nucleic acid and application thereof that suppress people RBBP6 genetic expression.

Background technology

RNA interference refers to the gene silencing phenomenon brought out by double-stranded RNA, mainly through hindering the translation of specific gene or transcribing inhibition of gene expression.Research shows, length is small RNA molecular (the small interfering RNA of 21-23nt, siRNA) be immediate cause (the Tuschl T causing RNA to disturb, Zamore PD, Sharp PA, Bartel DP.RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotideintervals.Cell 2000,101:25-33.).RNA interference has high efficiency, simplicity and specificity in inhibition of gene expression, has played vital role at present in gene functional research and disease treatment.In recent years, RNA interference had achieved some achievements (Uprichard, Susan L.The therapeutic potential of RNAinterference.FEBS Letters 2005,579:5996-6007) in oncotherapy.

RBBP6, full name is Retinoblastoma binding protein 6, is positioned human chromosomal 16p12.2.The protein localization of RBBP6 genes encoding is in nucleus.Because aminoterminal contains conservative Zinc finger domain, RBBP6 albumen has ubiquitin esterase activity, degraded (the Scott RE of Y-box associated proteins (the cell cycle positive regulation factor) can be promoted, GiannakourosT, Gao S, Peidis P.Functional potential of P2P-R:a role in the cell cycle and celldifferentiation related to its interactions with proteins that bind to matrix associated regionsof DNA? J Cell Biochem 2003; 90:6-12; Chibi M, Meyer M, Skepu A, G Rees DJ, Moolman-Smook JC, Pugh DJ.RBBP6interacts with multifunctional protein YB-1throughits RING finger domain, leading to ubiquitination and proteosomal degradation of YB-1.JMol Biol 2008; 384:908-16).RBBP6 can with tumor-inhibiting factor p53 and/or Rb, and nuclear skeleton attachment region binding factor forms complex body, the form relied on by cell cycle and cytodifferentiation affects genetic transcription, genetic expression and to nuclear adjustment (Scott RE, Giannakouros T, Gao S, Peidis P.Functional potential of P2P-R:arole in the cell cycle and cell differentiation related to its interactions with proteins that bindto matrix associated regions of DNA? J Cell Biochem 2003, 90:6-12., Li L, Deng B, Xing G, Teng Y, Tian C, Cheng X, Yin X, Yang J, Gao X, Zhu Y, Sun Q, Zhang L, Yang X, He F.PACT is a negative regulator of p53 and essential for cell growth and embryonicdevelopment.Proc Natl Acad Sci U S A 2007, 104:7951-6, Scott RE, White-Grindley E, Ruley HE, Chesler EJ, Williams RW.P2P-R expression is genetically coregulated withcomponents of the translation machinery and with PUM2, a translational repressor thatassociates with the P2P-R mRNA.J Cell Physiol 2005, 204:99-105).RBBP6 albumen is high level expression (Yoshitake Y in human esophageal carcinoma, Nakatsura T, Monji M, Senju S, Matsuyoshi H, TsukamotoH, Hosaka S, Komori H, Fukuma D, Ikuta Y, Katagiri T, Furukawa Y, Ito H, Shinohara M, Nakamura Y, Nishimura Y.Proliferation potential-related protein, an ideal esophagealcancer antigen for immunotherapy, identified using complementary DNA microarrayanalysis.Clin Cancer Res 2004, 10:6437-48).The RBBP6 albumen of process LAN can induce the cell-cycle arrest of osteosarcoma Saos2 cell in the mitotic prometaphase, cell death inducing, and chemosensitivity (the Scott RE of MCF-7 Breast Cancer Cell to antitumor drug camptothecine can be strengthened, Giannakouros T, Gao S, Peidis P.Functionalpotential of P2P-R:a role in the cell cycle and cell differentiation related to its interactionswith proteins that bind to matrix associated regions of DNA? J Cell Biochem 2003, 90:6-12, Scott RE, White-Grindley E, Ruley HE, Chesler EJ, Williams RW.P2P-R expression isgenetically coregulated with components of the translation machinery and with PUM2, atranslational repressor that associates with the P2P-R mRNA.J Cell Physiol2005, 204:99-105, Gao S, Scott RE.P2P-R protein overexpression restricts mitoticprogression at prometaphase and promotes mitotic apoptosis.J Cell Physiol 2002, 193:199-207, Gao S, Scott RE.Stable overexpression of specific segments of the P2P-Rprotein in human MCF-7cells promotes camptothecin-induced apoptosis.J Cell Physiol2003, 197:445-52, Scott RE, Gao S.P2P-R deficiency modifies nocodazoleinduced mitoticarrest and UV-induced apoptosis.Anticancer Res 2002,22:3837-42).Based on existing about the report of RBBP6 gene in the esophageal carcinoma, osteosarcoma and mammary cancer at present, can infer that RBBP6 has important function as an oncogene in tumour generation and progression, and be expected to the Effective target site becoming antineoplaston.

Summary of the invention

A first aspect of the present invention, provides the purposes of people RBBP6 gene in oncotherapy, and namely described people RBBP6 gene promotes tumor cell proliferation, can be used as the pharmacological agent target for tumour cell.Described medicine can be small-molecule chemical medicine, antibody medicine, also can be nucleic acid drug.Preferably, the RBBP6 gene of described tumour cell can be used as RNA interference medicament action target.

A second aspect of the present invention, provides people RBBP6 gene small molecule disturbance ribonucleic acid (siRNA) target sequence of separation, and wherein said target sequence is any sequence in SEQ ID NO 1-172.

Present invention also offers the nucleic acid construct and slow virus that comprise any sequence in SEQ ID NO 1-172.Preferably, the described nucleic acid construct (also referred to as rna interference vector) containing RBBP6 gene siRNA sequence is pGCSIL-GFP-RBBP6-siRNA (in expressed sequence table SEQ ID NO.4).

Present invention also offers small molecule disturbance ribonucleic acid (siRNA), it comprises just RNA fragment and antisense RNA fragment, described just RNA fragment comprises the RNA sequence of any sequence encoding in SEQ ID NO 1-172, wherein just RNA fragment and antisense RNA fragment can form double-stranded RNA, and can suppress the expression of people RBBP6 gene.Described just RNA fragment and antisense RNA fragment may reside on two different RNA chains, or are present on a RNA chain.Preferably, described Yeast Nucleic Acid is hair clip type single strand RNA molecule (shRNA), and the complementary region wherein between just RNA fragment and antisense RNA fragment forms double-stranded region.Yeast Nucleic Acid described in above-mentioned any one, wherein the length of just RNA fragment and antisense RNA fragment is 15-27 Nucleotide, is preferably 19-23 Nucleotide, is specially 19,20 or 21 Nucleotide.

Present invention also offers the application of above-mentioned any one in preparation or screening antineoplastic drugs, described tumour is selected from colorectal cancer or liver cancer.

In sum, the present invention devises 172 RNA interfered target sequences for people RBBP6 gene, build corresponding RBBP6shRNA expression vector, wherein the rna interference vector pGCSIL-GFP-RBBP6-siRNA of encoding sequence SEQ ID NO 4 significantly can lower the expression of RBBP6 gene at mRNA level in-site and protein level.Use slow virus (lentivirus, be abbreviated as Lv) carry rna interference vector pGCSIL-GFP-RBBP6-siRNA as genetic manipulation instrument the RNA interference sequence for RBBP6 gene can efficiently be imported people's colorectal carcinoma cell RKO and people's liver tumor cells SMMC-7721 target, reduce the expression level of RBBP6 gene, the propagation of the above-mentioned tumour cell of remarkable suppression and the speed of growth are the potential clinical non-operative treatment modes of colorectal carcinoma and liver neoplasm.Beneficial effect of the present invention

Small RNA provided by the invention or comprise the nucleic acid construct of small RNA sequence, slow virus specificity can suppress the expression of people RBBP6 gene, especially slow virus, efficiently can infect target cell, suppress the expression of RBBP6 gene in target cell expeditiously, and then the growth of inhibition tumor cell, significant in oncotherapy.

Accompanying drawing explanation

Fig. 1 represents pGCSIL-GFP Plasmid diagram.

Fig. 2 represents pGC-FU Plasmid diagram.

Fig. 3 represents that Lv-RBBP6-siRNA slow virus infected people's colorectal carcinoma cell RKO after 5 days, and the expression level of RBBP6mRNA significantly reduces.

Fig. 4 represents that Lv-RBBP6-siRNA slow virus infected people's liver tumor cells after SMMC-77215 days, and the expression level of RBBP6mRNA significantly reduces.

Fig. 5 represents that Lv-RBBP6-siRNA slow virus infected people's colorectal carcinoma cell RKO after 5 days, causes cell inhibitory effect.

Fig. 6 represents that Lv-RBBP6-siRNA slow virus infected people's liver tumor cells after SMMC-77215 days, causes cell inhibitory effect.

Embodiment

Contriver finds, can the propagation of inhibition tumor cell effectively after the expression of RBBP6 gene of transferring person under adopting RNA interference method, shows that RBBP6 gene is proto-oncogene, can be used as the target spot of oncotherapy.Contriver synthesizes further and tests the multiple siRNA for RBBP6 gene, filter out the expression that effectively can suppress RBBP6 and then the siRNA suppressing people colorectal carcinoma cell RKO and people's liver tumor cells SMMC-7721 process, complete the present invention on this basis.

The invention provides siRNA (siRNA) sequence of a series of interference people RBBP6 gene, constructing can the slow virus of the reticent RBBP6 genetic expression of specificity.The present invention studies discovery, for siRNA and the RNA interference slow virus of people RBBP6 gene design, and stable expression of also lowering RBBP6 gene specifically, and effectively suppress the propagation of human tumor cells.The present invention shows that RBBP6 gene can promote growth of tumour cell, is expected to the target spot becoming early diagnosis of tumor and treatment.And, by the expression of the reticent RBBP6 gene of RNA conflicting mode, can be used as the effective means of Tumor suppression development.

Mentality of designing of the present invention is:

The present invention screens by the following method and obtains a kind of people RBBP6 gene RNA interference slow virus: from Genbank, transfer people RBBP6 gene order; Prediction siRNA site; Synthesize the effective siRNA sequence for RBBP6 gene, two ends are containing the double-stranded DNA Oligo of restriction enzyme site cohesive end; Be connected with double-stranded DNA Oligo after lentiviral vectors double digestion; The RNA interference plasmid of construction expression RBBP6 gene siRNA sequence and the process LAN plasmid of expression RBBP6 gene; By RNA interference plasmid and RBBP6 gene overexpression plasmid co-transfection HEKC 293T; Western blot detects these RNA interference plasmids to the restraining effect of the protein expression level of RBBP6 gene, and screens effective interference plasmid according to restraining effect result; Assistant carrier (the Packing Mix of RNA interference plasmid and the slow virus packaging needs obtained will be screened, Sigma-aldrich company) cotransfection HEKC 293T, produce recombinant slow virus particle, the slow virus of efficient reticent RBBP6 gene can be obtained.

Based on aforesaid method, the invention provides the Effective target site (specifically as shown in SEQ ID NO1-172) of 172 interference RBBP6 genes, construct the slow virus of special interference people RBBP6 gene.

The present invention simultaneously also discloses a kind of people RBBP6 gene RNA interference slow virus (Lv-RBBP6-siRNA) and preparation and application thereof.

This research finds, utilizes the RNA interference method of lentivirus mediated, after reducing the expression in tumour cell of RBBP6 gene, and can the effectively propagation of inhibition tumor cell and growth.This research shows, RBBP6 gene is a proto-oncogene, tumor cell proliferation can be promoted, occur in tumour and in development, there is important biological function, RBBP6 gene can be the target of oncotherapy, and the RBBP6 gene specific silence of lentivirus mediated can be used as a kind of new tool of oncotherapy.

The present invention is set forth further below in conjunction with embodiment.Should be understood that embodiment only for illustration of the present invention, but not limit the scope of the invention.In embodiment, the experimental technique of unreceipted actual conditions and the reagent of undeclared formula are conveniently condition, as works such as [U.S.] Sambrook.J; Huang Peitang etc. translate.Molecular cloning texts guide, the third edition.Beijing: the condition of the condition described in Science Press 2002 or manufacturers's suggestion is carried out or configures.

Embodiment 1: for the preparation of people RBBP6 gene RNA interference slow virus

1. screening is for the effective siRNA target spot of people RBBP6 gene

RBBP6 (NM_006910.4, NM_018703.3, NM_032626.5) gene information is transferred from Genbank; Utilize the design software Genechem of Shanghai JiKai Gene Chemical Technology Co., Ltd design for the effective siRNA target spot of RBBP6 gene.In encoding sequence (CDS) region of RBBP6 gene (for 1041 of NM_006910.4 to 6419 bit bases), every the sequence of an initial acquisition of base 21 bases, table 1 lists wherein 172 effective siRNA target sequences for RBBP6 gene.

Table 1 target is in the siRNA target sequence of people RBBP6 gene

Numbering	Target sequence	Initiation site
			1	GTCCTGTGTGCATTATAAATT	1043
2	CCTCTAAACTCAACTATGATA	1066
			3	CTGCGACTTAAAGAAGCAGAT	1118
4	CGACTTAAAGAAGCAGATTAT	1121
			5	CACCAATGCGCAGACGAAAGA	1184
6	TGCGCAGACGAAAGAAGAATA	1190
			7	CAGACGAAAGAAGAATATACT	1194
8	GACGAAAGAAGAATATACTGA	1196
			9	GCTCTGATTCCTAAGAATTCT	1224
10	CTGATTCCTAAGAATTCTTCT	1227
			11	GAATTCCTATTGGAGGTGTTA	1261
12	CCTATTGGAGGTGTTAAATCT	1266
			13	GTGTTAAATCTACAAGCAAGA	1276
14	CTACAAGCAAGACATATGTTA	1285
			15	CTGAACCAGCGATGGCAACTA	1315
16	GATGACTCTTCCGCGTCTATT	1347
			17	GACTCTTCCGCGTCTATTTCT	1350
18	CTCTTCCGCGTCTATTTCTCT	1352
			19	CCAGCTTACAAAGACTGCCAA	1376
20	CAGCTTACAAAGACTGCCAAT	1377
			21	GCTTACAAAGACTGCCAATCT	1379
22	CCACCTCCATCTTACACGTGT	1503
			23	CACCTCCATCTTACACGTGTT	1504
24	CACGTGTTTCCGTTGTGGTAA	1517
			25	GAATCTGGTCCTAGGATTAAA	1587
26	CAGAAGTTTCATGATGGAAGT	1625
			27	GGAAGTGAAAGATCCTAATAT	1640
28	GATGCAGAAGCATATGCAATT	1710
			29	CCTCAGAAGAAGATGATCCTA	1777
30	CAGATGAATTGTTGTGTCTCA	1801
			31	GGATATTATGACTGATGCTGT	1829
32	GGAAACAGTTACTGTGATGAA	1866
			33	GAAACAGTTACTGTGATGAAT	1867

34	CTGGAATCAGATGAGCACACA	1905
			35	GTTTCTCCTGATGCTTTAATT	1950
36	CCTGATGCTTTAATTGCCAAT	1956
			37	CAGGCTGTAAATAACTTCAAA	1989
38	CTCCGAGACCACTGATTCAGA	2074
			39	GAGACCACTGATTCAGAGGAA	2078
40	CTGATTCAGAGGAACCTACAA	2085
			41	GAGGAACCTACAACCTCTGAT	2093
42	GGAACCTACAACCTCTGATGA	2095
			43	GAGATCTCCGATATCAAGACA	2114
44	CAAGACAACAAGATCCTCTTA	2128
			45	GACAACAAGATCCTCTTATGA	2131
46	CCAGCTCCGTCTATATCTTCA	2178
			47	CAGCTCCGTCTATATCTTCAT	2179
48	GCTCCGTCTATATCTTCATTA	2181
			49	CTCCGTCTATATCTTCATTAA	2182
50	CCTGTGTCTGGAAATCCGTCT	2229
			51	CTGTGTCTGGAAATCCGTCTT	2230
52	GTGTCTGGAAATCCGTCTTCT	2232
			53	CTCCAGCTCCTGTACCTGATA	2254
54	CCAGCTCCTGTACCTGATATA	2256
			55	GCTCCTGTACCTGATATAACT	2259
56	CCTGATATAACTGCAACAGTA	2268
			57	GAACCTCCTCAATTGCAATTA	2386
58	GGTTACCAGGTGCCTGTTCTT	2427
			59	CCATCTTTGCTTGGACAGTCA	2454
60	CTTTGCTTGGACAGTCATTAT	2458
			61	GTCATTATTGCATGGACAGTT	2471
62	CACAACTGGTCCAGTAAGAAT	2498
			63	GGTCCAGTAAGAATAAATACT	2505
64	GGTTTCTCCACCACAACAAAT	2582
			65	GTTTCTCCACCACAACAAATT	2583
66	GAGAGGAGCTGCTACAGAAGT	2613
			67	GAGGAGCTGCTACAGAAGTAT	2615
68	GGAGCTGCTACAGAAGTATAA	2617
			69	GAGCTGCTACAGAAGTATAAA	2618
70	CGACACCACAGCGAAAGATCA	2646
			71	CGAAAGATCACAGAGGACTCA	2657
72	CACTACCAGCAACTCCAGTCT	2686
			73	CCAGCAACTCCAGTCTTTGTA	2691
74	GCTCCTGCCAATTTATCAACA	2859
			75	CTGCCAATTTATCAACACCTT	2863
76	GACAGCTCATTCAAATACCAT	2903
			77	CAAGCTAGATGAGTTTACAAA	3008
78	GCTAGATGAGTTTACAAATGA	3011

79	CCTATAGTGGTTCTTCGTATT	3106
			80	GTGGTTCTTCGTATTCAAGAA	3112
81	GGTTCTTCGTATTCAAGAAGT	3114
			82	GGTTCAACACGTTCACGCTCT	3162
83	CAACACGTTCACGCTCTTATT	3166
			84	CACGCTCTTATTCTCGATCAT	3175
85	GCTCTTATTCTCGATCATTCA	3178
			86	CGATCATTCAGCCGCTCACAT	3189
87	GATCATTCAGCCGCTCACATT	3190
			88	GCTCACATTCTCGTTCCTATT	3202
89	CCTATTCACGGTCACCTCCAT	3217
			90	CTATTCACGGTCACCTCCATA	3218
91	CAGAGGCAAGAGCCGCAATTA	3251
			92	CCTTACAGACGCTATCATTCA	3324
93	GACGCTATCATTCACGATCAA	3331
			94	GACAGTCTCCTAATAAACGTA	3373
95	CAGTCTCCTAATAAACGTAAT	3375
			96	CACCATATGACATGAAAGCAT	3451
97	CCATATGACATGAAAGCATAT	3453
			98	GAGAAGTGTTGACTTTAGAGA	3479
99	CCTCAGCAAATAGAGAGAACT	3598
			100	CTCAGCAAATAGAGAGAACTT	3599
101	CCACTTAACATCAGGAATTCT	3639
			102	GCAAAGTCATAGAAGTCGAAA	3698
103	GTCGAAACATAGGTAGCAACT	3712
			104	CGAAACATAGGTAGCAACTAT	3714
105	GGTCACAATCAGAAGGATAAT	3759
			106	GTCACAATCAGAAGGATAATA	3760
107	CACAATCAGAAGGATAATACA	3762
			108	GAGACTTCTAGGAAATCAAGA	3903
109	GACTTCTAGGAAATCAAGAGA	3905
			110	CCAAACGGAAGAATGATGGAT	4096
111	CTCCTCGATCTGAACCTCCAA	4195
			112	CTCGATCTGAACCTCCAATTA	4198
113	CGATCTGAACCTCCAATTAAA	4200
			114	CCAAAGAGGAGACTCCGAAGA	4225
115	GGAGACTCCGAAGACTGACAA	4232
			116	GAGACTCCGAAGACTGACAAT	4233
117	CTCCGAAGACTGACAATACTA	4237
			118	CGAAGACTGACAATACTAAAT	4240
119	GTGAAGAAGGACTATTCCAAA	4368
			120	GACTATTCCAAAGATGTCAAA	4377
121	CCAAAGATGTCAAATCAGAAA	4384
			122	CTCGAAACTAGAAGTGACTGA	4541
123	GGAGTCAACATCTAGCAAAGT	4745

124	GTCAACATCTAGCAAAGTTAA	4748
			125	CAAAGGAAAGGTCAGACGAAA	4781
126	GGAACTGAAGGATCCAGCTCA	4809
			127	CCTGTGCGGAAATCTGAAGAA	4872
128	CTGTGCGGAAATCTGAAGAAA	4873
			129	CAGATACAAAGCGAACTGTGA	4897
130	GATACAAAGCGAACTGTGATT	4899
			131	CTCAATCCAAATGGGATAAAG	4990
132	GTTAAATCCACACAGCCTATA	5037
			133	CACACAGCCTATATCAAGTGT	5045
134	GAAAGTGAGCCATCCGAGAAA	5136
			135	CCAAGGACGTGAGCCATGAAA	5170
136	CAAGGACGTGAGCCATGAAAT	5171
			137	GTGAGCCATGAAATCATACAA	5178
138	GAGCCATGAAATCATACAACA	5180
			139	CGAGATTATTCAGTGTTGGAA	5256
140	CTCAGCCAGAGAAAGAGAGTA	5308
			141	CAGCCAGAGAAAGAGAGTAAT	5310
142	CGTCTGAATGAACAAGGAAAT	5337
			143	GAGGCTAGAACGTCAGATAAA	5385
144	GCTAGAACGTCAGATAAACAT	5388
			145	GAACGTCAGATAAACATGATT	5392
146	CCACTCGTGCTTCCTCAAATA	5413
			147	CACTCGTGCTTCCTCAAATAA	5414
148	GTTCCAAACGTAGAGATGAAA	5491
			149	GACTCTCCTTCTCGGAATAAA	5535
150	CCTCATGATCACAAAGCCACT	5664
			151	CTCATGATCACAAAGCCACTT	5665
152	CATGATCACAAAGCCACTTAT	5667
			153	CACAAAGCCACTTATGATACT	5673
154	CAAAGCCACTTATGATACTAA	5675
			155	GGATCGTGAGAAGCATGTATT	5741
156	GATCGTGAGAAGCATGTATTA	5742
			157	CGTGAGAAGCATGTATTAGAA	5745
158	GCATGTATTAGAAGCAAGGAA	5753
			159	CCACCAGAGACACAGGTTGAA	5817
160	CACCAGAGACACAGGTTGAAA	5818
			161	GCAAATTGACAAGAGTACTGT	5855
162	CAAGAGTACTGTCAAGCCTAA	5864
			163	CCTCTAGACTTTCCTCTGACT	5902
164	CTCTAGACTTTCCTCTGACTT	5903
			165	CTAGACTTTCCTCTGACTTAA	5905
166	GACTTTCCTCTGACTTAACTA	5908
			167	CCTCTGACTTAACTAGAGAAA	5914
168	GACTTAACTAGAGAAACTGAT	5919

169	CAGACTATAATGAAAGTGACA	5956
			170	GACTATAATGAAAGTGACAGT	5958
171	CAGAAAGTCAGGACAGCAAGA	6202
			172	GCACTGAAGTGGAATTGGAAA	6295

Double-stranded DNA Oligo sequence (table 2) of two ends containing Hpa I and Xho I restriction enzyme site cohesive end is synthesized for siRNA target spot (for SEQ ID NO 4); Act on pGCSIL-GFP carrier (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 1) with Hpa I and Xho I restriction enzyme, make its linearizing, agarose gel electrophoresis qualification endonuclease bamhi.

Table 2 two ends contain the double-stranded DNA Oligo of Hpa I and Xho I restriction enzyme site cohesive end

Numbering	5’	Neck	Ring	Neck	3’
						1-F	T	CGACTTAAAGAAGCAGATTAT	TTCAAGAGA	ATAATCTGCTTCTTTAAGTCG	TTTTTTC
1-R	TCGAGAAAAAA	CGACTTAAAGAAGCAGATTAT	TCTCTTGAA	ATAATCTGCTTCTTTAAGTCG	A

By T4DNA ligase enzyme, by double digestion linearizing, (double digestion reaction system is as shown in table 3,37 DEG C, react 1 hour) the carrier DNA double-stranded DNA Oligo good with purifying be connected, spend the night in 16 DEG C of connections in suitable buffer system (as shown in table 4), reclaim and connect product.By fresh competent escherichia coli cell (conversion operation reference: Molecular Cloning: A Laboratory guide second edition 55-56 page) prepared by connection product conversion calcium chloride.Growing bacterium clone surface at connection converted product is stained with, and be dissolved in 10 μ l LB substratum, mixing gets 1 μ l as template; The upstream and downstream of RNA interference sequence, designs general PCR primer (upstream primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' in lentiviral vectors; Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 '), carry out PCR identification experiment (, as table 5-1, reaction conditions is as table 5-2 for PGR reaction system).The clone positive to PCR qualification checks order and compare of analysis, and the clone that comparison is correct is the rna interference vector containing SEQ ID NO 4 sequence successfully constructed, called after pGCSIL-GFP-RBBP6-siRNA.

Table 3pGCSIL-GFP plasmid enzyme restriction reaction system

Reagent	Volume (μ l)
		PGCSIL-GFP plasmid (1 μ g/ μ l)	2
10×buffer	5
		100×BSA	0.5
Hpa I(10U/μl)	1
		Xho I(10U/μl)	1

H 2O	40.5
		total	50

Table 4 ligation system

Reagent	Positive control (μ l)	From connecting contrast (μ l)	Connecting groups (μ l)
				Linearizing carrier DNA (100ng/ μ l)	1	1	1
The double-stranded DNA Oligo (100ng/ μ l) of annealing	1	-	1
				10 × T4 phage DNA ligase enzyme damping fluid	1	1	1
T4 phage DNA ligase enzyme	1	1	1
				dd H2O	16	17	16
total	20	20	20

Table 5-1PCR reaction system

Reagent	Volume (μ l)
		10×buffer	2
dNTPs(2.5mM)	0.8
		Upstream primer	0.4
Downstream primer	0.4
		Taq polymerase	0.2
Template	1
		ddH 2O	15.2
Total	20

Table 5-2PCR reaction system program setting

From the cDNA library containing people RBBP6 gene, PCR method (, in Table 6-1, reaction system is in Table 6-2, and reaction conditions is in Table 6-3 for the primer used in PCR reaction) is utilized to fish and get goal gene.The pGC-FU over-express vector (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 2) of goal gene and linearization for enzyme restriction is carried out orientation connect, its product conversion bacterium competent cell.First carry out bacterium colony PCR qualification to the clone grown, then the clone positive to PCR qualification checks order and compare of analysis, the clone that comparison is correct is the RBBP6 process LAN plasmid successfully constructed.

Table 6-1cDNA over-express vector design of primers

Numbering	Primer sequence	Restriction enzyme site
			Upstream primer	5’-CCGCTCGAGATGTCCTGTGTGCATTATAAATTTTCCTCTAAAC-3’	Xho I
Downstream primer	5’-GGGGTACCGTCACAGTGACAGATTTCACTTTTTGGTCATCTTTC-3’	Kpn I

Table 6-2PCR reaction system

Reagent	Volume (μ l)
		5×PS Buffer	10
dNTP(2.5mM)	4
		Upstream primer (10uM)	1
Downstream primer (10uM)	1
		CDNA template	0.2
PrimerSTAR HS DNA polymerase&Taq DNA polymerase	0.5
		ddH 2O	43.3
Total	60

Table 6-3PCR reaction system program setting

By the pGCSIL-GFP-RBBP6-siRNA plasmid containing SEQ ID NO 4 sequence and RBBP6 process LAN plasmid co-transfection HEKC 293T cell.Well-grown 293T cell is 5 × 10 by density by first 1 day of transfection ⁴/ ml is inoculated in 24 orifice plates, when Growth of Cells to 80 ~ 90% merges, changes the opti-MEM1 of 400 μ l into.Two plasmid transfection consumption groups are set: (1) pGCSIL-GFP-RBBP6-siRNA:RBBP6 process LAN plasmid amount=0.25: 1 (low group); (2) pGCSIL-GFP-RBBP6-siRNA:RBBP6 process LAN plasmid amount=0.5: 1 (high group).Respectively according to aforementioned proportion, add pGCSIL-GFP-RBBP6-siRNA and RBBP6 process LAN plasmid (reaction system is in table 7), after transfection 6h, change fresh culture.After transfection 24h, fluorescence microscope, transfection efficiency (being greater than 70%), continue to cultivate 24h, collecting cell, carries out Western Blot detection.Subtract efficiency according to striking of RBBP6 albumen, screening is effectively for effective siRNA sequence of RBBP6 gene.

Table 7 plasmid co-transfection experiment group and dose design

Note: RBBP6 process LAN plasmid, adds 0.8 μ g; PGCSIL-GFP-Scr-siRNA is that RNA disturbs negative control plasmids (the same pGCSIL-GFP-RBBP6-siRNA of construction process, negative control siRNA sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ').

2. build RBBP6-siRNA slow virus:

Extract the DNA of RNA interference plasmid pGCSIL-GFP-RBBP6-siRNA with the plasmid extraction test kit of Qiagen company, be mixed with 100ng/ μ l storage liquid.24h before transfection, with the HEKC 293T cell of tryptic digestion logarithmic phase, with the DMEM perfect medium adjustment cell density containing 10% foetal calf serum for 1.5 × 10 ⁵cell/ml, is inoculated in 6 orifice plates, 37 DEG C, 5%CO ₂cultivate in incubator.Transfection is can be used for when cell density reaches 70%-80%.2h before transfection, the original substratum of sucking-off, adds the perfect medium that 1.5ml is fresh.According to the explanation of the MISSIONLentiviral Packaging Mix test kit of Sigma-aldrich company, Packing Mix (PVM) 20 μ l is added in a sterile centrifugation tube, PEI 12 μ l, plasma-free DMEM medium 400 μ l, get the plasmid DNA of the above-mentioned extracting of 20 μ l, add to above-mentioned PVM/PEI/DMEM mixed solution.Above-mentioned transfection miscellany is at room temperature hatched 15min, is transferred in the substratum of HEKC 293T cell, 37 DEG C, 5%CO ₂16h is cultivated in incubator.Discard the developing medium containing transfection miscellany, PBS solution is washed, and adds perfect medium 2ml, continues to cultivate 48h.Collecting cell supernatant liquor, Centricon Plus-20 centrifugal ultrafiltration unit (Millipore) purifying and concentrated (processing condition are in table 8) slow virus.

Table 8 slow virus purifying process condition

Embodiment 2: the silence efficiency that real-time fluorescence quantitative RT-PCR method detects RBBP6 gene is in people's colorectal carcinoma cell RKO of logarithmic phase and people's liver tumor cells SMMC-7721 and carries out trysinization, and (cell count is about 5 × 10 to make cell suspension ⁴/ ml) be inoculated in 6 orifice plates, be cultured to cytogamy degree and reach about 30%.According to infecting plural number (MOI) value, (the MOI value of SMMC-7721 cell is 20 to add the virus of sufficient quantity; The MOI value of RKO cell is 10), replaced medium after cultivation 24h, after time of infection reaches 5 days, collecting cell.According to the Trizol process specifications of Invitrogen company, extracted total RNA.According to the M-MLV process specifications of Promega company, RNA reverse transcription is obtained cDNA (reverse transcription reaction system is in table 9).The primer sequence detecting RBBP6 gene is: upstream primer: 5 '-CCAGCGATGGCAACTAC-3 ' and downstream primer: 5 '-ACCACAACGGAAACACG-3 '.By the proportional arrangement reaction system in table 10.Setting program is two-step approach Real-time PCR: denaturation 95 DEG C, 15s; Each step sex change 95 DEG C afterwards, 5s; Annealing extension 60 DEG C, 30s; Carry out 45 circulations altogether.Read light absorption value in the extension stage at every turn.After PCR terminates, 95 DEG C of sex change 1min, are then cooled to 55 DEG C, and DNA double chain is fully combined.To 95 DEG C from 55 DEG C, each walks increase by 0.5 DEG C, keeps 4s, reads light absorption value simultaneously, makes melting curve.Adopt 2-Δ Δ Ct analytical method to calculate and infect the gene expression abundance of RBBP6mRNA.Infect the cell of contrast virus (Lv-Scr-siRNA) in contrast.Experimental result (Fig. 3 and Fig. 4) shows, RKO cell infects RBBP6 expression level in group with SMMC-7721 cell Lv-RBBP6-siRNA slow virus and have dropped 39.3% and 79.6% respectively compared with control group.

Table 9 reverse transcription reaction system

Reagent	Volume (μ l)
		5×RT buffer	4
10mM dNTPs	2

RNasin	0.5
		M-MLV-RTase	1
DEPC H 2O	3.5
		Total	10

Note: above-mentioned system is at 42 DEG C of reaction 1h, and then water-bath 10min makes reversed transcriptive enzyme inactivation in 70 DEG C of water-baths.

Table 10Real-time PCR reaction system

Reagent	Volume (μ l)
		SYBR premix ex taq	10
Upstream primer (2.5 μMs)	0.5
		Downstream primer (2.5 μMs)	0.5
cDNA	1
		Water	8
Total	20

Embodiment 3: detect the multiplication capacity infecting the tumour cell of RBBP6-siRNA slow virus

The people's colorectal carcinoma cell RKO and the people's liver tumor cells SMMC-7721 that are in logarithmic phase carry out trysinization, and (cell count is about 5 × 10 to make cell suspension ⁴/ ml) be inoculated in 6 orifice plates, be cultured to cytogamy degree and reach about 30%.According to infecting plural number (MOI) value, (the MOI value of SMMC-7721 cell is 20 to add the virus of sufficient quantity; The MOI value of RKO cell is 10), replaced medium after cultivation 24h, after time of infection reaches 5 days, collecting cell.The resuspended one-tenth cell suspension (2 × 10 of perfect medium ⁴/ ml), be about 2000/hole with cell density, inoculate 96 orifice plates.Often organize 5 multiple holes, every hole 100 μ l.After completing plate, put 37 DEG C, 5%CO ₂incubator is cultivated.From after bed board second day, every day was detected with Cellomics instrument (Thermo Fisher) and reads plate once, and continuous detecting reads plate 5 days.By adjusting the input parameter of Cellomicsarrayscan, calculating the quantity of the cell of the band green fluorescence in each scanning orifice plate exactly, statistics being carried out to data and draws, draw cell proliferation curve (result as shown in Figure 5 and Figure 6).Result shows, slow virus is infected group cell proliferation rate and slows down, far below cellular control unit rate of propagation.

Claims (6)

1. the people RBBP6 gene small molecule disturbance ribonucleic acid target sequence be separated is preparing the application in antitumor drug, described application specifically by using RBBP6 gene as RNA interference medicament action target, wherein target sequence is SEQ ID NO 4, to reduce the expression level of RBBP6 gene, thus the propagation of inhibition tumor cell and the speed of growth, described tumour is selected from colorectal cancer.

2. apply as claimed in claim 1, it is characterized in that, the nucleic acid construct that described application is specially a kind of people RBBP6 gene small molecule disturbance ribonucleic acid target sequence of separation is preparing the application in antitumor drug.

3. the slow virus comprising a kind of nucleic acid construct of people RBBP6 gene small molecule disturbance ribonucleic acid target sequence of separation is preparing the application in antitumor drug, and described tumour is selected from colorectal cancer, and wherein said target sequence is SEQ ID NO 4.

4. a small molecule disturbance ribonucleic acid is preparing the application in antitumor drug, described tumour is selected from colorectal cancer, described small molecule disturbance ribonucleic acid its comprise just RNA fragment and antisense RNA fragment, described just RNA fragment is as shown in SEQ ID NO4, described just RNA fragment and described antisense RNA fragment can form double-stranded RNA, and this double-stranded RNA can suppress the expression of people RBBP6 gene.

5. apply as claimed in claim 4, wherein, described just RNA fragment and antisense RNA fragment are present on two different RNA chains or are present on same RNA chain.

6. apply as claimed in claim 5, wherein, described Yeast Nucleic Acid is hair clip type single strand RNA molecule, and the complementary region wherein between just RNA fragment and antisense RNA fragment forms double-stranded region.