miR-199a/b-3p inhibits HCC cell proliferation and invasion through a novel compensatory signaling pathway DJ-1\Ras\PI3K/AKT

Several studies have reported the effects of DJ-1 gene and miR-199a/b-3p on HCC development. However, whether miR-199a/b-3p regulates HCC progression through a novel compensatory signaling pathway involving DJ-1, Ras, and PI3K/AKT remains unknown. We used (TCGA, HPA, miRWalk and Target scan) databases, cancer and para-tissue HCC patients, dual-luciferase reporter gene analysis, proteomic imprinting, qPCR, cell proliferation, scratch, transport, and flow cytometry to detect the molecular mechanism of DJ-1 and miR-199a/b-3p co-expression in HCC cell lines. Bioinformatics analysis showed that DJ-1 was highly expressed in HCC ((P < 0.001) were closely associated with tumor stage (T), portal vein vascular invasion, OS, DSS, and PFI (P < 0.05); miR-199a/b-3p was lowly expressed in HCC (P < 0.001), which was the upstream regulator of DJ-1. Spearman coefficient r = −0.113, P = 0.031; Dual luciferase gene report verified the negative targeting relationship between them P< 0.001; Western blotting demonstrated that miR-199a/b-3p could inhibit the protein expression of DJ-1, Ras and AKT(P < 0.05); The results of CCK8, cell scratch, Transwell migration and flow cytometry showed that OE + DJ-1 increased the proliferation, migration and invasion ability of HepG2 cells, and decreased the apoptosis process, and the differences were statistically significant (P < 0.05), while miR-199a/b-3p had the opposite effect (P < 0.05).


Immunohistochemical detection of DJ-1 protein in hepatocellular carcinoma and paracancer tissues
5 pairs of liver cancer and adjacent tissue samples were collected from patients with hepatocellular carcinoma after surgery in the Department of Hepatobiliary Surgery of Ningxia Medical University General Hospital.The tissue specimens were fixed in formalin solution and embedded with paraffin wax.Each sample was then sectioned into 4um thick slices, which were subjected to standard dewaxing and hydration procedures.Antigen retrieval was performed using target search solution (Dako, CA), followed by blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide for 15 min.The slices were then blocked with goat serum, avidinin solution, and biotin solution, and incubated overnight at 4 °C with rabbit anti-human DJ-1 primary antibody (dilution 1:1000).High-sensitivity streptavidin-HRP conjugate detection and biotinylated goat anti-rabbit secondary antibody (Vector Laboratories CA) were used for detection.The slices were then incubated in Tris-HCl buffer with 1% H2O2 for 30 min, followed by DAB color development and counterstaining with hematoxylin QS (Vector Laboratories, CA).Finally, professional pathologists analyzed and interpreted the findings.
In accordance with the Declaration of Helsinki on Human Research, with the approval of the Ethics Committee of Ningxia Medical University General Hospital and the informed consent of patients and their guardians.In this paper, patient information is concealed.

Western blot detection
Collect the cultured cells and extract total cell protein using RIPA lysate.Quantify the protein concentration using BCA assay.Prepare a 10% SDS-PAGE gel for protein electrophoresis and transfer the protein onto a membrane.Block the membrane with 5% skim milk at room temperature, and then incubate with primary antibody (dilution ratio 1:1000) overnight at 4 °C.The next day, incubate the membrane with HRP-labeled secondary antibody (dilution ratio 1:5000), and wash with PBST three times for 10 min each.Finally, visualize the protein bands using SuperSignalWest Pico Plus and image with a BIO RAD gel imager.Analyze the grayscale values of the protein bands using Image Lab software.This method serves as an internal reference.

Real-time PCR detection
The experimental procedure involved the extraction of total RNA following the instructions of the TB Green Premix Ex TaqII (Tli RNaseh Plus) (Code No-RR820A) kit.The RNA was then reverse transcribed into cDNA using quantitative PCR, and real-time PCR (RT-qPCR) was performed using the LightCycler 480 system.The Ct value was obtained from the PCR instrument software, and the relative expression of mRNA was calculated using the 2-△△Ct method (△Ct = Ct target gene-△Ct internal control, △△Ct = △Ct experimental group-△Ct control group).Statistical analysis was carried out, and all experiments were repeated three times.

Transwell and cell scratch experiments
HepG2 cell migration and invasion ability detection using Transwell chamber, culture HepG2 cells, place 200L of cell suspension in transwell chamber, add 500L of complete medium containing 10% FBS to the 24-well plate, place the chamber in the plate, culture in the incubator for 48 h, wash off the medium with PBS, fix paraformaldehyde for 20 min, wash 2 times with PBS, stain 0.5% crystal violet for 10 min, wash off crystal violet, Under an inverted microscope, photograph the non-cellular seeding side.The experiment was carried out three times.Scratch experimental cell culture with the previous use of marker pen at the bottom of the 6-well plate evenly draw parallel straight lines, the cells are made into 2.5*105 / mL suspension, 2 mL is added to each well of the 6-well plate, the cells are crossed out with the tip along the direction perpendicular to the horizontal line of the marker's stroke, the scratched cells are removed, the corresponding serum-free medium is added, and the picture is taken under an inverted microscope to take pictures of the intersection of the horizontal line of the marker stroke and the horizontal line of the cell drawn by the tip of the gun, and this time point is recorded as 0 h.Continue to culture for 24 h, and take pictures again around the intersection point taken at 0 h, and this time point is recorded as 24 h.The experiment was repeated 3 times.

Cell flow cytometry to detect apoptosis
Culture HepG2 cells, digest the cells with EDTA-free pancrepsin, centrifuge the cells at 4 °C for 5 min, wash the cells with pre-chilled PBS 2 times, resuspend the cell pellet with 300uL of 1 × Binding Buffer, add 5uL Annexin V-FITC, mix and incubate in the dark for 10 min, add 5 uL PI, mix well and incubate in the dark for 5 min, machine detection within 1 h, FITC excitation 494 nm emission 520 nm, PI Excitation of 493 nm emits 636 nm.The experiment is repeated 3 times.www.nature.com/scientificreports/

Expression of DJ-1, Ras protein and AKT signaling protein molecules in PI3K/AKT signaling pathway
Same method 1.7 for cell culture and grouping,Then, protein imprinting experiments were used to detect the expression of AKT signaling protein molecules in DJ-1, Ras protein and PI3K/AKT signaling pathway in each group of cells.

Statistical methods
SPSS26.0 was used to analyze the experimental results; data conforming to the positive Pacific distribution were expressed inX-± S, the independent sample t-test was used to compare the two groups, and the Mann-WhitneyU test was used for the non-positive Pacific distribution sample, and P < 0.05 was considered statistically significant.

Ethic approval and consent to participate
This study was approved by the Ethics Committee of Ningxia Medical University General Hospital (approval number: KYLL 2021-85) .Patients were consented by an informed consent process that was reviewed by the Ethics Committee of Ningxia Medical University General Hospital and certify that the study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki.

Correlation analysis of DJ-1 expression with HCC
The expression level of the DJ-1(PARK7) in HCC were analyzed by using the TCGA-LIHC( including normal liver tissue 50 cases, paracancerous 50 cases, cancer tissue 374 cases), HPA database and immunohistochemical testing.Results showed that DJ-1 expression was significantly higher in cancer tissues of HCC patients compared to adjacent tissues and normal liver tissue, and the difference was statistically significant, P < 0.001 ( as shown in Fig. 1a, b ,c and d) .The expression levels of DJ-1 mRNA in TNM stage III and IV HCC tissues ,with vascular invasion ,and pathological grading of 3 and 4 were higher than those in TNM stage I and II tissues(P < 0.01), without vascular invasion, and grading of 1 and 2(P < 0.05).www.nature.com/scientificreports/(as shown in Table 1).The KM curve and ROC curve analysis showed that the expression level of DJ-1in HCC was significantly associated with overall survival (OS), disease-specific survival (DSS), and tumor progressionfree interval (PFI) (Fig. 2a-c).The diagnostic ROC curve demonstrated that DJ-1 had predictive value for clinical diagnosis, with an area under the curve of 79.6%, sensitivity of 84%, and specificity of 71.7% (Fig. 2d).

Prediction, validation, and molecular correlation analysis of miRNAs
Noncoding RNA can affect disease progression by regulating gene expression, but there are few studies on the process of its regulation of DJ-1 gene expression affecting HCC patients.To shed further light on the regulatory mechanism of DJ-1 in HCC patients, we conducted miRWalk and TargetScan predictive analysis using the human gene DJ-1 as the target gene and Wayne diagram for miRNAs screening(Fig.3a),We then analyzed the expression relationship of the overlapping miRNAs using TCGA-LIHC miRNA-seq and found that has-miR-199a/b-3p was poorly expressed in HCC tissues.Moreover, ROC curve analysis showed that has-miR-199a/b-3p had better diagnostic predictive value for HCC than other miRNAs, with an area under the curve of 88.2%, sensitivity of 90%, and specificity of 82.1% (Fig. 3b and c).Analysis of molecular correlation showed that hsa-miR-199a-3p exhibited a negative correlation with DJ-1, the Spearman correlation coefficients of r = −0.113and P = 0.031 (Fig. 3d).Based on the bioinformation results presented above, we conducted Rt-PCR experiments on LO2 and HepG2 cell lines.Our findings indicate that has-miR-199a/b-3p was statistically significant in HepG2 (0.6432 ± 0.4388), which was lower than in LO2 (1.1979 ± 0.9177) (P < 0.05) (Fig. 3e).Bisluciferase gene report verification showed that the diluciferase activity of the wild-type DJ-1 gene plasmid vector and mutant DJ-1 gene plasmid vector in the hsa-miR-199a-3 pmimics group was significantly reduced compared with the diluciferase Table 1.The correlation between DJ-1 expression and HCC TNM staging, pathological grading, and vascular invasion.Significant values are in bold.activity of the wild-type DJ-1 gene plasmid vector.This indicates that hsa-miR-199a-3p could bind to the wildtype DJ-1 gene and inhibit its expression, further supporting the existence of the targeted binding relationship mentioned above (Fig. 3f).

Effect of DJ-1 and miR-199a-3p on HepG2 cell function
The effects of DJ-1 gene, mi199a-3p and DJ-1 combined with miR199a-3p on HCC cell function were compared in HepG2 cell line.The detection methods and cell grouping were the same as 1.7.Cell function test results were shown in Fig. 4, The results showed that compared with the blank control group, overexpression of DJ-1 gene could promote the proliferation, migration and invasion of HepG2 cells and inhibit the process of apoptosis (as shown in groups 4a, b, c, d, e, f, D2 and F2).The intervention of miR-199a-3p mimics/inhibitors significantly inhibited the proliferation, migration and invasion of HepG2 cells.It promoted cell apoptosis (groups 4a,b,c,d,e,f, E2, H2), (groups 4a,b,c,d,e,f, G2, I2) and verified that miR199a-3p affected the cellular function of HepG2 cells by inhibiting the DJ-1 gene in both positive and negative aspects .

Molecular verification of the expression relationship between hsa-miR-199a-3p and DJ-1 gene in HepG2 cells and signaling pathway
By reading the literature, we predict that Ras and PI3K/AKT may be the compensatory circuits of DJ-1 gene function during HCC progression.To verify this hypothesis, HepG2 cells were transfected with miR-199a-3p mimics, inhibitors and OE-DJ-1, and HepG2 cells were untransfected as the control group.The protein expressions of DJ-1, Ras, AKT and β-actin as internal reference genes in different groups were detected, and the cell group was the same as 1.7.The detection method is the same as 1.8.The expression analysis of DJ-1 gene in the six groups of cells (Fig. 5 a, b) showed that the expression of DJ-1 gene in the E2 group (0.087 ± 0.03) was significantly lower than that in the D2 group (0.219 ± 0.08), and the difference was statistically significant (P = 0.023, P < 0.05).The DJ-1 gene expression in the F2 (0.648 ± 0.08), G2 (0.407 ± 0.03), H2 (0.393 ± 0.02), and I2 (0.832 ± 0.1) groups was significantly increased compared with that in the D2 group, and the difference was statistically significant (P < 0.05).This indicates that miR-199a-3p mimic can significantly inhibit the expression of DJ-1 gene in HepG2 cells, while overexpression of DJ-1 gene can partially reverse miR-199a-3p, and miR-199a-3p inhibitor has the opposite effect.The expression of Ras protein molecules showed that the E2 group (0.231 ± 0.02) was lower than the D2 group (0.362 ± 0.06), and the difference was statistically significant (P = 0.002, P < 0.05).The expression of Ras protein in the F2 (0.938 ± 0.02), G2 (0.819 ± 0.06), H2 (0.81 ± 0.04), and I2 (1.317 ± 0.03) groups was significantly enhanced compared with that in the D2 group, and the difference was statistically significant (P < 0.05) (Fig. 5 a, c).This indicates that overexpression of DJ-1 gene can promote the expression of Ras, while miR-199a-3p can inhibit its expression.The expression of AKT signaling molecules showed that the E2 www.nature.com/scientificreports/ group (0.121 ± 0.05) was lower than the D2 group (0.286 ± 0.07), and the difference was statistically significant (P = 0.009, P < 0.05).The expression of AKT protein in the F2 (0.543 ± 0.08), G2 (0.419 ± 0.08), H2 (0.429 ± 0.08), and I2 (0.754 ± 0.01) groups was higher than that in the D2 group, and the difference was statistically significant (P < 0.05) (Fig. 5 a, d).The above results showed that DJ-1 gene overexpression could activate Ras and AKT protein, miR-199a-3p mimics could increase the expression of DJ-1 gene, and miR-199a-3p could inhibit the activation of oncogene Ras and AKT protein molecules by inhibiting the expression of DJ-1 gene.

Discussion
Although the diagnosis and treatment of HCC has improved significantly, the five-year survival rate for HCC is still less than 15% 1-3 .The fundamental reason is that the molecular mechanism involved in the occurrence of HCC is extremely complex, and there is heterogeneity in HCC occurrence among patients, which may also be one of the reasons for the different treatment effect outcomes of patients.Therefore, comprehensively identifying the key target molecules involved in the progression of HCC, elucidating their pathogenesis and molecular mechanisms affecting drug sensitivity, and formulating precise individualized protocols according to patient heterogeneity may be effective methods to solve the poor prognosis and efficacy of HCC.molecule and inhibit the activation of PI3K/AKT signaling pathway key protein AKT.Conversely, increasing the expression of DJ-1 can reverse the inhibitory effect of miR-199a/b-3pminic on HepG2 cell function.In summary, these results show that miR-199a/b-3p can bind to DJ-1 gene targeting and inhibit the activation of PI3k/AKT signaling pathway by downregulating the expression of Ras protein, inhibit HepG2 cell proliferation, proliferation and migration invasion, and promote HepG2 cell apoptosis, which verifies our experimental hypothesis.To our knowledge, this is the first demonstration of the miR-199a/b-3p/DJ-1/Ras/PI3k/AKT axis as a study involved in regulating HepG2 cell proliferation, apoptosis, and migration invasion.In addition, our findings are significant because it provides a better understanding of the pathogenesis of HCC and provides a new approach to predicting and assessing prognosis and potential therapeutic targets for HCC.

Conclusions
In summary, our study suggests that miR-199a/b-3p, as an upstream regulatory molecule of DJ-1, may inhibit cell proliferation and invasion of HCC and promote cell apoptosis through a novel compensatory signaling pathway involving miR-199a/b-3p\DJ-1\Ras\PI3K/AKT.

Figure 1 .
Figure 1.DJ-1 expression was significantly higher in cancer tissues of HCC patients compared to adjacent tissues and normal liver tissue, and the difference was statistically significant, P < 0.001 (a,b) DJ-1 gene expression in HCC in TCGA database.(c) DJ-1 IHC results of HCC tissue and normal liver tissue in the HPA database,Patient information(Normal Patient No: 1720,1899,1846,Tumor Patient No:879,1163,983,Antibody: HPA004190); d:DJ-1 IHC results in three pairs of paired clinical HCC samples.

Figure 2 .
Figure 2. The correlation between DJ-1 expression and OS,DSS,PFI and value for clinical diagnosis in HCC.

Figure 5 .
Figure 5. Shows the expression of DJ-1 gene, Ras protein and AKT protein in HepG2 cell lines before and after the regulation of miR-199a-3p by Westenblot technology, β-acting as the internal reference gene, the results are expressed in X-± S, P < 0.05 is statistically significant, and the number of experimental replicates n = 3.