Association analysis of miRNA-related genetic polymorphisms in miR-143/145 and KRAS with colorectal cancer susceptibility and survival

Background: There is accumulating evidence of aberrant expression of miR-143 and miR-145 and their target gene KRAS in colorectal cancer (CRC). We hypothesize that single nucleotide polymorphisms (SNPs) within or near mRNA–microRNA (miRNA) binding sites may affect miRNA/target gene interaction, resulting in differential mRNA/protein expression and promoting the development and progression of CRC. Methods: We conducted a case–control study of 507 patients with CRC recruited from a tertiary hospital and 497 population-based controls to assess the association of genetic polymorphisms in miR-143/145 and the KRAS 3′ untranslated region (3′UTR) with susceptibility to CRC and patients’ survival. In addition, genetic variations of genomic regions located from 500 bp upstream to 500 bp downstream of the miR-143/miR-145 gene and the 3′UTR of KRAS were selected for analysis using the Haploview and HaploReg software. Results: Using publicly available expression profiling data, we found that miR-143/145 and KRAS expression were all reduced in rectal cancer tissue compared with adjacent non-neoplastic large intestinal mucosa. The rs74693964 C/T variant located 65 bp downstream of miR-145 genomic regions was observed to be associated with susceptibility to CRC (adjusted odds ratio (OR): 2.414, 95% CI: 1.385–4.206). Cumulative effects of miR-143 and miR-145 on CRC risk were observed (P_trend=0.03). Patients having CRC carrying variant genotype TT of KRAS rs712 had poorer survival (log-rank P=0.044, adjusted hazard ratio (HR): 4.328, 95% CI: 1.236–15.147). Conclusions: Our results indicate that miRNA-related polymorphisms in miR-143/145 and KRAS are likely to be deleterious and represent potential biomarkers for susceptibility to CRC and patients’ survival.     

Lessons from miR-143/145: the importance of cell-type localization of miRNAs

miR-143 and miR-145 are co-expressed microRNAs (miRNAs) that have been extensively studied as potential tumor suppressors. These miRNAs are highly expressed in the colon and are consistently reported as being downregulated in colorectal and other cancers. Through regulation of multiple targets, they elicit potent effects on cancer cell growth and tumorigenesis. Importantly, a recent discovery demonstrates that miR-143 and miR-145 are not expressed in colonic epithelial cells; rather, these two miRNAs are highly expressed in mesenchymal cells such as fibroblasts and smooth muscle cells. The expression patterns of miR-143 and miR-145 and other miRNAs were initially determined from tissue level data without consideration that multiple different cell types, each with their own unique miRNA expression patterns, make up each tissue. Herein, we discuss the early reports on the identification of dysregulated miR-143 and miR-145 expression in colorectal cancer and how lack of consideration of cellular composition of normal tissue led to the misconception that these miRNAs are downregulated in cancer. We evaluate mechanistic data from miR-143/145 studies in context of their cell type-restricted expression pattern and the potential of these miRNAs to be considered tumor suppressors. Further, we examine other examples of miRNAs being investigated in inappropriate cell types modulating pathways in a non-biological fashion. Our review highlights the importance of determining the cellular expression pattern of each miRNA, so that downstream studies are conducted in the appropriate cell type.     

MicroRNA-143 Inhibits Migration and Invasion of Human Non-Small-Cell Lung Cancer and Its Relative Mechanism

Background: MicroRNAs (miRNAs) play important roles in many biological processes, including cancer development. Among those miRNAs, miR-143 shows tumor-suppressive activity in some human cancers. However, the function and mechanism of miR-143 in lung cancer cells remains unknown. Here we explored the role of miR-143 in lung cancer. Results: According to qRT-PCR, we found that miR-143 was notably down-regulated in 19 NSCLC tissues and 5 cell lines. In vitro experiments showed us that miR-143 could significantly suppress the migration and invasion of NSCLC cell lines while it had no effects on the growth of NSCLC cell lines, and in vivo metastasis assay showed the same results. Finally, we found that the mechanism of miR-143 inhibiting the migration and invasion of NSCLC might be through targeting CD44v3. Conclusions: The up-regulated miR-143 in lung cancer could significantly inhibit cell migration and invasion, and this might work through targeting CD44v3, which was newly identified by us.     

MicroRNA-143 (miR-143) regulates cancer glycolysis via targeting hexokinase 2 gene

High glycolysis, well known as "Warburg effect," is frequently observed in a variety of cancers. Whether the deregulation of miRNAs contributes to the Warburg effect remains largely unknown. Because miRNA regulates gene expression at both mRNA and protein levels, we constructed a gene functional association network, which allows us to detect the gene activity instead of gene expression, to integratively analyze the microarray data for gene expression and miRNA expression profiling and identify glycolysis-related gene-miRNA pairs deregulated in cancer. Hexokinase 2 (HK2), coding for the first rate-limiting enzyme of glycolysis, is among the top list of genes predicted and potentially regulated by multiple miRNAs including miR-143. Interestingly, miR-143 expression was inversely associated with HK2 protein level but not mRNA level in human lung cancer samples. miR-143, down-regulated by mammalian target of rapamycin activation, reduces glucose metabolism and inhibits cancer cell proliferation and tumor formation through targeting HK2. Collectively, we have not only established a novel methodology for gene-miRNA pair prediction but also identified miR-143 as an essential regulator of cancer glycolysis via targeting HK2.     

EGFR signals downregulate tumor suppressors miR-143 and miR-145 in Western diet-promoted murine colon cancer: role of G1 regulators

Epidermal growth factor receptors (EGFR) contribute to colonic tumorigenesis in experimental models of colon cancer. We previously showed that EGFR was also required for colonic tumor promotion by Western diet. The goal of this study was to identify EGFR-regulated microRNAs that contribute to diet-promoted colonic tumorigenesis. Murine colonic tumors from Egfr(wt) and hypomorphic Egfr(wa2) mice were screened using micro RNA (miRNA) arrays and miR-143 and miR-145 changes confirmed by Northern, real-time PCR, and in situ analysis. Rodent and human sporadic and ulcerative colitis (UC)-associated colon cancers were examined for miR-143 and miR-145. Effects of EGFR on miR-143 and miR-145 expression were assessed in murine and human colonic cells and their putative targets examined in vitro and in vivo. miR-143 and miR-145 were readily detected in normal colonocytes and comparable in Egfr(wt) and Egfr(wa2) mice. These miRNAs were downregulated in azoxymethane and inflammation-associated colonic tumors from Egfr(wt) mice but upregulated in Egfr(wa2) tumors. They were also reduced in human sporadic and UC colon cancers. EGFR signals suppressed miR-143 and miR-145 in human and murine colonic cells. Transfected miR-143 and miR-145 inhibited HCT116 cell growth in vitro and in vivo and downregulated G(1) regulators, K-Ras, MYC, CCND2, cdk6, and E2F3, putative or established targets of these miRNAs. miRNA targets Ras and MYC were increased in colonic tumors from Egfr(wt) but not Egfr(wa2) mice fed a Western diet. EGFR suppresses miR-143 and miR-145 in murine models of colon cancer. Furthermore, Western diet unmasks the tumor suppressor roles of these EGFR-regulated miRNAs.     

The Viral Protein K7 Inhibits Biochemical Activities and Condensate Formation by the DEAD-box Helicase DDX3X

The DEAD-box RNA helicase DDX3X promotes translation initiation and associates with stress granules. A range of diverse viruses produce proteins that target DDX3X, including hepatitis C, dengue, vaccinia, and influenza A. The interaction of some of these viral proteins with DDX3X has been shown to affect antiviral intracellular signaling, but it is unknown whether and how viral proteins impact the biochemical activities of DDX3X and its physical roles in cells. Here we show that the protein K7 from vaccinia virus, which binds to an intrinsically disordered region in the N-terminus of DDX3X, inhibits RNA helicase and RNA-stimulated ATPase activities, as well as liquid–liquid phase separation of DDX3X in vitro. We demonstrate in HCT 116 cells that K7 inhibits association of DDX3X with stress granules, as well as the formation of aberrant granules induced by expression of DDX3X with a point mutation linked to medulloblastoma and DDX3X syndrome. The results show that targeting of the intrinsically disordered N-terminus is an effective viral strategy to modulate the biochemical functions and subcellular localization of DDX3X. Our findings also have potential therapeutic implications for diseases linked to aberrant DDX3X granule formation.     

The Ded1/DDX3 subfamily of DEAD-box RNA helicases

Abstract

In eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.     

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1

MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4–NOT deadenylase complex followed by the removal of the 5′ cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4–NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6–CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.     

miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E₂ (PGE₂) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE₂ levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE₂, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.     

miR-143 inhibits proliferation and metastasis of nasopharyngeal carcinoma cells via targeting FMNL1 based on clinical and radiologic findings

Mounting evidence has reported that microRNA-143 (miR-143) is involved in the development of multiple cancers. To investigate the underlying mechanisms of miR-143 regulating proliferation and metastasis in nasopharyngeal carcinoma (NPC) cells, we evaluated the levels of miR-143 and formin-like protein 1 (FMNL1) in NPC tissues. The results of qRT-PCR and Western blot analysis showed that the expression of miR-143 was decreased, while FMNL1 was increased in NPC tissues. The expression of miR-143 was significantly elevated in NPC cells compared with that of human nasopharyngeal epithelial cells. The results of MiRcode prediction, dual-luciferase reporter, and Western blot analysis assays indicated that miR-143 negatively regulated the expression of FMNL1 (r² = 0.4365P = 0.0001). Overexperssion of miR-143 or FMNL1 knockdown inhibited cell proliferation, migration, and invasion in NPC cells (P < 0.05). Ectopic expression of FMNL1 undermined the inhibition effect of miR-143 on proliferation, migration, and invasion in NPC cells. The findings of this study revealed that miR-143 functioned as a tumor suppressor and inhibited the NPC progression by targeting FMNL1.     

Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.     

miR-143-3p促进C2C12成肌细胞分化

MicroRNAs (miRNAs) 是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用 real-time PCR 检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p 的模拟物和特异性抑制剂分别处理细胞,采用 real-time PCR 和 Western印迹分别检测成肌因子 MyoG和成肌标志基因 MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达 miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC 的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分化被显著抑制.检测miR-143-3p对MyHC各亚型表达的影响发现,miR-143-3p表达的变化并不直接影响MyHC各亚型的表达.以上结果说明, miR-143-3p在骨骼肌和成肌细胞中均有表达,能够促进C2C12成肌细胞分化,但并不直接调控MyHCs的表达.