CN113304268B - Application of SPDEF gene inhibitor in preparation of medicines for enhancing sensitivity of lung mucus adenocarcinoma cells to cisplatin - Google Patents

Application of SPDEF gene inhibitor in preparation of medicines for enhancing sensitivity of lung mucus adenocarcinoma cells to cisplatin Download PDF

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CN113304268B
CN113304268B CN202110449016.0A CN202110449016A CN113304268B CN 113304268 B CN113304268 B CN 113304268B CN 202110449016 A CN202110449016 A CN 202110449016A CN 113304268 B CN113304268 B CN 113304268B
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倪振华
王雄彪
林玉华
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SHANGHAI PUTUO DISTRICT CENTRAL HOSPITAL
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Abstract

The invention discloses application of SPDEF gene inhibitor in preparation of a medicament for enhancing sensitivity of lung mucus adenocarcinoma cells to cisplatin, and the sensitivity of a patient to cisplatin treatment can be enhanced by inhibiting the expression level of SPDEF gene in the lung mucus adenocarcinoma cells of the patient. The invention also discloses a pharmaceutical composition for resisting lung mucous adenocarcinoma in a combined way, which comprises the following components in parts by weight: the combination of the SPDEF gene inhibitor and cisplatin can further promote apoptosis of lung mucus adenocarcinoma cells compared to the single use of cisplatin as an anticancer drug.

Description

Application of SPDEF gene inhibitor in preparation of medicines for enhancing sensitivity of lung mucus adenocarcinoma cells to cisplatin
Technical Field
The invention relates to the field of biotechnology, in particular to application of an SPDEF gene inhibitor in preparation of a medicament for enhancing sensitivity of lung mucus adenocarcinoma cells to cisplatin.
Background
Lung cancer is one of the most frequent malignant tumors today and has the highest mortality rate, and seriously threatens human health. Among the types of lung cancer, non-small cell lung cancer (NSCLC, including lung adenocarcinoma, lung squamous carcinoma and large cell carcinoma) is the most common pathological type of lung cancer, accounting for more than 70% of the total lung cancer, with the proportion of lung adenocarcinoma also increasing. Lung mucinous adenocarcinoma is a unique subtype of lung adenocarcinoma, accounting for about 5% of all lung adenocarcinomas. Unlike other lung adenocarcinomas, the treatment options for lung mucinous adenocarcinomas are limited, and most lung mucinous adenocarcinomas have "KRAS" mutations and poor efficacy, and patients have poor quality of life and prognosis for survival. Therefore, the development of new therapeutic targets and drugs remains an important approach to improve prognosis of lung mucinous adenocarcinoma patients.
The SAM tip domain containing E26 transformation specific factor (SAM pointed domain cintaining Ets transcription factor, SPDEF) is one of the members of the Ets transcription factor family. SPDEF is overexpressed in lung cancer and has been identified as a marker gene for lung mucinous adenocarcinoma. The current research reports that SPDEF and "KRAS" in transgenic mouse models G12D "Co-induces the formation of malignant lung mucinous adenocarcinoma, SPDEF gene ID:25803. overexpression of SPDEF and promotion of goblet cell differentiation mediate invasion, metastasis and proliferation of lung tumors, indicating that SPDEF plays an important role in the development and progression of lung mucinous adenocarcinoma. Thus, inhibition of SPDEF expression may be an effective method of treating lung mucinous adenocarcinoma.
Resveratrol (trans-3, 5,4' -trihydroxystillbene) is a polyphenol that is found in many plants, such as giant knotweed and grape. At present, the application of resveratrol in lung mucous adenocarcinoma is not reported, so that whether resveratrol can be used as a potential drug for treating lung mucous adenocarcinoma is further researched, and a new treatment scheme is provided for treating lung mucous adenocarcinoma.
Disclosure of Invention
The invention aims to provide application of SPDEF gene inhibitor in preparation of medicines for enhancing sensitivity of lung mucus adenocarcinoma cells to cisplatin, and aims to a patient insensitive to the lung mucus adenocarcinoma cells, and the sensitivity of the patient to cisplatin treatment can be enhanced by reducing the expression level of SPDEF gene in the lung mucus adenocarcinoma cells.
In order to achieve the above object, the present invention provides the use of SPDEF gene inhibitor in the preparation of a medicament for enhancing sensitivity of lung mucinous adenocarcinoma cells to cisplatin.
Optionally, the inhibitor comprises: resveratrol.
Optionally, the inhibitor comprises: at least one of an inhibitor targeting the promoter of the SPDEF gene, an inhibitor targeting the transcript of the SPDEF gene, and an inhibitor targeting the translational product of the SPDEF gene.
Optionally, the inhibitor targeting the SPDEF gene transcript comprises: shRNA, siRNA, dsRNA, cDNA, miRNA.
Alternatively, the siRNA sequence is selected from the group consisting of: 5'-UCAAGGAGUUGCUACUCAATT-3'.
The invention also provides a pharmaceutical composition for combined anti-lung mucous adenocarcinoma, which comprises the following components: SPDEF gene inhibitor and cisplatin.
Optionally, the SPDEF gene inhibitor comprises: resveratrol.
Optionally, the SPDEF gene inhibitor comprises: sirnas targeting SPDEF gene transcripts.
The invention also provides application of the primer for detecting SPDEF gene expression in identifying sensitivity of lung mucus adenocarcinoma cells to cisplatin.
Optionally, the primer sequence is as follows:
the forward primer is: 5'-CCTCAAGGAGTTGCTACTCAAG-3';
the reverse primer is as follows: 5'-CTGGGAGATGTCTGGCTTCC-3'.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides application of SPDEF gene inhibitor in preparing medicines for enhancing sensitivity of lung mucus adenocarcinoma cells to cisplatin, and the sensitivity of patients to cisplatin treatment can be enhanced by reducing the expression level of SPDEF gene in the lung mucus adenocarcinoma cells of the patients.
(2) The invention provides a medicine which can inhibit SPDEF gene expression, namely resveratrol, which can inhibit SPDEF gene expression and enhance sensitivity of lung mucus adenocarcinoma patients to cisplatin by inhibiting promoter activity of SPDEF gene.
(3) The invention provides a pharmaceutical composition for resisting lung mucous adenocarcinoma in a combined mode, which comprises the following components: the combination of the SPDEF gene inhibitor and cisplatin can further promote apoptosis of lung mucus adenocarcinoma cells compared to the single use of cisplatin as an anticancer drug.
Drawings
FIG. 1 is a graph showing the comparison of the relative expression levels of SPDEF gene mRNA in lung mucous adenocarcinoma cells and lung adenocarcinoma cells in TCGA database.
FIG. 2 is a graph showing the increase in SPDEF mRNA expression of A549 and H292 cell lines after lentiviral treatment.
FIG. 3 is a graph comparing the viable cell amounts of A549 SPDEF over cell lines and A549 NC over cell lines after cisplatin treatment.
FIG. 4 is a graph showing a comparison of A549 SPDEF over cells and A549 NC over cell mortality after cisplatin treatment as measured by flow cytometry.
FIG. 5 is a graph comparing A549 SPDEF over cells and A549 NC over cell mortality after cisplatin treatment.
FIG. 6 is a graph showing the relative expression amounts of SPDEF gene mRNA in A549 si SPDEF cells and A549 si NC cells.
FIG. 7 is a graph comparing the viable cell amounts of A549 si SPDEF cell lines and A549 si NC cell lines after cisplatin treatment.
FIG. 8 is a graph comparing the viable cell amounts of H292 SPDEF over cell lines and H292 NC over cell lines after cisplatin treatment.
FIG. 9 is a graph showing the relative expression levels of SPDEF gene mRNA in A549 cells after treatment with resveratrol at 25. Mu.M and 50. Mu.M.
FIG. 10 is a graph showing comparison of the amounts of SPDEF protein expressed in A549 cells after treatment with resveratrol at 25. Mu.M and 50. Mu.M using Western blot.
FIG. 11 is a graph showing comparison of SPDEF protein expression levels in A549 cells after treatment with resveratrol at 25. Mu.M and at 50. Mu.M.
FIG. 12 is a graph comparing the luciferase activity of A549 cells transfected with resveratrol-treated pGL3-SPDEF promoter vector.
FIG. 13 is a graph showing comparison of apoptosis rate of A549 cells after detection of DMSO, resveratrol 50. Mu.M, cisplatin 20. Mu.M, and combination of resveratrol 50. Mu.M and cisplatin 20. Mu.M by flow cytometry.
FIG. 14 is a graph showing comparison of apoptosis rate of A549 cells after DMSO, resveratrol 50. Mu.M, cisplatin 20. Mu.M, and combination of resveratrol 50. Mu.M and cisplatin 20. Mu.M.
FIG. 15 is a graph showing comparison of viable cell amounts of A549 cells after DMSO, resveratrol 50. Mu.M, cisplatin 20. Mu.M, and resveratrol 50. Mu.M and cisplatin 20. Mu.M were used in combination.
FIG. 16 is a graph comparing the amounts of living cells of A549 SPDEF over cells and A549 NC over cells after the combined use of DMSO, resveratrol 50. Mu.M, cisplatin 20. Mu.M, and resveratrol 50. Mu.M and cisplatin 20. Mu.M.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and examples. The experimental procedure, without specific conditions noted in the examples, is generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer.
Experimental materials: human lung adenocarcinoma cells A549 and H292 cell lines were purchased from Shanghai national academy of sciences life research center, overexpressed SPDEF lentiviruses were purchased from Jikai, anti SPDEF (# 11467-1-AP), purchased from Proteintech, cisplatin was purchased from Sigma, CCK-8 and Annexin V-633 kits, purchased from DOjindo, trizol was purchased from TaKaRa, china, human SPDEF gene interference siRNA:5'-UCAAGGAGUUGCUACUCAATT-3' from RiboBio Inc. in China, transIT-TKO from RiboBio Inc. in China, esayGeno cloning kit from Tiangen Bio Inc. in China, lipofectamine3000 from Thermo Fisher Scientific in the United states.
In the application, A549 SPDEF over cells are lentivirus-induced A549 cells which over express SPDEF genes and are used for simulating lung mucous adenocarcinoma cells, the simulation method has been reported in literature (Gene signature driving invasive mucinous adenocarcinoma of the lung.DOI10.15252/emm.20160611), and the A549 NC over cells are control group cells.
In the application, the A549si SPDEF cells are A549 cells with siRNA interfering with SPDEF gene low expression, and the A549si NC cells are control group cells.
The SPDEF gene is highly expressed in lung mucus adenocarcinoma
SPDEF has been found to be overexpressed in lung adenocarcinoma. Applicants used the TCGA database (https:// www.cancer.gov /) to query the data for lung adenocarcinoma and lung mucinous adenocarcinoma, extract SPDEF expression, and use GraphPad Prism8 for one-way anova test and chart. As shown in fig. 1, the results indicate that the SPDEF gene has a higher expression level in lung mucus adenocarcinoma cells compared to lung adenocarcinoma cells. In the figure: MUC-LUAD represents expression amount data of SPDEF gene mRNA of lung mucous adenocarcinoma cells in TCGA database; LUAD represents expression level data of SPDEF gene mRNA of lung adenocarcinoma cells in TCGA database.
(II) SPDEF Gene is related to chemosensitivity of lung mucinous adenocarcinoma cells to cisplatin (DDP)
(1) Construction of SPDEF Gene-overexpressed A549 and H292 cell lines
The applicant used lentiviruses that overexpress SPDEF to induce A549 and H292 cell lines to overexpress SPDEF genes (A549 SPDEF over cells and H292 SPDEF over cells) to simulate lung mucous adenocarcinoma cells, while a control group (the lentiviruses used in the control group were similar to those used in the SPDEF over cells except that they did not contain the SPDEF genes) was used to test the expression of the SPDEF genes in both cell lines using Real-time quantitative PCR (Real-time Quantitative PCR, qPCR), as shown in FIG. 2, lentiviruses that overexpress the SPDEF genes were able to successfully induce A549 cells to overexpress the SPDEF genes.
(2) Comparison of sensitivity of A549 SPDEF over cells and A549 NC over cells to cisplatin (DDP)
After the applicant used cisplatin (DDP) 20. Mu.M and 40. Mu.M to act on A549 SPDEF over cells and A549 NC over cells for 48 hours, the number of viable cells was detected with CCK-8 kit, and the apoptosis rate was detected with Annexin V-633 kit as flow cell. As shown in fig. 3-5, the mortality of a549 SPDEF over cells after cisplatin treatment is lower than that of a549 NC over cells, and the result shows that the overexpression of SPDEF can reduce the chemosensitivity of a549 cells to cisplatin and reduce the pro-apoptosis effect of cisplatin.
(3) Comparison of sensitivity of A549 si SPDEF cells and A549 si NC cells to cisplatin
The applicant synthesized 1 pair of human interfering si RNAs by designing from the SPDEF cDNA sequence searched in GeneBank:
SEQ ID NO 1：5'-UCAAGGAGUUGCUACUCAATT-3'。
further, the expression of SPDEF gene in A549 cells was inhibited by the above-mentioned siRNA, and after 24 hours, treatment was performed with 20. Mu.M cisplatin, and after 24 hours, the number of living cells was detected with CCK-8 reagent. After 24 hours of human interference with the si RNA, the mRNA levels of human SPDEF were detected by real-time quantitative PCR to verify if si RNA interference was successful, using the primer sequences:
SEQ ID NO：2：CCTCAAGGAGTTGCTACTCAAG；
SEQ ID NO：3：CTGGGAGATGTCTGGCTTCC。
as shown in fig. 6, human interfering si RNA was able to successfully inhibit expression of SPDEF in a549 cells. In the figure: si SPDEF represents an A549 cell strain in which the SPDEF gene was expressed under low levels, and si NC was used as a control group (the si RNA used in the control group did not contain a fragment binding to the mRNA of the SPDEF gene). As shown in fig. 7, mortality of si SPDEF cells after cisplatin treatment was higher than si NC cells, indicating that inhibiting expression of SPDEF genes increased chemosensitivity of a549 cells to cisplatin.
(4) H292 cell validation: the expression level of SPDEF gene is related to the chemosensitivity of lung mucus adenocarcinoma cells to cisplatin
Applicants used cisplatin at 20. Mu.M and 40. Mu.M to induce H292 cells that overexpressed SPDEF in lentiviruses for 48H and then tested the viable cell count using CCK-8 kit. As shown in FIG. 8, after the SPDEF gene is over-expressed, the death rate of the cisplatin treatment is lower than that of the cells of the control group, which indicates that the over-expression of the SPDEF gene can reduce the chemosensitivity of lung cancer cells to cisplatin and reduce the pro-apoptosis effect of cisplatin.
(III) resveratrol (Res) the expression of SPDEF can be reduced by inhibiting the promoter activity of SPDEF of A549 cells
(1) Resveratrol (Res) can inhibit expression of SPDEF gene
After the applicant treated a549 cells with resveratrol (Res) 25 μm or 50 μm for 48h, total RNA and total protein were extracted and subjected to q-PCR and Western blot, respectively, to detect SPDEF mRNA and protein expression levels. As shown in fig. 9-11, resveratrol at 25 μm and 50 μm inhibited SPDEF mRNA and protein expression.
(2) Resveratrol inhibits expression of SPDEF gene by inhibiting promoter activity of SPDEF gene
The applicant firstly amplifies SPDEF promoter sequences at-1 to-1371 positions, and then clones the amplified DNA fragments into plasmids through an EsayGeno cloning kit to construct pGL3-SPDEF vectors. A549 cells were plated in 24 wells, 50000/well, overnight prior to transfection. The following day, plasmid was transfected into cells using Lipofectamine3000 for 24 hours, and cells were treated with resveratrol 50 μm for another 24 hours, and luciferase activity was detected using a dual luciferase system. As shown in fig. 12, resveratrol treated a549 cells had a luciferase activity lower than untreated a549 cells, indicating that resveratrol reduced expression of SPDEF by inhibiting promoter activity of SPDEF, inhibiting transcription thereof.
(IV) resveratrol and cisplatin in combination can promote apoptosis of lung mucus adenocarcinoma cells
After applicant used DMSO, resveratrol 50 μm, cisplatin 20 μm, and a combination of resveratrol 50 μm and cisplatin 20 μm for 48 hours for a549 cells, the apoptosis rate was measured with Annexin V-633 kit as flow cells, and the number of living cells was measured with CCK-8 kit. As shown in fig. 13-15, resveratrol and cisplatin can inhibit cell viability of a549 and promote apoptosis, and the combination of resveratrol and cisplatin can effectively reduce cell proliferation and promote apoptosis.
Applicants used resveratrol 50 μm, cisplatin 20 μm, and a combination of resveratrol 50 μm and cisplatin 20 μm as a549 cells for over-expression of SPDEF, set up control groups, and after 48 hours, examined the number of viable cells with CCK-8 kit. As shown in fig. 16, the results demonstrate that the combination of resveratrol and cisplatin can further promote apoptosis of lung mucinous adenocarcinoma cells compared to the effect of cisplatin alone on lung mucinous adenocarcinoma cells, and that over-expression of SPDEF can reverse the effect of resveratrol in increasing cisplatin chemosensitivity.
The above experiments show that: (1) Inhibiting expression of the SPDEF gene can enhance chemosensitivity of lung mucus adenocarcinoma cells to cisplatin; (2) Resveratrol can inhibit expression of the SPDEF gene by inhibiting promoter activity of the SPDEF gene; (3) Compared with the single use of cisplatin acting on lung mucus adenocarcinoma cells, the combination of resveratrol and cisplatin can further promote apoptosis of lung mucus adenocarcinoma cells.
In summary, the invention provides the application of SPDEF gene inhibitor in preparing medicaments for enhancing the sensitivity of lung mucous adenocarcinoma cells to cisplatin, and the sensitivity of patients to cisplatin treatment can be enhanced by reducing the expression level of SPDEF genes in the lung mucous adenocarcinoma cells of the patients.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims (1)

  1. Use of an SPDEF gene inhibitor for the manufacture of a medicament for increasing sensitivity of lung mucinous adenocarcinoma cells a549 to cisplatin, characterized in that the SPDEF gene inhibitor is an siRNA targeting SPDEF gene transcription product: 5'-UCAAGGAGUUGCUACUCAATT-3'.
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