Application of TIM knockdown substances in the preparation of products for the treatment of non-small cell lung cancer

Abstract

The present invention discloses the use of a substance that knocks down TIM in the preparation of a product for treating non-small cell lung cancer. The present invention also discloses the use of a substance that knocks down TIM in the preparation of a product for inhibiting the proliferation of non-small cell lung cancer cells, inhibiting the migration of non-small cell lung cancer cells, increasing the responsiveness of non-small cell lung cancer cells to gefitinib, inhibiting the activation of EGFR and / or AKT and / or mTOR pathways in non-small cell lung cancer cells, downregulating the expression levels of EGFR-regulated oncogenes in non-small cell lung cancer cells, inhibiting the expression levels of SPHK1 and / or ATF3 in non-small cell lung cancer cells, and inhibiting the activation of the STAT3 pathway in non-small cell lung cancer cells. Compared with existing drugs for treating non-small cell lung cancer, the siRNA for knocking down TIM of the present invention is effective against a variety of non-small cell lung cancer tumor cells with different gene mutations, and has a more stable anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the use of a substance that knocks down TIM in the preparation of a product for treating non-small cell lung cancer. Background Art

[0002] Lung cancer is the most common cause of cancer-related deaths worldwide. Approximately 18 million people are diagnosed with lung cancer each year, and 16 million die from it, with a low 5-year survival rate. According to the global 2020 cancer new incidence and death data released in 2021 by the cancer journal CA Cancer J Clin, the number of new cases of lung cancer reached 2.2 million, ranking second in cancer incidence; the number of deaths reached 1.8 million, ranking first in cancer mortality. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer tissue subtypes and is the main type of lung cancer.

[0003] According to the 2022 NSCLC treatment guidelines published online by the National Comprehensive Cancer Network of the United States, the current drug treatment methods for NSCLC are mainly divided into: (1) Targeted therapy is used for patients with specific gene mutations. For example, for NSCLC patients with epidermal growth factor receptor (EGFR) sensitive mutations, such as exon 19 (in-frame) deletions and exon 21 point mutations (L858R), targeted therapy drugs such as gefitinib and osimertinib are used. However, as the duration of medication increases, most patients inevitably develop drug resistance, leading to treatment failure; (2) For patients who need urgent treatment but whose molecular test results are not yet clear, if their PD-L1 tumor cell positive proportion score is greater than 50%, one cycle of immunotherapy can be carried out first, but there are contraindications for treatment for patients who have been or are currently in the active stage of autoimmune diseases and / or have used immunosuppressive drugs in the past; (3) For NSCLC patients who do not meet the above treatment requirements, traditional chemotherapy is continued, but chemotherapy has great toxic side effects on patients and is likely to cause treatment interruption. Therefore, it remains crucial to clarify the mechanism of NSCLC occurrence and development and to find new and effective treatments. Summary of the Invention

[0004] One object of the present invention is to provide a novel use of a substance that inhibits the activity of a TIM protein or a substance that reduces the content of a TIM protein.

[0005] The present invention provides use of a substance that inhibits TIM protein activity or a substance that reduces TIM protein content in any of the following A1)-A8);

[0006] A1) preparing products for the prevention and / or treatment of non-small cell lung cancer;

[0007] A2) preparing a product for inhibiting the proliferation of non-small cell lung cancer cells;

[0008] A3) preparing a product for inhibiting the migration of non-small cell lung cancer cells;

[0009] A4) preparing a product that increases the responsiveness of non-small cell lung cancer cells to gefitinib;

[0010] A5) preparing products that inhibit activation of EGFR and / or AKT and / or mTOR pathways in non-small cell lung cancer cells;

[0011] A6) preparing a product that downregulates the expression levels of EGFR-regulated oncogenes c-MYC and AURKA in non-small cell lung cancer cells;

[0012] A7) preparing a product that inhibits the expression level of SPHK1 and / or ATF3 in non-small cell lung cancer cells;

[0013] A8) preparing a product that inhibits STAT3 pathway activation in non-small cell lung cancer cells.

[0014] Another object of the present invention is to provide a product, wherein the active ingredient of the product is a substance that inhibits the activity of TIM protein or a substance that reduces the content of TIM protein; the function of the product is any one of the following B1)-B8);

[0015] B1) Prevention and / or treatment of non-small cell lung cancer;

[0016] B2) inhibiting the proliferation of non-small cell lung cancer cells;

[0017] B3) inhibiting the migration of non-small cell lung cancer cells;

[0018] B4) improving the responsiveness of non-small cell lung cancer cells to gefitinib;

[0019] B5) inhibiting the activation of EGFR and / or AKT and / or mTOR pathways in non-small cell lung cancer cells;

[0020] B6) downregulating the expression levels of EGFR-regulated oncogenes c-MYC and AURKA in non-small cell lung cancer cells;

[0021] B7) inhibiting the expression level of SPHK1 and / or ATF3 in non-small cell lung cancer cells;

[0022] B8) Inhibits the activation of STAT3 pathway in non-small cell lung cancer cells.

[0023] In any of the above products or applications, the method of increasing the responsiveness of non-small cell lung cancer cells to gefitinib is to increase the responsiveness of non-small cell lung cancer cells to 1.0×10-5 Reactivity of gefitinib at 1 mol / L.

[0024] Any of the above products may be a medicine.

[0025] Another object of the present invention is to provide a method for improving the responsiveness of non-small cell lung cancer cells to gefitinib.

[0026] The method for improving the responsiveness of non-small cell lung cancer cells to gefitinib provided by the present invention comprises the following steps: introducing a substance that inhibits TIM protein activity or a substance that reduces TIM protein content into non-small cell lung cancer cells to obtain TIM knockdown non-small cell lung cancer cells; the TIM knockdown non-small cell lung cancer cells are responsive to 1.0×10 -5 The responsiveness of gefitinib to 1% gefitinib was higher than that of the non-small cell lung cancer cells.

[0027] In any of the above-mentioned products, applications or methods, the substance that inhibits the activity of TIM protein may be a protein, polypeptide or small molecule compound that inhibits the function of TIM protein.

[0028] The substance that reduces the content of TIM protein may be a substance that inhibits TIM protein synthesis or promotes TIM protein degradation or knocks down or knocks out the TIM gene.

[0029] Furthermore, the substance for knocking down the TIM gene may be siRNA that inhibits the expression of the TIM gene.

[0030] Furthermore, the siRNA that inhibits TIM gene expression consists of sequence 5 and sequence 6 in the sequence listing.

[0031] In any of the above-mentioned products, applications or methods, the non-small cell lung cancer may specifically be non-small cell lung adenocarcinoma.

[0032] The non-small cell lung cancer cells can specifically be H1975 cells or HCC827 cells.

[0033] In any of the above-mentioned products, applications or methods, the amino acid sequence of the TIM protein is shown in Sequence 1 in the sequence listing; the nucleotide sequence of the TIM gene is shown in Sequence 2 in the sequence listing.

[0034] The present invention provides a siRNA for knocking down TIM, which can effectively knock down TIM in non-small cell lung cancer cells and can be used to prepare products for preventing and / or treating non-small cell lung cancer, inhibiting non-small cell lung cancer cell proliferation, inhibiting non-small cell lung cancer cell migration, improving non-small cell lung cancer cell responsiveness to gefitinib, inhibiting EGFR and / or AKT and / or mTOR pathway activation in non-small cell lung cancer cells, downregulating EGFR-regulated oncogene expression levels in non-small cell lung cancer cells, inhibiting SPHK1 and / or ATF3 expression levels in non-small cell lung cancer cells, and inhibiting STAT3 pathway activation in non-small cell lung cancer cells. Compared with existing drugs for treating non-small cell lung cancer, the siRNA for knocking down TIM of the present invention is effective for a variety of non-small cell lung cancer tumor cells with different gene mutations (such as H1975 cells and HCC827 cells, etc.), and the anti-tumor effect is more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Confirmation of TIM siRNA knockdown efficiency. A: Western blot analysis of TIM siRNA knockdown in H1975 cells; B: Grayscale analysis of protein bands. siNC represents the control group; siTIM#1 represents the first TIM siRNA knockdown group; siTIM#2 represents the second TIM siRNA knockdown group; and siTIM#3 represents the third TIM siRNA knockdown group. Results are expressed as mean ± standard deviation (n = 3). Statistical analysis was performed using one-way ANOVA. **p < 0.01, compared with the siNC group. Experiments were repeated three times.

[0036] Figure 2 To inhibit H1975 cell proliferation, TIM knockdown was performed. The CCK8 assay was used to examine the effect of TIM knockdown on H1975 cell proliferation. The siNC group served as the control group, while the siTIM#3 group served as the TIM knockdown group. All results are presented as mean ± SD, n = 5. Statistical analysis was performed using the t-test. **p < 0.01, compared with the siNC group. Experiments were repeated three times.

[0037] Figure 3 Knockdown of TIM inhibits H1975 cell migration. A shows changes in cell migration ability after knockdown of TIM expression in H1975 cells using the Transwell assay. Magnification: 100×, scale bar: 100 μm. B shows quantitative analysis of cell number in the lower layer of the Transwell chamber. siNC represents the control group, and siTIM#3 represents the TIM knockdown group. All results are expressed as mean ± SD, n = 3. Statistical analysis was performed using the t-test. **p < 0.01, compared with the siNC group. Experiments were repeated three times.

[0038] Figure 4 Figure 3: Effects of different concentrations of gefitinib on H1975 cell proliferation and the effect of TIM knockdown on H1975 cell responsiveness to gefitinib. A: Effect of gefitinib on H1975 cell proliferation assessed by CCK8 assay; B: Effect of TIM knockdown on gefitinib efficacy in H1975 cells assessed by CCK8 assay. Control: control group; siTIM#3: TIM knockdown group. Results are expressed as mean ± SD, n = 5. Statistical analysis was performed using one-way ANOVA. **p < 0.01, compared with the control group. Experiments were repeated three times.

[0039] Figure 5 Knockdown of TIM expression inhibits EGFR activation and downstream pathway activation in H1975 and HCC827 cells. A: Western blot analysis of the effect of TIM knockdown in H1975 cells on EGFR activation; B: Grayscale analysis of protein bands in H1975 cells; C: Western blot analysis of the effect of TIM knockdown in H1975 cells on AKT / mTOR activation; D: Grayscale analysis of protein bands in H1975 cells; F: Western blot analysis of the effect of TIM knockdown in HCC827 cells on EGFR activation; G: Grayscale analysis of protein bands in HCC827 cells; H: Western blot analysis of the effect of TIM knockdown in HCC827 cells on AKT / mTOR activation; I: Grayscale analysis of protein bands in HCC827 cells. siNC represents the control group, and siTIM#3 represents the TIM knockdown group. Results are expressed as mean ± SD, n = 3. Statistical analysis was performed using the t-test, *p < 0.05, compared with the siNC group. The experiment was repeated three times independently.

[0040] Figure 6 Knockdown of TIM expression does not affect EGF and AREG mRNA levels in H1975 cells. A shows the effect of TIM knockdown on EGF mRNA levels in H1975 cells, as assessed by RT-qPCR; B shows the effect of TIM knockdown on AREG mRNA levels in H1975 cells, as assessed by RT-qPCR. The siNC group served as the control group, and the siTIM#3 group served as the TIM knockdown group. All results are presented as mean ± standard deviation (SD), with n = 3. Statistical analysis was performed using the t-test. ns: no significance, indicating no significant difference compared with the siNC group. Experiments were repeated three times.

[0041] Figure 7 Knockdown of TIM expression inhibits the expression of EGFR-related oncogenes c-MYC and AURKA in H1975 cells. AG represents the effect of TIM knockdown on EGFR-related oncogene mRNA levels in H1975 cells detected by RT-qPCR; H represents the effect of TIM knockdown on c-MYC protein expression in H1975 cells detected by Western blot; I represents the grayscale analysis of protein bands in H1975 cells. siNC represents the control group, and siTIM#3 represents the TIM knockdown group. All results are expressed as mean ± SD, n = 3. Statistical analysis was performed using the t-test. *p < 0.05, **p < 0.01, compared with the siNC group. The experiment was repeated three times.

[0042] Figure 8 Figure 3: Upregulation of SPHK1 mRNA levels in NSCLC lung adenocarcinoma tissues and inhibition of SPHK1 expression, STAT3 activation, and ATF3 expression in H1975 and HCC827 cells by TIM knockdown. A: RT-qPCR analysis of SPHK1 mRNA levels in NSCLC lung adenocarcinoma tissues and corresponding adjacent adjacent tissues. B: Western blot analysis of the effects of TIM knockdown on SPHK1 expression, STAT3 activation, and ATF3 expression in H1975 cells. C-E: Grayscale analysis of protein bands in H1975 cells. F: Western blot analysis of the effects of TIM knockdown on SPHK1 expression, STAT3 activation, and ATF3 expression in HCC827 lung adenocarcinoma cells. G-H: Grayscale analysis of protein bands in HCC827 cells. siNC was the control group, siTIM#1 was the first TIM siRNA knockdown group, siTIM#2 was the second TIM siRNA knockdown group, and siTIM#3 was the third TIM siRNA knockdown group. Results are presented as mean ± standard deviation (SD), n = 3. Statistical analysis was performed using t-test or one-way ANOVA. *p < 0.05; **p < 0.01, compared with the siNC group. Experiments were repeated three times.

[0043] Figure 9Knockdown of SPHK1 expression inhibits STAT3 and EGFR / AKT / mTOR activation in H1975 cells. Panels AC show the effects of SPHK1 knockdown on SPHK1 expression and STAT3 activation in H1975 cells, as well as grayscale analysis of the corresponding protein bands. Panels DF show the effects of SPHK1 knockdown on EGFR and downstream AKT / mTOR activation in H1975 cells, as well as grayscale analysis of the corresponding protein bands. siNC represents the control group, and siSPHK1 represents the SPHK1 knockdown group. DETAILED DESCRIPTION

[0044] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0045] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0046] The H1975 and HCC827 cells used in the following examples were purchased from the Cell Bank of the Chinese Academy of Sciences. H1975 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), while HCC827 cells were cultured in RPMI-1640. Both media contained 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. All cells were cultured in a 37°C, 5% CO2 incubator.

[0047] The TIM gene in the following embodiments is the clock gene TIMELESS. The nucleotide sequence of the TIM gene is shown in Sequence 2 in the sequence listing, and the amino acid sequence of the TIM protein encoded by the TIM gene is shown in Sequence 1 in the sequence listing.

[0048] Example 1: Use of substances that inhibit TIM activity in inhibiting NSCLC cell proliferation and migration

[0049] 1. Confirmation of TIM siRNA Knockdown Efficiency in NSCLC Cells

[0050] To investigate the effect of TIM on the proliferation and migration of NSCLC cells, the present invention used RNAi technology to knock down TIM in H1975 cells. The specific steps are as follows:

[0051] 1. Three siRNAs were designed and synthesized based on the TIM sequence. These three siRNAs were named siTIM#1, siTIM#2, and siTIM#3. A siRNA negative control (hereinafter referred to as siNC) was used as a control. The three siRNA sequences and the siNC sequence are shown in Table 1.

[0052] Table 1. siRNA sequences

[0053]

[0054]

[0055] 2. The siTIM#1, siTIM#2, siTIM#3 and siNC in step 1 were respectively introduced into H1975 cells to obtain TIM knockdown H1975 cells (siTIM#1), TIM knockdown H1975 cells (siTIM#2), TIM knockdown H1975 cells (siTIM#3) and TIM knockdown H1975 cells (siNC), and the TIM protein expression level in each TIM knockdown H1975 cell was detected by Western blot. The specific method of introducing each siRNA into H1975 cells comprises the following steps:

[0056] Tumor cells in the logarithmic growth phase were taken and washed twice with phosphate buffered saline (PBS), then digested with trypsin and perforated into single cells. 4 Cells were seeded into 6-well cell culture plates in DMEM or RPMI 1640 medium containing 10% (v / v) fetal bovine serum. When the cell confluence reached 60-80%, the medium in the plate was discarded, the cells were washed twice with PBS, and then replaced with DMEM or RPMI 1640 medium without fetal bovine serum. Lipofectamine was diluted with Opti-MEM medium. TM RNAiMAX and siRNA were then mixed in a 1:1 ratio to form a transfection solution and transfected into the tumor cell culture system. The transfection time was 8 hours, and then the cells were cultured in DMEM or RPMI1640 medium containing 10% (v / v) fetal bovine serum for 24-72 hours before subsequent experiments.

[0057] The results showed that compared with the other two TIM siRNAs, siTIM#3 had the most significant knockdown effect (p<0.01), reaching a knockdown efficiency of 53% ( Figure 1 ), so TIM knockdown H1975 cells (siTIM#3) were selected for subsequent experiments.

[0058] 2. Knockdown of TIM inhibits NSCLC cell proliferation

[0059] To investigate the effect of TIM on NSCLC cell proliferation, CCK8 assay was used to detect cell proliferation of TIM knockdown H1975 cells (siTIM#3) from 24 h to 72 h. TIM knockdown H1975 cells (siNC) were used as a control.

[0060] The results showed that knockdown of TIM significantly inhibited the cell proliferation ability of H1975 cells from 24h to 72h (p < 0.01) ( Figure 2 ).

[0061] 3. Knockdown of TIM inhibits NSCLC cell migration

[0062] To investigate the effect of TIM on NSCLC cell migration, the Transwell assay was used to detect changes in the migration ability of TIM-knockdown H1975 cells (siTIM#3). TIM-knockdown H1975 cells (siNC) were used as a control.

[0063] The results showed that after knocking down TIM expression, the number of cells crossing to the lower layer of the Transwell chamber was significantly reduced, indicating that knocking down TIM significantly inhibited the migration ability of H1975 cells (p < 0.01) ( Figure 3 ).

[0064] Example 2: Knockdown of TIM improves the responsiveness of NSCLC cells to gefitinib

[0065] 1. Effects of different concentrations of gefitinib on the proliferation of H1975 cells

[0066] In order to investigate whether gefitinib affects the proliferation of H1975 cells, different concentrations of gefitinib (1.0×10 -8 mol / L, 1.0×10 -7 mol / L、1.0×10 -6 mol / L, 1.0×10 -5 mol / L were used to treat H1975 cells for 48 h, and then the proliferation of H1975 cell lines after gefitinib treatment was detected by CCK8 method.

[0067] The results showed that gefitinib did not affect the proliferation of H1975 cells ( Figure 4 A).

[0068] 2. Knockdown of TIM expression promotes the responsiveness of H1975 cells to gefitinib

[0069] In order to investigate whether TIM affects the responsiveness of NSCLC cells to gefitinib, different concentrations of gefitinib (1.0×10 -6 mol / L or 1.0×10 -5 The TIM knockdown H1975 cells (siTIM#3) obtained in step 1 of Example 1 were treated with 30 mol / L for 48 h, and then the proliferation of the TIM knockdown H1975 cells (siTIM#3) after gefitinib treatment was detected by CCK8 method.

[0070] The results showed that knockdown of TIM expression did not affect the proliferation of H1975 cells to 1.0×10 -6 mol / L gefitinib, but significantly improved the efficacy of H1975 cells to 1.0×10 -5 mol / L gefitinib responsiveness (p<0.01) ( Figure 4 B).

[0071] Example 3: Knockdown of TIM inhibits EGFR and downstream AKT / mTOR pathway activation in NSCLC cells

[0072] In lung cancer, EGFR and its downstream PI3K / AKT / mTOR, RAS / RAF / MAPK pathways are abnormally active. In order to explore whether TIM affects the proliferation, migration and gefitinib responsiveness of NSCLC cells through the above pathways. The present invention first knocked down TIM expression in HCC827 cells using siTIM#3 according to the method in Example 1 to obtain TIM-knocked-down HCC827 cells (siTIM#3), and then used Western blot to detect the activation of EGFR (Genbank: NG_007726) and its downstream AKT / mTOR (AKT Genbank: NG_012188; mTOR Genbank: NG_033239) pathway activation in two cells: TIM-knocked-down H1975 cells (siTIM#3) and TIM-knocked-down HCC827 cells (siTIM#3).

[0073] The results showed that after knocking down TIM expression in H1975 cells, the p-EGFR level decreased and the ratio of p-EGFR to EGFR protein expression decreased significantly, indicating that EGFR activation was significantly reduced (p<0.05) ( Figure 5 A and Figure 5 B); p-AKT and p-mTOR levels decreased, the protein expression ratio of p-AKT to AKT decreased significantly, and the protein expression ratio of p-mTOR to mTOR decreased significantly, indicating that the activation of AKT and mTOR was also significantly reduced (p<0.05) ( Figure 5CE). Similarly, after knocking down TIM expression in HCC827 cells, the p-EGFR level decreased, and the ratio of p-EGFR to EGFR protein expression decreased significantly, indicating that EGFR activation was significantly reduced (p<0.05) ( Figure 5 F and Figure 5 G); p-AKT and p-mTOR levels decreased, the ratio of p-AKT to AKT protein expression decreased significantly, and the ratio of p-mTOR to mTOR protein expression decreased significantly, indicating that the activation of AKT and mTOR was significantly reduced (p<0.05) ( Figure 5 HJ).

[0074] Example 4: Effect of TIM knockdown on EGFR ligands in NSCLC cells

[0075] In lung cancer, stimulation of EGFR ligands is one of the pathways for EGFR activation. To investigate whether TIM affects EGFR ligands in NSCLC cells, the present invention employed RT-qPCR to detect changes in the mRNA levels of epidermal growth factor (EGF) and amphiregulin (AREG) in TIM-knockdown H1975 cells (siTIM#3) obtained in step 1 of Example 1. At the same time, TIM-knockdown H1975 cells (siNC) were used as a control. The primer sequences are as follows:

[0076] EGF-F: 5′-CATCATTGGCAAAACCAG-3′;

[0077] EGF-R: 5′-AACACCAAGCAGTTCCAAGC-3′;

[0078] AREG-F: 5′-ATATCACATTGGAGTCACTGCCCA-3′;

[0079] AREG-R: 5′-GGGTCCATTGTCTTATGATCCAC-3′.

[0080] The results showed that after knocking down TIM expression in H1975 cells, the mRNA levels of EGF and AREG were not affected ( Figure 6 ).

[0081] Example 5. Knockdown of TIM downregulates the levels of EGFR-regulated oncogenes in NSCLC cells

[0082] To investigate whether TIM affects the expression levels of EGFR-regulated oncogenes in NSCLC cells, the present invention employed RT-qPCR to detect changes in the mRNA levels of EGFR-related oncogenes BCRP (GenBank: AF098951.2), MYBL2 (sequence 3), CCND1 (sequence 4), c-MYC (GenBank: X66258.1), AURKA (GenBank: BC002499.2), PTGS2 (GenBank: KR709390.1), and TYMS (GenBank: NM_001354867.2) in H1975 cells (siTIM#3) knocked down with TIM obtained in step 1 of Example 1. Western blot was used to detect the expression level of c-MYC protein in H1975 cells (siTIM#3) knocked down with TIM obtained in step 1 of Example 1. Meanwhile, H1975 cells (siNC) knocked down with TIM were used as a control. The primer sequences for RT-qPCR detection are shown in Table 2.

[0083] Table 2. RT-qPCR primer sequences

[0084]

[0085]

[0086] The results showed that knockdown of TIM expression in H1975 cells could downregulate the mRNA levels of EGFR-related oncogenes in the nucleus, among which c-MYC and AURKA mRNA levels were significantly downregulated (p<0.05) ( Figure 7 AG); further detection revealed that the protein expression level of c-MYC was also significantly downregulated (p<0.01) ( Figure 7 H and Figure 7 I).

[0087] Example 6. Knockdown of TIM inhibits SPHK1 expression, STAT3 activation, and ATF3 expression in NSCLC cells. 1. SPHK1 mRNA levels are upregulated in tissues from NSCLC lung adenocarcinoma patients.

[0088] The present invention uses the RT-qPCR method to detect the SPHK1 mRNA expression level in cancer tissues and adjacent tissues of NSCLC lung adenocarcinoma patients.

[0089] SPHK1-F: 5′-CTGTCACCCATGAACCTGCT-3′;

[0090] SPHK1-R: 5′-TACAGGGAGGTAGGCCAGTC-3′.

[0091] The results showed that compared with adjacent adjacent tissues, SPHK1 mRNA levels were upregulated in NSCLC lung adenocarcinoma tissues ( Figure 8 A).

[0092] 2. Knockdown of TIM inhibits SPHK1 expression, STAT3 activation, and ATF3 expression in NSCLC cells

[0093] To investigate whether TIM affects the occurrence and development of NSCLC cells by affecting SPHK1, the present invention used Western blot to detect SPHK1 (GenBank: AAH08040.1) expression, STAT3 (GenBank: AAK17196.1) activation, and ATF3 (Genbank: CAG29330.1) expression levels in TIM knockdown H1975 cells (siTIM#3) and TIM knockdown HCC827 cells (siTIM#3).

[0094] The results showed that after knocking down TIM expression in H1975 cells, the expression level of SPHK1 was significantly decreased (p<0.05); the level of p-STAT3 was downregulated, and the protein expression ratio of p-STAT3 to STAT3 was significantly decreased, indicating that STAT3 activation was significantly reduced (p<0.05); the expression level of ATF3 was significantly decreased (p<0.01) ( Figure 8 BE). Similarly, in HCC827 cells, it was found that after knocking down TIM expression, the expression level of SPHK1 was significantly decreased (p<0.05); the level of p-STAT3 was downregulated, and the protein expression ratio of p-STAT3 to STAT3 was significantly decreased, indicating that STAT3 activation was significantly reduced (p<0.05); the expression level of ATF3 was also reduced ( Figure 8 FH).

[0095] Example 7: Knockdown of SPHK1 inhibits EGFR and downstream AKT / mTOR pathway activation in NSCLC cells

[0096] To investigate whether SPHK1 affects EGFR and downstream AKT / mTOR pathway activation in NSCLC cells, the present invention used RNAi interference technology to knock down SPHK1 expression in the H1975 cell line, generating SPHK1-knockdown H1975 cells. Western blot was then used to detect changes in EGFR activation and downstream AKT / mTOR pathway activation in the SPHK1-knockdown H1975 cells. The SPHK1 siRNA sequence is as follows:

[0097] SPHK1 siRNA sense strand sequence: 5′-GUGCACCCAAACUACUUCUTT-3′;

[0098] SPHK1 siRNA antisense strand sequence: 5′-AGAAGUAGUUUGGGUGCACTT-3′.

[0099] The results showed that after knocking down SPHK1 expression in H1975 cells, the p-STAT3 level decreased significantly, while the total STAT3 protein level did not change significantly, indicating that STAT3 activation was significantly reduced ( Figure 9 AC); p-EGFR level decreased, while EGFR total protein expression level did not change significantly, indicating that EGFR activation decreased; p-AKT and p-mTOR levels decreased, while AKT and mTOR total protein expression levels did not change significantly, indicating that downstream AKT / mTOR activation also decreased significantly ( Figure 9 DF).

[0100] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. The use of substances that inhibit TIM protein activity or reduce TIM protein content in the preparation of products that increase the responsiveness of non-small cell lung cancer cells to gefitinib; The substance that reduces the TIM protein content is siRNA that inhibits TIM gene expression; The siRNA for inhibiting TIM gene expression consists of sequence 5 and sequence 6 in the sequence listing.

2. The use according to claim 1, characterized in that: The method of increasing the responsiveness of non-small cell lung cancer cells to gefitinib is to increase the responsiveness of non-small cell lung cancer cells to 1.0×10 -5 Reactivity of gefitinib at 1 mol / L.

3. A method for improving the responsiveness of non-small cell lung cancer cells to gefitinib in vitro, comprising the following steps: introducing a substance that inhibits TIM protein activity or a substance that reduces TIM protein content into isolated non-small cell lung cancer cells to obtain isolated non-small cell lung cancer cells with TIM knockdown; the isolated non-small cell lung cancer cells with TIM knockdown are responsive to 1.0×10 -5 mol / L gefitinib was more reactive than the isolated non-small cell lung cancer cells; The substance that reduces the TIM protein content is siRNA that inhibits TIM gene expression; The siRNA for inhibiting TIM gene expression consists of sequence 5 and sequence 6 in the sequence listing.

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