# miR-654-5p Targets HAX-1 to Regulate the Malignancy Behaviors of Colorectal Cancer Cells

Authors: Fuda Huang; Xianjian Wu; Mingwei Wei; Houji Guo; Hua Li; Zesheng Shao; Yi Wu; Jian Pu
Journal: BioMed Research International
 (2020)

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## Abstract

Introduction. The biological roles of microRNA-654-5p (miR-654-5p) in cancers have been previously reported. However, its role in colorectal cancer (CRC) remains largely unknown. The purpose of this work was to investigate the roles and associated mechanisms in CRC. Methods. Quantitative Real-Time PCR (qRT-PCR) was utilized to explore the expression pattern of miR-654-5p in CRC cells. Cell Counting Kit-8 (CCK-8) assay, wound-healing assay, and transwell invasion assay were conducted to investigate the effects of miR-654-5p on CRC cell proliferation, migration, and invasion, respectively. Moreover, the mechanisms behind miR-654-5p regulates CRC progression were investigated. Results. Compared with normal cell line, miR-654-5p expression level was significantly suppressed in CRC cells. After overexpression of miR-654-5p, the malignancy behaviors of CRC cells including cell proliferation, migration, and invasion were remarkably decreased. Subsequently, we found hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) was a putative target for miR-654-5p. Rescue experiments showed overexpression of HAX-1 could partially reversed the effects of miR-654-4p on CRC cell events. Conclusion. miR-654-5p was reduced expression in CRC cells and could regulate CRC progression via targeting HAX-1.


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## Body

## 1. Introduction

About 1.2 million newly diagnosed colorectal cancer (CRC) patients and resulted in 551 thousand deaths of in 2018 at the worldwide range [1]. The incidence of CRC in Asian countries has rapidly increased in past decades [2]. Alterations in lifestyle and eating habits were considered as two major reasons to explain this phenomenon [2]. The pathogenesis of CRC is accompanied with the abnormal expression of multiple tumor suppressor genes and oncogenes [3]. Therefore, a deep investigation of the abnormal expressed genes is useful to understand mechanisms behind CRC development.MicroRNAs (miRNAs), with 18–25 nucleotides in length, are reported to exert biological effects by regulating target gene expression mainly through the 3′-untranslated region (3′-UTR) binding manner [4]. We have now understood that miRNAs have crucial roles in regulating cancer progression [5]. Importantly, it was found that miRNAs could affect almost all CRC cell behaviors including growth, metastasis, and angiogenesis [6]. Moreover, miRNAs were reported to have the potential to be developed as biomarkers for cancer diagnosis or prognosis prediction [6]. Hence, understanding of altered expression of miRNAs in CRC can help to develop miRNA-based therapeutic methods.The role of miR-654-5p in human cancers is confusing at present, in which it can be either the tumor suppressive gene or oncogene [7–9]. A recent report indicated miR-654-5p expression was downregulated in ovarian serous carcinoma, and its overexpression could inhibit tumor progression in vitro and in vivo via regulating pathways including MYC, WNT, and AKT pathways [7]. Besides that, Tan and co-authors showed low expression of miR-654-5p in breast cancer was closely associated with advanced tumor stages and poorer overall survival [8]. Moreover, they showed miR-654-5p functions a tumor suppressive role to regulate breast cancer cell growth and invasion via targeting epithelial stromal interaction 1 [8]. On the contrary, Lu and co-authors showed miR-654-5p was able to promote oral squamous cell carcinoma cell growth, metastasis, and chemoresistance through the targeting Grb-2-related adaptor protein/Ras/MAPK signaling pathway, indicating an oncogenic role of miR-654-5p [9].In this work, our purpose was to explore the biological functions of miR-654-5p in CRC and to deeply investigate the potential mechanisms involved in the miR-654-5p-mediated effects on CRC cells.


## 2. Materials and Methods

### 2.1. Cell Line

CRC cell lines (SW480 and SW620) and the normal colorectal cell (NCM460) were bought at the Cell Collection Center of Chinese Academy of Science (Shanghai, China). Cell incubation condition was as follows: DMEM supplemented with 10% fetal bovine serum (FBS, both bought from Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a 37 °C atmosphere contains 5% CO2.


### 2.2. Cell Transfection

miR-654-5p mimic and control miRNA (miR-con) were purchased from RiboBio (Guangzhou, Guangdong, China). pcDNA3.1 contains the coding sequence of hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1 and pHAX-1) was bought from GenScript (Nanjing, Jiangsu, China). Transfection was performed using Lipofectamine 2000 (cat # 11668-027, Invitrogen) on the basis of provided protocols.


### 2.3. Quantitative Real Time PCR (qRT-PCR)

RNA samples of cultured cells were extracted with Trizol reagent (Invitrogen). Complementary DNA was synthesized with PrimeScript RT Master Mix (cat # RR036A, Takara, Dalian, Liaoning, China) and subjected to qRT-PCR analysis on the ABI7500 system (Applied Biosystems, Foster City, CA, USA) using TB Green® Premix Ex Taq™ II (cat # RR820Q, Takara) with presynthesized primers (Sangon, Shanghai, China). Relative gene expression levels were calculated using the 2−ΔΔCt method with U6 snRNA as internal control.


### 2.4. Western Blot

Protein samples were isolated with RIPA lysis buffer (Invitrogen). After quantified with BCA kit (cat # P0012S, Beyotime, Haimen, Jiangsu, China), sample amount was subjected to 10% SDS-PAGE and then wet-transferred to the PVDF membrane. Following blocked with skim milk, membranes were incubated with rabbit anti-HAX-1 (ab137613) or rabbit anti-GAPDH (ab181602, Abcam, Cambridge, MA, USA) at 4°C overnight and then incubated with goat anti-rabbit secondary antibody (ab6721, Abcam). At length, BeyoECL Plus kit (cat #P0018S, Beyotime) was used to visualize band signals.


### 2.5. Cell Counting Kit-8 (CCK-8) Assay

5000 cells were seeded in the 96-well plate and incubated for 0 h, 24 h, 48 h, and 72 h. Cell proliferation rate was assessed through measuring optical density at 450 nm by adding the CCK-8 reagent (cat # C0037, Beyotime) to each well and further incubated for 4 h.


### 2.6. Wound-Healing Assay

500 cells were seeded in the 6-well plate and incubated to 100% confluence and treated with mitomycin C for 2 h before wound generation using a pipette tip. Cell images at 0 h and 48 h after wound creation were captured under a microscope (Olympus, Tokyo, Japan).


### 2.7. Transwell Invasion Assay

A Matrigel precoated chamber (BD Biosciences, San Jose, CA, USA) was used to analyze cell invasion ability. Cells in a serum-free medium were filled into the upper chamber, while the lower chamber was filled with a medium contains FBS. After incubation for 48 h, noninvasive cells were removed, while invasive cells were stained with crystal violet. At length, invasive cell numbers were counted under a microscope (Olympus).


### 2.8. Bioinformatical Analysis

The target of miR-654-5p was analyzed at TargetScan V_7.2 (http://www.targetscan.org/vert_72/). Among all these predicted targets, HAX-1 was selected for further analyses as it was reported to be overexpressed in CRC tissues [10].


### 2.9. Luciferase Activity Reporter Assay

To construct luciferase constructs, the wild type of HAX-1 contains the binding site for miR-654-5p was amplified from genome and inserted into pMIR-Report (Promega, Madison, WI, USA) to generate wt-HAX-1. Mutant luciferase construct (mt-HAX-1) was built from wt-HAX-1. Cells were cotransfected with miRNAs and luciferase vectors using Lipofectamine 2000. After 48 h transfection, relative luciferase activity was measured using the dual luciferase reporter assay system (Promega).


### 2.10. In Vivo Tumorigenesis

The animal experiment procedure was monitored by the Committee on Animal Care of Affiliated Hospital of Youjiang Medical University for Nationalities. CRC cells with miRNA transfection were injected into four-week-old male nude mice via tail vein. Tumor size was analyzed every 7 days and counted 4 times. Tumor volume was measured using the following formula: (length × width2)/2.


### 2.11. Statistical Analysis

Data obtained from at least three independent experiments were analyzed at SPSS 17.0 (SPSS Inc, Chicago, IL, USA) and then presented in the manner of mean ± SD. Student’st-test (for two groups) or one-way ANOVA (for three or above groups) were used for difference analysis. A P value less than 0.05 was believed to indicate statistically significant difference.


## 2.1. Cell Line

CRC cell lines (SW480 and SW620) and the normal colorectal cell (NCM460) were bought at the Cell Collection Center of Chinese Academy of Science (Shanghai, China). Cell incubation condition was as follows: DMEM supplemented with 10% fetal bovine serum (FBS, both bought from Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a 37 °C atmosphere contains 5% CO2.


## 2.2. Cell Transfection

miR-654-5p mimic and control miRNA (miR-con) were purchased from RiboBio (Guangzhou, Guangdong, China). pcDNA3.1 contains the coding sequence of hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1 and pHAX-1) was bought from GenScript (Nanjing, Jiangsu, China). Transfection was performed using Lipofectamine 2000 (cat # 11668-027, Invitrogen) on the basis of provided protocols.


## 2.3. Quantitative Real Time PCR (qRT-PCR)

RNA samples of cultured cells were extracted with Trizol reagent (Invitrogen). Complementary DNA was synthesized with PrimeScript RT Master Mix (cat # RR036A, Takara, Dalian, Liaoning, China) and subjected to qRT-PCR analysis on the ABI7500 system (Applied Biosystems, Foster City, CA, USA) using TB Green® Premix Ex Taq™ II (cat # RR820Q, Takara) with presynthesized primers (Sangon, Shanghai, China). Relative gene expression levels were calculated using the 2−ΔΔCt method with U6 snRNA as internal control.


## 2.4. Western Blot

Protein samples were isolated with RIPA lysis buffer (Invitrogen). After quantified with BCA kit (cat # P0012S, Beyotime, Haimen, Jiangsu, China), sample amount was subjected to 10% SDS-PAGE and then wet-transferred to the PVDF membrane. Following blocked with skim milk, membranes were incubated with rabbit anti-HAX-1 (ab137613) or rabbit anti-GAPDH (ab181602, Abcam, Cambridge, MA, USA) at 4°C overnight and then incubated with goat anti-rabbit secondary antibody (ab6721, Abcam). At length, BeyoECL Plus kit (cat #P0018S, Beyotime) was used to visualize band signals.


## 2.5. Cell Counting Kit-8 (CCK-8) Assay

5000 cells were seeded in the 96-well plate and incubated for 0 h, 24 h, 48 h, and 72 h. Cell proliferation rate was assessed through measuring optical density at 450 nm by adding the CCK-8 reagent (cat # C0037, Beyotime) to each well and further incubated for 4 h.


## 2.6. Wound-Healing Assay

500 cells were seeded in the 6-well plate and incubated to 100% confluence and treated with mitomycin C for 2 h before wound generation using a pipette tip. Cell images at 0 h and 48 h after wound creation were captured under a microscope (Olympus, Tokyo, Japan).


## 2.7. Transwell Invasion Assay

A Matrigel precoated chamber (BD Biosciences, San Jose, CA, USA) was used to analyze cell invasion ability. Cells in a serum-free medium were filled into the upper chamber, while the lower chamber was filled with a medium contains FBS. After incubation for 48 h, noninvasive cells were removed, while invasive cells were stained with crystal violet. At length, invasive cell numbers were counted under a microscope (Olympus).


## 2.8. Bioinformatical Analysis

The target of miR-654-5p was analyzed at TargetScan V_7.2 (http://www.targetscan.org/vert_72/). Among all these predicted targets, HAX-1 was selected for further analyses as it was reported to be overexpressed in CRC tissues [10].


## 2.9. Luciferase Activity Reporter Assay

To construct luciferase constructs, the wild type of HAX-1 contains the binding site for miR-654-5p was amplified from genome and inserted into pMIR-Report (Promega, Madison, WI, USA) to generate wt-HAX-1. Mutant luciferase construct (mt-HAX-1) was built from wt-HAX-1. Cells were cotransfected with miRNAs and luciferase vectors using Lipofectamine 2000. After 48 h transfection, relative luciferase activity was measured using the dual luciferase reporter assay system (Promega).


## 2.10. In Vivo Tumorigenesis

The animal experiment procedure was monitored by the Committee on Animal Care of Affiliated Hospital of Youjiang Medical University for Nationalities. CRC cells with miRNA transfection were injected into four-week-old male nude mice via tail vein. Tumor size was analyzed every 7 days and counted 4 times. Tumor volume was measured using the following formula: (length × width2)/2.


## 2.11. Statistical Analysis

Data obtained from at least three independent experiments were analyzed at SPSS 17.0 (SPSS Inc, Chicago, IL, USA) and then presented in the manner of mean ± SD. Student’st-test (for two groups) or one-way ANOVA (for three or above groups) were used for difference analysis. A P value less than 0.05 was believed to indicate statistically significant difference.


## 3. Results

### 3.1. miR-654-5p Expression Was Downregulated in CRC Cells

qRT-PCR was conducted to measure miR-654-5p expression level in CRC cells and normal cells. We found the levels of miR-654-5p were lower in CRC cells compared with normal cells (Figure1(a)).Figure 1
Ectopic miR-654-5p expression inhibits CRC cell proliferation, migration, and invasion. (a) miR-654-5p in CRC cells (SW480 and SW620) and one normal colorectal cell NCM460, (b) miR-654-5p expression, (c) cell proliferation, (d) cell migration, and (e) cell invasion were examined in CRC cells carrying miR-654-5p mimic or miR-con. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer; miR-con: control miRNA.
(a)
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### 3.2. Overexpression of miR-654-5p Suppresses CRC Cell Growth, Migration, and Invasion

Gain-of-function experiments were conducted to investigate the roles of miR-654-5p in CRC. We showed introduction of miR-654-5p mimic increased miR-654-5p levels in CRC cells (Figure1(b)). Results of the CCK-8 assay indicated miR-654-5p mimic transfection decreased cell proliferation rate compared with miR-con (Figure 1(c)). The wound-healing assay showed cell migration ability was significantly inhibited by miR-654-5p mimic (Figure 1(d)). The transwell invasion assay confirmed the tumor suppressive effect of miR-654-5p on CRC cell invasion (Figure 1(e)).


### 3.3. miR-654-5p Directly Targets HAX-1 in CRC

The bioinformatic tool showed HAX-1 was a putative target for miR-654-5p (Figure2(a)). The luciferase activity reporter assay was performed to validate the connection of miR-654-5p and HAX-1, and the results showed miR-654-5p mimic introduction decreased luciferase activity in cells transfected with wt-HAX-1 (Figure 2(b)). Moreover, we showed HAX-1 expression level in CRC cells was decreased by miR-654-5p mimic (Figure 2(c)).Figure 2
miR-654-5p targets HAX-1 in CRC. (a) Putative binding region between miR-654-5p and the 3′-UTR of HAX-1. (b) Relative luciferase activity was measured in CRC cells carrying luciferase reporter constructs or synthetic miRNAs. (c) HAX-1 expression in CRC cells carrying miR-654-5p mimic or miR-con. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer; miR-con: control miRNA; wt: wild-type; mt: mutant; HAX-1: hematopoietic cell-specific protein 1-associated protein X-1.
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### 3.4. HAX-1 Was Involved in the miR-654-5p Mediated Cell Growth, Migration, and Invasion of CRC Cells

Rescue experiments were utilized to confirm whether HAX-1 was a functional target of miR-654-5p in CRC. qRT-PCR showed pHAX-1 transfection increased HAX-1 expression in CRC cells and reversed the inhibitory effects of miR-654-5p mimic on HAX-1 (Figure3(a)). Functional assays showed HAX-1 overexpression exhibited tumor promoter roles on CRC cell behaviors (Figures 3(b)–3(d)). Moreover, we found overexpression of HAX-1 markedly reversed the effects of miR-654-5p on CRC cell growth, migration, and invasion (Figures 3(b)–3(d)).Figure 3
HAX-1 attenuates the inhibitory effects of miR-654-5p on CRC cell proliferation, migration, and invasion. (a) HAX-1 expression, (b) cell proliferation, (c) cell migration, and (d) cell invasion were examined in CRC cells carrying pHAX-1, pcDNA3.1, or pHAX-1 and miR-654-5p mimic. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer; HAX-1: hematopoietic cell-specific protein 1-associated protein X-1.
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### 3.5. miR-654-5p Inhibits CRC Tumor Growth In Vivo

At length, we explored whether miR-654-5p could affect tumor growthin vivo. We found miR-654-5p overexpression significantly decreased tumor volumes (Figure 4(a)) and tumor weights (Figure 4(b)). Moreover, miR-654-5p expression was also found elevated in the miR-654-5p mimic group (Figure 4(c)). These results suggested miR-654-5p overexpression inhibits CRC growth in vivo.Figure 4
miR-654-5p overexpression suppresses CRC growth in vivo. (a) Tumor volume was measured every 7 days. (b) Tumor weights were measured after 28 days. (c) Expression of miR-654-5p was analyzed in tumor tissues. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer.
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## 3.1. miR-654-5p Expression Was Downregulated in CRC Cells

qRT-PCR was conducted to measure miR-654-5p expression level in CRC cells and normal cells. We found the levels of miR-654-5p were lower in CRC cells compared with normal cells (Figure1(a)).Figure 1
Ectopic miR-654-5p expression inhibits CRC cell proliferation, migration, and invasion. (a) miR-654-5p in CRC cells (SW480 and SW620) and one normal colorectal cell NCM460, (b) miR-654-5p expression, (c) cell proliferation, (d) cell migration, and (e) cell invasion were examined in CRC cells carrying miR-654-5p mimic or miR-con. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer; miR-con: control miRNA.
(a)
(b)
(c)
(d)
(e)


## 3.2. Overexpression of miR-654-5p Suppresses CRC Cell Growth, Migration, and Invasion

Gain-of-function experiments were conducted to investigate the roles of miR-654-5p in CRC. We showed introduction of miR-654-5p mimic increased miR-654-5p levels in CRC cells (Figure1(b)). Results of the CCK-8 assay indicated miR-654-5p mimic transfection decreased cell proliferation rate compared with miR-con (Figure 1(c)). The wound-healing assay showed cell migration ability was significantly inhibited by miR-654-5p mimic (Figure 1(d)). The transwell invasion assay confirmed the tumor suppressive effect of miR-654-5p on CRC cell invasion (Figure 1(e)).


## 3.3. miR-654-5p Directly Targets HAX-1 in CRC

The bioinformatic tool showed HAX-1 was a putative target for miR-654-5p (Figure2(a)). The luciferase activity reporter assay was performed to validate the connection of miR-654-5p and HAX-1, and the results showed miR-654-5p mimic introduction decreased luciferase activity in cells transfected with wt-HAX-1 (Figure 2(b)). Moreover, we showed HAX-1 expression level in CRC cells was decreased by miR-654-5p mimic (Figure 2(c)).Figure 2
miR-654-5p targets HAX-1 in CRC. (a) Putative binding region between miR-654-5p and the 3′-UTR of HAX-1. (b) Relative luciferase activity was measured in CRC cells carrying luciferase reporter constructs or synthetic miRNAs. (c) HAX-1 expression in CRC cells carrying miR-654-5p mimic or miR-con. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer; miR-con: control miRNA; wt: wild-type; mt: mutant; HAX-1: hematopoietic cell-specific protein 1-associated protein X-1.
(a)
(b)
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## 3.4. HAX-1 Was Involved in the miR-654-5p Mediated Cell Growth, Migration, and Invasion of CRC Cells

Rescue experiments were utilized to confirm whether HAX-1 was a functional target of miR-654-5p in CRC. qRT-PCR showed pHAX-1 transfection increased HAX-1 expression in CRC cells and reversed the inhibitory effects of miR-654-5p mimic on HAX-1 (Figure3(a)). Functional assays showed HAX-1 overexpression exhibited tumor promoter roles on CRC cell behaviors (Figures 3(b)–3(d)). Moreover, we found overexpression of HAX-1 markedly reversed the effects of miR-654-5p on CRC cell growth, migration, and invasion (Figures 3(b)–3(d)).Figure 3
HAX-1 attenuates the inhibitory effects of miR-654-5p on CRC cell proliferation, migration, and invasion. (a) HAX-1 expression, (b) cell proliferation, (c) cell migration, and (d) cell invasion were examined in CRC cells carrying pHAX-1, pcDNA3.1, or pHAX-1 and miR-654-5p mimic. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer; HAX-1: hematopoietic cell-specific protein 1-associated protein X-1.
(a)
(b)
(c)
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## 3.5. miR-654-5p Inhibits CRC Tumor Growth In Vivo

At length, we explored whether miR-654-5p could affect tumor growthin vivo. We found miR-654-5p overexpression significantly decreased tumor volumes (Figure 4(a)) and tumor weights (Figure 4(b)). Moreover, miR-654-5p expression was also found elevated in the miR-654-5p mimic group (Figure 4(c)). These results suggested miR-654-5p overexpression inhibits CRC growth in vivo.Figure 4
miR-654-5p overexpression suppresses CRC growth in vivo. (a) Tumor volume was measured every 7 days. (b) Tumor weights were measured after 28 days. (c) Expression of miR-654-5p was analyzed in tumor tissues. miR-654-5p: microRNA-654-5p; CRC: colorectal cancer.
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## 4. Discussion

miRNAs were reported to regulate CRC development and could be used as diagnosis or prognosis biomarkers [11, 12]. For instance, miR-6716-5p expression was revealed to be upregulated in CRC tumor tissues and correlated with poorer overall survival [11]. Moreover, they found miR-6716-5p could regulate CRC cell migration and invasion via regulating N-acetyltransferase 10 (NAT10), suggesting the miR-6716-5p/NAT10 axis might be potential treatment targets for CRC [11]. Another work showed miR-125a overexpression hinders CRC tumor growth in vitro and in vivo by targeting Smad ubiquitin regulatory factor 1 [12].In this work, we showed miR-654-5p expression level was downregulated in CRC cells compared with normal cells. Force miR-654-5p expression significantly suppressed CRC cell proliferation, migration, and invasionin vitro. Abnormal status of cell behaviors and alternations in gene expression including oncogenes and tumor suppressor genes are considered as hallmarks for cancer [13]. Importantly, we showed overexpression of miR-654-5p inhibits tumor growth in vivo. Hence, our results suggested the antitumor effects of miR-654-5p in CRC, which is similar with its role in cancers including ovarian cancer and breast cancer [7, 8].Mounting studies indicated miRNA exerts its biological functions via regulating downstream target genes [14]. The bioinformatic analysis tool showed HAX-1 was a putative target for miR-654-5p. HAX-1, located at chromosome 1 (1q21.3), is reported to be highly expressed in tumors [10, 15, 16]. For example, HAX-1 was revealed to be overexpressed in hypopharyngeal squamous cell carcinoma and could promote cancer growth and migration [15]. Moreover, HAX-1 was found to be targeted by miR-100 to regulate the sensitivity of breast cancer cells to cisplatin [16]. In this work, we showed miR-654-5p could directly bind with the 3′-UTR of HAX-1. Rescue experiments further confirmed that overexpression of HAX-1 could partially abolish the effects of miR-654-5p on CRC cell behaviors.


## 5. Conclusion

Taken together, our results provided evidence that miR-654-5p functions as a tumor suppressive miRNA in CRC via regulating HAX-1. The miR-654-5p/HAX-1 axis identified in this work will advance our understanding regarding the mechanisms behind CRC pathogenesis.



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*Source: 4914707-2020-02-10.xml*