Application of PDLIM4 inhibitors in the preparation of drugs for regulating gastric cancer sensitivity to cisplatin

Abstract

The present invention relates to the field of biomedicine and provides the use of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer to cisplatin. The present invention uses a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer cells to cisplatin. By inhibiting the expression of PDLIM4 in gastric cancer cells, the expression of HSP70 is reduced, the DNA damage signaling pathway is promoted, and the sensitivity of gastric cancer cells to cisplatin is thereby enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer to cisplatin. Background Art

[0002] Gastric cancer is a malignant tumor that originates from the epithelial cells of the gastric mucosa and is one of the cancers with the highest morbidity and mortality rates worldwide. Chemotherapy is generally used as an adjuvant treatment before, during, and after surgery to achieve the following goals: localize the lesions to improve the surgical resection rate; reduce the chance of tumor cell dissemination and implantation during surgery; perform adjuvant chemotherapy after radical surgery to eliminate possible residual lesions and prevent metastasis and recurrence; and after palliative surgery, control the progression of the disease and prolong survival. Cisplatin is a platinum-based drug and the first-line drug for chemotherapy of gastric cancer. However, cisplatin resistance often occurs, affecting the effectiveness of chemotherapy. Therefore, it is of great significance to identify molecular targets that affect cisplatin resistance and intervene. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the present invention provides the use of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer to cisplatin, with the aim of solving the problems mentioned in the background art.

[0004] PDLIM4 (PDZ and LIM domain protein 4) mainly plays a role in the cytoskeleton and signal transduction, helping cells maintain their shape and function, and participating in various biological processes such as cell muscle mechanics, adhesion, and movement.

[0005] In a first aspect, the present invention provides use of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer cells to cisplatin.

[0006] Furthermore, the PDLIM4 inhibitor inhibits the expression of PDLIM4 in gastric cancer cells, thereby reducing the expression of HSP70 and promoting the DNA damage signaling pathway, thereby enhancing the sensitivity of gastric cancer cells to cisplatin.

[0007] Furthermore, the PDLIM4 inhibitor includes at least one of an RNA interference molecule or a small molecule compound that inhibits the expression of PDLIM4.

[0008] Furthermore, the RNA interference molecule includes at least one of shRNA, siRNA, dsRNA or miRNA.

[0009] Furthermore, the shRNA includes shRNA1 and shRNA2, the shRNA1-F sequence is shown in SEQ ID NO.1, the shRNA1-R sequence is shown in SEQ ID NO.2, the shRNA2-F sequence is shown in SEQ ID NO.3, and the shRNA2-R sequence is shown in SEQ ID NO.4.

[0010] Furthermore, the gastric cancer cells include MKN45 cells or HGC27 cells.

[0011] In a second aspect, the present invention provides a pharmaceutical composition for combined treatment of gastric cancer, comprising a PDLIM4 inhibitor and cisplatin.

[0012] Furthermore, the pharmaceutical composition is used to treat patients with cisplatin-resistant gastric cancer.

[0013] Furthermore, the PDLIM4 inhibitor includes shRNA1 and shRNA2, the shRNA1-F sequence is shown in SEQ ID NO.1, the shRNA1-R sequence is shown in SEQ ID NO.2, the shRNA2-F sequence is shown in SEQ ID NO.3, and the shRNA2-R sequence is shown in SEQ ID NO.4.

[0014] The present invention has the following technical effects:

[0015] (1) PDLIM4 inhibitors are used to prepare drugs that regulate the sensitivity of gastric cancer cells to cisplatin. By inhibiting the expression of PDLIM4 in gastric cancer cells, the expression of HSP70 is reduced, the DNA damage signaling pathway is promoted, and the sensitivity of gastric cancer cells to cisplatin is enhanced.

[0016] (2) PDLIM4 inhibitors are used in combination with cisplatin to treat gastric cancer, especially for patients with cisplatin-resistant gastric cancer, where PDLIM4 inhibitors serve as chemotherapy sensitizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] A more complete understanding of exemplary embodiments of the present invention may be obtained by referring to the following drawings:

[0018] Figure 1 The qRT-PCR and Western blotting results of Example 1 of the present invention are as follows:

[0019] Figure 1 a in the figure is the result of qRT-PCR experiment, *** indicates P < 0.001;

[0020] Figure 1 Figure b is the result of Western blotting experiment.

[0021] Figure 2 2 is a flow cytometry test result diagram of Example 2 of the present invention, wherein:

[0022] Figure 2 The a in the figure is the detection of cell apoptosis by flow cytometry, where the green fluorescence emission wavelength of Annexin V-FITC (Annexin V-fluorescein isothiocyanate) is 530 nm; the red fluorescence emission wavelength of PI (propidium iodide) is 620 nm;

[0023] Figure 2 b in the figure is a statistical graph of cell apoptosis ratio, ns indicates no significant difference, ** indicates P < 0.01, and *** indicates P < 0.001.

[0024] Figure 3 This is a graph showing the results of a clone formation experiment to detect cell proliferation ability in Example 3 of the present invention, wherein:

[0025] Figure 3 Figure a is the staining result of clone formation experiment;

[0026] Figure 3 b in the figure is a statistical graph of the number of colonies per well, and *** indicates P < 0.001.

[0027] Figure 4 This is a graph showing the results of a CCK-8 experiment to detect cell viability in Example 4 of the present invention, where ** indicates P < 0.01 and *** indicates P < 0.001.

[0028] Figure 5 1 is a diagram showing the results of an in vivo nude mouse tumor formation experiment in Example 5 of the present invention, wherein:

[0029] Figure 5 Figure a is a general picture of the tumor of a nude mouse after dissection;

[0030] Figure 5 b in the figure is the weight of the tumor after nude mouse dissection, *** indicates P < 0.001;

[0031] Figure 5 c in the figure is the volume of tumor after nude mouse dissection, ** indicates P < 0.01, *** indicates P < 0.001.

[0032] Figure 6 : This is a graph showing the experimental results of the PDLIM4 downstream target gene HSP70 according to Example 6 of the present invention, wherein:

[0033] Figure 6 a in the figure is a mass spectrometry analysis diagram;

[0034] Figure 6 b is the result of immunofluorescence experiment;

[0035] Figure 6 Figure c is the result of the CO-IP experiment;

[0036] Figure 6 d in the figure is the result of qRT-PCR experiment, ns means no significant difference;

[0037] Figure 6 Figure e is the result of Western blotting experiment.

[0038] Figure 7 2 is a diagram showing the CHX experimental results of Example 7 of the present invention.

[0039] Figure 8 This is a graph showing the experimental results of MG132 in Example 8 of the present invention.

[0040] Figure 9 This is a diagram showing the ubiquitination results of HSP70 in Example 9 of the present invention.

[0041] Figure 10 This is a diagram showing the immunoprecipitation results of Example 10 of the present invention.

[0042] Figure 11 The qRT-PCR and Western blotting results of Example 11 of the present invention are as follows:

[0043] Figure 11 a in the figure is the result of qRT-PCR experiment, *** indicates P < 0.001;

[0044] Figure 11 Figure b is the result of Western blotting experiment.

[0045] Figure 12 This is a graph showing the results of flow cytometry detection of cell apoptosis in Example 12 of the present invention.

[0046] Figure 13 This is a diagram showing the results of a clone formation experiment to detect cell proliferation ability in Example 13 of the present invention.

[0047] Figure 14 This is a graph showing the results of the CCK-8 experiment for detecting cell viability in Example 14 of the present invention.

[0048] Figure 15 This is a diagram showing the Western blotting results of Example 15 of the present invention.

[0049] Figure 16 This is a graph showing the protein blotting results of Example 16 of the present invention.

[0050] Figure 17 This is a graph showing the changes in γ-H2AX in Example 17 of the present invention. DETAILED DESCRIPTION

[0051] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terms used herein are only for the purpose of describing specific embodiments rather than limiting the present invention.

[0053] The embodiments of the present invention provide the use of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer cells to cisplatin.

[0054] In some embodiments, the PDLIM4 inhibitor inhibits the expression of PDLIM4 in gastric cancer cells, thereby reducing the expression of HSP70 and promoting the DNA damage signaling pathway, thereby enhancing the sensitivity of gastric cancer cells to cisplatin.

[0055] In some embodiments, the PDLIM4 inhibitor comprises at least one of an RNA interference molecule or a small molecule compound that inhibits PDLIM4 expression.

[0056] In some embodiments, the RNA interference molecule comprises at least one of shRNA, siRNA, dsRNA, or miRNA.

[0057] In some embodiments, the shRNA includes shRNA1 and shRNA2, the shRNA1-F sequence is shown in SEQ ID NO.1, the shRNA1-R sequence is shown in SEQ ID NO.2, the shRNA2-F sequence is shown in SEQ ID NO.3, and the shRNA2-R sequence is shown in SEQ ID NO.4.

[0058] In some embodiments, the gastric cancer cells include MKN45 cells or HGC27 cells.

[0059] In some embodiments, the present invention provides a pharmaceutical composition for the combined treatment of gastric cancer, comprising a PDLIM4 inhibitor and cisplatin.

[0060] In some embodiments, the pharmaceutical composition is used to treat patients with cisplatin-resistant gastric cancer.

[0061] In some embodiments, the PDLIM4 inhibitor includes shRNA1 and shRNA2, the shRNA1-F sequence is shown in SEQ ID NO.1, the shRNA1-R sequence is shown in SEQ ID NO.2, the shRNA2-F sequence is shown in SEQ ID NO.3, and the shRNA2-R sequence is shown in SEQ ID NO.4.

[0062] Experimental Materials:

[0063] (1) Cells: MKN45 cells (gastric cancer cells), HGC27 cells (gastric cancer cells), and HEK-293T cells (human embryonic kidney cells) were maintained under standard laboratory conditions. HEK-293T cells were cultured in DMEM medium, while MKN45 and HGC27 cells were cultured in RPMI 1640 medium. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide with medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0064] (2) The PDLIM4 shRNAs used include PDLIM4 shRNA1 and PDLIM4 shRNA2, and their sequences are as follows:

[0065] PDLIM4 shRNA1-F: 5'-GATCGCACACAGGATCCACATCGATCTCGAGATCGATGTGGATCCTGTGTGCTTTTTG-3' (SEQ ID NO.1);

[0066] PDLIM4 shRNA1-R: 5'-AATTCAAAAAGCACACAGGATCCACATCGATCTCGAGATCGATGTGGATCCTGTGTGC-3' (SEQ ID NO. 2);

[0067] PDLIM4 shRNA2-F: 5'-GATCGAACCTCAAGCAGCGTGGTTACTCGAGTAACCACGCTGCTTGAGGTTCTTTTTTG-3' (SEQ ID NO.3);

[0068] PDLIM4 shRNA2-R: 5'-AATTCAAAAAAGAACCTCAAGCAGCGTGGTTACTCGAGTAACCACGCTGCTTGAGGTTC-3' (SEQ ID NO. 4).

[0069] Example 1:

[0070] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#1 group: PDLIM4 knockdown using PDLIM4 shRNA1; shPDLIM4#2 group: PDLIM4 knockdown using PDLIM4 shRNA2. The mRNA level of PDLIM4 was detected by qRT-PCR (real-time quantitative reverse transcription-polymerase chain reaction), and the protein level of PDLIM4 was detected by western blotting.

[0071] The results of qRT-PCR experiments are as follows Figure 1 The results of Western blotting are shown in a. Figure 1 As shown in b, the results showed that: the MKN45 cells and HGC27 cells with PDLIM4 knockdown were successfully constructed at the mRNA level; the protein level detection results of PDLIM4 were as shown in Figure 1 As shown in b, the results showed that the construction of MKN45 cells and HGC27 cells with PDLIM4 knockdown was successfully verified at the protein level.

[0072] Example 2:

[0073] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#1 group: PDLIM4 knockdown using PDLIM4 shRNA1; shPDLIM4#2 group: PDLIM4 knockdown using PDLIM4 shRNA2; each group was treated with or without cisplatin (5 μg / ml) for 24 hours, and cell apoptosis was detected by flow cytometry.

[0074] Flow cytometry results Figure 2 The results showed that, compared with the control group, the proportion of apoptotic cells in the shPDLIM4#1 group and the shPDLIM4#2 group increased by 5.04% and 2.47% respectively in MKN45 cells without cisplatin treatment. In the presence of cisplatin, the proportion of apoptotic cells in the shPDLIM4#1 group and the shPDLIM4#2 group increased by 23.11% and 15.23% respectively in MKN45 cells. The same trend was observed in HGC27 cells. This indicates that knocking down PDLIM4 enhances the sensitivity of gastric cancer cells to cisplatin.

[0075] Example 3:

[0076] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#1 group: PDLIM4 knockdown using PDLIM4 shRNA1; shPDLIM4#2 group: PDLIM4 knockdown using PDLIM4 shRNA2; each group was treated with or without cisplatin (100 ng / ml) for 24 hours, and the cell proliferation ability was detected by clonogenic assay.

[0077] The results of the clone formation experiment to detect cell proliferation ability are as follows Figure 3 As shown, the results showed that the proliferation ability of MKN45 cells and HGC27 cells with PDLIM4 knockdown was weaker than that of the control group; and compared with the control group with cisplatin added, the proliferation ability of MKN45 cells and HGC27 cells with PDLIM4 knockdown was significantly weaker after the addition of cisplatin, indicating that knocking down PDLIM4 enhanced the sensitivity of gastric cancer cells to cisplatin.

[0078] Example 4:

[0079] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#1 group: PDLIM4 knockdown using PDLIM4 shRNA1; shPDLIM4#2 group: PDLIM4 knockdown using PDLIM4 shRNA2; each group was treated with different concentrations of cisplatin (0, 2, 4, 6, 8, 10 μg / ml) for 24 hours, and cell viability was detected by CCK-8 assay.

[0080] The results of CCK-8 experiment for detecting cell viability are shown in Figure 4. The results showed that the cell viability of MKN45 cells and HGC27 cells with PDLIM4 knockdown was significantly lower than that of the control group.

[0081] Example 5:

[0082] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#1 group: PDLIM4 knockdown by PDLIM4 shRNA1; shPDLIM4#2 group: PDLIM4 knockdown by PDLIM4 shRNA2, and then expanded and cultured, digested and centrifuged, and washed twice with phosphate buffer to prepare cell suspension. Eight 4-week-old bald-nude female mice with good growth and uniformity were selected from each group. Each nude mouse was injected with axilla containing Each mouse was divided into four groups with a volume of 200 μL of cell suspension. After the tumors grew and reached a maximum diameter of approximately 10 mm, cisplatin (4 mg / kg) was injected intraperitoneally every three days. Tumor growth was observed. When the maximum diameter reached 1.5 cm, the nude mice were sacrificed, and the tumors were removed, weighed, and measured.

[0083] The results of in vivo nude mouse tumor formation experiments were as follows Figure 5 As shown, the results showed that the tumor growth rate and tumor weight and volume of nude mice in the PDLIM4 knockdown group were lower than those in the control group. After treatment with cisplatin, the tumor volume of nude mice in the PDLIM4 knockdown group was more significantly reduced, indicating that knockdown of PDLIM4 synergistically increased the efficacy of cisplatin.

[0084] Example 6:

[0085] (1) If Figure 6 As shown in (a), genes interacting with PDLIM4 were detected by mass spectrometry analysis, and the intersection with the genes predicted by BioGRID (a universal repository for biological interaction datasets) was taken. Taking into account the protein function, HSP70 was selected as a candidate protein.

[0086] (2) Immunofluorescence experiments were performed on MKN45 and HGC27 cells using confocal microscopy;

[0087] The results of immunofluorescence experiments were as follows Figure 6 As shown in b, the results showed that PDLIM4 and HSP70 co-localized in the cytoplasm.

[0088] (3) In MKN45 and HEK-293T cells, PDLIM4 was overexpressed using SFB-PDLIM4, and HSP70 was overexpressed with or without HA-HSP70. Co-immunoprecipitation (CO-IP) experiments were performed using HA antibodies.

[0089] The results of the CO-IP experiment are as follows Figure 6 As shown in c, the results show that HSP70 can specifically immunoprecipitate PDLIM4, proving that there is an interaction between PDLIM4 and HSP70.

[0090] (4) MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#1 group: PDLIM4 knockdown was performed using PDLIM4 shRNA1; shPDLIM4#2 group: PDLIM4 knockdown was performed using PDLIM4 shRNA2. The mRNA level of HSP70 was detected by qRT-PCR, and the protein level of HSP70 was detected by western blotting.

[0091] The results of qRT-PCR experiments are as follows Figure 6 The results of Western blotting experiments are shown in d. Figure 6 As shown in Figure e, the results showed that PDLIM4 only affected the protein level of HSP70 but did not affect the mRNA level of HSP70.

[0092] In summary, HSP70 protein interacts with PDLIM4.

[0093] Example 7:

[0094] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#2 group: PDLIM4 knockdown was performed using PDLIM4 shRNA2, and then a CHX (cycloheximide) experiment was performed. The protein levels of PDLIM4 and HSP70 were detected by Western blotting (0, 8, 10, and 12 h).

[0095] CHX is a protein synthesis inhibitor that can specifically bind to the small subunit of the ribosome, thereby preventing the normal translation process of mRNA and leading to the interruption of protein synthesis.

[0096] The results of CHX experiment are as follows Figure 7 As shown, the results showed that after knocking down PDLIM4, the degradation rate of HSP70 was significantly faster than that of the control group.

[0097] Example 8:

[0098] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#2 group: PDLIM4 knockdown using PDLIM4 shRNA2, with or without 20 μM MG132 treatment for 6 hours, and the protein levels of PDLIM4 and HSP70 were detected by Western blotting.

[0099] MG132 is a proteasome inhibitor that blocks the degradation pathway of ubiquitinated proteins.

[0100] The experimental results of MG132 are as follows Figure 8 As shown, the results showed that after knocking down PDLIM4, the protein level of HSP70 was restored to a certain extent, indicating that PDLIM4 may affect the stability of HSP70 through the ubiquitin proteasome pathway.

[0101] Example 9:

[0102] HSP70 was overexpressed in HEK-293T cells using HA-HSP70, and PDLIM4 was overexpressed with or without SFB-PDLIM4. PDLIM4 was knocked down in MKN45 cells using PDLIM4 shRNA2, and a control was set up. All cells were treated with 20 μM MG132 for 6 hours, then immunoprecipitated and the ubiquitination of HSP70 was detected by Western blotting.

[0103] The results of HSP70 ubiquitination are as follows Figure 9 As shown, the results showed that after overexpression of PDLIM4 in HEK-293T cells and immunoprecipitation, the ubiquitination level of HSP70 was reduced; after knockdown of PDLIM4 in MKN45 cells and immunoprecipitation, the ubiquitination level of HSP70 was increased; this indicates that PDLIM4 affects the stability of HSP70 through the ubiquitin proteasome pathway.

[0104] Example 10:

[0105] HSP70 was overexpressed using HA-HSP70 in HEK-293T cells, and PDLIM4 was overexpressed using SFB-PDLIM4. HSP70 was truncated into different domains: HSP70-ΔN terminus (120–642aa), HSP70-ΔC terminus (1–428aa), and HSP70-ΔABD terminus (1–120aa+420–642aa). Immunoprecipitation was performed, and the specific domains of PDLIM4 binding to HSP70 were verified by Western blotting.

[0106] Immunoprecipitation results were as follows Figure 10 As shown, the results showed that PDLIM4 binds to the C-terminus of HSP70 and affects the ubiquitination of HSP70.

[0107] Example 11:

[0108] MKN45 cells and HGC27 cells were set up as follows: control + empty vector group: no PDLIM4 knockdown and HSP70 overexpression; shPDLIM4#2 + empty vector group: PDLIM4 was knocked down by PDLIM4 shRNA2, and HSP70 was not overexpressed; shPDLIM4#2 + overexpressed HSP70 group: PDLIM4 was knocked down by PDLIM4 shRNA2 and HSP70 was overexpressed. The mRNA level of HSP70 was detected by qRT-PCR, and the protein levels of HSP70 and PDLIM4 were detected by western blotting.

[0109] The results of qRT-PCR experiments are as follows Figure 11 The results of Western blotting are shown in a. Figure 11As shown in b, the results showed that HSP70 was successfully overexpressed at both mRNA and protein levels in MKN45 cells and HGC27 cells.

[0110] Example 12:

[0111] MKN45 cells and HGC27 cells were set up as follows: control + empty vector group: no PDLIM4 knockdown and HSP70 overexpression; shPDLIM4#2 + empty vector group: PDLIM4 was knocked down by PDLIM4 shRNA2, without HSP70 overexpression; shPDLIM4#2 + overexpression HSP70 group: PDLIM4 was knocked down by PDLIM4 shRNA2 and HSP70 was overexpressed. After treatment with different concentrations of cisplatin (0, 2, 4 μg / ml), cell apoptosis was detected by flow cytometry.

[0112] Flow cytometry detection of cell apoptosis results Figure 12 As shown, the results showed that gastric cancer cells with PDLIM4 knockdown had enhanced sensitivity to cisplatin and apoptosis rate was significantly increased, but this effect was restored after overexpression of HSP70 in gastric cancer cells with PDLIM4 knockdown, indicating that PDLIM4 affects the sensitivity of gastric cancer cells to cisplatin through HSP70.

[0113] Example 13:

[0114] MKN45 cells and HGC27 cells were set up as follows: control + empty vector group: no PDLIM4 knockdown and HSP70 overexpression; shPDLIM4#2 + empty vector group: PDLIM4 was knocked down by PDLIM4 shRNA2, and HSP70 was not overexpressed; shPDLIM4#2 + overexpressed HSP70 group: PDLIM4 was knocked down by PDLIM4 shRNA2 and HSP70 was overexpressed. Each group was treated with or without cisplatin (100 ng / ml) for 24 hours, and the cell proliferation ability was detected by clone formation experiment.

[0115] The results of the clone formation experiment to detect cell proliferation ability are as follows Figure 13 As shown, the results showed that the gastric cancer cells in the shPDLIM4#2+empty vector group had enhanced sensitivity to cisplatin, and their cell proliferation ability was significantly weakened, significantly lower than that of the gastric cancer cells in the control+empty vector group. However, this effect was restored in the gastric cancer cells in the shPDLIM4#2+overexpression HSP70 group. That is, compared with gastric cancer cells only knocking down PDLIM4, gastric cancer cells knocking down PDLIM4 and overexpressing HSP70 had enhanced cell proliferation ability. This indicates that PDLIM4 affects the sensitivity of gastric cancer cells to cisplatin through HSP70.

[0116] Example 14:

[0117] MKN45 cells and HGC27 cells were set up as follows: control + empty vector group: no PDLIM4 knockdown and HSP70 overexpression; shPDLIM4#2 + empty vector group: PDLIM4 was knocked down by PDLIM4 shRNA2, and HSP70 was not overexpressed; shPDLIM4#2 + overexpressed HSP70 group: PDLIM4 was knocked down by PDLIM4 shRNA2 and HSP70 was overexpressed. Each group was treated with different concentrations of cisplatin (0, 2, 4, 6, 8, 10 μg / ml) for 24 hours, and cell viability was detected by CCK-8 assay.

[0118] The results of CCK-8 assay to detect cell viability were as follows: Figure 14 As shown, the results showed that the cell viability of gastric cancer cells after knocking down PDLIM4 was significantly lower than that of gastric cancer cells without knocking down PDLIM4 and overexpressing HSP70, but this effect was restored after overexpressing HSP70 in gastric cancer cells with knocking down PDLIM4, indicating that PDLIM4 affects the sensitivity of gastric cancer cells to cisplatin through HSP70.

[0119] Example 15:

[0120] MKN45 cells and HGC27 cells were set up as follows: control group: no PDLIM4 knockdown; shPDLIM4#1 group: PDLIM4 knockdown using PDLIM4 shRNA1; shPDLIM4#2 group: PDLIM4 knockdown using PDLIM4 shRNA2. The protein levels of γ-H2AX (a DNA damage indicator), H2AX, and PDLIM4 were detected by Western blotting.

[0121] Western blot results Figure 15 As shown, the results showed that γ-H2AX was significantly increased after knocking down PDLIM4, indicating that PDLIM4 is related to the DNA damage pathway.

[0122] Example 16:

[0123] MKN45 cells and HGC27 cells were set up as follows: control + empty vector group: no PDLIM4 knockdown and HSP70 overexpression; shPDLIM4#2 + empty vector group: PDLIM4 knockdown by PDLIM4 shRNA2, without HSP70 overexpression; shPDLIM4#2 + overexpressed HSP70 group: PDLIM4 knockdown by PDLIM4 shRNA2 and HSP70 overexpression by HSP70-HA. The protein levels of γ-H2AX, H2AX, HSP70 and PDLIM4 were detected by western blotting.

[0124] Western blot results Figure 16 As shown, the results showed that after knocking down PDLIM4, γ-H2AX was significantly increased. This phenomenon was restored after overexpression of HSP70 (γ-H2AX was reduced), indicating that PDLIM4 affects DNA damage through HSP70.

[0125] Example 17:

[0126] MKN45 cells and HGC27 cells were set up as follows: control + empty vector group: no PDLIM4 knockdown and HSP70 overexpression; shPDLIM4#2 + empty vector group: PDLIM4 was knocked down by PDLIM4 shRNA2, and HSP70 was not overexpressed; shPDLIM4#2 + overexpressed HSP70 group: PDLIM4 was knocked down by PDLIM4 shRNA2 and HSP70 was overexpressed. Gastric cancer cells were treated with cisplatin (4ug / ml) for 0, 6, 12, and 24 hours, and the cisplatin was removed to observe the changes in γ-H2AX.

[0127] The changes of γ-H2AX are as follows Figure 17 As shown, the results showed that knocking down PDLIM4 and adding cisplatin caused more severe DNA damage, while overexpressing HSP70 restored DNA damage to a certain extent, indicating that PDLIM4 affects DNA damage through HSP70, ultimately affecting sensitivity to cisplatin.

[0128] Based on the analysis of the results of Examples 1-17, the present invention concludes that PDLIM4 inhibitors are used to prepare drugs that regulate the sensitivity of gastric cancer cells to cisplatin. By inhibiting PDLIM4 expression in gastric cancer cells, HSP70 expression is reduced, DNA damage signaling pathways are promoted, and the sensitivity of gastric cancer cells to cisplatin is enhanced. PDLIM4 inhibitors can be combined with cisplatin to treat gastric cancer, particularly in patients with cisplatin-resistant gastric cancer, with the PDLIM4 inhibitor acting as a chemotherapy sensitizer.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a PDLIM4 inhibitor in the preparation of a drug for enhancing the sensitivity of gastric cancer cells to cisplatin, characterized in that: The PDLIM4 inhibitor includes shRNA that inhibits PDLIM4 expression, and the shRNA includes shRNA1 and shRNA2. The shRNA1-F sequence is shown in SEQ ID NO.1, the shRNA1-R sequence is shown in SEQ ID NO.2, the shRNA2-F sequence is shown in SEQ ID NO.3, and the shRNA2-R sequence is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that: The PDLIM4 inhibitor inhibits the expression of PDLIM4 in gastric cancer cells, thereby reducing the expression of HSP70 and promoting the DNA damage signaling pathway, thereby enhancing the sensitivity of gastric cancer cells to cisplatin.

3. The use according to claim 1 or 2, characterized in that: The gastric cancer cells include MKN45 cells or HGC27 cells.

4. A pharmaceutical composition for combined treatment of cisplatin-resistant gastric cancer patients, characterized by: The pharmaceutical composition includes a PDLIM4 inhibitor and cisplatin, wherein the PDLIM4 inhibitor includes shRNA1 and shRNA2, wherein the shRNA1-F sequence is shown in SEQ ID NO.1, the shRNA1-R sequence is shown in SEQ ID NO.2, the shRNA2-F sequence is shown in SEQ ID NO.3, and the shRNA2-R sequence is shown in SEQ ID NO.4.

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