Small interfering RNA for treating cervical cancer and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical compositions, and relates to a small interfering RNA for treating cervical cancer and application thereof, the small interfering RNA comprises a first small interfering RNA and a second small interfering RNA, the first small interfering RNA is siE6 / E7, the siE6 / E7 is a nucleotide sequence as shown in a positive-sense strand of SEQ ID NO.7 and an antisense strand of SEQ ID NO.8, and the second small interfering RNA is a nucleotide sequence as shown in a positive-sense strand of SEQ ID NO.7 and an antisense strand of SEQ ID NO.8. The second small interfering RNA comprises a positive-sense strand and an antisense strand which is in reverse complementary pairing with the positive-sense strand, the positive-sense strand comprises nucleotide sequences as shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, and the antisense strand comprises nucleotide sequences as shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.6. The first small interfering RNA comprises a first small interfering RNA and a second small interfering RNA, and the second small interfering RNA comprises a positive-sense strand and an antisense strand which is in reverse complementary pairing with the positive-sense strand. The small interfering RNA designed by the invention can increase the anti-tumor activity of the human papilloma virus protein E6 / E7 small interfering RNA, and the anti-tumor activity of the E6 / E7 and CTBP co-knock-down treatment is obviously enhanced compared with the anti-tumor activity of the E6 / E7 and CTBP single knock-down treatment.

Description

Technical Field

[0001] This invention belongs to the field of drug combination technology and relates to a small interfering RNA for the treatment of cervical cancer and its application. Background Technology

[0002] The primary cause of cervical cancer is recurrent infection with high-risk human papillomavirus (HPV). The most common HPV types, HPV-16 and HPV-18, cause over 70% of cervical cancers and most HPV-positive head and neck cancers. Importantly, the highly conserved E6 and E7 oncogenes in HPV encode the E6 and E7 oncoproteins, which are crucial for the development and maintenance of HPV-related cancers by inhibiting numerous tumor suppressor factors, including the most studied p53. The E6 protein has been reported to interact with E6AP via the LxxLL motif, leading to proteasome-mediated p53 degradation.

[0003] Inhibition of E6 and E7 can lead to cell cycle arrest and apoptosis in HPV-positive cancer cells. Therefore, viral proteins E6 / E7 are excellent targets for anticancer therapy through RNA interference or small molecule inhibitors that directly target the E6 / p53 interaction.

[0004] CTBP is expressed at high levels during development and participates in cell proliferation. Some reports indicate that CTBP can bind to centrosomes and promote cell survival by maintaining mitotic fidelity. Furthermore, CTBP excision at DNA ends suggests that CTBP may play a crucial role in cell cycle regulation. CTBP has also been reported to interact with Hdm2 oncoprotein (a negative regulator of the cell cycle-associated protein p53) in an environment-dependent manner. However, the key roles of CTBP in the regulation of G1, S, and G2 phases remain unclear.

[0005] Treatment of human papillomavirus (HPV)-positive malignant tumors remains a global challenge. Drug therapies based on HPV viral proteins E6 / E7 have certain advantages. However, the small interfering RNA (sRNA) of E6 / E7 exhibits low antitumor activity, which is insufficient to meet the practical requirements of tumor treatment. Therefore, to better enhance antitumor activity, it is essential to find new drug combinations that can strengthen the antitumor activity of E6 / E7 sRNA. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a small interfering RNA (SRNA) for treating cervical cancer and its applications. The SRNA designed in this invention can increase the antitumor activity of human papillomavirus protein E6 / E7 SRNA, and the antitumor activity of co-knockdown treatment with E6 / E7 and CTBP is significantly enhanced compared to E6 / E7 knockdown treatment alone.

[0007] The technical solution provided by this invention is as follows: This invention provides a small interfering RNA (sRNA) for treating cervical cancer. The sRNA comprises a first sRNA and a second sRNA. The first sRNA is siE6 / E7, wherein siE6 / E7 has a sense strand as shown in SEQ ID NO.7 and an antisense strand as shown in SEQ ID NO.8. The second sRNA comprises a sense strand and an antisense strand that is inversely complementary to it. The sense strand comprises the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.3, and SEQ ID NO.5, and the antisense strand comprises the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6.

[0008] Specifically, the positive strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-ACGACUUCACCGUCAAGCA-3' (SEQ ID NO: 1); siCTBP2: 5'-GCGCUUGUGUCAGUAAUAG-3' (SEQ ID NO: 3); siCTBP: 5'-GGGAGGACCUGGAGAAGUU-3' (SEQ ID NO: 5).

[0009] Specifically, the antisense strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-UGCUUGACGGUGAAGUCGU-3' (SEQ ID NO: 2); siCTBP2: 5'-CAUUACUGACCAGGCGC-3' (SEQ ID NO: 4); siCTBP: 5'-AACUUCUCCAGGUCCUCCC-3' (SEQ ID NO: 6).

[0010] Specifically, the sense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CAUUUACCAGCCCGACGAG-3' (SEQ ID NO: 7); the antisense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CUCGUCGGGCUGGUAAAUG-3' (SEQ ID NO: 8).

[0011] Furthermore, the small interfering RNA also contains dangling bases, the number of which is 1-8, the dangling bases being deoxynucleosides, and the dangling bases being located at the 3' end of the sense strand and / or antisense strand of the small interfering RNA.

[0012] Specifically, the ends (e.g., the 3′ ends) of the sense and / or antisense strands of the aforementioned small interfering RNA (such as siRNA) molecules may also have n overhangs to increase the activity of the small interfering RNA. The overhangs can be the same or different deoxyribonucleotides (e.g., deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU), etc.), and n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), particularly an integer from 2 to 4. In one embodiment of the present invention, n = 2, and the overhangs can be dTdT, dTdC, or dUdU, etc.

[0013] Furthermore, both the sense and antisense strands of the aforementioned small interfering RNA molecules have dTdT dpole bases at their 3' ends.

[0014] Specifically, the positive strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-ACGACUUCACCGUCAAGCAdTdT-3'; siCTBP2: 5'-GCGCUUGUGUCAGUAAUAGdTdT-3'; siCTBP: 5'-GGGAGGACCUGGAGAAGUUdTdT-3'.

[0015] Specifically, the antisense strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-UGCUUGACCGGUGAAGUCGUdTdT-3'; siCTBP2: 5'-CAUUACUGACCAGGCGCdTdT-3'; siCTBP: 5'-AACUUCUCCAGGUCCUCCCdTdT-3'.

[0016] Specifically, the sense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CAUUUACCAGCCCGACGAGdTdT-3'; the antisense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CUCGUCGGGCUGGUAAAUGdTdT-3'.

[0017] Furthermore, the first small interfering RNA silences the E6 / E7 gene, and the second small interfering RNA silences the CTBP gene, wherein CTBP antagonizes E6 / E7 during the G1 / S transition phase through the cell cycle-related regulatory factor p21.

[0018] The present invention also provides the application of the small interfering RNA for treating cervical cancer as described above in the preparation of a drug for treating cervical cancer, wherein the small interfering RNA targets and downregulates the expression of E6 / E7 and CTBP genes in cervical cancer cells.

[0019] Furthermore, the small interfering RNA inhibits the proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells.

[0020] The present invention also provides a delivery system for small interfering RNA according to the above description, the delivery system comprising the small interfering RNA and the vector described above.

[0021] Specifically, the vector can be a viral vector or a non-viral vector.

[0022] The present invention also provides a pharmaceutical composition comprising the small interfering RNA or the delivery system described above, and pharmaceutically acceptable excipients.

[0023] The present invention also provides an application of the pharmaceutical composition described above in the preparation of a treatment for cervical cancer, wherein the pharmaceutical composition targets and downregulates the expression of E6 / E7 and CTBP genes in cervical cancer cells.

[0024] Furthermore, the pharmaceutical composition inhibits the proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells. Beneficial effects

[0025] The small interfering RNA designed in this invention can increase the antitumor activity of human papillomavirus protein E6 / E7 small interfering RNA. The antitumor activity of co-knockdown treatment of E6 / E7 and CTBP is significantly enhanced compared with E6 / E7 knockdown treatment alone.

[0026] CTBP antagonizes the E6 / E7 viral proteins in the G1 / S phase and also antagonizes the function of E6 / E7 in the transcriptional regulation of genes related to cell cycle regulation. Importantly, the upregulation of p21 expression caused by E6 / E7 knockdown is corrected by simultaneous knockdown of CTBP.

[0027] In this invention, knockdown of CTBP has different effects on cell cycle regulation. The siCTBP1 treatment group showed G1 phase arrest, while the siCTBP2 and siCTBP treatment groups showed S / G2 phase arrest, indicating that CTBP has a complex function in cell cycle regulation. Knockdown of E6 / E7 leads to a sharp cell cycle arrest in G1 phase. However, by further knockdown of CTBP, especially in the siCTBP2 and siCTBP groups, the G1 phase arrest caused by E6 / E7 knockdown was almost eliminated, indicating that CTBP antagonizes E6 / E7 in the G1 / S phase transition.

[0028] RT-qPCR experiments confirmed the regulatory role of CTBP on p21, indicating that CTBP antagonizes E6 / E7 by transcribedly regulating cell cycle-related genes, especially the G1 / S phase transition-related regulatory factor p21.

[0029] The pro-proliferative effects of CTBP family proteins are crucial for several cancers, including breast, prostate, and lung cancer, with most cancers exhibiting upregulation of CTBP proteins, particularly CTBP2. However, the role of CTBP in cervical cancer is rarely reported. This invention primarily investigates the pro-proliferative effect of CTBP in cervical cancer cells, aiming to better elucidate the molecular mechanisms by which CTBP influences cervical cancer proliferation.

[0030] There is a huge market demand for anti-tumor drugs. The drug combination of this invention exhibits significant anti-tumor activity against human papillomavirus-positive cervical cancer HeLa cells. Furthermore, the preparation of this drug combination is simple and easy, using abundant and readily available raw materials at low cost, and it can be applied as a novel anti-tumor drug for cancer treatment. This invention has market implementation potential, and its implementation will generate significant economic and environmental benefits. Attached Figure Description

[0031] Figure 1 This is an electron micrograph of the number of cells in HeLa cells 48 hours after transfection with siCTBP1, siCTBP2 and siCTBP in the embodiments of the present invention.

[0032] Figure 2 This is a statistical graph showing the number of cells transfected with siCTBP1, siCTBP2 and siCTBP in HeLa cells at 0 h, 24 h, 48 h and 72 h using Graphpad in an embodiment of the present invention.

[0033] Figure 3 This is a cell cycle detection diagram of HeLa cells transfected with siCTBP1, siCTBP2 and siCTBP for 48 h and 72 h, as analyzed by FACS in an embodiment of the present invention.

[0034] Figure 4This is a statistical graph of cell proliferation at 0h, 24h, 48h and 72h in HeLa cells co-transfected with knockdown E6 / E7 and knockdown CTBP, analyzed by FACS according to an embodiment of the present invention.

[0035] Figure 5 This is a cell cycle detection diagram of HeLa cells co-transfected with knockdown E6 / E7 and knockdown CTBP using FACS analysis in an embodiment of the present invention.

[0036] Figure 6 This embodiment of the invention uses Graphpad to target... Figure 5 A statistical graph of the cell cycle.

[0037] Figure 7 This is a graph showing the detection of apoptosis in HeLa cells transfected with CTBP knockdown, E6 / E7 knockdown, and E6 / E7 knockdown combined with CTBP knockdown, using Annexin V-FITC and PI combined with FACS analysis in an embodiment of the present invention.

[0038] Figure 8 This embodiment of the invention uses Graphpad to target... Figure 7 Statistical graph of cell apoptosis.

[0039] Figure 9 This is a schematic diagram illustrating the expression of CTBP1, CTBP2, p21, and p53 in HeLa cells transfected with knockdown CTBP, knockdown E6 / E7, and a combination of knockdown E6 / E7 and knockdown CTBP using RT-qPCR in an embodiment of the present invention. Detailed Implementation

[0040] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0041] As used herein, the terms “small interfering RNA” and “siRNA” have the same meaning. Each strand of an siRNA molecule contains about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). Example 1

[0042] This invention provides a small interfering RNA (sRNA) for treating cervical cancer. The sRNA includes a first sRNA and a second sRNA. The first sRNA is siE6 / E7, where siE6 / E7 is a sequence with a sense strand as shown in SEQ ID NO. 7 and an antisense strand as shown in SEQ ID NO. 8. The second sRNA includes one of the following sequences: a sequence with a sense strand as shown in SEQ ID NO. 1 and an antisense strand as shown in SEQ ID NO. 1, a sequence with a sense strand as shown in SEQ ID NO. 3 and an antisense strand as shown in SEQ ID NO. 4, and a sequence with a sense strand as shown in SEQ ID NO. 5 and an antisense strand as shown in SEQ ID NO. 6.

[0043] Small interfering RNAs can efficiently and specifically block the expression of homologous genes in vivo through RNA interference, promote the degradation of homologous mRNA, and induce cells to exhibit a phenotype with specific gene deletions.

[0044] Specifically, the positive strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-ACGACUUCACCGUCAAGCA-3' (SEQ ID NO: 1); siCTBP2: 5'-GCGCUUGUGUCAGUAAUAG-3' (SEQ ID NO: 3); siCTBP: 5'-GGGAGGACCUGGAGAAGUU-3' (SEQ ID NO: 5).

[0045] Specifically, the antisense strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-UGCUUGACGGUGAAGUCGU-3' (SEQ ID NO: 2); siCTBP2: 5'-CAUUACUGACCAGGCGC-3' (SEQ ID NO: 4); siCTBP: 5'-AACUUCUCCAGGUCCUCCC-3' (SEQ ID NO: 6).

[0046] Specifically, the sense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CAUUUACCAGCCCGACGAG-3' (SEQ ID NO: 7); the antisense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CUCGUCGGGCUGGUAAAUG-3' (SEQ ID NO: 8).

[0047] In this embodiment, the ends (e.g., the 3' ends) of the sense and / or antisense strands of the aforementioned interfering RNA (e.g., siRNA) molecule may also have n overhangs to increase the activity of the interfering RNA. The overhangs can be the same or different deoxyribonucleotides (e.g., deoxythymidine (dT), deoxycytidine (dC), deoxyuridine (dU), etc.), and n is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), particularly an integer from 2 to 4; in one embodiment of the invention, n = 2, and the overhangs can be dTdT, dTdC, or dUdU, etc.

[0048] In this embodiment, the 3' ends of both the sense and antisense strands of the aforementioned small interfering RNA molecule are provided with dTdT ...

[0049] Specifically, the positive strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-ACGACUUCACCGUCAAGCAdTdT-3'; siCTBP2: 5'-GCGCUUGUGUCAGUAAUAGdTdT-3'; siCTBP: 5'-GGGAGGACCUGGAGAAGUUdTdT-3'.

[0050] Specifically, the antisense strand of the second small interfering RNA contains or is composed of the following nucleotide sequences: siCTBP1: 5'-UGCUUGACCGGUGAAGUCGUdTdT-3'; siCTBP2: 5'-CAUUACUGACCAGGCGCdTdT-3'; siCTBP: 5'-AACUUCUCCAGGUCCUCCCdTdT-3'.

[0051] Specifically, the sense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CAUUUACCAGCCCGACGAGdTdT-3'; the antisense strand of siE6 / E7 contains or is composed of the following nucleotide sequence: siE6 / E7: 5'-CUCGUCGGGCUGGUAAAUGdTdT-3'.

[0052] In this embodiment, the first small interfering RNA silences the E6 / E7 gene, and the second small interfering RNA silences the CTBP gene. The CTBP antagonizes E6 / E7 during the G1 / S transition phase through the cell cycle-related regulatory factor p21.

[0053] In this embodiment, the positive strand of the selected negative control group siCtrl contains or is composed of the following nucleotide sequence: siCtrl: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO: 9); the antisense strand of siCtrl contains or is composed of the following nucleotide sequence: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO: 10).

[0054] Specifically, the selected negative control group had a dangling base dTdT at the 3' end of both the sense and antisense strands.

[0055] The technical solution of the present invention will be further explained below with reference to experiments.

[0056] 1. siRNA design and transfection

[0057] Based on the fundamental principles of siRNA target sequences, four-nucleotide siRNA sequences were designed using the human E6 / E7 gene transcript (Genbank: AF339137.1) and CTBP gene transcript (Genbank: NM 001377192.1), namely siE6 / E7, siCTBP1, siCTBP2, and siCTBP (indicating the joint knockdown of CTBP1 and CTBP2), including the sense and antisense strands. The base sequences are as follows: siCTBP1: Justice: 5'-ACGACUUCACCGUCAAGCAdTdT-3', Antonym: 5'-UGCUUGACGGUGAAGUCGUdTdT-3'; siCTBP2: Justice: 5'-GCGCUUGUGUCAGUAAUAGdTdT-3', Antonym: 5'-CAUUACUGACCAAGGCGCdTdT-3'; siCTBP: Justice: 5'-GGGAGGACCUGGAGAAGUUdTdT-3', Antonym: 5'-AACUUCUCCAGGUCCUCCCdTdT-3'; siE6 / E7: Justice: 5'-CAUUUACCAGCCCGACGAGdTdT-3', Antonym: 5'-CUCGUCGGGCUGGUAAAUGdTdT-3'.

[0058] The negative control group siCtrl base sequence selected in this embodiment is as follows (siCtrl, Qiagen, Germany): Justice: 5'-UUCUCCGAACGUGUCACGUdTdT-3', Antonym: 5'-ACGUGACACGUUCGGAGAAdTdT-3'.

[0059] According to an embodiment of the present invention, the commonly used cationic liposome Lipofectamine 2000 was selected as the transfection reagent.

[0060] The cell line used in this invention is HeLa cells. The cell transfection method is as follows: 1) Based on the siRNA synthesis report, add 125 μL of DEPC water to prepare a 1.00 OD solution. 260 Storage solution; 2) Seed cells in 12-well plates at a density of 7.5 × 10⁶ cells per well. 4 Spread the cells out in large quantities and culture them until they adhere to the wall. 3) Add 2 μL of siRNA and 2 μL of Lipofectamine 2000 to 200 μL of serum-free medium (Opti-MEM), mix thoroughly, and let stand at room temperature for 10 minutes. At this point, the ratio of siRNA to Lipofectamine 2000 and serum-free medium in the mixture is 1:1:100. 4) Add the transfection mixture from step 3) dropwise to the cell culture wells; 5) Continue culturing the cells for 48 hours in preparation for subsequent experiments.

[0061] 2. Cell proliferation and cell cycle analysis

[0062] HeLa cells (1×10⁻⁶) treated with siRNA for 48 h were collected after trypsin digestion. 5Cells were fixed overnight in 70% cold ethanol at 4˚C. Cell cycle progression was assessed by staining with 2 μg / mL RNase (Sigma, USA) and 50 μg / mL propidium iodide (PI). Samples were analyzed by flow cytometry using a FACS Calibur system (BD Biosciences, USA). The percentage of cell cycle phases was calculated using Mod-Fit LT software.

[0063] Figure 1 This is an electron micrograph of the number of HeLa cells after 48 h of transfection with siCTBP1, siCTBP2 and siCTBP according to the embodiments of the present invention; Figure 2 This is a statistical graph of the number of cells transfected with siCTBP1, siCTBP2 and siCTBP in HeLa cells at 0h, 24h, 48h and 72h using Graphpad in an embodiment of the present invention. The horizontal axis represents the number and the vertical axis represents the treatment time. Figure 3 This is a cell cycle detection diagram of HeLa cells transfected with siCTBP1, siCTBP2 and siCTBP 48 h and 72 h using FACS analysis according to an embodiment of the present invention; Figure 4 This is a statistical graph of cell proliferation in HeLa cells co-transfected with knockdown E6 / E7 and knockdown CTBP at 0h, 24h, 48h and 72h, analyzed by FACS in an embodiment of the present invention. The horizontal axis represents the number and the vertical axis represents the treatment time. Figure 5 This is a cell cycle detection diagram of HeLa cells co-transfected with knockdown E6 / E7 and knockdown CTBP using FACS analysis according to an embodiment of the present invention; Figure 6 This embodiment of the invention uses Graphpad to target... Figure 5 A statistical graph of the cell cycle.

[0064] Depend on Figure 1 and Figure 2 It can be seen that compared with the siCtrl control group, the number of cells in the siCTBP1, siCTBP2 and siCTBP treatment groups was significantly reduced. The reduction was more obvious in the siCTBP2 and siCTBP groups at 48 h and 72 h, respectively. This indicates that siCTBP1 and siCTBP2 are both necessary for the proliferation of HeLa cells, and the role of siCTBP2 may be more prominent.

[0065] Depend on Figure 3It was found that, compared with the siCtrl control group, the siCTBP1 treatment group showed a significant increase in G1 phase and a significant decrease in S phase at 48 h and 72 h, indicating that siCTBP1 induces G1 phase arrest. Conversely, cells treated with siCTBP2 and siCTBP showed a decrease in G1 phase and a significant increase in S phase at 72 h, indicating that siCTBP2 increased the number of cells in S phase. The siCTBP treatment group showed a significant decrease in G1 phase and a significant increase in G2 phase, with the increase in G2 phase being more pronounced at 72 h. This suggests that both CTBP1 and CTBP2 are essential for cell phase transitions, especially the G1 / S phase transition.

[0066] Depend on Figure 4 , Figure 5 and Figure 6 It can be seen that, compared with the SiCtrl control group, the cell proliferation in the siE6 / E7 treatment group was significantly reduced. Figure 4 The combined treatment of siE6 / E7 and siCTBP1 resulted in a slight decrease in cell proliferation at 72 h compared to the siE6 / E7 treatment group. The combined treatment of siE6 / E7 and siCTBP2 or siE6 / E7 and siCTBP2 significantly inhibited cell proliferation at both 48 h and 72 h. Figure 4 The knockdown of E6 / E7 induced severe G1 phase arrest. Figure 5 BC, Figure 6 In cells treated with CTBP in combination with E6 / E7, G1 phase arrest was almost eliminated, although in cells treated with CTBP1 or CTBP2 in combination with E6 / E7 knockdown, G1 phase arrest was slightly reduced. Figure 5 DF Figure 6 ).

[0067] 3. Apoptosis analysis

[0068] 1) Collect HeLa cells (1~5 x 10⁻⁵ cells) treated with siRNA for 48 h after trypsin digestion. 5 Centrifuge at 800 rpm, 4℃, for 5 min and remove the supernatant; 2) Wash with 1 mL of pre-cooled PBS, centrifuge at 800 rpm, 4℃, for 5 min and discard the supernatant; 3) Add 100 μL of 1xBinding Buffer to resuspend the cells; 4) Add 5 μL Annexin V-FITC and 10 μL PI Staining Solution to the cell suspension and mix gently; 5) After reacting at room temperature in the dark for 10-15 minutes, add 400 μL of 1x Binding Buffer, mix well, place on ice, and detect cell apoptosis by flow cytometry within 1 hour.

[0069] Figure 7 This is a graph showing the detection of apoptosis in HeLa cells transfected with CTBP knockdown, E6 / E7 knockdown, and E6 / E7 knockdown combined with CTBP knockdown using Annexin V-FITC and PI combined with FACS analysis in an embodiment of the present invention. Figure 8 This embodiment of the invention uses Graphpad to target... Figure 7 Statistical graph of cell apoptosis.

[0070] Depend on Figure 7 and Figure 8 It can be seen that: 48 hours after transfection, the co-silencing of E6 / E7 and CTBP induced strong apoptosis in HeLa cells. Figure 7 Furthermore, the proportion of apoptotic cells in the E6 / E7 and CTBP transient knockdown groups was significantly increased to super-high levels, reaching almost 50% in both groups. Figure 8 Therefore, the decrease in cell number may be due to apoptosis caused by the knockdown of E6 / E7 and CTBP.

[0071] 4. Effects of small interfering RNA targeting and silencing of E6 / E7 and CTBP genes on p21 and p53 expression

[0072] 4.1. RNA extraction

[0073] 4.1.1 48 h after transfection with siRNA, cells in 12-well plates were digested with 0.25% trypsin and transferred to 1.5 ml EP tubes. 200 μL of Trizol was added to fully lyse the cells. 4.1.2 Add 40 μL of chloroform, vortex for 30 s, and let stand for 3 minutes. Centrifuge at 12000 rpm for 15 min at 4℃, and transfer the supernatant to a new EP tube; 4.1.3 Add an equal volume of isopropanol, invert the container 6-8 times to mix thoroughly, let stand at room temperature for 10 min, then centrifuge at 12000 rpm for 10 min at 4℃. 4.1.4 Discard the supernatant, add 1 ml of 75% ethanol, and centrifuge at 10,000 rpm for 5 min at 4°C; 4.1.5 Discard the supernatant, air dry at room temperature for 2 min, add 30 μL of DEPC water to each tube, and dissolve at room temperature; 4.1.6 Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the concentration.

[0074] 4.2 Reverse transcribe the above RNA into cDNA

[0075] Reverse transcription was performed using the Prime Script™ RT Reagent Kit (Takara, Japan), configured on ice, as shown in Table 1.

[0076]

[0077] Specifically, add 4 μL of 4x gDNA wiper Mix and 500 ng of total RNA to a 200 μL EP tube, add DEPC water to make up to 16 μL, and incubate in a PCR instrument at 42℃ for 2 min. After removing the tube, add 4 μL of 5x HiScript IIqRT SuperMix II to each tube and incubate in a PCR instrument at 37℃ for 15 min and 85℃ for 5 s.

[0078] 4.3. Real-time quantitative PCR (RT-qPCR)

[0079] The obtained liquid was diluted 4-fold with DEPC water and then subjected to RT-qPCR analysis. The RT-qPCR reaction system was prepared on ice using SYBR GreenMaster Mix (Applied Biosystems, USA). (The primer sequences for CTBP1, CTBP2, TP53 or p21 and the internal control gene GAPDH are shown in the primer sequence section.) The preparation scheme is shown in Table 2.

[0080]

[0081] Specifically, add 1 μL of cDNA, 0.4 μL each of CTBP1, CTBP2, p53 or p21 forward and reverse primers, and 50x ROX Reference Dye1 to each well of a 96-well plate, along with 10 μL of 2x ChamQ SYBR qPCR Master Mix. Add DEPC water to bring the total volume to 20 μL, with three replicates per sample. Place the 96-well plate in a qPCR instrument for amplification under the following conditions: 95℃ for 30 s denaturation; 95℃ for 10 s; 60℃ for 30 s; 40 cycles. After amplification, perform melting curve analysis and collect the data at 95℃ for 15 s; 60℃ for 1 min; and 95℃ for 15 s.

[0082] 4.4 Analysis of RT-qPCR results

[0083] After the program finishes running, export the Ct values ​​of the target gene to a newly created Excel spreadsheet, using 2... -ΔΔCt The expression of relevant genes was analyzed. After normalizing the control group values, the relative expression levels of the target genes were calculated, and a gene expression bar chart was plotted.

[0084] The primer sequences are as follows: GAPDH: Forward: 5'-GCACCGTCAAGGCTGAGAAC-3' (SEQ ID NO.11) Reverse: 5'-ATGGTGGTGAAGACGCCAGT-3' (SEQ ID NO.12); CTBP1: Forward: 5'-AGCACAACCACCACCTCATCA-3' (SEQ ID NO.13), Reverse: 5'-CCCCTTGTCTCATCTGCTTGA-3' (SEQ ID NO.14); CTBP2: Forward: 5'-CCATAAAGCAGATGAGGCAGG-3' (SEQ ID NO.15) Reverse: 5'-GCTAAGGCTTTCTCGTCCACC-3' (SEQ ID NO.16); CDKN1A (p21): Forward: 5'-CGATGGAACTTCGACTTTGTCA-3' (SEQ ID NO.17) Reverse: 5'-GCACAAGGGTACAAGACAGTG-3' (SEQ ID NO.18); TP53 (p53): Forward: 5'-GAGGTTGGCTCTGACTGTACC-3' (SEQ ID NO.19) Reverse: 5'-TCCGTCCCAGTAGATTACCAC-3' (SEQ ID NO. 20).

[0085] 4.5 Experimental Results

[0086] like Figure 9 As shown, using GAPDH as an internal reference gene, through 2 -ΔΔCtRelative expression levels were calculated. Compared to the siCtrl control group, CTBP1 and CTBP2 were significantly reduced in knockdown cells. p21 transcription was significantly reduced in both the siCTBP2 and siCTBP treatment groups, consistent with the decrease in G1 phase. Knockdown of E6 / E7 led to a significant increase in p21, consistent with severe G1 phase arrest. Co-knockdown of CTBP and E6 / E7 resulted in p21 expression almost equal to that of siCtrl, confirming the antagonistic role of CTBP and E6 / E7 in p21 transcriptional regulation. p53 is a recognized upstream regulator of p21; compared to siCtrl, no significant changes in p53 transcription were found in all cells.

[0087] Example 2 This invention also provides the application of the small interfering RNA for treating cervical cancer according to Example 1 in the preparation of a drug for treating cervical cancer, wherein the small interfering RNA targets and downregulates the expression of E6 / E7 and CTBP genes in cervical cancer cells.

[0088] In this embodiment, the small interfering RNA inhibits the proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells.

[0089] Example 3 The present invention also provides a small interfering RNA delivery system according to Example 1, the delivery system comprising the small interfering RNA and vector described in Example 1.

[0090] Furthermore, the vector can be a viral vector or a non-viral vector.

[0091] Specifically, viral vectors include lentiviruses, retroviruses, adenoviruses, and herpes simplex viruses; non-viral vectors include liposomes, polymers, peptides, antibodies, aptamers, or combinations thereof.

[0092] Example 4 This invention also provides a pharmaceutical composition comprising the small interfering RNA described in Example 1 or the delivery system described in Example 3, and pharmaceutically acceptable excipients.

[0093] This pharmaceutical composition can be used to deliver small interfering RNA to the desired site in any manner. For example, the small interfering RNA of this embodiment can be administered via intramuscular, intraperitoneal, or intravenous injection. As a specific embodiment, the small interfering RNA is injected into the individual through a muscle in the limbs, such as the arm or leg.

[0094] Example 5 The present invention also provides an application of the pharmaceutical composition according to Example 4 in the preparation of a treatment for cervical cancer, wherein the pharmaceutical composition targets and downregulates the expression of E6 / E7 and CTBP genes in cervical cancer cells.

[0095] In this embodiment, the pharmaceutical composition inhibits the proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0097] The embodiments and methods described herein may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0098] In this invention, simply listing the steps of the method in a certain order does not constitute any restriction on the order of the method steps.

Claims

1. A small interfering RNA for treating cervical cancer, characterized in that, The small interfering RNA includes a first small interfering RNA and a second small interfering RNA. The first small interfering RNA is siE6 / E7, where siE6 / E7 is a nucleotide sequence of the positive strand as shown in SEQ ID NO.7 and an antisense strand as shown in SEQ ID NO.

8. The second small interfering RNA includes a positive strand and an antisense strand that is inversely complementary to it. The positive strand includes the nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.5, and the antisense strand includes the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.

6.

2. The small interfering RNA for treating cervical cancer according to claim 1, characterized in that, The small interfering RNA further comprises a dangling base, the number of which is 1-8, the dangling base being a deoxynucleoside, and the dangling base being located at the 3' end of the sense strand and / or antisense strand of the small interfering RNA.

3. The small interfering RNA for treating cervical cancer according to claim 2, characterized in that, The small interfering RNA molecule has a dangling base dTdT at the 3' end of both the sense and antisense strands.

4. The small interfering RNA for treating cervical cancer according to claim 1, characterized in that, The first small interfering RNA silences the E6 / E7 gene, and the second small interfering RNA silences the CTBP gene, wherein CTBP antagonizes E6 / E7 during the G1 / S transition phase through the cell cycle-related regulatory factor p21.

5. The use of the small interfering RNA for treating cervical cancer according to any one of claims 1-4 in the preparation of a drug for treating cervical cancer, characterized in that, The small interfering RNA targets and downregulates the expression of E6 / E7 and CTBP genes in cervical cancer cells.

6. The application according to claim 5, characterized in that, The small interfering RNA inhibits the proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells.

7. A delivery system, characterized in that, The delivery system comprises the small interfering RNA and vector as described in any one of claims 1-4.

8. A pharmaceutical composition, characterized in that, It includes the small interfering RNA according to any one of claims 1-4 or the delivery system according to any one of claims 6-7, and pharmaceutically acceptable excipients.

9. The application of the pharmaceutical composition according to claim 8 in the preparation of a treatment for cervical cancer, characterized in that, The drug composition targets and downregulates the expression of E6 / E7 and CTBP genes in cervical cancer cells.

10. The application of the pharmaceutical composition according to claim 9 in the preparation of a treatment for cervical cancer, characterized in that, The pharmaceutical composition inhibits the proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells.

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