Two antisense RNAs-AFAP1-AS1 and MLK7-AS1-promote colorectal cancer progression by sponging miR-149-5p and miR-485-5p.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Antisense RNAs (asRNAs) are closely associated with cancer malignancy. This study aimed to identify the action mechanism of asRNAs in controlling CRC malignancy. Analysis of the RNA sequencing data revealed that AFAP1-AS1 and MLK7-AS1 were upregulated in CRC patients and cell lines. High levels of both asRNAs were associated with poor prognosis in patients with CRC. Both in vitro and in vivo experiments revealed that the knockdown of the two asRNAs decreased the proliferative and metastatic abilities of CRC cells. Mechanistically, AFAP1-AS1 and MLK7-AS1 decreased the levels of miR-149-5p and miR-485-5p by functioning as ceRNAs. Overexpression of miRNAs by introducing miRNA mimics suppressed the expression of SHMT2 and IGFBP5 by directly binding to the 3' UTR of their mRNA. Knockdown of both asRNAs decreased the expression of SHMT2 and IGFBP5, which was reversed by inhibition of both miRNAs by miRNA inhibitors. In vivo pharmacological targeting of both asRNAs by small interfering RNA-loaded nanoparticles showed that knockdown of asRNAs significantly reduced tumor growth and metastasis. Our findings demonstrate that AFAP1-AS1 and MLK7-AS1 promote CRC progression by sponging the tumor-suppressing miRNAs miR-149-5p and miR-485-5p, thus upregulating SHMT2 and IGFBP5.

Two circPPFIA1s negatively regulate liver metastasis of colon cancer via miR-155-5p/CDX1 and HuR/RAB36.

BACKGROUND: Circular RNAs (circRNAs) play a critical role in colorectal cancer (CRC) progression, including metastasis. However, the detailed molecular mechanism is not fully understood. METHODS: Differentially expressed circRNAs between primary KM12C and liver metastatic KM12L4 colon cancer cells were identified by microarray. The expression of circRNAs was measured by semi-quantitative (semi-qPCR) and real time-quantitative PCR (RT-qPCR). Metastatic potential including invasive and migratory abilities, and liver metastasis were examined by transwell assays and intrasplenic injection, respectively. CircPPFIA1-associated microRNA (miRNA) and RNA-binding protein (RBP) were screened by an antisense oligonucleotide (ASO) pulldown experiment. The effects of circPPFIA1 on target gene expression were evaluated by RT-qPCR and western blot analyses. RESULTS: By analyzing circRNA microarray data, we identified two anti-metastatic circRNAs generated from PPFIA1 with different length, which named circPPFIA1-L (long) and -S (short). They were significantly downregulated in liver metastatic KM12L4 cells compared to primary KM12C cells. The knockdown of circPPFIA1s in KM12C enhanced metastatic potential and increased liver metastasis. Conversely, overexpression of circPPFIA1s weakened metastatic potential and inhibited liver metastasis. circPPFIA1s were found to function as sponges of oncogenic miR-155-5p and Hu antigen R (HuR) by an ASO pulldown experiment. circPPFIA1s upregulated tumor-suppressing CDX1 expression and conversely downregulated oncogenic RAB36 by decoying miR-155-5p and by sequestering HuR, respectively. CONCLUSION: Our findings demonstrate that circPPFIA1s inhibit the liver metastasis of CRC via the miR-155-5p/CDX1 and HuR/RAB36 pathways.

LINC00162 regulates cell proliferation and apoptosis by sponging PAQR4-targeting miR-485-5p.

Growing evidence indicates that long intergenic noncoding RNAs play an important role in cancer progression by affecting gene regulation at the transcriptional and posttranscriptional levels. Recent studies have shown that long intergenic noncoding RNA functions as a competitive endogenous RNA, which can interact with and mitigate the function of microRNA. In this study, we investigated the molecular mechanism by which LINC00162 regulates cell proliferation and apoptotic cell death. By analyzing RNA sequencing data, LINC00162 was identified to be a target of heterogeneous nuclear ribonucleoprotein K (hnRNPK). HnRNPK positively regulated LINC00162 expression through p38 mitogen-activated protein kinase. Lowering the level of either hnRNPK or LINC00162 decreased proliferation and colony formation while it increased apoptotic cell death. Small RNA sequencing followed by the antisense oligonucleotide pulldown, revealed that LINC00162 interacts directly with miR-485-5p which exhibited tumor-suppressing effects by suppressing cell proliferation and colony formation, and increasing apoptotic cell death. Through the bioinformatic approaches, progestin and adipoQ receptor 4 (PAQR4) was selected as a common target of LINC00162 and miR-485-5p. miR-485-5p decreased the expression of PAQR4 by directly binding to the 3'-untranslated region of PAQR4 messenger RNA. Knockdown of hnRNPK and LINC00162 increased the level of functional miR-485-5p, indicating that LINC00162 may compete for miR-485-5p, thereby derepressing PAQR4 expression. Overexpression of either hnRNPK or LINC00162, or inhibition of miR-485-5p, protected cells against etoposide-induced apoptotic death. Our findings demonstrate that a regulatory paradigm implicating hnRNPK, LINC00162, miR-485-5p, and PAQR4 plays an important role in cell proliferation and apoptosis, and is a promising target for cancer therapeutics.

hnRNPK-regulated LINC00263 promotes malignant phenotypes through miR-147a/CAPN2.

Malignant characteristics of cancers, represented by rapid cell proliferation and high metastatic potential, are a major cause of high cancer-related mortality. As a multifunctional RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK) is closely associated with cancer progression in various types of cancers. In this study, we sought to identify hnRNPK-regulated long intergenic non-coding RNAs (lincRNAs) that play a critical role in the regulation of cancer malignancy. We found that hnRNPK controlled malignant phenotypes including invasiveness, proliferation, and clonogenicity. RNA sequencing and functional studies revealed that LINC00263, a novel target of hnRNPK, is involved in the oncogenic functions of hnRNPK. Knockdown of LINC00263 mitigated the malignant capabilities. Conversely, increased malignant phenotypes were observed in LINC00263-overexpressing cells. Since LINC00263 was mainly localized in the cytosol and highly enriched in Argonaute 2-immunoprecipitation (Ago2-IP), we hypothesized that LINC00263 acts as a competitive endogenous RNA (ceRNA), and thus sought to identify LINC00263-associated microRNAs. Using small RNA sequencing followed by antisense oligonucleotide pull-down, miR-147a was selected for further study. We found that miR-147a negatively regulates LINC00263 via direct interaction, thus suppressing malignant capabilities. Moreover, knockdown of hnRNPK and LINC00263 upregulated miR-147a, indicating that LINC00263 serves as a ceRNA for miR-147a. By analyzing RNA sequencing data and miRNA target prediction, calpain 2 (CAPN2) was identified as a putative target of miR-147a. Ago2-IP and luciferase reporter assay revealed that miR-147a suppressed CAPN2 expression by directly binding to the 3'UTR of CAPN2 mRNA. In addition, we found that the weakened malignant capabilities following knockdown of hnRNPK or LINC00263 were restored by miR-147a inhibition or CAPN2 overexpression. Furthermore, our findings were validated in various other types of cancer cells including lung cancer, colorectal cancer, neuroblastoma, and melanoma. Collectively, we demonstrate that hnRNPK-regulated LINC00263 plays an important role in cancer malignancy by acting as a miR-147a decoy and thus upregulating CAPN2.

Dual-strand tumor suppressor miR-193b-3p and -5p inhibit malignant phenotypes of lung cancer by suppressing their common targets.

Emerging studies suggest that microRNAs (miRNAs) play multiple roles in cancer malignancy, including proliferation and acquisition of metastatic potential. Differentially expressed miRNAs responsible for the malignancy of lung cancer were searched by miRNA microarray using a previously established brain metastatic lung cancer model. Twenty-five miRNAs were down-regulated in brain metastatic lung cancer cells. Among those, miR-193b-3p and -5p were chosen for further studies. Their function in metastatic potential and proliferation was examined using Transwell invasion, wound healing, and colony forming assays. The underlying mechanism of tumor-suppressor miR-193b-3p and -5p was explored using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Western blot, Argonaute 2-RNA immunoprecipitation (Ago2-RIP), and reporter assays. Both strands of miR-193b were down-regulated in brain metastatic lung cancer cells and in tissues from lung cancer patients. Overexpression of miR-193b-3p and -5p inhibited invasive and migratory activities and diminished clonogenic ability. Conversely, inhibition of miR-193b-3p or -5p increased the metastatic potential and colony forming ability. Cyclin D1 (CCND1), Ajuba LIM Protein (AJUBA), and heart development protein with EGF like domains 1 (HEG1) were identified as common target genes of miR-193b-3p and -5p. A reporter assay and an Ago2-RIP experiment showed that both miRNAs directly bind to the 3' untranslated region (3'UTR) of the target mRNA. Knockdown of target gene reduced the proliferative and metastatic potential of primary and metastatic lung cancer cells. Our results demonstrate miR-193b is a dual-strand tumor suppressor and a novel therapeutic target for lung cancer.