Application of RPS6KA2 in the preparation of diagnostic reagents and therapeutic drugs for ovarian cancer

Abstract

In the invention of the application of RPS6KA2 in the preparation of diagnostic reagents and therapeutic drugs for ovarian cancer, the inventors found that RPS6KA2 was significantly downregulated in ovarian cancer tissues, and the low expression of RPS6KA2 in ovarian cancer patients was correlated with poor prognosis; overexpression of RPS6KA2 could inhibit cell proliferation in vitro and significantly inhibit tumor growth in vivo; RPS6KA2 inhibited the proliferation of ovarian cancer cells through the MAPK signaling pathway; the present invention also first revealed the biological function of circFAM169A as an upstream regulator of RPS6KA2 in ovarian cancer, and regulated RPS6KA2 through miR-106a-5p and miR-519d-3p; the present invention provides new clues for the treatment of ovarian cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical molecular biology, and particularly relates to the field of diagnosis and treatment of gynecological ovarian cancer. Background Art

[0002] Ovarian cancer remains the leading cause of death among gynecological cancers and the fifth leading cause of death among women. Due to the occult early symptoms of ovarian cancer and the difficulty of early diagnosis, many patients are already in the advanced stage at the time of diagnosis. Chemotherapy, radiotherapy, surgery, targeted therapy, and immunotherapy are the main treatment strategies for ovarian cancer at present. Despite the great progress of clinical technology, there are still many advanced patients who experience cancer recurrence. Therefore, it is crucial to identify new biomarkers and clarify the mechanisms of ovarian cancer progression.

[0003] The ribosomal protein S6 kinase (RPS6K) family is involved in multiple pathways, many of which play key roles in carcinogenesis. RPS6KA2 is a member of the RPS6K family. Different from other RPS6Ks, RPS6KA2 has a dual catalytic domain. The first domain is homologous to the cyclic AMP kinase family, while the second domain is homologous to the phosphorylase kinase family. Both of these domains are involved in growth factor-stimulated autophosphorylation of RPS6KA2. Some studies have shown that RPS6KA2 is related to the occurrence and development of prostate cancer. However, the exact functions of each subtype are still unclear. So far, the functional role of RPS6KA2 in ovarian cancer remains unclear.

[0004] Circular RNAs (circRNAs) are a subtype of non-coding RNAs. Different from traditional linear RNAs that contain 5' and 3' ends, circRNA molecules have a closed-loop circular structure and are less likely to be degraded by exonucleases. CircRNAs are related to cell stability, migration, differentiation, metabolism, and autophagy. Recent studies have found that circRNAs are abnormally expressed in breast cancer, ovarian cancer, and non-small cell lung cancer and affect the prognosis. CircFAM169A is a member of the circRNA family. Guo et al. found that abnormally regulated circFAM169A regulates intervertebral disc degeneration by targeting miR-583 and BTRC. However, the role of circFAM169A in the occurrence and development of ovarian cancer remains to be studied. Summary of the Invention

[0005] The present invention provides the following technical solutions through database bioinformatics prediction, in vitro cytological experiments, and detection experiments on clinical samples:

[0006] The first aspect of the present invention is to provide the use of RPS6KA2 in preparing a prognostic diagnostic marker for tumors, characterized in that RPS6KA2 is lowly expressed in tumor tissues, and the tumor patients have a poor prognosis; preferably, the tumor is a gynecological tumor, and more preferably, the tumor is ovarian cancer.

[0007] The second aspect of the present invention is to provide the use of RPS6KA2 in preparing a prognostic diagnostic kit for tumors, characterized in that RPS6KA2 is lowly expressed in tumor tissues; preferably, the tumor is a gynecological tumor, and more preferably, the tumor is ovarian cancer.

[0008] The third aspect of the present invention is to provide the use of RPS6KA2 in preparing a drug for treating tumors, characterized in that RPS6KA2 is lowly expressed in tumor tissues; preferably, the tumor is a gynecological tumor, and more preferably, the tumor is ovarian cancer. In a specific embodiment, the drug includes but is not limited to RPS6KA2 protein, a gene encoding RPS6KA2 protein, a vector containing the gene, and a pharmaceutically acceptable carrier.

[0009] The fourth aspect of the present invention provides the use of circFAM169A in promoting the expression of RPS6KA2, and the use is for non-therapeutic purposes.

[0010] The fifth aspect of the present invention provides the use of circFAM169A in preparing a reagent for promoting the expression of RPS6KA2. In a specific embodiment, circFAM169A spongily adsorbs miR-106a-5p and miR-519d-3p, reduces the targeted degradation of miR-106a-5p and miR-519d-3p on RPS6KA2 mRNA, and thus realizes the increase of the expression of RPS6KA2 in cells.

[0011] The sixth aspect of the present invention provides the use of circFAM169A in preparing a drug for treating ovarian cancer. In a specific embodiment, the drug includes but is not limited to the circFAM169A sequence, a gene encoding circFAM169A, a vector containing the gene, and a pharmaceutically acceptable carrier. In a specific embodiment, circFAM169A spongily adsorbs miR-106a-5p and miR-519d-3p, reduces the targeted degradation of miR-106a-5p and miR-519d-3p on RPS6KA2 mRNA, and thus realizes the increase of the expression of RPS6KA2 in cells.

[0012] The beneficial effects of the present invention are:

[0013] 1) RPS6KA2 was significantly downregulated in ovarian cancer tissues, and the low expression of RPS6KA2 in ovarian cancer patients was correlated with poor prognosis.

[0014] 2) Overexpression of RPS6KA2 could inhibit cell proliferation in vitro and significantly inhibit tumor growth in vivo, and it could be used as a potential drug for treating ovaries. It inhibited the proliferation of ovarian cancer cells through the MAPK signaling pathway.

[0015] 3) This study first revealed the biological function of circFAM169A as an upstream regulator of RPS6KA2 in ovarian cancer, and regulated RPS6KA2 through miR-106a-5p and miR-519d-3p.

[0016] The technical solution of the present invention provides new clues for the treatment of ovarian cancer. Brief Description of the Drawings

[0017] Figures 1A - 1G : The expression pattern of RPS6KA2 in ovarian cancer and its relationship with the survival prognosis of ovarian cancer;

[0018] Figures 2A - 2H : In vitro experiment: RPS6KA2 inhibits the proliferation of ovarian cancer cells;

[0019] Figures 3A - 3E : RPS6KA2 inhibits the proliferation of ovarian cancer cells through the MAKP pathway;

[0020] Figures 4A - 4D : miR-19a-39, miR-106a-5p and miR-519d-3p target RPS6KA2;

[0021] Figures 5A - 5F : CircFAM169A sponges miR-106a-5p and miR-519d-3p. Detailed Description of the Invention

[0022] The following further details the specific implementation manners and technical solutions of the present invention in conjunction with the accompanying drawings and specific embodiments. It should be clear that those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can clearly make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technical solutions of the present invention.

[0023] Example 1 Methods Used in the Present Invention

[0024] 1. Bioinformatics analysis

[0025] Pearson correlation analysis was used to extract ovarian cancer prognosis-related genes (|Pearson R|>0.5 and p<0.05) in each dataset. The prognosis-related genes screened from the TCGA database were cross-referenced with two GEO datasets (GSE26712, GSE26194) to obtain a shared prognosis-related gene (RPS6KA2). Then, the protein expression of RPS6KA2 in primary ovarian cancer (n = 426) and normal tissues (n = 88) was analyzed through the TCGA database. The KM-plot website (http: / / www.kmplot.com / ) showed the overall survival (OS) and recurrence-free survival (RFS). The Human Protein Atlas database (https: / / www.proteinatlas.org / ) was used to show the lower expression levels of RPS6KA2 in tumor tissues and normal tissues. Pathway analysis was performed through gene set enrichment analysis (GSEA, / / software.broadinstitute.org / gsea / index.isp). TargetScan (https: / / www.targetscan.org / vert_80 / ) was used to predict miRNAs targeting RPS6KA2. circRNAs were predicted by the circBank database (http: / / www.circbank.cn / index.html).

[0026] 2. Ovarian cancer specimens

[0027] All procedures involving human participants in this study complied with the ethical standards of the Declaration of Helsinki and its later amendments or similar ethical standards. The Ethics Committee of Zhejiang Cancer Hospital approved this study (NO. IRB-2021-124). Seventeen epithelial ovarian cancer samples and twenty-six normal ovarian samples were collected from the biobank of Zhejiang Cancer Hospital from June 2021 to December 2021.

[0028] 3. RNA extraction and quantitative real-time PCR (qRT-PCR)

[0029] Trizol reagent (Invitrogen) was used to isolate total RNA from clinical tissue specimens. Reverse transcription was performed using a reverse transcriptase cDNA synthesis kit according to the manufacturer's instructions. Then, the purity of total RNA was measured using a NanoDrop 2000 spectrophotometer. qRT-PCR was performed to detect the expression level of RPS6KA2. GAPDH represented the endogenous reference. The results were detected by the 2-ΔΔCT method. Each sample was tested in three independent experiments.

[0030] 4. Immunohistochemistry (IHC)

[0031] A standard procedure was performed for IHC staining. After deparaffinization and quenching of endogenous peroxidase, we incubated the sections with 1% bovine serum albumin (BSA) in PBS. Subsequently, the sections were treated with anti-RPS6KA2 antibody and then incubated with goat anti-rabbit peroxidase-conjugated secondary antibody. The DAB (3,3-diaminobenzidine) substrate was added, and the sections were counterstained with hematoxylin. Finally, the mounted sections were observed under a microscope (Leica, Germany).

[0032] 5. Cell culture

[0033] We purchased the human ovarian cancer cell lines IOSE386, HO8910, SKOV3, OVCAR3, and A2780 from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). All cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in 5% CO2.

[0034] 6. Cell proliferation assay

[0035] For the Cell Counting Kit-8 (Beyotime, China) assay, cells were seeded into 96-well plates and cultured at 2000 cells per well. After culturing the cells for 24 h, 48 h, 72 h, and 96 h respectively, 10 μg of CCK-8 reagent was added. After incubation for 2 h, the optical density (OD) value at an absorption wavelength of 450 nm of each well was measured in an enzyme-linked immunosorbent assay reader. For colony formation assay, cells were seeded into 6-well plates and cultured for 14 days. Finally, the colonies were fixed with 4% paraformaldehyde, stained with crystal violet, and photographed.

[0036] 7. Lentiviral transfection

[0037] Lentiviruses for RPS6KA2 overexpression and knockdown were constructed by BiolinkBioTECH (Shanghai, China). The lentiviruses were transfected into ovarian cancer cells (OVCAR3, SKOV3) using the ViaFect TM transfection reagent according to the manufacturer's instructions (Promega, USA), and then the expression levels of the transfected cells were compared with those of the empty vector-transfected cells.

[0038] 8. Western blotting

[0039] Radioimmuno-precipitation assay was used to lyse the total proteins of ovarian cancer cells. Bicinchoninic acid (BCA) protein assay kit was used to quantify the concentration of the extracted proteins. Protein samples were electrophoresed on polyacrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking overnight at 4 °C with 5% non-fat milk, the membranes were incubated with primary antibodies. Then, after rinsing with Tris-Buffered Saline and Tween buffer solution, the membranes were incubated with secondary antibodies for 1 hour at room temperature. Protein bands on the membranes were exposed by chemiluminescence.

[0040] 9. Subcutaneous xenograft nude mouse model

[0041] Nude mice were purchased and samples were collected according to the guidelines of Nanjing Medical University. Four-week-old female nude mice (5 mice / group) were subcutaneously injected with 1 × 10 7 ovarian cancer cells. All mice were sacrificed after 4 weeks, tumor lesions were excised and photographed. Tumor volume (mm3) was calculated as length × width2 / 2.

[0042] 10. Dual-luciferase reporter gene assay

[0043] The pMIR-REPORTTM system (Applied Biosystems, USA) was used to measure the interaction between mRNA, circRNA and miRNA. The expression plasmid of the transcription factor to be detected was co-transfected with the reporter plasmid into OVCAR3 and CAOV3 cell lines. Strictly according to the manufacturer's instructions, the dual-luciferase activity was tested using the Dual Luciferase Reporter Assay Kit (Promega, USA) after 48 hours.

[0044] 11. Statistical analysis

[0045] Experimental data were analyzed by GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA). Survival rate analysis was performed using Kaplan Meier survival analysis. Student's t-test was used for comparison between two groups, and one-way ANOVA was used for comparison among multiple groups. A p-value less than 0.05 was considered statistically significant.

[0046] Example 2. Expression pattern and function of RPS6KA2 in ovarian cancer

[0047] 1. The inventors first identified the top 100 prognosis-related genes from the TCGA database, 629 prognosis-related genes from the GSE26712 database, and 295 prognosis-related genes from the GSE26193 database. RPS6KA2 is the only gene related to prognosis in all three datasets, indicating that RPS6KA2 may play an important role in the occurrence, development, and prognosis of ovarian cancer ( Figure 1A ).

[0048] 2. The inventors evaluated the overall survival (OS) and recurrence-free survival (RFS) of RPS6KA2 through Kaplan-Meier curves ( Figure 1B and 1C ), and the results showed that ovarian cancer with high expression of RPS6KA2 had better OS and DFS.

[0049] 3. The expression of RPS6KA2 in ovarian cancer and normal tissues was verified through the RNA-seq data of TCGA and the ovarian cancer samples from the inventors' hospital ( Figure 1D -E). The expression level of RPS6KA2 mRNA in 17 tumor tissues and 26 normal tissues was detected by rt-PCR, and it was found that the expression level of RPS6KA2 mRNA in the tissues of ovarian cancer patients was significantly lower than that in normal tissues. In addition, the inventors analyzed the protein expression differences of RPS6KA2 in tumor tissues and normal tissues using the Human Protein Atlas database and immunohistochemical data of the inventors' ovarian cancer samples, and the results were consistent with those of mRNA ( Figure 1F and G).

[0050] 4. The inventors also analyzed the correlation between the expression level of RPS6KA2 and the clinicopathological characteristics of ovarian cancer patients. The expression level of RPS6KA2 was significantly correlated with tumor size and ascites (Table 1). These results indicate that low expression of RPS6KA2 may play a tumor-suppressive role in the development of ovarian cancer.

[0051] Table 1 Correlation between the expression level of RPS6KA2 and tumor size and ascites

[0052]

[0053] Example 3: In vitro experiment: RPS6KA2 inhibits the proliferation of ovarian cancer cells

[0054] To investigate the inhibitory effect of RPS6KA2 on ovarian cancer, the inventors detected the mRNA and protein expression of RPS6KA2 in four ovarian cancer cell lines (HO8910, SKOV3, OVCAR3, and AZ780) and a normal ovarian cancer cell line (IOSE386). Consistent with the results in ovarian cancer tumor tissues, the expression level of RPS6KA2 in ovarian cancer cells was lower than that in normal ovarian cells ( Figure 2A and 2B ). According to the expression of RPS6KA2, the inventors selected OVCAR3 and SKOV3 as representatives to study the relevant functional mechanisms. OVCAR3 was transfected with a negative control (NC) and an RPS6KA2 overexpression plasmid (OE), and SKOV3 was transfected with an empty vector control (NC) and an RPS6KA2 knockdown plasmid (Sh). First, WB and PCR were used to verify the transfection efficiency of the RPS6KA2-related plasmids, and it was found that the expression of RPS6KA2 in ovarian cancer cells could be effectively manipulated ( Figure 2C ). To further explore whether RPS6KA2 could inhibit the proliferation of ovarian cancer cells, the inventors conducted CCK-8 and colony formation assays. Compared with the control group cells, the cell viability of OVCAR3 cells in the OE group decreased, and the colony formation ability of ovarian cancer cells weakened. In contrast, the proliferation and colony formation ability of ovarian cancer cells were enhanced in SKOV3 cells transfected with RPS6KA2 ( Figure 2D ). A nude mouse subcutaneous tumorigenesis experiment was used to study the effect of RPS6KA2 on the in vivo proliferation of ovarian cancer. The inventors found that the tumor volume and weight were inhibited after overexpression of RPS6KA2 in the OVCAR3 group, and promoted after knockdown of RPS6KA2 in the SKOV3 group ( Figure 2E 、 2F 、2G). TUNEL apoptosis staining showed that the apoptosis rate in the OE group was higher than that in the NC group, while the apoptosis level in the Sh group was lower than that in the NC group ( Figure 2H ). The above results fully demonstrated that RPS6KA2 could inhibit the growth and proliferation of ovarian cancer cells.

[0055] Example 4 Mechanism of RPS6KA2 Inhibiting the Proliferation of Ovarian Cancer Cells

[0056] To explore the molecular mechanism of RPS6KA2 in ovarian cancer, the inventors performed gene set enrichment analysis according to the expression level of RPS6KA2. The top 8 pathways in KEGG were the calcium signaling pathway, cell cycle, DNA replication, ecm receptor interaction, MAPK signaling pathway, oxidative phosphorylation, RNA degradation, and vascular smooth muscle contraction ( Figure 3A ). Among these pathways, the MAPK pathway, which is usually associated with tumor development, was significantly enriched ( Figure 3B)。 The p-p38 / p38 and MAPK / p-MAPK proteins were detected in OVCAR3 cells transfected with NC and OE and in SKOV3 cells transfected with NC and Sh. Overexpression of RPS6KA2 decreased the expression of p-p38 and p-MAPK, while knockdown of RPS6KA2 increased the expression of p-p38 and p-MAPK( Figure 3C )。 This suggests that the MAKP signaling pathway may be involved in rps6ka2-mediated ovarian cancer progression. Further demonstrate the role of the MAKP signaling pathway in the regulation of ovarian cancer cell proliferation by RPS6KA2. The MAKP inhibitor SB239063 was added to OE-OVCAR3 cells and Sh-SKOV3 cells. Compared with the OE group, the proliferation ability of OVCAR3 cells was upregulated after treatment with SB239063. In SKOV3 cells, compared with the Sh group, the cell proliferation ability decreased after treatment with SB239063( Figure 3D and 3E )。 These data indicate that RPS6KA2 regulates ovarian cancer proliferation by inactivating the MAKP signaling pathway.

[0057] Example 4 miR-19a-39, miR-106a-5p and miR-519d-3p target RPS6KA2

[0058] To further explore the molecular mechanism of RPS6KA2, the inventors used TargetScan to predict related miRNAs. Among them, miR-19a-39, miR-106a-5p and miR-519d-3p were screened as prognosis-related targets. Figure 4A The prognostic relevance of these miRNAs to ovarian cancer was shown. In addition, the dual-luciferase reporter gene assay showed that miR-19a-39, miR-106a-5p and miR-519d-3p decreased the activity of the luciferase reporter gene fused to RPS6KA2( Figure 4B )。 The circBank database (http: / / www.circbank.cn) was screened to predict the ceRNAs of miR-19a-39, miR-106a-5p and miR-519d-3p. Then, circFAM169A was identified( Figure 4B )。 The inventors transfected CircFAM169A mimics into OVCAR3 cells and CircFAM169A inhibitors into SKOV3 cells. When circFAM169A mimicked OVCAR3 cells, its mRNA and protein levels increased, while when circFAM169A inhibited SKOV3 cells, its mRNA and protein levels decreased( Figure 4C Figure 4D )。

[0059] Example 5. CircFAM169A spongelike adsorbs miR-106a-5p and miR-519d-3p

[0060] CircFAM169A is derived from chr5:74,073,399-74162,776 of FAM169A pre RNA( Figure 5A ). Considering the connection between circFAM169A and miR-19a-39, miR-106a-5p and miR-519d-3p, the inventors hypothesized that circFAM169A is a sponge for miR-19a-39, miR-106a-5p and miR-519d-3p. Through FISH and nucleocytoplasmic fractionation experiments, circFAM169A is abundant in the cytoplasm( Figure 5B -C). The stability of circFAM169A, one of the characteristics of circRNAs, was verified by actinomycin D experiment. The results showed that FAM169A mRNA gradually degraded over time, while circFAM169A remained stable( Figure 5D ). Since miRNAs mainly function in an RNA-induced silencing complex (RISC)-dependent manner, which requires the participation of a key protein Argonaute 2 (AGO2). Therefore, it is very necessary to detect whether circFAM169A can bind to AGO2. The inventors conducted an RNA immunoprecipitation (RIP) experiment, and the results showed that the AGO2 antibody effectively pulled down circFAM169A( Figure 5E ). Finally, the dual-luciferase reporter gene experiment showed that miR-106a-5p and miR-519d-3p reduced the activity of the luciferase reporter gene fused with wild-type circFAM169A( Figure 5F ).

[0061] Through the above specific embodiments, the present invention confirmed the important role of RPS6KA2 in ovarian cancer. The inventors' data showed that circFAM169A, as a ceRNA of miR-106a-5p and miR-519d-3p, promoted RPS6KA2 expression and inhibited the proliferation of ovarian cancer cells. The inventors' research enriched the research on the molecular biological mechanism of ovarian cancer from the perspective of the circRNA-miRNA-mRNA network, and may provide a potential treatment method for ovarian cancer patients.

[0062] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. All those obtained by those skilled in the art in combination with the conventional techniques in the relevant fields are within the scope of the present invention.

Claims

1. The application of RPS6KA2 in preparing a prognostic diagnostic marker for tumors, characterized in that RPS6KA2 is lowly expressed in tumor tissues, and the patients from whom the tumors originate have poor prognoses. The tumors are ovarian cancers.

2. Use of RPS6KA2 in the preparation of a prognostic diagnosis kit for tumors, characterized in that RPS6KA2 is lowly expressed in tumor tissues, and the patients from whom the tumors originate have poor prognoses. The tumors are ovarian cancers.

3. Use of RPS6KA2 in the preparation of a medicament for treating tumors, characterized in that RPS6KA2 is lowly expressed in tumor tissues, and the patients from whom the tumors originate have poor prognoses. The tumors are ovarian cancers.

4. The application according to claim 3, wherein the drug comprises RPS6KA2 protein, a gene encoding RPS6KA2 protein, and a vector containing the gene; the drug further comprises a pharmaceutically acceptable carrier. Use of circFAM169A in the preparation of a drug for treating ovarian cancer, characterized in that, Among them, circFAM169A sponges miR-106a-5p and miR-519d-3p, reducing the targeted degradation of miR-106a-5p and miR-519d-3p on RPS6KA2 mRNA, thereby increasing the expression of RPS6KA2 in cells to achieve treatment.

6. The application according to claim 5, wherein the drug comprises a circFAM169A sequence, a gene encoding circFAM169A, and a vector containing the gene; the drug further comprises a pharmaceutically acceptable carrier.

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