Application of RBM4 in ovarian clear cell carcinoma

Abstract

The invention discloses an application of RBM4 in ovarian clear cell carcinoma, and researches find that a translation disorder event exists in the ovarian clear cell carcinoma (OCCC), and the translation disorder can promote the development of the OCCC; compared with normal tissues, the mRNA expression of RBM4 in OCCC tumor tissues is obviously reduced; compared with normal tissues, the protein expression level of RBM4 in OCCC tumor tissues is obviously increased; the knock-down of the RBM4 can inhibit the clone formation ability of the OCCC cells; the RBM4 is knocked down, so that the proliferation capacity of the OCCC cells can be inhibited; therefore, a possibility is provided for developing a novel OCCC treatment medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of RBM4 in ovarian clear cell carcinoma. Background Art

[0002] Ovarian cancer (OC) is one of the leading causes of death among women worldwide. [1] Ovarian clear cell carcinoma (OCCC) is a unique subtype of ovarian cancer. Compared with other populations, OCCC is significantly more common in East Asians and has unique clinical pathological and molecular biological characteristics. [2] OCCC is the second most common ovarian cancer and is insensitive to conventional treatments and has drug resistance. There is an urgent need to explore new treatments. [3-5] We conducted in-depth exploration of the pathogenesis of ovarian clear cell carcinoma through translationomics and transcriptomics, and found that a gene RBM4 has a higher translation efficiency in OCCC compared with normal tissues, affecting the proliferation and invasiveness of OCCC tumor cells. This provides a new direction for the development of new treatment methods for OCCC.

[0003] RNA binding motif 4 (RBM4) is an RNA binding factor that is mainly involved in post-transcriptional RNA regulatory processes such as mRNA alternative splicing, translation regulation, and RNA silencing. Previous studies have shown that RBM4 is a potential tumor suppressor for a variety of cancers, including lung cancer. [6] , breast cancer [7] , Gastric cancer [8] However, no studies have been conducted on ovarian cancer.

[0004] Yang Hongmei et al. evaluated the protein and mRNA expression levels of RBM4 in malignant gastric cancer and adjacent non-cancerous tissues and found that the levels in cancer tissues were significantly reduced and correlated with low survival rates. [9] . However, it did not conduct an in-depth study of the mechanism of action of RBM4, and only stayed at the level of statistical correlation. Zhou Guoxiong and others also found in their study of the molecular mechanism of liver cancer angiogenesis that RBM4 can directly bind to RelA / p65 mRNA, and recruit HNRNPM to form a heterotrimer to increase its stability, thereby activating the NF-kB signaling pathway to promote the expression and secretion of VEGF-A, thereby promoting angiogenesis in hepatocellular carcinoma and accelerating its malignant course.

[10] However, there is a lack of consideration of the effect of RBM4 itself on disease through the influence of translation regulation.

[0005] In summary, no researchers have discovered and explored the role of RBM4 in OCCC, a cancer with high incidence and mortality in Asia.

[0006] References:

[0007] [1]Torre LA,Bray F,Siegel RL,Ferlay J,Lortet-Tieulent J,Jemal A.Global cancer statistics,2012.CA Cancer J Clin.2015 Mar;65(2):87-108.

[0008] [2]Mabuchi S,Sugiyama T,Kimura T.Clear cell carcinoma of the ovary:molecular insights and future therapeutic perspectives.J Gynecol Oncol.2016 May;27(3):e31.

[0009] [3]Takano M,Kikuchi Y,Yaegashi N,Kuzuya K,Ueki M,Tsuda H,Suzuki M,Kigawa J,Takeuchi S,Tsuda H,Moriya T,Sugiyama T.Clear cell carcinoma of the ovary:a retrospective multicentre experience of 254 patients with complete surgical staging.Br J Cancer.2006 May 22;94(10):1369-74.

[0010] [4]Oliver KE,Brady WE,Birrer M,Gershenson DM,Fleming G,Copeland LJ,Tewari K,Argenta PA,Mannel RS,Secord AA,Stephan JM,Mutch DG,Stehman FB,MuggiaFM,Rose PG,Armstrong DK,Bookman MA,Burger RA,Farley JH.An evaluation ofprogression free survival and overall survival of ovarian cancer patientswith clear cell carcinoma versus serous carcinoma treated with platinumtherapy:An NRG Oncology / Gynecologic Oncology Group experience.GynecolOncol.2017Nov;147(2):243-249.

[0011] [5]Pectasides D,Fountzilas G,Aravantinos G,Kalofonos C,Efstathiou H,Farmakis D,Skarlos D,Pavlidis N,Economopoulos T,Dimopoulos MA.Advanced stageclear-cell epithelial ovarian cancer:the Hellenic Cooperative Oncology Groupexperience.Gynecol Oncol.2006Aug;102(2):285-91.

[0012] [6]Li S,Qi Y,Yu J,Hao Y,He B,Zhang M,Dai Z,Jiang T,Li S,Huang F,ChenN,Wang J,Yang M,Liang D,An F,Zhao J,Fan W,Pan Y,Deng Z,Luo Y,Guo T,Peng F,HouZ,Wang C,Zheng F,Xu L,Xu J,Wen Q,Jin B,Wang Y,Liu Q.Nuclear Aurora Kinase Switches m6A Reader YTHDC1 to Enhance Oncogenic RNA Splicing of Tumor Suppressor RBM4.Signal Transduct Target Ther.2022Apr 17(1):97.

[0013] [7]Zhang Y,Yong H,Fu J,Gao G,Shi H,Zhou X,Fu M.miR-504PromotedGastric Cancer Cell Proliferation and Inhibited Cell Apoptosis by TargetingRBM4.J Immunol Res.2021Jun 4,2021:5555950.

[0014] [8]Lin JC,Lin CY,Tarn WY,Li FY.Elevated SRPK1 induces apoptosis in breast cancer cells through RBM4-regulated splicing events.RNA.2014Oct920(10):1621-31.

[0015] [9]Yong H, Zhu H, Zhang S, Zhao W, Wang W, Chen C, Ding G, Zhu L, Zhu Z, LiuH, Zhang Y, Wen J, Kang X, Zhu J, Feng Z, Liu B. Prognostic value of decreased expression of RBM4 in human gastric cancer.

[0016]

[10] Han H, Lin T, Wang Z, Song J, Fang Z, Zhang J, You Summary of the invention

[0017] The first purpose of the present invention is to explore the effect of changes in RBM4 translation efficiency on the occurrence and development of OCCC, a rare cancer subtype of ovarian cancer. Another purpose of the present invention is to provide a new direction for the exploration of the treatment of OCCC, which is still under conventional treatment and has a high incidence of drug resistance. The specific technical scheme of the present invention is as follows:

[0018] First, investigate the differences in the expression of Ribo-level genes in normal tissues and OCCC tumor tissues

[0019] The distribution of the expression abundance of genes at the translation level (Ribo) in normal tissue samples and OCCC tumor tissue samples was statistically analyzed, and the genes in the two groups were compared at the Ribo level. The results showed that the Ribo level genes were expressed differently in normal tissues and OCCC tumor tissues, preliminarily indicating that translation dysregulation events exist in OCCC.

[0020] Second, analysis of genes with differential translation efficiency between OCCC tissues and normal tissues

[0021] Results: Compared with normal tissues, 226 genes were upregulated and 67 genes were downregulated in TE in OCCC tumor tissues; GO and KEGG pathway analysis of differentially expressed genes showed that cell growth, immune response, intercellular junction and kinase activity, mTOR signaling pathway, MAPK signaling pathway, etc. were upregulated in OCCC tumor tissues, while DNA damage repair and T cell-mediated immunity were downregulated, indicating that translation dysregulation events exist in OCCC and this translation dysregulation may promote the development of OCCC.

[0022] Third, investigate the mRNA and protein expression of RBM4 in OCCC tissues and normal tissues

[0023] qPCR detection was used to investigate the expression level of RBM4 mRNA in normal tissues and OCCC tumor tissues. The results showed that compared with normal tissues, the mRNA expression of RBM4 in OCCC tumor tissues was significantly decreased;

[0024] Western Blot was used to examine the expression of RBM4 protein in normal tissues and OCCC tumor tissues. The results showed that the protein expression level of RBM4 in OCCC tumor tissues was significantly increased compared with that in normal tissues.

[0025] Fourthly, investigate the effect of RBM4 on the clone formation ability of OCCC cells

[0026] Results: Compared with ES2 cells without RBM4 knockdown, the cell clone-forming ability of ES2 cells with RBM4 knockdown was significantly reduced; compared with TOV21G cells without RBM4 knockdown, the cell clone-forming ability of TOV21G cells with RBM4 knockdown was significantly reduced, indicating that knocking down RBM4 can inhibit the proliferation and survival of OCCC cells.

[0027] Fifth aspect: investigate the effect of RBM4 on the proliferation ability of OCCC cells

[0028] Results: Compared with ES2 cells without RBM4 knockdown, the cell proliferation ability of ES2 cells with RBM4 knockdown was significantly decreased after 96 hours of culture, P<0.0001; compared with TOV21G cells without RBM4 knockdown, the cell proliferation ability of TOV21G cells with RBM4 knockdown was significantly decreased after 72 hours and 96 hours of culture, P<0.0001, indicating that RBM4 knockdown can inhibit the proliferation ability of OCCC cells.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. Translation disorders exist in OCCC and may promote the development of OCCC;

[0031] 2. Compared with normal tissues, the mRNA expression of RBM4 in OCCC tumor tissues was significantly decreased; compared with normal tissues, the protein expression level of RBM4 in OCCC tumor tissues was significantly increased;

[0032] 3. Knockdown of RBM4 can inhibit the clonal formation ability of OCCC cells;

[0033] 4. Knockdown of RBM4 can inhibit the proliferation ability of OCCC cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 , Heat map display of Ribo-level differentially expressed genes in normal (N) and tumor (T) in OCCC;

[0035] Figure 2 , volcano plot display of TE differentially expressed genes in normal tissues and OCCC tumor tissues;

[0036] Figure 3 GO pathway enrichment analysis of TE differentially expressed genes in normal tissues and OCCC tumor tissues;

[0037] Figure 4 KEGG pathway enrichment analysis of TE differentially expressed genes in normal tissues and OCCC tumor tissues, among which,

[0038] A is the upstream pathway KEGG enrichment analysis,

[0039] B is the KEGG enrichment analysis of downstream pathways;

[0040] Figure 5 , the relative expression comparison statistical diagram of RBM4 mRNA in normal tissues and OCCC tumor tissues, where N is normal tissue and T is OCCC tumor tissue;

[0041] Figure 6 , the expression of RBM4 protein in normal tissues and OCCC tumor tissues, among which,

[0042] The left side shows the Western Blot test results.

[0043] The right side shows the statistical graph of Western Blot test results, N is normal tissue, and T is OCCC tumor tissue;

[0044] Figure 7 , Statistical graph comparing the relative expression of RBM4 mRNA between the control group and the ES2 cell group with RBM4 knockdown (left), Statistical graph comparing the relative expression of RBM4 mRNA between the control group and the TOV21G cell group with RBM4 knockdown (right);

[0045] Figure 8 , Comparison of Western Blot results of RBM4 protein expression in ES2 cell group with control and RBM4 knockdown (left), Comparison of Western Blot results of RBM4 protein expression in TOV21G cell group with control and RBM4 knockdown (right);

[0046] Fig. 9 , Comparison of cell clone formation ability between the control group and the ES2 cell group with RBM4 knockdown (top), Comparison of cell clone formation ability between the control group and the TOV21G cell group with RBM4 knockdown (bottom);

[0047] Fig.10 , Comparison of the absorbance at 450nm between the control group and the ES2 cell group with RBM4 knockdown at 0h, 24h, 48h, 72h, and 96h of culture (left), and comparison of the absorbance at 450nm between the control group and the TOV21G cell group with RBM4 knockdown (right).

[0048] in Figure 1-Figure 10 middle,

[0049] *, **, *** and **** represent P<0.05, P<0.01, P<0.001 and P<0.0001, respectively, indicating that the difference between the two groups was statistically significant. DETAILED DESCRIPTION

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0053] Reagents and Materials

[0054]

[0055]

[0056]

[0057] instrument

[0058]

[0059] Buffer and culture medium components

[0060] 1. Lysis buffer: 5mM Tris-HCl pH7.4, 1.5mM KCl, 5mM MgCl2, 0.5% Triton X-100, 0.5% Sodium deoxycholate, 1mM DTT, 100μg / mL Cycloheximide, 10U / mL Turbo DNase, 100μM RNaseOUT, 1× cOmplete protease inhibitor mixture;

[0061] 2. 1M sucrose buffer: 40mM Tris-acetate pH 8.0, 100mM KCl, 15mM MgCl2, 10mM 2-mercaptoethanol, 100μg / mL Cycloheximide;

[0062] 3. Precipitation buffer: 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 5 mM EGTA, 100 mM NaCl and 1% SDS;

[0063] 4. RNA extraction buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl and 0.1% SDS;

[0064] 5. Complete culture medium: ES2, McCoy's 5A + 10% fetal bovine serum;

[0065] 6. TOV21G, MCDB 105 medium (SIGMA-117-500): 199 medium mixed in a 1:1 ratio + 10% fetal bovine serum.

[0066] Example 1: Ribo-seq sequencing of OCCC tissue and normal tissue

[0067] 1. Methods

[0068] Ribo-seq sequencing was performed on 14 OCCC tissues and 8 normal tissues using the following method:

[0069] 1. Extraction

[0070] After CHX fixation, tissues were snap-frozen in liquid nitrogen, ground in liquid nitrogen, and extracted by resuspending in 900 μL of lysis buffer and incubating on ice for 15 min;

[0071] The composition of the lysis buffer: 5 mM Tris-HCl pH 7.4, 1.5 mM KCl, 5 mM MgCl2, 0.5% Triton X-100, 0.5% Sodium deoxycholate, 1 mM dithiothreitol DTT, 100 μg / mL Cycloheximide, 10 U / mL Turbo DNase, 100 μM RNaseOUT, 1× cOmplete protease inhibitor cocktail;

[0072] 2. Measure absorbance

[0073] After incubation on ice, the cells were centrifuged at 14,000 g for 10 min at 4°C, and the cell lysate was transferred to a new reaction tube, and the UV absorbance was measured at 260 nm in a spectrophotometer;

[0074] 3. Digestion

[0075] For nuclease digestion, 24 U of RNase I was used per AU260 of lysate and digested at 20°C for 15 min with slow rotation; the reaction was stopped by adding 100 U of SUPERase·In RNase inhibitor;

[0076] 4. Centrifugation to separate RNA

[0077] It was then overlaid on 330 μL of 1 M sucrose buffer and centrifuged at 131,000 g for 1.5 h at 4 °C using a TLA-55 rotor in an ultracentrifuge;

[0078] The composition of 1 M sucrose buffer was 40 mM Tris-acetate pH 8.0, 100 mM KCl, 15 mM MgCl2, 10 mM 2-mercaptoethanol, and 100 μg / mL cycloheximide;

[0079] 5. Extraction of ribosome footprints (RPFs)

[0080] After removing the centrifugation supernatant, the pellet was resuspended in 600 μL of precipitation buffer, and ribosome footprints (RPFs) were extracted from the precipitation buffer using 1 mL of TRIzol;

[0081] The components of the precipitation buffer were: 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 5 mM EGTA, 100 mM NaCl and 1% SDS;

[0082] RNA samples were denatured for 90 s at 80°C. RPFs were separated by 15% (wt / vol) polyacrylamide TBE-urea gel at 200 V for 60 min;

[0083] The RPFs region of 20 to 40 nt was excised;

[0084] RNA was extracted from polyacrylamide gel slices by adding 600 μL of RNA extraction buffer and gently mixing overnight at 4°C, RNA was recovered by phenol / chloroform / isoamyl alcohol (25:24:1), and the 5′ end was phosphorylated using T4 PNK to remove rRNA, following the human-mouse-rat Ribo-Seq riboPOOL (siTOOLs, dp-K012-50);

[0085] RNA extraction buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl and 0.1% SDS;

[0086] 6. Ribo-seq sequencing

[0087] The samples were then subjected to sequencing library preparation using the Mich small RNA-seq kit, briefly, 3′ adapter ligation, 5′ adapter ligation, reverse transcription, and PCR amplification, followed by 150 bp paired-end sequencing on the Illumina platform;

[0088] 7. Quality control of sequencing results

[0089] The sequencing results were quality controlled and it was found that the RPF was distributed between 26-30nt. Due to differences between individual samples, the RPF of some samples may shift by 1-2nt. The analysis of the percentage of three bases of the reading frame matched by the P site in each sample found that it was in line with the three-base movement rule. The above results prove that the sequencing data is reliable and can be used for subsequent analysis.

[0090] 2. Results

[0091] The distribution of the expression abundance of genes at the translation level (Ribo) in normal tissue samples and OCCC tumor tissue samples was statistically analyzed, and the genes in the two groups were compared at the Ribo level, such as Figure 1 As shown, Ribo level genes are expressed differently in normal tissues and OCCC tumor tissues.

[0092] Conclusion

[0093] Preliminary results indicate that translational dysregulation events exist in OCCC.

[0094] Example 2: Analysis of genes with differential translation efficiency and pathway enrichment between OCCC tissues and normal tissues

[0095] 1. Methods

[0096] The changes in gene translation efficiency (ΔTE) in 14 OCCC tissues and 8 normal tissues were calculated, and GO and KEGG pathway analysis was performed on the differentially expressed genes.

[0097] 1. Change in translation efficiency (ΔTE)

[0098] We introduce interaction terms into the statistical model of DESeq2 to detect differentially translated genes (DTEGs). We call this method ΔTE. The fold change of the interaction term is equivalent to the change of TE, which can detect DTEGs more accurately.

[0099] 2. GO and KEGG pathway analysis of differentially expressed genes

[0100] The R package clusterProfiler 4.2.2 was used to perform enrichment analysis based on the gene list. The enrichment threshold pvalue≤0.5&qvalue≤0.5 was used to screen the enriched pathways, and GO and KEGG pathway analysis was performed on the differentially expressed genes.

[0101] 2. Results

[0102] like Figure 2 As shown, compared with normal tissues, 226 genes were upregulated and 67 genes were downregulated in TE in OCCC tumor tissues;

[0103] like Figure 3 and Figure 4 As shown, GO and KEGG pathway analysis of differentially expressed genes showed that cell growth, immune response, intercellular junction and kinase activity, mTOR signaling pathway, MAPK signaling pathway, etc. were upregulated in OCCC tumor tissues, while DNA damage repair and T cell-mediated immunity were downregulated.

[0104] Conclusion

[0105] There are translational dysregulation events in OCCC, and this translational dysregulation may promote the development of OCCC.

[0106] Example 3: Verification of mRNA and protein expression of RBM4 in OCCC

[0107] 1. Methods

[0108] The mRNA and protein expressions of RBM4 were verified using 8 normal tissues and 13 OCCC tissues;

[0109] 1. Expression levels of RBM4 mRNA in normal tissues and OCCC tumor tissues

[0110] qPCR detection was performed as follows:

[0111] After RNA extraction from tissue, prepare the following reaction system for every 2-3 μg RNA:

[0112] Element volume Oligo(dT) Primer 1μL dNTP(10mM) 1μL RNase free water Make up to 14 μL

[0113] After preparation, mix well with RNA, let stand at 65°C for 5 min, and then immediately place on ice for 2 min.

[0114] The above completed reaction system is continued to be added according to the following system components:

[0115] Element volume 5×First-Strand buffer 4μL DTT(0.1M) 2μL RNase Inhibitor 1μL M-MLV 1μL

[0116] React at 37℃ for 1h, then at 70℃ for 15min, then take out and store at -20℃ for future use.

[0117] GAPDH was used as the internal reference, and 5 replicates were set for each reaction. The reaction system was as follows:

[0118]

[0119]

[0120] Set the temperature and number of cycles for denaturation, annealing, extension, etc. on the qPCR instrument, run the program, and analyze the data.

[0121] 2. Expression of RBM4 protein in normal tissue and OCCC tumor tissue

[0122] Western Blot detection was performed as follows:

[0123] (1) Equipment preparation: The glass plate and hole-making comb should be cleaned in advance and dried in an oven. Note that the residual glue on the glass plate should be cleaned and checked for impurities before use. The glue-making clips and racks should be cleaned with water and checked for impurities before use.

[0124] (2) Assemble the plastic plate: After aligning the glass plate, put it into the plastic clamp and check whether the bottom is aligned with the finger. The clamp is clamped on the rack. A 1mL pipette can be used to strengthen the fixation at the white clamp to prevent the plastic plate from loosening and leaking.

[0125] (3) Prepare the separation gel. Use 2 mL of isopropanol to flatten the separation gel mixture to make the liquid surface flat. After about half an hour, the washing boundary line can be seen. At this time, the separation gel is solidified. The isopropanol can be poured out at an angle. Keep the tilted state and use filter paper to absorb the residual liquid. Prepare the compressed gel. After adding the compressed gel mixture, immediately insert the hole-making comb and add 1 mL of the compressed gel mixture to prevent the introduction of bubbles when inserting the hole-making comb. After about 30 minutes, the separation gel can also be solidified.

[0126] (4) Check for bubbles and impurities. If necessary, re-prepare the glue.

[0127] (5) Remove the gel from the rack and clamps and store it in a 4°C refrigerator (put it in a bag and add water to 1 / 3 of the plate height). Prepare and check the electrophoresis samples (stored at -20°C), take out the samples in advance and place them at room temperature, shake them evenly after they are thawed, and centrifuge them for immediate use.

[0128] (6) Clean the electrophoresis tank and check whether the electrodes, circuits, etc. are usable. Take out the gel and put it into the electrophoresis tank. Push it upward with both hands and fasten the clamps on both sides. Pour some electrophoresis liquid and check whether there is any leakage.

[0129] (7) When the inner tank is full of electrophoresis solution, pull out the well comb with both hands to ensure that the sample wells are complete and vertical.

[0130] (8) Aspirate residual glue: Use a 10 μL pipette to aspirate the residual glue in the well until the square at the bottom of the sample well is aspirated.

[0131] (9) Sample loading: Be accurate and consistent. Do not generate bubbles when adding samples to prevent bubbles from rising in the electrophoresis solution and causing sample overflow. If the outer wall of the gun tip is contaminated with sample after aspiration, it must be cleaned to prevent inaccurate sample loading and excessive target protein.

[0132] (10) Perform electrophoresis at 80 V until the sample reaches the lower edge of the concentrate. After it is flattened to the upper edge of the separation gel, increase the voltage to 120 V and continue electrophoresis.

[0133] (11) It is not recommended to run the electrophoresis for too long (total duration < 1 h). Stop the electrophoresis when the target protein is clearly separated according to the protein marker.

[0134] (12) Prepare electroporation buffer and store in a 4°C refrigerator until the temperature drops, then pour into the electroporation tank.

[0135] (13) Clean the sponge, transfer tray and other transfer equipment, and prepare one PVDF membrane, six filter papers and tweezers.

[0136] (14) First place the electrophoresis tank in a basin and add ice around it (this will prevent subsequent movement from affecting the transfer).

[0137] (15) Pour an appropriate amount of methanol into a clean dish, place the PVDF membrane in it, activate it for >1 min, and place it in the transfer buffer for later use.

[0138] (16) Pour the electroporation solution used last time (recovered in a 4 °C refrigerator) into a tray and soak the filter paper. Use tweezers to transfer the PVDF membrane to the tray and gently shake it in the liquid to remove the methanol on the surface.

[0139] (17) Remove the gel sheet, cut off the upper concentrated gel and the surrounding part of the separation gel, and mark the left band at the upper left corner.

[0140] (18) According to the principle of "black glue and white film", place a white plywood on the palm of your left hand, spread a sponge and 3 layers of filter paper on it, use tweezers to spread the PVDF membrane, spread the glue block, spread 3 layers of filter paper and sponge, and cover with a black plywood. Note: There should be no bubbles throughout the process, especially between the glue block and the PVDF membrane. You can use a glass tube to pour water on it and check for bubbles under the light. The filter paper can be spread by hand, and the PVDF membrane must be spread with tweezers. When closing the black and white transfer plates, they must be aligned and pinched with both hands to prevent displacement and sliding.

[0141] (19) Install the slot: Hold the "sandwich" tightly with both hands and slowly push it into the slot. Pour the electro-transfer solution into the slot until it covers the PVDF membrane. Put a small ice pack in the slot. The electrodes should be assembled according to the principle of "black to black" and "red to red".

[0142] (20) Perform electrophoresis at 250 mA. Remove the ice at one corner of the electrophoresis tank and check for bubbles (start electrophoresis if bubbles are present). The electrophoresis time is determined by the size of the protein. Generally, 1 h is required for proteins with a size of 45 kDa and 2 h is required for proteins with a size of 72 kDa or more.

[0143] (21) Prepare a blocking solution of 5% skim milk. For example, to prepare 50 mL: weigh 2.5 g of milk powder, add it to 50 mL of 1× TBST, mix well, and place on a shaker at room temperature. Thaw the required primary antibody and place it at 4°C.

[0144] (22) Ice should be added to maintain a low temperature environment during the entire electrophoresis process.

[0145] (23) After the electrotransfer is completed, place the white plate in the palm of your left hand and remove the gel. Leave the gel, membrane, and one or two layers of filter paper under the membrane. First, use tweezers to remove a corner of the gel and observe whether the protein marker on the gel has been completely transferred to the PVDF membrane. Then pinch the "gel, membrane, and filter paper" (you can gently pinch the gel block with your hand, and there is no sample on the gel at this time), and use scissors to cut it into the required size, paying attention to the upper left corner mark.

[0146] (24) Discard the gel block and place the membrane in 1× TBST using tweezers. Gently shake to clean the electroporation solution.

[0147] (25) Transfer the membrane to the blocking solution and shake gently on a shaker for 1 h. At the same time, recover the electroporation solution and place it in a 4°C refrigerator until next use.

[0148] (26) Pour off the blocking solution and wash the membrane with 1× TBST until there are no white milky filaments. Otherwise, residual milk will deteriorate after the primary antibody is recovered.

[0149] (27) Add the primary antibody solution to the primary antibody incubation box first so that the membrane can be transferred to the primary antibody solution immediately after cutting. Cut the PE gloves, use tweezers to take out the membrane from the box and place it in the inner layer of the gloves. Use an art knife and ruler to cut out the required strips. Remember to mark the left corner. Place the strips in the corresponding primary antibody and check whether the strips are completely immersed in the solution.

[0150] (28) Incubate on a shaker at 4°C overnight.

[0151] (29) Recover the primary antibody: Use a pipette to suck the primary antibody back to the original storage tube (generally 4 to 6 times. If the result band becomes lighter, add new antibody in time). Wash 3 times with 1×TBST, shaking for 5 minutes each time, with a slightly faster shaking speed to achieve the purpose of washing. When adding new 1×TBST after each wash, be careful not to pour it directly on the membrane.

[0152] (30) When cleaning the primary antibody, prepare the secondary antibody solution: add 5% skim milk (10 mL in large compartment and 5 mL in small compartment) to 1× secondary antigen solution (stock solution 10000×). For example, to prepare 10 mL: add 0.5 g milk powder and 1 μL secondary antibody to 10 mL of 1× TBST. Note that rabbit / mouse antibodies should be used according to the resistance of the primary antibody. Shake gently on a shaker for 1 h at room temperature.

[0153] (31) Wash three times with 1× TBST, shaking for 5 min each time, with a slightly faster shaking speed to achieve the purpose of washing. When adding new 1× TBST after each wash, be careful not to pour it directly on the membrane. After washing, add new 1× TBST to the small box, place the membrane in it, and prepare for development. Prepare the developer and pre-cool the instrument during this period.

[0154] (32) Instrument precooling: Run the software, the machine will automatically initialize and cool to -20℃.

[0155] (33) Prepare the developer. According to experience, about 500 to 700 μL (depending on the size of the strip) is needed to cover one membrane strip. The developer ratio is 1:1. Invert and mix thoroughly, then keep away from light and prepare for development.

[0156] (34) Clean the surface of the developer. Grasp the edge of the strip with tweezers, take it out, and gently place the edge and back of the strip on toilet paper to absorb the TBST. Lay it flat on the developer surface.

[0157] (35) Check the strip positioning and whether it is skewed under white light. Gently add developer in mid-air without impacting the membrane surface. After development, take a photo and save the image.

[0158] (36) Grasp the edge of the strip with tweezers, take it out, and gently place the edge and back of the strip on toilet paper to absorb the developer to prevent the developer from burning the membrane, and then put it back into the TBST box.

[0159] (37) The membrane can be stored in 1× TBST at 4 °C and the elution process can be continued.

[0160] Data processing and analysis.

[0161] 2. Results

[0162] like Figure 5 As shown, the relative expression of RBM4 mRNA in OCCC tumor tissues was significantly decreased compared with normal tissues, P < 0.0001;

[0163] like Figure 6 As shown in the figure, compared with normal tissues, the protein expression level of RBM4 in OCCC tumor tissues was significantly increased, P < 0.05.

[0164] Conclusion

[0165] Compared with normal tissues, the mRNA expression of RBM4 in OCCC tissues was significantly decreased, while its protein expression level was significantly increased.

[0166] Example 4: Gene Editing of OCCC Cell Lines

[0167] 1. Methods

[0168] 1. Reduce the expression of RBM4 in ES2 cells and TOV21G cells

[0169] ES2 and TOV21G cells were infected by RBM4 lentivirus infection method to obtain RBM4 knockdown ES2 cells and TOV21G cells, thereby reducing the expression of RBM4 in ES2 cells and TOV21G cells.

[0170] 2. The expression of RBM4 mRNA in ES2 cells and TOV21G cells with RBM4 knockdown was detected by qPCR, using the same method as Example 3.

[0171] 3. The expression of RBM4 protein in ES2 cells and TOV21G cells with RBM4 knockdown was detected by Western Blot, using the same method as in Example 3.

[0172] 2. Results

[0173] like Figure 7 As shown, compared with the control group, the expression of RBM4 mRNA in the ES2 cell group with RBM4 knockdown and the TOV21G cell group with RBM4 knockdown was significantly decreased;

[0174] like Figure 8 As shown, compared with the control group, the expression of RBM4 protein in the ES2 cell group with RBM4 knockdown and the TOV21G cell group with RBM4 knockdown was significantly decreased.

[0175] Conclusion

[0176] In ES2 cells and TOV21G cells with RBM4 knockdown, the mRNA and protein expressions of RBM4 were significantly decreased compared with the control group.

[0177] Example 5: Effect of RBM4 on OCCC cell clone formation ability

[0178] 1. Methods

[0179] 1. Cell Preparation

[0180] Starting from ES2 cells and TOV21G cells with RBM4 knockdown and without knockdown in the logarithmic growth phase, the cells were trypsinized, completely suspended in complete medium, and counted;

[0181] The control group consisted of ES2 cells and TOV21G cells without RBM4 knockdown, and the sample group consisted of ES2 cells and TOV21G cells with RBM4 knockdown;

[0182] 2. Vaccination

[0183] In a 6-well plate, control the cell number at 1,000 cells / well and seed the cells;

[0184] 3. Cultivation

[0185] Continue culturing for 7 days, replace the culture medium every 3 days, and observe the cell status;

[0186] 4. Fixation and staining

[0187] Use 1 mL of 4% paraformaldehyde to fix the cells for 15 minutes, then wash with PBS. Then add 1 mL of crystal violet stain to each well and control the staining time to 20 minutes;

[0188] 5. Take photos and count

[0189] The cells were photographed and recorded.

[0190] 2. Results

[0191] like Fig. 9 As shown,

[0192] Compared with the control group, the cell clone formation ability of the ES2 cell group with RBM4 knockdown was significantly reduced;

[0193] Compared with the control group, the cell clone formation ability of TOV21G cell group with RBM4 knockdown was significantly decreased.

[0194] Conclusion

[0195] The cell clone-forming ability of ES2 cells and TOV21G cells with RBM4 knockdown was reduced, indicating that knocking down RBM4 can inhibit the proliferation and survival ability of OCCC cells.

[0196] Example 6: Effect of RBM4 on the proliferation ability of OCCC cells

[0197] 1. Methods

[0198] 1. Cell seeding

[0199] ES2 cells and TOV21G cells with or without RBM4 knockdown were inoculated in 96-well culture plates, respectively, with 100 μL per well, and the cell density was controlled at 1000 cells / well, and cultured at 37°C and 5% CO2 for 24 hours;

[0200] 2. Observe cell growth

[0201] Observe the cell growth status under a microscope and select well-grown and evenly distributed wells for subsequent experiments;

[0202] 3. Prepare sample solution

[0203] Three replicate wells were set up for each of the four cell types and culture continued;

[0204] Control group: ES2 cells and TOV21G cells without knockdown of RBM4;

[0205] Sample group: ES2 cells and TOV21G cells with RBM4 knockdown

[0206] 4. CCK8 test to detect the proliferation ability of OCCC cells

[0207] Perform CCK8 test according to the instructions of CCK8 kit;

[0208] (1) Use of CCK8 reagent

[0209] Add 10 μL of CCK-8 reagent to each well and incubate at 37°C and 5% CO2 for 2 hours;

[0210] (2) Absorbance measurement

[0211] The absorbance of the sample solution was measured using an enzyme-labeled instrument at a wavelength of 450 nm.

[0212] 2. Results

[0213] like Fig.10 As shown,

[0214] Compared with the control group, the absorbance at 450nm of ES2 cells with RBM4 knockdown was significantly reduced at 96h of culture, P < 0.0001, indicating that its cell proliferation ability was significantly reduced;

[0215] Compared with the control group, the absorbance at 450nm of TOV21G cells with RBM4 knockdown was significantly reduced at 72h and 96h of culture, P < 0.0001, indicating that its cell proliferation ability was significantly reduced.

[0216] Conclusion

[0217] RBM4 knockdown inhibited the proliferation ability of OCCC.

[0218] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of an agent for reducing RBM4 expression in the preparation of a drug for treating ovarian clear cell carcinoma, characterized in that: The reagent uses the RBM4 lentivirus infection method to reduce the expression of RBM4 in ovarian clear cell carcinoma cancer cells.

2. The use according to claim 1, characterized in that The reagent reduces the clone-forming ability of ovarian clear cell carcinoma cells, thereby inhibiting the proliferation and survival ability of ovarian clear cell carcinoma cells.

3. The use according to claim 1, characterized in that The agent reduces the ability of ovarian clear cell carcinoma cancer cells to proliferate.

4. The use according to claim 3, characterized in that The ability of the reagent to reduce the proliferation of ovarian clear cell carcinoma cells is detected by a CCK8 kit.

5. The use according to claim 1, characterized in that In the ovarian clear cell carcinoma cancer cells in which the RBM4 expression was reduced, the mRNA and protein expressions of RBM4 were significantly decreased compared to the control group.

6. The use according to claim 1, characterized in that Compared with normal tissues, the relative expression of RBM4 mRNA was significantly decreased in ovarian clear cell carcinoma tumor tissues.

7. The use according to claim 6, characterized in that The relative expression of RBM4 mRNA was detected by qPCR.

8. The use according to claim 1, characterized in that Compared with normal tissues, the protein expression of RBM4 was significantly increased in ovarian clear cell carcinoma tumor tissues.

9. The use according to claim 8, characterized in that The protein expression of RBM4 was detected by Western Blot.

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