CN110787181A - Group of miRNA and application thereof in biological targeted therapy of breast cancer - Google Patents

Abstract

The invention relates to a group of miRNA and application thereof in biological targeted therapy of breast cancer. The miRNA comprises: miR-9-5p, miR-10b-5p, miR-21-5p, miR-23a-3p, miR-29a-3p, miR-155-5p, miR-222-3p, miR-301a-3p and miR-373-3 p. Interfering the expression and function of the miRNA, and inhibiting the proliferation and transfer activity of TNBC and non-TNBC cells; meanwhile, the function of the 9 miRNAs is interfered, the proliferation activity of TNBC and non-TNBC cancer cells can be obviously inhibited, and the tumor growth of a TNBC cell nude mouse transplantation tumor model is obviously inhibited. The invention provides a new candidate therapeutic strategy for breast cancer, particularly highly malignant refractory TNBC.

Description

Group of miRNA and application thereof in biological targeted therapy of breast cancer

Technical Field

The invention relates to the technical field of biomedicine and molecular biology, in particular to a group of miRNA and application thereof in biological targeted therapy of breast cancer.

Background

Breast cancer is a leading cause of cancer death in women. In the last decade, due to the combination of chemotherapy, endocrine therapy and the exploration and application of Her-2 targeted therapy, the curative effect of clinical treatment of breast cancer is obviously improved, and the death rate is reduced year by year. However, one type of breast cancer is very specific, accounting for about 15-20%, and is characterized by the fact that cancer cells do not express three markers, Estrogen Receptor (ER), Progesterone Receptor (PR), and human epidermal growth factor receptor 2 (HER-2), and is therefore called Triple Negative Breast Cancer (TNBC). TNBC is not sensitive to hormonal therapy and no targeted drugs are currently available, so cytotoxic chemotherapy is the only treatment option for TNBC. Overall, TNBC patients have poor clinical treatment efficacy and poor overall prognosis compared to other breast cancer subtypes, and there is a great need to develop effective treatment methods.

miRNAs have small molecules, large quantity and wide distribution and are important core components in a gene regulation network. A considerable number of human genes are regulated by miRNAs and are closely related to the occurrence and development of a plurality of major diseases (such as tumors, AIDS, cardiovascular system diseases, nervous system diseases and the like) of human beings.

At present, a large number of documents show that miRNAs promote or inhibit the growth and differentiation of tumor cells in breast cancer, and have certain clinical application potential in the aspects of gene diagnosis and treatment of breast cancer and guidance of breast cancer personalized medicine. For example: patent document CN109439748A, published Japanese 2019.03.08, discloses the application of miR-9-5p as a breast cancer distant metastasis monitoring marker. The invention proves that the expression level of miR-9-5p in a breast cancer patient is obviously increased and can be secreted into serum, the miR-9-5p level in the serum is closely related to the distant metastasis of the breast cancer, and the serum in the patient with the distant metastasis of the breast cancer is obviously increased compared with the serum in the patient with the non-distant metastasis of the breast cancer. In 2018, the university of south China university Master academic thesis, "screening and verification of early diagnosis microRNA markers for breast cancer based on big data", downloading high-throughput microRNA-seq data (including expression information of 2589 microRNAs in total) of 409 early breast cancer tissue samples with clinical pathology staging information and 7 paracancer samples serving as healthy controls from a TCGA cancer database, removing microRNAs with data deletion rates larger than 10%, and further preliminarily screening 20 candidate microRNAs capable of serving as early diagnosis markers according to expression difference analysis results, wherein p-value <0.05, expression change multiple | fold-change | >3.5 and average expression abundance mean _ C >100 of samples in a control group: miR-99a-5p, miR-486-5p, miR-451a, miR-378a-5p, miR-378a-3p, miR-375, miR-337-3p, miR-21-5p, miR-21-3p, miR-203a-3p, miR-200a-5p, miR-183-5p, miR-182-5p, miR-145-5p, miR-144-5p, miR-141-3p, miR-139-5p, miR-125b-5p and miR-10b-5 p. Si and the like (SI M L, ZHU S, WU H, et al, miR-21-mediated tumor growth [ J ]. Oncogene, 2007, 26(19):2799-2803.) designs that antisense oligonucleotides aiming at miRNA-21 are transferred into a breast cancer cell strain MCF-7, and the cell growth is obviously inhibited. However, determining the key miRNAs and constructing efficient miRNAs-based breast cancer drugs, particularly drugs aiming at refractory breast cancer TNBC, are in need of research.

Disclosure of Invention

The invention aims to provide a miRNA for biological targeted therapy of breast cancer and application thereof, aiming at the defects in the prior art.

In a first aspect, the present invention provides an application of a miRNA combination or an inhibitor of a miRNA combination in the preparation of a medicament for treating breast cancer, the miRNA combination comprising: miR-9-5p, miR-10b-5p, miR-21-5p, miR-23a-3p, miR-29a-3p, miR-155-5p, miR-222-3p, miR-301a-3p and miR-373-3 p.

As a preferred example, the inhibitor of the miRNA combination is a substance that decreases the expression level, activity, stability or effective duration of action of each miRNA in the miRNA combination.

More preferably, the inhibitor of the miRNA combination is selected from:

a) a nucleic acid inhibitor, protein inhibitor, nuclease or nucleic acid binding molecule;

b) a plasmid vector carrying or expressing any one of a); or

c) A host carrying or expressing any one of a).

More preferably, the inhibitor of the miRNA combination is selected from:

a) the method comprises the following steps of (1) interfering LncRNA, wherein a sequence for coding the interfering LncRNA is n copies of a sequence shown in 16-189bp in SEQ ID NO.10, and n is an integer more than or equal to 1;

b) expressing the plasmid vector of a); or

c) Carrying the virus of a).

As another preferred example, the breast cancer is a triple negative breast cancer.

In a second aspect, the invention provides an interfering LncRNA, wherein the sequence coding for the LncRNA is n copies of the sequence shown as 16-189bp in SEQ id No.10, and n is an integer greater than or equal to 1.

In a third aspect, the present invention provides a nucleic acid molecule encoding LncRNA as described above.

As a preferred example, the nucleic acid molecule comprises a sequence shown as 16-189bp in SEQ ID NO. 10.

In a fourth aspect, the present invention provides a virus having integrated into its genome an expression cassette expressing LncRNA as described above.

The invention has the advantages that:

1. according to the invention, through miRNA Microarray and qRT-PCR screening of breast cancer tissues and breast cancer cell lines, 9 high-expression Oncorirs (miR-9-5p, miR-10b-5p, miR-21-5p, miR-23a-3p, miR-29a-3p, miR-155-5p, miR-222-3p, miR-301a-3p and miR-373-3p) related to TNBC are determined in miRNA of breast cancer differential expression. Representative target genes (PTEN, PDCD4, FOXO3a, RhoA, TRPS1) were also predicted by the miRanda-mirSVR database, literature reports and mRNA Microarray analysis and confirmed in cytological experiments. Interfering the expression and function of the OncomiR can up-regulate the target gene to express the cancer suppressor and inhibit the proliferation and transfer activity of TNBC and non-TNBC cells.

2. The invention designs an interference lncRNA (lncRNAi9) artificially, expresses with high copy and high efficiency under the mediation of a tumor specific proliferation adenovirus vector, and can simultaneously interfere 9 OncoriR functions, thereby establishing a TNBC treatment strategy. The tumor specific proliferation adenovirus AdSVP-lncRNAi9 infects breast cancer cells and proliferates and replicates in the cells at high copy number, the maximum copy number can reach more than 5 ten thousand times, the expressed interference lncRNA (lncRNAi9) forms a spongy net frame structure in the cells, competitively combines a plurality of OncoriR to inhibit the function, protects all target genes, enables the target genes to express cancer suppressor factors, and plays a role in resisting cancer. Cell experiments prove that in TNBC and non-TNBC cancer cells infected by AdSVP-lncRNAi9, the expression levels of OncomiR target genes PTEN, PDCD4, FOXO3a, RhoA and TRPS1 are obviously up-regulated, the EMT of the cancer cells is reversed, and the proliferation activity of the cancer cells is obviously inhibited. The curative effect of the TNBC treatment strategy is verified by establishing a nude mouse transplantation tumor model of TNBC cells. As a result, the AdSVP-lncRNAi9 has obvious anticancer effect, and the tumor inhibition rate is 73.84% 35 days after the treatment of the transplanted tumor.

Drawings

FIG. 1. differences in expression of OncomiRs in TNBC and non-TNBC cell lines. .

FIGS. 2 and 3. inhibition of Oncorirs function to reverse the course of EMT in breast cancer cells.

FIG. 4 inhibition of Oncorirs function inhibits the proliferative activity of breast cancer cells.

FIG. 5 proliferation of AdSVP-lncRNAi9 virus interfering with Oncorirs function and its inhibition of proliferative activity in breast cancer cells.

Figure 6. TNBC treatment strategies interfering with OncomiRs function an observation of the efficacy of TNBC animal models.

FIG. 7.pENTR11dual shuttle vector map.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1

Materials and methods

1.MiRNA expression profile chip detection for breast cancer tissues

8 breast cancer tissue samples of clinical operation of a hospital in 11 months in 2014 1-2014 were taken, and 3 breast fibroadenoma samples of surgical excision were taken, and normal lobular tissue was taken as a control beside the tumor. Extracting total RNA from a part of fresh tissues, and preparing the total RNA as miRNA Microarray; the other part of the tissue is subjected to conventional fixing, embedding and flaking and is prepared for ER, PR and HER2 immunohistochemistry. All patients do not undergo radiotherapy, chemotherapy, endocrine treatment and targeted treatment before operation, and all cases have complete clinical pathological data.

2. qRT-PCR detection of breast cancer cell line miRNA expression

TNBC cell line MDA-MB-231[ ER-, PR-, HER2/Neu-, EGFR +, TGF- α receptor + and WNT7B oncogene + ], MDA-MB-468[ ER-, PR-, HER2/Neu-, EGFR + +, and p53mutant R2073H ], and MCF-7[ ER (+), PR (+), HER-2(-), WNT7B oncogene expressing, SK-BR-3[ HER2 overexpressing ], as well as normal mammary duct epithelial cell line MCF10A.Western Blotting identified ER, PR, HER2 expression, miRNA expression was detected using qRT-PCR (miR-9-5p, miR-10b-5p, miR-21-5p, miR-23a-3p, miR-29a-3p, miR-155-5p, miR-222-3p, miR-301a-3p, miR-373-3 p).

Western Blotting to detect the influence of Oncorirs inhibitor on the gene expression of breast cancer cells

MDA-MB-231, MDA-MB-468, MCF-7, SK-BR-3 and MCF10A cells were plated in 96-well plates at 1 × 10 per well⁴The cells were counted and allowed to adhere after 24h, the culture medium was changed, the OncomiR inhibitor (see Table 1, Shanghai GenecheCo., LTD.) was added to a final concentration of 20nM, and the culture was continued for 72 h. An inCtrl negative control group was set. Collecting cells, extracting total protein, and detecting the expression of Oncorirs target genes and EMT related markers by a Western Blotting method. And (3) analyzing and selecting representative target genes of Oncorirs according to a miRanda-mirSVR database, a literature report and miRNA Microarray experiment results.

TABLE 1 OncomiR inhibitors

CCK-8 detection of Breast cancer cell proliferative Activity

The experimental cell line was seeded in 96-well culture plates at 1X 10 per well⁴Cell number, after 24h incubation, the OncomiR inhibitor was added to a final concentration of 20nM per well, 8 replicate wells per group. Cell proliferation activity assays were performed at 24h, 48h, 72h, and 96h time points, respectively, as specified in the CellCounting Kit-8(CCK-8) Kit (Dojindo Molecular Technologies, Inc., Shanghai, China).

Transwell detection of metastatic Activity of Breast cancer cells

Transwell chambers (Corning, Tewksbury, USA) were placed in 24-well plates, the upper chamber was covered with 50. mu.l of 1:6diluted matrigel (BD Biosciences, San Jose, USA), and the experimental cell lines were added, 5X 10 per chamber⁴Cell count/100. mu.l, 500. mu.l of medium containing 10% fetal bovine serum was added to the lower chamber. After 24h incubation, OncomiR inhibitor was added to the upper chamber to a final concentration of 20nM per well. After further culturing for 48h, the cells were removed, wiped off the upper layer of the chamber, stained with 0.1% crystal violet for 15min, and counted and photographed under an optical microscope with 5 fields (x 200 times) at random.

6. Construction and cytological experiment of tumor specific proliferation virus interfering OncomiR function

We designed an open reading frame (SEQ ID NO: 10) encoding LncRNA, which contains the complementary sequence of 9 OncoriR seed sequences highly expressed in TNBC and repeats 10 copies in tandem (lncRNAi9), and inserted into EcoRI/SalI sites of pENTR11dual shuttle vector (vector map is shown in FIG. 7) of thermo Fisher company, and recombined with pAdSVPE3RC, which is an adenovirus backbone constructed by us, to obtain the required AdSVP-lncRNAi9 (the complete genome sequence of AdSVP-lncRNAi9 is shown in SEQ ID NO: 11). And simultaneously constructing a proliferative adenovirus AdSVP-EGFP and a non-proliferative adenovirus Ad5-EGFP which express a green fluorescent protein reporter gene (EGFP) gene as control viruses. All recombinant adenoviruses are amplified in HEK293 cells, the adenoviruses are purified by cesium chloride gradient centrifugation, and the virus titer is detected by a TCID50 method.

Experimental cells plated in 96-well plates, 1X 10⁴A hole, after the cells are attached to the wall, the serum-free culture solution is replaced; the experimental and control viruses were added at MOI ═ 1 pfu/cell. Changing 5% serum culture solution after 2h of virus infection, continuously culturing for 0h, 24h, 48h and 72h, collecting cells, detecting virus titer by a TCID50 method, extracting protein, detecting protein expression by a WesternBlotting method, and detecting the proliferation activity of experimental cells by a CCK-8 method.

7. Animal experiments

20 healthy and pure BALB/C nude mice are 5 weeks old, female, provided by Shanghai Si Laike laboratory animal center in Chinese academy of sciences, and have the qualification number SCXK (Shanghai) 2018-. Injecting MDA-MB-231 cell suspension in logarithmic growth phase into the subcutaneous tissue of the right axilla of the nude mouse at 1 × 10⁷The number of cells was 100. mu.l/cell. The tumor formation rate is 100% 14 days after inoculation, and the diameter of the transplanted tumor is about 0.5-0.8 cm. The groups were randomly divided into 4 groups (AdSVP-lncRNAi9, AdSVP-EGFP, Ad5-EGFP, blank control group) of 5 individuals each. The virus treatment group was given corresponding adenovirus intratumoral multiple injections, each at 2X 10 doses⁸pfu/100. mu.l once every other day for 5 times; the blank control group was injected with 100. mu.l each of virus stock solution simultaneously. After treatment, the tumor size is measured by regular observation and measurement, and the maximum diameter is multiplied by the minimum diameter²And (5) calculating the volume of the tumor body by a multiplied by 0.5' formula, and drawing a tumor growth curve.

After termination of the experimental observations, mice were euthanized, tumor bodies were fixed in 10% neutral buffered formalin, and paraffin-embedded sections were used for immunohistochemical detection of the expression of oncocomir representative target genes (PTEN, PDCD4), EMT markers (E-cadherin, Vimentin), and the expression level of EpCAM. The number of positive cells in 5 fields was counted under a 20-fold objective lens for each section and analyzed statistically.

Second, result in

1. Differential expression profiles of miRNAs in breast cancer tissue

8 breast cancer patients were identified by immunohistochemistry, 4 were TNBC, 2 were all positive for ER, PR, HER2, 1 was positive for ER, HER2, PR, and 1 was negative for ER, PR, and HER 2. By miRNA microarray profile detection, compared with normal breast tissues, 24 miRNA are highly expressed and 13 miRNA are lowly expressed in 8 breast cancer tissues. In 4 cases of TNBC, 7 miRNAs are highly expressed and 29 miRNAs are lowly expressed in TNBC compared with 4 cases of non-TNBC.

2. Expression of miRNAs in breast cancer cell lines

Western Blotting identified that MDA-MB-231 and MDA-MB-468 are confirmed to be TNBC, MCF-7 cell ER (+)/PR (+)/Her-2(-), SK-BR-3 cell ER (-)/PR (-)/Her-2(+), MCF10A cell ER (-)/PR (-)/Her-2 (+/-). According to qRT-PCR identification, 5 of miRNA (miR-10b-5p, miR-21-5p, miR-29a-3p, miR-222-3p and miR-301a-3p) are expressed in TNBC cell lines MDA-MB-231 and MDA-MB-468 higher than the expression levels of the miRNA in MCF-7 and SK-BR-3 (figure 1).

3. Inhibition of Oncorirs expression reverses the EMT process in TNBC cells

MDA-MB-231, MDA-MB-468, MCF-7, SK-BR-3, MCF10A cells were treated with 9 OncomiR inhibitors (negative controls inCtrl, inhibitor control), respectively, and it was found that miR-21-5p inhibitors increased PTEN and PDCD4 expression, miR-155-5p inhibitors increased FOXO3a, RhoA expression, miR-222-3p and miR-373-3p inhibitors increased TRPS1 expression, and miR-301a-3p and miR-10b-5p inhibitors increased PTEN expression (FIG. 2). All Oncorirs inhibitors were able to increase E-cadherin expression and decrease Vimentin expression (FIG. 3). All OncomiRs inhibitors had no significant effect on the expression of normal mammary duct epithelial cell MCF10A gene.

4. Interference with Oncomirs function in inhibiting proliferation of breast cancer cells

To verify the effect of multiple OncomiR inhibitors on breast cancer cell proliferative activity, we performed cell proliferative activity assays using the CCK-8 assay. Compared with the parental cells, 9 OncomiR inhibitors can reduce the survival rate of 4 breast cancer cell lines, but have no effect on MCF10A cells. Wherein the inhibition effect of the inhibitor of miR-10b-5p, miR-21-5p, miR-29a-3p, miR-301a-3p and miR-373-3p on the proliferation of TNBC cells is stronger than that of non-TNBC cells, and the inhibition effect of the inhibitor of miR-23a-3p and miR-155-5p on the non-TNBC cells is stronger than that of the inhibitor of the non-TNBC cells (figure 4). The control inCtrl had no effect on all cell proliferation activities.

5. TNBC treatment strategy that interferes with OncomiRs function

The function of 9 Oncorirs can be simultaneously interfered by constructing tumor-specific adenovirus AdSVP-lncRNAi9 and expressing artificially designed LncRNA (lncRNAi 9). After cells are infected by the AdSVP-lncRNAi9 and the control virus, virus titer detection shows that the AdSVP-lncRNAi9 and the AdSVP-EGFP are highly replicated in breast cancer cell lines, and the replication is up to more than 5 ten thousand times, but is below 50 times in MCF10A (A in FIG. 5). The proliferation activity of the AdSVP-lncRNAi 9-infected cancer cells is obviously inhibited, and the survival rate of the AdSVP-EGFP cancer cells is reduced to a certain extent (B in figure 5). AdSVP-lncRNAi9 and AdSVP-EGFP have no obvious influence on the survival rate of MCF10A, and Ad5-EGFP has no influence on the proliferation of all cells.

Anticancer therapy of AdSVP-lncRNAi9 against TNBC nude mouse transplanted tumor

After the nude mice MDA-MB-231 are transplanted with tumor and become tumor, experimental adenovirus and control adenovirus are given for treatment. The result of 28 days of observation shows that AdSVP-lncRNAi9 has obvious anticancer effect and the tumor inhibition rate reaches 81.6%. While AdSVP-EGFP also had some inhibitory effect, Ad5-EGFP had no effect on tumor growth (A in FIG. 6). Immunohistochemical detection revealed that AdSVP-lncRNAi9 treatment group E-cadherin up-regulated expression, Vimentin down-regulated expression (B in FIG. 6), and PTEN and PDCD4 all up-regulated expression (C in FIG. 6).

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> second people hospital in Hefei city

<120> a group of miRNA and application thereof in biological targeted therapy of breast cancer

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gagcctgagc ccgagccaga accggagcct gcaagaccta cccgccgtcc taaaatggcg 2160

cctgctatcc tgagacgccc gacatcacct gtgtctagag aatgcaatag tagtacggat 2220

agctgtgact ccggtccttc taacacacct cctgagatac acccggtggt cccgctgtgc 2280

cccattaaac cagttgccgt gagagttggt gggcgtcgcc aggctgtgga atgtatcgag 2340

gacttgctta acgagcctgg gcaacctttg gacttgagct gtaaacgccc caggccataa 2400

gtcgacgcgg ccgcaagctt ctagagttcg agcaacttgt ttattgcagc ttataatggt 2460

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 2520

agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcgt ctagcatcga 2580

agatccaata acttcgtata gcatacatta tacgaagtta taagtactga attcggatct 2640

gggcgtggtt aagggtggga aagaatatat aaggtggggg tcttatgtag ttttgtatct 2700

gttttgcagc agccgccgcc gccatgagca ccaactcgtt tgatggaagc attgtgagct 2760

catatttgac aacgcgcatg cccccatggg ccggggtgcg tcagaatgtg atgggctcca 2820

gcattgatgg tcgccccgtc ctgcccgcaa actctactac cttgacctac gagaccgtgt 2880

ctggaacgcc gttggagact gcagcctccg ccgccgcttc agccgctgca gccaccgccc 2940

gcgggattgt gactgacttt gctttcctga gcccgcttgc aagcagtgca gcttcccgtt 3000

catccgcccg cgatgacaag ttgacggctc ttttggcaca attggattct ttgacccggg 3060

aacttaatgt cgtttctcag cagctgttgg atctgcgcca gcaggtttct gccctgaagg 3120

cttcctcccc tcccaatgcg gtttaaaaca taaataaaaa accagactct gtttggattt 3180

ggatcaagca agtgtcttgc tgtctttatt taggggtttt gcgcgcgcgg taggcccggg 3240

accagcggtc tcggtcgttg agggtcctgt gtattttttc caggacgtgg taaaggtgac 3300

tctggatgtt cagatacatg ggcataagcc cgtctctggg gtggaggtag caccactgca 3360

gagcttcatg ctgcggggtg gtgttgtaga tgatccagtc gtagcaggag cgctgggcgt 3420

ggtgcctaaa aatgtctttc agtagcaagc tgattgccag gggcaggccc ttggtgtaag 3480

tgtttacaaa gcggttaagc tgggatgggt gcatacgtgg ggatatgaga tgcatcttgg 3540

actgtatttt taggttggct atgttcccag ccatatccct ccggggattc atgttgtgca 3600

gaaccaccag cacagtgtat ccggtgcact tgggaaattt gtcatgtagc ttagaaggaa 3660

atgcgtggaa gaacttggag acgcccttgt gacctccaag attttccatg cattcgtcca 3720

taatgatggc aatgggccca cgggcggcgg cctgggcgaa gatatttctg ggatcactaa 3780

cgtcatagtt gtgttccagg atgagatcgt cataggccat ttttacaaag cgcgggcgga 3840

gggtgccaga ctgcggtata atggttccat ccggcccagg ggcgtagtta ccctcacaga 3900

tttgcatttc ccacgctttg agttcagatg gggggatcat gtctacctgc ggggcgatga 3960

agaaaacggt ttccggggta ggggagatca gctgggaaga aagcaggttc ctgagcagct 4020

gcgacttacc gcagccggtg ggcccgtaaa tcacacctat taccgggtgc aactggtagt 4080

taagagagct gcagctgccg tcatccctga gcaggggggc cacttcgtta agcatgtccc 4140

tgactcgcat gttttccctg accaaatccg ccagaaggcg ctcgccgccc agcgatagca 4200

gttcttgcaa ggaagcaaag tttttcaacg gtttgagacc gtccgccgta ggcatgcttt 4260

tgagcgtttg accaagcagt tccaggcggt cccacagctc ggtcacctgc tctacggcat 4320

ctcgatccag catatctcct cgtttcgcgg gttggggcgg ctttcgctgt acggcagtag 4380

tcggtgctcg tccagacggg ccagggtcat gtctttccac gggcgcaggg tcctcgtcag 4440

cgtagtctgg gtcacggtga aggggtgcgc tccgggctgc gcgctggcca gggtgcgctt 4500

gaggctggtc ctgctggtgc tgaagcgctg ccggtcttcg ccctgcgcgt cggccaggta 4560

gcatttgacc atggtgtcat agtccagccc ctccgcggcg tggcccttgg cgcgcagctt 4620

gcccttggag gaggcgccgc acgaggggca gtgcagactt ttgagggcgt agagcttggg 4680

cgcgagaaat accgattccg gggagtaggc atccgcgccg caggccccgc agacggtctc 4740

gcattccacg agccaggtga gctctggccg ttcggggtca aaaaccaggt ttcccccatg 4800

ctttttgatg cgtttcttac ctctggtttc catgagccgg tgtccacgct cggtgacgaa 4860

aaggctgtcc gtgtccccgt atacagactt gagaggcctg tcctcgagcg gtgttccgcg 4920

gtcctcctcg tatagaaact cggaccactc tgagacaaag gctcgcgtcc aggccagcac 4980

gaaggaggct aagtgggagg ggtagcggtc gttgtccact agggggtcca ctcgctccag 5040

ggtgtgaaga cacatgtcgc cctcttcggc atcaaggaag gtgattggtt tgtaggtgta 5100

ggccacgtga ccgggtgttc ctgaaggggg gctataaaag ggggtggggg cgcgttcgtc 5160

ctcactctct tccgcatcgc tgtctgcgag ggccagctgt tggggtgagt actccctctg 5220

aaaagcgggc atgacttctg cgctaagatt gtcagtttcc aaaaacgagg aggatttgat 5280

attcacctgg cccgcggtga tgcctttgag ggtggccgca tccatctggt cagaaaagac 5340

aatctttttg ttgtcaagct tggtggcaaa cgacccgtag agggcgttgg acagcaactt 5400

ggcgatggag cgcagggttt ggtttttgtc gcgatcggcg cgctccttgg ccgcgatgtt 5460

tagctgcacg tattcgcgcg caacgcaccg ccattcggga aagacggtgg tgcgctcgtc 5520

gggcaccagg tgcacgcgcc aaccgcggtt gtgcagggtg acaaggtcaa cgctggtggc 5580

tacctctccg cgtaggcgct cgttggtcca gcagaggcgg ccgcccttgc gcgagcagaa 5640

tggcggtagg gggtctagct gcgtctcgtc cggggggtct gcgtccacgg taaagacccc 5700

gggcagcagg cgcgcgtcga agtagtctat cttgcatcct tgcaagtcta gcgcctgctg 5760

ccatgcgcgg gcggcaagcg cgcgctcgta tgggttgagt gggggacccc atggcatggg 5820

gtgggtgagc gcggaggcgt acatgccgca aatgtcgtaa acgtagaggg gctctctgag 5880

tattccaaga tatgtagggt agcatcttcc accgcggatg ctggcgcgca cgtaatcgta 5940

tagttcgtgc gagggagcga ggaggtcggg accgaggttg ctacgggcgg gctgctctgc 6000

tcggaagact atctgcctga agatggcatg tgagttggat gatatggttg gacgctggaa 6060

gacgttgaag ctggcgtctg tgagacctac cgcgtcacgc acgaaggagg cgtaggagtc 6120

gcgcagcttg ttgaccagct cggcggtgac ctgcacgtct agggcgcagt agtccagggt 6180

ttccttgatg atgtcatact tatcctgtcc cttttttttc cacagctcgc ggttgaggac 6240

aaactcttcg cggtctttcc agtactcttg gatcggaaac ccgtcggcct ccgaacggta 6300

agagcctagc atgtagaact ggttgacggc ctggtaggcg cagcatccct tttctacggg 6360

tagcgcgtat gcctgcgcgg ccttccggag cgaggtgtgg gtgagcgcaa aggtgtccct 6420

gaccatgact ttgaggtact ggtatttgaa gtcagtgtcg tcgcatccgc cctgctccca 6480

gagcaaaaag tccgtgcgct ttttggaacg cggatttggc agggcgaagg tgacatcgtt 6540

gaagagtatc tttcccgcgc gaggcataaa gttgcgtgtg atgcggaagg gtcccggcac 6600

ctcggaacgg ttgttaatta cctgggcggc gagcacgatc tcgtcaaagc cgttgatgtt 6660

gtggcccaca atgtaaagtt ccaagaagcg cgggatgccc ttgatggaag gcaatttttt 6720

aagttcctcg taggtgagct cttcagggga gctgagcccg tgctctgaaa gggcccagtc 6780

tgcaagatga gggttggaag cgacgaatga gctccacagg tcacgggcca ttagcatttg 6840

caggtggtcg cgaaaggtcc taaactggcg acctatggcc attttttctg gggtgatgca 6900

gtagaaggta agcgggtctt gttcccagcg gtcccatcca aggttcgcgg ctaggtctcg 6960

cgcggcagtc actagaggct catctccgcc gaacttcatg accagcatga agggcacgag 7020

ctgcttccca aaggccccca tccaagtata ggtctctaca tcgtaggtga caaagagacg 7080

ctcggtgcga ggatgcgagc cgatcgggaa gaactggatc tcccgccacc aattggagga 7140

gtggctattg atgtggtgaa agtagaagtc cctgcgacgg gccgaacact cgtgctggct 7200

tttgtaaaaa cgtgcgcagt actggcagcg gtgcacgggc tgtacatcct gcacgaggtt 7260

gacctgacga ccgcgcacaa ggaagcagag tgggaatttg agcccctcgc ctggcgggtt 7320

tggctggtgg tcttctactt cggctgcttg tccttgaccg tctggctgct cgaggggagt 7380

tacggtggat cggaccacca cgccgcgcga gcccaaagtc cagatgtccg cgcgcggcgg 7440

tcggagcttg atgacaacat cgcgcagatg ggagctgtcc atggtctgga gctcccgcgg 7500

cgtcaggtca ggcgggagct cctgcaggtt tacctcgcat agacgggtca gggcgcgggc 7560

tagatccagg tgatacctaa tttccagggg ctggttggtg gcggcgtcga tggcttgcaa 7620

gaggccgcat ccccgcggcg cgactacggt accgcgcggc gggcggtggg ccgcgggggt 7680

gtccttggat gatgcatcta aaagcggtga cgcgggcgag cccccggagg tagggggggc 7740

tccggacccg ccgggagagg gggcaggggc acgtcggcgc cgcgcgcggg caggagctgg 7800

tgctgcgcgc gtaggttgct ggcgaacgcg acgacgcggc ggttgatctc ctgaatctgg 7860

cgcctctgcg tgaagacgac gggcccggtg agcttgagcc tgaaagagag ttcgacagaa 7920

tcaatttcgg tgtcgttgac ggcggcctgg cgcaaaatct cctgcacgtc tcctgagttg 7980

tcttgatagg cgatctcggc catgaactgc tcgatctctt cctcctggag atctccgcgt 8040

ccggctcgct ccacggtggc ggcgaggtcg ttggaaatgc gggccatgag ctgcgagaag 8100

gcgttgaggc ctccctcgtt ccagacgcgg ctgtagacca cgcccccttc ggcatcgcgg 8160

gcgcgcatga ccacctgcgc gagattgagc tccacgtgcc gggcgaagac ggcgtagttt 8220

cgcaggcgct gaaagaggta gttgagggtg gtggcggtgt gttctgccac gaagaagtac 8280

ataacccagc gtcgcaacgt ggattcgttg atatccccca aggcctcaag gcgctccatg 8340

gcctcgtaga agtccacggc gaagttgaaa aactgggagt tgcgcgccga cacggttaac 8400

tcctcctcca gaagacggat gagctcggcg acagtgtcgc gcacctcgcg ctcaaaggct 8460

acaggggcct cttcttcttc ttcaatctcc tcttccataa gggcctcccc ttcttcttct 8520

tctggcggcg gtgggggagg ggggacacgg cggcgacgac ggcgcaccgg gaggcggtcg 8580

acaaagcgct cgatcatctc cccgcggcga cggcgcatgg tctcggtgac ggcgcggccg 8640

ttctcgcggg ggcgcagttg gaagacgccg cccgtcatgt cccggttatg ggttggcggg 8700

gggctgccat gcggcaggga tacggcgcta acgatgcatc tcaacaattg ttgtgtaggt 8760

actccgccgc cgagggacct gagcgagtcc gcatcgaccg gatcggaaaa cctctcgaga 8820

aaggcgtcta accagtcaca gtcgcaaggt aggctgagca ccgtggcggg cggcagcggg 8880

cggcggtcgg ggttgtttct ggcggaggtg ctgctgatga tgtaattaaa gtaggcggtc 8940

ttgagacggc ggatggtcga cagaagcacc atgtccttgg gtccggcctg ctgaatgcgc 9000

aggcggtcgg ccatgcccca ggcttcgttt tgacatcggc gcaggtcttt gtagtagtct 9060

tgcatgagcc tttctaccgg cacttcttct tctccttcct cttgtcctgc atctcttgca 9120

tctatcgctg cggcggcggc ggagtttggc cgtaggtggc gccctcttcc tcccatgcgt 9180

gtgaccccga agcccctcat cggctgaagc agggctaggt cggcgacaac gcgctcggct 9240

aatatggcct gctgcacctg cgtgagggta gactggaagt catccatgtc cacaaagcgg 9300

tggtatgcgc ccgtgttgat ggtgtaagtg cagttggcca taacggacca gttaacggtc 9360

tggtgacccg gctgcgagag ctcggtgtac ctgagacgcg agtaagccct cgagtcaaat 9420

acgtagtcgt tgcaagtccg caccaggtac tggtatccca ccaaaaagtg cggcggcggc 9480

tggcggtaga ggggccagcg tagggtggcc ggggctccgg gggcgagatc ttccaacata 9540

aggcgatgat atccgtagat gtacctggac atccaggtga tgccggcggc ggtggtggag 9600

gcgcgcggaa agtcgcggac gcggttccag atgttgcgca gcggcaaaaa gtgctccatg 9660

gtcgggacgc tctggccggt caggcgcgcg caatcgttga cgctctaccg tgcaaaagga 9720

gagcctgtaa gcgggcactc ttccgtggtc tggtggataa attcgcaagg gtatcatggc 9780

ggacgaccgg ggttcgagcc ccgtatccgg ccgtccgccg tgatccatgc ggttaccgcc 9840

cgcgtgtcga acccaggtgt gcgacgtcag acaacggggg agtgctcctt ttggcttcct 9900

tccaggcgcg gcggctgctg cgctagcttt tttggccact ggccgcgcgc agcgtaagcg 9960

gttaggctgg aaagcgaaag cattaagtgg ctcgctccct gtagccggag ggttattttc 10020

caagggttga gtcgcgggac ccccggttcg agtctcggac cggccggact gcggcgaacg 10080

ggggtttgcc tccccgtcat gcaagacccc gcttgcaaat tcctccggaa acagggacga 10140

gccccttttt tgcttttccc agatgcatcc ggtgctgcgg cagatgcgcc cccctcctca 10200

gcagcggcaa gagcaagagc agcggcagac atgcagggca ccctcccctc ctcctaccgc 10260

gtcaggaggg gcgacatccg cggttgacgc ggcagcagat ggtgattacg aacccccgcg 10320

gcgccgggcc cggcactacc tggacttgga ggagggcgag ggcctggcgc ggctaggagc 10380

gccctctcct gagcggtacc caagggtgca gctgaagcgt gatacgcgtg aggcgtacgt 10440

gccgcggcag aacctgtttc gcgaccgcga gggagaggag cccgaggaga tgcgggatcg 10500

aaagttccac gcagggcgcg agctgcggca tggcctgaat cgcgagcggt tgctgcgcga 10560

ggaggacttt gagcccgacg cgcgaaccgg gattagtccc gcgcgcgcac acgtggcggc 10620

cgccgacctg gtaaccgcat acgagcagac ggtgaaccag gagattaact ttcaaaaaag 10680

ctttaacaac cacgtgcgta cgcttgtggc gcgcgaggag gtggctatag gactgatgca 10740

tctgtgggac tttgtaagcg cgctggagca aaacccaaat agcaagccgc tcatggcgca 10800

gctgttcctt atagtgcagc acagcaggga caacgaggca ttcagggatg cgctgctaaa 10860

catagtagag cccgagggcc gctggctgct cgatttgata aacatcctgc agagcatagt 10920

ggtgcaggag cgcagcttga gcctggctga caaggtggcc gccatcaact attccatgct 10980

tagcctgggc aagttttacg cccgcaagat ataccatacc ccttacgttc ccatagacaa 11040

ggaggtaaag atcgaggggt tctacatgcg catggcgctg aaggtgctta ccttgagcga 11100

cgacctgggc gtttatcgca acgagcgcat ccacaaggcc gtgagcgtga gccggcggcg 11160

cgagctcagc gaccgcgagc tgatgcacag cctgcaaagg gccctggctg gcacgggcag 11220

cggcgataga gaggccgagt cctactttga cgcgggcgct gacctgcgct gggccccaag 11280

ccgacgcgcc ctggaggcag ctggggccgg acctgggctg gcggtggcac ccgcgcgcgc 11340

tggcaacgtc ggcggcgtgg aggaatatga cgaggacgat gagtacgagc cagaggacgg 11400

cgagtactaa gcggtgatgt ttctgatcag atgatgcaag acgcaacgga cccggcggtg 11460

cgggcggcgc tgcagagcca gccgtccggc cttaactcca cggacgactg gcgccaggtc 11520

atggaccgca tcatgtcgct gactgcgcgc aatcctgacg cgttccggca gcagccgcag 11580

gccaaccggc tctccgcaat tctggaagcg gtggtcccgg cgcgcgcaaa ccccacgcac 11640

gagaaggtgc tggcgatcgt aaacgcgctg gccgaaaaca gggccatccg gcccgacgag 11700

gccggcctgg tctacgacgc gctgcttcag cgcgtggctc gttacaacag cggcaacgtg 11760

cagaccaacc tggaccggct ggtgggggat gtgcgcgagg ccgtggcgca gcgtgagcgc 11820

gcgcagcagc agggcaacct gggctccatg gttgcactaa acgccttcct gagtacacag 11880

cccgccaacg tgccgcgggg acaggaggac tacaccaact ttgtgagcgc actgcggcta 11940

atggtgactg agacaccgca aagtgaggtg taccagtctg ggccagacta ttttttccag 12000

accagtagac aaggcctgca gaccgtaaac ctgagccagg ctttcaaaaa cttgcagggg 12060

ctgtgggggg tgcgggctcc cacaggcgac cgcgcgaccg tgtctagctt gctgacgccc 12120

aactcgcgcc tgttgctgct gctaatagcg cccttcacgg acagtggcag cgtgtcccgg 12180

gacacatacc taggtcactt gctgacactg taccgcgagg ccataggtca ggcgcatgtg 12240

gacgagcata ctttccagga gattacaagt gtcagccgcg cgctggggca ggaggacacg 12300

ggcagcctgg aggcaaccct aaactacctg ctgaccaacc ggcggcagaa gatcccctcg 12360

ttgcacagtt taaacagcga ggaggagcgc attttgcgct acgtgcagca gagcgtgagc 12420

cttaacctga tgcgcgacgg ggtaacgccc agcgtggcgc tggacatgac cgcgcgcaac 12480

atggaaccgg gcatgtatgc ctcaaaccgg ccgtttatca accgcctaat ggactacttg 12540

catcgcgcgg ccgccgtgaa ccccgagtat ttcaccaatg ccatcttgaa cccgcactgg 12600

ctaccgcccc ctggtttcta caccggggga ttcgaggtgc ccgagggtaa cgatggattc 12660

ctctgggacg acatagacga cagcgtgttt tccccgcaac cgcagaccct gctagagttg 12720

caacagcgcg agcaggcaga ggcggcgctg cgaaaggaaa gcttccgcag gccaagcagc 12780

ttgtccgatc taggcgctgc ggccccgcgg tcagatgcta gtagcccatt tccaagcttg 12840

atagggtctc ttaccagcac tcgcaccacc cgcccgcgcc tgctgggcga ggaggagtac 12900

ctaaacaact cgctgctgca gccgcagcgc gaaaaaaacc tgcctccggc atttcccaac 12960

aacgggatag agagcctagt ggacaagatg agtagatgga agacgtacgc gcaggagcac 13020

agggacgtgc caggcccgcg cccgcccacc cgtcgtcaaa ggcacgaccg tcagcggggt 13080

ctggtgtggg aggacgatga ctcggcagac gacagcagcg tcctggattt gggagggagt 13140

ggcaacccgt ttgcgcacct tcgccccagg ctggggagaa tgttttaaaa aaaaaaaagc 13200

atgatgcaaa ataaaaaact caccaaggcc atggcaccga gcgttggttt tcttgtattc 13260

cccttagtat gcggcgcgcg gcgatgtatg aggaaggtcc tcctccctcc tacgagagtg 13320

tggtgagcgc ggcgccagtg gcggcggcgc tgggttctcc cttcgatgct cccctggacc 13380

cgccgtttgt gcctccgcgg tacctgcggc ctaccggggg gagaaacagc atccgttact 13440

ctgagttggc acccctattc gacaccaccc gtgtgtacct ggtggacaac aagtcaacgg 13500

atgtggcatc cctgaactac cagaacgacc acagcaactt tctgaccacg gtcattcaaa 13560

acaatgacta cagcccgggg gaggcaagca cacagaccat caatcttgac gaccggtcgc 13620

actggggcgg cgacctgaaa accatcctgc ataccaacat gccaaatgtg aacgagttca 13680

tgtttaccaa taagtttaag gcgcgggtga tggtgtcgcg cttgcctact aaggacaatc 13740

aggtggagct gaaatacgag tgggtggagt tcacgctgcc cgagggcaac tactccgaga 13800

ccatgaccat agaccttatg aacaacgcga tcgtggagca ctacttgaaa gtgggcagac 13860

agaacggggt tctggaaagc gacatcgggg taaagtttga cacccgcaac ttcagactgg 13920

ggtttgaccc cgtcactggt cttgtcatgc ctggggtata tacaaacgaa gccttccatc 13980

cagacatcat tttgctgcca ggatgcgggg tggacttcac ccacagccgc ctgagcaact 14040

tgttgggcat ccgcaagcgg caacccttcc aggagggctt taggatcacc tacgatgatc 14100

tggagggtgg taacattccc gcactgttgg atgtggacgc ctaccaggcg agcttgaaag 14160

atgacaccga acagggcggg ggtggcgcag gcggcagcaa cagcagtggc agcggcgcgg 14220

aagagaactc caacgcggca gccgcggcaa tgcagccggt ggaggacatg aacgatcatg 14280

ccattcgcgg cgacaccttt gccacacggg ctgaggagaa gcgcgctgag gccgaagcag 14340

cggccgaagc tgccgccccc gctgcgcaac ccgaggtcga gaagcctcag aagaaaccgg 14400

tgatcaaacc cctgacagag gacagcaaga aacgcagtta caacctaata agcaatgaca 14460

gcaccttcac ccagtaccgc agctggtacc ttgcatacaa ctacggcgac cctcagaccg 14520

gaatccgctc atggaccctg ctttgcactc ctgacgtaac ctgcggctcg gagcaggtct 14580

actggtcgtt gccagacatg atgcaagacc ccgtgacctt ccgctccacg cgccagatca 14640

gcaactttcc ggtggtgggc gccgagctgt tgcccgtgca ctccaagagc ttctacaacg 14700

accaggccgt ctactcccaa ctcatccgcc agtttacctc tctgacccac gtgttcaatc 14760

gctttcccga gaaccagatt ttggcgcgcc cgccagcccc caccatcacc accgtcagtg 14820

aaaacgttcc tgctctcaca gatcacggga cgctaccgct gcgcaacagc atcggaggag 14880

tccagcgagt gaccattact gacgccagac gccgcacctg cccctacgtt tacaaggccc 14940

tgggcatagt ctcgccgcgc gtcctatcga gccgcacttt ttgagcaagc atgtccatcc 15000

ttatatcgcc cagcaataac acaggctggg gcctgcgctt cccaagcaag atgtttggcg 15060

gggccaagaa gcgctccgac caacacccag tgcgcgtgcg cgggcactac cgcgcgccct 15120

ggggcgcgca caaacgcggc cgcactgggc gcaccaccgt cgatgacgcc atcgacgcgg 15180

tggtggagga ggcgcgcaac tacacgccca cgccgccacc agtgtccaca gtggacgcgg 15240

ccattcagac cgtggtgcgc ggagcccggc gctatgctaa aatgaagaga cggcggaggc 15300

gcgtagcacg tcgccaccgc cgccgacccg gcactgccgc ccaacgcgcg gcggcggccc 15360

tgcttaaccg cgcacgtcgc accggccgac gggcggccat gcgggccgct cgaaggctgg 15420

ccgcgggtat tgtcactgtg ccccccaggt ccaggcgacg agcggccgcc gcagcagccg 15480

cggccattag tgctatgact cagggtcgca ggggcaacgt gtattgggtg cgcgactcgg 15540

ttagcggcct gcgcgtgccc gtgcgcaccc gccccccgcg caactagatt gcaagaaaaa 15600

actacttaga ctcgtactgt tgtatgtatc cagcggcggc ggcgcgcaac gaagctatgt 15660

ccaagcgcaa aatcaaagaa gagatgctcc aggtcatcgc gccggagatc tatggccccc 15720

cgaagaagga agagcaggat tacaagcccc gaaagctaaa gcgggtcaaa aagaaaaaga 15780

aagatgatga tgatgaactt gacgacgagg tggaactgct gcacgctacc gcgcccaggc 15840

gacgggtaca gtggaaaggt cgacgcgtaa aacgtgtttt gcgacccggc accaccgtag 15900

tctttacgcc cggtgagcgc tccacccgca cctacaagcg cgtgtatgat gaggtgtacg 15960

gcgacgagga cctgcttgag caggccaacg agcgcctcgg ggagtttgcc tacggaaagc 16020

ggcataagga catgctggcg ttgccgctgg acgagggcaa cccaacacct agcctaaagc 16080

ccgtaacact gcagcaggtg ctgcccgcgc ttgcaccgtc cgaagaaaag cgcggcctaa 16140

agcgcgagtc tggtgacttg gcacccaccg tgcagctgat ggtacccaag cgccagcgac 16200

tggaagatgt cttggaaaaa atgaccgtgg aacctgggct ggagcccgag gtccgcgtgc 16260

ggccaatcaa gcaggtggcg ccgggactgg gcgtgcagac cgtggacgtt cagataccca 16320

ctaccagtag caccagtatt gccaccgcca cagagggcat ggagacacaa acgtccccgg 16380

ttgcctcagc ggtggcggat gccgcggtgc aggcggtcgc tgcggccgcg tccaagacct 16440

ctacggaggt gcaaacggac ccgtggatgt ttcgcgtttc agccccccgg cgcccgcgcg 16500

gttcgaggaa gtacggcgcc gccagcgcgc tactgcccga atatgcccta catccttcca 16560

ttgcgcctac ccccggctat cgtggctaca cctaccgccc cagaagacga gcaactaccc 16620

gacgccgaac caccactgga acccgccgcc gccgtcgccg tcgccagccc gtgctggccc 16680

cgatttccgt gcgcagggtg gctcgcgaag gaggcaggac cctggtgctg ccaacagcgc 16740

gctaccaccc cagcatcgtt taaaagccgg tctttgtggt tcttgcagat atggccctca 16800

cctgccgcct ccgtttcccg gtgccgggat tccgaggaag aatgcaccgt aggaggggca 16860

tggccggcca cggcctgacg ggcggcatgc gtcgtgcgca ccaccggcgg cggcgcgcgt 16920

cgcaccgtcg catgcgcggc ggtatcctgc ccctccttat tccactgatc gccgcggcga 16980

ttggcgccgt gcccggaatt gcatccgtgg ccttgcaggc gcagagacac tgattaaaaa 17040

caagttgcat gtggaaaaat caaaataaaa agtctggact ctcacgctcg cttggtcctg 17100

taactatttt gtagaatgga agacatcaac tttgcgtctc tggccccgcg acacggctcg 17160

cgcccgttca tgggaaactg gcaagatatc ggcaccagca atatgagcgg tggcgccttc 17220

agctggggct cgctgtggag cggcattaaa aatttcggtt ccaccgttaa gaactatggc 17280

agcaaggcct ggaacagcag cacaggccag atgctgaggg ataagttgaa agagcaaaat 17340

ttccaacaaa aggtggtaga tggcctggcc tctggcatta gcggggtggt ggacctggcc 17400

aaccaggcag tgcaaaataa gattaacagt aagcttgatc cccgccctcc cgtagaggag 17460

cctccaccgg ccgtggagac agtgtctcca gaggggcgtg gcgaaaagcg tccgcgcccc 17520

gacagggaag aaactctggt gacgcaaata gacgagcctc cctcgtacga ggaggcacta 17580

aagcaaggcc tgcccaccac ccgtcccatc gcgcccatgg ctaccggagt gctgggccag 17640

cacacacccg taacgctgga cctgcctccc cccgccgaca cccagcagaa acctgtgctg 17700

ccaggcccga ccgccgttgt tgtaacccgt cctagccgcg cgtccctgcg ccgcgccgcc 17760

agcggtccgc gatcgttgcg gcccgtagcc agtggcaact ggcaaagcac actgaacagc 17820

atcgtgggtc tgggggtgca atccctgaag cgccgacgat gcttctgaat agctaacgtg 17880

tcgtatgtgt gtcatgtatg cgtccatgtc gccgccagag gagctgctga gccgccgcgc 17940

gcccgctttc caagatggct accccttcga tgatgccgca gtggtcttac atgcacatct 18000

cgggccagga cgcctcggag tacctgagcc ccgggctggt gcagtttgcc cgcgccaccg 18060

agacgtactt cagcctgaat aacaagttta gaaaccccac ggtggcgcct acgcacgacg 18120

tgaccacaga ccggtcccag cgtttgacgc tgcggttcat ccctgtggac cgtgaggata 18180

ctgcgtactc gtacaaggcg cggttcaccc tagctgtggg tgataaccgt gtgctggaca 18240

tggcttccac gtactttgac atccgcggcg tgctggacag gggccctact tttaagccct 18300

actctggcac tgcctacaac gccctggctc ccaagggtgc cccaaatcct tgcgaatggg 18360

atgaagctgc tactgctctt gaaataaacc tagaagaaga ggacgatgac aacgaagacg 18420

aagtagacga gcaagctgag cagcaaaaaa ctcacgtatt tgggcaggcg ccttattctg 18480

gtataaatat tacaaaggag ggtattcaaa taggtgtcga aggtcaaaca cctaaatatg 18540

ccgataaaac atttcaacct gaacctcaaa taggagaatc tcagtggtac gaaactgaaa 18600

ttaatcatgc agctgggaga gtccttaaaa agactacccc aatgaaacca tgttacggtt 18660

catatgcaaa acccacaaat gaaaatggag ggcaaggcat tcttgtaaag caacaaaatg 18720

gaaagctaga aagtcaagtg gaaatgcaat ttttctcaac tactgaggcg accgcaggca 18780

atggtgataa cttgactcct aaagtggtat tgtacagtga agatgtagat atagaaaccc 18840

cagacactca tatttcttac atgcccacta ttaaggaagg taactcacga gaactaatgg 18900

gccaacaatc tatgcccaac aggcctaatt acattgcttt tagggacaat tttattggtc 18960

taatgtatta caacagcacg ggtaatatgg gtgttctggc gggccaagca tcgcagttga 19020

atgctgttgt agatttgcaa gacagaaaca cagagctttc ataccagctt ttgcttgatt 19080

ccattggtga tagaaccagg tacttttcta tgtggaatca ggctgttgac agctatgatc 19140

cagatgttag aattattgaa aatcatggaa ctgaagatga acttccaaat tactgctttc 19200

cactgggagg tgtgattaat acagagactc ttaccaaggt aaaacctaaa acaggtcagg 19260

aaaatggatg ggaaaaagat gctacagaat tttcagataa aaatgaaata agagttggaa 19320

ataattttgc catggaaatc aatctaaatg ccaacctgtg gagaaatttc ctgtactcca 19380

acatagcgct gtatttgccc gacaagctaa agtacagtcc ttccaacgta aaaatttctg 19440

ataacccaaa cacctacgac tacatgaaca agcgagtggt ggctcccggg ttagtggact 19500

gctacattaa ccttggagca cgctggtccc ttgactatat ggacaacgtc aacccattta 19560

accaccaccg caatgctggc ctgcgctacc gctcaatgtt gctgggcaat ggtcgctatg 19620

tgcccttcca catccaggtg cctcagaagt tctttgccat taaaaacctc cttctcctgc 19680

cgggctcata cacctacgag tggaacttca ggaaggatgt taacatggtt ctgcagagct 19740

ccctaggaaa tgacctaagg gttgacggag ccagcattaa gtttgatagc atttgccttt 19800

acgccacctt cttccccatg gcccacaaca ccgcctccac gcttgaggcc atgcttagaa 19860

acgacaccaa cgaccagtcc tttaacgact atctctccgc cgccaacatg ctctacccta 19920

tacccgccaa cgctaccaac gtgcccatat ccatcccctc ccgcaactgg gcggctttcc 19980

gcggctgggc cttcacgcgc cttaagacta aggaaacccc atcactgggc tcgggctacg 20040

acccttatta cacctactct ggctctatac cctacctaga tggaaccttt tacctcaacc 20100

acacctttaa gaaggtggcc attacctttg actcttctgt cagctggcct ggcaatgacc 20160

gcctgcttac ccccaacgag tttgaaatta agcgctcagt tgacggggag ggttacaacg 20220

ttgcccagtg taacatgacc aaagactggt tcctggtaca aatgctagct aactacaaca 20280

ttggctacca gggcttctat atcccagaga gctacaagga ccgcatgtac tccttcttta 20340

gaaacttcca gcccatgagc cgtcaggtgg tggatgatac taaatacaag gactaccaac 20400

aggtgggcat cctacaccaa cacaacaact ctggatttgt tggctacctt gcccccacca 20460

tgcgcgaagg acaggcctac cctgctaact tcccctatcc gcttataggc aagaccgcag 20520

ttgacagcat tacccagaaa aagtttcttt gcgatcgcac cctttggcgc atcccattct 20580

ccagtaactt tatgtccatg ggcgcactca cagacctggg ccaaaacctt ctctacgcca 20640

actccgccca cgcgctagac atgacttttg aggtggatcc catggacgag cccacccttc 20700

tttatgtttt gtttgaagtc tttgacgtgg tccgtgtgca ccggccgcac cgcggcgtca 20760

tcgaaaccgt gtacctgcgc acgcccttct cggccggcaa cgccacaaca taaagaagca 20820

agcaacatca acaacagctg ccgccatggg ctccagtgag caggaactga aagccattgt 20880

caaagatctt ggttgtgggc catatttttt gggcacctat gacaagcgct ttccaggctt 20940

tgtttctcca cacaagctcg cctgcgccat agtcaatacg gccggtcgcg agactggggg 21000

cgtacactgg atggcctttg cctggaaccc gcactcaaaa acatgctacc tctttgagcc 21060

ctttggcttt tctgaccagc gactcaagca ggtttaccag tttgagtacg agtcactcct 21120

gcgccgtagc gccattgctt cttcccccga ccgctgtata acgctggaaa agtccaccca 21180

aagcgtacag gggcccaact cggccgcctg tggactattc tgctgcatgt ttctccacgc 21240

ctttgccaac tggccccaaa ctcccatgga tcacaacccc accatgaacc ttattaccgg 21300

ggtacccaac tccatgctca acagtcccca ggtacagccc accctgcgtc gcaaccagga 21360

acagctctac agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat 21420

taggagcgcc acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctagagacac 21480

tttcaataaa ggcaaatgct tttatttgta cactctcggg tgattattta cccccaccct 21540

tgccgtctgc gccgtttaaa aatcaaaggg gttctgccgc gcatcgctat gcgccactgg 21600

cagggacacg ttgcgatact ggtgtttagt gctccactta aactcaggca caaccatccg 21660

cggcagctcg gtgaagtttt cactccacag gctgcgcacc atcaccaacg cgtttagcag 21720

gtcgggcgcc gatatcttga agtcgcagtt ggggcctccg ccctgcgcgc gcgagttgcg 21780

atacacaggg ttgcagcact ggaacactat cagcgccggg tggtgcacgc tggccagcac 21840

gctcttgtcg gagatcagat ccgcgtccag gtcctccgcg ttgctcaggg cgaacggagt 21900

caactttggt agctgccttc ccaaaaaggg cgcgtgccca ggctttgagt tgcactcgca 21960

ccgtagtggc atcaaaaggt gaccgtgccc ggtctgggcg ttaggataca gcgcctgcat 22020

aaaagccttg atctgcttaa aagccacctg agcctttgcg ccttcagaga agaacatgcc 22080

gcaagacttg ccggaaaact gattggccgg acaggccgcg tcgtgcacgc agcaccttgc 22140

gtcggtgttg gagatctgca ccacatttcg gccccaccgg ttcttcacga tcttggcctt 22200

gctagactgc tccttcagcg cgcgctgccc gttttcgctc gtcacatcca tttcaatcac 22260

gtgctcctta tttatcataa tgcttccgtg tagacactta agctcgcctt cgatctcagc 22320

gcagcggtgc agccacaacg cgcagcccgt gggctcgtga tgcttgtagg tcacctctgc 22380

aaacgactgc aggtacgcct gcaggaatcg ccccatcatc gtcacaaagg tcttgttgct 22440

ggtgaaggtc agctgcaacc cgcggtgctc ctcgttcagc caggtcttgc atacggccgc 22500

cagagcttcc acttggtcag gcagtagttt gaagttcgcc tttagatcgt tatccacgtg 22560

gtacttgtcc atcagcgcgc gcgcagcctc catgcccttc tcccacgcag acacgatcgg 22620

cacactcagc gggttcatca ccgtaatttc actttccgct tcgctgggct cttcctcttc 22680

ctcttgcgtc cgcataccac gcgccactgg gtcgtcttca ttcagccgcc gcactgtgcg 22740

cttacctcct ttgccatgct tgattagcac cggtgggttg ctgaaaccca ccatttgtag 22800

cgccacatct tctctttctt cctcgctgtc cacgattacc tctggtgatg gcgggcgctc 22860

gggcttggga gaagggcgct tctttttctt cttgggcgca atggccaaat ccgccgccga 22920

ggtcgatggc cgcgggctgg gtgtgcgcgg caccagcgcg tcttgtgatg agtcttcctc 22980

gtcctcggac tcgatacgcc gcctcatccg cttttttggg ggcgcccggg gaggcggcgg 23040

cgacggggac ggggacgaca cgtcctccat ggttggggga cgtcgcgccg caccgcgtcc 23100

gcgctcgggg gtggtttcgc gctgctcctc ttcccgactg gccatttcct tctcctatag 23160

gcagaaaaag atcatggagt cagtcgagaa gaaggacagc ctaaccgccc cctctgagtt 23220

cgccaccacc gcctccaccg atgccgccaa cgcgcctacc accttccccg tcgaggcacc 23280

cccgcttgag gaggaggaag tgattatcga gcaggaccca ggttttgtaa gcgaagacga 23340

cgaggaccgc tcagtaccaa cagaggataa aaagcaagac caggacaacg cagaggcaaa 23400

cgaggaacaa gtcgggcggg gggacgaaag gcatggcgac tacctagatg tgggagacga 23460

cgtgctgttg aagcatctgc agcgccagtg cgccattatc tgcgacgcgt tgcaagagcg 23520

cagcgatgtg cccctcgcca tagcggatgt cagccttgcc tacgaacgcc acctattctc 23580

accgcgcgta ccccccaaac gccaagaaaa cggcacatgc gagcccaacc cgcgcctcaa 23640

cttctacccc gtatttgccg tgccagaggt gcttgccacc tatcacatct ttttccaaaa 23700

ctgcaagata cccctatcct gccgtgccaa ccgcagccga gcggacaagc agctggcctt 23760

gcggcagggc gctgtcatac ctgatatcgc ctcgctcaac gaagtgccaa aaatctttga 23820

gggtcttgga cgcgacgaga agcgcgcggc aaacgctctg caacaggaaa acagcgaaaa 23880

tgaaagtcac tctggagtgt tggtggaact cgagggtgac aacgcgcgcc tagccgtact 23940

aaaacgcagc atcgaggtca cccactttgc ctacccggca cttaacctac cccccaaggt 24000

catgagcaca gtcatgagtg agctgatcgt gcgccgtgcg cagcccctgg agagggatgc 24060

aaatttgcaa gaacaaacag aggagggcct acccgcagtt ggcgacgagc agctagcgcg 24120

ctggcttcaa acgcgcgagc ctgccgactt ggaggagcga cgcaaactaa tgatggccgc 24180

agtgctcgtt accgtggagc ttgagtgcat gcagcggttc tttgctgacc cggagatgca 24240

gcgcaagcta gaggaaacat tgcactacac ctttcgacag ggctacgtac gccaggcctg 24300

caagatctcc aacgtggagc tctgcaacct ggtctcctac cttggaattt tgcacgaaaa 24360

ccgccttggg caaaacgtgc ttcattccac gctcaagggc gaggcgcgcc gcgactacgt 24420

ccgcgactgc gtttacttat ttctatgcta cacctggcag acggccatgg gcgtttggca 24480

gcagtgcttg gaggagtgca acctcaagga gctgcagaaa ctgctaaagc aaaacttgaa 24540

ggacctatgg acggccttca acgagcgctc cgtggccgcg cacctggcgg acatcatttt 24600

ccccgaacgc ctgcttaaaa ccctgcaaca gggtctgcca gacttcacca gtcaaagcat 24660

gttgcagaac tttaggaact ttatcctaga gcgctcagga atcttgcccg ccacctgctg 24720

tgcacttcct agcgactttg tgcccattaa gtaccgcgaa tgccctccgc cgctttgggg 24780

ccactgctac cttctgcagc tagccaacta ccttgcctac cactctgaca taatggaaga 24840

cgtgagcggt gacggtctac tggagtgtca ctgtcgctgc aacctatgca ccccgcaccg 24900

ctccctggtt tgcaattcgc agctgcttaa cgaaagtcaa attatcggta cctttgagct 24960

gcagggtccc tcgcctgacg aaaagtccgc ggctccgggg ttgaaactca ctccggggct 25020

gtggacgtcg gcttaccttc gcaaatttgt acctgaggac taccacgccc acgagattag 25080

gttctacgaa gaccaatccc gcccgccaaa tgcggagctt accgcctgcg tcattaccca 25140

gggccacatt cttggccaat tgcaagccat caacaaagcc cgccaagagt ttctgctacg 25200

aaagggacgg ggggtttact tggaccccca gtccggcgag gagctcaacc caatcccccc 25260

gccgccgcag ccctatcagc agcagccgcg ggcccttgct tcccaggatg gcacccaaaa 25320

agaagctgca gctgccgccg ccacccacgg acgaggagga atactgggac agtcaggcag 25380

aggaggtttt ggacgaggag gaggaggaca tgatggaaga ctgggagagc ctagacgagg 25440

aagcttccga ggtcgaagag gtgtcagacg aaacaccgtc accctcggtc gcattcccct 25500

cgccggcgcc ccagaaatcg gcaaccggtt ccagcatggc tacaacctcc gctcctcagg 25560

cgccgccggc actgcccgtt cgccgaccca accgtagatg ggacaccact ggaaccaggg 25620

ccggtaagtc caagcagccg ccgccgttag cccaagagca acaacagcgc caaggctacc 25680

gctcatggcg cgggcacaag aacgccatag ttgcttgctt gcaagactgt gggggcaaca 25740

tctccttcgc ccgccgcttt cttctctacc atcacggcgt ggccttcccc cgtaacatcc 25800

tgcattacta ccgtcatctc tacagcccat actgcaccgg cggcagcggc agcggcagca 25860

acagcagcgg ccacacagaa gcaaaggcga ccggatagca agactctgac aaagcccaag 25920

aaatccacag cggcggcagc agcaggagga ggagcgctgc gtctggcgcc caacgaaccc 25980

gtatcgaccc gcgagcttag aaacaggatt tttcccactc tgtatgctat atttcaacag 26040

agcaggggcc aagaacaaga gctgaaaata aaaaacaggt ctctgcgatc cctcacccgc 26100

agctgcctgt atcacaaaag cgaagatcag cttcggcgca cgctggaaga cgcggaggct 26160

ctcttcagta aatactgcgc gctgactctt aaggactagt ttcgcgccct ttctcaaatt 26220

taagcgcgaa aactacgtca tctccagcgg ccacacccgg cgccagcacc tgtcgtcagc 26280

gccattatga gcaaggaaat tcccacgccc tacatgtgga gttaccagcc acaaatggga 26340

cttgcggctg gagctgccca agactactca acccgaataa actacatgag cgcgggaccc 26400

cacatgatat cccgggtcaa cggaatccgc gcccaccgaa accgaattct cttggaacag 26460

gcggctatta ccaccacacc tcgtaataac cttaatcccc gtagttggcc cgctgccctg 26520

gtgtaccagg aaagtcccgc tcccaccact gtggtacttc ccagagacgc ccaggccgaa 26580

gttcagatga ctaactcagg ggcgcagctt gcgggcggct ttcgtcacag ggtgcggtcg 26640

cccgggcagg gtataactca cctgacaatc agagggcgag gtattcagct caacgacgag 26700

tcggtgagct cctcgcttgg tctccgtccg gacgggacat ttcagatcgg cggcgccggc 26760

cgtccttcat tcacgcctcg tcaggcaatc ctaactctgc agacctcgtc ctctgagccg 26820

cgctctggag gcattggaac tctgcaattt attgaggagt ttgtgccatc ggtctacttt 26880

aaccccttct cgggacctcc cggccactat ccggatcaat ttattcctaa ctttgacgcg 26940

gtaaaggact cggcggacgg ctacgactga acaagtttgt acaaaaaagc aggcttcgaa 27000

ggagatagaa ccaattctct aaggaaatac ttaaccatgg tcgactttat ttggatcttc 27060

gatgctagac gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag 27120

aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 27180

cattataagc tgcaataaac aagttgctcg aattactaaa gtgcttcgat tttagtagtg 27240

caatagtatt gtctgcataa tgctaatcgt gatagctaaa gcttatcaga ctgagatagc 27300

accatctgaa atgctgaccc tgtagaaccg aatttgtata tcacattgcc agggctggct 27360

acatctggct actgcgcttt ggttatctag cgaagtgctt cgattttagt agtgcaatag 27420

tattgtctgc ataatgctaa tcgtgatagc taaagcttat cagactgaga tagcaccatc 27480

tgaaatgctg accctgtaga accgaatttg tatatcacat tgccagggct ggctacatct 27540

ggctactgcg ctttggttat ctagcgaagt gcttcgattttagtagtgca atagtattgt 27600

ctgcataatg ctaatcgtga tagctaaagc ttatcagact gagatagcac catctgaaat 27660

gctgaccctg tagaaccgaa tttgtatatc acattgccag ggctggctac atctggctac 27720

tgcgctttgg ttatctagcg aagtgcttcg attttagtag tgcaatagta ttgtctgcat 27780

aatgctaatc gtgatagcta aagcttatca gactgagata gcaccatctg aaatgctgac 27840

cctgtagaac cgaatttgta tatcacattg ccagggctgg ctacatctgg ctactgcgct 27900

ttggttatct agcgaagtgc ttcgatttta gtagtgcaat agtattgtct gcataatgct 27960

aatcgtgata gctaaagctt atcagactga gatagcacca tctgaaatgc tgaccctgta 28020

gaaccgaatt tgtatatcac attgccaggg ctggctacat ctggctactg cgctttggtt 28080

atctagcgaa gtgcttcgat tttagtagtg caatagtatt gtctgcataa tgctaatcgt 28140

gatagctaaa gcttatcaga ctgagatagc accatctgaa atgctgaccc tgtagaaccg 28200

aatttgtata tcacattgcc agggctggct acatctggct actgcgcttt ggttatctag 28260

cgaagtgctt cgattttagt agtgcaatag tattgtctgc ataatgctaa tcgtgatagc 28320

taaagcttat cagactgaga tagcaccatc tgaaatgctg accctgtaga accgaatttg 28380

tatatcacat tgccagggct ggctacatct ggctactgcg ctttggttat ctagcgaagt 28440

gcttcgattt tagtagtgca atagtattgt ctgcataatg ctaatcgtga tagctaaagc 28500

ttatcagact gagatagcac catctgaaat gctgaccctg tagaaccgaa tttgtatatc 28560

acattgccag ggctggctac atctggctac tgcgctttgg ttatctagcg aagtgcttcg 28620

attttagtag tgcaatagta ttgtctgcat aatgctaatc gtgatagcta aagcttatca 28680

gactgagata gcaccatctg aaatgctgac cctgtagaac cgaatttgta tatcacattg 28740

ccagggctgg ctacatctgg ctactgcgct ttggttatct agcgaagtgc ttcgatttta 28800

gtagtgcaat agtattgtct gcataatgct aatcgtgata gctaaagctt atcagactga 28860

gatagcacca tctgaaatgc tgaccctgta gaaccgaatt tgtatatcac attgccaggg 28920

ctggctacat ctggctactg cgctttggtt atctagcgca tggtggcgaa ttcgcggccg 28980

cactcgagat atctagaccc agctttcttg tacaaagtgg tctaataaaa aaaaataata 29040

aagcatcact tacttaaaat cagttagcaa atttctgtcc agtttattca gcagcacctc 29100

cttgccctcc tcccagctct ggtattgcag cttcctcctg gctgcaaact ttctccacaa 29160

tctaaatgga atgtcagttt cctcctgttc ctgtccatcc gcacccacta tcttcatgtt 29220

gttgcagatg aagcgcgcaa gaccgtctga agataccttc aaccccgtgt atccatatga 29280

cacggaaacc ggtcctccaa ctgtgccttt tcttactcct ccctttgtat cccccaatgg 29340

gtttcaagag agtccccctg gggtactctc tttgcgccta tccgaacctc tagttacctc 29400

caatggcatg cttgcgctca aaatgggcaa cggcctctct ctggacgagg ccggcaacct 29460

tacctcccaa aatgtaacca ctgtgagccc acctctcaaa aaaaccaagt caaacataaa 29520

cctggaaata tctgcacccc tcacagttac ctcagaagcc ctaactgtgg ctgccgccgc 29580

acctctaatg gtcgcgggca acacactcac catgcaatca caggccccgc taaccgtgca 29640

cgactccaaa cttagcattg ccacccaagg acccctcaca gtgtcagaag gaaagctagc 29700

cctgcaaaca tcaggccccc tcaccaccac cgatagcagt acccttacta tcactgcctc 29760

accccctcta actactgcca ctggtagctt gggcattgac ttgaaagagc ccatttatac 29820

acaaaatgga aaactaggac taaagtacgg ggctcctttg catgtaacag acgacctaaa 29880

cactttgacc gtagcaactg gtccaggtgt gactattaat aatacttcct tgcaaactaa 29940

agttactgga gccttgggtt ttgattcaca aggcaatatg caacttaatg tagcaggagg 30000

actaaggatt gattctcaaa acagacgcct tatacttgat gttagttatc cgtttgatgc 30060

tcaaaaccaa ctaaatctaa gactaggaca gggccctctt tttataaact cagcccacaa 30120

cttggatatt aactacaaca aaggccttta cttgtttaca gcttcaaaca attccaaaaa 30180

gcttgaggtt aacctaagca ctgccaaggg gttgatgttt gacgctacag ccatagccat 30240

taatgcagga gatgggcttg aatttggttc acctaatgca ccaaacacaa atcccctcaa 30300

aacaaaaatt ggccatggcc tagaatttga ttcaaacaag gctatggttc ctaaactagg 30360

aactggcctt agttttgaca gcacaggtgc cattacagta ggaaacaaaa ataatgataa 30420

gctaactttg tggaccacac cagctccatc tcctaactgt agactaaatg cagagaaaga 30480

tgctaaactc actttggtct taacaaaatg tggcagtcaa atacttgcta cagtttcagt 30540

tttggctgtt aaaggcagtt tggctccaat atctggaaca gttcaaagtg ctcatcttat 30600

tataagattt gacgaaaatg gagtgctact aaacaattcc ttcctggacc cagaatattg 30660

gaactttaga aatggagatc ttactgaagg cacagcctat acaaacgctg ttggatttat 30720

gcctaaccta tcagcttatc caaaatctca cggtaaaact gccaaaagta acattgtcag 30780

tcaagtttac ttaaacggag acaaaactaaacctgtaaca ctaaccatta cactaaacgg 30840

tacacaggaa acaggagaca caactccaag tgcatactct atgtcatttt catgggactg 30900

gtctggccac aactacatta atgaaatatt tgccacatcc tcttacactt tttcatacat 30960

tgcccaagaa taaagaatcg tttgtgttat gtttcaacgt gtttattttt caattgcaga 31020

aaatttcaag tcatttttca ttcagtagta tagccccacc accacatagc ttatacagat 31080

caccgtacct taatcaaact cacagaaccc tagtattcaa cctgccacct ccctcccaac 31140

acacagagta cacagtcctt tctccccggc tggccttaaa aagcatcata tcatgggtaa 31200

cagacatatt cttaggtgtt atattccaca cggtttcctg tcgagccaaa cgctcatcag 31260

tgatattaat aaactccccg ggcagctcac ttaagttcat gtcgctgtcc agctgctgag 31320

ccacaggctg ctgtccaact tgcggttgct taacgggcgg cgaaggagaa gtccacgcct 31380

acatgggggt agagtcataa tcgtgcatca ggatagggcg gtggtgctgc agcagcgcgc 31440

gaataaactg ctgccgccgc cgctccgtcc tgcaggaata caacatggca gtggtctcct 31500

cagcgatgat tcgcaccgcc cgcagcataa ggcgccttgt cctccgggca cagcagcgca 31560

ccctgatctc acttaaatca gcacagtaac tgcagcacag caccacaata ttgttcaaaa 31620

tcccacagtg caaggcgctg tatccaaagc tcatggcggg gaccacagaa cccacgtggc 31680

catcatacca caagcgcagg tagattaagt ggcgacccct cataaacacg ctggacataa 31740

acattacctc ttttggcatg ttgtaattca ccacctcccg gtaccatata aacctctgat 31800

taaacatggc gccatccacc accatcctaa accagctggc caaaacctgc ccgccggcta 31860

tacactgcag ggaaccggga ctggaacaat gacagtggag agcccaggac tcgtaaccat 31920

ggatcatcat gctcgtcatg atatcaatgt tggcacaaca caggcacacg tgcatacact 31980

tcctcaggat tacaagctcc tcccgcgtta gaaccatatc ccagggaaca acccattcct 32040

gaatcagcgt aaatcccaca ctgcagggaa gacctcgcac gtaactcacg ttgtgcattg 32100

tcaaagtgtt acattcgggc agcagcggat gatcctccag tatggtagcg cgggtttctg 32160

tctcaaaagg aggtagacga tccctactgt acggagtgcg ccgagacaac cgagatcgtg 32220

ttggtcgtag tgtcatgcca aatggaacgc cggacgtagt catatttcct gaagcaaaac 32280

caggtgcggg cgtgacaaac agatctgcgt ctccggtctc gccgcttaga tcgctctgtg 32340

tagtagttgt agtatatcca ctctctcaaa gcatccaggc gccccctggc ttcgggttct 32400

atgtaaactc cttcatgcgc cgctgccctg ataacatcca ccaccgcaga ataagccaca 32460

cccagccaac ctacacattc gttctgcgag tcacacacgg gaggagcggg aagagctgga 32520

agaaccatgt tttttttttt attccaaaag attatccaaa acctcaaaat gaagatctat 32580

taagtgaacg cgctcccctc cggtggcgtg gtcaaactct acagccaaag aacagataat 32640

ggcatttgta agatgttgca caatggcttc caaaaggcaa acggccctca cgtccaagtg 32700

gacgtaaagg ctaaaccctt cagggtgaat ctcctctata aacattccag caccttcaac 32760

catgcccaaa taattctcat ctcgccacct tctcaatata tctctaagca aatcccgaat 32820

attaagtccg gccattgtaa aaatctgctc cagagcgccc tccaccttca gcctcaagca 32880

gcgaatcatg attgcaaaaa ttcaggttcc tcacagacct gtataagatt caaaagcgga 32940

acattaacaa aaataccgcg atcccgtagg tcccttcgca gggccagctg aacataatcg 33000

tgcaggtctg cacggaccag cgcggccact tccccgccag gaaccttgac aaaagaaccc 33060

acactgatta tgacacgcat actcggagct atgctaacca gcgtagcccc gatgtaagct 33120

ttgttgcatg ggcggcgata taaaatgcaa ggtgctgctc aaaaaatcag gcaaagcctc 33180

gcgcaaaaaa gaaagcacat cgtagtcatg ctcatgcaga taaaggcagg taagctccgg 33240

aaccaccaca gaaaaagaca ccatttttct ctcaaacatg tctgcgggtt tctgcataaa 33300

cacaaaataa aataacaaaa aaacatttaa acattagaag cctgtcttac aacaggaaaa 33360

acaaccctta taagcataag acggactacg gccatgccgg cgtgaccgta aaaaaactgg 33420

tcaccgtgat taaaaagcac caccgacagc tcctcggtca tgtccggagt cataatgtaa 33480

gactcggtaa acacatcagg ttgattcatc ggtcagtgct aaaaagcgac cgaaatagcc 33540

cgggggaata catacccgca ggcgtagaga caacattaca gcccccatag gaggtataac 33600

aaaattaata ggagagaaaa acacataaac acctgaaaaa ccctcctgcc taggcaaaat 33660

agcaccctcc cgctccagaa caacatacag cgcttcacag cggcagccta acagtcagcc 33720

ttaccagtaa aaaagaaaac ctattaaaaa aacaccactc gacacggcac cagctcaatc 33780

agtcacagtg taaaaaaggg ccaagtgcag agcgagtata tataggacta aaaaatgacg 33840

taacggttaa agtccacaaa aaacacccag aaaaccgcac gcgaacctac gcccagaaac 33900

gaaagccaaa aaacccacaa cttcctcaaa tcgtcacttc cgttttccca cgttacgtaa 33960

cttcccattt taagaaaact acaattccca acacatacaa gttactccgc cctaaaacct 34020

acgtcacccg ccccgttccc acgccccgcg ccacgtcaca aactccaccc cctcattatc 34080

atattggctt caatccaaaa taaggtatat tattgatgat 34120

Claims (9)

1. Use of a miRNA combination or an inhibitor of a miRNA combination for the preparation of a medicament for the treatment of breast cancer, wherein the miRNA combination consists of: miR-9-5p, miR-10b-5p, miR-21-5p, miR-23a-3p, miR-29a-3p, miR-155-5p, miR-222-3p, miR-301a-3p and miR-373-3 p.
2. 2. The use of claim 1, wherein the inhibitor of the combination of mirnas is an agent that reduces the expression level, activity, stability or effective duration of action of each miRNA in the combination of mirnas.
3. 3. The use according to claim 2, wherein the inhibitor of the combination of mirnas is selected from:

   a) a nucleic acid inhibitor, protein inhibitor, nuclease or nucleic acid binding molecule;

   b) a plasmid vector carrying or expressing any one of a); or

   c) A host carrying or expressing any one of a).
4. 4. The use according to claim 3, wherein the inhibitor of the combination of miRNAs is selected from the group consisting of:

   a) the method comprises the following steps of (1) interfering LncRNA, wherein a sequence for coding the interfering LncRNA is n copies of a sequence shown in 16-189bp in SEQ ID NO.10, and n is an integer more than or equal to 1;

   b) expressing the plasmid vector of a); or

   c) Carrying the virus of a).
5. 5. The use of claim 1, wherein the breast cancer is triple negative breast cancer.
6. 6. An interfering LncRNA, characterized in that the sequence coding the LncRNA is n copies of the sequence shown as 16-189bp in SEQ ID NO.10, and n is an integer more than or equal to 1.
7. 7. A nucleic acid molecule encoding the LncRNA of claim 6.
8. 8. The nucleic acid molecule of claim 7, wherein the sequence of said nucleic acid molecule comprises the sequence shown as 16-189bp of SEQ id No. 10.
9. 9. A virus having integrated into its genome an expression cassette for expressing LncRNA of claim 6.

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