CN112089840A

CN112089840A - Application of inhibitor of KTN1-AS1 in preparation of medicines for treating bladder cancer

Info

Publication number: CN112089840A
Application number: CN202011167530.7A
Authority: CN
Inventors: 曹科; 王占旺; 胡雪莹; 何东; 向亮
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2020-12-18
Anticipated expiration: 2040-10-27
Also published as: CN112089840B

Abstract

The invention discloses application of an inhibitor of KTN1-AS1 in preparation of a medicament for treating bladder cancer. The research of the invention shows that KTN1-AS1 is obviously up-regulated in BUC cells and tissues and is negatively related to the prognosis of patients, and the expression of knocking out and up-regulating KTN1-AS1 can respectively enhance or inhibit the proliferation of BUC cells in vitro and in vivo and respectively inhibit or promote the apoptosis of the cells in vitro and in vivo; in addition, we demonstrated that up-regulated KTN1-AS1 recruits the EP300 to KTN1 promoter, activates KTN1 expression, and accelerates the proliferation and development of bladder cancer by activating the KTN1/Rho GTPase pathway. Therefore, the inhibitor of KTN1-AS1 can be applied to preparation of a targeted negative regulation KTN1 expression preparation. Meanwhile, the inhibitor of KTN1 can also be applied to the preparation of medicines for treating bladder cancer.

Description

Application of inhibitor of KTN1-AS1 in preparation of medicines for treating bladder cancer

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of an inhibitor of KTN1-AS1 in preparation of a medicine for treating bladder cancer.

Background

Bladder cancer is the tenth most common cancer worldwide with high morbidity and mortality. According to the 2018 global cancer statistics, about 549,000 new cases of bladder cancer are diagnosed each year, and as many as 200,000 bladder cancer patients die each year. Notably, while 70% to 80% of bladder cancers diagnosed are non-muscle invasive tumors, 20% to 30% are muscle invasive tumors. However, a non-muscle invasive tumor of about 1/3 will eventually develop into a muscle invasive tumor or develop metastases, which will lead to progression of the tumor. Even in patients with bladder cancer who receive optimal treatment (surgery and chemotherapy), the incidence of distant metastasis is high and clinical outcome and prognosis are poor. Therefore, there is an urgent need for a better understanding of the molecular mechanisms that drive the development and progression of bladder cancer.

An increasing number of studies have shown that long non-coding rnas (lncrnas) are involved in a variety of physiological and pathological conditions, such as embryonic development, cardiovascular disease and cancer. LncRNA are specific RNA transcripts greater than 200 nucleotides in length with no (or limited) protein coding potential. Functionally, aberrant expression of lncrnas may lead to a variety of biological effects, such as cell proliferation, differentiation and apoptosis. Mechanistically, lncRNA is a versatile molecule with the potential to interact with DNA, RNA, or proteins, and can be used as a prototype for signaling, decoys, guides, and scaffolds. LncRNA KTN1 antisense RNA 1 (KTN 1-AS 1) is located on chromosome 14q22.3 and maps to the antisense DNA strand in the upstream region of KTN 1. The lncRNA was first reported to be highly expressed in colorectal cancer cells and tissues and was positively correlated with tumorigenesis and poor clinical outcome. However, it is not clear whether KTN1-AS1 is upregulated in bladder cancer and its associated underlying mechanisms. Therefore, the function and action mechanism of KTN1-AS1 in BUC needs to be studied extensively.

Disclosure of Invention

The invention aims to provide a new target point for preparing a medicament for treating bladder cancer.

The invention provides application of an inhibitor of KTN1-AS1 in preparation of a medicament for treating bladder cancer. The bladder cancer is BUC.

The invention provides application of a pre-testing agent KTN1-AS1 in preparation of a marker for predicting recurrence of bladder cancer.

The invention provides an application of a target site detection reagent of KTN1-AS1 in preparation of a molecular marker for treating bladder cancer.

The invention provides application of KTN1-AS1 in preparation of targeted negative regulation KTN1 and Rho GTPase preparations.

The invention provides application of KTN1 as a target site in preparation of a medicament for treating bladder cancer. The bladder cancer is BUC.

The invention provides application of a KTN1-AS1/KTN1/Rho GTPase regulating and controlling preparation in preparation of a medicine for treating bladder cancer. The bladder cancer is BUC.

The research of the invention shows that the expression of KTN1-AS1 is up-regulated in bladder cancer cells and tissues, and the expression of KTN1-AS1 is observed to be higher in bladder cancer patients from stage II to stage IV than in bladder cancer patients from stage I, and the prognosis of the patients with higher expression of KTN1-AS1 is worse. Knock-out or overexpression of KTN1-AS1 enhanced or inhibited proliferation and invasion of BUC cells in vitro and in vivo, respectively. In addition, the up-regulation of KTN1-AS1 promoted the expression level of KTN 1. Mechanistic studies have shown that KTN1-AS1 can activate expression of KTN1 in cis by recruiting EP300 to the KTN1 promoter region. Therefore, the results of the above studies indicate that KTN1-AS1/KTN1 is an important participant in the proliferation and invasion of BUC, and KTN1-AS1/KTN1 can be used AS a novel biomarker and a therapeutic target for BUC patients.

The present invention also reveals for the first time that in bladder cancer, the expression of KTN1 was significantly elevated in cancer tissues compared to normal tissues, and that upregulation of the expression of KTN1 was also observed in both datasets (GSE 3167 and GSE 138118) in the GEO database. The knock-out or over-expression of KTN1-AS1 can affect the expression of mRNA and protein of KTN1, and the suggestion is that KTN1-AS1 plays a role in cis. By analyzing the expression correlation between KTN1-AS1 and KTN1 in different databases, we found that the expression of KTN1-AS1 and KTN1 is in a significant positive correlation. Since subcellular fractionation analysis showed that KTN1-AS1 was expressed in the nucleus above the cytosol, we hypothesized that KTN1-AS1 might interact with nuclear proteins, regulating the expression of KTN1 at the transcriptional level.

The research explores the action mechanism of KTN1-AS1 in bladder cancer, and in order to better understand the relationship between KTN1-AS1 and KTN1, the UCSC online database (http:// genome. UCSC. edu /) analysis is used to find that the KTN1-AS1 and the KTN1 promoter region are highly overlapped, the region is highly enriched with the H3K27Ac peak, and the H3K27Ac is a marker of an activity enhancer. Therefore, we used the predictive RNAct algorithm to look for KTN1-AS1 binding proteins with Histone Acetyltransferase (HAT) activity. We predict that EP300 (HAT) binds KTN1-AS 1. We performed RIP experimental analysis in RT4 and T24 cells (empty vector transfected or overexpressing KTN1-AS 1). The results show that KTN1-AS1 can recruit the modification indirectly responsible for H3K27Ac through EP 300. To demonstrate our hypothesis, we performed ChIP-qPCR analysis. After we knocked out KTN1-AS1 in RT4 and T24 cells, H3K27Ac in the KTN1 promoter region was deleted. In contrast, H3K27Ac was highly enriched in RT4 and T24 cells overexpressing KTN1-AS1, a phenotype that disappeared in the case of EP300 knockdown. The above results indicate that KTN1-AS1 can promote the expression level of KTN1 by recruiting EP300 and enriching H3K27Ac in the KTN1 promoter region.

The invention further discusses whether KTN1-AS1 promotes the progress of bladder cancer in a KTN 1-dependent manner, and a series of function acquisition/loss experiments are carried out. Consistent with the results obtained after the KTN1-AS1 knock-out, bladder cancer cells transfected with sh-KTN1 showed significantly lower levels of proliferation, invasion and migration. Similarly, overexpression of KTN1-AS1 promoted migration and invasion of bladder cancer cells. However, this phenotype was partially lost when KTN1 was simultaneously silenced in cells overexpressing KTN1-AS 1. These results indicate that KTN1 plays an important role in the development of bladder cancer. Bioinformatic analysis of data retrieved from the STRING and inBioMap databases revealed that KTN1 might interact with several proteins (RHOA, RAC1, RHOG, CDC42, KLC1 and EEF 1D), of which four proteins (RAC 1, RHOA, RHHOG and CDC 42) are members of the Rho GTPase family. Previous research reports that Rho GTPases are involved in physiological and pathological activities such as cell migration, cell polarity, cell cycle regulation, cytoskeleton remodeling and the like. Therefore, we explored whether KTN1-AS1 has an effect on the Rho GTPase signaling pathway by modulating KTN1 expression. Western blot results showed that the protein levels of RAC1, RHOA and CDC42 were significantly reduced in cells that silenced KTN 1; in contrast, levels of KTN1, RAC1, RHOA and CDC42 proteins increased with overexpression of KTN1-AS 1. In cells overexpressing KTN1-AS1, the expression of KTN1, RAC1, RHOA and CDC4 was partially reduced after simultaneous knock-out of KTN 1. Thus, the results of the above studies indicate that KTN1-AS1 can promote the progression of bladder cancer by modulating the KTN1/Rho GTPase signaling pathway.

In order to further study whether KTN1-AS1 regulates the growth of bladder cancer in vivo, a subcutaneous xenograft mouse model is established. T24 cells were stably transfected with control vectors KTN1-AS1 or KTN1-AS1 + sh-KTN1 and injected subcutaneously into nude mice. Overexpression of KTN1-AS1 in bladder cancer cells significantly increased the growth rate of bladder cancer (AS measured by tumor volume) compared to controls, while simultaneous knock-out of KTN1 in the case of overexpression of KTN1-AS1 in T24 cells abolished this effect. The proliferation marker Ki-67 is observed by using an IHC (IHC assay) method, so that the tumorigenic potential of KTN1-AS1 is further proved. The results show that the Ki-67 levels in the KTN1-AS1 overexpressing group were significantly increased to a much greater extent than the Ki-67 levels in the KTN1 knockout mice, and that the expression of KTN1 and RAC1 (also assessed by IHC) was consistent with the in vitro results described above. These results indicate that KTN1-AS1 can participate in the occurrence and development of bladder cancer through the KTN1/Rho GTPase signal axis.

In conclusion, the research describes an expression model of KTN1-AS1 up-regulated in BUC in detail, and confirms the role of KTN1-AS1 in the occurrence and development of bladder cancer and the molecular mechanism of the KTN1-AS 1. The high-expression KTN1-AS1 can promote the proliferation and invasion of bladder cancer through cis-activation of KTN1 and the signaling cascade reaction of Rho GTPase mediated by the cis-activation of KTN1, and shows that KTN1-AS1 and KTN1 play important roles in the development of bladder cancer. Therefore, the inhibitor of KTN1-AS1 can be applied AS a target site in the preparation of medicines for treating bladder cancer. Meanwhile, the inhibitor of KTN1 can also be used as a target site to be applied to the preparation of medicines for treating bladder cancer.

Drawings

FIG. 1 KTN1-AS1 was highly expressed in bladder cancer tissue. (A) Expression of KTN1-AS1 in cancer cells. (B, C) differentially expressed KTN1-AS1 was analyzed by the starBase and TANRIC databases. (D) The expression of KTN1-AS1 in different bladder cancer tumor grades was assessed using the TANRIC database. (E) KTN1-AS1 expression was detected in 6 pairs of bladder cancer and adjacent non-tumor tissues using RT-qPCR. (F) cellular localization of KTN1-AS1 was determined in RT4 and T24 cells using RT-qPCR. P <0.05, P <0.01, P <0.001, P < 0.0001.

FIG. 2 knock-out of KTN1-AS1 inhibited proliferation, migration and invasion of bladder cancer cells. KTN1-AS1 was silenced by sh-RNA in RT4 and T24 cells. (A) Silencing efficiency was confirmed using RT-qPCR. (B) MTT assay was performed to assess cell proliferation in RT4 and T24 cells that silenced KTN1-AS 1. (C) after sh-KTN1-AS1 transfection, a clonogenic assay was performed with RT4 and T24 cells. (D) Scratch experiments were performed to assess migration of RT4 and T24 cells after knock-out of KTN1-AS 1. (E) The invasion capacity of RT4 and T24 cells after knocking out KTN1-AS1 was studied by using a Transwell experiment. P <0.05, P <0.01, P < 0.001.

FIG. 3 overexpression of KTN1-AS1 promoted proliferation, migration and invasion of bladder cancer cells. (A) The overexpression efficiency was confirmed by RT-qPCR. (B) The MTT method detects cell proliferation in RT4 and T24 cells overexpressing KTN1-AS 1. (C) After overexpression of KTN1-AS1, colony formation experiments were performed with RT4 and T24 cells. (D) RT4 and T24 cell migration after over-expression of KTN1-AS1 was evaluated using a scratch test. (E) The invasive capacity of RT4 and T24 cells after overexpression of KTN1-AS1 was investigated using a Transwell assay. P <0.05, P <0.01, P < 0.001.

FIG. 4 KTN1 was highly expressed in bladder cancer and positively correlated with KTN1-AS 1. (A, B) mRNA and protein expression levels of KTN1 were measured in 6 pairs of bladder cancer and adjacent non-tumor tissues using RT-qPCR and WB method. (C, D) analyzing the expression of KTN1 in the GEO database. (E-H) mRNA and protein expression of KTN1 in RT4 and T24 cells were determined after downregulation or upregulation of KTN1-AS1 using RT-qPCR and WB method. (I-L) search of GEPIA and GEO databases to analyze the correlation between KTN1-AS1 and KTN1 levels. P <0.05, P <0.01, P < 0.001.

FIG. 5 KTN1-AS1 recruits the EP300 to KTN1 promoter and is positively correlated with KTN1 expression. (a) observing the positional relationship between KTN1-AS1 and KTN1 using UCSC database. (B) The RNAct algorithm was used to predict potential KTN1-AS1 binding proteins, particularly histone acetyltransferases. (C) RIP experiments were performed to identify the interaction profile of KTN1-AS1 with the EP300 protein. (D, E) search GEPIA and GEO databases for correlation between EP300 and KTN 1. (F) After EP300 knock-out, protein expression of EP300 and KTN1 was detected in RT4 and T24 cells. (G) The ChIP-qPCR method detects H3K27Ac, especially in the KTN1 promoter region. P <0.05, P <0.01, P < 0.001.

FIG. 6 KTN1-AS1 promoted the proliferation, migration and invasion of bladder cancer cells in a KTN 1-dependent manner. (A) The down-and/or up-regulation of the target gene (transfection efficiency) was confirmed using RT-qPCR. (B) MTT method was used to evaluate the proliferation of bladder cancer cells transfected with sh-KTN1, KTN1-AS1 and KTN1-AS1 + sh-KTN 1. (C) The detection of clonogenic experiments was performed using RT4 and T24 cells transfected with different constructs. (D) migration of RT4 and T24 cells transfected with different constructs was examined using a scratch assay. (E) The Transwell experiment explored the variation in invasiveness of RT4 and T24 cells transfected with different constructs. P <0.05, P <0.01, P < 0.001.

FIG. 7 KTN1-AS1 regulates the KTN1/Rho GTPase axis. (A-C) predicted potential interaction protein KTN1 using STRING and inBio-Map databases. (D) The expression conditions of KTN1, RAC1, RHOA, CDC42, RHOG and beta-actin in RT4 and T24 cells transfected with sh-KTN1, KTN1-AS1 and KTN1-AS1 + sh-KTN1 are detected by a WB method. P <0.05, P <0.01, P < 0.001.

FIG. 8 KTN1-AS1 promoted the development of bladder cancer in vivo mediated by KTN1 expression. (A) Nude mice were injected subcutaneously with T24 control cells (transfected with empty vector), T24 cells transfected with KTN1-AS1 or T24 cells co-transfected with KTN1-AS1 and sh-KTN 1. (B) Tumor volume was measured every 3 days from day 7 to day 25 after cell injection. (C) Ki-67, KTN1 and RAC1 expression in tumors was detected by immunohistochemistry. P <0.05, P <0.01, P < 0.001.

Fig. 9 KTN1-AS1 promoted tumorigenesis of bladder cancer by cis-activating KTN1 and its mediated signaling cascade of Rho GTPase: schematic representation.

(A-C) differential expression of KTN1-AS1 at different bladder Cancer tumor stages (compared to adjacent normal tissues) was analyzed based on the Cancer RNA-Seq Nexus database.

FIG. 11 knock-down efficiency of KTN1-AS1 was determined in RT4 cells.

(A, B) correlation cases between KTN1-AS1 and KTN1 levels in datasets retrieved from GEO databases.

Fig. 13 KTN1 is positively correlated with EP 300. (A) The catapid database was used to predict the interaction between KTN1-AS1 and EP 300. Correlation between KTN1 and EP300 in different data sets retrieved by the (B-O) GEO database.

Fig. 14 (a, B) interference efficiency of KTN1 and KTN1-AS1 in RT4 and T24 cells.

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, immunohistochemistry assay, MTT assay, scratch assay, Transwell assay, RNA pulldown, RIP, nuclear matrix separation assay, chromatin co-immunoprecipitation, dual luciferase assay, all of which are existing methods and will not be described herein.

2. Results

2.1 upregulation of LncRNA KTN1-AS1 in bladder cancer

To reveal whether KTN1-AS1 is upregulated in bladder cancer, we first retrieved and analyzed the expression data of KTN1-AS1 from different databases including the TCGA database. We found that KTN1-AS1 was significantly elevated not only in bladder cancer, but also in several other cancers (FIGS. 1A-C). Similar results were also found in stage II to IV bladder cancer (FIG. 1D and FIGS. 10A-C, respectively). In addition, bladder cancer tissues analyzed in this study also showed a significant increase in KTN1-AS1 compared to adjacent non-tumorous bladder tissues (FIG. 1E). In addition, we found that KTN1-AS1 is localized mainly in the nucleus (fig. 1F), indicating that KTN1-AS1 function is associated with transcriptional regulation. Taken together, these results indicate that KTN1-AS1 may exert carcinogenic effects.

2.2 knock-out KTN1-AS1 inhibited proliferation, migration and invasion of bladder cancer cells

To assess the potential role of KTN1-AS1 in bladder cancer, we performed a series of in vitro silencing experiments using two different bladder cancer cell lines. Notably, KTN1-AS1 silencing was demonstrated in both RT4 and T24 cells (fig. 2A and fig. 11, respectively). First, we evaluated the effect of knockdown KTN1-AS1 on bladder cancer cell growth. MTT experiments showed that silencing KTN1-AS1 significantly reduced the proliferation of bladder cancer cells (FIG. 2B). Also, clonogenic experiments showed that knock-out KTN1-AS1 significantly inhibited the clonogenic capacity of bladder cancer cells (FIG. 2C). In addition, to investigate the role of KTN1-AS1 in cancer metastasis, we performed scratch experiments and Transwell experiments, and the results showed that knockdown of KTN1-AS1 significantly inhibited wound healing capacity and invasion potential of bladder cancer cells (see fig. 2D, 2E, respectively). These results indicate that knock-out KTN1-AS1 inhibits the proliferation and metastasis of bladder cancer cells.

2.3 overexpression promotes migration and invasion of bladder cancer cells

To further investigate the carcinogenic effect of KTN1-AS1, we established cell lines (stably) overexpressing KTN1-AS 1. We confirmed the efficiency of KTN1-AS1 overexpression in both RT4 and T24 cells using RT-qPCR (fig. 3A). In contrast to the experimental results observed with the silent KTN1-AS1, the over-expression of KTN1-AS1 significantly improved cell viability and increased cell colony formation (FIGS. 3B and 3C, respectively). Scratch experiments showed a significant increase in the mobility of bladder cancer cells after overexpression of KTN1-AS1 (FIG. 3D). Furthermore, according to the results of transwell experiments, over-expression of KTN1-AS1 significantly enhanced the invasive potential of bladder cancer cells compared to the control group (FIG. 3E). These findings indicate that overexpression of KTN1-AS1 significantly promoted proliferation and invasion of bladder cancer cells.

2.4 upregulation in bladder cancer and Positive correlation with KTN1-AS1 expression

In view of the potential role of KTN1-AS1 in bladder cancer, we hypothesized whether lncRNA can act in cis or trans, particularly affecting the expression of its neighboring gene KTN1 (KTN 1-AS1 maps to the antisense DNA strand KTN1 of the upstream promoter region). We examined the expression of KTN1 in the collected bladder cancer samples, and the expression of KTN1 was significantly increased in both mRNA and protein levels in cancer tissues compared to normal tissues (FIGS. 4A and B, respectively). In addition, the two datasets in the GEO database (GSE 3167 and GSE 138118) also showed a significant upregulation of KTN1 in bladder cancer (FIGS. 4C and D, respectively). Notably, we observed that knock-out or overexpression of KTNI-AS1 affected mRNA and protein levels of KTN1 (fig. 4E-H), suggesting that lncRNA may act in cis. We further analyzed the correlation between KTN1-AS1 and KTN1 expression. Analysis of the data retrieved from the different databases showed that KTN1-AS1 was confirmed to be in significant positive correlation with KTN1 expression (fig. 4I-L), which is consistent with the analysis results in the GEO database (fig. 12A, 12B). Since cell localization analysis showed that KTN1-AS1 was expressed in the nucleus more than in the cytoplasm (fig. 1F), we hypothesized that KTN1-AS1 might interact with nucleoproteins, regulating the expression of KTN1 at the transcriptional level.

2.5 Regulation of KTN1 expression by recruitment of EP300 protein and downstream epigenetic modifications

To better understand the relationship between KTN1-AS1 and KTN1, we used UCSC database analysis. The results show a high overlap between the KTN1-AS1 and the KTN1 promoter region, which is highly enriched for the H3K27Ac peak (fig. 5A); notably, H3K27Ac is a marker for an activity enhancer. This observation led us to use the predictive RNAct algorithm to look for KTN1-AS1 binding proteins with Histone Acetyltransferase (HAT) activity. We predict that EP300 (HAT) binds KTN1-AS1 (FIG. 5B). To validate this hypothesis, we performed RIP analysis in the presence of RT4 and T24 cells (empty vector transfected or overexpressing KTN1-AS 1). The results show that KTN1-AS1 can be pulled down together with EP300, and that the abundance of overexpressed KTN1-AS1 is greater compared to negative control cells (fig. 5C). Then, we studied other correlations using the retrieved TCGA and GEO databases, with the expression of KTN1 in bladder cancer samples being significantly positively correlated with EP300 (fig. 5D and E, respectively). Notably, we also observed a decrease in KTN1 protein level expression following EP300 knock-out, further confirming the relationship between EP300 and KTN1 (fig. 5F). Therefore, we speculate that KTN1-AS1 is indirectly responsible for the modification of H3K27Ac by recruiting EP 300. To demonstrate our hypothesis, we performed ChIP-qPCR analysis and the results show that H3K27Ac in the KTN1 promoter region is deleted in the case of KTN1-AS1 knock-out in RT4 and T24 cells (fig. 5G). In contrast, H3K27Ac showed high enrichment in RT4 and T24 cells overexpressing KTN1-AS1, a phenomenon that disappeared in the case of knockout EP300 (fig. 5G). Taken together, these data indicate that KTN1-AS1 promotes the expression level of KTN1 by enriching H3K27Ac by recruiting EP300 to the KTN1 promoter region.

2.6 promotion of bladder cancer progression through the KTN1/Rho GTPase signaling pathway

To further investigate whether KTN1-AS1 promotes the progression of bladder cancer in a KTN 1-dependent manner, we performed a series of gain/loss of function experiments. Consistent with the results obtained after the KTN1-AS1 knock-out, bladder cancer cells transfected with sh-KTN1 showed lower levels of proliferation, invasion and migration (FIGS. 6B-E). Similarly, overexpression of KTN1-AS1 promoted migration and invasion of bladder cancer cells. However, this phenotype was partially lost in knockout KTN1 in cells overexpressing KTN1-AS1 (fig. 6B-E). These results indicate that KTN1 is also an oncogene in bladder cancer. Bioinformatic analysis of the data retrieved from the STRING and inBioMap databases showed that KTN1 might interact with several proteins (RHOA, RAC1, RHOG, CDC42, KLC1 and EEF 1D) (FIGS. 7A-C). Among them, there are four proteins (RAC 1, RHOA, RHOG and CDC 42) which are members of the Rho GTPase family. There is increasing evidence that Rho GTPases are involved in cell migration, cell polarity, cell cycle regulation and cytoskeletal remodeling pathophysiological processes. Therefore, we examined whether KTN1-AS1 affected the Rho GTPase signaling pathway by modulating KTN1 expression. WB experimental results showed that protein levels of RAC1, RHOA and CDC42 were significantly reduced in KTN1 knock-out cells; however, this was not the case for RHOG protein levels (fig. 7D). In contrast, levels of KTN1, RAC1, RHOA and CDC42 proteins increased with overexpression of KTN1-AS 1. Consistent with our previous speculation, expression of KTN1, RAC1, RHOA and CDC4 was partially restored following sh-KTN1 transfection in cells overexpressing KTN1-AS1 (fig. 7D). In sum, KTN1-AS1 can promote the progression of bladder cancer by modulating the KTN1/Rho GTPase signaling pathway.

2.7 promoting KTN1 mediated tumorigenesis of bladder cancer in vivo

To further investigate whether KTN1-AS1 modulates the growth of bladder cancer in vivo, we established a subcutaneous xenograft mouse model. T24 cells were stably transfected with control vectors KTN1-AS1 or KTN1-AS1 + sh-KTN1 and injected subcutaneously into nude mice (FIG. 8A). Notably, overexpression of KTN1-AS1 in bladder cancer cells significantly increased the growth rate of the cancer (AS measured by tumor volume) compared to controls, while knock-out of KTN1 in the presence of KTN1-AS1 overexpression in T24 cells partially abolished the observed phenomenon. Promotion of cancer (fig. 8B). The tumorigenic potential was further confirmed by observing the proliferation marker Ki-67 using the IHC assay. Consistent with the above results, Ki-67 levels were significantly increased in the group of animals overexpressing KTN1-AS1 to a much greater extent than in the horizontal cells of animals receiving KTN1 silencing, KTN1-AS1 overexpression (FIG. 8C). In addition, the expression of KTN1 and RAC1 (also assessed by IHC) was consistent with the in vitro results described above (fig. 8C). Taken together, these results indicate that KTN1-AS1 may be involved in tumorigenesis of bladder cancer via the KTN1/Rho GTPase axis.

Claims

The application of an inhibitor of KTN1-AS1 in preparing a medicament for treating bladder cancer.
2. The use of claim 1, wherein the bladder cancer is BUC.
Use of a pretest agent of KTN1-AS1 in the preparation of a marker for predicting recurrence of bladder cancer.
Application of a target site detection reagent of KTN1-AS1 in preparation of molecular markers for treating bladder cancer.
5, application of KTN1-AS1 in preparation of targeted negative regulation KTN1 and Rho GTPase preparations.
The application of KTN1 as a target site in preparing a medicament for treating bladder cancer.
7. The use of claim 6, wherein the bladder cancer is BUC.
The application of the regulation and control preparation of KTN1-AS1/KTN1/Rho GTPase axis in the preparation of the medicine for treating bladder cancer.
9. The use of claim 8, wherein the bladder cancer is BUC.