Melanoma verofenib drug-resistant cell strain as well as construction method and application thereof

Abstract

The invention relates to the field of cell engineering, in particular to a melanoma verofenib drug-resistant cell strain as well as a construction method and application thereof. According to the invention, a parent human melanoma cell line A375 is used as a parent cell strain, verofenib is adopted to simulate an in-vivo treatment process, continuous treatment is carried out for 3 months, the drug resistance index of the cell strain to verofenib is detected through CCK-8, the cell strain resistant to verofenib is successfully screened out, the cell strain is named as the drug-resistant melanoma cell strain A375R homo sapiens, and the cell strain has the advantages that the drug resistance index of the drug-resistant melanoma cell strain A375R homo sapiens is shown in the description; the compound can be used for exploring a molecular mechanism or a signal channel for promoting or mediating the drug resistance of melanoma cells to the virofenib, and is used for exploring a molecular target spot for regulating and controlling the drug resistance of the melanoma virofenib, so that a targeted drug for reversing the drug resistance of the melanoma virofenib is developed. And the method is used for screening a drug resistance reversal agent of melanoma and searching a medication scheme for synergistically inhibiting drug resistance of a melanoma cell strain.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell engineering, and particularly relates to a melanoma verolifibine drug-resistant cell strain, a construction method and application thereof. BACKGROUND

[0002] It is a difficult point in the field of tumor research to explore the mechanism of tumor drug resistance and to find drugs and methods to delay or even reverse tumor drug resistance. BRAF is a serine / threonine kinase, and its gene mutation occurs in various cancers, including melanoma, lung cancer, colon cancer, gastric cancer, ovarian cancer and thyroid cancer. BRAF V600E Point mutations are the main mutation form, especially in melanoma, accounting for about 50% of all patients. BRAF V600E Mutations can activate BRAF kinase activity, and then activate the downstream MEK-ERK signaling pathway, which is composed of a group of sequentially activated protein kinases such as RAS / RAF / MEK / MAPK, and is involved in the regulation of important physiological functions such as cell proliferation, differentiation and survival, and plays an important role in the occurrence and development of various diseases such as tumors, and is an important target for tumor treatment.

[0003] Verolifibine is a specific BRAF inhibitor (BRAFi). Clinical trials have shown that the effective rate of verolifibine alone in the treatment of BRAF V600E mutant advanced melanoma patients can reach 50-60%, however, the efficacy usually lasts for about 7 months, and tumor cells are prone to rapidly develop drug resistance. BRAF and MEK inhibitor combination can improve the overall survival and progression-free survival of patients, but drug resistance still occurs. Therefore, reversing the drug resistance of molecular targeted drugs is a key strategy for the clinical treatment of melanoma, and studying the mechanism is a key to improving the treatment effect. In order to enhance the clinical effect, it is urgent to understand the mechanism of melanoma resistance to BRAF inhibitors and to find drugs and methods to delay or even reverse drug resistance, which not only has important significance for the basic research of drug genetics, but also has important significance for the clinical treatment of melanoma and all tumors. However, there is still a lack of effective drug resistance reversing agents or treatment programs to combat drug resistance. Therefore, the construction of melanoma cell strains resistant to verolifibine is of great significance for studying the mechanism of drug resistance, finding anti-drug resistance targets, and evaluating treatment programs after drug resistance.

[0004] Tumor drug-resistant cell strains are important research tools for analyzing the drug resistance mechanism of tumor cells and developing new drugs to combat tumor drug resistance. Although many tumor drug-resistant cell strains have been established at home and abroad, such as hepatocellular carcinoma sorafenib-resistant cell strains and esophageal cancer cisplatin-resistant cell strains, there is still no melanoma cell strain resistant to verolifibine. Therefore, it is of great scientific significance and clinical value to establish a melanoma verolifibine-resistant cell strain. SUMMARY

[0005] The present application aims at overcoming the deficiencies of the prior art, and proposes a melanoma verolfibine drug-resistant cell strain, a construction method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the technical scheme specifically adopted by the present application is as follows: The present application takes the parental human melanoma cell line A375 as the parental cell strain, uses verolfibine to simulate the in-vivo treatment process, continuously treats for 3 months, detects the drug resistance index of the cell strain to verolfibine through CCK-8, successfully screens out the cell strain resistant to verolfibine, the cell strain is named as drug-resistant melanoma cell strain A375R homo sapiens, and is preserved in the China Center for Type Culture Collection on September 24, 2025, the preservation number is CCTCC NO: C2025188, and the preservation address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The present application also provides a construction method of the above-mentioned melanoma verolfibine drug-resistant cell strain, comprising the following steps: S1, inoculate the parental human melanoma cell line A375 in a culture solution, when the cell strain growth density is fused to 80%-90%, carry out cell subculture, and obtain the parental A375 cell strain; S2, treat the parental A375 cell strain with 2 μM verolfibine, replace the fresh culture solution and re-add 2 μM verolfibine every 48 hours until the cell strain can stably proliferate in the culture medium containing 2 μM verolfibine, and obtain the drug-resistant melanoma cell strain A375R homo sapiens.

[0008] Further, in the step S1, the parental human melanoma cell line A375 is placed in the DMEM culture solution containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and is cultured in a cell culture box at 37℃ and 5% CO2, and the liquid is replaced every 48 hours until the cell strain growth density is fused to 80%-90%.

[0009] Further, in the step S2, the parental A375 cell strain is cultured in the complete DMEM culture solution containing 2 μM verolfibine, and the fresh culture solution is replaced and 2 μM verolfibine is re-added every 48 hours.

[0010] The melanoma vemurafenib drug-resistant cell strain can be used to explore the molecular mechanism or signal pathway that promotes or mediates the drug resistance of melanoma cells to vemurafenib. Specifically, researchers can use transcriptomics, metabolomics, single-cell genomics and other technologies, combined with conventional molecular biology experiments including Western blotting, immunofluorescence, real-time quantitative PCR, etc., to explore the molecular mechanism or signal pathway that promotes or mediates the drug resistance of melanoma cells to vemurafenib, with the parent cell strain as a reference.

[0011] The melanoma vemurafenib drug-resistant cell strain can be used to explore the molecular mechanism or signal pathway that promotes or mediates the drug resistance of melanoma cells to vemurafenib. Specifically, researchers can use transcriptomics, metabolomics, single-cell genomics and other technologies, combined with conventional molecular biology experiments including Western blotting, immunofluorescence, real-time quantitative PCR, etc., to explore the molecular mechanism or signal pathway that promotes or mediates the drug resistance of melanoma cells to vemurafenib, with the parent cell strain as a reference.

[0012] The melanoma vemurafenib drug-resistant cell strain can be used to screen drug resistance reversing agents for melanoma, and find drug regimens that can synergistically inhibit the drug resistance of melanoma cell strains. Specifically, researchers can use the drug-resistant cell strain, combined with CCK8, clonal formation and other cell proliferation detection methods, to screen some target drugs from existing chemotherapeutic drugs and compound libraries, explore the anti-proliferation effect of the target drugs on the vemurafenib drug-resistant cell strain, and obtain candidate drugs that can reverse the vemurafenib drug resistance of melanoma cells. At the same time, researchers can also screen multiple target drugs from the compound library, combined with CCK8, clonal formation and other cell proliferation detection methods, to explore whether multiple drugs can synergistically inhibit the proliferation of drug-resistant cell strains when combined with vemurafenib, thereby providing a theoretical basis for finding treatment regimens to combat vemurafenib drug resistance.

[0013] The present application uses human melanoma parent A375 cell strain, which is induced by vemurafenib for a long time, to obtain a new melanoma cell strain resistant to vemurafenib. The drug resistance index of the drug-resistant melanoma cell strain A375R homo sapiens meets the evaluation requirements of drug-resistant strains, and has important application prospects and scientific significance in the study of melanoma drug resistance mechanism, reversal of melanoma drug resistance and guidance of patient medication.

[0014] The drug-resistant melanoma cell strain A375R homo sapiens prepared by the present application can be used to screen key genes, proteins or RNAs (such as microRNAs, long noncoding RNAs, circular RNAs, etc.) that regulate the vemurafenib drug resistance of melanoma, to explore molecular targets that regulate the vemurafenib drug resistance of melanoma, and to develop related molecular targeted drugs.

[0015] The drug-resistant melanoma cell line A375R homo sapiens obtained by this invention is beneficial for exploring key signaling pathways that promote melanoma resistance to vemurafenib; screening candidate compounds that can reverse vemurafenib resistance in melanoma; and on this basis, exploring the inhibitory effect of two or more drugs in combination on drug-resistant melanoma cell lines, and further exploring drug regimens to reverse vemurafenib resistance in melanoma. Attached Figure Description

[0016] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The inhibition rate and IC50 value of different concentrations of vemurafenib on parental A375 cell line and drug-resistant A375R cell line were determined.

[0017] Figure 2 A375V vs A375C, A375RV vs A375RC, A375RC vs A375C and A375RV vs Differential expression gene test results for A375V.

[0018] Figure 3 The levels of ERK1 / 2 and p-ERK1 / 2 were determined after treatment with 2 μM vemurafenib on sensitive A375 cells and drug-resistant melanoma cell line A375Rhomo sapiens for 24 h. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0020] In the following examples, the DMEM culture medium used was a DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The DMEM (Dulbecco) culture medium contained... , The modified eagle medium (DMEM) was purchased from Gibco. Example 1

[0021] The construction of the vemurafenib-resistant human melanoma cell line A375R includes the following steps: (1) Culture of parental A375 cell line The parental human melanoma cell line A375 purchased from Shanghai Cell Bank of Chinese Academy of Sciences was placed in DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, and cultured in a cell incubator at 37°C, 5% CO2. The medium was changed every 48 hours, and when the cell density reached 80-90% confluence, the cells were subcultured. When the cells were subcultured, the original culture medium was first discarded, 5 mL of 1x PBS solution was added to wash the cells to remove residual serum. Then 1 mL of 0.25% trypsin was added, and the cells were digested at room temperature and observed under an inverted microscope. When the cells became round, retracted, and began to float, 6 mL of DMEM culture medium was immediately added to terminate the digestion, and the cells were gently blown into a single cell suspension and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was discarded, 8 mL of DMEM culture medium was added to resuspend the cells, and then the cells were inoculated in a 100 cm 6 culture dish at a density of 8 mL of DMEM culture medium per dish and 2x10 2 cells per dish.

[0022] (2) Construction of drug-resistant cell line A375R The parental A375 cell line was cultured in DMEM culture medium containing 2 μM vemurafenib (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin), and fresh culture medium was changed every 48 hours and 2 μM vemurafenib was added again. When the cell density reached 80% confluence, the cells were subcultured, and the subculture procedure was the same as that of the parental A375 cell line. The cells were continuously cultured for about 3 months until the cell characteristics were stable (after the addition of vemurafenib, the cell proliferation was no longer inhibited, and the IC50 value of A375R was more than 10 times that of the parental A375 cells), and the drug-resistant melanoma cell line A375R homo sapiens was successfully constructed. The successfully constructed drug-resistant melanoma cell line A375R homo sapiens was continuously cultured in DMEM culture medium containing 2 μM vemurafenib, and the cells were changed every two days and fresh vemurafenib was added to a final concentration of 2 μM.

[0023] (3) CCK-8 method for detecting drug resistance The parental A375 cell line and the drug-resistant melanoma cell line A375R homo sapiens in good growth condition and in logarithmic growth phase were respectively inoculated in 96-well plates at a density of 7x10 3Cells were seeded at a density of 100 μL in 96-well cell culture plates, with 100 μL of DMEM culture medium added to each well. The plates were incubated overnight at 37°C with 5% CO2. The following day, 100 μL of DMEM culture medium supplemented with vemurafenib was added to each well to bring the final concentrations of vemurafenib to 0 μM and 10 μM respectively. -4 μM, 10 -3 μM, 10 -2 μM, 10 -1 μM, 1μM, 10μM, 10 2 For the control group, DMEM culture medium containing an equal volume of DMSO was added. Each group had 5-6 replicates and was cultured for 72 h. 2.5 h before the end of the culture period, 10 μL of CCK-8 reagent was added, and incubation continued for another 2.5 h before termination of culture. Cell proliferation was measured using an absorbance (OD value) at 450 nm using a microplate reader (Saxony, BioTEK, USA). Cell proliferation rate was calculated using the following formula: Cell proliferation rate % = (A test -A blank ) / (A control -A blank ) × 100%, each experiment was repeated 3 times.

[0024] The IC50 values ​​of vemurafenib on the proliferation inhibition of parental A375 cell line and drug-resistant A375R cell line after 72 h were calculated using Graphpad Prism, and the resistance index (RI) was calculated as follows: RI = IC50 of drug-resistant A375R cell line / IC50 of parental A375 cell line. The results are as follows: Figure 1 As shown, the IC50 value of vemurafenib against A375 parental cells was 0.312 μM, while the IC50 value against A375R resistant cells was 30.67 μM, a difference of 98 times, indicating that the resistant cell subline was successfully constructed. Example 2

[0025] Transcriptome sequencing analysis of drug resistance To analyze the drug resistance of A375R to vemurafenib, the parental A375 cell line and the drug-resistant melanoma cell line A375R homo sapiens in good growth condition and logarithmic growth phase were treated with 2 μM vemurafenib for 24 hours, respectively, and then the cells were collected for transcriptome sequencing analysis. The experiment was divided into 4 groups (3 repeated samples in each group): A375 cell control group (A375 Control, A375C, treated with equal volume of DMSO (8 mL of culture solution + 1.6 μL of DMSO) for 24 hours), A375 vemurafenib treatment group (A375 Vem, A375V, treated with vemurafenib (8 mL of culture solution + 1.6 μL of 10 mM vemurafenib) for 24 hours), A375R cell control group (A375R Control, A375RC, treated with equal volume of DMSO for 24 hours) and A375R vemurafenib treatment group (A375R Vem, A375RV, treated with 2 μM vemurafenib for 24 hours). DESeq method was used to analyze the differentially expressed genes of A375V vs A375C, A375RV vs A375RC, A375RC vs A375C and A375RV vs A375V. The results are shown in Figure 2 A375V and A375C had 13243 differentially expressed genes, including 6483 up-regulated genes and 6760 down-regulated genes; A375RV and A375RC had 2652 differentially expressed genes, including 1082 up-regulated genes and 1570 down-regulated genes; A375RC and A375C had 7145 differentially expressed genes, including 3659 up-regulated genes and 3486 down-regulated genes; A375RV and A375V had 13852 differentially expressed genes, including 6757 up-regulated genes and 7095 down-regulated genes. Obviously, the number of differentially expressed genes of A375RV and A375RC was significantly less than that of A375V and A375C, indicating that the sensitivity of A375R cells to vemurafenib was significantly lower than that of A375 cells, and A375R cells indeed had drug resistance to vemurafenib, further indicating that the A375R drug-resistant cell line was successfully constructed. Example 3

[0026] Detection of ERK signal changes in A375R cells to evaluate drug resistance Vemurafenib treatment can inhibit the activation of ERK signaling in sensitive melanoma cells, and the reactivation of ERK signaling in drug-resistant melanoma cells treated with vemurafenib is the mechanism known for the drug resistance of melanoma to vemurafenib. To evaluate the changes in ERK signaling in A375R cells, sensitive A375 cells, i.e. the parental A375 cell line (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) and the drug-resistant melanoma cell line A375R homo sapiens were treated with 2 μM vemurafenib for 24 hours, respectively, and the levels of ERK1 / 2 and p-ERK1 / 2 were detected by Western blot. The results are shown in Figure 3 Figure 1: Compared with sensitive A375 cells, the levels of p-ERK1 and p-ERK2 in A375R cells were up-regulated (ERK1 and ERK2 were used as internal controls, respectively), indicating that A375R cells had drug resistance characteristics, and the drug-resistant melanoma cell line A375R homo sapiens was successfully constructed.

[0027] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only preferred examples of the present application and are not intended to limit the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A melanoma vemurafenib-resistant cell line, characterized in that, The drug-resistant cell line is A375R homo sapiens, a drug-resistant melanoma cell line, with accession number CCTCC NO:C2025188.

2. The application of the vemurafenib-resistant melanoma cell line as described in claim 1 in targeted drugs for vemurafenib-resistant melanoma.

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