阅读说明：本技术 Mafg-as1作为靶位点的检测试剂在制备治疗膀胱癌药物中的应用 (Application of MAFG-AS1 AS target site detection reagent in preparation of medicines for treating bladder cancer ) 是由 曹科 肖梦卿 向亮 何东 朱煜星 曾庆海 于 2019-11-23 设计创作，主要内容包括：本发明研究表明,原发性BUC组织中lncRNA MAFG-AS1显著过表达,上调或下调MAFG-AS1的表达分别增强或抑制体外和体内BUC细胞的增殖转移与侵袭能力。因此,MAFG-AS1可在制备治疗膀胱癌药物中作为靶位点。机制研究表明,MAFG-AS1可以直接结合HuR,并正向调控HuR/PTBP1轴来促进BUC细胞的增殖转移与侵袭能力。因此,MAFG-AS1可以在制备靶向负向调控HuR/PTBP1轴表达制剂中应用。(The research of the invention shows that lncRNA MAFG-AS1 in the primary BUC tissue is obviously over-expressed, and the expression of up-regulating or down-regulating MAFG-AS1 respectively enhances or inhibits the proliferation, metastasis and invasion capacities of BUC cells in vitro and in vivo. Therefore, MAFG-AS1 can be used AS a target site in the preparation of a medicament for treating bladder cancer. Mechanistic studies indicate that MAFG-AS1 can directly bind to HuR and positively regulate the HuR/PTBP1 axis to promote the proliferation, metastasis and invasion capacity of BUC cells. Therefore, MAFG-AS1 can be applied to preparation of a targeted negative regulation HuR/PTBP1 axis expression preparation.)

Use of MAFG-AS1 AS a detection reagent for a target site in the preparation of a medicament for the treatment of bladder cancer.

2. Use of MAFG-AS1 AS a reagent for the detection of a target site for the preparation of a molecular marker for the treatment of bladder cancer according to claim 1.

3. Use of MAFG-AS1 AS a predictor in the preparation of a marker for predicting bladder cancer recurrence according to claim 1.

4. The use of claim 1, wherein the bladder cancer is BUC.

Application of MAFG-AS1 in preparation of a targeted negative regulation HUR/PTBP1 axis expression preparation.

6. The use of claim 5, wherein the bladder cancer is BUC.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a detection reagent with MAFG-AS1 AS a target site in preparation of a medicine for treating bladder cancer.

Background

Bladder Urothelial Cancer (BUC) is the most common malignancy in the urinary system, and both incidence and mortality have increased year by year in recent years, with about 43 million new diagnosed cases worldwide each year, resulting in 165000 patient deaths each year. Bladder cancer is divided into non-muscle invasive bladder cancer (NMIBC) and Muscle Invasive Bladder Cancer (MIBC), where NMIBC accounts for 70% -80% of bladder cancer cases, and is mainly characterized by rapid progression and easy metastasis, with nearly 75% of high-grade NMIBC relapsing, progressing or dying within 5 years after diagnosis; MIBC usually requires radical cystectomy in combination with neoadjuvant chemotherapy or radiation therapy, but even if MIBC is treated with radical cystectomy, the mortality rate increases from 18.6% to 77.6% within 5 years. Despite the significant progress made in recent years in the treatment of bladder cancer, the problem of bladder cancer being susceptible to metastasis and recurrence remains to be further solved. Therefore, further research on the pathogenesis of bladder cancer, especially the distant metastasis mechanism, is of great clinical significance.

Long non-coding RNA (lncRNA) is RNA that contains more than 200 nucleotides and lacks or exhibits limited ability to encode proteins. Research has shown that lncRNA can regulate tumor cell proliferation, invasion and migration and the like through various action modes to influence the generation and development of tumors, for example, the lncRNA can be used as a sponge adsorbent miRNA to regulate the expression of related mRNA, can coordinate transcription factors to activate the expression of related genes, and can also be combined with protein to prevent protein degradation to stabilize the function of the protein. More and more researches in recent years show that the interaction of lncRNA and protein plays an important role in the occurrence and development of malignant tumors, such as BLACAT2 up-regulates VEGF-C expression by directly combining with WDR5 so as to promote the lymphatic metastasis of bladder cancer; wang Z et al demonstrated that lncRNA EPIC1 in breast cancer could directly bind to Myc protein and form a positive feedback loop to promote cancer, and therefore, the intensive study of the specific molecular mechanism between the interaction of dysregulated lncRNAs and proteins in bladder cancer is very important for elucidating the pathogenesis of bladder cancer, especially the distant metastasis mechanism.

Disclosure of Invention

The invention aims to provide a detection reagent for preparing a new target of a medicine for treating bladder cancer. We found that MAFG-AS1 was significantly high expressed in bladder cancer, and its high expression correlated with patient prognosis and TNM staging; further results showed that MAFG-AS1 was able to bind directly to HuR protein and stabilize HuR expression by recruiting the deubiquitinase USP 5; binding of the protein to HuR by MAFG-AS1 up-regulates the translational expression of PTBP1mRNA, thereby promoting tumor cell proliferation, EMT, and metastatic invasion. Our findings are highly likely to provide novel target detection reagents for bladder cancer therapy.

The research of the invention shows that compared with normal tissues, the expression of MAFG-AS1 in the primary BUC tissues is obviously up-regulated, the high expression of MAFG-AS1 is obviously and negatively related to the overall survival time (OS) and the disease-free survival time (DFS) of bladder cancer patients, and the expression level of MAFG-AS1 in the bladder cancer patients with stage III-IV is higher compared with that of the bladder cancer patients with stage II in clinical staging. Up-or down-regulating the expression of MAFG-AS1 enhances or inhibits the proliferative metastasis and invasion capacity of BUC cells in vitro and in vivo, respectively. In addition, MAFG-AS1 promoted Epithelial Mesenchymal Transition (EMT) of BUC cells. Mechanistic studies indicate that MAFG-AS1 can directly bind to the HuR protein and stabilize the expression of HuR, and MAFG-AS1 can positively regulate the HuR/PTBP1 axis to promote BUC proliferation metastasis and invasion. Collectively, our data indicate that high expression of MAFG-AS1 is associated with recurrent metastasis and low survival in BUC patients. Moreover, targeting MAFG-AS1 may be a new strategy to improve treatment and survival in BUC patients.

In order to explore the important and molecular mechanism of lncRNAs in the occurrence and development of bladder cancer, we found that MAFG-AS1 is highly expressed in bladder cancer tissues for the first time, and found that MAFG-AS1 plays a role in promoting the proliferation, invasion and metastasis and EMT of bladder cancer in vitro and in vivo experiments, which suggests that MAFG-AS1 is very likely to play a role in deducing oncogenes in bladder cancer. Further analysis revealed that high expression of MAFG-AS1 was significantly negatively correlated with bladder cancer patient prognosis, and that expression of MAFG-AS1 was closely correlated with TNM staging of bladder cancer patients. Therefore, the research may provide a new molecular target for bladder cancer prognosis.

The HuR is a ubiquitous member of a Hu/ELAV (human/embryonic lethality abnormal vision) RBP family, can regulate the processes of mRNA stability, translation, miRNA generation and the like to play a role, and the activity of the HuR is mainly influenced by nuclear translocation, phosphorylation, ubiquitination and the like. Recently, it has been shown that lncRNA can also be involved in regulation of HuR function, e.g., lincRNA-UFC1 can bind directly to HuR protein and stabilize its expression, while lncRNA OCC-1 can regulate the level of HuR protein by promoting its ubiquitination degradation at the post-translational level, and lncRNA HCG22 can promote HuR protein degradation in bladder cancer. We found that MAFG-AS1 can directly bind to HuR protein by experimental means such AS CHIRP, PRM and RIP. To search for the molecular mechanism between MAFG-AS1 and HuR, we found that the expression level of HuR protein was significantly increased after over-expressing MAFG-AS1, which indicates that the combination of MAFG-AS1 and HuR protein most likely stabilized the expression of HuR protein. The stability of the protein is often related to the ubiquitination level of the protein, and the increase of the ubiquitination level of the protein can cause the ubiquitination degradation of the protein. USP5 is an important member of the deubiquitinase family, and has been reported to function as oncogenes in a variety of cancer species. Next, experiments such AS COIP and the like prove that USP5 can be directly combined with HuR, the ubiquitination level of the HuR protein is reduced after MAFG-AS1 is over-expressed, and the HuR protein is obviously fallen back after USP5 is knocked out while MAFG-AS1 is over-expressed. Thus, the present invention demonstrates that MAFG-AS1 can directly bind to HuR protein in bladder cancer and stabilize HuR expression by recruiting deubiquitinase USP 5.

In cells, HuR can bind to ARE in the 3' untranslated region of mRNA, conferring transcript stability and facilitating translation, and we therefore considered whether HuR can exert its effect by affecting some key mRNA molecule in bladder cancer. The RIP and RNA stability tests show that the HuR can combine and stabilize PTBP1mRNA, so that the translation expression of PTBP1 is promoted, and further in vitro and in vivo function recovery tests show that after the HuR or PTBP1 is knocked out while MAFG-AS1 is overexpressed, the proliferation and transfer capacity of the tumor are reduced, and the EMT process is inhibited. These results suggest that: in bladder cancer, MAFG-AS1 can bind to and stabilize the HuR protein to up-regulate PTBP1 expression, thereby promoting bladder cancer proliferation, invasive metastatic ability and EMT progression. Therefore, our studies indicate that targeting the MAFG-AS1/HuR/PTBP1 pathway may be a new strategy for improving the therapeutic efficacy and prognosis of BUC patients, and that new agents or inhibitors may be developed.

Drawings

FIG. 1 MAFG-AS1 is highly expressed in bladder cancer and negatively correlated with patient prognosis. (A) Screening a workflow diagram of MAFG-AS 1. (B) Expression of MAFG-AS1 in BUC tissue and non-tumor tissue. (C) High expression of MAFG-AS1 correlates with shorter overall and disease-free survival. (D) The relationship between MAFG-AS1 expression and late disease stage (clinical, T, N and M stages) in patients with bladder cancer was further validated. Bars represent standard deviation, p < 0.05, p < 0.01, p < 0.001.

FIG. 2: MAFG-AS1 promotes BUC cell proliferation, metastatic invasion and EMT. (A) qTR-PCR method detected the expression level of MAFG-AS1 in different bladder cancer cell lines (BIU87,5637, T24, EJ and RT 4). (B, C) qTR-PCR method examined the amount of expression of MAFG-AS1 after transfection of T24 cells with different MAFG-AS1 targeting si-RNA (si-1, si-2, si-3) and after transfection of RT4 cells with the MAFG-AS1 overexpression plasmid. (D, E) cell proliferation was measured in T24 and RT4 cells using MTT method and colony formation assay. (F, G) scratch test and Transwell test the metastatic invasion capacity of cells was determined in T24 and RT 4. (H) The WB experiment examined the effect of MAFG-AS1 on EMT progression. (I) The method comprises the following steps Representative tumor images of mice subcutaneous neoplasia (J): immunohistochemistry detected the expression of ki67 in each group. (K) Representative images and number of metastatic nodules in lung metastases in vivo animal models after HE staining in each group. Each set of histogram data is the average of three independent replicates; bars denotes the standard deviation of the signals, ^*p＜0.05， ^**p＜0.01， ^***p＜0.001。

FIG. 3: MAFG-AS1 binds directly to the HuR protein. (A) The FISH assay was used to detect the subcellular localization of MAFG-AS1 in T24 and RT4 cells. (B) Flow chart of CHIRP experiment. (C) CHIRP-MS detects proteins to which MAFG-AS1 might bind. (D) MAFG-AS1 might bind to the HuR protein. (E) PRM experiments confirmed the proteins to which MAFG-AS1 might bind. (F) RIP experiments reverse-validated that the HuR protein could bind to MAFG-AS 1.

FIG. 4: MAFG-AS1 stabilized the expression of HuR protein by recruiting deubiquitinase USP 5. (A) WB experiments examined the expression level of the HuR protein in bladder cancer cell lines (BIU87,5637, T24, EJ and RT 4). (B) WB test shows that 6 pairs of HuR protein expression levels in tumor tissues and tissues beside the tumor tissues of patients with bladder cancer are detected. (C) And 6, immunohistochemical detection is carried out to detect the expression level of the HuR protein in the tumor tissues and the tissues beside the tumor of the bladder cancer patient. (D) WB measures the effect of MAFG-AS1 on HuR protein levels. (E) Effect of MAFG-AS1 on HuR protein stability, expression levels of HuR were measured by WB at 0, 3, 6, 12, 24, 48 hours after transfection of blank plasmid or sh-MAFG-AS1 plasmid in T24/RT4 cells with MG132 (10 μ M). (F) WB detects HuR protein expression after knockdown of deubiquitinase in T24 and RT4 cell lines. (G) WB detected HuR protein levels following simultaneous knockdown of USP5 with over-expression of MAFG-AS1 and over-expression of MAFG-AS 1. (H) The HuR protein may bind to USP 5. (I) After HuR was used as a precipitating antibody, the expression level of USP5 was measured using COIP. (J) Effect of MAFG-AS1 on the degradation of the ubiquitination of HuR proteins, co-IP examined the ubiquitination levels of HuR after overexpression of MAFG-AS1 and overexpression of MAFG-AS1 in T24/RT4 cells while knocking down USP 5. Bars represent standard deviation, p < 0.05, p < 0.01, p < 0.001.

FIG. 5: MAFG-AS1 may facilitate translation of PTBP1 by stabilizing the HuR protein. A WB experiment detects the expression level of the PTBP1 protein in bladder cancer cell lines (BIU87,5637, T24, EJ and RT 4). (B) WB test shows that 6 pairs of HuR protein expression levels in tumor tissues and tissues beside the tumor tissues of patients with bladder cancer are detected. And (C) immunohistochemical detection 6 of the expression level of PTBP1 protein in the tumor tissues and the tissues beside the tumor of the bladder cancer patient. (D) RIP assay detection indicated that the HuR protein could bind directly to PTBP1 mRNA. (E) qTR-PCR method to examine the effect of MAFG-AS1 and HuR on PTBP1mRNA levels. The (F, G) WB experiment examined the effect of MAFG-AS1 and HuR on PTBP1 protein levels, showing that MAFG-AS1 can up-regulate PTBP1 protein levels by stabilizing HuR expression. (H) The effect of MAFG-AS1 on PTBP1mRNA stability was examined by transfecting over-expressing MAFG-AS1 or si-MAFG-AS1 plasmids into T24/RT4 cells, showing that MAFG-AS1 can stabilize PTBP1mRNA levels. Bars represent standard deviation, p < 0.05, p < 0.01, p < 0.001.

FIG. 6: MAFG-AS1/HuR/PTBP1 can promote the proliferative transfer of BUC and EMT at the cellular level. (A, B) MTT method and clone formation experiment to detect the influence of MAFG-AS1/HuR/PTBP1 axis on cell proliferation ability. (C, D) scratch test and Transwell test were carried out to examine the influence of the axis MAFG-AS1/HuR/PTBP1 on the invasion capacity of cell metastasis. (E) The expression of HuR, PTBP1, E-Cadherin and Vimentin was tested by WB experiment. Bars represent standard deviation, p < 0.05, p < 0.01, p < 0.001.

FIG. 7 MAFG-AS1/HuR/PTBP1 promoted the proliferative transfer of BUC and EMT at the animal level. (A) mouse xenograft tumor model and tumor body volume of each group. (B) WB detects the expression level of EMT molecular markers in mouse transplanted tumor tissues. (C) Immunohistochemistry detected the expression of ki67 in each group. Bars represent 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, immunoprecipitation, immunofluorescence, cell scratch repair assay, colony formation assay, all of which are conventional methods and will not be described herein.

Patient tissue specimen

In this study, 30 histopathological diagnoses of 2016-2018 tumor hospitals affiliated to Hunan ya medical college, Hunan ya three Hospital, Zhongnan university and Hunan ya medical college were used as paraffin-embedded specimens of patients with bladder cancer (BUC) concurrent Radical Cystectomy (RC) (Table 1). 6 Radical Cystectomy (RC) BUC tissue specimens and corresponding adjacent non-tumor tissue specimens were immediately stored at-80 ℃ and used for qRT-PCR and/or Western blot.

And the results obtained

3.1 MAFG-AS1 is highly expressed in bladder cancer and negatively correlated with prognosis

We intersected the GEO database (GSE 31189) with the TCGA database and found that MAFG-AS1 was the only long non-coding RNA that was significantly differentially expressed in both databases (fig. 1A). In comparison to normal tissue, MAFG-AS1 was significantly high expressed in cancer tissue (fig. 1B), and high expression of MAFG-AS1 was significantly negatively correlated with OS and DFS in bladder cancer patients (fig. 1C), we further validated the relative expression of MAFG-AS1 in bladder cancer patients at different stages. AS in fig. 1D, MAFG-AS1 expression was significantly increased in bladder cancer tissue obtained from patients in clinical stage (III-IV, N = 292), T stage (T3-4, N = 269), N stage (N1-2, N = 174), M stage (M1-2, N = 219). The above results indicate that MAFG-AS1 is highly expressed in bladder cancer and is associated with prognosis, clinical staging and TNM staging of bladder cancer patients.

Can promote proliferation and metastasis invasion of tumor cells

Since MAFG-AS1 is highly expressed in BUC, the expression of which is closely related to the prognosis of the patient, we further investigated the function of MAFG-AS1 in the bladder. We examined the expression of MAFG-AS1 in 5 BUC cell lines (BIU87,5637, T24, EJ and RT4) by qRT-PCR. The results showed that MAFG-AS1 was expressed relatively high in T24 cells and relatively low in RT4 cells (fig. 2A). We intervened in MAFG-AS1 knockdown and overexpression, respectively, in two T24/RT4 cells (FIG. 2B, C). The proliferation capacity of tumor cells was significantly reduced after MAFG-AS1 knock-down AS shown by clonogenic and MTT experiments, and the metastatic invasion capacity of tumor cells was reduced after MAFG-AS1 knock-down AS shown by scarification and Transwell chamber experiments, in contrast to the overexpression of MAFG-AS1 (FIGS. 2D-G). We further found that knocking out MAFG-AS1 promoted the expression of the epithelial marker E-cadherin, whereas over-expressing MAFG-AS1 promoted the expression of the mesenchymal marker Vimentin, suggesting that MAFG-AS1 promotes the progression of EMT in bladder cancer (FIG. 2H). To validate the function of MAFG-AS1, we further tested the effect of MAFG-AS1 on subcutaneous neoplasia of bladder cancer cells and distant lung metastases in vivo experiments. We found that compared to the control group, the sh-MAFG-AS1 group had significantly reduced tumor volume (fig. 2I), significantly reduced positive rate of Ki67, a tumor proliferation-related index (fig. 2J), and significantly reduced number of lung metastatic nodules, in contrast to the over-expressed group (fig. 2K). The above results demonstrate that MAFG-AS1 increases the proliferative metastasis, invasiveness and EMT progression of bladder cancer both in vivo and in vitro.

Can be directly combined with HuR protein

MAFG-AS1 was found to be expressed in both the nucleus and cytoplasm by FISH (FIG. 3A). Since lncRNA can often act by interacting with proteins, we followed a CHIRP-MS experiment on a high throughput screen of binding proteins for MAFG-AS1 (FIG. 3B), which showed that a total of 26 proteins were targeted by the MAFG-AS 1-specific probe set (FIG. 3C), of which the HuR protein is one. The HuR protein is an RNA binding protein of a member of the ELAVL family, which is involved in a variety of biological processes and is implicated in a number of diseases, including cancer. We found that MAFG-AS1 presents a binding site with HuR (panel D). Further PRM validation of CHIRP-MS results revealed that strong signals for HuR were detected in the protein pulled down by the MAFG-AS 1-specific probe (FIG. 3E). To further confirm this interaction, we validated this interaction between MAFG-AS1 and HuR by RNA immunoprecipitation (RNA-IP). The results show a significant enrichment of binding of MAFG-AS1 to HuR compared to the non-specific IgG control group (FIG. 3F). Taken together, these results suggest that MAFG-AS1 can bind HuR directly, but the specific relationship between the two remains to be elucidated further.

3.4 MAFG-AS1 expression of the HuR protein was stabilized by recruitment of deubiquitinase USP5

To investigate the specific relationship between MAFG-AS1 and HuR protein, we first examined the expression level of HuR protein in bladder cancer cells and tissues, AS shown by the relatively high expression level of HuR in T24 cells and relatively low expression level in RT4 cells (fig. 4A), and the HuR protein level in cancer tissues was significantly higher than in normal tissues (fig. 4B), which we further verified by immunohistochemical methods (fig. 4C). We found by WB experiments that expression levels of HuR protein were significantly down-regulated after knockdown of MAFG-AS1, while levels of HuR protein were significantly elevated after overexpression of MAFG-AS1 (fig. 4D). This suggests that we may influence the stability of the HuR protein by MAFG-AS 1. We knocked down MAFG-AS1 in T24 and RT4 cells, followed by treatment with Cycloheximide (CHX) to inhibit de novo protein synthesis, while adding proteasome inhibitor MG132 to retard protein degradation, the sh-MAFG-AS1 group reduced HuR protein stability in T24 and RT4 cells compared to the control group (fig. 4E). To further investigate the mechanism of MAFG-AS1 to increase HuR stability, we found that HuR has multiple ubiquitination sites through the photosite website, and we have reported that lncRNA can affect ubiquitination degradation of HuR protein, and we guessed whether MAFG-AS1 stabilized its expression by affecting ubiquitination degradation of HuR protein. Protein expression levels of HuR were most significantly reduced following knockdown of deubiquitinase USP5 by WB experiments (fig. 4F), and were down-regulated relative to the levels of HuR in the group overexpressing MAFG-AS1 following simultaneous knockdown of USP5 by overexpressing MAFG-AS1 (fig. 4G). Surprisingly, we found that HuR binds directly to USP5 protein and MAFG-AS1 also has a binding site with USP5 (fig. H), and that HuR was used AS a precipitating antibody and USP5 expression was detected by the COIP method, which showed that HuR binds directly to USP5 protein (fig. I), and that after overexpression of MAFG-AS1, the level of ubiquitination of HuR decreased, while after overexpression of MAFG-AS1 and simultaneous knock-down of deubiquitinase USP5, the level of ubiquitination of HuR protein increased (fig. 4J). These results suggest that MAFG-AS1 may recruit deubiquitinase USP5 to prevent ubiquitination degradation of HuR, thereby stabilizing expression of HuR protein.

MAFG-AS1 can promote translational expression of PTBP1 by stabilizing the HuR protein

We found that PTBP1 was expressed relatively high in T24 cells with high metastatic invasion and relatively low in RT4 cells with low metastatic invasion (fig. 5A) and was expressed relatively high in bladder cancer tissue (fig. 5B, C). we further verified that the HuR protein could bind to PTBP1mRNA by RIP experiments (fig. 5D). to verify whether MAFG-AS1 could promote expression of PTBP1 by stabilizing HuR, we over-expressed MAFG-AS1, PTBP1mRNA expression level was significantly increased, whereas upon simultaneous knock-down of HuR with over-expression of MAFG-AS1 (fig. 5E) pt 1mRNA level had a significant drop-off (fig. 5E). we further followed by walking experiments, after overexpression of MAFG-AS1, PTBP1 protein level expression was increased (fig. 5F), whereas upon over-expression of MAFG-AS1, the protein was also knocked down with USP 7 or after knock-down of MAFG-AS1, the protein level was significantly decreased by comparison with mRNA synthesis of MAFG-ap-AS 1, 1mRNA after stable mRNA inhibition by mRNA expression of MAFG-1, after T-AS 1, after cross-RNA synthesis by mRNA inhibition by mRNA (fig. 1H).

The signal axis can promote the proliferation and metastasis of bladder cancer and EMT

To verify the effect of the MAFG-AS1/HuR/PTBP1 signal axis on the proliferation and metastasis invasion capacity of bladder cancer, cell experiments show that after the MAFG-A1 is over-expressed, the survival rate and the clone formation rate of the cells are obviously improved (FIGS. 6A and B), the invasion capacity of the metastasis is obviously enhanced (FIGS. 6C and D), however, after over-expressing MAFG-AS1 and knocking down the HuR or PTBP1, the survival rate and the clone formation rate of cells are reduced and the invasion and transfer capacity of the cells is also obviously reduced compared with the group over-expressing MAFG-AS1, and further WB finds that the E-Cadherin protein is obviously reduced when over-expressing MAFG-AS1, while the Vimentin protein is obviously increased, after over-expressing MAFG-AS1 and knocking down the HuR or PTBP1, the expression of the E-Cadherin protein is increased, while the expression of Vimentin protein is reduced (FIG. 6E), which suggests that the MAFG-AS1/HuR/PTBP1 signal axis can promote the proliferation and invasion capacity of bladder cancer and the EMT process. We further validated this result in vivo experiments AS shown in fig. 7A, B, that upon knockdown of MAFG-AS1, the tumorigenic capacity of the cells decreased and EMT progression was inhibited, whereas upon overexpression of MAFG-AS1, the tumorigenic capacity of the cells increased and EMT progression was promoted (fig. 7A, B). Furthermore, we knocked down the expression of HuR or PTBP1 while overexpressing MAFG-AS1, and AS a result, found that the tumorigenic capacity of the cells was reduced compared to the group overexpressing MAFG-AS1 and the EMT progression was inhibited (fig. 7A, B). Immunohistochemical detection of the tumor proliferation-related index Ki67 also confirmed this result (fig. 7C). Therefore, combining the above results, the MAFG-AS1/HuR/PTBP1 signal axis can increase the proliferation and metastatic invasion capacity of BUC cells and promote the EMT process of bladder cancer.