Application of TMEM147-AS1 inhibitors in the preparation of combination targeted drugs for the treatment of ovarian cancer

Abstract

This invention relates to the field of lncRNA and protein technology, and particularly to the application of a TMEM147-AS1 inhibitor in the preparation of combination targeted drugs for the treatment of ovarian cancer. The TMEM147-AS1 inhibitor is characterized by its sequence GCAGCUUCUUGUCCUCAUA dTdT. After methylation and cholesterol modification, the TMEM147-AS1 inhibitor specifically inhibits TMEM147-AS1 expression in a human xenograft model of ovarian cancer. Compared with existing technologies, this invention studies the expression differences of TMEM147-AS1 in normal human ovaries and ovarian cancer tissues, finding that TMEM147-AS1 is specifically highly expressed in human ovarian cancer tissues and cell lines. Therefore, this invention proposes that TMEM147-AS1 can serve as a potential drug target for combination therapy in ovarian cancer, thus providing the application of a TMEM147-AS1 inhibitor in the preparation of combination targeted drugs for the treatment of ovarian cancer, providing important experimental evidence and application guidance for the treatment of ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of lncRNA and protein technology, and in particular to the application of a TMEM147-AS1 inhibitor in the preparation of a combination targeted drug for the treatment of ovarian cancer. Background Technology

[0002] Ovarian cancer is one of the most deadly malignant tumors worldwide, with the highest mortality rate among all gynecological cancers. Recent reports show that ovarian cancer ranks fifth in mortality among female cancer patients, with a five-year survival rate of 45%. Epithelial ovarian cancer accounts for the highest proportion of all ovarian cancer cases, exceeding 50%. Currently, the main methods used clinically for ovarian cancer diagnosis include pelvic examination, vaginal ultrasound, and serum tumor marker CA125 testing. The main treatment is staging surgery combined with platinum / paclitaxel-based chemotherapy. Because ovarian cancer is prone to developing chemotherapy resistance during treatment, its prognosis is poor and recurrence rate is high.

[0003] Studies have shown that lncRNAs regulate gene expression at multiple levels in organisms and have become a recent research hotspot due to their important regulatory roles in cells. TMEM147-AS1, located on human chromosome 19q13.12, has a transcript length of 3300 bp and is the antisense lncRNA of the gene TMEM147. Antisense lncRNAs are generally non-coding RNA molecules transcribed from the antisense strand of a protein-coding gene and have sequence overlap with the mRNA of that gene. More importantly, antisense lncRNAs are usually associated with the expression of their sense strand genes, indicating that antisense lncRNAs can participate in the regulation of protein-coding gene expression. To date, no research reports have been found regarding the biological function of TMEM147-AS1 in ovarian cancer.

[0004] Nearly 20% of ovarian cancer patients develop resistance to standard platinum-based chemotherapy, making chemotherapy resistance one of the most critical issues in ovarian cancer treatment. Patients receiving chemotherapy face the risk of disease recurrence and drug resistance. More importantly, there are usually no effective treatments for acquired resistance. Currently, research on lncRNAs and ovarian cancer drug resistance is still in its early stages. Only a small number of lncRNAs have been found to be associated with chemotherapy resistance, and the related regulatory mechanisms include only DNA damage and apoptosis. Therefore, further research is needed to explore more potential lncRNAs that regulate chemotherapy resistance. Summary of the Invention

[0005] Given the lack of more potential lncRNAs that can regulate chemotherapy resistance in existing technologies, this invention provides the TMEM147-AS1 inhibitor for use in the preparation of combination targeted drugs for the treatment of ovarian cancer.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] This invention first provides a TMEM147-AS1 inhibitor targeting TMEM147-AS1, the sequence of which is GCAGCUUCUUGUCCUCAUA dTdT (3' overhang). The TMEM147-AS1 inhibitor sequence is shown in SEQ ID NO.1. After methylation and cholesterol modification, the TMEM147-AS1 inhibitor can specifically inhibit the expression of TMEM147-AS1 in a human xenograft model of ovarian cancer.

[0008] This invention further provides the application of TMEM147-AS1 inhibitor in the preparation of combination targeted drugs for the treatment of ovarian cancer.

[0009] In one embodiment of the present invention, the TMEM147-AS1 inhibitor is used in the preparation of a drug for inhibiting the expression of AURKA / DDX5 protein to inhibit the tumor growth of ovarian cancer.

[0010] In one embodiment of the present invention, the TMEM147-AS1 inhibitor is used in the preparation of a drug that inhibits the cell proliferation of HEY and SKOV3 cells.

[0011] In one embodiment of the present invention, the TMEM147-AS1 inhibitor is used in the preparation of a drug that inhibits the cell cycle transition of ovarian cancer cells G2 / M.

[0012] In one embodiment of the present invention, the TMEM147-AS1 inhibitor is used in the preparation of a drug that inhibits lipid droplet formation in HEY and SKOV3 cells.

[0013] In one embodiment of the present invention, the TMEM147-AS1 inhibitor is used in the preparation of a drug that inhibits lipophage in HEY and SKOV3 cells to promote cisplatin sensitivity in ovarian cancer.

[0014] In one embodiment of the present invention, the TMEM147-AS1 inhibitor and the AURKA inhibitor VX-680 are used in the preparation of a combination drug for promoting cisplatin sensitivity in ovarian cancer.

[0015] In one embodiment of the present invention, the TMEM147-AS1 inhibitor is used in the preparation of a medicament for non-small cell lung cancer.

[0016] In one embodiment of the present invention, the TMEM147-AS1 inhibitor, the AURKA inhibitor VX-680, and cisplatin are used in the preparation of a combination drug to inhibit the growth of ovarian cancer.

[0017] Compared with existing technologies, this invention studies the expression differences of TMEM147-AS1 in normal human ovaries and ovarian cancer tissues, and finds that TMEM147-AS1 is specifically highly expressed in human ovarian cancer tissues and cell lines. Therefore, this invention proposes that TMEM147-AS1 can be used as a potential drug target for combination therapy of ovarian cancer, and further provides the application of TMEM147-AS1 inhibitors in the preparation of combination targeted drugs for the treatment of ovarian cancer, providing important experimental evidence and application guidance for the treatment of ovarian cancer. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the principle of using TMEM147-AS1 inhibitor as a combination targeted drug for the treatment of ovarian cancer;

[0019] Figure 2. TMEM147-AS1 shows a high expression trend in epithelial ovarian cancer cell lines and tissues.

[0020] Figure 2 includes Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E , Figure 2F , Figure 2G , Figure 2H , Figure 2I , Figure 2J ;

[0021] A. Based on previous research, construct [systems] in OVCA-420 and OVCA-429 respectively. AURKA Overexpression / knockdown stable cell lines.

[0022] B. By analyzing the RNA-Seq results and screening for... AURKA lncRNAs that are expressed with positive and negative correlations.

[0023] C. Transcript length and chromosomal location of the selected lncRNAs.

[0024] D. Validation and confirmation of TMEM147-AS1 with qRT-PCR AURKA The expression shows a positive correlation.

[0025] E. Expression of TMEM147-AS1 in normal ovarian epithelial cell line HOSEpiC and human ovarian cancer cell lines A2780, HEY, SKOV3, OVCA-420, OVCA-429 and OVCA-433.

[0026] F. Expression of TMEM147-AS1 in 71 normal ovarian tissues and 60 epithelial ovarian cancer tissues.

[0027] The association between G. TMEM147-AS1 and overall survival in epithelial ovarian cancer.

[0028] The association between H. TMEM147-AS1 and progression-free survival in epithelial ovarian cancer.

[0029] I. Immunohistochemical detection of AURKA expression in 4 normal ovarian tissues and 4 epithelial ovarian cancer tissues.

[0030] J. In situ hybridization was used to detect the expression of TMEM147-AS1 in four normal ovarian tissues and four epithelial ovarian cancer tissues.

[0031] Figure 3. Knockdown of TMEM147-AS1 inhibits the proliferation of epithelial ovarian cancer cells and G2 / M cell cycle transition.

[0032] Figure 3 includes Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, and 3K;

[0033] A. qRT-PCR detection of changes in the expression level of TMEM147-AS1 in TMEM147-AS1 knockdown stable cell lines.

[0034] B. The CCK-8 assay confirmed that knocking down TMEM147-AS1 can inhibit the proliferation of epithelial ovarian cancer cells.

[0035] C. qRT-PCR was used to detect changes in the expression level of TMEM147-AS1 in TMEM147-AS1 overexpression stable cell lines.

[0036] D. The CCK-8 assay confirmed that overexpression of TMEM147-AS1 can promote the proliferation of epithelial ovarian cancer cells.

[0037] E&F. Clonogenesis assays confirmed that knocking down TMEM147-AS1 can inhibit the clonal ability of epithelial ovarian cancer cells.

[0038] G&H. clonogenic assays confirmed that overexpression of TMEM147-AS1 can promote the clonal ability of epithelial ovarian cancer cells.

[0039] I. Overexpression of TMEM147-AS1 can upregulate the IC50 value of VX-680 in ovarian cancer.

[0040] J&K. Overexpression of TMEM147-AS1 can reverse the blocking effect of VX-680 on the G2 / M phase.

[0041] Figure 4. TMEM147-AS1 can induce lipid droplet formation and lipophage to promote cisplatin resistance in epithelial ovarian cancer.

[0042] Figure 4 includes Figure 4A, Figure 4B, Figure 4C , Figure 4D , Figure 4E , Figure 4F , Figure 4G , Figure 4H

[0043] A. Knocking down TMEM147-AS1 can downregulate the IC50 value of cisplatin in ovarian cancer.

[0044] B. Overexpression of TMEM147-AS1 can upregulate the IC50 value of cisplatin in ovarian cancer.

[0045] C&E. Knockdown of TMEM147-AS1 promotes apoptosis in cisplatin-treated ovarian cancer.

[0046] D&F. Overexpression of TMEM147-AS1 can inhibit the apoptosis rate in cisplatin-treated ovarian cancer.

[0047] G. TMEM147-AS1 can induce lipid droplet formation in ovarian cancer cells.

[0048] H. TMEM147-AS1 can mediate lipophage in ovarian cancer cells to resist cisplatin treatment.

[0049] Figure 5. TMEM147-AS1 drives ovarian cancer tumor growth in vivo by activating AURKA / DDX5.

[0050] Figure 5 includes Figure 5A , Figure 5B , Figure 5C , Figure 5D , Figure 5E , Figure 5F , Figure 5G , Figure 5H

[0051] A. Knockdown of TMEM147-AS1 inhibits tumor formation in ovarian cancer cells.

[0052] B. Overexpression of TMEM147-AS1 promotes tumor formation in ovarian cancer cells.

[0053] Statistical analysis of tumor volume in the C. TMEM147-AS1 knockdown group.

[0054] Statistical analysis of tumor volume in the D. TMEM147-AS1 overexpression group.

[0055] Statistical analysis of tumor weight in the E. TMEM147-AS1 knockdown group.

[0056] Statistical analysis of tumor weight in the F. TMEM147-AS1 overexpression group.

[0057] G. TMEM147-AS1 knockdown tumor tissue was stained with hematoxylin and eosin solution. IHC was used to detect the protein levels of Ki-67, AURKA, and DDX5. In situ hybridization was used to detect the RNA level of TMEM147-AS1.

[0058] H. Tumor tissues overexpressing TMEM147-AS1 were stained with hematoxylin and eosin. IHC was used to detect the protein levels of Ki-67, AURKA, and DDX5. In situ hybridization was used to detect the RNA level of TMEM147-AS1.

[0059] Figure 6. The combination therapy of “TMEM147-AS1 inhibitor + VX-680 + cisplatin” can inhibit in vivo tumor formation in an ovarian cancer PDX model.

[0060] Figure 6 includes Figure 6A , Figure 6B , Figure 6C , Figure 6D , Figure 6E

[0061] A. Illustration of the injection of in vivo TMEM147-AS1 inhibitor, VX680 and cisplatin in a PDX model.

[0062] B. Tumor images of four groups of mice 21 days after combined intratumoral and intraperitoneal injection, including siNC+0.9% saline+CDDP, siTMEM147-AS1+0.9% saline+CDDP, siNC+VX-680+CDDP, and 50% siTMEM147-AS1+50% VX-680+CDDP.

[0063] C. Measure tumor volume twice a week and analyze it to construct a growth curve.

[0064] D. Collect the final tumor tissue and weigh it to determine the tumor weight.

[0065] E. Tumor tissues of the PDX model were stained with hematoxylin and eosin solution. Protein levels of Ki-67, AURKA, DDX5, ACC1, and LC3B were detected by IHC. RNA levels of TMEM147-AS1 were detected by in situ hybridization. Detailed Implementation

[0066] This invention first screens and identifies the long non-coding RNA TMEM147-AS1 as an important regulatory molecule in the downstream signaling pathway of AURKA. The steps include:

[0067] 1) Based on the expression levels of AURKA in different ovarian cancer cell lines in previous studies, construct ovarian cancer cell lines with stable overexpression or knockdown of AURKA.

[0068] 2) Then, collect relevant stable cells and perform RNA-Seq next-generation sequencing. Analyze the sequencing results and screen for lncRNAs that are positively or negatively correlated with the expression of AURKA.

[0069] 3) The expression level of the screened lncRNAs was detected by qRT-PCR, and the results of RNA-Seq analysis were summarized to determine that the final research object was TMEM147-AS1.

[0070] This invention also investigates the biological functions of TMEM147-AS1 in promoting ovarian cancer cell proliferation, G2 / M cell cycle transition, and cisplatin resistance. The specific methods are as follows:

[0071] 1) Based on the expression level of TMEM147-AS1 in different ovarian cancer cell lines, construct ovarian cancer cell lines with stable overexpression or knockdown of TMEM147-AS1.

[0072] 2) In vitro and in vivo experiments using Cell Counting Kit-8, clonogenic and BALB / c nude mouse subcutaneous tumor models demonstrated that TMEM147-AS1 plays an important regulatory role in the proliferation of ovarian cancer cells.

[0073] 3) In vitro experiments, including administration of the AURKA inhibitor VX-680 and flow cytometry analysis of the cell cycle, demonstrated that TMEM147-AS1 plays an important regulatory role in the G2 / M phase transition of ovarian cancer cells.

[0074] 4) In vitro and in vivo experiments, including IC50 and flow cytometry analysis of cell apoptosis cycle and cisplatin-injected BALB / c nude mouse subcutaneous tumor model, demonstrated that TMEM147-AS1 can regulate cisplatin resistance in ovarian cancer cells.

[0075] 5) Oil red staining was used to demonstrate that TMEM147-AS1 can promote lipid droplet formation in ovarian cancer cells.

[0076] This invention investigates the biological function of TMEM147-AS1 inhibitor combined with AURKA inhibitor VX-680 in promoting cisplatin sensitivity in ovarian cancer. The method is as follows:

[0077] An ovarian cancer PDX model was constructed, and TMEM147-AS1 inhibitor was injected intratumorally into the PDX model, while AURKA inhibitor VX-680 and cisplatin were injected intraperitoneally. The experiment demonstrated that the combination of "TMEM147-AS1 inhibitor + VX-680 + cisplatin" could inhibit the growth of ovarian cancer in the PDX model.

[0078] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, specific experimental conditions or methods are generally performed under conventional conditions—that is, the conditions described in "Molecular Cloning" (3rd ed.) or as recommended by the manufacturer.

[0079] Example 1: Screening and identification of the long non-coding RNA TMEM147-AS1 as an important regulatory molecule in the downstream signaling pathway of AURKA.

[0080] 1) AURKA Construction and stable transfection of expression plasmids and shRNA plasmids

[0081] Primers were designed (first upstream primer F: 5'-GCTCTAGAATGGACCGATCTAAAGAAAACT-3', sequence shown in SEQ ID NO.2; first downstream primer R: 5'-CGGGATCCCTAAGACTGTTTGCTAGCTG-3', sequence shown in SEQ ID NO.3), and PCR amplification was performed. AURKA The cDNA was used to construct the recombinant expression plasmid pCDH-puro- AURKA According to published articles from the laboratory, the synthesis is used for knockdown... AURKA The shRNA sequence (second upstream primer F: 5'-CCGGGTCTTGTGTCCTTCAAATTCTCGAGAATTTGAAGGACACAAGACTTTTTG-3', sequence shown in SEQ ID NO.4; second downstream primer R: 5'-AATTCAAAAAGTCTTGTGTCCTTCAAATTCTCGAGAATTTGAAGGACACAAGAC-3', sequence shown in SEQ ID NO.5) was obtained and the knockdown plasmid pLKO.1-puro- was constructed. AURKAReference laboratory detection in common ovarian cancer cells. AURKA The expression is constructed in OVCA-420 and OVCA-429 respectively. AURKA Stable cell lines were overexpressed and knocked down. Viral fluid was collected after transfection using a lentiviral packaging system, followed by infection of relevant cells for 24 h, and then selection using culture medium containing puromycin. The desired stable cell lines were identified by Western blotting.

[0082] 2) To AURKA RNA-Seq and qRT-PCR validation were performed on the stable cell lines.

[0083] Successfully built AURKA After overexpression and knockdown of stable cell lines, the cells were sent to CrystalBio (Shanghai) Co., Ltd. for RNA-Seq next-generation sequencing. The sequencing results were used to screen for altered lncRNAs, with screening criteria of fold increase (FC) > 1.2 or fold decrease (FC) < 0.8 and P < 0.05. Primers were designed and the screening results were verified by qRT-PCR, confirming that the final lncRNA for study was TMEM147-AS1.

[0084] 3) The expression of TMEM147-AS1 in common ovarian cancer cell lines and ovarian cancer tissues was detected using qRT-PCR and in situ hybridization.

[0085] We collected normal ovarian epithelial cell line HOSEpiC and various epithelial ovarian cancer cell lines, including A2780, HEY, SKOV3, OVCA420, OVCA429, and OVCA433, and extracted RNA from each cell line and reverse transcribed it.

[0086] Figure 2 shows the high expression trend of TMEM147-AS1 in epithelial ovarian cancer cell lines and tissues. A. Based on previous research, TMEM147-AS1 was constructed in OVCA-420 / OVCA-429, respectively. AURKA A. Overexpression / knockdown stable cell lines. B. Analyze RNA-Seq results and screen for lines that are compatible with... AURKA C. LncRNAs showing positive and negative correlations in expression. D. Transcript lengths and chromosomal locations corresponding to the screened lncRNAs. Evidence and confirmation of TMEM147-AS1 and its expression by qRT-PCR. AURKAE. Expression of TMEM147-AS1 in normal ovarian epithelial cell line HOSEpiC and human ovarian cancer cell lines A2780, HEY, SKOV3, OVCA-420, OVCA-429, and OVCA-433. F. Expression of TMEM147-AS1 in 71 normal ovarian tissues and 60 epithelial ovarian cancer tissues. G. Correlation between TMEM147-AS1 and overall survival in epithelial ovarian cancer. H. Correlation between TMEM147-AS1 and progression-free survival in epithelial ovarian cancer. I. Immunohistochemical detection of AURKA expression in 4 normal ovarian tissues and 4 epithelial ovarian cancer tissues. J. In situ hybridization detection of TMEM147-AS1 expression in 4 normal ovarian tissues and 4 epithelial ovarian cancer tissues.

[0087] qRT-PCR analysis revealed that TMEM147-AS1 showed a high expression trend in various epithelial ovarian cancer cell lines, compared to the normal ovarian epithelial cell line HOSEpiC. Simultaneously, tissue samples from 40-50 normal ovarian tissues and 40-50 epithelial ovarian cancer tissues were collected, and RNA was extracted and reverse transcribed. qRT-PCR analysis confirmed that TMEM147-AS1 showed a high expression trend in epithelial ovarian cancer samples compared to normal ovarian tissues. To further validate the qRT-PCR results, a specific probe for TMEM147-AS1 was designed, and its expression abundance in four normal ovarian tissues and four epithelial ovarian cancer tissues was detected using in situ hybridization (ISH).

[0088] Example 2: Biological function study of TMEM147-AS1 in promoting ovarian cancer cell proliferation, G2 / M cell cycle transition, and cisplatin resistance.

[0089] 1) Construction and stable transfection of TMEM147-AS1 expression plasmid and shRNA plasmid

[0090] The shRNA sequences for knocking down TMEM147-AS1 were designed and synthesized (third upstream primer F: 5'-CCGGGGGCCAGAACACGTGGCTTCCCTCGAGGGAAGCCACGTGTTCTGGCCCTTTTTG-3', sequence as shown in SEQ ID NO.6; third downstream primer R: 5'-AATTCAAAAAGGGCCAGAACACGTGGCTTCCCTCGAGGGAAGCCACGTGTTCTGGCCC-3', sequence as shown in SEQ ID NO.7; fourth upstream primer F: 5'-CCGGGGAACAGGAGTCAGAACTTAGCTCGAGCTAAGTTCTGACTCCTGTTCCTTTTTG-3', sequence as shown in SEQ ID NO.8; fourth downstream primer R: 5'-AATTCAAAAAGGAACAGGAGTCAGAACTTAGCTCGAGCTAAGTTCTGACTCCTGTTCC-3', sequence as shown in SEQ ID NO.9; fifth upstream primer F: The sequence of the 5'-CCGGGCAGCTTCTTGTCCTCATACCCTCGAGGGTATGAGGACAAGAAGCTGCTTTTTG-3' (as shown in SEQ ID NO.10) and the fifth downstream primer R: 5'-AATTCAAAAAGCAGCTTCTTGTCCTCATACCCTCGAGGGTATGAGGACAAGAAGCTGC-3' (as shown in SEQ ID NO.11) were used to construct the knockdown plasmid pLKO.1-puro-TMEM147-AS1. The TMEM147-AS1 overexpression plasmid was synthesized and constructed by Suzhou Genewiz Biotechnology Co., Ltd. into the recombinant expression plasmid pCDH-GFP-TMEM147-AS1, which was then identified by sequencing. Simultaneously, stable TMEM147-AS1 overexpression and knockdown cell lines were constructed in HEY and SKOV3 cells, respectively. The cells were transfected using a lentiviral packaging system, and the viral fluid was collected. The cells were then infected with the viral fluid for 24 h. The knockdown cell lines were screened using culture medium containing puromycin. Overexpressing cell lines were sorted for GFP using flow cytometry. After identification by qRT-PCR, the desired stable cell lines were selected.

[0091] 2) The effect of TMEM147-AS1 on cell proliferation was detected using the CCK-8 assay.

[0092] Cells were digested normally and counted. In a 96-well plate, 1,500–2,000 cells / well and 150 μL of cell suspension / well were added. Measurements were taken at 24 h, 48 h, 72 h, and 96 h, with four replicates per measurement. Before each measurement, the old culture medium was removed, and 100 µL of the working assay solution (5 µL CCK-8 + 95 µL serum-free medium) was added to each well. Four blank wells were added only to the working assay solution as blank controls. The plates were incubated in the dark for 2.5 h. 90 µL of the assay solution was transferred from each well to a clean 96-well ELISA plate, and the OD450 value was measured.

[0093] 3) Detection of the effect of TMEM147-AS1 on cell colony formation ability

[0094] Logarithmic growth phase cells were digested with trypsin to prepare a single-cell suspension. 500-600 cells were seeded into 6-well cell culture plates and cultured overnight. After 10-14 days, cells were fixed with methanol, stained with crystal violet, and the number of cell clones was counted to determine the effect of TMEM147-AS1 overexpression and knockdown on cell colony formation ability.

[0095] 4) Detection using the CCK-8 method AURKA IC50 values ​​after treatment with inhibitor VX-680 or cisplatin

[0096] Cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension and counted. In a 96-well plate, 10,000 cells / well and 150 μL of cell suspension / well were added, with four replicates per well. Nine concentration gradients were designed for the VX-680 group: 0 nM, 0.5 nM, 1 nM, 2 nM, 16 nM, 32 nM, 64 nM, 128 nM, and 256 nM; ten concentration gradients were designed for the cisplatin group: 0 μM, 0.0128 μM, 0.064 μM, 0.32 μM, 1.6 μM, 8 μM, 40 μM, 100 μM, 200 μM, and 100 μM. Cells were incubated at 37°C for 48 hours before detection. For the assay, remove the old culture medium and add 100 µL of the working assay solution (5 µL LCK-8 + 95 µL serum-free culture medium) to each well. Add only the working assay solution to four blank wells as blank controls. Incubate in the dark for 1.5 h. Transfer 90 µL of the assay solution from each well to a clean 96-well microplate and measure the OD450 value. Process the measured OD450 values ​​and perform curve fitting to calculate the final IC50 value.

[0097] 5) Detect the effect of TMEM147-AS1 on the cell cycle using flow cytometry.

[0098] Cells were digested and mixed with an equal volume of cell culture medium. The mixed cell suspension was transferred to 15 mL centrifuge tubes and centrifuged at 800 rpm for 5 min at room temperature to remove the cell supernatant. The cell pellet was pipetted with fresh cell culture medium containing 10% FBS to obtain a homogeneous cell suspension, and the total cell count was determined using a cell counter. Cells were seeded in 6-well cell culture plates at a seeding density of 2 × 10⁶ cells / well. 5 The cells were then statically cultured at 37°C with 5% CO2. Once the cells reached a suitable density, cell cycle synchronization and 10 nM [a specific treatment / treatment] were performed. AURKA Treatment with inhibitor VX-680. Cells were routinely digested and centrifuged at 1000 rpm for 5 min at room temperature, then the supernatant was discarded. Cells were resuspended in 1 mL PBS and washed twice. Cells were then resuspended in 0.3 mL PBS, followed by slow addition of pre-chilled anhydrous ethanol to the cell suspension until a final concentration of 70% was reached. The cell suspension was then inverted and gently mixed, and fixed overnight at -20°C. The next day, 20 µL of FBS was added to each tube to aid in cell precipitation after fixation, followed by centrifugation for 5 min at 4°C and 1000 rpm. The supernatant was discarded. Cells were washed once with PBS at 4°C, followed by centrifugation for 5 min at the same temperature and speed, and the supernatant was discarded. 0.2 mL of RNase A (100 ng / mL) was added to each well, and the cells were incubated at room temperature for 20 min for digestion. Add 200 µL of 2×PI solution to each well, mix thoroughly, and then incubate at room temperature for 15 min in the dark. Filter the cells using a 200-mesh filter and then analyze them.

[0099] 6) The effect of cisplatin-treated TMEM147-AS1 on apoptosis was detected by flow cytometry.

[0100] Cells were digested and mixed with an equal volume of cell culture medium. The mixed cell suspension was transferred to 15 mL centrifuge tubes and centrifuged at 800 rpm for 5 min at room temperature to remove the cell supernatant. The cell pellet was pipetted with fresh cell culture medium containing 10% FBS to obtain a homogeneous cell suspension, and the total cell count was determined using a cell counter. Cells were seeded in 6-well cell culture plates at a seeding density of 2 × 10⁶ cells / well. 5Cells were collected and then statically cultured at 37°C with 5% CO2. After 12 h, cisplatin was added to an appropriate concentration based on the IC50 value, and incubation continued for another 48 h. The cell supernatant was collected, and the cells were routinely digested. The cells were centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant was removed. The cells were resuspended in 1 mL PBS and washed once. Apoptosis of the collected cells was detected by flow cytometry using a PI / Annexin V-FITC or 7-AAD / Annexin V-APC apoptosis kit.

[0101] 7) Detection of the effect of TMEM147-AS1 on lipid droplet formation using Oil Red staining.

[0102] An appropriate amount of cells were seeded onto cell slides and co-cultured with oleic acid in the culture medium for 48 h, followed by starvation induction for 24 h. After 72 h of conditioned treatment, the cells were fixed and stained with Oil Red, and the formation of lipid droplets was observed under a microscope.

[0103] 8) Construct a subcutaneous tumor model in nude mice

[0104] TMEM147-AS1 overexpressing and knockdown stable cell lines in logarithmic growth phase were prepared into single-cell suspensions by trypsin digestion, with 3 × 10⁶ HEY cells included. 6 / 0.1 mL of cells and 5 × 10⁶ SKOV3 6 0.1 mL of cells were implanted subcutaneously into 5-week-old female BALB / c nude mice to construct a subcutaneous tumor model. Tumor size was measured weekly after injection. At week 6, the BALB / c mice were sacrificed, and the tumors were removed and weighed. A portion of the tumor tissue was homogenized in liquid nitrogen, RNA was extracted and reverse transcribed, and the expression of TMEM147-AS1, let-7b-5p, and let-7c-5p was detected by qRT-PCR. The remaining tumor tissue was stored in liquid nitrogen for subsequent Western blotting, in situ hybridization, and immunohistochemical experiments.

[0105] Figure 3 illustrates that knockdown of TMEM147-AS1 inhibits the proliferation of epithelial ovarian cancer cells and G2 / M cell cycle transition. A. qRT-PCR detection of TMEM147-AS1 expression level changes in TMEM147-AS1 knockdown stable cell lines. B. CCK-8 assay confirms that TMEM147-AS1 knockdown inhibits the proliferation of epithelial ovarian cancer cells. C. qRT-PCR detection of TMEM147-AS1 expression level changes in TMEM147-AS1 overexpression stable cell lines. D. CCK-8 assay confirms that TMEM147-AS1 overexpression promotes the proliferation of epithelial ovarian cancer cells. E&F. Clonogenic assay confirms that TMEM147-AS1 knockdown inhibits the clonal ability of epithelial ovarian cancer cells. G&H. Clonogenic assay confirms that TMEM147-AS1 overexpression promotes the clonal ability of epithelial ovarian cancer cells. I. Overexpression of TMEM147-AS1 upregulated the IC50 value of VX-680 in ovarian cancer. J&K. Overexpression of TMEM147-AS1 reversed the blocking effect of VX-680 on the G2 / M phase.

[0106] Figure 4 illustrates how TMEM147-AS1 induces lipid droplet formation and lipophage to promote cisplatin resistance in epithelial ovarian cancer. A. Knockdown of TMEM147-AS1 downregulates the IC50 value of cisplatin in ovarian cancer. B. Overexpression of TMEM147-AS1 upregulates the IC50 value of cisplatin in ovarian cancer. C & E. Knockdown of TMEM147-AS1 promotes apoptosis in cisplatin-treated ovarian cancer. D & F. Overexpression of TMEM147-AS1 inhibits apoptosis in cisplatin-treated ovarian cancer. G. TMEM147-AS1 induces lipid droplet formation in ovarian cancer cells. H. TMEM147-AS1 mediates lipophage in ovarian cancer cells to resist cisplatin treatment.

[0107] Figure 5 illustrates how TMEM147-AS1 drives ovarian cancer tumor growth in vivo by activating AURKA / DDX5. A. Knockdown of TMEM147-AS1 inhibits tumor formation in ovarian cancer cells. B. Overexpression of TMEM147-AS1 promotes tumor formation in ovarian cancer cells. C. Statistical analysis of tumor volume in the TMEM147-AS1 knockdown group. D. Statistical analysis of tumor volume in the TMEM147-AS1 overexpression group. E. Statistical analysis of tumor weight in the TMEM147-AS1 knockdown group. F. Statistical analysis of tumor weight in the TMEM147-AS1 overexpression group. G. TMEM147-AS1 knockdown tumor tissue stained with hematoxylin and eosin. IHC detection of Ki-67, AURKA, and DDX5 protein levels. In situ hybridization detection of TMEM147-AS1 RNA level. H. TMEM147-AS1 overexpression tumor tissue stained with hematoxylin and eosin. IHC was used to detect the protein levels of Ki-67, AURKA, and DDX5. In situ hybridization was used to detect the RNA levels of TMEM147-AS1.

[0108] Example 3: Constructing an ovarian cancer PDX model to implement a combination therapy regimen of "TMEM147-AS1 inhibitor + VX-680 + cisplatin".

[0109] Fresh ovarian cancer tissue was implanted subcutaneously into 5-week-old female BALB / c nude mice to construct a subcutaneous tumor model. After tumor formation (approximately 4-6 weeks), the BALB / c mice were sacrificed, and the tumor was excised and divided into equal-sized pieces. The excised tumor pieces were washed with sterile PBS and then implanted subcutaneously into a new batch of 5-week-old female nude mice. Tumor size was measured weekly for 2-3 weeks after implantation. Subsequently, the BALB / c mice within the same group were double-blindly divided into four groups and administered the following treatments: siNC + 0.9% saline + CDDP, siTMEM147-AS1 + 0.9% saline + CDDP, siNC + VX-680 + CDDP, and 50% siTMEM147-AS1 + 50% VX-680 + CDDP. Tumor size was measured every three days for 3-4 weeks based on tumor growth rate. At the end of the experiment, the BALB / c mice were sacrificed, and the tumors were excised and weighed. Some tumor tissue was ground in liquid nitrogen, RNA was extracted and reverse transcribed, and the expression of TMEM147-AS1, let-7b-5p and let-7c-5p was detected by qRT-PCR. The remaining tumor tissue was stored in liquid nitrogen for subsequent Western blotting, in situ hybridization and immunohistochemical experiments.

[0110] Figure 6 illustrates how the combination therapy of "TMEM147-AS1 inhibitor + VX-680 + cisplatin" can inhibit in vivo tumor formation in an ovarian cancer PDX model. A. Illustration of the injection of TMEM147-AS1 inhibitor, VX680, and cisplatin into the PDX model. B. Tumor images of four groups of mice 21 days after combined intratumoral and intraperitoneal injections, including siNC + 0.9% saline + CDDP, siTMEM147-AS1 + 0.9% saline + CDDP, siNC + VX-680 + CDDP, and 50% siTMEM147-AS1 + 50% VX-680 + CDDP. C. Tumor volume was measured twice weekly and analyzed to construct growth curves. D. The final tumor tissue was collected and weighed to determine tumor weight. E. Tumor tissue of the PDX model was stained with hematoxylin and eosin. IHC was used to detect the protein levels of Ki-67, AURKA, DDX5, ACC1, and LC3B. In situ hybridization was used to detect the RNA levels of TMEM147-AS1.

[0111] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of TMEM147-AS1 inhibitor, AURKA inhibitor VX-680, and cisplatin in the preparation of a combination therapy for ovarian cancer, characterized in that... The TMEM147-AS1 inhibitor sequence is GCAGCUUCUUGUCCUCAUA dTdT, where dTdT represents the 3' overhang; The combined medication is used to inhibit the formation of tumors in ovarian cancer in vivo.

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