LncRNA NEAT1 knockdown attenuates autophagy to elevate 5-FU sensitivity in colorectal cancer via targeting miR-34a.

Colorectal carcinoma (CRC) is a common malignant tumor. Increasing evidences indicated that CRC showed a resistance to 5-fluorouracil (5-FU) and further resulted in a poor prognosis. In this study, we aim to investigate the effect of long noncoding RNA nuclear paraspeckle assembly transcript 1 (LncRNA NEAT1) on cell viability, sensitivity to 5-FU, and autophagy of CRC cell lines. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) was used to detect cell viability, immunofluorescent staining was used to detect autophagy puncta, and luciferase reporter system was used to determine binding ability between miR-34a and NEAT1 or putative targets. Additionally, indicated mRNAs and protein expressions were determined by qRT-PCR or western blotting, respectively. We found that NEAT1 expression was increased in CRC tissues and cells, which showed a negative correlation with miR-34a expression. In addition, NEAT1 knockdown noticeably inhibited the proliferation of CRC cells and enhanced 5-FU sensitivity. It revealed that NEAT1 knockdown suppressed the LC3 puncta and the expressions of Beclin-1, ULK1, and ratio of LC3II/I. Overexpression of miR-34a showed similar trends with NEAT1 knockdown. miR-34a was validated to target the putative binding sites in 3'-UTR of HMGB1, ATG9A, and ATG4B, which are involved in the activation of autophagy. Inhibition of miR-34a or overexpression of HMGB1 could effectively reverse elevated 5-FU sensitivity upon NEAT1 knockdown. In addition, 3-MA reversed NEAT1 overexpression-induced resistance in HT29 cells. These findings indicate that LncRNA NEAT1 could target miR-34a and promote autophagy to facilitate 5-FU chemoresistance in CRC.

Liu F ，Ai FY ，Zhang DC ，Tian L ，Yang ZY ，Liu SJ ... -  《Cancer Medicine》

Inhibition of lncRNA-NEAT1 sensitizes 5-Fu resistant cervical cancer cells through de-repressing the microRNA-34a/LDHA axis.

Cervical cancer is one of the most diagnosed malignancies among females. The 5-fluorouracil (5-Fu) is a widely used chemotherapeutic agent against diverse cancers. Despite the initially encouraging progresses, a fraction of cervical cancer patients developed 5-Fu resistance. We detected that nuclear-rich transcripts 1 (NEAT1) was significantly up-regulated in cervical cancer tissues and cell lines. Moreover, NEAT1 was positively associated with 5-Fu resistance. Furthermore, expression of NEAT1 was significantly up-regulated in 5-Fu resistant CaSki cervical cancer cells. Knocking down NEAT1 by shRNA dramatically promoted the sensitivity of 5-Fu resistant CaSki cells. We observed a negative correlation between long noncoding RNA (lncRNA)-NEAT1 and miR-34a in cervical cancer patient tissues. Overexpression of miR-34a significantly sensitized 5-Fu resistant cells. Bioinformatics analysis uncovered that NEAT1 functions as a competitive endogenous RNA (ceRNA) of miR-34a in cervical cancer cells via sponging it at multiple sites to suppress expression of miR-34a. This negative association between NEAT1 and miR-34a was further verified in cervical cancer tissues. We found the 5-Fu resistant cells displayed significantly increased glycolysis rate. Overexpression of miR-34a suppressed cellular glycolysis rate and sensitized 5-Fu resistant cells through direct targeting the 3'-untranslated region (UTR) of LDHA, a glycolysis key enzyme. Importantly, knocking down NEAT1 successfully down-regulated LDHA expressions and glycolysis rate of cervical cancer cells by up-regulating miR-34a, a process could be further rescued by miR-34a inhibition. Finally, we demonstrated inhibition of NEAT1 significantly sensitized cervical cancer cells to 5-Fu through the miR-34a/LDHA pathway. In summary, the present study suggests a new molecular mechanism for the NEAT1-mediated 5-Fu resistance via the miR-34a/LDHA-glycolysis axis.

Shao X ，Zheng X ，Ma D ，Liu Y ，Liu G ... -  《-》

lncRNA NEAT1 regulates gastric carcinoma cell proliferation, invasion and apoptosis via the miR‑500a‑3p/XBP‑1 axis.

Zhou Y ，Sha Z ，Yang Y ，Wu S ，Chen H ... -  《-》

MicroRNA-149 Increases the Sensitivity of Colorectal Cancer Cells to 5-Fluorouracil by Targeting Forkhead Box Transcription Factor FOXM1.

Previously, we have shown that microRNA (miR)-149 suppresses the migration and invasion of colorectal cancer (CRC) cells by targeting forkhead box transcription factor (FOXM1). However, the roles of miR-149 in the chemoresistance of CRC cells to 5-Fluorouracil (5-FU) is unclear. The aim of this study is to investigate whether miR-149 targets FOXM1 to regulate the 5-FU resistance of CRC. The qRT-PCR assay was performed to detect the expression of miR-149 in 5-FU-resistant CRC cells (HCT-8/5-FU and LoVo/5-FU) and their parental CRC cells (HCT-8 and LoVo). Also, the effects of miR-149 expression on the sensitivity of CRC cells to 5-FU were determined by gain- and loss-of-function assays. Finally, whether miR-149 regulates the 5-FU resistance of CRC cells by targeting the mammalian Forkhead Box M1 (FOXM1) was investigated. The expression of miR-149 was significantly downregulated in 5-FU-resistant CRC cells in comparison with their parental CRC cells. Re-expression of miR-149 could enhance the 5-FU sensitivity of 5-FU-resistant CRC cells by increasing 5-FU-inducing apoptosis, while downregulation of miR-149 could decrease the 5-FU sensitivity of parental CRC cells by decreasing 5-FU-inducing apoptosis. In addition, the luciferase assay indicated that miR-149 could bind to the 3'-UTR sequence of FOXM1 mRNA. The silencing of FOXM1 could mimic the effect of miR-149 upregulation on the 5-FU resistance of 5-FU-resistant CRC cells. Furthermore, the expression of miR-149 in the 5-FU-responding CRC tissues was significantly higher than that in the non-responding tissues and inversely correlated with FOXM1 mRNA level. MiR-149 reverses the resistance of CRC cells to 5-FU by directly targeting FOXM1. Thus, targeting miR-149/FOXM1 signaling will be a potential strategy in the treatment of 5-FU-chemoresistant CRC.

Liu X ，Xie T ，Mao X ，Xue L ，Chu X ，Chen L ... -  《-》

Upregulation of microRNA-135b and microRNA-182 promotes chemoresistance of colorectal cancer by targeting ST6GALNAC2 via PI3K/AKT pathway.

MicroRNAs (miRNAs) are increasingly involved in the development of drug resistance, including 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC). Aberrant sialylation is correlated with human CRC. The study was to explore whether miR-135b and miR-182 modulated 5-FU chemoresistance of CRC by targeting ST6GALNAC2 via PI3K/AKT pathway. MiR-135b and miR-182 were found to be up-regulated in CRC tissues and 5-FU resistant CRC cell lines. Forced miR-135b and miR-182 expression also affected ST6GALNAC2 levels. Using reporter-gene assay, ST6GALNAC2 was identified as direct target of miR-135b and miR-182, while ST6GALNAC2 expression exhibited patterns opposite to that of miR-135b and miR-182 in CRC samples and cell lines. Interestingly, up-regulation of miR-135b or miR-182 increased drug resistance and proliferation, but decreased apoptosis in 5-FU resistant CRC cell lines. Suppression of these miRNAs implicated an inverse function, while altered expression of ST6GALNAC2 mediated CRC progression upon transfection with miR-135b/-182 mimic or inhibitor. Furthermore, miR-135b and miR-182 were clarified to regulate the activity of phosphoinositide-3 kinase (PI3K)/AKT pathway. Inhibition of the PI3K/AKT pathway enhanced the chemosensitivity to 5-FU in HCT-8/5-FU and LoVo/5-FU. Taken together, miR-135b and miR-182 may reverse the resistance to 5-FU in CRC cells by targeting ST6GALNAC2 via PI3K/AKT pathway, which render potential chemotherapy targets for the treatment of CRC.

Liu B ，Liu Y ，Zhao L ，Pan Y ，Shan Y ，Li Y ，Jia L ... -  《-》