Application of lncRNA SVIL-AS1 as a target in AKT1 E17K mutant tumors

Abstract

This invention discloses the application of lncRNA SVIL-AS1 as a target in AKT1E17K mutant tumors, belonging to the field of molecular biology technology. This invention provides the application of lncRNA SVIL-AS1-related biomaterials in the preparation of products for regulating the function of AKT1E17K mutant cells. This invention verifies that SVIL-AS1 can specifically bind to AKT1E17K mutants and inhibit their phosphorylation level and kinase function, thereby inhibiting the proliferation of AKT1E17K mutant cells, revealing the role of SVIL-AS1 in the treatment of AKT1E17K mutant tumors. This invention applies lncRNA SVIL-AS1 to the treatment of AKT1E17K mutant tumors, not only providing a new source for the preparation of materials for the prevention and treatment of AKT1E17K mutant tumors, but also exploring new pharmaceutical value of lncRNA SVIL-AS1.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to the application of lncRNA SVIL-AS1 as a target in AKT1E17K mutant tumors. Background Technology

[0002] AKT1, also known as protein kinase B, is a serine / threonine protein kinase. As a key node in the PI3K-AKT-mTOR signaling pathway, it plays a central role in responses to stimuli from hormones, growth factors, and cytokines. Due to its diverse substrates and broad functional scope, AKT1 is crucial for cell growth, survival, and energy metabolism. In malignant tumors, various forms of abnormal activation of AKT1 are frequently observed, with AKT1 gene mutations being one of the most significant contributing factors.

[0003] The most common AKT1 mutation is the AKT1 E17K mutation, where glutamic acid at position 17 is replaced by lysine. Multiple studies have detected the AKT1 E17K mutation in various cancers, including breast cancer, bladder cancer, endometrial cancer, colorectal cancer, and acute lymphoblastic leukemia. Although the mutation frequency is approximately 2%-7%, the AKT1 E17K mutation is associated with malignant transformation of tumors and poor patient prognosis.

[0004] Given the crucial role of AKT in tumors, various small-molecule inhibitors of AKT have been developed, and several targeted drugs targeting the PI3K-AKT-mTOR signaling pathway are either on the market or in clinical trials. Targeting key molecules in tumor cell signaling pathways has yielded some success in cancer treatment. Although studies have shown that patients carrying AKT1 E17K mutations can benefit from AKT inhibitor therapy, not only do mutated patients exhibit varying sensitivities to different types of AKT inhibitors, but their responses to PI3K and mTOR inhibitors also differ from those of AKT1 wild-type patients. This suggests that the activation mechanism of AKT1 E17K and its regulatory roles with molecules such as PI3K or mTOR remain unclear. The limitations of current drug candidates underscore the necessity and urgency of discovering new therapeutic targets and treatment methods. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of lncRNA SVIL-AS1 as a target in AKT1E17K mutant tumors.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is: the application of lncRNA SVIL-AS1 related biological materials in the preparation of products for regulating the function of AKT1 E17K mutant cells.

[0007] Currently, patients carrying the AKT1 E17K mutation can be treated with AKT inhibitors. However, different AKT1 E17K mutation patients have varying sensitivities to AKT inhibitors, and some patients exhibit resistance. Through extensive research, the inventors of this application have discovered that lncRNA SVIL-AS1 can specifically bind to the AKT1 E17K mutant and inhibit its phosphorylation level and kinase function, thereby suppressing the proliferation of AKT1 E17K mutant cells. This suggests that lncRNA SVIL-AS1 could serve as a novel therapeutic target for AKT1 E17K mutant tumors.

[0008] As a preferred embodiment of the application described in this invention, the sequence of the lncRNA SVIL-AS1 is shown in SEQ NO:1.

[0009] As a preferred embodiment of the application described in this invention, the lncRNA SVIL-AS1-related biological material includes substances that knock down lncRNA SVIL-AS1 expression.

[0010] As a preferred embodiment of the application described in this invention, the regulation of AKT1 E17K mutant cell function includes inhibiting the proliferation of AKT1 E17K mutant cells.

[0011] The present invention also provides the use of the reagent that targets and knocks down lncRNA SVIL-AS1 in at least one of the following (a)-(b): (a) preparing a product for treating AKT1 E17K mutant tumors; (b) preparing a product for improving drug resistance in AKT1 E17K mutant tumors.

[0012] This invention also provides the application of reagents that target and knock down lncRNA SVIL-AS1 in combination with AKT pathway inhibitors in the preparation of products for treating AKT1 E17K mutant tumors.

[0013] The inventors of this application have discovered that knocking down SVIL-AS1 can improve the reduced sensitivity of AKT1 E17K mutant cells to PI3K inhibitors and AKT allosteric inhibitors. Therefore, in the future treatment of AKT1E17K mutant tumors, targeting SVIL-AS1 in combination with PI3K inhibitors or AKT allosteric inhibitors may be an effective treatment strategy.

[0014] The present invention also provides a pharmaceutical composition for treating AKT1 E17K mutant tumors, the pharmaceutical composition comprising a substance that targets and knocks down lncRNA SVIL-AS1.

[0015] The present invention also provides a reagent for detecting the expression level of lncRNA SVIL-AS1 in the preparation of products for predicting the prognosis of breast cancer.

[0016] In a preferred embodiment of the application described in this invention, subjects with high expression of lncRNA SVIL-AS1 have a relatively poor prognosis, while subjects with low expression of lncRNA SVIL-AS1 have a relatively good prognosis.

[0017] The inventors of this application have found that lncRNA SVIL-AS1 is expressed in breast cancer, and that patients with high expression of SVIL-AS1 have shorter overall survival and disease-free survival.

[0018] The present invention also provides a breast cancer prognostic kit, the kit comprising reagents for detecting the expression level of lncRNA SVIL-AS1.

[0019] Beneficial effects of this invention: This invention provides the application of lncRNA SVIL-AS1 as a target in AKT1 E17K mutant tumors. This invention verifies that SVIL-AS1 can specifically bind to AKT1 E17K mutants and inhibit their phosphorylation level and kinase function, thereby inhibiting the proliferation of AKT1 E17K mutant cells, revealing the role of SVIL-AS1 in the treatment of AKT1 E17K mutant tumors. This invention applies lncRNA SVIL-AS1 to the treatment of AKT1E17K mutant tumors, not only providing a new source for the preparation of materials for the prevention and treatment of AKT1 E17K mutant tumors, but also exploring new pharmaceutical value of lncRNA SVIL-AS1. Attached Figure Description

[0020] Figure 1 AB represents the expression of lncRNA SVIL-AS1 in MDA-MB-231 cells, MCF-10A WT cells, MCF-10AAKT1 E17K cells, IHH4 cells, and KU-19-19 cells detected by qRT-PCR; CD represents the changes in AKT phosphorylation level and downstream PRAS40 phosphorylation in AKT1 E17K mutant cell lines MCF-10A AKT1 E17K, KU-19-19, and IHH-4 cells after SVIL-AS1 overexpression or knockdown, detected by Western blot.

[0021] Figure 2A: Colony formation assay to detect the number of colonies formed in MCF-10A wild-type cells, MCF-10AAKT1 E17K, KU-19-19, and IHH-4 cell lines after SVIL-AS1 knockdown; B: MTS assay to detect the number of colonies formed in MCF-10A wild-type cells, MCF-10AAKT1 E17K, KU-19-19, and IHH-4 cell lines after SVIL-AS1 knockdown. The cell lines E17K, KU-19-19, and IHH-4 show proliferation; C shows tumor volume changes in the MCF-10A cell line with stable SVIL-AS1 knockdown; D shows tumor weight changes in the MCF-10A cell line with stable SVIL-AS1 knockdown; E shows tumor growth changes in the MCF-10A cell line with stable SVIL-AS1 knockdown; F shows tumor volume changes in the KU-19-19 cell line with stable SVIL-AS1 knockdown; G shows tumor weight changes in the KU-19-19 cell line with stable SVIL-AS1 knockdown; H shows tumor growth changes in the KU-19-19 cell line with stable SVIL-AS1 knockdown.

[0022] Figure 3 A represents the dose-response curves of MK2206 to MCF-10A wild-type and AKT1 E17K mutant cells; B represents the dose-response curves of BYL719 to MCF-10A wild-type and AKT1 E17K mutant cells; CH represents the effect of SVIL-AS1 knockdown combined with MK2206 / BYL719 in treating AKT1 E17K mutant subcutaneous tumors; I represents the Ki67 expression when SVIL-AS1 knockdown combined with MK2206 / BYL719 is used to treat AKT1 E17K mutant subcutaneous tumors; JK represents the phosphorylation level of AKT1 in SVIL-AS1 knockdown combined with MK2206 / BYL719 in treating AKT1 E17K mutant subcutaneous tumors.

[0023] Figure 4 A shows the expression of SVIL-AS1 in breast cancer detected by immunohistochemistry; B shows the statistical correlation between SVIL-AS1 and T stage, N stage and recurrence of breast cancer; CE shows the Kaplan-Meier analysis of breast cancer case cohorts; F shows the correlation between SVIL-AS1 and AKT1 phosphorylation. Detailed Implementation

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0025] Example 1

[0026] In this embodiment, SVIL-AS1 was overexpressed or knocked down in MCF-10A AKT1 E17K cell lines carrying the AKT E17K mutation, IHH-4 cell lines, and KU-19-19, while MCF-10A wild-type cell lines were used as controls, and Western blot analysis was performed.

[0027] The specific experimental methods are as follows: Following the instructions for use of Lipofectamine RNAiMAX (#13778150, Invitrogen), siRNA was transfected into cells. Lentiviral solution was prepared by transfecting shRNA plasmid / overexpression SVIL-AS1 plasmid and lentiviral plasmid vectors (pMD2.G, pSPAX2) into HEK293FT cells according to the instructions for use of Lipofectamine 3000 transfection reagent. The viral solution was collected at 48h and 72h. The culture medium for the cells to be infected was discarded, and the virus solution was added, along with polybrene. After 12-24h of infection, the culture medium was replaced with ordinary complete culture medium. At 72h, antibiotics such as puromycin or G418 were added for selection based on the different resistances of the virus.

[0028] The siRNA sequence used for SVIL-AS1 knockdown is as follows:

[0029] Sense(5'-3'):GCUACUGCCUCAGUCACUUTT;

[0030] Anti-Sense(5'-3'):AAGUGACUGAGGCAGUAGCTT.

[0031] The sequence of the shRNA is: AAGUGACUGAGGCAGUAGCTT.

[0032] Extraction and quantification of total cellular protein: Cells were discarded from the culture medium, washed twice with pre-cooled PBS, and lysed with RIPA lysis buffer containing protease and phosphatase inhibitors (adjusting the lysis buffer volume according to the bottom area of ​​the culture dish). After lysing on ice for 20 min, the cell lysate was transferred to a clean 1.5 ml EP tube using a cell scraper. The tubes were centrifuged at 12000 g for 20 min at 4°C, and the supernatant was transferred to a new EP tube. Protein concentration was determined using the BCA method. The required lysis buffer volume was calculated based on the target loading amount, and loading buffer was added. After mixing, the cells were denatured at 98°C for 10 min.

[0033] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): Mix all reagent components according to the kit instructions and pour into the gel preparation plate. Add an appropriate amount of protein sample and use a pre-stained protein marker as a molecular size indicator. Electrophore at a constant voltage of 60-80V until the protein enters the separating gel, then adjust the voltage to 120V and continue electrophoresis at a constant voltage until the bromophenol blue reaches the bottom of the separating gel.

[0034] Protein transfer: Activate the PVDF membrane by soaking it in methanol for 1 min and then place it in electroporation buffer for later use. After electrophoresis, remove the gel and assemble the electroporation apparatus in a pan containing electroporation buffer, following this order: cathode - sponge - filter paper - electrophoresis gel - PVDF membrane - filter paper - sponge - anode. Place the clamped electroporation clamp into the electroporation tank, pour in the electroporation buffer, and place the electroporation tank in an ice-water mixture. Electroporate at a constant current of 250 mA for 2 h.

[0035] Blocking and antibody incubation: After electroporation, remove the PVDF membrane and place it in an incubation chamber containing an appropriate amount of blocking buffer (TBST buffer containing 5% BSA or skim milk powder). Incubate at room temperature on a horizontal decolorizing shaker for 1 hour. Dilute the primary antibody with 5% BSA at a certain ratio and incubate overnight at 4°C on a decolorizing shaker. Wash three times with rapid shaking in TBST buffer, 10 minutes each time. Incubate the secondary antibody working solution at room temperature with slow shaking for 1 hour. Wash three times with rapid shaking in TBST buffer, and then proceed with subsequent development and photographic recording using ultrasensitive ECL chemiluminescence reagent.

[0036] The results are as follows Figure 1 As shown. By Figure 1 AB analysis showed that SVIL-AS1 was successfully overexpressed or knocked down in MDA-MB-231 cells, MCF-10A WT cells, MCF-10A AKT1 E17K cells, IHH4 cells, and KU-19-19 cells; Figure 1 C indicates that overexpression of SVIL-AS1 resulted in a slight increase in the phosphorylation levels of AKT and its downstream substrate PRAS40 in the MCF-10A wild-type cell line, while all cells carrying the AKT1 E17K mutation showed a significant increase in AKT1, AKT1 phosphorylation levels, and PRAS40 phosphorylation levels. Figure 1 As can be seen from D, after knocking down SVIL-AS1, the phosphorylation levels of AKT1 and its downstream substrate PRAS40 in the MCF-10A wild-type cell line did not change significantly or were slightly inhibited, while all cells carrying the AKT1 E17K mutation showed significantly inhibited AKT1, AKT1 phosphorylation levels, and PRAS40 phosphorylation levels.

[0037] Example 2

[0038] In this embodiment, to further investigate the effect of SVIL-AS1 on tumor cell function, SVIL-AS1 was knocked down in the cell lines MCF-10A AKT1 E17K, IHH4 cells and KU-19-19 cells carrying the AKT1E17K mutation, and MTS experiments were performed.

[0039] The specific experimental method is as follows: 1000-3000 cells / 200 μL were seeded into 96-well plates, with 3 replicates per group. Prepare MTS working solution (20 μL MTS + 100 μL culture medium) / well and discard the culture medium in the test wells. Add 120 μL of the prepared MTS working solution / well and incubate in the dark for 2 hours. Use a multi-mode microplate reader (wavelength 492 nm) to detect the blank control wells; the difference between the OD value of the test well and the OD value of the blank well is the final value for each well. When detecting the IC50 of the drug, multiple concentration gradient groups were set up, and the results were detected 24 hours after drug addition. When detecting cell proliferation, measurements were taken at 6 time points: day 0, day 1, day 2, day 3, day 4, and day 5. Finally, a cell proliferation curve was plotted.

[0040] The test results are as follows Figure 2 As shown. By Figure 2 As shown in Figure A, knockdown of SVIL-AS1 significantly inhibited the proliferation of MCF-10A AKT1 E17K, IHH4, and KU-19-19 cells carrying the AKT1 E17K mutation, while the proliferation inhibition of MCF-10A wild-type cells was significantly weaker than that of cells carrying the AKT1 E17K mutation; Figure 2 B shows that after SVIL-AS1 knockdown, the number of colonies formed on plate in the three AKT1 E17K mutant cell lines was significantly reduced compared to the control group. The effect of SVIL-AS1 knockdown on colony formation in MCF-10A wild-type cells was significantly weaker than that in cells carrying the AKT1 E17K mutation. In MCF-10A and KU-19-19 cells, SVIL-AS1 knockdown significantly inhibited the growth rate and size of xenografts in nude mice compared to the control group, and the inhibition rate of tumor growth was even higher in the context of AKT1 E17K mutation. Figure 2 CH). As can be seen from the above, knocking down SVIL-AS1 may be used to treat tumors with AKT1 E17K mutations.

[0041] Example 3

[0042] This embodiment studies the effect of SVIL-AS1 on the drug resistance process of AKT1 E17K mutant cells through experiments. The specific experimental method is as follows:

[0043] (1) Detection of IC50 of MCF-10A wild-type and AKT1 E17K mutant cells against AKT-PI3K pathway inhibitors MK2206 and BYL719: 1000-3000 cells / 200 μL were seeded into 96-well plates, with 3 replicates per group. MTS working solution (20 μL MTS + 100 μL culture medium) / well was prepared, and the culture medium in the test wells was discarded. 120 μL of the prepared MTS working solution / well was added to each well, and the plates were incubated in the dark for 2 hours. Blank control wells were tested using a multi-mode microplate reader (wavelength 492 nm). The difference between the OD value of the test well and the OD value of the blank well was the final value for each well. Multiple concentration gradient groups were set up, and the results were detected 24 hours after drug addition. Results are as follows: Figure 3 As shown in AB, cells carrying the AKT1 E17K mutation are less sensitive to BYL719 and MK2206.

[0044] (2) Further in vivo experiments:

[0045] Tumorigenesis experiment of KU-19-19 stable cell line: Thirty immunodeficient female nude mice aged 4-5 weeks were randomly divided into 6 groups of 5 mice each. Three groups of mice were subcutaneously inoculated with KU-19-19sh-NC cells on their flanks (one group received saline, one received BYL719, and one received MK2206), while the other three groups were inoculated with KU-19-19sh-SVIL-AS1 cells (one group received saline, one received BYL719, and one received MK2206). The inoculation dose was 5 × 10⁻⁶ cells / mL. 6 / 100μl. After injecting tumor cells, observe weekly for tumor formation. Once tumors have formed, measure the longest and shortest diameters of the mass weekly using calipers. Calculate the volume using the formula: Tumor volume = 0.5 × longest diameter × shortest diameter. 2 Calculate the tumor volume.

[0046] Tumorigenicity experiment of stable MCF-10A AKT1 E17K (KRAS G12V) cell lines: Thirty immunodeficient female nude mice aged 4-5 weeks were randomly divided into 6 groups of 5 mice each. MCF-10A and MCF-10AAKT1 E17K cells were first infected with KRAS G12V to obtain tumorigenicity. Then, sh-NC and sh-SVIL-AS1 cells were constructed using the two cell lines respectively. Three groups of nude mice were subcutaneously inoculated with MCF-10A (KRAS G12V) sh-NC cells (subjected to saline, BYL719, and MK2206), while the other three groups were subcutaneously inoculated with MCF-10A AKT1 E17K (KRAS G12V) sh-SVIL-AS1 cells (subjected to saline, BYL719, and MK2206). The inoculation amount was 5 × 10⁶ cells / mL. 6 / 100μl. After injecting tumor cells, observe for tumor formation every three days. Once tumor formation occurs, measure the longest and shortest diameters of the mass weekly using calipers. Calculate the volume using the formula: Tumor volume = 0.5 × longest diameter × shortest diameter. 2 Calculate the tumor volume.

[0047] like Figure 3 As shown in the CH study, knocking down SVIL-AS1 while administering MK2206 or BYL719 significantly reduced the size of subcutaneous tumors. Figure 3 I indicates that the expression of Ki-67 is reduced, and the phosphorylation level of AKT1 is also reduced. Figure 3 As shown above, knocking down SVIL-AS1 can increase the sensitivity of AKT1 E17K tumors to MK2206 and BYL719.

[0048] Example 4

[0049] This embodiment investigates the expression of SVIL-AS1 in breast cancer through experiments. The specific experimental methods are as follows: In situ hybridization detection experiment: Paraffin sections were dried in a 60℃ oven for 2 hours. The dried paraffin sections were then rapidly dewaxed by immersing them in xylene for 10 minutes twice. The sections were then fixed and washed sequentially in 100% ethanol I, 100% ethanol II, 95% ethanol, 80% ethanol, 75% ethanol, and distilled water, 5 minutes each time. Endogenous peroxidase was inactivated by treating with 3% H2O2 at room temperature for 10 minutes. In situ hybridization detection was performed using the Sensitivity Enhanced In situ Hybridization Detection Kit I (POD) (BOSTER MK1030). SVIL-AS1 staining depth was categorized as 0 (negative), 1 (light), 2 (medium), and 3 (dark), and staining area was categorized as 0, 1 (0-25%), 2 (25-50%), 3 (50-75%), and 4 (≥75%). The median of 6 was used as the cutoff value. Values ​​greater than 6 indicated high SVIL-AS1 expression, while values ​​less than or equal to 6 indicated low SVIL-AS1 expression.

[0050] The results are as follows Figure 4 As shown. By Figure 4 As shown in Figure A, high expression of SVIL-AS1 was statistically correlated with T stage (P = 0.027), N stage (P = 0.021), relapse (P = 0.014), metastasis (P < 0.001), and survival (P = 0.037). Figure 4 B). In the TCGA database, the 5-year cumulative survival rate and the 5-year disease-free survival rate were 93.2% and 80.4%, respectively. Figure 4 C). In the case cohort, Kaplan-Meier analysis also found that patients with high SVIL-AS1 expression had shorter overall survival and disease-free survival. Figure 4DE). Furthermore, SVIL-AS1 levels are positively correlated with phosphorylated AKT1 levels. Figure 4 F).

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A substance for knocking down the expression of lncRNA SVIL-AS1 in the preparation of a product for regulating the function of AKT1 E17K mutant cells, characterized in that, The substance for knocking down expression of the lncRNA SVIL-AS1 is siRNA or shRNA; a sequence of a sense strand of the siRNA is GCUACUGCCUCAGUCACUUTT, and a sequence of an antisense strand is AAGUGACUGAGGCAGUAGCTT; a sequence of the shRNA is AAGUGACUGAGGCAGUAGCTT; the AKT1 E17K mutant cell function is inhibited is inhibiting proliferation of the AKT1 E17K mutant cell; the mutant cell is a breast cancer cell, a bladder cancer cell or a thyroid cancer cell; and a sequence of the lncRNA SVIL-AS1 is shown in SEQ NO:

1.

2. Application of a substance for targetedly knocking down expression of the lncRNA SVIL-AS1 in at least one of (a)-(b): (a) preparing a product for treating AKT1 E17K mutant tumor; (b) a product that increases the sensitivity of AKT1 E17K mutant tumor patients to AKT inhibitors; characterized in that, The substance for knocking down expression of the lncRNA SVIL-AS1 is siRNA or shRNA; a sequence of a sense strand of the siRNA is GCUACUGCCUCAGUCACUUTT, and a sequence of an antisense strand is AAGUGACUGAGGCAGUAGCTT; a sequence of the shRNA is AAGUGACUGAGGCAGUAGCTT; a sequence of the lncRNA SVIL-AS1 is shown in SEQ NO:1; the mutant tumor is breast cancer or bladder cancer; and the AKT inhibitor is MK2206 or BYL719.

3. The use of a substance targeting the knockdown of lncRNA SVIL-AS1 expression in combination with an AKT pathway inhibitor in the preparation of a product for treating AKT1 E17K mutant tumors, characterized in that, The substance for knocking down expression of the lncRNA SVIL-AS1 is siRNA or shRNA; a sequence of a sense strand of the siRNA is GCUACUGCCUCAGUCACUUTT, and a sequence of an antisense strand is AAGUGACUGAGGCAGUAGCTT; a sequence of the shRNA is AAGUGACUGAGGCAGUAGCTT; a sequence of the lncRNA SVIL-AS1 is shown in SEQ NO:1; the tumor is breast cancer or bladder cancer; and the AKT pathway inhibitor is MK2206 or BYL719.

4. A pharmaceutical composition for treating AKT1 E17K mutant tumors, comprising, The pharmaceutical composition comprises a substance for targetedly knocking down the lncRNA SVIL-AS1; the substance for targetedly knocking down the lncRNA SVIL-AS1 is siRNA or shRNA; a sequence of a sense strand of the siRNA is GCUACUGCCUCAGUCACUUTT, and a sequence of an antisense strand is AAGUGACUGAGGCAGUAGCTT; and a sequence of the shRNA is AAGUGACUGAGGCAGUAGCTT.

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