Overcoming stemness and chemoresistance in colorectal cancer through miR-195-5p-modulated inhibition of notch signaling.

MiR-195-5p has been shown to have a regulatory role in a variety of cancers. Its influence on colorectal cancer (CRC), however, has never been evaluated. In the present study, we found that miR-195-5p expression was significantly decreased as compared to paired, tumor-adjacent normal colorectal tissues. We demonstrated that miR-195-5p inhibited the stem-like capacity of CRC cells. We established 5-FU-resistant SW620 and HT-29 cell lines and performed a variety of functional assays following exposure to miR-195-5p and anti-miR-195-5p. In 5-FU-resistant cells, expression of miR-195-5p, P-gp and ABCG2 was decreased. MiR-195-5p significantly increased cancer cell apoptosis and decreased tumor sphere formation. In order to determine the mechanism by which miR-195-5p reduced CRC cell stemness and chemoresistance, we first identified potential targets of miR-195-5p. The 3' UTR of Notch signaling proteins Notch2 and RBPJ, which are essential genes in CRC cell stemness and chemoresistance, possessed double putative binding sites of miR-195-5p. qRT-PCR and western blot assays demonstrated significant decreases in Notch2 and RBPJ when CRC cell lines were exposed to miR-195-5p and significant increases when exposed to anti-miR-195-5p. These findings indicate that miR-195-5p has the potential to improve standard therapeutic approaches to CRC.

Jin Y ，Wang M ，Hu H ，Huang Q ，Chen Y ，Wang G ... -  《-》

GDPD5, a target of miR-195-5p, is associated with metastasis and chemoresistance in colorectal cancer.

Successful treatment of colorectal cancer (CRC) is greatly impeded by metastasis and chemoresistance, particularly to 5-fluoruracil (5-Fu), one of the staples of clinical intervention in advanced CRC. The purpose of this study is to determine whether the gene glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) contributes to CRC cell metastasis and 5-Fu resistance. Ultimately, we evaluated the ability of microRNA 195-5p (miR-195-5p) to reduce GDPD5 expression and thus to enhance CRC cell susceptibility to chemotherapy. We obtained human primary CRC and adjacent normal tissues from 15 patients who underwent CRC resection. Quantitative real-time polymerase chain reactions (qRT-PCR), western blot, and immunohistochemistry (IHC) were used to determine GDPD5 expression levels in the human CRC tissues, four CRC cell lines, and two 5-Fu resistant CRC cell lines. To measure the impact of GDPD5 on CRC cell chemoresistance, we silenced GDPD5 using GDPD5 siRNA and measured cell survival and apoptosis using MTT assays and TUNEL staining, respectively. Transwell Matrigel invasion assays were performed to explore whether GDPD5 affects the invasion capabilities of 5-Fu resistant CRC cells. Finally, to assess the effects of miR-195-5p on GDPD5 levels and CRC cell chemoresistance, we generated luciferase reporter plasmids with either the wild-type 3'UTR miR-195-5p potential binding sites or mutant binding sites of GDPD5. We first determined that GDPD5 mRNA and protein are overexpressed in human primary CRC tissues as compared to adjacent normal tissues. GDPD5 overexpression was also evident in four CRC cell lines (Caco-2, HCT8, HCT116 and SW480), as well as in the two 5-Fu resistant CRC cell lines that we generated (HCT116/5-Fu and SW480/5-Fu). Using MTT assays and TUNEL staining of HCT116/5-Fu and SW480/5-Fu cells, we found that GDPD5 silencing sensitizes 5-Fu resistant CRC cells to 5-Fu. Furthermore, GDPD5 silencing in 5-Fu resistant CRC cells reduced the epithelial-to-mesenchymal transition (EMT) and cell invasion, both of which are crucial for CRC metastasis. We then used the bioinformatics algorithm TargetScan to identify the miRNA miR-195-5p, which targets two regions of GDPD5 3'UTR. By generating luciferase reporter plasmids with the 3'UTR miR-195-5p binding sites, we ultimately determined that miR-195-5p increases chemosensitivity and cell apoptosis in 5-Fu resistant CRC cells. This study identifies the gene GDPD5 as an effector of chemoresistance and metastasis in CRC. Furthermore, our results demonstrate that miR-195-5p is a potent suppressor of GDPD5 and that, as such, it significantly increases chemosensitivity and apoptosis in chemoresistant CRC cells. This study thus not only identifies potential prognostic biomarkers of CRC, but it also opens the possibility for incorporating miR-195-5p into current therapeutic regimens to overcome barriers to successful CRC treatment.

Feng C ，Zhang L ，Sun Y ，Li X ，Zhan L ，Lou Y ，Wang Y ，Liu L ，Zhang Y ... -  《-》

miR-139-5p sensitizes colorectal cancer cells to 5-fluorouracil by targeting NOTCH-1.

Multidrug resistance (MDR), a phenomenon that often occurs with drug treatment and is characterized by relapse or attenuation of drug efficacy, is almost unavoidable in colorectal cancer (CRC) patients receiving 5-fluorouracil (5-FU)-based chemotherapy. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression. Our previous study has identified miR-139-5p as a potential tumor suppressor in CRC, but its role in chemoresistance of CRC has not been elucidated. In this study, we demonstrated that miR-139-5p was down-regulated either in CRC tumors receiving chemotherapy or in 5-FU-resistant CRC cell lines (HCT-8/5-FU and HCT-116/5-FU). Ectopic expression of miR-139-5p sensitized CRC cells to 5-FU by increasing 5-FU-induced apoptosis. In addition, miR-139-5p inhibited the expression of the miR-139-5p target gene NOTCH-1 and its downstream molecules MRP-1 and BCL-2, two key MDR-associated genes. Furthermore, silencing NOTCH-1 expression promoted the chemotherapeutic effects of 5-FU, and up-regulation of NOTCH-1 abrogated miR-139-5p-mediated sensitization to 5-FU in LoVo and HCT-116 cells. Taken together, our data indicate a new role of miR-139-5p/NOTCH-1 pathway in the drug resistance of CRC cells to 5-FU, which may be a promising therapeutic target for the anti-MDR treatment of CRC.

Liu H ，Yin Y ，Hu Y ，Feng Y ，Bian Z ，Yao S ，Li M ，You Q ，Huang Z ... -  《-》

MiR-30-5p suppresses cell chemoresistance and stemness in colorectal cancer through USP22/Wnt/β-catenin signaling axis.

Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell-like characteristics (stemness) and chemoresistance. MiR-30-5p has been shown to function as a tumor suppressor by targeting the Wnt/β-catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR-30-5p exerts anti-oncogenic effects in CRC by regulating the USP22/Wnt/β-catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR-30-5p family expression. After identifying the 3'UTR of USP22 as a potential binding site of miR-30-5p, we constructed a luciferase reporter containing the potential miR-30-5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR-30-5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR-30-5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR-30-5p is normally decreased in CD133 + CRC cells, miR-30-5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR-30-5p expression is normally reduced in 5-fluorouracil (5-FU) resistant CRC cells, whereas miR-30-5p overexpression in 5-FU resistant cells reduces sphere formation and cell viability. Inhibition of miR-30-5p reversed the process. Finally, we determined that miR-30-5p attenuates the expression of Wnt/β-catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and β-catenin protein levels in CRC stem cells.

Jiang S ，Miao D ，Wang M ，Lv J ，Wang Y ，Tong J ... -  《-》

The long non-coding RNA HOTAIRM1 suppresses cell progression via sponging endogenous miR-17-5p/ B-cell translocation gene 3 (BTG3) axis in 5-fluorouracil resistant colorectal cancer cells.

5-Fluorouracil (5-FU)-based chemotherapy has always been the first-line treatment of colorectal cancer (CRC). However, the occurrence of clinical 5-FU resistance is a major reason for CRC therapy failure. This study intended to explore the possible role of long non-coding RNA HOTAIRM1 (HOTAIRM1) in the pathogenesis of 5-FU resistant CRC and its underlying mechanism. Our data showed that HOTAIRM1 was downregulated in CRC tissues and cell lines (HCT116 and SW480), and even lower in 5-FU resistant CRC tissues and cell lines (HCT116/5-FU and SW480/5-FU). In vitro, effects of HOTAIRM1 dysregulation in 5-FU resistant CRC cells were investigated and its overexpression could reduce cell viability, invasion, migration, and multi-drug resistance as evidenced by MTT assay, Transwell assay, epithelial-mesenchymal transition (EMT), and western blot analyzing expression of drug-resistant genes MRP1 and MDR1, respectively. Mechanically, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) identified that HOTAIRM1 and B-cell translocation gene 3 (BTG3) were target genes of miR-17-5p. Moreover, miR-17-5p was upregulated and BTG3 was downregulated in HCT116/5-FU and SW480/5-FU cells. Silencing of miR-17-5p showed suppressive role on cell viability, invasion, migration, and multi-drug resistance in HCT116/5-FU and SW480/5-FU cells, which could be abolished by HOTAIRM1 knockdown. Similarly, ectopic expression of miR-17-5p reversed BTG3-mediated inhibition on cell viability, invasion, migration, and multi-drug resistance. In vivo, the tumorigenesis of HCT116/5-FU cells when highly expressed HOTAIRM1 by lentivirus infection was inhibited through downregulating miR-17-5p and upregulating BTG3. In conclusion, HOTAIRM1 might act as a tumor-suppressor in 5-FU resistant CRC cells in vitro and in vivo through downregulating miR-17-5p/BTG3 pathway and inhibiting multi-drug resistance.

Ren T ，Hou J ，Liu C ，Shan F ，Xiong X ，Qin A ，Chen J ，Ren W ... -  《-》