1. Specific siRNA for inhibiting FOXO1 gene expression, wherein the specific siRNA is a fragment corresponding to 19 continuous nucleotide bases in human FOXO1 mRNA sequence; the specific siRNA is siFX1, and the sequence is as follows:

siFX1-s：5'-GCGCCGACUUCAUGAGCAA dTdT-3'；

siFX1-as：5'-UUGCUCAUGAAGUCGGCGC dTdT-3'。

2. the specific siRNA of claim 1, wherein said specific siRNA inhibits the transcriptional expression of FOXO1 gene in human tissue cells comprising FOXO1 gene by transfection.

3. Specific siRNA according to claim 2, wherein the specific siRNA inhibits transcriptional expression of FOXO1 gene in human acinar-type rhabdomyosarcoma cells.

4. Specific siRNA according to claim 1, wherein the specific siRNA inhibits or reduces transcriptional expression of FOXO1 gene in non-human primate-derived tissues and cells.

5. Use of a specific siRNA according to any of claims 1-4 in the basic study of tissue cells containing the FOXO1 gene.

6. Use of a specific siRNA according to any one of claims 1-4 in the basic study of a cell or disease containing the FOXO1 fusion gene.

7. Use of specific siRNA according to any one of claims 1-4 in the basic study of alveolar rhabdomyosarcoma.

8. The use of claim 7, wherein the specific siRNA inhibits the transcriptional expression of the human FOXO1 gene by transfection into acinar-type rhabdomyosarcoma cells.

9. The use according to claim 8, wherein the final concentration of the specific siRNA transfected into acinar-type rhabdomyosarcoma cells is 10-30 nM; preferably 20 nM.

10. A kit for inhibiting the transcription or/and expression of FOXO1 gene in human beings comprising the specific siRNA of any one of claims 1-4 or any other improved or optimized method, tool or research system based on said siRNA.

Background

FOXO1(FKHR), an important transcription factor in the forkhead (forkhead) protein O (FoxO) family, is involved in the growth, metabolism, tumor formation and the like of organisms by regulating various physiological processes such as oxidative stress, proliferation and apoptosis of cells. The expression of the FOXO1 gene has space-time difference, is widely expressed in various tissues and organs of adults, including heart, brain tissue, placenta, esophagus, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte and the like, is involved in the expression regulation of various disease genes, and may play an important role in regulating various physiological metabolic functions of cells, such as growth, differentiation, aging and the like. Therefore, the research on the FOXO1 gene has the promotion effect on the research and treatment of various related diseases, for example, the regulation and control effect on the islet beta cells can make the FOXO1 gene a new target gene for treating type II diabetes; in recent years, PAX3-FOXO1 fusion protein as a high-efficiency transcription factor can activate a plurality of downstream genes and also provide more targets for treating acinar type rhabdomyosarcoma.

The siRNA (small interfering RNA or short interfering RNA) interference technology for inhibiting the normal expression of a target gene by a short-segment double-stranded RNA molecule is an important tool and a method which are widely used in gene expression regulation and function research. At present, various siRNA double-stranded nucleotide sequences are verified and applied in the research of various protein genes and the treatment of diseases. However, in these studies and applications, siRNA duplex interference also has certain limitations, such as instability, short-term expression silencing, cell membrane permeability, and the presence of off-target effects, among others. Therefore, one siRNA directed against the same target gene cannot be used in the study of different content, purpose and condition backgrounds, and thus it is necessary to find and use different interfering sequences.

Combining the importance of the transcription factor FOXO1 in organisms, a new siRNA double-stranded short fragment sequence is screened out aiming at the design of the transcription factor FOXO 1. We found that this sequence has a strong knock-down or inhibitory effect on both the transcriptional and translational expression of the human FOXO1 gene. It has important application value in basic research of acinar type rhabdomyosarcoma, cells or diseases containing FOXO1 fusion gene, etc. Furthermore, based on the results of sequence alignment analysis, the siFX1siRNA sequence is also suitable for basic research work on FOXO1 gene in primates such as macaques, gorilla, and the like.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a novel specific siRNA (siFX1) for inhibiting FOXO1 gene expression and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the first aspect of the invention provides a specific siRNA for inhibiting FOXO1 gene expression, wherein the specific siRNA is a fragment corresponding to 19 continuous nucleotide bases in human FOXO1 mRNA sequence; the specific siRNA is siFX1, and the sequence is as follows:

siFX1-s：5'-GCGCCGACUUCAUGAGCAA dTdT-3'；

siFX1-as：5'-UUGCUCAUGAAGUCGGCGC dTdT-3'。

further, the specific siRNA inhibits transcriptional expression of FOXO1 gene in human tissue cells containing FOXO1 gene by transfection.

Further, the specific siRNA inhibits the transcriptional expression of FOXO1 gene in human acinar-type rhabdomyosarcoma cells.

Further, the specific siRNA can also inhibit transcriptional expression of FOXO1 gene derived from non-human primates such as macaque, orangutan, etc. by sequence alignment analysis.

The second aspect of the invention provides the application of the specific siRNA in basic research of tissue cells containing FOXO1 gene.

The third aspect of the invention is to provide the application of the specific siRNA in basic research of cells or diseases containing FOXO1 fusion gene.

The fourth aspect of the present invention provides the use of the specific siRNA described above in the basic study of alveolar rhabdomyosarcoma.

Further, the specific siRNA inhibits transcriptional expression of the human FOXO1 gene by transfection into acinar-type rhabdomyosarcoma cells.

Further, the final concentration of the specific siRNA transfected into the acinar-type rhabdomyosarcoma cells is 10-30 nM.

Further, the final concentration of the specific siRNA transfected into acinar-type rhabdomyosarcoma cells was 20 nM.

The fifth aspect of the invention is to provide a kit for inhibiting the transcription or/and expression of human FOXO1 gene comprising the above specific siRNA or any other improved and optimized method, tool and research system based on the siRNA.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the specific siRNA for inhibiting FOXO1 gene expression has extremely strong specificity, does not have off-target effect, and has stronger inhibiting and knocking-down effects; meanwhile, the specific siFX1 has little influence on cell morphology and growth, so the sequence has important application value in the research on FOXO1 gene, especially in the basic research on acinar rhabdomyosarcoma and cells or diseases containing FOXO1 fusion gene; in addition, according to sequence alignment analysis, the siFX1 sequence is also applicable to basic research on FOXO1 gene transcription expression in primates other than human, including macaques, orangutan and the like.

Drawings

FIG. 1 shows the results of quantitative PCR (QRT-PCR) analysis of the human FOXO1 gene after transfection of siFX1 with acinar-type rhabdomyosarcoma cells RH4 in accordance with an embodiment of the present invention; wherein GAPDH is an internal reference gene;

FIG. 2 shows the Western Blot results (panel A) and the relative quantitative analysis results (panel B) of the protein expression of FOXO1 and PAX3-FOXO1 genes after transfection of siFX1 with acinar rhabdomyosarcoma cells RH4 according to an embodiment of the present invention; wherein the alpha-Tubulin is an internal reference protein.

FIG. 3 shows the Western Blot results (panel A) and the relative quantitative analysis results (panel B) of the protein expression of FOXO1 and PAX3-FOXO1 genes after transfection of siFX4 with acinar rhabdomyosarcoma cells RH4 according to an embodiment of the present invention; wherein, the alpha-Tubulin is an internal reference protein;

FIG. 4 shows the cell morphology change after transfection of alveolar rhabdomyosarcoma cell RH4 with different siRNA interfering sequences in one embodiment of the invention.

Detailed Description

The invention provides a specific siRNA for inhibiting FOXO1 gene expression, which has important significance for fundamental research of acinar rhabdomyosarcoma and cells or diseases containing FOXO1 fusion gene, in particular, the specific siRNA, namely siFX1 is 19 continuous nucleotide base sequences in human FOXO1 mRNA sequence, and dTdT is suspended at the 3' end. Specifically, the nucleotide sequence of siFX1 is as follows:

siFX1-s:5'-GCGCCGACUUCAUGAGCAA dTdT-3'(SEQ ID No.1)；

siFX1-as:5'-UUGCUCAUGAAGUCGGCGC dTdT-3'(SEQ ID No.2)。

the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention is further described with reference to the following specific examples and figures, but is not to be construed as being limited thereto.

Example 1

This example demonstrates the inhibitory effect of siFX1 on the transcription level of FOXO1 gene in acinar type rhabdomyosarcoma cell line, and the specific experimental steps and results are as follows:

representative cell line RH4(SJ-RH4) of acinar rhabdomyosarcoma cells was inoculated in a 6-well plate or 60mm cell culture dish, grown overnight to a cell density of 60-80% in DMEM medium containing 10% FBS (fetal bovine serum) but no antibiotics at 37 ℃ under 5% carbon dioxide. siFX1 and siCON (control siRNA, siCON-s (sense strand): 5 '-CUAUAGCGAACCGUGAUCUdT-3' (SEQ ID No.3), siCON-as (antisense strand): 5 '-AGAUCACGUUCGUAUAGdT-3' (SEQ ID No.4)) were transfected into cells using Lipofectamine 2000 reagent (final concentration of siRNA was 20nM), and after 6-8 hours, the culture was replaced with DMEM containing 0.3% FBS and 1% antibiotic and cultured at 37 ℃ under 5% carbon dioxide for 72 hours. Cells were collected, extracted and total RNA concentration determined. Mu.g of total RNA was taken and reverse-transcribed into cDNA, quantitative PCR reaction was performed using the following human-derived specific primers, the variation of FOXO1 gene in transcription level was detected, and the effect of siFX1 sequence on FOXO1 gene transcription was analyzed, the results are shown in FIG. 1.

Primer sequences for qRT PCR reaction of human FOXO1 gene:

a forward primer: 5'-GACACCAGTTGATCCTGGGG-3' (SEQ ID No.5)

Reverse primer: 5'-CTGGCTGCCATAGGTTGACA-3', respectively; (SEQ ID No.6)

Reference sequence of human GAPDH gene for q RT PCR reaction:

a forward primer: 5'-CTGGGCTACACTGAGCACC-3' (SEQ ID No.7)

Reverse primer R: 5'-AAGTGGTCGTTGAGGGCAATG-3' (SEQ ID No. 8).

As can be seen from FIG. 1, the human FOXO1 gene was significantly reduced in transcription level after transfection of siFX1 into acinar rhabdomyosarcoma cell RH 4.

Example 2

This example demonstrates the inhibitory effect of siFX1 and known specific FOXO 1siRNA sequences (siFX4, siFX4-s (sense strand): 5 '-GGAGGUAUGAGUGACUAADTdT-3' (SEQ ID No.9), siFX4-as (antisense strand): 5 '-UACUGACUCAUACCUCCTCTdT-3' (SEQ ID No. 10); reference "FOXO 1 inhibiting and stabilizing ZEB2-induced epithelial-transcriptional transition", Tianxu Dong et al, Oncotarget, volume 8, phase 1, page 3-1713, 20170103) on the FOXO1 gene at the protein level in an acinar type rhabdomyosarcoma cell strain, the results of the experimental procedures and the results of the following:

as in example 1, siFX1 or siCON was transfected into RH4 cells, and after culturing for 72 hours under serum-free or low-concentration fetal bovine serum (0.3%), the cells were collected and total protein was extracted. 30-50. mu.g of total protein was subjected to SDS-PAGE gel electrophoresis, transferred to a PVDF membrane, and incubated with antibodies against FOXO1, α -tubulin, etc., to examine the effect of siFX1siRNA on FOXO1 expression in cells, as shown in FIG. 2.

As can be seen from fig. 2, siFX1 inhibited only transcription and protein expression of FOXO1 of acinar rhabdomyosarcoma cells, did not inhibit expression of the fusion genes PAX3-FOXO1, and had no off-target effect. And comparing with fig. 3, it can be seen that siFX1 has higher interference efficiency and stronger specificity than the existing FOXO 1siRNA compared with the known siRNA sequence.

It was also found that upon transfection of siFX1siRNA, there was an increase in PAX3-FOXO1 expression in cells, but this was not an off-target effect, but rather a reflection of the inherent interaction between FOXO1 and PAX3-FOXO 1.

Example 3

This example demonstrates the effect of siFX1 and the known positive control siFX4 on cell morphology, with specific experimental procedures and results as follows:

in the same manner as in example 1, siFX1, siFX4 siRNA and siCON were transfected into RH4 cells, respectively, cultured for 60-72 hours under the condition of low-concentration fetal bovine serum (0.3% FBS), and different time points were selected, and the difference in the change of cell morphology was observed under a microscope and recorded by photographing, and the results are shown in FIG. 4.

As can be seen in FIG. 4, the effect of the siFX1 interference sequence on cell morphology was different from the known control siFX4 interference sequence. Upon knock-down of FOXO1 expression by transfection of siRNA sequences, siFX1 had little effect on cell morphology and growth under low serum culture conditions, whereas siFX4 severely affected its morphology and cell growth, resulting in the cell eventually becoming filamentous, or even cell death. It can be seen that the known control interfering sequences, such as siFX4, may have off-target effects on cells or other effects not yet fully understood.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be appreciated by those skilled in the art that any equivalent modifications and substitutions are within the scope of the present invention. Therefore, equivalent changes, modifications and applications of the technology including shRNA (short hairpin RNA) without departing from the spirit and scope of the present invention are intended to be covered by the present invention.

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