Application of targeted intervention of HHIPL2-HNF1A regulatory axis in the preparation of anti-non-small cell lung cancer drugs

Abstract

The present invention relates to the application of targeted intervention of HHIPL2-HNF1A regulatory axis in the preparation of anti-non-small cell lung cancer drugs. The HHIPL2-HNF1A regulatory axis includes HHIPL2 gene and HNF1A gene, and the mRNA nucleotide sequences of HHIPL2 gene and HNF1A gene are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The present invention first discovered that the activity of HHIPL2-HNF1A signaling pathway can be more effectively inhibited than the single interference with the expression level of HHIPL2 or HNF1A, and the cell proliferation, migration and invasion of non-small cell lung cancer can be more effectively inhibited. The siMIX designed by the present invention can specifically knock down the mRNA levels of HHIPL2 and HNF1A at the same time. When the same dose of siRNA is transfected, the functional experiment of non-small cell lung cancer cells shows that siMIX has a more effective anti-tumor effect than siHHIPL2 or siHNF1A. Therefore, the HHIPL2-HNF1A regulatory axis can be used as a drug target and applied to the research and development and preparation of anti-tumor drugs for non-small cell lung cancer.

Description

Technical Field

[0001] The invention relates to application of targeted intervention of HHIPL2-HNF1A regulatory axis in preparing anti-non-small cell lung cancer drugs, and belongs to the technical field of biomedicine. Background Art

[0002] Lung cancer is one of the common human malignancies. Its high invasiveness and high tumor heterogeneity pose a serious threat to human health. Lung cancer is mainly divided into two pathological types, small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC accounts for about 85% of confirmed lung cancer cases and can be further divided into squamous cell carcinoma, adenocarcinoma and large cell carcinoma. The existing treatments for NSCLC mainly include surgical resection, adjuvant chemoradiotherapy, targeted therapy and immunotherapy. In recent years, targeted drugs targeting gene mutations such as EGFR and KRAS have brought definite survival benefits to patients in the personalized treatment of NSCLC. However, existing targeted drugs often lead to the development of tumor cell resistance during treatment, thereby accelerating NSCLC patients to enter the terminal stage of cancer. Therefore, it is extremely important to discover new driver genes or signaling pathway axes and develop new intervention drugs based on them to improve the prognosis of NSCLC patients.

[0003] Small interfering RNA (siRNA) is a double-stranded RNA composed of 19-25 pairs of nucleotides and is a key component in RNA interference technology. After siRNA is transfected into cells, it binds to the RNA-induced silencing complex (RISC), and then one of the positive strands of siRNA is degraded. The remaining antisense strand will guide RISC to bind to the target mRNA based on the principle of base complementary pairing, and finally induce the degradation of the mRNA, thereby achieving the purpose of inhibiting the expression of the target gene. Designing siRNA with high specificity and high interference efficiency will play an important role in the targeted treatment of NSCLC. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an application of targeted intervention of the HHIPL2-HNF1A regulatory axis in the preparation of an anti-non-small cell lung cancer drug.

[0005] The technical solution of the present invention is as follows:

[0006] Application of the HHIPL2-HNF1A regulatory axis as a drug target in the preparation of anti-non-small cell lung cancer drugs.

[0007] Preferably according to the present invention, the HHIPL2-HNF1A regulatory axis includes the HHIPL2 gene and the HNF1A gene.

[0008] Preferably according to the present invention, the mRNA nucleotide sequence of the HHIPL2 gene is shown as SEQ ID NO.1, and the mRNA nucleotide sequence of the HNF1A gene is shown as SEQ ID NO.2.

[0009] Preferably, according to the present invention, the anti-non-small cell lung cancer drug uses HHIPL2 and HNF1A genes as intervention targets and interferes with HHIPL2 and HNF1A mRNA as a basis, and can efficiently and specifically inhibit the expression levels of HHIPL2 and HNF1A genes.

[0010] Preferably according to the present invention, the anti-non-small cell lung cancer drug is siRNA, shRNA, microRNA or HHIPL2 and HNF1A mRNA chemical inhibitor.

[0011] Application of siRNA that specifically interferes with HHIPL2 gene expression and siRNA that specifically interferes with HNF1A gene expression in the preparation of anti-non-small cell lung cancer drugs.

[0012] Preferably according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown as SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown as SEQ ID NO.4.

[0013] Preferably according to the present invention, the molar ratio of the siRNA that specifically interferes with the expression of the HHIPL2 gene to the siRNA that specifically interferes with the expression of the HNF1A gene in the anti-non-small cell lung cancer drug is 1:1.

[0014] An anti-non-small cell lung cancer drug comprises siRNA that specifically interferes with HHIPL2 gene expression and siRNA that specifically interferes with HNF1A gene expression.

[0015] Preferably according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown as SEQ ID NO.3, and the nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown as SEQ ID NO.4.

[0016] Beneficial effects:

[0017] The present invention discovered for the first time that the activity of the HHIPL2-HNF1A signaling pathway can be more effectively inhibited by targeted inhibition, compared with the interference of HHIPL2 or HNF1A expression levels alone, cell proliferation, migration and invasion of non-small cell lung cancer can be more effectively inhibited. The siMIX designed by the present invention can simultaneously and specifically knock down the levels of HHIPL2 and HNF1A mRNA. When the same dose of siRNA is transfected, the functional experiment of non-small cell lung cancer cells shows that siMIX has a more effective anti-tumor effect than siHHIPL2 or siHNF1A. Therefore, the HHIPL2-HNF1A regulatory axis can be used as a drug target and applied to the research and development and preparation of anti-tumor drugs for non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A549 cells were selected and transfected with the same dose of siMIX or siNC. Reverse transcription-real-time fluorescence quantitative PCR was used to detect the changes in HHIPL2 and HNF1A mRNA levels. The statistical significance of the differences among the groups was determined by Student's t test.

[0019] Figure 2 The same doses of siHHIPL2, siHNF1A, siMIX or siNC were transfected into A549 cells, and the proliferation ability of A549 cells was evaluated by CCK-8 assay. The statistical significance of the differences among the groups was verified by two-way ANOVA test.

[0020] Figure 3 A549 cells were selected and transfected with siHHIPL2, siHNF1A, siMIX or siNC, respectively. The transfection dose of each siRNA was the same. The migration and invasion ability of A549 cells were detected by Transwell assay, and the statistical significance of the differences among the groups was determined by one-way ANOVA. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0022] Human non-small cell lung cancer cell line A549 cells are available from Shanghai Fuheng Biotechnology Co., Ltd.

[0023] siHHIPL2 targeting sequence: 5′-gttctgccaatctatgcttat-3′;

[0024] siHNF1A targeting sequence: 5′-cgaagatggtcaagtccta-3′.

[0025] Example 1

[0026] The inventors' previous studies found that the HHIPL2-HNF1A pathway axis was abnormally activated in non-small cell lung cancer, and then further studied its mechanism and found that the HHIPL2-HNF1A pathway axis was closely related to the malignant phenotype of tumor cells. Therefore, the inventors believe that knocking down the expression levels of HHIPL2 and HNF1A mRNA at the same time will play a better anti-cancer effect in non-small cell lung cancer, and the HHIPL2-HNF1A regulatory axis can be used as a drug target for the development and preparation of anti-tumor drugs for non-small cell lung cancer.

[0027] Example 2

[0028] The mRNA nucleotide sequences of the HHIPL2 gene and the HNF1A gene were obtained using the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. According to the above sequences, siRNAs (siHHIPL2 and siHNF1A) that specifically knock down the expression levels of HHIPL2 and HNF1A mRNA were designed, and their target sequences were shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The negative control of the above siRNAs was siNC, which was a conventional existing sequence. siHHIPL2 and siHNF1A were mixed at a molar ratio of 1:1 to obtain siMIX. The same dose (volume 3 μL, concentration 10 μM) of siNC and siMIX were transfected into A549 cells, and the cells were cultured for 48 hours before RNA was extracted. The mRNA levels of HHIPL2 and HNF1A were detected by reverse transcription-real-time fluorescence quantitative PCR. The specific results are shown in the figure. Figure 1 shown.

[0029] Depend on Figure 1 It can be seen that transfection of siMIX can significantly interfere with the expression of HHIPL2 and HNF1A mRNA in A549 cells and reduce the expression levels of HHIPL2 and HNF1A mRNA.

[0030] The specific implementation process is as follows:

[0031] (1) A549 cells were seeded into 6-well plates. When the cell growth density reached about 60%, siNC and siMIX were transfected into A549 cells using the transfection reagent Lipofectamine RNAiMAX Reagent (Life technologies, 13778-150). The siRNA dose used was the same (volume 3 μL, concentration 10 μM);

[0032] (2) 48 hours after transfection, the cells were collected and RNA was extracted using the RNA-Quick Purification Kit (Shanghai Yishan, RN001). The integrity of the extracted RNA was evaluated by agarose gel electrophoresis, and the concentration of the extracted RNA was detected by NanoDrop2000.

[0033] (3) Using the above-mentioned RNA as a template, and using the reverse transcription kit LunaScript TM RT SuperMix Kit (NEB, E3010) was used for reverse transcription and synthesis of cDNA products. Green Master Mix (ThermoFisher Scientific, 4367659) kit in QuantStudio TM Fluorescence quantitative PCR was performed on a 5System (Thermo Fisher Scientific) PCR instrument, and the ACTB mRNA level was selected as the internal reference. -ΔΔCT The relative expression levels of HHIPL2 and HNF1A mRNA were calculated by the formula.

[0034] Example 3

[0035] A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNF1A and siMIX, respectively. The CCK-8 assay was used to detect the interference effects of the above siRNAs on A549 cell proliferation. The specific results are as follows: Figure 2 shown.

[0036] Depend on Figure 2 It can be seen that compared with siNC, transfection of siHHIPL2, siHNF1A or siMIX can significantly inhibit the proliferation of A549 cells, but the application of siMIX shows lower cell viability than the same dose of siHHIPL2 or siHNF1A. This shows that simultaneous interference with the expression of HHIPL2 and HNF1A genes shows a better proliferation inhibitory effect than interfering with the expression level of any one of the genes alone.

[0037] The specific implementation process is as follows:

[0038] (1) siNC, siHHIPL2, siHNF1A and siMIX were transfected into A549 cells respectively. The siRNA dose used was the same (volume 3 μL, concentration 10 μM). The specific steps are detailed in Example 2. After 48 hours of transfection, the cells were collected and counted. The different transfected cells were re-seeded in a 96-well plate (2,000 cells / well) and divided into 4 groups (Day 0, 1, 2, 3). Each group included 4 types of transfected cells, siNC, siHHIPL2, siHNF1A and siMIX, and each type of cell was plated in 3 replicate wells.

[0039] (2) About 2 to 4 hours after cell plating, CCK-8 reagent (TargetMol, C0005) was added to the Day 0 group, 10 μL per well, and placed in a 37°C incubator for 1 hour. The cells were briefly shaken and placed in a microplate reader to measure the absorbance at 450 nm. This was the reading for Day 0 (initial cell planting).

[0040] (3) After completing the absorbance measurement on Day 0, the absorbance values ​​of the remaining Day 1, 2, and 3 groups were measured every 24 hours, and the relative level of cell viability in each group was calculated using the Day 0 group as a reference.

[0041] Example 4

[0042] The same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNF1A and siMIX were transfected into A549 cells, and the Transwell assay was used to detect the changes in cell migration and invasion abilities. The specific results are as follows Figure 3 shown.

[0043] Depend on Figure 3 It can be seen that under the condition of transfection of the same dose of siRNAs, siMIX has a more significant inhibitory effect on the migration and invasion of A549 cells than siHHIPL2 or siHNF1A, indicating that knocking down the expression levels of HHIPL2 and HNF1A genes at the same time has a better anti-cancer effect than knocking down the expression of either gene alone.

[0044] The specific implementation process is as follows:

[0045] (1) A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNF1A and siMIX, respectively, using the method described in Example 2;

[0046] (2) Select Transwell chambers (Corning, 3422). The chambers without Matrigel are used to examine cell migration, and the chambers pre-coated with Matrigel are used to measure cell invasion ability. Complete culture medium (600 μL / well) is added to a 24-well plate, and the Transwell chambers are placed in the well plate;

[0047] (3) 48 hours after transfection, cells were collected and counted, the cell concentration was adjusted to 100,000 cells / mL, 200 μL of the cell suspension was added to the Transwell chamber, and cultured in a cell incubator (37°C, 5% CO2) for 48 hours;

[0048] (4) The Transwell chambers were collected and the culture medium was discarded. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then stained with crystal violet (0.1%) overnight. The non-migrated and non-invaded cells in the chamber were scraped off with a cotton swab, and the migrated or invaded cells were observed under an inverted microscope and photographed for record.

Claims

1. Use of siRNA that specifically interferes with HHIPL2 gene expression and siRNA that specifically interferes with HNF1A gene expression in the preparation of an anti-non-small cell lung cancer drug, characterized in that: The targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown in SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that The molar ratio of the siRNA that specifically interferes with the HHIPL2 gene and the siRNA that specifically interferes with the HNF1A gene in the anti-non-small cell lung cancer drug is 1:1.

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