Inhibition of KHSRP sensitizes colorectal cancer to 5-fluoruracil through miR-501-5p-mediated ERRFI1 mRNA degradation

K-homology (KH)-type splicing regulatory protein (KHSRP) is an RNA binding protein that participates in RNA variable splicing and stability, and facilitates the biogenesis of miRNAs that target mRNA. However, to date, the role of KHSRP in colorectal cancer (CRC) progression has not been reported. In this study, the function of KHSRP in CRC proliferation and 5-fluoruracil (5-FU) resistance was investigated. The upregulation of KHSRP expression was confirmed in CRC patient tissues and two CRC cell lines. Manipulating KHSRP expression altered cell proliferation and 5-FU resistance in CRC cells. ERRFI1, a downstream effector of KHSRP in CRC cells, reduced CRC cell proliferation. Sensitivity to 5-FU mediated by KHSRP knockdown was reversed by ERRFI1 knockdown. We found that KHSRP decreased ERRFI1 mRNA expression indirectly. By screening KHSRP-regulated miRNAs, we further found that miR-501-5p directly combines with KHSRP in CRC cells. Mechanistically, the results of a luciferase assay suggested that miR-501-5p directly binds to the ERRFI1 3′-untranslated region. Taken together, our data indicated that modification of ERRFI1 by KHSRP occurs through miR-501-5p, an essential mechanism driving CRC proliferation and 5-FU resistance. Insight into this mechanism may provide novel targets for overcoming drug resistance in CRC.     

miR-138-5p targets MCU to inhibit mitochondrial biogenesis and colorectal cancer growth

miR-138-5p has been identified as a novel cancer-related miRNA molecule in a variety of malignancies. However, the functions and mechanisms underlying miR-138-5p in colorectal carcinoma (CRC) remains largely unknown. In the present study, we analysed the biological effects and clinical significance of miR-138-5p in CRC. miR-138-5p expression was analysed by quantitative real-time PCR in CRC tissues and cell lines. The effects of miR-138-5p on CRC cell growth was detected by cell proliferation, colony formation, cell cycle and cell apoptosis assays in vitro and in vivo. Our data showed that miR-138-5p was significantly downregulated in CRC. Downregulated miR-138-5p was related with poor prognosis in patients with CRC. miR-138-5p suppressed CRC growth but promoted cell death both in vitro and in vivo. Online predictions and integrated experiments identified that miR-138-5p targeted MCU, and downregulated miR-138-5p promoted mitochondrial biogenesis in CRC. In the light of the underlying mechanisms, our results indicated that downregulated miR-138-5p led to increased expression of MCU, which subsequently increased the production of ROS to promote CRC growth. Our results indicated that downregulated miR-138-5p strengthened mitochondrial biogenesis through targeting MCU, thus contributing to CRC cell growth, which may provide a potential therapeutic target for CRC.     

Circ_0005615 promotes cervical cancer cell growth and metastasis by modulating the miR-138-5p/KDM2A axis

Cervical cancer (CC) is a highly fatal gynecological malignancy due to its high metastasis and recurrence rate. Circular RNA (circRNA) has been regarded as a regulator of CC. However, the underlying molecular mechanism of circ_0005615 in CC remains unclear. The levels of circ_0005615, miR-138-5p, and lysine demethylase 2A (KDM2A) were measured using qRT-PCR or western blot. Cell proliferation was assessed by Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine, and colony formation experiments. Cell invasion and migration were tested by transwell assay and wound healing assay. Flow cytometry and Caspase-Glo 3/7 Assay kit were used to analyze cell apoptosis. The expression of proliferation-related and apoptosis-related markers was detected by western blot. The binding relationships among circ_0005615, miR-138-5p, and KDM2A were verified by dual-luciferase reporter assay or RNA immunoprecipitation assay. Xenograft assay was applied to detect the effect of circ_0005615 in vivo. Circ_0005615 and KDM2A were upregulated, while miR-138-5p was downregulated in CC tissues and cells. Circ_0005615 knockdown retarded cell proliferation, migration, and invasion, while promoting apoptosis. Besides, circ_0005615 sponged miR-138-5p, and miR-138-5p could target KDM2A. miR-138-5p inhibitor reversed the regulation of circ_0005615 knockdown on CC cell growth and metastasis, and KDM2A overexpression also abolished the inhibitory effect of miR-138-5p on CC cell growth and metastasis. In addition, we also discovered that circ_0005615 silencing inhibited CC tumor growth in vivo. Circ_0005615 acted as a tumor promoter in CC by regulating the miR-138-5p/KDM2A pathway.     

Circ-ERBB2 knockdown sensitized colorectal cancer cells to 5-FU via miR-181a-5p/PTEN/Akt pathway

Colorectal cancer (CRC) is the fourth most deadly cancer worldwide, drug resistance impedes treatment of CRC. It is still urgent to find new molecular targets to improve the sensitivity of chemotherapeutic drugs. In this study, circ-ERBB2 was upregulated in CRC cells. Upregulation of circ-ERBB2 promoted CRC cells proliferation and clone formation, but inhibited apoptosis. We identified miR-181a-5p as circ-ERBB2's target. The effect of miR-181a-5p on CRC cells was contrary to circ-ERBB2, miR-181a-5p downregulation abolished the function of circ-ERBB2 silencing in CRC cells. In addition, phosphatase and tensin homolog (PTEN) was verified as miR-181a-5p's downstream target, circ-ERBB2 activates the Akt pathway and inhibits cell apoptosis through modulating miR-181a-5p/PTEN. Circ-ERBB2 silencing significantly reduced CRC cell resistance to 5-FU. miR-181a-5p downregulation abolished the role of circ-ERBB2 knockdown in CRC cell resistance to 5-FU. In conclusion, upregulation of circ-ERBB2 promoted the malignancy of CRC and reduced CRC cell resistance to 5-FU. Besides, additional mechanism study provided a novel regulatory pathways that circ-ERBB2 knockdown promoted CRC cell sensitivity to 5-FU by regulating miR-181a-5p/PTEN/Akt pathway. This research indicated that circ-ERBB2 may be a valuable biomarker for the diagnosis and treatment of CRC.     

microRNA-16-5p-containing exosomes derived from bone marrow-derived mesenchymal stem cells inhibit proliferation,migration, and invasion,while promoting apoptosis of colorectal cancer cells by downregulating ITGA2

Colorectal cancer (CRC) is a form of cancer developing from either the colon or rectum. Nowadays, research supports the functionality of exosome expressing microRNAs (miRNAs) as potential biomarker for various cancers including CRC. This study was performed with the intent of investigating the roles of both bone marrow-derived mesenchymal stem cells (BMSCs) and exosomal miR-16-5p in CRC by regulating integrin α2 (ITGA2). A microarray-based analysis was conducted to screen the CRC-associated differentially expressed genes (DEGs) as well as potential regulatory miRNAs. Next, the role of miR-16-5p in terms of its progression in association with CRC was determined. Subsequently, CRC cells were exposed to exosomes secreted by BMSCs transfected with miR-16-5p, isolated and cocultured with CRC cells in an attempt to identify the role of exosomes. Effects of BMSCs-derived exosomes overexpressing miR-16-5p on biological functions of CRC cells and tumorigenicity were all subsequently detected. Effects of miR-16-5p treated with CRC cells in regard to CRC in vivo were also measured. ITGA2 was overexpressed, while miR-16-5p was poorly expressed in CRC cells and miR-16-5p targeted ITGA2. The in vitro experiments revealed that the BMSCs-derived exosomes overexpressing miR-16-5p inhibited proliferation, migration, and invasion, while simultaneously stimulating the apoptosis of the CRC cells via downregulation of ITGA2. Furthermore, the results of in vivo experiments confirmed that the BMSCs-derived exosomes overexpressing miR-16-5p repressed the tumor growth of CRC. Collectively, BMSCs-derived exosomes overexpressing miR-16-5p restricted the progression of CRC by downregulating ITGA2.     

microRNA-16-5p suppresses cell proliferation and angiogenesis in colorectal cancer by negatively regulating forkhead box K1 to block the PI3K/Akt/mTOR pathway

MicroRNAs (miRNAs/miRs) have aroused increasing attention in colorectal cancer (CRC) therapy. This study is designed for a detailed analysis of the roles of miR-16-5p and forkhead box K1 (FOXK1) in cell angiogenesis and proliferation during CRC in addition to their underlying mechanisms. CRC tissues and colon cancer cell lines (SW620 and HCT8) were investigated. qRT-PCR and Western blot were utilized to evaluate miR-16-5p and FOXK1 expression. Following gain- and loss-of-function assays on miR-16-5p or FOXK1, the effects of miR-16-5p and FOXK1 were assessed on cell angiogenesis and proliferation in CRC cells. A dual-luciferase reporter assay was employed to evaluate the binding relationship of miR-16-5p and FOXK1. Western blot was used to determine the effects of miR-16-5p and FOXK1 on key molecules of the PI3K/Akt/mTOR pathway. Highly expressed FOXK1 and lowly expressed miR-16-5p were observed in CRC cells and tissues. miR-16-5p overexpression or FOXK1 knockdown reduced CRC cell proliferation and angiogenesis of human umbilical vein endothelial cells co-cultured with the supernatant of CRC cells, whereas miR-16-5p silencing or FOXK1 upregulation caused opposite trends. Additionally, miR-16-5p negatively modulated FOXK1 expression. The blockade of the PI3K/Akt/mTOR pathway was triggered by miR-16-5p overexpression or FOXK1 silencing. In conclusion, miR-16-5p hampers cell angiogenesis and proliferation during CRC by targeting FOXK1 to block the PI3K/Akt/mTOR pathway.Key words: microRNA-16-5p, forkhead box K1, PI3K/Akt/mTOR pathway, colorectal cancer, proliferation, angiogenesis     

circ-ZEB1 regulates epithelial-mesenchymal transition and chemotherapy resistance of colorectal cancer through acting on miR-200c-5p

Circular RNAs (circRNAs) have been demonstrated to be important regulators in human malignant tumors, including colorectal cancer (CRC). While the role circ-ZEB1 played in CRC remains unclear. In this study, we aim to explore the biological function and the underlying mechanism of circ-ZEB1 in CRC. RNAscope was used to analyze the expression and localization of circ-ZEB1 in CRC tissues. Loss of function experiments were conducted, including CCK-8, transwell assays, flow cytometry analysis, and murine xenograft models, so as to detect the effect of circ-ZEB1 on CRC cells. IC50 assay was used to evaluate the influence of circ-ZEB1 on the chemoresistance of CRC cells. Epithelial-mesenchymal transition (EMT) related markers were detected. The relationship between circ-ZEB1 and miR-200c-5p was investigated by FISH, dual-luciferase reporter assay, and RIP assay. We found in our study that circ-ZEB1 was significantly upregulated in CRC tissues. Downregulation of circ-ZEB1 inhibited cell proliferation, colony formation, as well as cell migration and invasion abilities of CRC cell lines. In vivo experiments indicated that knockdown of circ-ZEB1 suppressed tumorigenesis and distant metastasis of CRC cells in nude mice. What's more, EMT and chemoresistance of CRC cells were also attenuated following circ-ZEB1 knockdown. Mechanistically, we proved that circ-ZEB1 could directly bind with miR-200c and functioned as miR-200c sponge to exert its biological functions in CRC cells. In conclusion, circ-ZEB1 could promote CRC cells progression, EMT, and chemoresistance via acting on miR-200c, elucidating a potential therapeutic target to inhibit CRC progression.     

MicroRNA‐425‐5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10

Acquired chemoresistance represents a major obstacle in cancer treatment, the underlying mechanism of which is complex and not well understood. MiR‐425‐5p has been reported to be implicated tumorigenesis in a few cancer types. However, its role in regulating chemoresistance has not been investigated in colorectal cancer (CRC) cells. Microarray analysis was performed in isogenic chemosensitive and chemoresistant HCT116 cell lines to identify differentially expressed miRNAs. miRNA quantitative real‐time PCR was used to detect miR‐425‐5p expression levels between drug resistant and parental cancer cells. MiR‐425‐5p mimic and inhibitor were transfected, followed by CellTiter‐Glo^® assay to examine drug sensitivity in these two cell lines. Western Blot and luciferase assay were performed to investigate the direct target of miR‐425‐5p. Xenograft mouse models were used to examine in vivo function of miR‐425‐5p. Our data showed that expression of miR‐425‐5p was significantly up‐regulated in HCT116‐R compared with parental HCT116 cells. Inhibition of miR‐425‐5p reversed chemoresistance in HCT116‐R cells. Programmed cell death 10 (PDCD10) is the direct target of miR‐425‐5p which is required for the regulatory role of miR‐425‐5p in chemoresistance. MiR‐425‐5p inhibitor sensitized HCT116‐R xenografts to chemo drugs in vivo. Our study demonstrated that miR‐425‐5p regulates chemoresistance of CRC cells by modulating PDCD10 expression level both in vitro and in vivo. MiR‐425‐5p may represent a new therapeutic target for the intervention of CRC.     

Long noncoding RNA PRKCQ-AS1 promotes CRC cell proliferation and migration via modulating miR-1287-5p/YBX1 axis

Colorectal cancer (CRC) brings more than 600 000 deaths every year around the globe, making itself the third most frequently occurred carcinoma. The great progress human achieved in diagnosis and treatment of various cancers has failed to reverse this trend. Fortunately, growing evidence has implied the relationship between lncRNAs and cancer progression. Long noncoding RNA (lncRNA) PRKCQ-AS1 was heightened in CRC cells and tissues and related with dismal prognosis of CRC patients. Knockdown of PRKCQ-AS1 would induce a decrease in proliferative and migrating ability of CRC cells. Also, PRKCQ-AS1 enriched in cytoplasm of CRC cells and negatively regulated miR-1287-5p level. More important, PRKCQ-AS1 could bind to argonaute 2 and function in the RNA-induced silencing complex with miR-1287-5p. Therefore, PRKCQ-AS1 was a competing endogenous RNA for miR-1287-5p. Subsequently, it was validated that miR-1287-5p could suppress the proliferative and migratory functions in CRC. Furthermore, PRKCQ-AS1 could upregulate the mRNA and protein level of YBX1 targeted by miR-1287-5p. And YBX1 expression was elevated in CRC cells and tissues. Rescue assays in vitro and in vivo showed that overexpression of YBX1 could partly offset the effect of CRC progression induced by knocking down PRKCQ-AS1, demonstrating PRKCQ-AS1 mediating CRC progression via miR-1287-5p/YBX1 pathway.     

MicroRNA-218 Inhibits Cell Cycle Progression and Promotes Apoptosis in Colon Cancer by Downregulating BMI1 Polycomb Ring Finger Oncogene

Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3′ untranslated region (3′UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3′UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.     

Long noncoding RNA FOXD3-AS1 promotes colon adenocarcinoma progression and functions as a competing endogenous RNA to regulate SIRT1 by sponging miR-135a-5p

More and more documents have proved that the abnormal expression of long noncoding RNAs (lncRNAs) are correlated with the initiation and progression of colorectal cancer (CRC). lncRNA FOXD3-AS1 has been reported in glioma for its oncogenic property. According to the survival analysis of The Cancer Genome Atlas database, FOXD3-AS1 upregulation implied lower survival rate of patients with CRC. Quantitative real-time polymerase chain reaction showed the overexpression of FOXD3-AS1 in both CRC tissues and cells. The Kaplan–Meier method demonstrated the prognostic value of FOXD3-AS1 for patients with CRC. To explore the effect of FOXD3-AS1 on CRC progression, loss-of-function experiments were carried out, whose results indicated that knockdown of FOXD3-AS1 suppressed cell proliferation, migration, and invasion, inhibited cell cycle and promoted cell apoptosis in vitro. In vivo experiments affirmed that FOXD3-AS1 affected tumor growth. FOXD3-AS1 expression was enriched in the cytoplasm of CRC cells. Mechanism experiments revealed that FOXD3-AS1 served as a competing endogenous RNA to upregulate SIRT1 by sponging miR-135a-5p. In addition, SIRT1 silencing also restrained cell proliferation and motility. Rescue assays revealed the biological function of FOXD3-AS1/miR-135a-5p/SIRT1 axis in CRC progression. In conclusion, FOXD3-AS1 promotes CRC progression by regulating miR-135a-5p/SIRT1 axis, shedding lights on the way to CRC treatments.     

CircPRMT5 circular RNA promotes proliferation of colorectal cancer through sponging miR-377 to induce E2F3 expression

CircPRTM5 is associated with cell proliferation and migration in many kinds of malignancies. However, the functions and mechanisms of CircPRTM5 in CRC progression remain unclear. We explored the role and the mechanisms of CircPRTM5 in the development of CRC. Tissues of CRC patients and matched adjacent non-tumour tissues were collected to evaluate the expression of CircPRTM5. The expression of CircPRTM5 in CRC tissues was significantly higher than that in adjacent tissues. The biological functions of CircPRTM5 in CRC were determined by overexpression and down-regulation of CircPRTM5 in CRC cells in vitro and in vivo. The results indicate that knockdown of CircPRTM5 can significantly inhibit the proliferation of CRC cells. The potential mechanisms of CircPRTM5 in CRC development were identified by RT-qPCR, Western blotting analysis and luciferase reporter assay. CircPRTM5 competitively regulates the expression of E2F3 by capillary adsorption of miR-377. CircPRMT5 regulates CRC proliferation by regulating the expression of E2F3, which affects the expression of the cell cycle-associated proteins cyclinD1 and CDK2. CircPRTM5 exerts critical regulatory role in CRC progression by sponging miR-377 to induce E2F3 expression.     

The expression of long noncoding RNA CRCAL-3 in colorectal cancer and its impacts on cell proliferation and migration

Long noncoding RNAs (lncRNAs) have been implicated in colorectal cancer (CRC). And lncRNA RP11-138J23.1 (CRCAL-3) was previously reported as a candidate regulator of CRC development. But its regulating functions have not been fully elucidated. Here, we analyzed RNA sequencing data from the Cancer Genome Atlas (TCGA) and 253 CRC patients treated in our hospital to assess expression dysregulation of CRCAL-3, and the correlation between CRCAL-3 expression and disease progression. Further, polymerase chain reaction (PCR) assay on different cell lines and knockdown experiments by small interfering RNA were performed to assess functions of CRCAL-3 in proliferation and migration of CRC cells. As a result, analyses on TCGA datasets showed an upregulated CRCAL-3 expression in 14 solid tumors, including CRC. PCR assay on 253 cases of CRC tissue and 114 cases of normal adjacent tissue confirmed this expression upregulation. Also, CRCAL-3 expression was exhibited by survival analyses on the 253 CRC patients, to have a negative correlation with patients' overall and progression-free survivals. PCR assay on different cell lines showed that CRC cells expressed a higher level of CRCAL-3, compared with normal colonic epithelial cells. In vitro knockdown of CRCAL-3 resulted in an obvious retardation of proliferation and migration in two CRC cell lines (HCT116 and DLD-1). Moreover, CRCAL-3 knockdown was observed in xenograft models to repress cell proliferation and enhance cisplatin sensitivity. Taking these results together, CRCAL-3 emerged as a biomarker for early diagnosis, prognosis prediction, and individualized treatment of CRC.     

LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53.     

LncRNA OIP5-AS1 regulates radioresistance by targeting DYRK1A through miR-369-3p in colorectal cancer cells

Object

This study aimed to investigate the role of lncRNA OIP5-AS1 in regulating radioresistance of colorectal cancer (CRC) cells.