circ_0052184 promote colorectal cancer progression via targeing miR-604/HOXA9 axis

doi:10.21203/rs.3.rs-1607376/v1

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circ_0052184 promote colorectal cancer progression via targeing miR-604/HOXA9 axis

https://doi.org/10.21203/rs.3.rs-1607376/v1

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Background:

Mortality rate colorectal cancer (CRC) ranks second. In a variety of diseases CircRNAs are abnormal expression, and their dysregulation is associated with cancer progression. Recent studies have shown that the malignant progression of colorectal cancer cannot be separated from the abnormal expression of circRNAs

Methods:

First, the circ_0052184 expression in clinical tissue and cell samples was analyzed by database analysis and qRT-PCR. qRT-PCR also assessed Construction of circ_0052184-silenced colorectal cancer cells. Furthermore, the proliferation ability of cells was detected by MTT method. Cell migration ability was tested by wound healing assay. Cell migration and invasion ability were detected by transwell method.

Results:

In cell lines and tissues of colorectal cancer circ_0052184 is significantly overexpressed. Proliferation, migration and invasion of colorectal cancer cell are inhibited by circ_0052184 silencing. The existence of the circ_0052184/miR-604/HOXA9 regulatory network was verified. Moreover, the existence of this regulatory network has an impact on the occurrence and development of colorectal cancer.

Conclusions:

Our study demonstrated the molecular mechanism by which circ_0052184 regulates the miR-604/HOXA9 axis for the first time. This mechanism promotes the malignant progression of colorectal cancer cells.

circ_0052184

colorectal cancer

cancer progression

miR-604

HOXA9

Colorectal cancer (CRC) has the second highest mortality rate in the world and the third most common cancer worldwide[1]. According to the National Comprehensive Cancer Network (NCCN) report: As of December 1, 2021, there were 104,270 new cases of colon cancer worldwide and 52,980 annual deaths [2]. People's diet, weight, lifestyle and other factors often affect the incidence of CRC. However, the specific pathogenesis of CRC is complex and the prevention and treatment forms are severe. In recent years, the clinical treatment of colorectal cancer has made great progress. However, the survival rate of patients with advanced colorectal cancer within 5 years is still very low. And the most critical cause of death in CRC patients is the development and metastasis of CRC cells [3]. It is indispensable to further explore the mechanism of CRC progression to identify new targets.

circular RNA (circRNA) is endogenous non-coding RNAs (ncRNAs) [4]. Covalently closed loops of circRNAs are formed by back-splicing, making this structure highly stable and conserved [5]. CircRNAs can act as miRNA sponges to regulate the expression of key genes, or function as transcriptional regulators [6]. It is worth noting that these important roles of circRNAs exist in physiological processes, and tumor malignant progression has also been shown to be associated with the dysregulation of circRNAs [7].

miRNA can be completely or incompletely paired with the 3' end of target mRNA, degrade the target mRNA or inhibit its translation into protein [8]. Many experiments have shown that the malignant development of human diseases is often associated with miRNAs. Many miRNAs related to colorectal cancer have been discovered. For example, Yu et al found that colorectal cancer migration and invasion are regulated by miR-17-5p [9]. Ding et al found that apoptosis and cell cycle in colorectal cancer were regulated by miR-515-5p targeted by MAD2L1 [10]. Kang et al. demonstrated that colorectal cancer development and patient prognosis are affected by low expression of miR-138 [11]. These studies all suggest the impact of miRNAs on colorectal cancer.

Many studies have found that there are many mechanisms by which circRNAs exert their functions. Among them, circRNAs are the most common as competing endogenous RNAs. At this time, the circRNAs sponge absorbs miRNAs, thereby regulating the pathways where the downstream target genes of miRNAs are located. [12]. Interestingly, we found that circ_0052184 had the most elevated expression in colorectal cancer through bioinformatics analysis, and circ_0052184 may be a molecular sponge of miR-604. We speculate that circ_0052184 may regulate the malignant development of colorectal cancer through the miR-604/HOXA9 axis. This paper aims to verify the establishment of this hypothesis. In our study, circ_0052184 was found to promoted the development of colorectal cancer through the miR-604/HOXA9 axis.

Clinical sample collection

The colorectal cancer patients in our hospital were recruited as the research subjects, and the case detection results of the research subjects were all colorectal cancer. Cancer tissue and paracancerous tissue were collected during surgery. After sampling, the samples were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for subsequent operations. All subjects in the study had not received other radiotherapy and chemotherapy before the experiment. This study was approved by the ethics committee of our hospital, and all participating patients were signed the informed consent. All experimental manipulations involved were performed in compliance with the Declaration of Helsinki.

Cell culture

All cell lines were purchased from BFB Cell Culture Bank (Shanghai, China). The cell lines involved can be divided into two types: 1. NCM460 (normal colorectal cell line). 2. LoVo, HT29, HTC116 and SW480 (colorectal cancer cell line). These cell lines were cultured in DMEM (Waltham, MA, USA). The medium contained fetal bovine serum (10%), 100 U/mL penicillin and 100 µg/mL streptomycin (Thermo fisher Scientific, MA, USA). Constant temperature incubators were used for the cultivation of cell lines. Conditions were as follows: 37°C 5% CO2. Subsequent experiments were mainly performed on cells in logarithmic growth phase. Medium exchange rate: once every 3 days.

Bioinformatic analysis

The online bioinformatics analysis website circinteractome was used to analyze the binding sites between circ_0052184 and miRNA. The possible binding sites between miR-604 and HOXA9 was used to analyze the bioinformatics analysis website Starbase (https://starbase.sysu.edu.cn/).

qRT-PCR

TRIzol (Invitrogen, California, US) was used to extract RNA from CRC tissues and cells. Then SuperScript IV reverse transcription kit (Thermo fisher scientific) was used to synthesize cDNA. LabScript Two-Steps SYBR Green qRT-PCR Kit is used for qRT-PCR detection. Primer list is detailed in Table 2.

GAPDH or U6 were used as controls, and the relative expression levels were normalized.

Reaction conditions: pre-denaturation at 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min for 30 s.

Relative expression levels of related genes were calculated by 2^-△△CT method.

Transwell assay

The successfully transfected Lovo and HT29 cell lines were collected. These cells were suspended in serum-free medium. Cell suspension was added to the upper chamber of the Transwell. The lower chamber of Transwell is medium supplemented with 20% fetal bovine serum. Remove the cell compartment from the incubator after 24 hours of culture.. 4% paraformaldehyde (Beyotime Biotechnology, Shanghai, China) fixed the migrated cells. After fixation, cells were stained with 0.1% crystal violet (Beyotime Biotechnology), rinsed, and photographed for counting after drying. In the invasion experiment, a layer of Millipore matrigel (MA, USA) was coated on the surface of the Transwell chamber. Other steps were equally the migration experiment steps.

MTT assay

The transfected cells in logarithmic growth phase were taken, and the concentration of the cell suspension was adjusted to about 3×10 4 cells/mL. Add 100 μL of the cell suspension to a 96-well plate, incubate at 37°C for about 24 hours until the cells grow to a monolayer, aspirate the supernatant, wash with PBS, add 20 μL MTT solution (5 mg/mL) and 80 μL serum-free medium , placed in a 37°C cell incubator for 4h. Add 100ul of Formanzan lysis solution to each well, and continue to incubate for about 4h in the cell culture incubator. The absorbance at 570 nm was read in a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Dual-luciferase reporter assay

The wild-type and mutant circ_0052184 and HOXA9 luciferase reporter vectors were constructed. After the colorectal cancer cell lines LoVo and HT29 were seeded in 96-well plates for a period of time, the Wt or Mut luciferase reporter plasmids and miR-604 mimic or NC were co-transfected into LoVo and HT29 cells by Lipofectamine TM 2000 (Invitrogen). Cells were cultured after 48h. The cellular luciferase activity of the different groups was detected by a Renilla-Firefly Luciferase Dual Assay Kit (MCE, Shanghai, China).

RIP

Targeting relationships between genes were examined using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit(Millipore, Massachusetts, US). After lysis of transfected cells with RIP lysis buffer, Incubate anti-ago2 antibody or negative control IgG-conjugated magnetic beads with RIP-added buffer. Next, added proteinase K to the mixture. and incubated with shaking. qRT-PCR verifies targeting relationships between genes in purified RNA.

Wound-healing assay

The successfully transfected colorectal cancer cell lines were diluted and placed in 6-well plates for 24 hours. And then streaked with a sterilized pipette tip. Photographs were taken at 24h and 48h to record the wound closure. Relative cell migration ratio = (0h scratch area-48h scratch area)/0h scratch area.

Western blotting assay

RIPA lysis buffer (Beyotime, Shanghai, China) was used to extract total protein from tissues and cell lines. SDS-PAGE and Polyvinylidene fluoride (PVDF) separate target proteins. Finally, the ECL kit (Beyotime) was used to detect total protein.

Statistical analysis

Graphpad Prism 8.0 (CA, USA) was used to analyze all data involved in this experiment. In order to better demonstrate the authenticity of the data, all experiments were performed at least three times. Mean ± standard deviation (X ± SD) represents measurement data. Two groups were compared by t-test or one-way ANOVA. Pearson correlation analysis was performed to analyze the correlation between genes. The difference was statistically significant with P<0.05.

circ_0052184 expression in CRC tissues and cells

First, we elucidate the important role of circ_0052184 in the occurrence and development of CRC cells. The expression of circ_0052184 in cancer tissues and corresponding adjacent tissues of 42 CRC patients was analyzed by qRT-PCR. The patient details are shown in Table 1. The expression of circ_0052184 was increased in colorectal cancer tissues (Fig. 1A). circ_0052184 expression in CRC cell lines was higher than that in human normal colorectal cell lines (Fig. 1B). We selected colorectal cancer cell lines (Lovo and HT29) with significantly increased expression of circ_0052184 for subsequent cell experiments.

Downregulation of circ_0052184 affects colorectal cancer cell proliferation, migration and invasion

circ_0052184 was silenced in Lovo and HT29, respectively. Then qRT-PCR detection of transfection efficiency showed that the expression of circ_0052184 was significantly reduced in the transfected cell line, which could be used for subsequent experiments (Fig. 2A). MTT experiments showed that Lovo and HT29 cell lines proliferation ability were inhibited after circ_0052184 was silenced (Fig. 2B). Wound healing experiments and transwell experiments showed that after silencing circ_0052184, the migration ability of Lovo and HT29 cell lines was reduced (Fig. 2C, 2D). In addition, the detection of invasion by transwell showed that the invasion ability of Lovo and HT29 cell lines was inhibited after silencing of circ_0052184 (Fig. 2E). Therefore, the above experiments all indicated that silencing circ_0052184 inhibited the malignant development of colorectal cancer cells.

circ_0052184 targets miR-604

To further explore the mechanism of circ_0052184 regulating the development of colorectal cancer cells. The target genes of circ_0052184 were predicted through the online bioinformatics analysis website circinteractome. The results showed that there was a binding site between circ_0052184 and miR-604 (Fig. 3A). The dual-luciferase reporter gene assay and RIP assay further confirmed the targeting relationship between circ_0052184 and miR-604.. In the dual-luciferase reporter assay, overexpression of miR-604 significantly reduced the luciferase activity of circ_0052184, but had no effect on the luciferase activity of circ_0052184-MUT (Fig. 3B). Meanwhile, RIP experiment showed that circ_0052184 and miR-604 were highly enriched in the RNA-Ago2 antibody complex (Fig. 3C). The results showed that the expression of miR-604 was lower in colorectal cancer tissues compared with paracancerous tissues (Fig. 3D). The expression of circ_0052184 in colorectal cancer cell lines was lower than that in human normal colorectal cancer cell lines (Fig. 3E). When the expression of circ_0052184 in Lovo and HT29 cell lines was inhibited, the detection results of qRT-PCR showed that the expression of miR-604 was increased (Fig. 3F). Pearson correlation analysis clarified that circ_0052184 expressions and miR-604 expressions were negatively correlated (Fig. 3G). In conclusion, circ_0052184 can directly bind to miR-604 to inhibit the expression of miR-604, and miR-604 abnormal expression may be related to colorectal cancer.

circ_0052184 regulates CRC cell development by targeting miR-604

A series of in vitro experiments were conducted to explore the regulatory effect of circ_0052184 on miR-604 on CRC cells. The results of MTT experiments showed that inhibiting the expression of miR-604 promoted the proliferation of colorectal cancer cells, while inhibiting the expression of circ_0052184 reversed the proliferation of colorectal cancer cells(Fig. 4A). Next, the results of Wound-healing assay and transwell assay showed that inhibiting the expression of miR-604 promoted the migration of CRC cells, while inhibiting the expression of circ_0052184 reversed the migration of CRC cells (Figure 4B, C). Finally, transwell assays showed that when miR-604 expression was inhibited, it promoted CRC cell invasion. However, when inhibiting the expression of circ_0052184 reversed the CRC cells invasion (Fig. 4D). From the above results, it can be concluded that abnormal expression of miR-604 can promote CRC malignant progression, and inhibiting the expression of circ_0052184 can reverse this process. In short, the circ_0052184/miR-604 axis regulates CRC cells development.

circ_0052184 regulates HOXA9 expression by targeting miR-604

Next, we further elucidate the downstream molecular mechanism of circ_0052184/miR-604. We predicted the downstream target genes of miR-604 through the online bioinformatics analysis website Starbase (https://starbase.sysu.edu.cn/). The prediction results showed that, There was 8 base pair complementation between miR-604 and HOXA9, and we deduced that HOXA9 might be a potential downstream target gene of miR-604 (Fig. 5A). We validated our inferences by constructing a dual-luciferase reporter assay system co-transfected with HOXA9-MT or HOXA9-WT reporter systems with miR-604 mimics and negative controls, as shown in the Dual-Luciferase assay in Figure 5B, the upregulation of miR-604 inhibited HOXA9-WT luciferase activity, but not HOXA9-MT activity. Next, qRT-PCR detected HOXA9 expression in CRC tissues and cells, and the results showed that the expression of HOXA9 was increased in CRC tissues and cells (Fig. 5C, D). qRT-PCR and western blotting analyzed HOXA9 mRNA and protein expressions after miR-604 down-regulation. And the results showed that when miR-604 was down-regulated, the expression of HOXA9 increased (Fig. 5E, F). At the same time, Pearson correlation showed that HOXA9 expression was inversely correlated with that of miR-604 (Fig. 5G).. In conclusion, there is a circ_0052184/miR-604/HOXA9 axis in colorectal cancer, which can regulate the occurrence and development of colorectal cancer (Fig. 6).

Colorectal cancer is a common digestive tract cancer that menaces people's health [13]. At present, the morbidity and mortality of CRC patients are on the rise, and 5-year survival rate is low for advanced CRC patients [14]. Therefore, exploring the molecular mechanism of CRC progression and clarifying the regulation of the process of advanced CRC progression will help to develop new clinical treatment strategies for advanced CRC patients. Dysregulation of circRNAs is related to CRC progression of in existing studies. Various CRC tumor markers belong to circRNAs, and circRNAs can regulate the development of CRC [15]. Our article is the first to explorethe specific ways in which CRC progression is facilitated by circ_0052184.
Non-coding circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) expressed in a variety of diseases and widely present in eukaryotic cells. CircRNAs form covalently closed loops by back-splicing. This structure makes them highly stable and conserved [16]. The role of circRNAs in colorectal cancer has received increasing attention. A variety of circRNAs have been confirmed to be related to CRC development. Numerous studies have investigated the role of circRNAs in colorectal cancer. circ_0014130 can act as a molecular sponge for miR-197-3p and participate in the drug sensitivity and development of colorectal cancer by regulating the miR-197-3p/PFKFB3 axis [17]. Downregulation of Circ_0011385 in colorectal cancer can inhibit cell proliferation, and can promote apoptosis by regulating the miR-330-3p/MYO6 axis [18]. Circ_0001666 can inhibit the progression of colorectal cancer by regulating the miR-576-5p/PCDH10 axis [19]. However, the affect of circ_0052184 on CRC is unknown. In our work, we verified that circ_0052184 is upregulated in colorectal cancer tissues and cells. We also determined the effect of circ_0052184 on colorectal cancer: silencing circ_0052184 in CRC cells inhibited the proliferation and migration and invasion of CRC cells in vitro. This revealed that the expression of circ_0052184 played a crucial key in regulating the normal occurrence of CRC cells, but its specific underlying mechanism still needs to be further explored.
The knockdown or overexpression of circRNAs has an impact on the occurrence and development of cancer cells. There are various mechanisms for the regulation of circRNAs on tumors, such as: miRNA sponges [20], interaction with proteins [21], etc. One of the most common regulatory mechanisms is that circRNAs, which are endogenous competitive RNAs [22]. Therefore, the role of circ_0052184 in colorectal cancer should be further explored. Firstly, we identified the downstream target of circ_0052184 as miR-604 through circinteractome website prediction, dual luciferase reporter gene experiments and RIP experiments. Rectal cancer cells expression and tissues expression were lower. The expression of circ_0052184 was suppressed in CRC cell lines. Pearson correlation analysis showed that circ_0052184 expression and miR-604 expression were negatively correlated. Recent research results have shown that miR-604 is abnormally expressed in tumors such as NSCLC [23], ovarian cancer [24] and gastric cancer [25]. Qiu et al. SNP found abnormal expression of miR-604 [26]. Our research also proves this. And the results of MTT assay, wound healing assay and transwell assay indicated that miR-604 could inhibit the cell proliferation, migration and invasion of colorectal cancer cells. Regulating the expression of circ_0052184 can reverse this phenomenon, and it is speculated that miR-604 is a tumor suppressor gene. In conclusion, circ_0052184 regulates the occurrence and development of colorectal cancer by targeting miR-604. Our study demonstrates that miR-604 is a target gene of circ_0052184, and inhibition of miR-604 can attenuate the development of colorectal cancer cells promoted by circ_0052184.
HOX genes are a subfamily of the homeobox gene family and are evolutionarily highly conserved [27]. Recent studies have confirmed that abnormal HOX gene expression can induce malignant transformation, proliferation and invasiveness of cells, leading to tumorigenesis [28]. HOXA9 gene is a cluster A HOX gene located on chromosome 7. Most studies have shown that the progression of multiple malignancies is often associated with aberrant HOXA9 expression [29]. qRT-PCR detected HOXA9 mRNA level in colorectal cancer tissues and cells, and the results were the same as the study by Bhatlekar et al. HOXA9 is highly expressed in CRC, which reveals that HOXA9 promotes CRC progression [30]. We determined that there is a specific binding region between HOXA9 and miR-604 by Starbase prediction and dual luciferase reporter gene experiments. qRT-PCR, Western blot analysis and Pearson correlation indicated that HOXA9 interacted with miR-604 expression. It is suggested that the miR-604/HOXA9 axis can regulate the malignant development of CRC and is regulated by circ_0052184.
Taken together, our findings suggest that circ_0052184 promotes colorectal cancer progression by targeting the miR-604/HOXA9 axis (Fig. 6). circ_0052184 is a newly discovered circRNA, and no studies have shown the regulatory effect of circ_0052184 on colorectal cancer. To explore the regulatory mechanism of circ_0052184 to provide a theoretical basis for further improving the clinical targeted therapy of colorectal cancer. At the same time, our study will provide valuable reference for the mechanism of action of circ_0052184 in other tumors, and also provide strong evidence for circ_0052184 as a clinical target for treatment. Of course, our study has some limitations. In vitro experiments are the focus of our research, and further in vivo experiments are needed for verification. Other downstream target genes of circ_0052184 need to be screened by follow-up research.

The molecular mechanism of CRC is further refined by our work. This study confirmed that circ_0052184 can regulate the miR-604/HOXA9 axis, and determined that the mechanism promotes the malignant development of CRC cells(Fig. 6). Our work provides evidence that circ_0052184 can be a therapeutic target for CRC. And provided a theoretical basis for the exploration of CRC targeted therapy sites and biomarkers for CRC diagnosis. The research contributes to the development of targeted drugs for colorectal cancer.

Funding

None.

Authors' contributions

Feng K contributed to the study concept and design; Huang YD and Bai QY performed the main experiments and wrote and revised the manuscript; Wang ZL, Yu HB, Li YR, Lu H, Kang HM, and Shi XW partly contributed to the experiments, data analysis, and interpretation. All authors read and approved the final manuscript.

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Table 1

Correlation of HOXA9 protein expression with clinical characteristics of CRC (n = 42)

		HOXA9 protein expression
Category	Number	Low (n = 21)	High (n = 21)	chi-square	P-value
Sex				0.398	0.533
Male	18	8	10
Female	24	13	11
Age				0.0988	0.753
≥55	17	8	9
<55	25	13	12
Tumor size(cm)				5.08	0.024^*
<5	15	11	4
≥5	27	10	17
Lymph node metastasis				4.71	0.030^*
Negative	19	13	6
Positive	23	8	15
TNM Stage				6.46	0.011^*
I+II	16	12	4
III+IV	26	9	17

Table 2

Primers for circ_0052184, miR-604 and HOXA9.

Primers	Forward	Reverse
circ_0052184	5'-CGGTGGCAGCGGGAC-3'	5'-CCTCCAGGTGCTGGACT-3'
miR-604	5'-GCCGTATCCGCAATGTGTTAT-3'	5'-CGACGCTTGCGCGAG-3'
HOXA9	5'-CCCCGACTTCAGTCCTTGC-3'	5'-GATGCACGTAGGGGTGGTG-3'
U6	5'-CTCGCTTCGGCAGCACA-'3	5'-AACGCTTCACGAATTTGCGT-3'
GAPDH	5' -ACTCCTCCACCTTTGACGC-3'	5'-GCTGTAGCCAAATTCGTTGTC-3'

No competing interests reported.

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circ_0052184 promote colorectal cancer progression via targeing miR-604/HOXA9 axis

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