LncRNA CARLo-7 facilitates proliferation, migration, invasion, and EMT of bladder cancer cells by regulating Wnt/β-catenin and JAK2/STAT3 signaling pathways

Abstract

Background: Aberrant expression of long noncoding RNAs (lncRNAs) has been found to enroll in the initiation and progression of bladder cancer (BC). Earlier results show cancer-associated region long noncoding RNA-7 (CARLo-7) can be a prognostic marker for BC, but its biological function and the underlying mechanism is still to be discovered. Our study aims to explore the effects of CARLo-7 on the initiation and progression of BC and the potential mechanisms.

Methods: The expression of CARLo-7 in BC tissues and cell lines was determined by quantitative real-time polymerase chain reaction (qRT-PCR). T24 and HT1197 cells were transfected with CARLo-7 expression vector or sh-CARLo-7, then cell viability assay, BrdU assay, flow cytometry, Transwell cell migration, and invasion assay, and western blot were conducted to evaluate cell proliferation, apoptosis, invasion, migration, and epithelial-mesenchymal transition (EMT).

Results: CARLo-7 was dramatically upregulated in BC tissues and cell lines. Silencing CARLo-7 by sh-CARLo-7 significantly suppressed proliferation and induced apoptosis of BC cells, while enforced CARLo-7 expression promoted cell proliferation. Meanwhile, silencing CARLo-7 attenuated migration, invasion, and EMT of BC cells, while CARLo-7 overexpression had the contrary effects. The β-catenin, p-JAK2 and p-STAT3 levels were decreased by CARLo-7 knockdown, while activation of Wnt/β-catenin or JAK2/STAT3 pathways abolished the effects of CARLo-7 knockdown on cell proliferation and migration.

Conclusions: Collectively, CARLo-7 plays a critical role in regulating BC development by regulating cell proliferation, migration, invasion, and EMT through Wnt/β-catenin and JAK2/STAT3 signaling. Therefore, CARLo-7 might be a promising therapeutic target for BC.

Keywords: Cancer-associated region long noncoding RNA-7 (CARLo-7); JAK2/STAT3; Wnt/β-catenin; bladder cancer (BC); metastasis; proliferation.

Figure 1

CARLo-7 is upregulated in bladder cancer tissues and cell lines. (A) The expression level of CARLo-7 was evaluated by qRT-PCR in tumor tissues of bladder cancer patients (n=143) and paired adjacent normal tissues. (B) The CARLo-7 expression level in human bladder cancer cell lines (T24 and HT1197 cell lines) or human epithelial SV40 immortalized uroepithelium cell line (SV-HUC-1) was evaluated by qRT-PCR. *P<0.05. CARLo-7, cancer-associated region long noncoding RNA-7; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2

Enforced CARLo-7 expression promoted proliferation while silencing CARLo-7 suppressed proliferation and induced apoptosis of bladder cancer cells. (A) T24 and HT1197 cells were transfected with pEX-CARLo-7, pEX-NC, sh-CARLo-7, or sh-NC, then the expression levels of CARLo-7 were evaluated by qRT-PCR. Parental T24 or HT1197 cells were used as a control group. (B) T24 and HT1197 cells were transfected with showed vectors. Then cell viability determined cell viability assay. (C,D) T24 and HT1197 cells were transfected with showed vectors, then used for BrdU assay. Represent images (C), and the percentage of BrdU positive cells (D) were shown. DAPI (Blue) was used to mark the nucleus, scale bar =500 μm. (E) T24 and HT1197 cells were transfected with pEX-CARLo-7, sh-CARLo-7, or sh-NC control, then the percentage of apoptosis cells (Annexin V and PI positive) was evaluated by flow cytometry. *P<0.05 compare to the control group. CARLo-7, cancer-associated region long noncoding RNA-7.

Figure 3

Enforced CARLo-7 expression facilitated migration, invasion, and EMT of bladder cancer cells while silencing CARLo-7 had the contrary effects. T24 and HT1197 cells were transfected with pEX-CARLo-7, pEX-NC, sh-CARLo-7, or sh-NC, then cells were used for Transwell cell migration (A) or invasion (B) assay, or collected cell lysates for western blot (C,D). Parental T24 or HT1197 cells were used as a control group. The protein expression of E-cadherin, N-cadherin, and vimentin was decided by Western blot (C), and protein expression normalized to β-actin was shown (D). *P<0.05 compare to the control group. CARLo-7, cancer-associated region long noncoding RNA-7; EMT, epithelial-mesenchymal transition.

Figure 4

Wnt/β-catenin and JAK2/STAT3 signaling pathways took part in regulating cell proliferation and migration by CARLo-7. (A,B) T24 and HT1197 cells were transfected with pEX-CARLo-7, pEX-NC, sh-CARLo-7, or sh-NC, then β-catenin, p-JAK2, and p-STAT3 levels were determined by Western blot (A). The ratio of β-catenin, p-JAK2, and p-STAT3 normalized to β-actin has been calculated (B). (C,D) T24 cells were transfected with indicated vectors and treated with XAV-939, AG490, BML-24 or IL-6, cell viability (C), and migration (D) were evaluated. The magnification is 800 × for photos of migration cells. *P<0.05 vs. control group; ^#P<0.05 vs. sh-CARLo-7 group. CARLo-7, cancer-associated region long noncoding RNA-7.