Application of shRNA and recombinant viral vectors that inhibit BCL-9 in the treatment of retinal angiogenesis-related diseases

Abstract

The present invention provides the use of shRNA and recombinant viral vectors that inhibit BCL-9 in the treatment of retinal angiogenesis-related diseases. The present invention screened and obtained shRNA that can effectively target and inhibit the expression of the BCL-9 gene, and constructed a recombinant viral vector containing the shRNA. By introducing the shRNA or recombinant viral vector into human retinal vascular endothelial cells by injection to specifically inhibit the expression of the BCL-9 gene, the activity, proliferation ability and tube-forming ability of human retinal vascular endothelial cells can be effectively reduced, thereby inhibiting retinal angiogenesis. Therefore, shRNA and recombinant viral vectors that specifically inhibit the expression of the BCL-9 gene can be used to treat retinal angiogenesis-related diseases, including diabetic retinopathy, neovascular age-related retinopathy and retinopathy of prematurity. The present invention provides a new target for the treatment of retinal angiogenesis-related diseases and develops a new approach.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of shRNA and recombinant viral vectors that inhibit BCL-9 in treating retinal angiogenesis-related diseases. Background Art

[0002] Angiogenesis refers to the formation of new blood vessels from existing capillaries or post-capillary veins. The main processes include: degradation of the vascular basement membrane during the activation period, activation, proliferation, migration, and reconstruction of endothelial cells to form new blood vessels and vascular networks. It is a complex process involving multiple cells and molecules, and the mechanisms regulating angiogenesis vary greatly in different organs and diseases.

[0003] Retinal neovascularization is a key pathological feature of retinal fundus diseases and is associated with a variety of retinal diseases, such as diabetic retinopathy, neovascular age-related retinopathy, and retinopathy of prematurity. Retinal neovascularization is an adaptive biological response to retinal ischemia and hypoxia. Hyperglycemia can also damage the retinal endothelium, leading to disruption of the integrity of the blood-retinal barrier (BRB), increased retinal microvascular permeability, and subsequent neovascularization. However, retinal neovascularization caused by ischemia, hypoxia, and hyperglycemia does not function like normal blood vessels. The high permeability of the neovascular walls can lead to subretinal hemorrhage, lipid exudation, detachment of the retinal pigment epithelium and choroid, and the formation of fibrotic scars, resulting in irreversible vision damage.

[0004] Retinal neovascularization is a complex process involving diverse pathways. Ultimately, these pathways influence the proliferation, migration, adhesion, and differentiation of vascular cells through proteins related to vascular development, generation, and leakage, such as vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1), angiopoietin (ANGPT), and intercellular adhesion molecule 1 (ICAM-1), leading to retinal neovascularization and vascular leakage. Effectively controlling retinal neovascularization and preventing its onset early is crucial for treating numerous retinal diseases.

[0005] Currently, there are limited effective treatments for retinal neovascularization, primarily intravitreal anti-VEGF injections, retinal laser photocoagulation, and vitrectomy. Commonly used treatments are incurable. Identifying the underlying mechanisms and pathways of retinal neovascularization, identifying novel targets, and addressing the underlying causes through gene therapy are currently hot topics. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide the use of shRNA and recombinant viral vectors that inhibit BCL-9 in the treatment of retinal angiogenesis-related diseases, wherein the shRNA and recombinant viral vectors can effectively inhibit retinal angiogenesis.

[0007] In a first aspect, the present invention provides a shRNA for inhibiting BCL-9 gene expression, wherein the nucleotide sequence of the shRNA is shown in SEQ ID NO. 2 or SEQ ID NO. 3.

[0008] In a second aspect, the present invention provides a recombinant viral vector for inhibiting the expression of the BCL-9 gene, wherein the recombinant viral vector contains the shRNA as described above.

[0009] In some embodiments, the backbone of the recombinant viral vector is a lentivirus, an adeno-associated virus, or an adenovirus.

[0010] The third aspect of the present invention provides use of the shRNA or recombinant viral vector described above in the preparation of a drug for treating retinal angiogenesis-related diseases.

[0011] In some embodiments, the use comprises inhibiting the expression of the BCL-9 gene and then reducing the expression of the ESM1 gene.

[0012] In some embodiments, the retinal angiogenesis-related diseases include diabetic retinopathy, neovascular age-related retinopathy, and retinopathy of prematurity.

[0013] A fourth aspect of the present invention provides a drug for treating retinal angiogenesis-related diseases, wherein the active ingredient of the drug includes the shRNA and / or recombinant viral vector as described above.

[0014] In some embodiments, the drug is in the form of an injection.

[0015] In some embodiments, the drug is in the form of an ocular injection.

[0016] A fifth aspect of the present invention provides a method for inhibiting retinal angiogenesis for non-therapeutic purposes, comprising the following steps: introducing the shRNA and / or recombinant viral vector described above into a test sample to inhibit the expression of the BCL-9 gene in the test sample.

[0017] Due to the special nature of the retina, there are currently limited effective clinical treatments for retinal neovascularization diseases, mainly intravitreal injection of anti-VEGF protein, retinal laser photocoagulation, and vitrectomy. However, these commonly used clinical treatments cannot cure the disease.

[0018] The present invention has discovered BCL-9, a target for gene therapy of retinal neovascular diseases, and screened and obtained shRNA that can effectively target and inhibit BCL-9 gene expression, as well as constructed a recombinant viral vector containing the shRNA. According to the particularity of ophthalmic medication, the recombinant viral vector can be prepared into an injectable dosage form and directly injected into the eye. The shRNA is introduced into human retinal vascular endothelial cells through the recombinant viral vector and specifically inhibits the expression of the BCL-9 gene, which can effectively reduce the activity, proliferation and tube-forming ability of human retinal vascular endothelial cells, thereby inhibiting retinal angiogenesis and treating retinal angiogenesis-related diseases, including diabetic retinopathy, neovascular age-related retinopathy and retinopathy of prematurity. The present invention provides a new target for the treatment of retinal angiogenesis-related diseases and develops a new approach.

[0019] Further studies have shown that, unlike anti-angiogenesis strategies based on anti-VEGF proteins, the present invention uses the BCL-9 gene as a therapeutic target. By directly introducing shBCL9#3 into human retinal endothelial cells via injection, the targeted inhibition of BCL-9 gene expression can effectively reduce the expression of the endothelial cell-specific molecule (ESM1) while having no significant effect on the expression of vascular endothelial growth factor (VEGFA). This indicates that the direct introduction of the shRNA specifically targeting the BCL-9 gene into human retinal endothelial cells via injection can effectively inhibit retinal angiogenesis by reducing the expression of ESM1. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 2 is a structural diagram of each recombinant lentiviral vector.

[0021] Figure 2 This figure shows the results of knocking down the expression of BCL9 in HREC using recombinant lentiviral vector.

[0022] Figure 3 The results show the number of tube-forming connection points and the amount of tube-forming in HREC cells with BCL9 knockdown.

[0023] Figure 4The results of VEGFA expression detection in HREC cells with BCL9 knockdown.

[0024] Figure 5 The results show that knocking down BCL9 affects the activity of HREC cells.

[0025] Figure 6 The results show that knocking down BCL9 affects the proliferation ability of HREC cells.

[0026] Figure 7 These are the results of detecting the expression of BCL9 around new blood vessels caused by hypoxia in the mouse retina.

[0027] Figure 8 This is the RNA-seq sequencing result. DETAILED DESCRIPTION

[0028] Experimental procedures in the following examples, unless otherwise specified, generally followed conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0031] Example 1

[0032] This example used a recombinant lentiviral vector to knock down BCL9 in human retinal microvascular endothelial cells (HRECs). In vitro studies, including tube formation assays, monoclonal crystal violet staining, CCK-8 assays, and qPCR, were performed. In vivo experiments were conducted after establishing an OIR model mouse model to illustrate the role of recombinant lentiviral vector knockdown in regulating tube formation in human retinal microvascular endothelial cells. Molecular biology experiments were also used to clarify the mechanism by which BCL9 affects retinal neovascularization.

[0033] 1. Knockdown of BCL9 expression in HRECs using recombinant lentiviral vectors

[0034] Recombinant lentiviral vector was used to specifically knock down BCL9 expression in HRECs.

[0035] Yunzhou Biotechnology (Guangzhou) Co., Ltd. was commissioned to connect the shRNA and NC sequence of the BCL9 gene to the lentiviral backbone to obtain a recombinant lentiviral vector. In this embodiment, a variety of shRNAs targeting the BCL9 gene were designed, and two shRNAs that can effectively inhibit the expression of the BCL9 gene in HREC were screened. Taking shRNA1 to 3 as an example, the nucleotide sequence of shRNA1 targeting the BCL9 gene is shown in SEQ ID NO.1, the nucleotide sequence of shRNA2 is shown in SEQ ID NO.2, the nucleotide sequence of shRNA3 is shown in SEQ ID NO.3, and the NC sequence is shown in SEQ ID NO.4.

[0036]

[0037] The recombinant lentiviral vectors containing the above shRNA1-3 and NC sequences were named shBCL9#1, shBCL9#2, shBCL9#3, and si-NC, respectively. The structures and numbers of each recombinant lentiviral vector are as follows: Figure 1 As shown, experiments were performed using the above-mentioned recombinant lentiviral vector.

[0038] 1.1 Experimental steps

[0039] A. On the first day, HREC cells were plated at 1.5×10 5 Cells were seeded at 100 cells / well in a 6-well plate and cultured for 24 hours until approximately 50% of the cells were transfected with a recombinant lentiviral vector. The experimental groups were set as: blank control group (control), blank vector group (si-NC), and BCL9 knockdown group (three knockdown sequences: shBCL9#1, shBCL9#2, and shBCL9#3), to verify the knockdown effects of the three different knockdown sequences.

[0040] B. On the second day, mix complete culture medium and transfection reagent (Hitrans GA): culture medium volume = number of wells × 960ul transfection reagent volume + number of wells × 40ul. Mix the culture medium and transfection reagent evenly and add 1ml of the mixture to each well.

[0041] C. Calculate the volume of virus added: Volume of virus added per well = Number of cells * MOI / Titer. The MOI is 10. That is, Volume of virus added per well = (Number of cells × 10) / (Viral titer). Add the appropriate volume of virus to each well.

[0042] D. After 24 hours of incubation at 37°C, replace the culture medium with 2 ml of fresh medium per well. 48 hours after transfection, cells can be harvested for further experiments.

[0043] E. Extract the transfected cells, extract RNA (QIAGEN, 74104), reverse transcribe (Takara, RR036A), and then verify the knockdown effect of BCL9 in the cells by qPCR (Takara, RR420A). The operation was carried out strictly according to the instructions. The detection primers for the BCL9 gene are: Upstream primer: Downstream primer:

[0044] 1.2 Experimental Results

[0045] Results: As Figure 1 As shown, compared with the control group, green fluorescence appeared in a large number of HRECs in the si-NC group and the shBCL9#3 group, indicating that the recombinant lentiviral vector was successfully transfected.

[0046] qPCR test results ( Figure 2 ) showed that both the shhBCL9#2 and shBCL9#3 knockdown groups effectively knocked down BCL9 expression in HRECs, while shBCL9#1 had no significant knockdown effect. The shBCL9#3 knockdown group achieved a knockdown efficiency exceeding 50%, demonstrating greater efficacy in knocking down BCL9 expression in HRECs compared to other shRNAs.

[0047] 2. Investigate the effect of knocking down BCL9 gene expression on the tube-forming ability of HREC.

[0048] 2.1 Experimental steps

[0049] A, HRECs were transfected with recombinant lentiviral vectors for 48 hours. Groups: blank control group (control), blank vector group (si-NC), and BCL9 knockdown group (shBCL9#3).

[0050] B. Coming Matrigel M Incubate the basement membrane matrix (catalog number 354230) on ice for at least 10 minutes until the matrix gel becomes liquid.

[0051] C. Tube formation experiments were performed on u-slide angiogenesis slides (ibidi, 81506). 10 μL Matrigel was added to each well and then incubated at 37°C for 30 minutes.

[0052] D. Digest the cells 48 hours after transfection and adjust the cell density to 2×105 cells / ml, and prepare cell suspension.

[0053] E. Add 50 μl of cell suspension per well (10,000 cells per well) on top of the Matrigel. Incubate at 37°C for 1, 6, and 24 hours, then photograph each well under a microscope.

[0054] F. The number of endothelial lumens in triplicate wells was counted using the Angiogenesis Analyzer in ImageJ version 1.48 software and statistically analyzed. Data are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA (*P < 0.05).

[0055] 2.2 Experimental Results

[0056] Results: As Figure 3 As shown, 6 hours after plating, the number of tube-forming connection points and the amount of tube-forming in HREC cells after BCL9 knockdown in the shBCL9#3 group were significantly reduced compared with those in the si-NC group, that is, the tube-forming ability of HREC cells after BCL9 knockdown was significantly decreased.

[0057] 3. Effect of recombinant lentiviral vector transfection and knockdown of BCL9 in HREC on VEGFA

[0058] 3.1 Experimental steps

[0059] (1) HREC were transfected with the recombinant lentiviral vector for 48 hours. Groups: blank control group (control), blank vector group (si-NC), and BCL9 knockdown group (shBCL9#3).

[0060] (2) Lentivirus transfection MOI: 10. Take fresh complete medium, add diluted lentivirus, and mix thoroughly. Add 1 ml of complete medium containing lentivirus to each well. Add 1 ml of complete medium without lentivirus to the blank control group. Place the 6-well plate in an incubator at 37°C, 5% CO2, and incubate for 48 h.

[0061] (3) HREC RNA was extracted 48 hours after transfection, and the expression of BCL9 and VEGFA genes after lentiviral transfection was detected by qPCR after reverse transcription. The detection kit was the same as above. The detection primers for the VEGFA gene were: Upstream primer: Downstream primer:

[0062] 3.2 Experimental Results

[0063] Results: As Figure 4As shown in Figure A, compared with the si-NC group, BCL9 in HRECs of the shBCL9#3 knockdown group was effectively knocked down, but there was no statistical difference in the expression of VEGFA ( Figure 4 Therefore, the effect of shBCL9#3 knockdown on BCL9 on retinal angiogenesis was not significantly correlated with VEGFA.

[0064] 4. CCK-8 assay to examine the effect of BCL9 knockdown on HREC cell viability

[0065] 4.1 Experimental steps

[0066] A. HRECs were transfected with a recombinant lentiviral vector for 48 hours. Groups: blank control group (control), blank vector group (si-NC), and BCL9 knockdown group (shBCL9#3). Lentiviral transfection MOI: 10.

[0067] B. Digest the cells and prepare a cell suspension. Use a spray gun to seed the cells into a 96-well plate, with 100 μl of cell suspension per well and a cell density of 1.5×10 5 Cells / ml. Add 100 μl of PBS to each well surrounding the cell well to prevent evaporation of the culture medium. Place the 96-well plate in a 37°C, 5% CO2 incubator for 48 hours and 5 days, respectively. Replace the complete culture medium with fresh culture medium every two days.

[0068] C. After culturing for 48 hours and 5 days, respectively, remove the 96-well plate, aspirate the original culture medium, and replace it with 90 μl of fresh culture medium. At the same time, add 90 μl of fresh complete culture medium to the wells without cells as CCK-8 blank control wells. Then add 10 μl of CCK-8 reagent to each well. Protect from light and place in an incubator at 37°C, 5% CO2 for incubation for 2 hours.

[0069] D. Remove the culture plate from light and measure the absorbance at 450 nm on a microplate reader.

[0070] E. Export data analysis and calculate cell viability. The calculation formula is as follows:

[0071] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%

[0072] As: absorbance of experimental wells (containing knockdown BCL9 cells, culture medium, and CCK-8 solution);

[0073] Ac: absorbance of control wells (containing NC group cells, culture medium, and CCK-8 solution);

[0074] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but no cells or drugs).

[0075] 4.2 Experimental Results

[0076] Results: As Figure 5 As shown in the figure, after knocking down BCL9, the cell activity of HREC was 0.6235 of that of the blank vector group after 48 hours (P < 0.0001), and the cell activity after 5 days was 0.1977 of that of the blank vector group (P < 0.0001), that is, after knocking down BCL9, the cell activity of HREC was significantly reduced.

[0077] 5. Crystal violet staining of monoclonal cells to observe the effect of knockdown of BCL9 on the proliferation ability of HREC

[0078] 5.1 Experimental steps

[0079] A, HRECs were transfected with recombinant lentiviral vectors for 48 hours. Groups: blank control group (control), blank vector group (si-NC), and BCL9 knockdown group (shBCL9#3).

[0080] B. Prepare cell suspension and perform cell culture. Seed cells into 6-well plates at a density of 1000 cells per well. Add complete culture medium to 3 ml per well.

[0081] C. Place the cells in a cell culture incubator at 37°C and 5% CO2 for 5 days.

[0082] D. After 5 days of normal culture, distinct cell colonies should be visible. Remove the 6-well plate, discard the original culture medium, and carefully wash the cells three times with PBS. Aspirate the cBS.

[0083] E. Add 1 ml of methanol to each well, fix the cells for 15 minutes, and discard the methanol.

[0084] F. Add 1 ml of 0.1% crystal violet to each well and stain for 30 minutes. Remove the crystal violet by aspiration, wash the cells three times with PBS, and dry in air.

[0085] G. Photographs were taken under a microscope, and clones were counted. The experiment was repeated four times. Statistical analysis was performed. Data are presented as mean ± SD. One-way ANOVA was used for statistical analysis (*P < 0.05).

[0086] 5.2 Experimental Results

[0087] Results: As Figure 6 As shown in the results, after knocking down BCL9 in HREC cells, the number of cell monoclonal clusters was significantly reduced compared with the blank control group and the blank vector group, and the number of surviving monoclonal cell clusters was significantly reduced compared with the control group. In other words, after knocking down BCL9, the proliferation ability of HREC was significantly reduced (P < 0.001).

[0088] 6. Effect of BCL9 on retinal neovascularization induced by hypoxia (oxygen-induced retinopathy, OIR) in mice

[0089] 6.1 Experimental steps

[0090] (1) Preparation of OIR model mice: Newborn C57BL / 6J pups and their nursing mothers were placed in an oxygen chamber and subjected to alternating high and low oxygen cycles (50% ± 2% and 10% ± 2% O2, respectively) daily for 14 days. The mice were then returned to room air until they were 18 days old.

[0091] (2) The mouse eyeball was soaked in 4% paraformaldehyde for 1 hour, then replaced with pre-chilled PBS and placed on ice. A 30G needle was used to puncture the cornea to reduce intraocular pressure. A circle of cornea was cut off and the lens and vitreous were removed. The ciliary body was left as much as possible when cutting the cornea. The choroid and retina were carefully peeled off. The retina was then cut into four radial petals and transferred to a slide. The retina was soaked in chilled PBS.

[0092] (3) Incubate the tissue with 1% Triton-X (1:100 Triton-X:PBS) at room temperature for 30 minutes and wash the tissue three times with PBS.

[0093] (4) Block the retina with 5% blocking solution at room temperature for 1 hour at 4°C.

[0094] (5) 1% isolectin B4 (4 μg / mL, Alexa Fluor 488, catalogue no. I21411; Life Technologies Australia, Mulgrave, VIC, Australia) and anti-BCL9 primary antibody (abcam, ab37305) were diluted 1:500 in 5% blocking buffer, overlaid on the retina, and incubated overnight at 4°C in the dark.

[0095] (6) The next day, the retina was washed three times with PBS and incubated with secondary antibodies at room temperature for 2 h.

[0096] (7) Wash the retina three times with PBS, wipe the water on the slide with a diameter, seal the slide with anti-fluorescence quenching mounting medium containing DAPI and transparent nail polish, and take pictures under a fluorescence microscope.

[0097] 6.2 Experimental Results

[0098] Results: As Figure 7As shown, hypoxia-induced neovascularization (red) occurred in the mouse retina. High expression of BCL9 (green) was observed around these neovascularizations. This indicates that BCL9 expression is positively correlated with neovascularization.

[0099] 7. RNA-sequencing (RNA-seq) technology detected that BCL9 can affect angiogenesis through ESM1

[0100] 7.1 Experimental steps

[0101] (1) Cell culture and grouping: HREC were transfected with recombinant lentiviral vector for 48 hours. Groups: blank control group (control), blank vector group (si-NC), and BCL9 knockdown group (shBCL9#3).

[0102] (2) 72 hours after lentiviral transfection, cells were harvested and RNA was extracted.

[0103] (3)RNA-seq method

[0104] RNA-seq was commissioned to Genewise Biotechnology Co., Ltd. and included the following steps:

[0105] 1) 1 μg of total RNA was used to prepare the following library.

[0106] 2) Isolation of poly(A) mRNA using Oligo(dT) beads.

[0107] 3) Use divalent cations and high temperature to fragment mRNA.

[0108] 4) Primer screening was performed using random primers.

[0109] 5) Synthesize first-strand cDNA and second-strand cDNA, then treat the purified double-stranded cDNA, repair both ends and add dA-tailing in one reaction, followed by TA ligation and adding adapters to both ends.

[0110] 6) Size selection of the adapter-ligated DNA was performed using DNA cleaning beads. PCR amplification of each sample was then performed using primers P5 and P7, and the PCR products were verified.

[0111] 7) Libraries with different indices were multiplexed and loaded onto an Illumina HiSeq / Illumina Novaseq / MGI2000 instrument and sequenced using a 2x150 paired-end (PE) configuration according to the instrument's instructions.

[0112] 7.2 Experimental Results

[0113] Results: RNA-seq sequencing was used to analyze the differentially expressed genes in HRECs after shBCL9#3 knocked down BCL9 expression in HRECs. We analyzed the top 20 mRNAs with reduced expression. The results showed that the endothelial cell-specific molecule (ESM1) associated with angiogenesis was reduced ( Figure 8 , NC-1 to NC-4 in the figure represent four repeated NC samples, and shBCL9-1 to shBCL9-4 represent four repeated shBCL9#3 samples). Therefore, it can be seen that, unlike anti-angiogenesis strategies based on anti-VEGF proteins, the present invention uses the BCL-9 gene as a therapeutic target. After shBCL9#3 is directly introduced into human retinal vascular endothelial cells via injection to target and inhibit BCL-9 gene expression, it can effectively reduce ESM1 expression and effectively inhibit retinal angiogenesis at the genetic level.

[0114] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0115] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of a recombinant viral vector for inhibiting BCL-9 gene expression in the preparation of a drug for treating retinal angiogenesis-related diseases, characterized in that: The recombinant viral vector contains shRNA, the nucleotide sequence of the shRNA is shown in SEQ ID NO. 3, and the retinal angiogenesis-related diseases are diabetic retinopathy, neovascular age-related retinopathy, and retinopathy of prematurity.

2. The use according to claim 1, characterized in that The backbone of the recombinant viral vector is lentivirus, adeno-associated virus or adenovirus.

3. The use according to claim 1, characterized in that The use includes inhibiting retinal angiogenesis.

4. The use according to claim 3, characterized in that The application includes reducing the expression of ESM1 gene after inhibiting the expression of BCL-9 gene.

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