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

Abstract 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.

does not develop. The fluoropyrimidine fluorouracil (5-FU) is a first-line adjuvant chemotherapeutic often given as part of a regimen with other cytotoxic drugs including irinotecan, 4 oxaliplatin 5 and leucovorin. 6 Understanding the mechanisms of chemotherapeutic resistance to particular agents is crucial to developing treatment strategies that improve responses to first-line regimens. 7 In the present study, we explored the role of microRNA 30-5p (miR-30-5p) in CRC progression and determined that it inhibits CRC stemness and chemoresistance by negatively regulating the ubiquitin-specific peptidase 22 (USP22)/Wnt/β-catenin signaling axis.
MiRNAs are short, non-coding, evolutionarily conserved RNAs that post-transcriptionally regulate gene expression, mainly by binding the 3' untranslated regions (3'UTRs) of mRNAs. 8 They were originally recognized for their indispensable roles in the maintenance of crucial cellular process, including cell fate determination, cell growthand stress responses. 9 Increasingly, however, miRNAs are being appreciated for their effects on pathological processes, including oncogenesis and drug resistance. [10][11][12] In fact, miRNA activity can function as a prognostic biomarker that predicts the response of CRC to neoadjuvant chemoradiotherapy. 13 MiR-30 is the microRNA precursor of the mature strands miR-30-5p and miR-30-3p. MiR-30 as well as members of the miR-30-5p family (miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-30d-5pand miR-30e-5p) have been shown to play crucial roles in breast cancer and have the potential to serve as cancer-related biomarkers. 14,15 However, based on miRNA network studies, it is likely that miR-30 and its mature strands act either antior pro-oncogenically in other cancers as well. 16 So far, the majority of research into the miR-30 family has focused on miR-30a, which has been shown to negatively regulate TGF-β1-induced epithelial-tomesenchymal transition (EMT), 17 increase cisplatin sensitivity of gastric cancer cells, 18 and suppress oncogenesis and metastasis in CRC. 19,20 Interestingly, miR-30a has also been shown to serve as both oncogene or onco-suppressor in different cancer types. 21 Apart from miR-30a, miR-30b-5p functions as a tumor suppressor in renal cell carcinoma by inhibiting EMT, cell proliferationand metastasis. 22 Given the potentially conflicting effects of the miR-30 family on oncogenesis and the fact that the miR-30-5p mature strand has not been extensively explored in CRC, we decided to investigate the effects of miR-30-5p family members on CRC stemness and chemoresistance.
In our previous study, 23 we found that ubiquitin-specific peptidase 22 (USP22) promotes CRC stemness and chemoresistance through the Wnt/β-catenin signaling pathway. In the present study, after determining that miR-30-5p expression is reduced in CRC tissues and cell lines, we identified the 3'UTR of USP22 as a binding site of miR-30-5p. USP22 positively regulates a number of oncogenic signaling pathways that cause a variety of lethal cancer phenotypes. 24 In 2017, Li et al. demonstrated that USP22 also promotes EMT, thereby increasing CRC invasion and metastasis. 25 Mechanistically, there is evidence that USP22 promotes cell cycle progression by increasing β-catenin nuclear localization, which is necessary for Wnt pathway activation. 26 The Wnt/β-catenin pathway is an evolutionarily conserved signal transducer responsible for regulating a host of normal physiological processes such as cell proliferation, cell differentiationand cell polarity. 27,28 However, like many other signaling pathways, it also contributes to disease, including numerous cancers. 29 Specifically in CRC, the Wnt/β-catenin pathway promotes cancer stem cell (CSC) maintenance, tumorigenesisand chemoresistance. [30][31][32] In the present study, we have determined that miR-30-5p not only targets USP22 but also attenuates the Wnt/β-catenin pathway, thereby negatively regulating CRC stemness and chemoresistance.

| Reverse transcription PCR (RT-PCR)
Total RNA was isolated from CRC tissues and cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using Superscript First-Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. Expression of miRNA was detected using the TaqMan ® microRNA Reverse Transcription Kit (Applied Biosystem, Foster City, CA, USA), in accordance with the manufacturer's protocol. GAPDH or snRNAU6 were used as an internal control. The primers were used as previously described. 23 PCR conditions were: initial denaturation at 95°C for 2 minutes, followed by 30 cycles of amplification at 95°C for 30 seconds, 55°C for 45 seconds and 72°C for 1 minute, and a final extension at 72°C for 15 minutes. The fold-change was calculated using 2 −ΔΔCt method.

| Luciferase assay
Wild-type and mutant USP22 3'UTR was constructed into the psi-CHECK2 reporter vector. HEK 293T cells were seeded and cultured in 96-well plate overnight. Then the cells were transfected with wild-type USP22 reporter plasmid and miR-30-5p or miR-control.
After 48 hours of transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Relative luciferase activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity.

| MTT assays
Cell viability was assayed using the CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (CellTiter96; Promega) according to the manufacturer's instructions. Briefly, the cells were seeded onto 96well plates and cultured for up to 7 days. At the end of each period, 10 μL MTT solution was added and the cells were incubated for an F I G U R E 1 Expression of miR-30-5p family is decreased in CRC tissues and cell lines. A, MiR-30-5p family (miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-30d-5p and miR-30e-5p) expression in 30 CRC tissues compared with adjacent normal tissues. P < 0.001 compared with normal tissues. B, MiR-30-5p expression in normal CRC tissues and six CRC cell lines (Caco2, HT29, HCT15, HCT116, SW620 and SW480). *P < 0.05 compared with normal CRC tissues additional 4 hours, after which 150 μL dimethyl sulfoxide (DMSO) was added to each well and mixed thoroughly. The optical density of each well was measured with a spectrophotometer (UV5100, Shanghai).

| 5-FU resistant cell generation
5-FU resistant CRC cells were generated by continuous exposure to increasing concentrations of 5-FU (from 5 to 30 μg/mL) with repeated subculture until fully resistant to 5-FU. Cells were first cultured in growing medium with 5 μg/mL 5-FU for 2 months, and the concentration of 5-FU increased 5 μg/mL every 2 months.

| Statistical analysis
Statistical analysis was performed using the GraphPad software (version 5.0). The differences between paired groups were analysed by F I G U R E 2 MiR-30-5p directly targets USP22. A Schematic representation of miR-30-5p binding sites in the USP22 3'UTR (SIP1 3'UTR WT). B, Luciferase activity in 293T cells co-transfected with wild type (WT) or mutant (MUT) USP22 3'UTR luciferase reporter plasmid and miR-30-5p. *P < 0.05 compared with miR-control. C, USP22 protein levels assessed by western blotting in Caco2 cells transiently transfected with miR-control, miR-30-5p or miR-30-5p inhibitor (miR-30-5p-in) Student's t-test; differences between multiple groups were analysed by one-way ANOVA. P values less than 0.05 were considered statistically significant. The data are expressed as mean ± standard error of the mean (SD).

| MiR-30-5p directly targets USP22
To identify the target of miR-30-5p, the TargetScan target prediction algorithm was used to predict putative target genes with the miR-30-5p seed region sequence. The 3'UTR of USP22 was predicted to be a binding site of miR-30-5p (Figure 2A). In our previous report, we found that USP22 plays an important role in CRC development. 23 We therefore investigated whether USP22 is the target of miR-30-5p. A luciferase reporter plasmid of wild type USP22 3'UTR containing the potential miR-30-5p binding sites or mutant was transfected into HEK293T cells along with miR-30-5p or control miRNA. MiR-30-5p significantly decreased the luciferase activity of wild type USP22 3'UTR and failed to affect mutant USP22 3'UTR ( Figure 2B). We then transfected miR-30-5p, miR-30-5p inhibitor or control miRNA into Caco2 cells and found that miR-30-5p significantly reduced USP22 protein expression and miR-30-5p inhibitor promoted its expression ( Figure 2C).

| MiR-30-5p is decreased in CRC stem cells and overexpression of miR-30-5p reduces CRC cell stemness
Previously, we demonstrated that USP22 maintains stemness in CRC stem cells. 23 To evaluate the effects of miR-30-5p on CRC cell stemness, we isolated CD133 + Caco2 and HCT15 cells using microbeads. 23 As shown in Figure 3A, miR-30-5p was significantly downregulated in CD133 + Caco2 stem cells. After inducing CD133 + HCT15 stem cells to differentiate, we observed that they also had decreased miR-30-5p expression ( Figure 3B). To further study the role of miR-30-5p in CRC cell stemness, we transfected miR-30-5p into Caco2 and HCT15 stem cells. RT-PCR confirmed the transfection's effectiveness ( Figure 4A). Using RT-PCR and western blot, we found that miR-30-5p transfection significantly decreased expression of stem cell markers CD133 and Sox2 in Caco2 and HCT15 stem cells ( Figure 4B,C). We then performed sphere formation assays using miR-30-5p overexpression Caco2 cells. As shown F I G U R E 3 MiR-30-5p is decreased in CRC stem cells. A, RT-PCR analysis of miR-30-5p in CD133-and CD133 + Caco2 stem cells. *P < 0.05 compared with CD133-cells. B, HCT15 CD133 + stem cells were induced to differentiate. RT-PCR analysis of miR-30-5p levels was assessed before and after cell differentiation. *P < 0.05 compared with stem cells F I G U R E 4 Overexpression of miR-30-5p reduces CRC cell stemness. A, MiR-30-5p levels assessed by RT-PCR in Caco2 and HCT15 stem cells transiently transfected with miR-control or miR-30-5p. **P < 0.01 compared with miR-control. B, C, MiR-30-5p overexpression cells were subjected to RT-PCR (B) and western blot (C) for CD133 and Sox2 mRNA and protein expression, respectively. *P < 0.05 compared with miRcontrol. D, MiR-30-5p overexpression Caco2 stem cells were subjected to sphere formation assays. The number of spheres was quantified. *P < 0.05 compared with miR-control cells. E, MTT analysis of miR-30-5p overexpression HCT15 stem cells in Figure 4D, miR-30-5p overexpression reduced the number of CD133 + Caco2 cell spheres. Furthermore, miR-30-5p overexpression significantly inhibited HCT15 stem cell proliferation, as determined by MTT assays ( Figure 4E). In addition, we performed above experiments using miR-30-5p inhibitor. Opposite results were observed in CRC cells (Supplemental Figure S1).

| Overexpression of miR-30-5p inhibits chemoresistance in CRC cells
Our previous results demonstrated that USP22 is required for CRC cell chemoresistance. 23 We therefore sought to determine whether miR-30-5p inhibits CRC cell chemoresistance by targeting USP22. We generated 5-FU resistant Caco2 cells, 23 and RT-PCR showed that miR-30-5p expression was decreased in these cells ( Figure 5A). We then increased USP22 expression in Caco2 and HCT15 cells with 5-FU resistance. Sphere formation and cell viability assays revealed that overexpression of USP22 significantly reduced the rate of sphere formation and viability of CRC cells ( Figure 5B,C). We further examined cell chemoresistance by miR-30-5p transfection and found inhibition of miR-30-5p promoted CRC chemoresistance (Supplemental Figure S2).

| MiR-30-5p regulates CRC cells through USP22
To investigate whether miR-30-5p plays its function by targeting USP22 in the CRC cells, a rescue experiment was performed to analyse whether USP22 was involved in the miR-30-5p-mediated malignant phenotypes of CRC cells. Caco2 stem cells were transfected miR-30-5p or miR-control with USP22 overexpression plasmid. Western blot assay was used to confirm USP22 expression ( Figure 7A).
MTT assays showed that co-transfection with the USP22 successfully rescued cell proliferation reduced by miR-30-5p ( Figure 7B). We performed tumor sphere assays and found that the inhibited chemoresistance by miR-30-5p in CRC cells was partially abolished by USP22 overexpression ( Figure 7C). Next, we performed TOPflash luciferase assays. As shown in Figure 7D, inhibited effect of luciferase by miR-30-5p was partially reversed by USP22.

| DISCUSSION
In the present study, we have identified miR-30-5p as a crucial negative regulator of CRC stemness and chemoresistance induced  Stemness and chemoresistance are two major barriers to advancing CRC treatment regimens and represent the most pressing limitation for curing advanced disease. 33 Beyond the individual harmful effects of stemness and chemoresistance, there is increasing evidence that they perpetuate each other. The theory that chemoresistance arises from cancer stem cells (CSCs) and then provides a more favorable milieu for CSCs to propagate is well supported for a variety of cancers, including CRC. [34][35][36] Drug resistance can be acquired, inherent, or can result from a combination of both. 37 Because CSCs are slowcycling, have superior DNA repair abilityand express ATP-binding cassette (ABC) transporters that cause drug efflux, they give rise to inherent drug resistance. 38,39 Acquired resistance likely occurs when CSC subpopulations that survive a course of chemotherapy accumulate mutations that confer a chemoresistant phenotype. 40 In both cases, increases in CSC populations and chemoresistance occur through a variety of deregulated signaling pathways, including Hedgehog/TGF-β, EGFand Wnt/β-catenin. 37,39,41 In CRC specifically, the Wnt signaling pathway plays an outsized role in disease progression, with loss of Wnt pathway negative regulator adenomatous polyposis coli (APC) serving as a hallmark of human CRC, with more than 80% of patients having such mutations. 42,43 Therefore, any curative first-line therapeutic for CRC will likely need to target the Wnt pathway.
To our knowledge, the present study is the first to investigate the role of miR-30-5p as a negative regulator of the Wnt/β-catenin pathway in CRC. In our previous study, 23 we determined that USP22 overexpression significantly enhances CRC stemness and chemoresistance by promoting Wnt/β-catenin activity. At the molecular level, USP22 causes β-catenin localization, which is ultimately necessary for the expression of Wnt target genes. 26,44,45 In normal physiological states, Wnt signaling is activated at the base of intestinal crypts to maintain stem cell populations and intestinal epithelium homeostasis. 46 When the pathway is aberrantly upregulated, however, both nonhypermutated microsatellite stable (MSS) and hypermutated microsatellite instability (MSI) CRCs can arise. 47 Even though aberrant Wnt signaling is extremely prominent in CRC progression, given its importance in normal physiology, future studies that explore the use of exogenous miR-30-5p as a treatment should consider the appropriate amount to deliver without interfering with normal intestinal functioning. In addition to the heightened oncogenic gene expression caused directly by Wnt pathway overactivation, there are stepwise accumulations of gene mutations, such as TGF-β, p53and PI3K, which cause cancer progression. 47,48 Targeting the USP22/Wnt/β-catenin pathway should therefore be considered as one component of a more comprehensive treatment strategy.

| CONCLUSIONS
MiRNAs are increasingly being appreciated for their roles in CRC as prognostic biomarkers and treatment possibilities. Given the significant challenges that aggressive phenotypes, including stemness and chemoresistance, pose in CRC, the use of exogenous miRNAs to target oncogenic signaling pathways in a multifactorial manner is a promising avenue for augmenting current treatment regimens. While future studies are necessary to determine miR-30-5p's range of effects in pathological and normal physiological states, the present study provides compelling evidence that miR-30-5p provides an effective means of targeting the oncogenic USP22/Wnt/β-catenin signaling axis.

ACKNOWLEDG EMENTS
This study was supported by the General Project of Heilongjiang Health Department (No. 2013081).

CONFLI CTS OF INTEREST
All authors declared no conflicts of interest in this work.