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Lactate Induces Aberration in the miR-30a-DBF4 Axis to Promote the Development of Gastric Cancer and Weakens the Sensitivity to 5-Fu


 Background: Gastric cancer (GC) is one of the most common malignancies, molecular mechanism of which is still not clear. Aberrant expression of tumor-associated genes is the major cause of tumorigenesis. DBF4 is an important factor in cancers, although there is yet no report on its function and molecular mechanism in GC.Methods: The expression of DBF4 in tumor tissues or cells of GC was detected by RT-PCR and western blotting. Gastric cancer cell line MGC-803 and AGS were transfected with DBF4 siRNA or overexpression vector to detect the function of DBF4 in proliferation and migration with CCK-8 assay, colony formation assay, transwell assay and wound healing assay. miR-30a was found to be the regulator of DBF4 by online bioinformatics software and confirmed with RT-PCR, western blot and dual-luciferase reporter assays.Results: In our study, increased expression of DBF4 in GC tissues was first identified through The Cancer Genome Atlas (TCGA) and later confirmed using specimens from GC patients. Furthermore, functional experiments were applied to demonstrate that DBF4 promotes cell proliferation and migration in GC cell lines, moreover weakens the sensitivity of MGC803 and AGS cells to 5-Fu. We further demonstrated that miR-30a showed significantly lower expression in GC cells and inhibited the expression of DBF4 through 3ʹ-UTR suppression. Furthermore, rescue experiments revealed that the miR-30a-DBF4 axis regulated the GC cell proliferation and migration. The important composition in tumor microenvironment, lactate, may be the primary factor that suppressed miR-30a to strengthen the expression of DBF4. Conclusions: Taken together, our study was the first to identify DBF4 as a regulator of cell proliferation and migration in GC. Furthermore, our study identified the lactate-miR-30a-DBF4 axis as a crucial regulator of tumor progression and the tumor sensitivity to 5-Fu, which maybe serve useful for the development of novel therapeutic targets.


Introduction
Gastric cancer (GC) is the fth most common malignancy and ranks as the third leading cause of cancerrelated death worldwide, with over 1 million estimated new cases each year [1][2][3]. Although there have been recent advances in GC diagnosis and therapy, the 5-year survival rate remains below 30% for GC patients [4]. Therefore, it necessary to determine the underlying pathogenic molecular mechanisms of GC to develop effective and novel strategies for treatment [5,6].
A hallmark of cancer cells is aberrations in DNA replication, a major cause of genomic instability and tumorigenesis [7]. DNA replication is initiated by assembly of the checkpoint effector enzyme cell division cycle 7-related protein kinase (CDC7), which then phosphorylates the minichromosome maintenance (MCM) helicase complex on the MCM2 and MCM4 subunits [8][9][10]. The main regulatory activation subunit of CDC7 is DBF4, which is a highly conserved protein that represents an essential checkpoint at the G1/S transition [11]. DBF4 is reported to be scarcely expressed in normal tissues but signi cantly (Invitrogen, Carlsbad, CA, USA) was used to perform cell transfection, in accordance with the manufacturer's protocol.

Dual-luciferase reporter assay
The interaction between miR-30a and DBF4 was predicted by the Starbase bioinformatics database. MGC-823 and AGS cells were seeded in 24-well plates. GC cell were co-transfected with reporter plasmid and miR-30a mimics or negative control using Lipofectamine 2000. After 48 h, cells were lysed with passive lysis buffer for 10 min at 4°C. Luciferase activity was assessed using the Dual Luciferase Assay Kit (Promega, Madison, WI, USA), according to the manufacturer's protocol. Fire y luciferase was normalized to Renilla luciferase activity.

CCK-8 assay
The treated cells were seeded in 96-well plates and were incubated with 100 µL DMEM, containing CCK-8 solution (90 µL/well) for 30 min at 37°C. The absorbance was measured at 450 nm with a spectrophotometer (Molecular Devices, San Jose, CA, USA). All experiments were repeated three times in triplicate.

Transwell assay
Transwell chambers with 8-µm pore lters (BD Biosciences, San Jose, CA, USA) were used to determine the invasion and migration of transfected GC cells. Transfected GC cells at a density of 5×10 4 cells/chamber were added to the upper chamber of each transwell containing 1% FBS medium. Medium with 10% FBS was added to the bottom chamber as a chemoattractant. The cells were nurtured at 37°C in a 5%-CO2 humidi ed atmosphere. The migration of transfected GC cells onto the lower surface of the membrane were visualized by staining with 0.1% Crystal Violet for 30 min. The number of migrating and invading transfected GC cells was counted using an inverted microscope (Olympus, Tokyo, Japan).

Wound healing assay
The transfected cells were re-digested and seeded into a 24-well cell plate. After the cells adhere to the wall, a 1 ml sterile pipette tip was used to draw a straight line in the cell well. Rinse the oating cells repeatedly with sterile PBS and take pictures under the microscope. Later, change the medium to DMEM medium containing 1% FBS and incubate for 48 hours under 5% CO2 37°C. After 48h, rinse repeatedly with sterile PBS, and take pictures under the microscope after removing the non-adherent cells. After the experiment was repeated three times, cell migration was expressed as the migration rate: (original scratch width-new scratch width)/original scratch width ×100%.
According to the manufacturer's instructions, cells were plated in 96-well plates at an initial number of 5×103 cells per well. Then, 10µL of CCK-8 solution was added to each well followed by incubation at 37℃for 2hours. Sample absorbances were measured at 450nm using a microplate reader (Bio-Rad, Hercules, CA). Then CCK-8 solution was thrown away and cells were treated with different concentrations of 5-Fu for 48hours. After that absorbances of each well were measured using the same way. Taken cells without 5-Fu treatment as a control group, the relative cell viability was calculated according to the formula: relative cell viability (%) = OD experiment/OD control x100%.

Statistical analysis
Comparisons between different groups were analyzed with GraphPad Prism v8.0 software (GraphPad Software, La Jolla, CA, USA) using paired or unpaired t-tests. Data are presented as means ± standard deviation. Linear regression analysis was used to analyze the correlation between DBF4 and miR-30a expression in GC samples. Statistical signi cance was reported as highly signi cant using * P < 0.05, ** P < 0.01, *** P < 0.001, or **** P < 0.0001.

Results
DBF4 is upregulated in human gastric cancer.
To explore gene expression pro les in GC, we performed gene set enrichment analysis (GSEA) with data from The Cancer Genome Atlas (TCGA; Fig. 1A, 1B). The analysis showed that the CELL_CYCLE signatures were signi cantly enriched in gastric tumor cells compared with adjacent tissues cells. We chose one of the most differentially expressed genes, DBF4, as our candidate for further experiments. DBF4 is a principle regulator of DNA replication, but its signi cant has not been previously explored in GC. Statistical analysis of DBF4 expression in 32 adjacent tissues and 375 tumor tissues from TCGA showed that DBF4 was dramatically upregulated in human GC (Fig. 1C). To further con rm the results from the TCGA analysis, an additional cohort with four pairs of GC tissues and adjacent normal tissues were collected to analyze DBF4 expression by western blots. As shown in Fig. 1D, DBF4 was increased signi cantly in GC tumor tissues, in accordance with the results from TCGA database. qRT-PCR was then employed to evaluate DBF4 expression in GC cells and normal cells, nding that DBF4 was potently upregulated in GC cell lines (Fig. 1E).Collectively, these ndings evinced that DBF4 was highly expressed in GC.
DBF4 promotes the proliferation of gastric cancer cells.
To investigate the biological role of DBF4 in GC, we designed siRNAs to speci cally target DBF4 and transfected the siRNAs into MGC-803 and AGS cells. The qRT-PCR and western blot analyses showed that the expression of DBF4 was signi cantly decreased in DBF4-siRNA-transfected cells ( Fig. 2A, 2B). Next, we used CCK-8 assays and colony formation assays to detect proliferation of GC cells with decreased expression of DBF4. These assays showed that GC cell proliferation was decreased in the si-DBF4 group compared with the control group (P < 0.01; Fig. 2C, 2D). Expression of PCNA and cyclin-A was also decreased in GC cells transfected with si-DBF4 (Fig. 2E). To determine whether DBF4 overexpression had the opposite effect, we constructed the DBF4 overexpression vector, PEX-DBF4, and transfected this vector or the control vector (PEX) into GC cell lines (MGC-803 and AGS). The transfection e ciency was validated by qRT-PCR and western blotting (Fig. 2F, 2G). Functionally, the CCK-8 assay and colony formation assay suggested that overexpression of DBF4 signi cantly enhanced GC cell proliferation (Fig. 2H, 2I). Expression of PCNA and cyclin-A was increased in GC cells with DBF4 overexpression, as detected by western blotting (Fig. 2J). Together, these data demonstrated that DBF4 signi cantly enhanced GC cell proliferation.
Expression of DBF4 weakens the sensitivity of MGC803 and AGS cells to 5-Fu 5-uorouracil (5-Fu) is considered as one of the rst-line chemotherapy drugs in advanced gastric cancer(GC). First, the effect of 5-Fu treatment on cellular proliferation-associated MGC803 and AGS GC cells was assessed by CCK-8 kit. As shown in Fig. 3A, MGC803 and AGS cells were exposed to a series of concentrations of 5-Fu for 48hours, taking the cell viability of the control sample (without 5-Fu) as 100%, viability of MGC803 and AGS cells was inhibited signi cantly after 5-Fu treatment with the half-maximal inhibitory concentration (IC50) values of 0.4111 and 0.3952 mM respectively. Then, DBF4 overexpression was performed in vitro based on MGC803 and AGS cells by plasmid transfection and IC 50 values of DBF4-overexpressing MGC803-DBF4 and AGS-DBF4 cells were detected by the same way with 0.821 and 1.269 mM separately (Fig. 3B). Meanwhile, short interference siRNA for DBF4(siDBF4) were applied to knockdown the expression of DBF4 inMGC803 and AGS cells. IC 50 values of MGC803-siDBF4 and AGS-siDBF4 cells were detected by the same way with 0.1453 and 0.1677 mM separately (Fig. 3C). Taken together, these results demonstrate that as an oncogene, DBF4 weakens the sensitivity of MGC803 and AGS GC cells to 5-Fu.
DBF4 promotes the migration of gastric cancer cells.
To investigate the biological function of DBF4 in GC, transwell assays and woud healing assay were used to determine the migration of MGC-803 and AGS cells. As shown in Fig. 4A, 4B, silencing of DBF4 repressed the migration of both gastric cancer cells. To further con rm the results, DBF4 was overexpressed in MGC-803 and AGS cells and the migration of both GC were detected by transwell assays and woud healing assay. Consistent with the results above, the migratory ability of MGC-803 and AGS cells was strongly enhanced following DBF4 overexpression (Fig. 4C,4D). Collectively, these experiments indicated that DBF4 also perform an important role in migration of GC cells.
The miR-30a inhibits the expression of DBF4.
The miRNAs are involved in the regulation of various biological processes such as cell proliferation [19], they regulate expression of many oncogenes and tumor suppressor genes, and they have been reported to play key roles in various human cancers, including GC [16,17]. We rst analyzed the miRNA expression pro le in 436 GC tissues and 41 adjacent tissues tissues from TCGA. A total of 1881 miRNAs were expressed in these tissues and 139 showed differential expression. Of these, 79 miRNAs were downregulated and 60 were upregulated in GC (fold change ≥ 2 or ≤ 0.5, P < 0.05; Fig. 5A). A heat map was created showing differentially expressed microRNAs in GC tissues, relative to matched normal tissues (Fig. 5A). To determine the target regulatory miRNA for DBF4, two databases were screened, including TargetScan (https://www.targetscan.org) and TarBase (http://starbase.sysu.edu.cn/starbase2/). The only overlapping result from the two databases was miR-30a-5p (Fig. 5B). As shown in Fig. 5A and 4B, miR-30a-5p also shows signi cant differential expression in GC. Statistical analysis showed that miR-30a expression was signi cantly decreased in GC tumor tissue (Fig. 5C). In addition, the expression level of DBF4 was negatively correlated with the expression level of miR-30a in GC tissues, based on the data from TCGA (Fig. 5D). To further con rm the effect of miR-30a on DBF4 expression, both MGC-803 and AGS cells were transfected with miR-30a mimics and negative controls. DBF4 expression was analyzed by qRT-PCR and western blotting assays, which showed that DBF4 expression was signi cantly downregulated in GC cells transfected with miR-30a mimics. This suggested that DBF4 may be the target of miR-30a in GC (Fig. 5E). Moreover, bioinformatics analysis predicted a putative 8-mer-binding site with miR-30a in the 3'-UTR of the DBF4 transcript (Fig. 5F). Dualluciferase reporter assays were performed to determine whether miR-30a directly targets DBF4.
Overexpression of miR-30a signi cantly decreased the luciferase activity of wild-type DBF4 in GC cells, but had no effect on luciferase activity of mutant DBF4 in GC cells (Fig. 5G), demonstrating that miR-30a speci cally binds to the 3' UTR of miR-30a. Taken together, these data suggested that miR-30a, one of the most differentially downregulated miRNAs in GC, directly regulated DBF4 expression.
The miR-30a-DBF4 axis regulates the proliferation of gastric cancer cells.
Having con rmed the interaction of miR-30a and DBF4, next we performed rescue experiments to determine whether miR-30a functions as a tumor suppressor gene via regulation of DBF4. Previous reports suggested that miR-30a may inhibit the proliferation and invasion of GC [22]. In agreement with these previous ndings, MGC-803 and AGS gastric cell lines exhibited reduced proliferation after transfection with miR-30a mimics (Fig. 6B, 6C). qRT-PCR analysis showed that DBF4 expression was suppressed in the miR-30a mimic group and upregulated in the DBF4 overexpression group (Fig. 6A).
CCK-8 and colony formation assays showed that the suppressive effect of miR-30a overexpression on cell proliferation was abrogated by overexpression of DBF4 (Fig. 6B,6C). In order to further determine the effect of miR-30a-DBF4 axis on cell migration, woud healing assay and t Transwell assays were used, and the results indicated that suppressive effect of miR-30a overexpression on cell migration was abrogated following DBF4 overepression. (Fig. 6D and 6E). Taken together, these results suggested that miR-30a restored the effects of DBF4 on proliferation and migration of AGS and MGC-803 cells.
Lactate in the tumor microenvironment induces aberrant expression of miR-30a and DBF4.
In addition to dysregulated cell proliferation, tumors exhibit another dimension of complexity in that they contain a repertoire of recruited immune cells, metabolites such as lactate, and in ammatory factors such as interleukin-6 (IL-6). To determine the primary factor that induced the aberrant expression of miR-30a and DBF4, we stimulated GC cells with lactate and IL-6 in MGC-803 and AGS cells. As shown in Fig. 7A, lactate suppressed miR-30a expression and IL-6 showed a weak effect on miR-30a expression. By qRT-PCR analysis, we determined that lactate was the main factor inducing the aberrant expression of DBF4 (Fig. 7B). Together, our ndings demonstrated that accumulation of lactate in the tumor microenvironment inhibited miR-30a expression and increased DBF4 expression.

Discussion
Although gastric cancer (GC) is one of the most aggressive malignancies worldwide, there are limited treatment options available and the disease has a poor prognosis after local progression and metastasis [23]. Arguably one of the most fundamental traits of cancer cells is their ability to undergo continued proliferation, but little is known about the precise that regulate this process. Using the TCGA database and GSEA analysis, we identi ed the cell cycle pathway as one of the most enriched pathways in GC tissues. We also found that DBF4 had signi cantly higher expression in GC compared to normal tissues. Two GC cell lines, MGC-803 and AGS, were used in this study. CCK-8, colony formation, and western blotting assays were used to analyze GC cell proliferation, would healing and transwell assay were performed to detect GC cells migration, following transfection with DBF4 siRNA or a DBF4 overexpression vector. Cell proliferation assay were applied to con rm that DBF4 weakens the sensitivity of GC cells to 5-Fu. Furthermore, miR-30a, one of the most signi cantly downregulated miRNAs in GC tissue, was predicted and con rmed to be negatively correlated with DBF4 expression. qRT-PCR and western blots veri ed that miR-30a inhibited DBF4 expression, and a dual-luciferase reporter assay demonstrated that miR-30a directly inhibited the 3'-UTR region of DBF4. Resulting from the Weinberg effect of tumor cells, a large amount of lactate accumulates in the tumor microenvironment. qRT-PCR experiments veri ed that lactate inhibited the expression of miR-30a and signi cantly increased the expression of DBF4. Ultimately, using TCGA databases for GC, we identi ed DBF4 as an important regulator of proliferation and migration in GC cells. Moreover, through prediction and experimental veri cation, we demonstrated the important role of the lactate-miR-30a-DBF4 axis in the development of GC, providing new targets and important theoretical guidance for the treatment of late-stage GC.
CDC7-DBF4 is a conserved serine/threonine kinase that plays an important role in initiation of DNA replication and DNA damage tolerance. DBF4 is the regulatory subunit, which is necessary for kinase activity and for targeting of various substrates [24]. In the last decade, the CDC7-DBF complex has emerged as a potential novel chemotherapeutic target. CDC7 kinase de ciency can induce signi cant apoptosis of tumor cells, while normal cells undergo a reversible cell cycle arrest [25][26][27]. However, the effect of DBF4 on GC proliferation and migration and the precise mechanisms involved remained unclear. To our knowledge, our study is the rst to report that increased expression of DBF4 potentiated GC cell proliferation, as demonstrated by the CCK-8, colony formation, and western blot assays in two GC cell lines transfected with si-DBF4 or DBF4 overexpression vectors. 5-Fu is considered as one of the rst-line chemotherapy drugs in advanced gastric cancer(GC). And we also gured out that DBF4 weakens the sensitivity of GC cells to 5-Fu, which suggested that DBF4 may become a novel therapeutic targets in GC treatment. Furthermore, our results suggested that DBF4 also play an important role in the regulation of cancer cells migration.
The miRNAs are small non-coding RNAs that post-transcriptionally regulate the expression of many genes to control physiological and pathological processes, including the occurrence, growth, and progression of cancer [28]. Until now, approximately 200 different miRNAs have been implicated in the occurrence and treatment of GC, and numerous studies have suggested that miRNAs may play an important role in various types of cancer, including GC [28][29][30]. Deregulation of miR-30a has been shown to play a role in many human cancers, and signi cant downregulation of miR-30a has been detected in anaplastic thyroid carcinomas and non-small cell lung cancer [31,32]. In contrast, upregulation of miR-30a has been reported in glioma [33], which suggests a context-speci c role for miR-30a across different cancer types. In the present study, miR-30a signi cantly inhibited the proliferation of GC cells, which is in agreement with the previous reports [22]. Furthermore, we demonstrated miR-30a regulated GC cells by inhibiting the expression of DBF4. Dual-luciferase reporter assays showed that miR-30a signi cantly suppressed the 3'-UTR of DBF4. However, additional studies are required to determine the precise mechanism of this regulation. miR-30a was recently observed to be downregulated in GC, resulting from high promoter methylation induced by DNA methyltransferase [34]. In our study, we found that accumulation of lactate in the tumor microenvironment reduced the expression of miR-30a, suggesting that miR-30a may also be regulated by lactylation. Additional future experiments are needed to con rm this nding.
Our study is the rst time to identify DBF4 as a critical tumor suppressor in GC. Downregulation of DBF4 increases proliferation of GC cells. Mechanistically, miR-30 suppressed the 3'-UTR of DBF4 and inhibited its expression. Lactate in the tumor microenvironment was the primary factor that induced aberrant expression of miR-30a and DBF4. In conclusion, the lactate-miR-30a-DBF4 axis was a critical regulator of GC cell proliferation, migration, sensitivity to 5-Fu and may have potential to serve as a target for the treatment of gastric cancer.      Overexpression of DBF4 restores the effects of miR-30a upregulation on GC cells. MGC-803 and AGS cells were transfected with miR-NC, miR-30a, miR-30a+PEX, or miR-30a+PEX-DBF4. (A) qRT-PCR analysis was used to determine DBF4 mRNA expression levels in MGC-803 (above) and AGS (below) cells. (B, C)

Declarations
The proliferation of MGC-803 and AGS cells was assessed using CCK-8 (B) and colony formation (C) assays. Transwell assay (D) and wound healing assay (E) were performed to detect the migration of gastric cancer cells. Data are presented as the mean ± SD (n=3). **P <0.01