Application of the Targeting HNRNPC-HNF1A Regulatory Axis in the Treatment of Non-Small Cell Lung Cancer

Abstract

The present invention relates to the application of targeting the HNRNPC-HNF1A regulatory axis in the treatment of non-small cell lung cancer. The HNRNPC-HNF1A regulatory axis includes the HNRNPC gene and the HNF1A gene, and the nucleotide sequences of the mRNA of the HNRNPC gene and the mRNA of the HNF1A gene are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively. The present invention discovers for the first time that simultaneously targeting and intervening the key genes of the HNRNPC-HNF1A signaling pathway shows a more effective anti-proliferation and anti-metastasis effect on non-small cell lung cancer compared with separately inhibiting the expression levels of the HNRNPC or HNF1A gene. The present invention designs siRNA (siMIX) that can specifically and efficiently knockdown the mRNA levels of HNRNPC and HNF1A. The results of cell function experiments show that when the same dose of siRNA is transfected into non-small cell lung cancer cells, siMIX has a better anti-cell proliferation, migration and invasion effect than siHNRNPC or siHNF1A. Therefore, targeted inhibition of the HNRNPC-HNF1A pathway axis can be applied to the research and preparation of anti-tumor drugs for non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the application of the targeted HNRNPC-HNF1A regulatory axis in the treatment of non-small cell lung cancer, belonging to the field of biomedical technology. Background Art

[0002] Lung cancer is one of the common human malignancies, and its high invasiveness and high tumor heterogeneity pose a serious threat to human health. Lung cancer is mainly divided into two major pathological types, small cell lung cancer and non-small cell lung cancer (NSCLC), among which NSCLC accounts for about 85% of the confirmed cases of lung cancer and can be further divided into squamous cell carcinoma of the lung, adenocarcinoma of the lung and large cell carcinoma of the lung. The existing treatment methods for NSCLC mainly include surgical resection, adjuvant radiotherapy and chemotherapy, targeted therapy and immunotherapy, etc. In recent years, targeted drugs for gene mutations such as EGFR and KRAS have brought definite survival benefits to patients in the individualized treatment of NSCLC. However, the existing targeted drugs often lead to the generation of tumor cell drug resistance during the treatment process, thereby accelerating NSCLC patients into the terminal stage of cancer. Therefore, discovering new driver genes or signaling pathway axes and developing new intervention drugs based on them are extremely important for improving 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 transfection into cells, siRNA binds to the RNA-induced silencing complex (RISC). Subsequently, one sense strand of siRNA degrades, and the remaining antisense strand will guide RISC to bind to the target mRNA according to the base complementary pairing principle, and finally induce the degradation of this mRNA, thereby achieving the purpose of inhibiting the expression of the target gene. Designing siRNAs 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 the application of the targeted HNRNPC-HNF1A regulatory axis in the treatment of non-small cell lung cancer.

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

[0006] The application of the HNRNPC-HNF1A regulatory axis as a drug target in the preparation of drugs for treating non-small cell lung cancer.

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

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

[0009] Preferably according to the present invention, the drug for treating non-small cell lung cancer takes the HNRNPC and HNF1A genes as intervention targets, and is based on interfering with HNRNPC and HNF1A mRNA, and can efficiently and specifically inhibit the expression levels of the HNRNPC and HNF1A genes.

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

[0011] Use of siRNA specifically interfering with the expression of the HNRNPC gene and siRNA specifically interfering with the expression of the HNF1A gene in the preparation of a drug for treating non-small cell lung cancer.

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

[0013] Preferably according to the present invention, the molar ratio of the siRNA specifically interfering with the expression of the HNRNPC gene to the siRNA specifically interfering with the expression of the HNF1A gene in the drug for treating non-small cell lung cancer is 1:1.

[0014] A drug for treating non-small cell lung cancer, the drug for treating non-small cell lung cancer comprises siRNA specifically interfering with the expression of the HNRNPC gene and siRNA specifically interfering with the expression of the HNF1A gene.

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

[0016] Beneficial effects:

[0017] The present invention for the first time discovers key genes that simultaneously target and interfere with the HNRNPC-HNF1A signaling pathway, which shows a more effective anti-proliferation and anti-metastasis effect on non-small cell lung cancer compared to separately inhibiting the expression levels of HNRNPC or HNF1A genes. The present invention designs siRNA (siMIX) that can specifically and efficiently knockdown the mRNA levels of HNRNPC and HNF1A. The results of cell function experiments show that when the same dose of siRNA is transfected into non-small cell lung cancer cells, siMIX has a better anti-cell proliferation, migration and invasion effect than siHNRNPC or siHNF1A. Therefore, targeted inhibition of the HNRNPC-HNF1A pathway axis can be applied to the research and preparation of anti-tumor drugs for non-small cell lung cancer. Description of the Drawings

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

[0019] Figure 2 siHNRNPC, siHNF1A, siMIX or siNC were transfected into A549 cells respectively, and the same transfection dose was used for each siRNA. The CCK-8 assay was used to detect the proliferation ability of A549 cells, and two-way analysis of variance was used to verify the statistical significance of the differences between groups.

[0020] Figure 3 A549 cells were selected and transfected with the same dose of siHNRNPC, siHNF1A, siMIX or siNC respectively. The Transwell assay was used to evaluate the changes in the migration and invasion abilities of A549 cells, and one-way analysis of variance was used to determine the statistical significance of the differences between groups. Detailed Embodiments

[0021] The technical solutions of the present invention will be further described below in combination 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 ordinary commercially available products unless otherwise specified.

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

[0023] siHNRNPC targeting sequence: 5'-gcgcttgtctaagatcaaatt-3';

[0024] siHNF1A targeting sequence: 5'-tgggatacagtcttcttacttgg-3'.

[0025] Example 1

[0026] In previous studies, the inventors found that the HNRNPC protein was abnormally accumulated in the cytoplasm of non-small cell lung cancer, and up-regulated the expression level of HNF1A by binding to HNF1A mRNA, thereby promoting the proliferation and metastasis of non-small cell lung cancer. Therefore, targeted inhibition of the HNRNPC-HNF1A pathway axis will play a key role in the treatment of non-small cell lung cancer.

[0027] Example 2

[0028] The nucleotide sequences of HNRNPC and HNF1A mRNA were obtained using the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2. Specific siRNAs (siHNRNPC and siHNF1A) that interfere with the expression levels of HNRNPC or HNF1A mRNA were designed for the above sequences, and their targeting sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The above two siRNAs were mixed at a molar ratio of 1:1 to obtain siMIX. A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC (siRNAs negative control, a conventional existing sequence) and siMIX respectively. After 48 hours of transfection, the cells were collected and RNA was extracted. Reverse transcription-real-time fluorescence quantitative PCR was used to detect the levels of HNRNPC and HNF1A mRNA. The specific results are as Figure 1 shown.

[0029] As Figure 1 can be seen, transfection with siMIX can significantly reduce the levels of HNRNPC and HNF1A mRNA in A549 cells compared with siNC.

[0030] The specific implementation process is as follows:

[0031] (1) A549 cells were collected and plated in 6-well plates. When the cell growth density reached about 60%, siMIX and siNC were transfected into A549 cells using the transfection reagent Lipofectamine RNAiMAX Reagent (Life technologies, 13778-150). The same dose of siRNA was used (volume 3 μL, concentration 10 μM), and the cells were cultured for another 48 hours;

[0032] (2) The above cells were collected, and total RNA was extracted using the RNA-Quick Purification Kit (Shanghai Yishan, RN001) kit. The concentration of the extracted RNA was detected using NanoDrop2000, and the integrity of the extracted RNA was evaluated using agarose gel electrophoresis;

[0033] (3) Apply the reverse transcription kit LunaScript TM RT SuperMix Kit (NEB, E3010) and use the extracted RNA as a template to perform the reverse transcription step to synthesize cDNA products. Use Power Green Master Mix (Thermo Fisher Scientific, 4367659) kit and the QuantStudio TM 5 System (Thermo Fisher Scientific) PCR instrument to perform fluorescence quantitative PCR. Take the ACTB mRNA level as an internal reference, and calculate the relative expression levels of HNRNPC and HNF1A mRNA according to the 2 -ΔΔCT formula.

[0034] Example 3

[0035] Select A549 cells and transfect them with the same dose (volume 3 μL, concentration 10 μM) of siNC, siHNRNPC, siHNF1A, and siMIX respectively. Apply the CCK-8 assay to detect the effects of the above transfections on the proliferation ability of A549 cells. The specific results are as Figure 2 shown.

[0036] As Figure 2 can be seen, transfection with siHNRNPC, siHNF1A, or siMIX can significantly weaken the proliferation ability of A549 cells compared with siNC. However, the siMIX group shows lower cell viability compared with the siHNRNPC or siHNF1A group at the same dose. This indicates that simultaneous knockdown of the expression of HNRNPC and HNF1A genes has a better tumor growth inhibitory effect than single interference with the expression level of either gene.

[0037] The specific implementation process is as follows:

[0038] (1) Transfect the same dose (volume 3 μL, concentration 10 μM) of siNC, siHNRNPC, siHNF1A, and siMIX into A549 cells respectively. For the specific method, please refer to Example 2. After 48 hours of transfection, collect the transfected cells and continue to culture the transfected cells (2,000 cells / well) in a 96-well plate. A total of 4 groups are divided (Day 0, 1, 2, 3). Each group includes 4 types of transfected cells: siNC, siHNRNPC, siHNF1A, and siMIX. Each type of cell is plated in 3 replicate wells;

[0039] (2) After culturing in a cell incubator for 2 - 4 hours, add 10 μL of CCK-8 reagent (TargetMol, C0005) to each well of the Day 0 group, place it in an incubator at 37 °C for 1 hour, gently shake, and then measure the absorbance value (450 nm) using a microplate reader. This is the absorbance value of the initially seeded cells (Day 0).

[0040] (3) Measure the absorbance values (450 nm) of the Day 1, 2, and 3 groups every 24 hours, and calculate the change in cell viability of each group with reference to the absorbance value measured for the Day 0 group.

[0041] Example 4

[0042] Using A549 cells, transfect siNC, siHNRNPC, siHNF1A, and siMIX respectively, with the same dosage (volume 3 μL, concentration 10 μM). Use the Transwell experiment to detect the migration and invasion abilities of the cells. The specific results are as Figure 3 shown.

[0043] As Figure 3 can be seen, under the condition of transfecting the same dosage of siRNAs, the migration and invasion abilities of the cells in the siMIX group are significantly weaker than those in the siHNRNPC or siHNF1A group. Such results indicate that simultaneously interfering with the expression levels of HNRNPC and HNF1A has a stronger anti-cancer effect compared to individually inhibiting the expression of the HNRNPC or HNF1A gene.

[0044] The specific implementation process is as follows:

[0045] (1) Use a transfection reagent to transfect the same dosage (volume 3 μL, concentration 10 μM) of siNC, siHNRNPC, siHNF1A, and siMIX into A549 cells respectively, using the method described in Example 2;

[0046] (2) Select a Transwell chamber (Corning, 3422). The chamber without pre-coated Matrigel is used to detect cell migration ability, and the chamber pre-coated with Matrigel is used to evaluate cell invasion ability. Select a 24-well plate, add 600 μL of complete medium to each well in advance, and then place the Transwall chamber in the 24-well plate for standby;

[0047] (3) After 48 hours of transfection, collect the cell pellet, resuspend it with serum-free medium, adjust the cell concentration to 100,000 cells / mL, aspirate 200 μL of the cell suspension and add it into the Transwall chamber, and place the 24-well plate in an incubator (37 °C, 5% CO 2 ) and continue to culture for 48 hours;

[0048] (4) Remove the Transwell chamber and discard the culture medium. Fix the cells with 4% paraformaldehyde at room temperature for 15 minutes, then stain with crystal violet (0.1%) overnight. Scrape off the cells that have not migrated or invaded on the inner surface of the chamber, and observe and count the cells that have migrated or invaded to the outer side of the chamber under an inverted microscope.

Claims

1. Use of siRNA that specifically interferes with HNRNPC gene expression and siRNA that specifically interferes with HNF1A gene expression in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC 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.

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

1.

3. A drug for treating non-small cell lung cancer, characterized in that: The drug for treating non-small cell lung cancer comprises siRNA that specifically interferes with the expression of the HNRNPC gene and siRNA that specifically interferes with the expression of the HNF1A gene; The nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown in SEQ ID NO.3, and the nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown in SEQ ID NO.4.

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