Application of combined interference with abnormal expression of HHIPL2, HNRNPC and HNF1A mRNA in the treatment of human non-small cell lung cancer

Abstract

The present invention relates to the application of combined interference with abnormal expression of HHIPL2, HNRNPC and HNF1A mRNA in the treatment of human non-small cell lung cancer. The mRNA nucleotide sequence of the HHIPL2 gene is shown in SEQ ID NO.1, the mRNA nucleotide sequence of the HNRNPC gene is shown in SEQ ID NO.2, and the mRNA nucleotide sequence of the HNF1A gene is shown in SEQ ID NO.3. The present invention first discovered that combined interference with the expression of HHIPL2 gene, HNRNPC gene and HNF1A gene can effectively inhibit the malignant biological behavior of non-small cell lung cancer compared with the single interference with the expression level of any one of the genes. The present invention designs siRNA (siMIX) that can efficiently and specifically knock down the expression of HHIPL2, HNRNPC and HNF1A genes, and transfects the same dose of siRNA into non-small cell lung cancer cells. siMIX exhibits an anti-tumor effect that is superior to siHHIPL2, siHNRNPC or siHNF1A.

Description

Technical Field

[0001] The invention relates to application of combined interference with abnormal expression of HHIPL2, HNRNPC and HNF1A mRNA in treating human non-small cell lung cancer, and belongs to the technical field of biomedicine. Background Art

[0002] Lung cancer is one of the most common human malignancies and the leading cause of cancer-related death. Non-small cell lung cancer (NSCLC) is the most common pathological subtype of lung cancer, accounting for about 85% of confirmed lung cancer cases. The traditional treatment of NSCLC is mainly surgery, radiotherapy, and chemotherapy. In recent decades, with the rapid development of sequencing technology and the discovery of related oncogenic driver genes, targeted drugs targeting EGFR, KRAS, and ALK sensitive mutations have been applied to clinical treatment and have brought certain prognosis improvements to NSCLC patients. However, the high tumor heterogeneity of NSCLC often leads to tumor resistance during targeted therapy, which ultimately leads to cancer cell recurrence and metastasis. Therefore, continuing to explore new NSCLC driver genes and clarifying their oncogenic molecular mechanisms will point the way for the development of new targeted drugs.

[0003] Small interfering RNA (siRNA), also known as silencing RNA or short interfering RNA, has many uses in biology. The most studied and widely used one is its role in regulating gene expression. siRNA is a double-stranded RNA composed of 19-25 pairs of nucleotides. It enters cells under the action of transfection reagents, participates in the formation of RNA-induced silencing complexes, and binds to target gene mRNA based on the principle of base complementary pairing, thereby inducing the degradation of the mRNA or inhibiting its translation, and ultimately reducing the protein level translated by the target gene mRNA. How to improve the efficiency of targeted interference and reduce its off-target effects are key issues that must always be considered in the basic research related to NSCLC and potential targeted therapeutic applications of siRNA. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an application of jointly interfering with abnormal expression of HHIPL2, HNRNPC and HNF1A mRNA in treating human non-small cell lung cancer.

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

[0006] The application of mRNA of HHIPL2 gene, HNRNPC gene and HNF1A gene as drug targets in the preparation of targeted drugs for the treatment of non-small cell lung cancer.

[0007] Preferably according to the present invention, the mRNA nucleotide sequence of the HHIPL2 gene is shown as SEQ ID NO.1, the mRNA nucleotide sequence of the HNRNPC gene is shown as SEQ ID NO.2, and the mRNA nucleotide sequence of the HNF1A gene is shown as SEQ ID NO.3.

[0008] Preferably according to the present invention, the targeted treatment drug for non-small cell lung cancer takes HHIPL2 gene, HNRNPC gene and HNF1A gene as intervention targets, based on interfering with HHIPL2, HNRNPC and HNF1A mRNA, and can efficiently and specifically inhibit the expression levels of HHIPL2, HNRNPC and HNF1A genes.

[0009] Preferably according to the present invention, the drug for the targeted treatment of non-small cell lung cancer is siRNA, shRNA, microRNA or chemical inhibitors of HHIPL2, HNRNPC and HNF1A mRNA.

[0010] The application of siRNA specifically interfering with HHIPL2 gene, siRNA specifically interfering with HNRNPC gene and siRNA specifically interfering with HNF1A gene in the preparation of drugs for targeted treatment of non-small cell lung cancer.

[0011] 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.4, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown as SEQ ID NO.5, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown as SEQ ID NO.6.

[0012] Preferably according to the present invention, the molar ratio of the siRNA that specifically interferes with the expression of the HHIPL2 gene, the siRNA that specifically interferes with the expression of the HNRNPC gene and the siRNA that specifically interferes with the expression of the HNF1A gene in the targeted treatment drug for non-small cell lung cancer is 1:1:1.

[0013] A targeted drug for treating non-small cell lung cancer comprises siRNA that specifically interferes with HHIPL2 gene expression, siRNA that specifically interferes with HNRNPC gene expression, and siRNA that specifically interferes with HNF1A gene expression.

[0014] 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.4, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown as SEQ ID NO.5, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown as SEQ ID NO.6.

[0015] Beneficial effects:

[0016] The present invention discovered for the first time that combined interference with the expression of HHIPL2, HNRNPC and HNF1A genes can effectively inhibit the malignant biological behavior of non-small cell lung cancer compared to interfering with the expression level of any one of these genes alone. The present invention designed siRNA (siMIX) that can efficiently and specifically knock down the expression of HHIPL2, HNRNPC and HNF1A genes. When the same dose of siRNA was transfected into non-small cell lung cancer cells, siMIX showed an anti-tumor effect that was superior to siHHIPL2, siHNRNPC or siHNF1A. Therefore, the HHIPL2-HNRNPC-HNF1A axis can be used as a targeted pathway for the development and preparation of anti-tumor drugs for non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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 mRNA levels of HHIPL2, HNRNPC and HNF1A. The Student's t test was used to clarify the statistical significance of the differences among the groups.

[0018] Figure 2 The same dose of siHHIPL2, siHNRNPC, siHNF1A, siMIX or siNC was 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.

[0019] Figure 3 A549 cells were selected and transfected with siHHIPL2, siHNRNPC, 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

[0020] 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.

[0021] Human non-small cell lung cancer cell line A549 cells are available from Shanghai Fuheng Biotechnology Co., Ltd.

[0022] siHHIPL2 targeting sequence: 5′-gctctgccttccattctaa-3′;

[0023] siHNRNPC targeting sequence: 5′-caacgggactattatgata-3′;

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

[0025] Example 1

[0026] The inventors' previous studies have found that the expression level of HHIPL2 is abnormally upregulated in non-small cell lung cancer and is significantly negatively correlated with the poor prognosis of patients; molecular mechanism studies have revealed that HHIPL2 protein upregulates the level of HNF1A mRNA by binding to HNRNPC, thereby promoting the proliferation and metastasis of non-small cell lung cancer. Based on this, the inventors presume that simultaneous interference with the expression levels of HHIPL2, HNRNPC and HNF1A mRNA will play a significant anti-cancer role in non-small cell lung cancer.

[0027] Example 2

[0028] The nucleotide sequences of HHIPL2, HNRNPC and HNF1A mRNA were obtained from the NCBI database, as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively. siRNAs (siHHIPL2, siHNRNPC and siHNF1A) that specifically knock down the expression levels of HHIPL2, HNRNPC or HNF1A mRNA were designed for the above sequences, and their targeting sequences were shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively. The above three siRNAs were mixed at a molar ratio of 1:1:1 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. After transfection for 48 hours, the cells were collected and RNA was extracted. Reverse transcription-real-time fluorescence quantitative PCR was used to detect the mRNA levels of HHIPL2, HNRNPC and HNF1A. The specific results are shown in the following table. Figure 1 shown.

[0029] Depend on Figure 1 It can be seen that transfection of siMIX can effectively knock down the mRNA levels of HHIPL2, HNRNPC and HNF1A in A549 cells.

[0030] The specific implementation process is as follows:

[0031] (1) A549 cells were selected and cultured in a 6-well plate. When the cell growth density reached about 60%, the same dose (volume 3 μL, concentration 10 μM) of siMIX and siNC were transfected into A549 cells using the transfection reagent Lipofectamine RNAiMAX Reagent (Life technologies, 13778-150);

[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) Application of reverse transcription kit LunaScript TM RT SuperMix Kit (NEB, E3010) was used to perform reverse transcription to synthesize cDNA products using the extracted RNA as a template. Green Master Mix Kit (ThermoFisher Scientific, 4367659) and QuantStudio TM 5SystemPCR instrument (Thermo Fisher Scientific) was used for fluorescence quantitative PCR, with ACTB mRNA level as the internal reference, according to 2 -ΔΔCT The relative expression levels of HHIPL2, HNRNPC and HNF1A mRNA were calculated by the formula.

[0034] Example 3

[0035] The same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNRNPC, siHNF1A and siMIX were transfected into A549 cells, and the CCK-8 assay was used to evaluate the effects of the above siRNAs on cell proliferation. The specific results are shown in Figure 2 shown.

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

[0037] The specific implementation process is as follows:

[0038] (1) The same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNRNPC, siHNF1A and siMIX were transfected into A549 cells respectively. The specific method is described in Example 2. The transfected cells were collected 48 hours after transfection, and the different transfected cells were re-cultured in 96-well plates (2,000 cells / well) and divided into 4 groups (Day 0, 1, 2, 3). Each group included 5 types of transfected cells, including siNC, siHHIPL2, siHNRNPC, siHNF1A and siMIX, and each type of cell was plated in 3 replicate wells;

[0039] (2) After the cells adhere to the wall (about 2-4 hours), add CCK-8 reagent (TargetMol, C0005) to the Day 0 group, 10 μL per well, place in a 37°C incubator for 1 hour, shake briefly, place in a microplate reader and measure the absorbance at 450 nm. This is the absorbance value of the initial seeded cells (Day 0);

[0040] (3) After completing the initial cell absorbance measurement, measure the absorbance values ​​of Day 1, 2, and 3 groups every 24 hours, and calculate the changes in cell viability of each group with Day 0 group as a reference.

[0041] Example 4

[0042] A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNRNPC, siHNF1A or siMIX, and the cell migration and invasion abilities were evaluated by Transwell assay. The specific results are shown in Figure 3 shown.

[0043] Depend on Figure 3It can be seen that under the condition of transfection of the same dose of siRNAs, siMIX has the most significant inhibitory effect on the migration and invasion ability of A549 cells compared with siHHIPL2, siHNRNPC or siHNF1A, indicating that knocking down HHIPL2, HNRNPC and HNF1A at the same time has a better anti-cancer effect than knocking down the expression of any one of the genes 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, siHNRNPC, siHNF1A and siMIX, respectively, using the method described in Example 2;

[0046] (2) Transwell chambers (Corning, 3422) were selected. The chambers without pre-coated Matrigel were used to evaluate cell migration, and the chambers pre-coated with Matrigel were used to detect cell invasion ability. The Transwell chambers were placed in 24-well plates (600 μL complete culture medium was pre-added to each well);

[0047] (3) 48 hours after transfection, the cell pellet was collected and resuspended in serum-free medium to adjust the cell concentration to 100,000 cells / mL. 200 μL of the cell suspension was added to the Transwell chamber and incubated at 37°C and 5% CO. 2 The cells were cultured in an incubator for 48 h;

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

Claims

1. Use of siRNA that specifically interferes with HHIPL2 gene expression, siRNA that specifically interferes with HNRNPC gene expression, and siRNA that specifically interferes with HNF1A gene expression in the preparation of a drug for targeted treatment of non-small cell lung cancer, 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.4, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown in SEQ ID NO.5, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown in SEQ ID NO.6; The mRNA nucleotide sequence of the HHIPL2 gene is shown in SEQ ID NO.1, the mRNA nucleotide sequence of the HNRNPC gene is shown in SEQ ID NO.2, and the mRNA nucleotide sequence of the HNF1A gene is shown in SEQ ID NO.

3.

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

1.

3. A drug for targeted treatment of non-small cell lung cancer, characterized in that: The targeted treatment drug for non-small cell lung cancer comprises siRNA that specifically interferes with HHIPL2 gene expression, siRNA that specifically interferes with HNRNPC gene expression, and siRNA that specifically interferes with HNF1A gene expression; Among them, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown as SEQ ID NO.4, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown as SEQ ID NO.5, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNF1A gene is shown as SEQ ID NO.6.

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