CN113101369B - Application of circular RNA circ-RNF13 as target site inhibitor - Google Patents

Application of circular RNA circ-RNF13 as target site inhibitor Download PDF

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CN113101369B
CN113101369B CN202110391054.5A CN202110391054A CN113101369B CN 113101369 B CN113101369 B CN 113101369B CN 202110391054 A CN202110391054 A CN 202110391054A CN 113101369 B CN113101369 B CN 113101369B
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rnf13
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nasopharyngeal carcinoma
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CN113101369A (en
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曹科
程雅新
何东
陈兴宇
王占旺
张瑶
陈浩田
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Third Xiangya Hospital of Central South University
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Abstract

The invention discloses application of a circular RNA circ-RNF13 as an inhibitor of a target site in preparation of a paclitaxel sensitizing drug for treating nasopharyngeal carcinoma. The research of the invention shows that the cyclic RNA circ-RNF13 in the nasopharyngeal darcinoma taxol-resistant cell strain is highly expressed, and the circ-RNF13 is knocked out to increase the taxol medicine sensitivity of nasopharyngeal darcinoma cells. Therefore, the circ-RNF13 can be used as a target site in the preparation of a nasopharyngeal carcinoma paclitaxel sensitization medicine and is also a molecular marker for clinically predicting the paclitaxel drug sensitivity of a nasopharyngeal carcinoma patient. Mechanism research shows that the circ-RNF13 with high expression can promote ubiquitination degradation of p53 protein by ubiquitination ligase TRIM41 protein to cause the drug resistance of nasopharyngeal carcinoma cells to paclitaxel.

Description

Application of circular RNA circ-RNF13 as target site inhibitor
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of an inhibitor taking circular RNA circ-RNF13 as a target site in preparation of a nasopharyngeal carcinoma paclitaxel sensitizing medicine.
Background
Nasopharyngeal carcinoma is a malignant tumor originating from the epithelial tissue of the nasopharynx, and its morbidity and mortality are among the head and neck tumors. At present, radiotherapy is mainly used for treating nasopharyngeal carcinoma; for patients staged above T1N0M0, NCCN guidelines recommend chemotherapy combination therapy. Paclitaxel, one of the most widely used chemotherapy drugs in clinical practice, is also used in the treatment of patients with nasopharyngeal carcinoma in middle and late stages, however, the anticancer effect of paclitaxel is greatly limited due to the generation of drug resistance during the treatment process. Therefore, the molecular mechanism of the nasopharyngeal carcinoma tumor cells for generating drug resistance to the paclitaxel is further researched, a possible treatment target is searched, and the curative effect of the paclitaxel is very urgent.
Circular RNA (circRNA) is a Non-coding RNA (ncRNA) molecule with a closed circular structure formed by reverse splicing, which is widely present in various eukaryotes. Initially, circRNA was considered an aberrant byproduct of splicing. With the rapid development of RNA sequencing technologies and bioinformatic analysis, thousands of types of circRNAs have been discovered. More than 25,000 circRNAs were detected in fibroblasts using the whole genome RNase R enrichment method as in Jeck et al; memczak et al identified 1950 circRNAs in humans, 1903 circRNAs in mice and 724 circRNAs in caenorhabditis elegans using RNA sequencing in combination with human leukocyte database analysis; circRNAs are also found expressed in fungi, plants, and prokaryotes. In addition to broad expression, circRNA is more stable than linear RNA in vivo due to its covalently closed loop structure and lack of free ends, which stability results in a circRNA that is not affected by exonucleases and is not easily degraded. Furthermore, circRNA also shows high levels of conservation across species, with circRNA sequences of approximately 15,000 humans detectable in the genome of mice or rats. Several circRNA formation models have been proposed, including ALU direct reverse splicing, inverted repeat complementation, exon lasso, and models mediated by RNA binding proteins (ADAR, MBL and QKI, etc.). In addition, circRNA was found to be involved in several biological functions: the sponge can adsorb microRNA and regulate downstream target genes thereof, and can interact with protein to regulate gene expression, gene transcription and protein translation.
As research progresses, more and more circRNAs have been demonstrated to play an indispensable role in various diseases, especially in the development of tumors and chemotherapy resistance. For example, the Cdr1as which is down-regulated in tissues and cell lines of patients with cisplatin resistance can sensitize ovarian cancer cells to cisplatin by regulating miR-1270/SCAI signal channels; circPAN3 can promote AML resistance by modulating autophagy and affecting apoptosis-related protein expression through the AMPK/mTOR signaling pathway; the hsa _ circ _0025202 realizes tumor inhibition and tamoxifen sensitization through miR-182-5p/FOXO3a axis. However, there are currently few studies on the sensitivity of circRNA to paclitaxel in nasopharyngeal carcinoma.
Disclosure of Invention
The invention aims to provide a new target spot for preparing a nasopharyngeal carcinoma paclitaxel sensitizing medicament. In this study, inhibition of circ-RNF13 expression increased the killing of nasopharyngeal carcinoma cells by paclitaxel. In the present study, we found for the first time that circ-RNF13 highly expressed in nasopharyngeal carcinoma cells can induce ubiquitination degradation of p53 protein by binding TRIM41 protein and p53 protein. The circ-RNF13 is proved to be a key participant of drug resistance of the nasopharyngeal carcinoma paclitaxel for the first time, and is a valuable novel biomarker and a treatment target in the field of nasopharyngeal carcinoma treatment.
The invention provides application of a circular RNA circ-RNF13 as an inhibitor of a target site in preparation of a paclitaxel sensitizing drug for treating nasopharyngeal carcinoma.
The invention also provides application of the circular RNA circ-RNF13 as an inhibitor of a target site in preparation of a molecular marker for predicting nasopharyngeal carcinoma paclitaxel sensitivity.
The invention also provides application of the ubiquitin ligase TRIM41 in preparing a medicine for sensitizing paclitaxel for treating nasopharyngeal carcinoma.
The invention carries out the circRNA microarray analysis and screening on two pairs of cell strains CNE-1 parent strains of nasopharyngeal carcinoma and corresponding taxol-resistant cell strains (CNE-1/P and CNE-1/T), HNE-2 parent cell lines and corresponding taxol-resistant cell lines (HNE-2/P and HNE-2/T), and finds that the circ-RNF13 has differential high expression in the two pairs of cell strains. We confirmed the stability of the circ-RNF13 loop structure by RNase R enzyme and actinomycin D; qRT-PCR and RNA fluorescence in situ hybridization confirmed the distribution of circ-RNF13 in both the nucleus and cytoplasm, mainly in the cytoplasm. In vivo and in vitro experiments prove that the circ-RNF13 can promote the drug resistance of nasopharyngeal carcinoma cell paclitaxel.
The invention further researches a specific molecular mechanism of circ-RNF13 for regulating and controlling the drug resistance of nasopharyngeal carcinoma cell paclitaxel, and FISH/IF double-staining fluorescence in situ hybridization, a co-IP experiment, an IP experiment and an ubiquitination experiment show that the circ-RNF13 can mediate ubiquitination degradation of p53 protein. In vitro experiments show that the reduction of p53 expression can promote the drug resistance of nasopharyngeal carcinoma cells to paclitaxel; overexpression of p53 reverses the resistance of nasopharyngeal carcinoma cells to paclitaxel.
According to prediction of cataPID and Ubibroswer websites, RIP experiments, RNA pull down experiments, co-IP experiments, IP experiments and ubiquitination experiments are combined, and the fact that the ubiquitin ligase TRIM41 protein can be successfully identified to be combined with circ-RNF13 and regulate ubiquitination degradation of p53 protein is successfully identified. The over-expression of circ-RNF13 reduces the expression level of p53 protein in a nasopharyngeal carcinoma paclitaxel drug-resistant cell strain, and the expression level of p53 protein is recovered after knocking down TRIM 41. MTT experiment, cell scratch repair experiment, cell invasion experiment and apoptosis experiment further confirm that TRIM41 can mediate the drug resistance of nasopharyngeal carcinoma cell taxol caused by circ-RNF 13.
We found that the RNA and protein levels of TRIM41 were not different between the nasopharyngeal carcinoma parental cell line and the corresponding paclitaxel resistant cell line; although there was no difference in the RNA level of p53, the levels of p53 protein in CNE-1/T and HNE-2/T cells were significantly reduced compared to the parental cell lines. And neither overexpression nor knock-down of circ-RNF13 affected the RNA and protein expression levels of TRIM 41; while the expression level of circ-RNF13 was not related to the mRNA level of p53, but was negatively related to the p53 protein level.
In conclusion, the invention discovers for the first time that in nasopharyngeal carcinoma cells, circ-RNF13 can be combined with TRIM41 protein and p53 protein, and the level of p53 protein is reduced through the ubiquitination degradation of p53 protein mediated by TRIM41 protein, so that the drug resistance of the nasopharyngeal carcinoma cells to paclitaxel is promoted. The Circ-RNF13 can be used as a target site in the preparation of a nasopharyngeal carcinoma paclitaxel sensitizing drug, is also a molecular marker for clinically predicting the sensitivity of the nasopharyngeal carcinoma patient paclitaxel drug, and can be a new strategy for improving the treatment and survival of the nasopharyngeal carcinoma patient. Meanwhile, TRIM41 is also used as a target site to be applied to the preparation of medicaments for treating nasopharyngeal carcinoma.
Drawings
FIG. 1: the circ-RNF13 is highly expressed in the nasopharyngeal darcinoma paclitaxel drug-resistant cell strain. (a-b) there were 42 circRNAs highly expressed and 15 circRNAs low expressed simultaneously in CNE-1 and HNE-2 paclitaxel resistant cell lines compared to the parent strain. The screening threshold is | multiple change | ≧ 1.5 and P < 0.05. (c) Of the 42 circrnas with significantly high expression, 5 circrnas were derived from the same maternal gene, RNF13 (red marker). (d) qRT-PCR detects the expression of hsa _ circ _0006801, hsa _ circ _0067717, hsa _ circ _0006697, hsa _ circ _0067716, hsa _ circ _0001346 and hsa _ circ _0001346 in nasopharyngeal carcinoma cell parent strains and paclitaxel-resistant cell strains. The hsa _ circ _0067717 difference was most significant. (e) The pattern diagram for circ-RNF13 (hsa _ circ _0067717, consisting of exons 3-6 of RNF13, NM _ 007282). (f) And (3) detecting mRNF13 expression in the nasopharyngeal carcinoma cell parent strain and the paclitaxel drug-resistant cell strain by qRT-PCR. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 2: the loop structure and cellular localization of circ-RNF13 were confirmed. (a) And qRT-PCR detects the expression of circ-RNF13 and mRNF13 after RNase R treatment. (b) After treatment with actinomycin D, the relative RNA levels of circ-RNF13 and mRNF13 in CNE-1/T cells and HNE-2/T cells were determined by qRT-PCR. (c) The expression level of circ-RNF13 in cytoplasm and nucleus was examined by qRT-PCR in CNE-1/T cells and HNE-2/T cells. Beta-actin and U1 were used as positive controls in the cytoplasm and nucleus, respectively. (d) The Fluorescent In Situ Hybridization (FISH) of RNA from circ-RNF13 showed a distribution of circ-RNF13 throughout the nucleus pulposus. Nuclei were stained with DAPI. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 3: in vitro experiments prove that the circ-RNF13 can promote the drug resistance of the nasopharyngeal carcinoma to the paclitaxel. (a-d) cell scratch repair experiments, MTT experiments, cell invasion experiments and apoptosis experiments prove that si-circ-RNF13 improves the sensitivity of CNE-1/T cells and HNE-2/T cells to paclitaxel. Taxol: IC25 concentration of paclitaxel in CNE-1/T and HNE-2/T cells. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 4: in vivo experiments prove that the circ-RNF13 is used for promoting the drug resistance of the nasopharyngeal carcinoma paclitaxel. (a) The nude mouse subcutaneous tumor model showed that knocking down circ-RNF13 enhanced the effect of paclitaxel in inhibiting tumor volume growth. (b-c) immunohistochemical staining to detect the expression levels of Caspase-3 and Ki 67. Taxol: IC25 concentration of paclitaxel in CNE-1/T and HNE-2/T cells. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 5: the circ-RNF13 may bind to the p53 protein. (a) The p53 pathway showed significant enrichment when circ-RNF13 was knocked down in CNE-1/T cells. (b) Gene Set Enrichment Analysis (GSEA) of the p53 signaling pathway. (c) RNA FISH and immunofluorescent staining assays showed localization of circ-RNF13 (red fluorescent marker), p53 (green fluorescent marker) and DAPI nuclear staining (blue fluorescent marker) in CNE-1 and HNE-1 parental and paclitaxel-resistant cell lines. (d) RIP experiment detects the combination of circ-RNF13 and p53 in the drug-resistant cell strain of nasopharyngeal carcinoma taxol. IgG served as a negative control. P53 was enriched with more circ-RNF13 in the circ-RNF13 over-expressed group compared to the control group. (e) RNA pull down experiments show that after circ-RNF13 is over-expressed, the biotin-labeled circ-RNF13 probe (bio-circ) is enriched with more p53 protein. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 6: circ-RNF13 may modulate the protein stability of p 53. (a-b) qRT-PCR, western blot results showed that overexpression or knockdown of circ-RNF13 did not affect the expression of p53 RNA in CNE-1/T and HNE-2/T cells, but that circ-RNF13 was negatively associated with p53 protein levels. (c) Under the treatment of intracellular protein synthesis inhibitor CHX, MG132 can stabilize endogenous p53, indicating that p53 protein is affected by ubiquitination proteasome system. (d) Under the treatment of intracellular protein synthesis inhibitor CHX, the degradation rate of p53 protein is obviously slowed down after knocking down circ-RNF13 compared with that of a control group. (e) Exogenous ubiquitination experiments showed that circ-RNF13 enhances ubiquitination levels of p53 protein. CHX: a cycloheximide; MG 132: a proteasome inhibitor; bio-circ: biotin-labeled circ-RNF13 probe. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 7: reduction of p53 expression promotes chemical resistance of nasopharyngeal carcinoma cells to taxol. (a-d) MTT experiment, cell scratch repair experiment, cell invasion experiment and apoptosis experiment show that the sensitivity of nasopharyngeal carcinoma cells to paclitaxel is weakened after p53 is knocked down, and the sensitivity of nasopharyngeal carcinoma cells to paclitaxel is enhanced after p53 is over-expressed. The histogram data for each group is the average of three independent replicate samples. Taxol: IC25 concentration of paclitaxel in CNE-1/T and HNE-2/T cells. Bars denotes the standard deviation; p <0.05, P <0.01, P < 0.001.
FIG. 8: TRIM41 is a key factor of circ-RNF13 in mediating ubiquitination of p53 protein. (a) Combining the prediction results of RNAct website and UbiBrowser website, 6 genes which can be combined with circ-RNF13 and target ubiquitination of p53 protein are found, wherein HECW1, RNF123 and TRIM41 are ubiquitination E3 ligase which is well-identified. (b) Western blot detection of HECW1, RNF123 and TRIM41 protein expression. (c) RNA pull down experiments show that circ-RNF13 is combined with TRIM41 protein more obviously, combined with RNF123 less strongly and combined with HECW1 and beta-actin not obviously. (d) The catapid website predicts the mutual binding region between circ-RNF13 and TRIM41 proteins. (e) Spatial binding scheme of TRIM41 protein and circ-RNF 13. Blue represents TRIM41 protein, and green represents the 101-200 nt region of circ-RNF 13. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 9: expression of TRIM41 and p53 in nasopharyngeal carcinoma parent strains and paclitaxel resistant strains. (a-b) qRT-PCR and western blot showed that overexpression or knock-down of circ-RNF13 did not affect the expression levels of TRIM41 RNA and protein in CNE-1/T and HNE-2/T cells. (c-d) qRT-PCR assay showed no difference in expression of TRIM41 and p53 in nasopharyngeal carcinoma parental strain and paclitaxel resistant cell strain. (e) Western blot detection of the expression levels of TRIM41 protein and p53 protein. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 10: ubiquitination ligase TRIM41 induced p53 ubiquitination. (a) The prediction of the ubibrower website indicates that p53 is the gene most likely to be targeted by TRIM41 for ubiquitination. (b) HDOCK website predictions show a 3D spatial binding map between TRIM41 protein (green) and p53 protein (blue). The protein junction was marked orange. (c) Co-IP experiments prove that the p53 protein can be combined with the TRIM41 protein. Beta-actin served as an internal control. (d) The ubiquitination level of p53 was greater in CNE-1/T and HNE-2/T cells compared to the parental strain. (e) IP experiments showed that knocking down TRIM41 decreased the ubiquitination level of p53 protein. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 11: the circ-RNF13 can influence the binding ability of TRIM41 protein and p53 protein. (a) Co-IP measures p53 protein ubiquitination level and the binding ability of TRIM41 protein to p53 protein after knocking down or over-expressing circ-RNF 13. (b) Co-IP assays showed that p53 protein binds to TRIM41 protein less after knockdown of circ-RNF 13. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 12: circ-RNF13 promoted ubiquitination degradation of p53 protein by TRIM 41. (a) qRT-PCR showed that neither overexpression of circ-RNF13 nor knock-down of TRIM41 affected the expression of p53 RNA in CNE-1/T and HNE-2/T cells. (b) The reversion experiment shows that knocking-down TRIM41 can reverse the reduction of the expression quantity of the p53 protein caused by the overexpression of circ-RNF 13. (c) In the case of CHX (10. mu.g/mL), western blot analysis showed that knock-down of TRIM41 reversed the rapid degradation of p53 protein by circ-RNF13, slowing down the rate of ubiquitination degradation of p53 protein. (d) Co-IP experiments show that highly expressed circ-RNF13 enhances p53 protein ubiquitination levels, while decreasing p53 ubiquitination levels after addition of si-TRIM 41. (e) Exogenous ubiquitination experiments showed that p53 protein had enhanced levels of ubiquitination when circ-RNF13 was overexpressed. The histogram data for each group is the average of three independent replicate samples. CHX: and (3) cycloheximide. Bars denotes the standard deviation; p <0.05, P < 0.01.
FIG. 13: circ-RNF13 is resistant to paclitaxel by TRIM41 village nasopharyngeal carcinoma cells. (a) MTT experiment, (b) cell scratch repair experiment, (c) cell invasion experiment and (d) apoptosis experiment show that the reduction of TRIM41 can reverse taxol chemical resistance caused by circ-RNF13 overexpression. Taxol: IC25 concentration of paclitaxel in CNE-1/T and HNE-2/T cells. Bars denotes the standard deviation; p <0.05, P <0.01, P < 0.001.
Detailed Description
The invention will be further explained and illustrated with reference to the drawings and experimental data
1. Materials and methods
Cell culture and transfection, qRT-PCR analysis, Western blot analysis, immunohistochemical assay, MTT assay, flow cytometry, immunoprecipitation, immunofluorescence, cell scratch repair experiments, all of which are conventional methods and will not be described herein.
And the results obtained
2.1 high expression of Circ-RNF13 in nasopharyngeal carcinoma paclitaxel cell line
To identify circRNA associated with paclitaxel resistance in nasopharyngeal carcinoma, we performed circRNA microarray analysis on two nasopharyngeal carcinoma cell lines: CNE-1 and HNE-2 parental cell lines (CNE-1/P and HNE-2/P) and corresponding paclitaxel resistant cell lines (CNE-1/T and HNE-2/T). We identified 57 differentially expressed circrnas in two pairs of cell lines (| fold change | ≧ 1.5 and P < 0.05), 42 in the drug-resistant cell lines and 15 in the drug-resistant cell lines (fig. 1 a-b). Of the 42 circRNAs with significant upregulation, 5 were transcribed from RNF13, including hsa _ circ _0006801, hsa _ circ _0067717, hsa _ circ _0006697, hsa _ circ _0067716, and hsa _ circ _0001346 (FIG. 1 c). We designed specific primers for these 5 circRNAs and showed by qRT-PCR analysis that hsa _ circ _0067717 showed the highest differential expression between nasopharyngeal carcinoma parental cells and the paclitaxel-resistant cell line (FIG. 1 d). Therefore, we chose hsa _ circ _0067717 for subsequent studies and called circ-RNF 13. It is transcribed from exon 3-6 of the RNF13 gene (FIG. 1 e). However, the expression of linear RNF13 (mRNA) was not different between nasopharyngeal carcinoma parental cells and paclitaxel resistant cell lines (FIG. 1 f).
To confirm the circular form of circ-RNF13, its stability was assessed by treatment with exonuclease (RNase R). The results showed that circ-RNF13 was resistant to RNase R, whereas mRNF13 was digested by RNase R degradation (FIG. 2 a). Furthermore, we used actinomycin D to inhibit gene transcription to measure the half-lives of circ-RNF13 and mRNA in CNE-1/T and HNE-2/T cells, indicating that circ-RNF13 is more stable than mRNF13 (FIG. 2 b). In addition, both qRT-PCR experiments and RNA FISH experiments showed that circ-RNF13 was distributed in both the nucleus and cytoplasm, mostly in the cytoplasm (FIGS. 2 c-d). Taken together, these data indicate that circ-RNF13 is a circular and stable transcript with significantly high expression in nasopharyngeal carcinoma paclitaxel-resistant cells.
In vivo and in vitro experiments prove that circ-RNF13 can promote taxol resistance of nasopharyngeal carcinoma cells
To investigate the role of circ-RNF13 in paclitaxel resistance in nasopharyngeal carcinoma, we designed specific reverse-ligated siRNA oligonucleotides (si-circ-RNF 13) and a circ-RNF13 overexpression vector against circ-RNF13 to further evaluate the role of circ-RNF13 in nasopharyngeal carcinoma. Cell scratch test, MTT test and migration test showed that knocking down circ-RNF13 increased the ability of paclitaxel to inhibit cell proliferation and invasion, thereby reversing the drug resistance of paclitaxel, compared to the control group (fig. 3 a-c). At the same time, highly expressed circ-RNF13 decreased the ability of paclitaxel to mediate apoptosis, making nasopharyngeal carcinoma cells more resistant to paclitaxel (fig. 3 d).
To assess the role of nasopharyngeal carcinoma cells in paclitaxel sensitivity in vivo, we constructed a model of subcutaneous tumor formation in nude mice and treated with paclitaxel at IC25 concentration, and found that tumor volume growth was slower in si-circ-RNF13 group compared to control group (fig. 4 a). In addition, immunohistochemistry results indicated that Ki-67 expression levels were reduced in the si-circ-RNF13 group tumor tissues, but Caspase-3 expression, the most prominent terminal-cutter during apoptosis, was increased (FIGS. 4 b-c). Overall, the above results indicate that highly expressed circ-RNF13 enhances the resistance of nasopharyngeal carcinoma cells to paclitaxel.
Can regulate the stability of p53 protein
To elucidate the specific mechanism by which circ-RNF13 reduces the sensitivity to paclitaxel in nasopharyngeal carcinoma, we found by microarray analysis on mRNA that when circ-RNF13 was knocked down, the downstream genes were likely to participate in the p53 signaling pathway (FIG. 5 a); and GSEA results showed significant expression of the activity of the p53 signaling pathway (fig. 5 b). Subsequently, RNA FISH experiments indicated that p53 may bind to circRNF13, and it can be seen that expression of p53 was lower in the paclitaxel-resistant cell line than in its parental cell line, while expression of circ-RNF13 was more abundant in the resistant cell line than in the parental cell line (fig. 5 c). RIP experiments further confirmed the binding of both, with circ-RNF13 binding more p53 protein in nasopharyngeal carcinoma paclitaxel-resistant cells compared to CNE-1 and HNE-2 cells (FIG. 5 d). RNA pull down experiments also confirmed that the circ-RNF13 over-expressed group was enriched for more p53 protein than the control group (FIG. 5 e).
Surprisingly, however, the results of the qRT-PCR and western blot experiments showed that circ-RNF13 did not affect the RNA level of p53, but only negatively correlated with the p53 protein level (FIGS. 6 a-b). In CNE-1/T and HNE-2/T cells, we found that the degradation rate of p53 protein was slowed when treated with the proteasome inhibitor MG132 (FIG. 6 c), suggesting that p53 protein was affected by UPS system. Subsequently, we found that knocking down circ-RNF13 significantly improved the stability of p53 protein (fig. 6 d), and exogenous ubiquitination experiments showed that circ-RNF13 overexpression promoted ubiquitination of p53 protein (fig. 6 e). In combination with the above experimental results, we believe that circ-RNF13 binds to p53 protein and promotes its protein ubiquitination degradation.
To investigate the role of p53 in paclitaxel resistance in nasopharyngeal carcinoma, we transfected the constructed p53 overexpression plasmid and p53 siRNA (si-p 53) into CNE-1/T and HNE-2/T cells and confirmed the success of transfection by western blot experiments. MTT experiment, cell scratch repair experiment, cell invasion experiment and apoptosis experiment further show that after the p53 is knocked down, the abilities of inhibiting proliferation and invasion and transfer of nasopharyngeal carcinoma cells and promoting apoptosis of the nasopharyngeal carcinoma cells are weakened. In contrast, overexpression of p53 enhanced the sensitivity of nasopharyngeal carcinoma cells to paclitaxel (FIGS. 7 a-d).
Ubiquitination of p53 induced by the enzyme TRIM41, ubiquitination E3
From the above experiments, we found that circ-RNF13 can bind to p53 and affect its stability. To explore the specific mechanism by which circ-RNF13 affects p53 ubiquitin degradation, we used the ubibrower website to predict 141 possible E3 enzymes targeting the p53 protein. At the RNAct prediction site, we downloaded the first 500 proteins (2 without gene name) that could potentially bind to circ-RNF 13. We found by alignment that there were 6 proteins that could target both the p53 protein and the circ-RNF13 binding. These 6 proteins are PCGF6, BAZ1B, GEMIN5, RNF123, HECW1 and TRIM41, respectively. However, only HECW1, RNF123 and TRIM41 are well-characterized E3 ubiquitin protein ligases, as described by the ubibrower website and Genecard database (fig. 8 a). Western blot analysis showed that there was no difference in protein expression between the 3 ubiquitin ligases in nasopharyngeal carcinoma parental cells and the corresponding paclitaxel-resistant cells (FIG. 8 b). The results of RNA pull down experiments showed that circ-RNF13 did not bind to HECW1 protein, weakly bound to RNF123 protein, but strongly bound to TRIM41 protein in paclitaxel-resistant cells (FIG. 8 c). We use cataPID [26] website prediction to show that TRIM41 protein and circ-RNF13 have high binding capacity, and a 3D space binding diagram visually shows the binding condition between the two (FIG. 8D-e). In conclusion, we conclude that TRIM41 may be a key factor in circ-RNF13 mediated ubiquitination degradation of p 53.
We found by qRT-PCR and western blot experiments that neither circ-RNF13 overexpression nor knockdown affected the RNA and protein expression levels of TRIM41 (FIGS. 9 a-b). And the RNA and protein levels of TRIM41 were not different between nasopharyngeal carcinoma parental cells and the corresponding paclitaxel-resistant cells (fig. 9 c); although there was no difference in the RNA level of p53 (FIG. 9 d), the levels of p53 protein were significantly reduced in CNE-1/T and HNE-2/T cells compared to parental cells (FIG. 9 e), which is consistent with the results of previous FISH, IF double-stain fluorescence in situ hybridization (FIG. 5 c) experiments.
The ubibrower website predicts that p53 is the most likely ubiquitination substrate for TRIM41 (fig. 10 a). The HDock website [31] predicted that TRIM41 protein could bind to p53 protein in the protein interaction space model (FIG. 10 b). Co-IP and IP experiments further confirmed that TRIM41 protein was enriched more in p53 and increased ubiquitination of p53 protein in paclitaxel-resistant cell lines (FIG. 10 c) than in parental cell lines (FIG. 10 d). In addition, p53 protein ubiquitination was decreased and expression of p53 protein was increased in cells after knock-down of TRIM41 compared to control group (fig. 10 e). In summary, these experiments showed that ubiquitin ligase TRIM41 binds more p53 protein in paclitaxel-resistant cell lines, promoting its ubiquitination degradation.
Can promote the degradation of p53 protein ubiquitin by TRIM41 and induce the drug resistance of nasopharyngeal carcinoma cells to taxol
To test whether circ-RNF13 is involved in the binding of p53 protein to TRIM41 protein, and thus affects ubiquitination degradation of p53, we found by co-IP experiments that in CNE-1/T and HNE-2/T cells, knocking down circ-RNF13 reduces ubiquitination level of p53 protein and weakens the binding of TRIM41 protein and p53 protein, and over-expressing circ-RNF13 enhances ubiquitination level of p53 protein and strengthens the binding of TRIM41 protein and p53 protein (FIGS. 11 a-b).
We found by qRT-PCR experiments that either overexpression of circ-RNF13 or knock-down of TRIM41 hardly affected p53 RNA levels. However, western blots showed that silencing of TRIM41 reversed the reduction in p53 protein levels caused by overexpression of circ-RNF13 (FIGS. 12 a-b). Under CHX treatment, overexpression of circ-RNF13 reduced the level of p53 protein in nasopharyngeal carcinoma paclitaxel-resistant cells. However, p53 protein levels were recovered after knock-down of TRIM41 (fig. 12 c). This indicates that circ-RNF13 negatively regulates p53 protein levels by TRIM 41. Co-IP experiments and IP experiments show that overexpression of circ-RNF13 enhances p53 ubiquitination and binding ability of p53 to TRIM 41. However, in nasopharyngeal carcinoma paclitaxel-resistant cells, silencing TRIM41 reversed the effect of circ-RNF13 overexpression (FIG. 12 d). Exogenous ubiquitination experiments show that in nasopharyngeal carcinoma paclitaxel-resistant cells, co-transfection of TRIM41 and p53 promotes ubiquitination degradation of p53 protein, and addition of circ-RNF13 makes ubiquitination level of p53 protein stronger (FIG. 12 e). In conclusion, the highly expressed circ-RNF13 enhances the binding of TRIM41 protein to p53 protein and promotes TRIM 41-mediated ubiquitination degradation of p 53.
We found that in nasopharyngeal carcinoma paclitaxel-resistant cells, silencing TRIM41 reversed paclitaxel chemical resistance caused by high expression of circ-RNF13, and results of MTT experiments, cell scratch repair experiments, cell invasion experiments and apoptosis experiments prove that TRIM41 is a key factor of paclitaxel resistance of nasopharyngeal carcinoma cells caused by circ-RNF13 (FIGS. 13 a-d). In combination with the above experimental results, we believe that circ-RNF13 highly expressed in the paclitaxel-resistant cell line can promote p53 ubiquitination degradation mediated by TRIM41 by enhancing the binding between TRIM41 and p53, thereby inducing the chemical resistance of nasopharyngeal carcinoma cells to paclitaxel.

Claims (3)

1. The application of an inhibitor of circular RNA circ-RNF13 in preparing a medicament for treating taxol-resistant nasopharyngeal carcinoma is disclosed, wherein the inhibitor of circular RNA circ-RNF13 is si-circ-RNF 13.
2. The application of the circular RNA circ-RNF13 as a molecular marker in preparing a reagent for predicting the sensitivity of nasopharyngeal carcinoma paclitaxel.
3. The application of an inhibitor of ubiquitin-ligase TRIM41 in preparing a medicament for treating taxol-resistant nasopharyngeal carcinoma is disclosed, wherein the inhibitor of ubiquitin-ligase TRIM41 is si-TRIM 41.
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