The effect of LNCRNA SHANK3 on the malignant development of gastric cancer cells by regulating the miR-4530/MNX1

Highlights

• •LNCRNA SHANK3 is highly expressed in gastric cancer and is associated with poor prognosis.
• •LNCRNA SHANK3 sponge adsorbs miR-4530 to inhibit its expression, while mine-4530 inhibits its expression by binding to 3 'UTR of MNX1.
• •LNCRNA SHANK3 regulates the proliferation, migration, and invasion of gastric cancer cells by regulating miR-4530/MNX1.
• •This study has the hope of providing possible diagnostic and therapeutic targets and theoretical support for the design and development of GC preventive drugs.

Abstract

Gastric cancer (GC) has become the first malignant tumor with highest incidence rate and mortality of cancer in China, finding therapeutic targets for gastric cancer is of great significant for improving the survival rate of patients with GC. Recently, many of studies have shown that LncRNAs is involved in multiple biological progresses in the development of GC. This study, we screened for abnormally high expression of LncSHANK3 in GC through the TCGA database, and found that LncSHANK3 sponge adsorbs miR-4530, further competing with MNX1 and binding to miR-4530. We demonstrated the interaction between LncSHANK3 and miR-4530 through luciferase reporting analysis, with miR-4530 negatively regulating MNX1.Through CCK8, colony formation, transwell, and wound healing assays, it was found that LncSHANK3 affects the occurrence of GC through cell proliferation, migration and invasion. In conclusion, LncSHANK3/miR-4530/MNX1 axis is a potential mechanism for the treatment of GC.

Introduction

Gastric cancer is a common malignant tumor of the digestive tract, with the second highest mortality rate among various types of tumors worldwide [1]. Incidence of GC has declined worldwide over the past 50 years, despite efforts to prevent and treat Helicobacter Pylori infection and improve dietary habits [2]. Currently, the common treatment for gastric cancer include surgery, chemotherapy, radiation chemotherapy, laser therapy and combination therapy [3]. Although surgical resection is a treatment for gastric cancer, and recent advances in chemotherapy have also improved progression-free survival and overall survival, the progression of patients is still poor, with a 5-year survival rate of only about 20% for advanced patients [4].

The classification of GC has traditionally followed the customized criteria based on the histopathological characteristics of GC by Lauren et al. in 1965 [5]. According to Lauren criteria, GC is divided into two main histological subtypes: enteric and diffuse. Intestinal type GC is the most common,accounting for 54% of all GC cases, and is characterized by a distinct glandular duct structure with strong adhesion characteristics [6]. Intestinal type GC is statistically more common in the elderly, while diffuse GC is more common in younger and woman patients [7] .

MiRNA is highly conserved endogenous non-coding RNA consisting of about 19–25 nucleotides that function by forming complementary sequences with partial sequences of target mRNA [[8], [9], [10]]. So far, more than 2600 miRNAs have been found in the human genome, and numerous studies have demonstrated their abnormal expression is closely related to tumor proliferation, migration and apoptosis [[11], [12], [13], [14], [15]]. Has-miR-4530 is located on chromosome 19. Some studies have shown that over-expression of miR-4530 in breast cancer will inhibit cell proliferation and promote cell apoptosis [16].

Compared with miRNA, lncRNA length greater than 200 nucleotides and also a class of RNA without the ability to encode proteins [[17], [18], [19], [20]]. At the transcription level, lncRNA regulates mRNA expression by interacting with transcription complexes [[21], [22], [23]]. LncRNA are also involved in the process of regulating post-transcription modification of genes. For example, LncRNA can act as molecular sponges for miRNA on target genes [[24], [25], [26]].

MNX1 is a member of the EHG homologous gene family, which also includes EN1, EN2, GBX1, GBX2 [27]. MNX1 is a key development gene that is commonly expressed in neurons as well as pancreatic and lymphoid cells. It is also involved in motor neuron differentiation and pancreatic βcell development. Currarino syndrome is mainly caused by MNX1 deletion or mutation, suggestion that MNX1 affects human development and cell transformation [[28], [29], [30]]. In2016, a study found that MNX1 is a novel prostate carcinogen gene, which partially plays a role by promoting lipid synthesis [[31], [32], [33]]. In summary, up-regulation of MNX1 is associated with the progression of multiple cancers, suggesting that MNX1 may be an oncogene.

After analyzing on the miRDB website, we found that miR-4530 can bind to the 3′UTR sequence of MNX1, and LncSHANK3 can competitively bind to miR-4530 with MNX1, thereby regulating the expression of MNX1 and promoting the proliferation, migration, invasion, and other functions of gastric cancer cells. These findings illustrate our understanding of the pathogenesis of GC from a novel perspective and provide new research thinking and method for inhibiting the progression of GC.

Results

LncRNA SHANK3 and MNX1 are highly expressed in GC tissues

In order to identify important LncRNA that may be associated with gastric cancer (GC), we analyzed the expression of SHANK3 and MNX1 in gastric cancer patients using the Public Database (TCGA) (Fig. 1a,b). Compared with para-cancerous tissue, the expression of LncSHANK3 and MNX1 in GC tissue is significantly increased, so it is was selected for further study . Next, by performing a volcanic map of differentially expressed genes in gastric cancer, it was found that MNX1 is differentially upregulated in gastric cancer (Fig. 1c). We detected the expression of SHANK3 and MNX1 in 5 pairs of gastric cancer patient tissues and adjacent normal tissues. We observed that the expression of SHANK3 and MNX1 was significantly higher in gastric cancer tissues than in adjacent normal tissues. As shown in Fig. 1d,e, and through TCGA database analysis, it was found that miR-4530 is underexpressed in gastric cancer tissue(Fig. 1f). We analyzed the relationship between the expression levels of LncSHANK3 and MNX1 and clinical factors in patients with GC. As shown in the KM-plotter survival curve, the ensemble survival rate of GC patients in the high expression groups of LncSHANK3 and MNX1 is evidently below than that in the low expression groups of LncSHAK3 and MNX1 (Fig. 1g,h). In summary, these results indicate that LncSHANK3 and MNX1 are highly expressed in GC and are associated with poor prognosis in GC. In order to explore the important role of LncSHANK3 in the progression of GC, we conducted pathway enrichment analysis on genes related to LncSHANK3 in gastric cancer using the KEGG database. The results indicate that LncSHANK3 may play a role by affecting the proliferation and invasion of GC cells (Fig. 1i).

Fig. 1.

LncSHANK3 and MNX1 are upregulated in GC. a,b Representative LncSHANK3 and MNX1 expression in GC analyzed by TCGA database (Fold change >2 p < 0.05). c Volcano map displaying the expression profile of MNX1 in GC patients in TCGA (Fold change >2 p < 0.05).d,e Representative LncRNA SHANK3 and MNX1 expression in tumor sections of 5 patients with GC were detected by qRT-PCR. f Representative miR-4530 expression in GC analyzed by TCGA database (Fold change >2 p < 0.05) g,h Survival rate of GC patients was analyzed by Kaplan–Meier method. Log-rank tests were used to determine statistical significance. *** p < 0.001. i Enrichment analysis of genes related pathways of LncSHANK3 through KEGG pathway enrichment in gastric cancer.

SHANK3 and MNX1 regulated GC cell proliferation, migration/ invasion ability

In order to confirm the role of LncSHANK3 and MNX1 in GC. First, we checked the endogenous expression of SHANK3 and MNX1 at the RNA level through qRT-PCR in 5 GC cell lines (GES1, SGC7901, MGC803, AGC, HGC27) and selected high expression MGC803 and low expression HGC27 GC cells for the next experiment (Fig. 2a). In subsequent experiments, we performed gene knockdown in MGC803 and over-expression of this factor in HGC27 (Fig. 2b). QRT-PCR was used to detected the knockdown efficiency of LncSHANK3 and MNX1 in MGC803. The knockdown efficiency of sh-SHANK3#1,sh-MNX1#2 was significant, so it was selected for the next experiment (called sh-SHANK3 and sh-MNX1from now on) (Fig. 2c). Next, we analyzed the mRNA expression level of MNX1 in MGC803 cell with LncSHANK3 knockdown and in HGC27 cell with LncSHANK3 over-expression (Fig. 2d). The results showed that SHANK3 over-expression could promote the expression of MNX1 in HGC27 cell, whereas SHANK3 knockdown had the opposite effect in MGC803 cell. According to previous pathway enrichment analysis, SHANK3 regulates the occurrence of GC by influencing the proliferation, migration and invasion of cells. Therefore we conducted CCK8 experiments, colony formation, wound healing experiments and Transwell assays to investigate its function (Fig. 2e-j). Over-expression of SHANK3 or MNX1 in HGC27 cells can promotes the proliferation, migration and invasion of GC cells, but knockdown SHANK3 or MNX1 expression in MGC803 cells has the opposite effect. What is more important, MNX1 knockdown can eliminate the effects of SHANK3 over-expression on the proliferation, migration and invasion ability of HGC27 cell. Conversely, over-expression of MNX1 in MGC803 could restore the inhibitory effects of knockdown of SHANK3 on cell proliferation, migration and invasion. In conclusion, these outcomes suggest SHANK3 and MNX1 are involved in the proliferation, migration and invasion of GC cells.

Fig. 2.

SHANK3 and MNX1 regulate GC cell proliferation, migration/invasion in vitro. a LncSHANK3 and MNX1 expression in four GC cell lines (HGC27, MGC803, AGS, and sgc7901), and in a normal human gastric mucosa epithelial cell line GES1 were analyzed by qRT-PCR (n = 3). b The efficiency of LncSHANK3 knockdown and overexpression were detected by qRT-PCR. Knockdown of LncSHANK3 using four different shRNAs (sh-SHANK3-#1, sh-SHANK3-#2, sh-SHANK3-#3, sh-SHANK3-#4) or a control shRNA (sh-NC) in MGC803 cells, overexpression of SHANK3 were in HGC27 cells (n = 3). C The efficiency of MNX1 knockdown and overexpression were detected by qRT-PCR. Knockdown of MNX1 using two different shRNAs (sh-MNX1-#1, sh-MNX1-#2,sh-MNX1#3,sh-MNX1#4) or a control shRNA (sh-NC) in MGC803 cells, overexpression of MNX1 were in HGC27 cells (n = 3). d qRT-PCR analysis of MNX1 expression levels in HGC27 cells and MGC803 cells, HGC27 cells were transfected with plvx /SHANK3, while MGC803 cells were transfected with plko.1/sh-SHANK3(n = 3). e-j HGC27 cells were co-transfected with SHANK3 and sh-MNX1, while MGC803 cells were co-transfected with sh-SHANK3/MNX1 (n = 3). e-f CCK8 and colony-forming growth assays were performed to determine the proliferation of HGC27 and MGC803 cells harboring the different vectors indicated (n = 3). g Transwell assays were performed to determine the migration and invasion capacity in HGC27 and MGC803 cells (n = 3). Scale bar = 50 μm. h Wound healing assays were performed to assess the migratory capacity in HGC27 and MGC803 cells (n = 3). i, j Migration and invasion abilities (fold change of migrated or invaded) were calculated, compared with the different vectors in HGC27 and MGC803 cells. The migratory activity (wound healing) was calculated and compared with those at 0 h (n = 3).

SHANK3 and MNX1 promotes GC tumor growth in vivo

In order to investigate whether LncSHANK3 and MNX1affect the growth of gastric cancer tumors in vivo, cells over-expression or knockdown LncSHANK3 and MNX1 would be used 1 × 10⁷ was injected into 4-week-old nude mice, and the tumor weight and volume were regularly observed and measured. When injected into mice with MGC803 cells that down-regulated the expression of LncSHANK3, the mass and volume of the tumor decreased significantly, while over-expression of MNX1 restored the tumor's proliferation capacity (Fig. 3a).In contrast, over-expression of LncSHANK3 in HGC27 cells significantly raised tumor mass and volume, and this effect was reversed by injection of knockdown MNX1 cells (Fig. 3b).

Fig. 3.

LncSHANK3 and MNX1regulate GC cell growth in vivo. a Tumor weight and growth curves (n = 6) were measured after injection of MGC803 cells transfected with sh-SHANK3 and MNX1. b Tumor weight and growth curves (n = 6) were measured after injection of HGC27 cells transfected with LncSHANK3 and sh-MNX1. c,d Western blot and qRT-PCR analyses of MNX1 expression levels in HGC27 and MGC803 cells (n = 6). *p < 0.05 and **p < 0.01.

Finally, the transcription level mRNA expression and translation level protein expression of MNX1 were detected in HGC27 and MGC803 cells (Fig. 3c, d). We found that over-expression of LncSHANK3 in HGC27 cells also increased MNX1 expression, and this effect was offset by knockdown of MNX1 expression. Similarly, knockdown the expression of LncSHANK3 in MGC803 cells also reduces the expression of MNX1, which can be canceled by over-expression of MNX1. In summary, it is shown that the expression of MNX1 is positively correlated with the expression of LncSHANK3. When the expression of MNX1 is reduced, the effect of LncSHANK3 on the proliferation of gastric cancer cells will also be reduced in turn.

LncSHANK3 acts in GC cells by sponge adsorption miR-4530

Recently, many LncRNAs have been reported as competitive RNA to regulate miRNA expression and affect biological functions. Therefore, we speculated that whether LncSHANK3 also regard as a miRNA competitive RNA to affect the expression of downstream target gene mRNA. Trough the search of DIANA Lncbase database, it was found that LncSHANK3 and miR-4530 had interaction binding sites(Fig. 4a). Therefore, we selected miR-4530 as the miRNA downstream of LncSHANK3, and the mutation occurred at the miR-4530 binding site as shown in Fig. 4b. Co-transfection of SHANK3-Wt, SHANK3-Mut or empty vector with miR-4530 mimic/mimic-NC or miR-4530 inhibitor /inhibitor-NC in MGC803 and HGC27 cells was performed to detect luciferase reporting activity 48 h after transfection (Fig. 4c-e). We found that in SHANK3-Wt cells over-expression miR-4530, luciferase activity was relatively low, while transfected with miR-4530 inhibitors, luciferase activity doubled. We evaluated the interaction between lncRNA SHANK3 and miR-4530 using RIP analysis. Compared with mimic NC, cells transfected with miR-4530 mimic were enriched for both AGO2 and negative control IgG. The result is placed in the supplementary figure, as shown in Fig. 4f.When LncRNA SHANK3 is overexpressed in HGC27 cells, the expression of miR-4530 decreases, while when LncRNA SHANK3 is knocked down in MGC803, the expression of miR-4530 increases. (Fig. 4,h). In addition, when transfected with miR-4530 inhibitors or inhibitors NC in HGC27 cells, compared to the control, the expression of LncRNA SHANK3 was increased. On the contrary, when transfected with miR-4530 mimc or mimic NC in MGC803 cells, the expression of LncRNA SHANK3 in cells transfected with miR-4530mic decreased compared to the control group.(Fig 4i,j) To investigate the effects of SHANK3 and miR-4530 on the proliferation, migration, and invasion of GC cells, we conducted CCK8 experiments, colony formation experiments, Transwell experiments, and wound healing experiments to verify their effectiveness (Fig. 4k,o). MiR-4530 mimic can counteract the promoting effect of SHANK3 overexpression on cell proliferation, migration, and invasion, while miR-4530 inhibitors can reverse the inhibitory effect of SHANK3 gene knockdown on cell proliferation, migration, and invasion. The above results indicate that miR-4530 is a downstream miRNA of SHANK3, which promotes the proliferation, migration, and invasion of GC cells by targeting miR-4530.

Fig. 4.

LncSHANK3 is a target of miR-4530 and regulates its expression. a Predicting the combination of SHANK3 and miR-4530 through DIANA website. b Wild-type (SHANK3-WT) and mutant (SHANK3-MUT) SHANK3 with mutations at the predicted miR-4530 binding site (n = 3). c,d A luciferase reporter vector carrying SHANK3-WT or SHANK3-MUT (or the empty vector) was co-transfected into MGC803 cells with miR-4530 mimic or mimic-NC (c) or miR-4530 inhibitor or inhibitor-NC (d) as indicated into HGC27. Relative luciferase activity was measured at 48 h after transfection (n = 3). e The luciferase reporter vector carrying MNX1 3′-UTR-WT was co-transfected into MGC803 cells with miR-4530 mimic, mimic-NC, pcDNA3.1-SHANK3, and pcDNA3.1-NC, alone or in combination as indicated. Relative luciferase activity was measured at 48 h after transfection (n = 3). f 293T cells were transfected with miR-4530 mimic or mimic-NC for 48 h, and association between LncSHANK3 and miR-4530 was assessed by RNA immunoprecipitation assay (n = 3) g,h QRT PCR analysis of 4530 expression when SHANK3 is overexpressed or knocked down. i,j qRT-PCR analysis of SHANK3 expression during transfection with miR-4530 inhibitors or miR-4530 mimic. k-m CCK8 and colony-forming growth assays were performed to determine the proliferation of MGC803 and HGC27 cells (n = 3). n Transwell assays were performed to determine the migration and invasion capacity of MGC803 and HGC27 cells (n = 3). Scale bars = 50 μm. The migration and invasion abilities calculated (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns not significance.l o Wound healing assays were performed to assess the migratory capacity of MGC803 and HGC27 cells (n = 3) The migratory activity (wound healing) were calculated (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns not significance.

MiR-4530 regulates proliferation, migration/invasion of GC cells through targeting 3′-UTR of MNX1

We used miRDB, TargetScan and miRTarBase database to analyze the intersection, and we found that 17 genes were potential downstream targets of miR-4530 (Fig. 5a). We found that the gene MNX1 was highly expression in GC through qRT-PCR analysis.(Fig 5.b)To further verify the function between MNX1 and miR-4530, we obtained through the analysis of miRDB website that miR-4530 acted on the 3′UTR of MNX1, so the mutation was introduced into the binding site of MNX1 and miR-4530 through site-specific mutation (Fig. 5c). When wild-type and mutant MNX1 were co-transfected with miR-4530 mimics, it was found that the luciferase activity in wild-type cells was decreased after the addition of miR-4530 mimics, but there was no difference in the luciferase activity of mutant MNX1 cells (Fig. 5d). We detected the expression of miR-4530 in 5 pairs of gastric cancer patient tissues and adjacent normal tissues, and observed that the expression of 4530 was significantly lower in gastric cancer tissues than in adjacent normal tissues. As shown in Fig. 4e.We then detected the endogenous mRNA expression of miR-4530 in GC cells, and found that compared with normal GES1 cell, MGC803 cell showed significantly lower expression of miR-4530 (Fig 5.f). Furthermore, MNX1 expression was evaluated in MGC803 and HGC27 cells, and the results indicated that MNX1 protein expression was significantly decreased in the presence of miR-4530 mimic and OIP5-AS1 knockdownMGC803 cells.Conversely, MNX1 expression was increased in the presence of miR-4530 inhibitor and SHANK3 overexpressing HGC27 cells, demonstrating that SHANK3 is a target of miR-4530(Fig 5g,h) However, when miR-4530 was inhibitor in HGC27 cells, the expression of MNX1 was increased (Fig. 5i). Next, we tested the proliferation, migration and invasion ability of miR-4530 in GC cells through CCK8, colony formation, Transwell and wound healing. In HGC27 cells, inhibitor miR-4530 significantly increased cell proliferation, migration and invasion, while knocking down MNX1 expression can offset these functions. Similarly, adding miR-4530 mimics to MGC803 cell can inhibit the proliferation, migration,invasion ability of cell caused by overexpression of MNX1 (Fig. 5j-n). In summary, MNX1 is the downstream target gene of miR-4530, which inhibits its transcription by binding to MNX1 3′UTR, thereby inhibiting the proliferation, migration, invasion of gastric cancer cells.

Fig. 5.

MNX1 is a direct target of miR-4530 and is involved in miR-4530-mediated cell proliferation and migration/invasion. a TargetScan, miRDB and miRTarBase predict the target genes of miR-4530, using Funrich to create a Venn map. b Target genes expression in GC cell lines were analyzed by qRT-PCR (n = 3). c WT 3′-UTR of MNX1 (MNX1 3′-UTR-WT) and a mutant 3′- UTR of MNX1 with mutations at the predicted miR-4530 binding site (MNX1 3′-UTR-MUT) (n = 3). d A luciferase reporter vector carrying MNX1 3′-UTR-WT or MNX1 3′-UTR-MUT (or the empty vector) was co-transfected with miR-4530 mimic or mimic-NC, and relative luciferase activity was measured 48 h post transfection (n = 3). e Representative miR-4530 expression in tumor sections of 5 patients with GC were detected by qRT-PCR. f qRT-PCR analysis of miR-4530 expression in four GC cell lines (SGC7901, MGC803, AGS, and HGC27), and in GES1 cells (n = 3). g,h Western blot analysis of MNX1 protein levels following treatment of MGC803 cells with miR-4530 mimic or si-SHANK3, and HGC27 cells with miR-4530 inhibitor or pcDNA3.1-SHANK3. GAPDH was used as an internal control (n = 3). i Western blot and qRT-PCR analyses of MNX1expression levels in HGC27 cells transfected with inhibitor-NC or miR-4530 inhibitor (n = 3). j-l CCK8 and colony-forming growth assays were performed to determine the proliferation of MGC803 and HGC27 cells (n = 3). m Transwell assays were performed to determine the migration and invasion capacity of MGC803 and HGC27 cells (n = 3). Scale bars =50 μm On the right is a quantitative statistical chart of transwell and migration results. nWound healing assays were performed to assess the migratory capacity of MGC803 and HGC27 cells (n = 3) On the right is a quantitative statistical chart of and migration results.

Discussion

Recently, a large amount of evidence has shown that LncRNA plays an significant role in the pathogenesis and development of GC, showing the potential of application or targeting LncRNA in the treatment of GC. This paper mainly focuses on LncSHANK3 /miR-4530/MNX1 signal axis to study the development and mechanism of GC (Fig. 6).

Fig. 6.

LncSHANK3 /miR-4530/MNX1 signal axis to study the development and mechanism of GC.

Many studies have shown confirmed that LncRNA play important role in the proliferation, migration, and invasion of gastric cancer, indicating the potential of application or targeting of LncRNA in the treatment of GC [34,35]. ZEB1-AS1 is highly expression in gastric cancer, and it's over-expression indicates poor prognosis and promotes metastasis [36]. LncRNA DANCR accelerate migration and invasion of GC cells through inhibiting LncRNA LET [37]. LncRNA UCA1 can aggravate the progression of GC by regulating the PI3K-Akt-mTOR signaling and it's downstream products [38]. LncRNA MTM may target MTIF and promote the growth of GC cells [39]. Ectopic of LncRNA XLOC010235 enhances epithelial mesenchymal transformation (EMT) and promotes metastasis of GC by binding to Snail1 in GC [40].

So, it's extremely that reveal the biological activity of LncRNAs and include the identification of new LncRNA in cancers.

In this article, we found that LncSHANK3 is highly expression in GC patients through TCGA database screening and through KM-plotter database analysis, it was observed that the expression of LncSHANK3 is closely related to prognosis with GC. High expression has a low survival rate, while low expression has the opposite effect. We have demonstrated through a series of cell experiments and in vivo animal experiments that LncSHANK3 boosts proliferation, migration, invasion of gastric cancer cells. All the results indicate that LncSHANK3 is expected to become a new biomarker molecule in GC.

Therefore, to further explore its specific action pathway in GC, we predicted the miRNA that might interact with LncSHANK3 through the analysis of DIANA and miRDB online database, and found that miR-4530 had highly complementary binding sites with LncSHANK3. Next, we verified the interaction between LncSHANK3 and miR-4530 by luciferase reporting experiment. MiR-4530 was identified as the downstream molecule of SHANK3. The next step is to analyze which downstream target genes SHANK3 and miR-4530 affect the progression of GC. First, we calculate the possible downstream target genes of miR-4530 by miRDB and miRTarBase database analysis, and 17 downstream target genes were obtained by interaction. And then detected the endogenous expression of these target genes mRNA in GC, the results found that MNX1 was highly increased in GC cells compared to normal GC epithelial cells, and western blot assay also detected that MNX1 was remarkably expressed in GC cells at protein level. Therefore, the interaction between miR-4530 and MNX1 is further studied, and the luciferase report analysis experiments shows that the two interact closely.

Conclusion

In summary, all the results in this paper indicate that LncSHANK3 is an LncRNA significantly correlated with the occurrence of GC, which is not only markedly highly expressed in GC cells, but also is closely related to the prognosis of GC patients. From the perspective of mechanism, SHANK3, as a competitive RNA combining with miR-4530, promotes the expression of MNX1, thus affecting the proliferation, migration and invasion ability of MGC803 and HGC27 cells. Although the regulatory axis of LncSHANK3/miR-4530/MNX1 is a new clinical theoretical basis for the progression of GC, how MNX1, as a transcription factor, affects the specific regulatory mechanisms and molecular pathway of downstream target genes is still worthy of further studies discussion. It is hoped that future studies can provide possible diagnosis and treatment targets and theoretical support for the design and development of preventive drugs for GC.

Materials and methods

Tissue collection

GC tissue and adjacent tissues were obtained from 5 patients at the Affiliated Hospital of Huazhong University of Science and Technology, and were obtained from October to the end of December 2023 after receiving informed consent from the patients. This plan complies with the ethical standards of the Helsinki Declaration and has been approved by the Medical Ethics Committee of Wuhan University of Science and Technology. At least two pathologists have confirmed the diagnosis of gastric cancer pathology

Cell lines culture

Human gastric cancer cell lines MGC803, HGC27 correctly identified by short tandem repeat certification were got from type keep in store in US. HGC27 and MGC803 were cultivated in Roswell Park Memorial Institute-1640 (Invitrogen) supplemented with 10% fetal bovine serum (Every Green, China) streptomycin, and penicillin, and placed in an incubator at 5% CO₂ and 37 °C.

Plasmid construction and cell transfection

For over-expression of LncSHANK3 and MNX1, the following primers were used for amplification: LncSHANK3: forward: 5′-ACTAGTCCAGTGTGGTGGGACCCTGTCCCATTACAGAGC-3′; reverse: 5′-GTGCTGGATATCTGCAGAATTGTCGTAGGAGCCCACTGTGTAGTG-3′. MNX1: forward: 5′-CCACTAGTCCAGTGTGGTGGATGGAAAAATCCAAAAATTTCCGC-3′; reverse: 5′-CTGGATATCTGCAGAATTCTACTGGGGCGCGGGCTG-3′. Then cloning the full length cDNA of human Lnc-SHANK3 and MNX1 onto the EcoRI and Nhel site of plvx mammalian expression vector (Invitrogen) to construct plvx-SHANK3/MNX1. Using PurePlasmid Mini Kit (China CWBIO) to purify all plasmids and screen stable transfected plasmids with puromycin. Cell transfection using empty vector plvx (plvx-NC) as a negative control. For the knockdown plasmid of Lcn-SHANK3 and MNX1, two shRNAs: sh-SHANK3#1 (5′-TGACCGTCATGTGGGTGTTTGTGTT-3′), sh-SHANK3#2 (5′-TCCCATTACAGAGCTTCCTTGCATT-3′), sh-MNX1#1 (5′-TGATCCTGCCTAAGATGCCCGACTT-3′), sh-MNX1#2 (5′-GCGGATGAAATGGAAACGCAGCAAA-3′) were designed and cloned into plko.1plasmid (Addgene). For the overexpression and knockdown of miR-4530, miR-4530 mimics, miR-4530 inhibitor and two corresponding scramble siRNA were synthesized by Ribobio (China). The wild-type (WT) and mutant (MUT) SHANK3 sequences and the MNX1 3′UTR wild type and mutant fragments containing the assumed miR-4530 binding site were obtained through site-specific mutagenesis using Vazyme's Mut Express MultiS Fast Mutagenesis Kit. The primers used were as follows: WT-pmir-SHANK3 forward: 5′-TAGTTGTTTAAACGAGCTCGGACCCTGTCCCATTACAGAGC-3′; reverse: 5′-ACTCTAGACTCGAGGCTAGGTCGTAGGAGCCCACTGTGTA-3′. MUT-pmir-SHANK3 forward: 5′-CGGGGTGATTACGGCACTTCTGCAGCTTCTCCATTGGA-3′; reverse: 5′-AGTGCCGTAATCACCCCGCAATGCAAGGAAGCTCTGTA-3′. WT-pmir-MNX1 forward: 5′-TAGTTGTTTAAACGAGCTCGGAGCCCCGCGGCCCAGCA-3′; reverse: 5′-TTTGAGATTCTTGATGATTTGAACGCTCGTGACATAATC-3′. MUT-pmir-MNX1 forward: 5′-CGTTCAAATCATCAAGAATCTCAAAACTGTACTGTCTTTATTTTTG-3′; reverse: 5′-TTTGAGATTCTTGATGATTTGAACGCTCGTGACATAATC-3′. The constructed fragment was cloned into the pmirGLO double luciferase reporter vector downstream of the luciferase reporter gene to produce SHANK3-WT, SHANK3-Mut, MNX1-WT, MNX1-Mut luciferase vectors. According to manufacturer's instructions, all transfections were performed in HEK293T cells using Lipofectamine 2000 (Invitrogen).

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA and miRNA were extracted using miRNeasy Mini Kit (QIAGEN)and cDNA was synthesized by HiScript II Q RT SuperMix for qPCR (+gDNA wiper (Vazyme China) using 1ug RNA as a template. Quantitative real-time PCR (qPCR) using Hieff qPCR SYBR Green Master Mix (Yeasen China) and all amplification assays were performed on the CFX96 system. (Bio-Rad).Using 2^−∆∆Ct method measures relative transcriptional abundance and normalizes mRNA expression using GAPDH. U6 was used to normalize the samples. The primers sequence for qPCR are as follows: LncSHANK3 forward: 5′-TACAGGGTCAGTTCCGCTTC-3′; reverse: 5′-CGGTATGAGGGACAGACACA-3′. MNX1 forward: 5′-CTCCTACTCGTACCCGCAG-3′; reverse: 5′-CTCCTACTCGTACCCGCAG-3′. GAPDH forward: 5′-GGAGCGAGATCCCTCCAAAA-3′; reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′. MiR-4530 forward: 5′-CGGCCCCAGCAGGACG-3′; reverse: 5′-ACTGCAGGGTCCGAGGTATT-3′. U6 forward: 5′-CTCGCTTCGGCAGCACA-3′; reverse: 5′- AACGCTTCACGAATTTGCGT-3′. For miR-4530 and U6, special stem-loop primers are needed to reverse transcribe. MiR-4530 (5′-GTCGTATCGACTGCAGGGTCCGAGGTATTCGCAGTCGATACGACGTCC-3′); U6 (5′- AACGCTTCACGAATTTGCGT-3′).

RNA immunoprecipitation assay

Transfect MGC803 cells with miR-4530 mimic or mimic control for 48 h, wash with pre cooled PBS, and then lyse with RNA immunoprecipitation lysis buffer. Incubate the cell lysate with magnetic beads bound to anti AGO2 antibodies or mouse immunoglobulin as a negative control. Collect small magnetic beads, wash them, extract RNA in the presence of protease K, and detect SHANK3 using qRT PCR. Cell lysate is used as input

Western blot assayFor the extraction of total protein, the first step is to lyse cells with RIPA lysate added with protease inhibitors and quantified using BCA. Protein sample (30ug) was segregated by 12% SDS-PAGE and subsequently transferred to a polyvinyl fluoride (PVDF) membrane. After being enclosed in 5% skim milk for 3 h, the membrane was enclosed overnight with the primary antibody of MNX1 (ABclonal, A19690, 1:l000), AGO2 (ABclonal, A6802, 1:1000), IgG (ABclonal, AC005, 1:200) and GAPDH (ABclonal, A19056, 1:10,000) at 4 °C, and incubated with the secondary antibody with horseradish peroxidase (HRP) for 1 h. Finally, the membrane was placed in the ECL luminescent solution, and then imaging was carried out. The level of target protein expression in each sample was normalized by GAPDH.

Luciferase reporter assay

The constructed luciferase reporter plasmid and the internal reference plasmid were co-transfected into the target cells, and 48 h later, the lysate was added into the kit for 20 min. The detection work was carried out according to the instructions of the luciferase reporter activity analysis kit. The absorbance was determined using the a multifunctional enzyme maker, and the fluorescence value was divided by the protein content to represent the relative luciferase activity.

Cell proliferation assays

Cell suspension was inoculated into 96-well plates, with 2000 cells contained in 100ul culture medium in each well. After culturing the cells until they adhere to the wall, add 10ul of CCK8 solution to each wall, incubate for 2 h, and then measure the absorbance at 450 nm.The absorbance at 450 nm of cells at 0 h, 24, 48, 72, 96 h was measure at the same time every day

colony formation assays

The stable transfected cell count was planted on a 6-well plate with 500 cells per well, and 2ML medium was added. The growth of the cells was observed daily until a significant number of cell colonies were formed and in good condition. The cells were fixed with polyformaldehyde, stained with crystal violet, washed with PBS, and photographed to calculate the clone formation rate.

Transwell assays

The stably transfected well cell was planted in 6-well plates, and the cell density was ensured to reach 40–50% within 8–10 h. Discard the original medium when the density is appropriate, add serum-free medium, starve for 8–12 h, spread matrix glue when hungry, add 30ul to each cell. Place in a 37°incubator to solidify and spread cell. Each chamber was added with 200ul medium containing 5 × 10⁴ cells, and 500ul complete medium having 20% serum was put in below chamber so that nutrient concentration difference was formed between the upper and lower chambers, and place them in a constant temperature incubator for continued culture. After 24 h, take out the chamber, discard the culture medium, wash it 2–3 times with PBS, and fix it at room temperature with 4% paraformaldehyde for 20 min, stained with crystal violet at room temperature for 10 min, cleaned several times with 1 × PBS and dried. Finally, the cells were photographed under the microscope and counted in 5 random fields.

Wound healing assay

Several types of stably transfected gastric cancer were selected and placed in the 6-well plate, and the cell density was guaranteed to reach 80–90% in 8–10 h. The black lines perpendicular to the marks outside the 6-well plate were drawn in the petri dish with a 200ul gun head, washed 3 times with 1 × PBS, washed off the dead cells, then continued to culture with 2% FBS and double antibody medium, photographed under the microscope and recorded for 0 h, continued to be placed in the incubator for culture, photographed and recorded for 24 h.

Tumor formation assay

The animal experiment has been approved by the Experimental Animal Center and the Ethics Review Committee of Experimental Animals at Wuhan University of Science and Technology. Nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.Four week old mice were randomly divided into six groups. Replace pcDNA3.1, SHANK3, SHANG3 +sh-MNX1 stable transfection of into HGC27 cells, stable transfection of sh-NC, sh-SHANK3, sh-SHANK3+MNX1 into MGC803 cells, and then, these stable transfected cells were transfected with 1 × 10⁶ density was inoculated subcutaneously in 4-week-old female mice, and the tumor size and volume of the mice were regularly checked. After 30 days of implantation, the mice were euthanized through cervical dislocation and the tumor was taken for further analysis.

Statistical analysis

Snapgene software (3.2.1) was used for primer design, Image Lab software (5.2.1) was used for Western blot result analysis, and Image J software (1.51j8) is used to gray-scale analysis of Western blot results. GraphPad prism software (8.0.2) is used to convert experimental data into visual charts and perform significance analysis, while exporting charts. The analysis of experimental results was represented by Mean±SD or Mean±SEM. For experimental results, student-t-test was used to analyze the significance between the two groups of data, and one-way ANOVA was used to analyze the significance between multiple groups of data.

Funding

This work was financially supported by National Natural Science Foundation of China (No. 31501149, 31770815, 31570764, 82203497, 82203497), Hubei Natural Science Foundation 2021CFB230, 2022CFB026 and 2024AFB905), the Wuhan Health and Family Planning Scientific Research Project (WX21Q49), Educational Commission of Hubei (B2020001), Hubei Province Health and Family Planning Scientific Research Project (WJ2021Q051, WJ2019M255), Hubei Province Key R&D Program (No. 2022BCA007) and Frontier project of applied basic research in Wuhan (2020020601012250), The 14th Five Year Plan Hubei Provincial advantaged characteristics disciplines (groups) project of Wuhan University of Science and Technology (No. 2023C0303), China Postdoctoral Science Foundation (2021M702538).

CRediT authorship contribution statement

Li-Li Zhao: Writing – review & editing, Writing – original draft, Data curation, Conceptualization. Yuan Xiang: Writing – original draft, Software, Conceptualization. Jin-Xuan Wang: Visualization, Software, Data curation. Chao Shen: Resources, Project administration, Data curation. Hui Liu: Visualization, Validation, Supervision, Software. Qi-Bei Zong: Investigation, Data curation. Hui-Min Zhang: Visualization, Software. Jia-Peng Li: Conceptualization, Writing – review & editing. Cong Wang: Conceptualization, Writing – review & editing. Fan Sun: Conceptualization, Writing – review & editing. Xing-Hua Liao: Conceptualization, Project administration, Validation, Writing – review & editing.

Declaration of competing interest

We declare that we have no financial or personal relationships with other people or organizations that improperly affect our work, nor do we have any nature or professional or other ongoing interests, such as any products, services, and/or companies, that may be interpreted as affecting the position in this initial draft or the review of this manuscript.