Application of PDLIM4 inhibitor in preparation of medicine for regulating and controlling sensitivity of gastric cancer to cis-platinum

Abstract

The invention relates to the technical field of biological medicine, and provides application of a PDLIM4 inhibitor in preparation of a medicine for regulating and controlling sensitivity of gastric cancer to cis-platinum. According to the present invention, the PDLIM4 inhibitor is applied to the preparation of the drug for regulating the sensitivity of the gastric cancer cells to the cis-platinum, and the expression of the PDLIM4 in the gastric cancer cells is inhibited so as to reduce the expression of the HSP70 and promote the DNA damage signal channel, such that the sensitivity of the gastric cancer cells to the cis-platinum is enhanced;

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of PDLIM4 inhibitors in the preparation of drugs for regulating the sensitivity of gastric cancer to cisplatin. Background Art

[0002] Gastric cancer is a malignant tumor originating from gastric mucosal epithelial cells and is one of the cancers with relatively high incidence and mortality rates globally. Chemotherapy is generally used as adjuvant treatment before, during, and after surgery, and can achieve the following purposes: making the lesion limited to improve the surgical resection rate; reducing the chance of intraoperative tumor cell dissemination and implantation; adjuvant chemotherapy after radical surgery to eliminate possible residual lesions and prevent metastasis and recurrence; after palliative surgery, it can control the progression of the disease and prolong the survival period. Cisplatin is a platinum-based drug and is a first-line drug for gastric cancer chemotherapy. However, cisplatin resistance often occurs, affecting the chemotherapy effect. Therefore, it is of great significance to find molecular targets affecting cisplatin resistance and intervene. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the present invention provides the application of PDLIM4 inhibitors in the preparation of drugs for regulating the sensitivity of gastric cancer cells to cisplatin, aiming to solve 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, helps cells maintain their shape and function, and participates in various biological processes such as cell muscle mechanics, adhesion, and movement.

[0005] In the first aspect, the present invention provides the application of PDLIM4 inhibitors in the preparation of drugs for regulating the sensitivity of gastric cancer cells to cisplatin.

[0006] Further, the PDLIM4 inhibitor reduces the expression of HSP70 by inhibiting the expression of PDLIM4 in gastric cancer cells, promotes the DNA damage signaling pathway, and thereby enhances the sensitivity of gastric cancer cells to cisplatin.

[0007] Further, the PDLIM4 inhibitor includes at least one of RNA interference molecules or small molecule compounds that inhibit the expression of PDLIM4.

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

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

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

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

[0012] Further, the pharmaceutical composition is used for treating gastric cancer patients with cisplatin resistance.

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

[0014] The present invention has the following technical effects: (1) Applying the PDLIM4 inhibitor to 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, reducing the expression of HSP70, promoting the DNA damage signaling pathway, and thus enhancing the sensitivity of gastric cancer cells to cisplatin.

[0015] (2) The combined treatment of gastric cancer with the PDLIM4 inhibitor and cisplatin can especially treat gastric cancer patients with cisplatin resistance, where the PDLIM4 inhibitor serves as a chemotherapy sensitizer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] By referring to the following drawings, the exemplary embodiments of the present invention can be more completely understood:

[0017] Figure 1 are the experimental results of qRT-PCR and Western blotting in Example 1 of the present invention, where: Figure 1 a in is the experimental result diagram of qRT-PCR, *** indicates P < 0.001; Figure 1 b in is the experimental result diagram of Western blotting.

[0018] Figure 2 is the flow cytometry detection result diagram of Example 2 of the present invention, where: Figure 2 In a, the apoptosis of cells was detected by flow cytometry. The green fluorescence emission wavelength of Annexin V-FITC (Annexin V-fluorescein isothiocyanate) was 530 nm; the red fluorescence emission wavelength of PI (propidium iodide) was 620 nm. Figure 2 In b, it is a statistical chart of the cell apoptosis ratio. ns indicates no significant difference, ** indicates P < 0.01, and *** indicates P < 0.001.

[0019] Figure 3 It is a result graph of the clone formation experiment in Example 3 of the present invention to detect the cell proliferation ability, where: Figure 3 In a, it is a staining result graph of the clone formation experiment; Figure 3 In b, it is a statistical chart of the number of colonies per well, and *** indicates P < 0.001.

[0020] Figure 4 It is a result graph of the CCK-8 experiment in Example 4 of the present invention to detect the cell viability, ** indicates P < 0.01, and *** indicates P < 0.001.

[0021] Figure 5 It is a result graph of the in vivo tumorigenesis experiment of nude mice in Example 5 of the present invention, where: Figure 5 In a, it is a gross graph of the tumor of the nude mouse after dissection; Figure 5 In b, it is the weight of the tumor of the nude mouse after dissection, and *** indicates P < 0.001; Figure 5 In c, it is the volume of the tumor of the nude mouse after dissection, ** indicates P < 0.01, and *** indicates P < 0.001.

[0022] Figure 6 It is a result graph of the experiment on the downstream target gene HSP70 of PDLIM4 in Example 6 of the present invention, where: Figure 6 In a, it is a mass spectrometry analysis graph; Figure 6 In b, it is a result graph of the immunofluorescence experiment; Figure 6 In c, it is a result graph of the CO-IP experiment; Figure 6 In d, it is a result graph of the qRT-PCR experiment, and ns indicates no significant difference;

[0023] Figure 6 In e, it is a result graph of the Western blotting experiment.

[0024] Figure 7 It is the CHX experimental result diagram of Embodiment 7 of the present invention.

[0025] Figure 8 It is the MG132 experimental result diagram of Embodiment 8 of the present invention.

[0026] Figure 9 It is the result diagram of the ubiquitination of HSP70 in Embodiment 9 of the present invention.

[0027] Figure 10 It is the immunoprecipitation result diagram of Embodiment 10 of the present invention.

[0028] Figure 11 It is the experimental results of qRT-PCR and Western blotting in Embodiment 11 of the present invention, where: Figure 11 a in is the qRT-PCR experimental result diagram, *** indicates P < 0.001; Figure 11 b in is the Western blotting experimental result diagram.

[0029] Figure 12 It is the result diagram of flow cytometry for detecting cell apoptosis in Embodiment 12 of the present invention.

[0030] Figure 13 It is the result diagram of the clone formation experiment for detecting cell proliferation ability in Embodiment 13 of the present invention.

[0031] Figure 14 It is the result diagram of the CCK-8 experiment for detecting cell viability in Embodiment 14 of the present invention.

[0032] Figure 15 It is the Western blotting detection result diagram in Embodiment 15 of the present invention.

[0033] Figure 16 It is the Western blotting detection result diagram in Embodiment 16 of the present invention.

[0034] Figure 17 It is the result diagram of the change of γ-H2AX in Embodiment 17 of the present invention. Detailed implementation manners

[0035] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention will be 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 used to limit the present invention.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs; the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0037] An embodiment of the present invention provides an application of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer cells to cisplatin.

[0038] In some embodiments, the PDLIM4 inhibitor reduces the expression of HSP70 by inhibiting the expression of PDLIM4 in gastric cancer cells, promotes the DNA damage signaling pathway, and thereby enhances the sensitivity of gastric cancer cells to cisplatin.

[0039] In some embodiments, the PDLIM4 inhibitor includes at least one of an RNA interference molecule or a small molecule compound that inhibits the expression of PDLIM4.

[0040] In some embodiments, the RNA interference molecule includes at least one of shRNA, siRNA, dsRNA, or miRNA.

[0041] In some embodiments, the shRNA includes shRNA1 and shRNA2. The shRNA1-F sequence is as shown in SEQ ID NO.1, the shRNA1-R sequence is as shown in SEQ ID NO.2, the shRNA2-F sequence is as shown in SEQ ID NO.3, and the shRNA2-R sequence is as shown in SEQ ID NO.4.

[0042] In some embodiments, the gastric cancer cells include MKN45 cells or HGC27 cells.

[0043] In some embodiments, the present invention provides a pharmaceutical composition for the combined treatment of gastric cancer, and the pharmaceutical composition includes a PDLIM4 inhibitor and cisplatin.

[0044] In some embodiments, the pharmaceutical composition is used for treating gastric cancer patients with cisplatin resistance.

[0045] In some embodiments, the PDLIM4 inhibitor includes shRNA1 and shRNA2. The shRNA1-F sequence is as shown in SEQ ID NO.1, the shRNA1-R sequence is as shown in SEQ ID NO.2, the shRNA2-F sequence is as shown in SEQ ID NO.3, and the shRNA2-R sequence is as shown in SEQ ID NO.4.

[0046] Experimental materials: (1)Cells: MKN45 cells (gastric cancer cells), HGC27 cells (gastric cancer cells), and HEK-293T cells (human embryonic kidney cells) were preserved under standard laboratory conditions. HEK-293T cells were cultured in DMEM medium, while MKN45 and HGC27 cells were both cultured in RPMI 1640 medium. The cells were incubated at 37 °C with medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in a humidified environment containing 5% carbon dioxide.

[0047] (2)The PDLIM4 shRNAs used included PDLIM4 shRNA1 and PDLIM4 shRNA2, and the sequences were as follows: PDLIM4 shRNA1-F: 5’-GATCGCACACAGGATCCACATCGATCTCGAGATCGATGTGGATCCTGTGTGCTTTTTG-3’ (SEQ ID NO.1); PDLIM4 shRNA1-R: 5’-AATTCAAAAAGCACACAGGATCCACATCGATCTCGAGATCGATGTGGATCCTGTGTGC-3’ (SEQ ID NO.2); PDLIM4 shRNA2-F: 5’-GATCGAACCTCAAGCAGCGTGGTTACTCGAGTAACCACGCTGCTTGAGGTTCTTTTTTG-3’ (SEQ ID NO.3); PDLIM4 shRNA2-R: 5’-AATTCAAAAAAGAACCTCAAGCAGCGTGGTTACTCGAGTAACCACGCTGCTTGAGGTTC-3’ (SEQ ID NO.4).

[0048] Example 1: MKN45 cells and HGC27 cells were respectively set as follows: Control group: no knockdown of PDLIM4; shPDLIM4#1 group: knockdown of PDLIM4 using PDLIM4 shRNA1; shPDLIM4#2 group: knockdown of PDLIM4 using PDLIM4 shRNA2. The mRNA level of PDLIM4 was detected by qRT-PCR (quantitative real-time reverse transcription-polymerase chain reaction), and the protein level of PDLIM4 was detected by Western blotting.

[0049] The results of the qRT-PCR experiment were as shown in Figure 1 a in the figure, and the results of the Western blotting experiment were as shown in Figure 1As shown in b in [reference], the results showed that MKN45 cells and HGC27 cells with knocked-down PDLIM4 were successfully constructed at the mRNA level; the protein level detection results of PDLIM4 are as Figure 1 shown in b in [reference], and the results showed that MKN45 cells and HGC27 cells with knocked-down PDLIM4 were successfully constructed at the protein level.

[0050] Example 2: MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#1 group: knockdown of PDLIM4 using PDLIM4 shRNA1; shPDLIM4#2 group: knockdown of PDLIM4 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.

[0051] The results of flow cytometry detection are as Figure 2 shown. The results showed that in MKN45 cells without cisplatin treatment, compared with the control group, the proportion of apoptotic cells in the shPDLIM4#1 group increased by 5.04%, and the proportion of apoptotic cells in the shPDLIM4#2 group increased by 2.47%; in MKN45 cells with cisplatin treatment, compared with the control group, the proportion of apoptotic cells in the shPDLIM4#1 group increased by 23.11%, and the proportion of apoptotic cells in the shPDLIM4#2 group increased by 15.23%; the same trend was observed in HGC27 cells. This indicates that knocking down PDLIM4 enhances the sensitivity of gastric cancer cells to cisplatin.

[0052] Example 3: MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#1 group: knockdown of PDLIM4 using PDLIM4 shRNA1; shPDLIM4#2 group: knockdown of PDLIM4 using PDLIM4 shRNA2; each group was treated with or without cisplatin (100 ng / ml) for 24 hours, and cell proliferation ability was detected by colony formation assay.

[0053] The results of detecting cell proliferation ability by colony formation assay are as Figure 3 shown. The results showed that the proliferation ability of MKN45 cells and HGC27 cells with knocked-down PDLIM4 was weaker than that of the control group; and compared with the control group added with cisplatin, the proliferation ability of MKN45 cells and HGC27 cells with knocked-down PDLIM4 was significantly weaker after adding cisplatin, which indicates that knocking down PDLIM4 enhances the sensitivity of gastric cancer cells to cisplatin.

[0054] Example 4: The MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#1 group: knockdown of PDLIM4 using PDLIM4 shRNA1; shPDLIM4#2 group: knockdown of PDLIM4 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.

[0055] The results of detecting cell viability by CCK-8 assay are shown in Figure 4, and the results show that the cell viability of MKN45 cells and HGC27 cells with knockdown of PDLIM4 was significantly lower than that of the control group.

[0056] Example 5: The MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#1 group: knockdown of PDLIM4 using PDLIM4 shRNA1; shPDLIM4#2 group: knockdown of PDLIM4 using PDLIM4 shRNA2, and then expanded in culture, washed twice with phosphate buffer after digestion and centrifugation, and cell suspensions were prepared. 8 4-week-old balc-nude female mice with good and uniform growth status were taken from each group, and 200 μL of cell suspension containing cells was injected into the axilla of each nude mouse and divided into 4 groups. After the tumor grew out and the maximum diameter reached about 10 mm, cisplatin (4 mg / kg) was injected intraperitoneally once every 3 days, the growth of the tumor was observed, and when the maximum diameter reached 1.5 cm, the nude mice were sacrificed, the tumors were taken, and the weight and volume were measured.

[0057] The results of in vivo experiments on nude mouse tumorigenesis are as Figure 5 shown, and the results show that the growth rate, weight and volume of the tumors in nude mice in the PDLIM4 knockdown group were smaller than those in the control group. After cisplatin treatment, the tumor volume in the PDLIM4 knockdown group decreased more significantly, indicating that knockdown of PDLIM4 synergistically increased the efficacy of cisplatin.

[0058] Example 6: (1) As shown in a of Figure 6 , genes interacting with PDLIM4 were detected by mass spectrometry analysis, and the intersection was taken with the genes predicted by BioGRID (a general repository for biological interaction datasets). Considering protein function comprehensively, HSP70 was selected as a candidate protein.

[0059] (2) Immunofluorescence experiments were performed on MKN45 cells and HGC27 cells using a confocal microscope; The results of the immunofluorescence experiment are as Figure 6As shown in b of , the results showed that PDLIM4 and HSP70 were co-localized in the cytoplasm.

[0060] (3)In MKN45 and HEK-293T cells, PDLIM4 was overexpressed using SFB-PDLIM4, and HSP70 was overexpressed with or without HA-HSP70. A CO-IP (co-immunoprecipitation) experiment was performed using an HA antibody. The results of the CO-IP experiment were as Figure 6 shown in c of , and the results showed that HSP70 could specifically immunoprecipitate PDLIM4, demonstrating an interaction between PDLIM4 and HSP70.

[0061] (4)MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#1 group: knockdown of PDLIM4 using PDLIM4 shRNA1; shPDLIM4#2 group: knockdown of PDLIM4 using PDLIM4 shRNA2. The mRNA level of HSP70 was detected by qRT-PCR, and the protein level of HSP70 was detected by Western blotting.

[0062] The results of the qRT-PCR experiment were as Figure 6 shown in d of , and the results of the Western blotting experiment were as Figure 6 shown in e of , and the results showed that PDLIM4 only affected the protein level of HSP70 and did not affect the mRNA level of HSP70.

[0063] In summary, the HSP70 protein interacts with PDLIM4.

[0064] Example 7: MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#2 group: knockdown of PDLIM4 using PDLIM4 shRNA2. Then, after a CHX (cycloheximide) experiment, the protein levels of PDLIM4 and HSP70 were detected by Western blotting (0, 8, 10, 12 h).

[0065] CHX is a protein synthesis inhibitor that can specifically bind to the small subunit of ribosomes, thereby preventing the normal translation process of mRNA and resulting in the interruption of protein synthesis.

[0066] The results of the CHX experiment were as Figure 7 shown, and the results showed that after knocking down PDLIM4, the degradation rate of HSP70 was significantly faster than that of the control group.

[0067] Example 8: The MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#2 group: knockdown of PDLIM4 using PDLIM4 shRNA2. After treatment with or without 20 μM MG132 for 6 hours, the protein levels of PDLIM4 and HSP70 were detected by Western blotting.

[0068] MG132 is a proteasome inhibitor that blocks the degradation pathway of ubiquitinated proteins.

[0069] The experimental results of MG132 are as Figure 8 shown. The results showed that after knockdown of 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.

[0070] Example 9: In HEK-293T cells, HSP70 was overexpressed using HA-HSP70, and PDLIM4 was overexpressed using SFB-PDLIM4 with or without; in MKN45 cells, PDLIM4 was knocked down using PDLIM4 shRNA2 and a control was set; after treatment with 20 μM MG132 for 6 hours, immunoprecipitation was performed, and the ubiquitination of HSP70 was detected by Western blotting.

[0071] The results of the ubiquitination of HSP70 are as Figure 9 shown. The results showed that after overexpression of PDLIM4 and immunoprecipitation in HEK-293T cells, the ubiquitination level of HSP70 decreased; after knockdown of PDLIM4 and immunoprecipitation in MKN45 cells, the ubiquitination level of HSP70 increased; this indicates that PDLIM4 affects the stability of HSP70 through the ubiquitin-proteasome pathway.

[0072] Example 10: In HEK-293T cells, HSP70 was overexpressed using HA-HSP70, and PDLIM4 was overexpressed using SFB-PDLIM4. HSP70 was truncated into different domains: HSP70-ΔN terminus (120–642 aa), HSP70-ΔC terminus (1–428 aa), and HSP70-ΔABD terminus (1–120 aa + 420–642 aa). Immunoprecipitation was performed, and the specific domain of the binding of PDLIM4 to HSP70 was verified by Western blotting.

[0073] The results of immunoprecipitation are as Figure 10 shown. The results showed that PDLIM4 binds to the C terminus of HSP70 and thus affects the ubiquitination of HSP70.

[0074] Example 11: The MKN45 cells and HGC27 cells were respectively set as follows: control + empty vector group: no knockdown of PDLIM4 and overexpression of HSP70; shPDLIM4#2 + empty vector group: knockdown of PDLIM4 using PDLIM4 shRNA2, no overexpression of HSP70; shPDLIM4#2 + overexpression of HSP70 group: knockdown of PDLIM4 using PDLIM4 shRNA2 and overexpression of HSP70. The mRNA level of HSP70 was detected by qRT-PCR, and the protein levels of HSP70 and PDLIM4 were detected by Western blotting.

[0075] The experimental results of qRT-PCR are as shown in Figure 11 a in, and the experimental results of Western blotting are as shown in Figure 11 b in. The results showed that HSP70 was successfully overexpressed at both the mRNA and protein levels in MKN45 cells and HGC27 cells.

[0076] Example 12: The MKN45 cells and HGC27 cells were respectively set as follows: control + empty vector group: no knockdown of PDLIM4 and overexpression of HSP70; shPDLIM4#2 + empty vector group: knockdown of PDLIM4 using PDLIM4 shRNA2, no overexpression of HSP70; shPDLIM4#2 + overexpression of HSP70 group: knockdown of PDLIM4 using PDLIM4 shRNA2 and overexpression of HSP70. After treatment with different concentrations of cisplatin (0, 2, 4 μg / ml), cell apoptosis was detected by flow cytometry.

[0077] The results of flow cytometry for detecting cell apoptosis are as shown in Figure 12 shown. The results showed that the sensitivity of gastric cancer cells with knockdown of PDLIM4 to cisplatin was enhanced, and the apoptosis rate increased significantly. However, this effect was restored after overexpression of HSP70 in gastric cancer cells with knockdown of PDLIM4, indicating that PDLIM4 affects the sensitivity of gastric cancer cells to cisplatin through HSP70.

[0078] Example 13: The MKN45 cells and HGC27 cells were respectively set as follows: control + empty vector group: no knockdown of PDLIM4 and overexpression of HSP70; shPDLIM4#2 + empty vector group: knockdown of PDLIM4 using PDLIM4 shRNA2, no overexpression of HSP70; shPDLIM4#2 + overexpression of HSP70 group: knockdown of PDLIM4 using PDLIM4 shRNA2 and overexpression of HSP70. Each group was treated with or without cisplatin (100 ng / ml) for 24 hours, and the cell proliferation ability was detected by colony formation assay.

[0079] The results of detecting the cell proliferation ability by colony formation assay were as Figure 13 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, which was 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 of HSP70 group, that is, compared with the gastric cancer cells with only knockdown of PDLIM4, the cell proliferation ability was enhanced after knockdown of PDLIM4 and overexpression of HSP70 in the gastric cancer cells. This indicates that PDLIM4 affects the sensitivity of gastric cancer cells to cisplatin through HSP70.

[0080] Example 14: The MKN45 cells and HGC27 cells were respectively set as follows: control + empty vector group: no knockdown of PDLIM4 and overexpression of HSP70; shPDLIM4#2 + empty vector group: knockdown of PDLIM4 using PDLIM4 shRNA2, no overexpression of HSP70; shPDLIM4#2 + overexpression of HSP70 group: knockdown of PDLIM4 using PDLIM4 shRNA2 and overexpression of HSP70. Each group was treated with different concentrations of cisplatin (0, 2, 4, 6, 8, 10 μg / ml) for 24 hours, and the cell viability was detected by CCK-8 assay.

[0081] The results of detecting the cell viability by CCK-8 assay were as Figure 14 shown. The results showed that the cell viability of gastric cancer cells after knockdown of PDLIM4 was significantly lower than that of gastric cancer cells without knockdown of PDLIM4 and overexpression of HSP70. However, this effect was restored after overexpression of HSP70 in the gastric cancer cells with knockdown of PDLIM4. This indicates that PDLIM4 affects the sensitivity of gastric cancer cells to cisplatin through HSP70.

[0082] Example 15: The MKN45 cells and HGC27 cells were respectively set as follows: control group: no knockdown of PDLIM4; shPDLIM4#1 group: knockdown of PDLIM4 using PDLIM4 shRNA1; shPDLIM4#2 group: knockdown of PDLIM4 using PDLIM4 shRNA2. The protein levels of γ-H2AX (DNA damage index), H2AX and PDLIM4 were detected by Western blotting.

[0083] The results of Western blotting detection were as Figure 15 shown. The results showed that after knockdown of PDLIM4, γ-H2AX increased significantly, indicating that PDLIM4 is related to the DNA damage pathway.

[0084] Example 16: The MKN45 cells and HGC27 cells were respectively set as follows: control + empty vector group: no knockdown of PDLIM4 and no overexpression of HSP70; shPDLIM4#2 + empty vector group: knockdown of PDLIM4 using PDLIM4 shRNA2, no overexpression of HSP70; shPDLIM4#2 + overexpression of HSP70 group: knockdown of PDLIM4 using PDLIM4 shRNA2 and overexpression of HSP70 using HSP70-HA. The protein levels of γ-H2AX, H2AX, HSP70 and PDLIM4 were detected by Western blotting.

[0085] The results of Western blotting detection were as Figure 16 shown. The results showed that after knockdown of PDLIM4, the phenomenon of significant increase in γ-H2AX was restored (γ-H2AX decreased) after overexpression of HSP70, indicating that PDLIM4 affects DNA damage through HSP70.

[0086] Example 17: The MKN45 cells and HGC27 cells were respectively set as follows: control + empty vector group: no knockdown of PDLIM4 and no overexpression of HSP70; shPDLIM4#2 + empty vector group: knockdown of PDLIM4 using PDLIM4 shRNA2, no overexpression of HSP70; shPDLIM4#2 + overexpression of HSP70 group: knockdown of PDLIM4 using PDLIM4 shRNA2 and overexpression of HSP70. After treating gastric cancer cells with cisplatin (4 μg / ml) for 0, 6, 12, 24 hours respectively and then removing cisplatin, the changes of γ-H2AX were observed.

[0087] The change results of γ-H2AX were as Figure 17As shown, the results indicate that DNA damage is more severe after knocking down PDLIM4 and adding cisplatin. After overexpressing HSP70, the DNA damage is partially restored, suggesting that PDLIM4 affects DNA damage through HSP70 and ultimately affects the sensitivity to cisplatin.

[0088] From the result analysis of Examples 1 - 17, the present invention draws the following conclusion: The application of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer cells to cisplatin can inhibit the expression of PDLIM4 in gastric cancer cells, reduce the expression of HSP70, promote the DNA damage signaling pathway, and thereby enhance the sensitivity of gastric cancer cells to cisplatin. The combination of a PDLIM4 inhibitor and cisplatin for treating gastric cancer, especially for treating gastric cancer patients with cisplatin resistance, where the PDLIM4 inhibitor acts as a chemosensitizer.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a PDLIM4 inhibitor in the preparation of a drug for regulating the sensitivity of gastric cancer cells to cisplatin.

2. The application according to claim 1, wherein: The PDLIM4 inhibitor reduces the expression of HSP70 by inhibiting the expression of PDLIM4 in gastric cancer cells, promotes the DNA damage signaling pathway, and thereby enhances the sensitivity of gastric cancer cells to cisplatin.

3. The application according to claim 1, wherein: The PDLIM4 inhibitor includes at least one of an RNA interference molecule or a small molecule compound that inhibits the expression of PDLIM4.

4. The application according to claim 3, wherein: The RNA interference molecule includes at least one of shRNA, siRNA, dsRNA, or miRNA.

5. The application according to claim 4, wherein: The shRNA includes shRNA1 and shRNA2. The shRNA1-F sequence is as shown in SEQ ID NO.1, the shRNA1-R sequence is as shown in SEQ ID NO.2, the shRNA2-F sequence is as shown in SEQ ID NO.3, and the shRNA2-R sequence is as shown in SEQ ID NO.

4.

6. The application according to claim 1, wherein: The gastric cancer cells include MKN45 cells or HGC27 cells.

7. A pharmaceutical composition for the combined treatment of gastric cancer, characterized in that: The pharmaceutical composition includes a PDLIM4 inhibitor and cisplatin.

8. The pharmaceutical composition for the combined treatment of gastric cancer according to claim 7, wherein: The pharmaceutical composition is used for treating gastric cancer patients with cisplatin resistance.

9. The pharmaceutical composition for the combined treatment of gastric cancer according to claim 8, wherein: The PDLIM4 inhibitor includes shRNA1 and shRNA2. The shRNA1-F sequence is as shown in SEQ ID NO.1, the shRNA1-R sequence is as shown in SEQ ID NO.2, the shRNA2-F sequence is as shown in SEQ ID NO.3, and the shRNA2-R sequence is as shown in SEQ ID NO.4.

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