Identification of Tumor-Suppressive miR-139-3p-Regulated Genes: TRIP13 as a Therapeutic Target in Lung Adenocarcinoma

Simple Summary Based on the miRNA expression signature of LUAD, we focused on miR-139-3p, a passenger strand, and clarified its tumor-suppressive function in lung adenocarcinoma (LUAD) cells. A total of the 21 target genes (KRT80, CENPM, SPC24, ORC1, MYEOV, TRIP13, GPX8, ARHGEF39, MKI67, KIF18B, CHAF1B, CP, GPRIN1, UCK2, CHEK1, HELLS, CTSV, FAM111B, SLC16A3, MELK, and CENPF) were identified as miR-139-3p targets in LUAD, and the expression of these genes was as independent prognostic factor for patient survival. Moreover, inhibition of TRIP13 using a specific inhibitor (DCZ0415) enhanced the sensitivity of LUAD cells to anticancer drugs. Abstract Analyses of our microRNA (miRNA) expression signature combined with The Cancer Genome Atlas (TCGA) data revealed that both strands of pre-miR-139 (miR-139-5p, the guide strand, and miR-139-3p, the passenger strand) are significantly downregulated in lung adenocarcinoma (LUAD) clinical specimens. Functional analyses of LUAD cells ectopically expressing miR-139-3p showed significant suppression of their aggressiveness (e.g., cancer cell proliferation, migration, and invasion). The involvement of the passenger strand, miR-139-3p, in LUAD pathogenesis, is an interesting finding contributing to the elucidation of unknown molecular networks in LUAD. Of 1108 genes identified as miR-139-3p targets in LUAD cells, 21 were significantly upregulated in LUAD tissues according to TCGA analysis, and their high expression negatively affected the prognosis of LUAD patients. We focused on thyroid hormone receptor interactor 13 (TRIP13) and investigated its cancer-promoting functions in LUAD cells. Luciferase assays showed that miR-139-3p directly regulated TRIP13. siRNA-mediated TRIP13 knockdown and TRIP13 inhibition by a specific inhibitor (DCZ0415) attenuated the malignant transformation of LUAD cells. Interestingly, when used in combination with anticancer drugs (cisplatin and carboplatin), DCZ0415 exerted synergistic effects on cell proliferation suppression. Identifying the molecular pathways regulated by tumor-suppressive miRNAs (including passenger strands) may aid in the discovery of diagnostic markers and therapeutic targets for LUAD.


Introduction
Lung cancer is the leading cause of cancer-related death worldwide, with approximately 2.3 million new cases of lung cancer and 1.8 million lung cancer-related deaths each year [1].Patient prognosis is extremely poor, with a 5-year overall survival rate of only 5% for patients with metastatic cancer.Even in patients whose tumors are localized to the lungs, the overall survival rate ranges from 33% to 60% [2].According to the histological classification of lung cancer, approximately 85% of all lung cancers are non-small-cell lung cancer (NSCLC), the majority of which are lung adenocarcinoma (LUAD) [3].The treatment methods for LUAD include surgery, radiotherapy, chemotherapy, molecular targeted therapy, and immunotherapy, and the treatment strategy is determined by the disease stage and the presence or absence of driver gene mutations [3].Molecularly targeted drugs that address genetic mutations in driver genes are being developed rapidly, and many patients will benefit from these drugs [3].However, in the past, cytotoxic anticancer drugs were the mainstay of drug therapy for advanced-stage LUAD in which molecularly targeted drugs were not applicable.Recently, the effectiveness of immunotherapy has been confirmed, and immunotherapy alone or in combination with anticancer drugs is being used to treat LUAD [4].It is essential to develop new treatment regimens that increase the effectiveness of anticancer drugs for advanced-stage patients without driver gene mutations.
In the post-genomic era, it has been discovered that the human genome contains an extremely large number of functional RNAs that do not encode proteins.We realized the importance of clarifying their functions in normal and diseased cells [5].microRNAs (miRNAs) are short (~22 nucleotides) non-coding RNAs that act mainly as gene expression regulators at the post-transcriptional level in a sequence-dependent manner [6].A single miRNA controls the expression of many different types of genes, and miRNAs play important roles in physiological cellular processes [7].
Numerous studies have shown that the aberrant expression of miRNAs is closely involved in cancer cell malignant transformation, e.g., proliferation, invasion, metastasis, and drug resistance [8].Traditionally, it was thought that only the guide strand of miRNAs derived from pre-miRNAs was functional in cells, and therefore, cancer research has been focused on the guide strands.Our recent studies revealed that the passenger strand of miRNAs derived from pre-miRNAs is intricately involved in the molecular pathogenesis of human cancers, including lung cancer [9,10].We can uncover new molecular pathways in cancer cells by examining the molecules regulated by both strands of miRNAs derived from pre-miRNAs.
Recently, we created a miRNA expression signature based on LUAD tissues using RNA sequencing [10].Analysis of this signature showed that both the guide and passenger strands of pre-miR-139 (miR-139-5p and miR-139-3p, respectively) were downregulated in LUAD tissues.Focusing on miR-139-3p, we have identified its tumor-suppressive roles and cancer-promoting target genes in various cancer types (e.g., bladder cancer, renal cell carcinoma, oral cancer, and colorectal cancer) [11][12][13][14].A unique feature of miRNAs is that the target genes of a specific miRNA differ depending on the cell.Our previous studies confirmed that the same tumor-suppressive miRNA regulates different genes depending on the cancer type [11][12][13][14].The aim of this study was to confirm the tumor-suppressive function of miR-139-3p and to clarify the molecular networks controlled by miR-139-3p in LUAD cells.Ectopic expression of miR-139-3p in cancer cells significantly blocked proliferation and induced cell cycle arrest and apoptosis.Regarding miR-139-3p target genes, thyroid hormone receptor interactor 13 (TRIP13) was found to be directly regulated by tumor-suppressive miR-139-3p in LUAD cells.Aberrant expression of TRIP13 facilitated the malignant transformation of LUAD cells.Importantly, a TRIP13-specific inhibitor (DCZ0415) exerted synergistic effects on the suppression of cell proliferation when used in combination with various anticancer drugs (e.g., cisplatin and carboplatin).
Exploring the oncogenic networks controlled by tumor-suppressive miRNAs, including the passenger strands, will facilitate the identification of therapeutic target molecules for LUAD.

Functional Assay of miRNAs and miRNA Target Genes in LUAD Cells
Two LUAD cell lines, A549 and H1299, were used in the functional assays in this study (American Type Culture Collection, Manassas, VA, USA).Small RNAs (miRNAs and siRNAs) were transfected into LUAD cell lines, and cell proliferation, migration, and invasion were evaluated and compared with those of control cells.The transfection procedures of miRNAs and siRNAs were described in our previous studies [10,[15][16][17].All miRNA precursors were transfected at 10 nM, and all siRNAs were transfected at 5 nM into A549 and H1299 cell lines using RNAiMAX (Invitrogen, Carlsbad, CA, USA).Mock was a group without precursors or siRNAs.Cell cycle analysis was performed using a flow cytometer (BD FACSCelestaTM Flow Cytometer, BD Biosciences, Franklin Lakes, NJ, USA).Details of the cell functional assays are described in our previous paper [10,[15][16][17].The reagents used for these analyses are listed in Table S1.
We used GeneCodis4 software to infer the molecular functions of the miR-139-3p target genes [18].Gene set enrichment analysis software was used to infer the molecular pathways controlled by these genes [19,20].

Dual-Luciferase Reporter Assay
We conducted a dual-luciferase reporter assay to confirm that miR-139-3p binds directly to the 3 UTR of the TRIP13 gene.The miR-139-3p-binding sequence cloned into the psiCHEK2 vector (C8021; Promega, Madison, WI, USA) is shown in Figure S1.The dualluciferase reporter assay procedure was described in our previous studies [10,[15][16][17].Transfection of the purified plasmid vectors into LUAD cells was performed using Lipofectamine 2000 (Invitrogen) at 50 ng/well.After 72 h of transfection, we conducted dual-luciferase reporter assays using the Dual Luciferase Reporter Assay System (Promega).The reagents used for these analyses are listed in Table S1.

Anticancer Effects of DCZ0415 in LUAD Cells
To determine the anticancer effects of cisplatin, carboplatin, and DCZ0415 (TRIP13 inhibitor) in LUAD cells, XTT assays were conducted.XTT assays using Cell Proliferation Kits (catalog no.: 20-300-1000, Biological Industries, Beit-Haemek, Israel) were performed to assess cell proliferation.LUAD cells were plated at 4.0 × 104 cells per well in 96-well plates.Then, the half-maximal inhibitory concentration (IC50) values of these drugs were calculated using GraphPad Prism8 software.

Statistical Analysis
In this study, statistical analyses were performed using JMP Pro 16 (SAS Institute Inc., Cary, NC, USA).Analysis of differences between the two groups used Welch's t-test.Differences among multiple groups were analyzed using Dunnett's test.Patient survival rates were analyzed using Kaplan-Meier survival curves and the log-rank test.

Tumor-Suppressive Function of miR-139-3p in LUAD Cells
Analysis of our miRNA expression signature created by RNA sequencing showed that both strands of pre-miR-139 were downregulated in LUAD tissues (Figure 1A).According to the miRNA database miRBase, miR-139-5p is annotated as the guide strand derived from pre-miR-139 and miR-139-3p as the passenger strand (Figure 1B).Downregulation of these miRNAs in LUAD clinical specimens was confirmed by analysis of TCGA-LUAD datasets from TCGA (Figure 1C).A positive correlation was detected between miR-139-5p and miR-139-3p expression levels by Spearman's rank analysis (r = 0.513, p < 0.001; Figure 1D).
Our research interests were to elucidate the function of the passenger strands of miRNAs and identify novel oncogenic pathways regulated by tumor-suppressive miRNAs in LUAD cells.The tumor-suppressive roles of miR-139-3p were evaluated by transient transfection assays using two LUAD cell lines, A549 and H1299.We also investigated the functional significance of miR-139-5p (the guide strand) in LUAD cells.Surprisingly, compared to miR-139-3p, miR-139-5p had poor tumor suppressive function in LUAD cells (Figure S2).
Cancer cell proliferation was significantly suppressed by ectopic expression of miR-139-3p in A549 and H1299 cells (Figure 2A).Cell cycle assays demonstrated an increased proportion of cells in the G0/G1 phase after the induction of miR-139-3p expression (Figure 2B).Furthermore, cell invasion and migration were significantly suppressed by ectopic expression of miR-139-3p in LUAD cells (Figure 2C,D).Typical images from the invasion and migration assays after miR-139-3p transfection are shown in Figure S3.
These results suggest that miR-139-3p acts as a tumor-suppressive miRNA in LUAD cells.

Identification of Cancer-Promoting Genes Regulated by miR-139-3p in LUAD Cells
Next, we aimed to clarify the molecular network regulated by tumor-suppressive miR-139-3p in LUAD cells.Our strategy for identifying miR-139-3p targets is shown in Figure 3.
In a search of the TargetScanHuman database (release 8.0), 3145 genes were identified as having miR-139-3p-binding sequences within their 3 UTR.We applied microarray analysis to search for genes with suppressed expression after miR-139-3p transfection in A549 cells.A total of 7608 genes were downregulated by miR-139-3p transfection.Gene expression data were deposited in the GEO database (accession number GSE242241).We integrated these two datasets to narrow down the gene list, and 1108 genes remained as candidate miR-139-3p targets in LUAD cells.Among these target genes, 52 were upregulated in LUAD tissues according to the analysis of the TCGA-LUAD dataset (Table 1), of which 21 genes (Bold) were significantly upregulated (Figure 4).Notably, high expression of these 21 genes was negatively associated with the prognosis of LUAD patients (Figure 5).Moreover, we investigated the extent to which expression of a negative correlation between miR-139-3p and their target genes in LUAD clinical specimens (Figure S4).Spearman's rank test indicated negative correlations between the expression levels of miR-139-3p and 13 target genes (CENPM, SPC24, ORC1, TRIP13, ARHGEF39, MKI67, KIF18B, CHAF1B, CHEK1, HELLS, FAM111B, MELK, and CENPF).

Direct Regulation of TRIP13 by miR-139-3p in LUAD Cells
To identify new therapeutic targets for LUAD from among the 52 genes (Table 1), we functionally classified these genes using GeneCodis4 software (https://genecodis.genyo.es/(accessed on 24 May 2023)) [17].Among these 52 genes, 13 are involved in "cell cycle" and "DNA replication" (Table 2), and 2 (TRIP13 and CENPF) are involved in cell division checkpoints.Some genes (e.g., MCM2, CHEK1, GINS2, ADAM8, and THY1) were involved in multiple molecular pathways.Analysis of these genes is a critical issue for understanding the malignant transformation of LUAD.However, genes that did not affect prognosis were excluded from further study.Since an inhibitor is available for TRIP13, we conducted further analyses using the TRIP13 inhibitor DCZ0415.GEO accession number: GSE242241).A total of 1108 genes were identified as putative miR-139-3p targets.Furthermore, we searched for genes that were associated with the prognosis of LUAD patients using two databases: GEPIA (http://gepia2.cancer-pku.cn/#analysis(accessed on 17 January 2023)) and OncoLnc (http://www.oncolnc.org(accessed on 17 January 2023)).Of the miR-139-3p target genes, 21 were upregulated in LUAD tissues, and these 21 genes were analyzed further.First, we confirmed that TRIP13 mRNA and protein levels were suppressed by ectopic expression of miR-139-3p in LUAD cells (Figure 6A,B).Full-size images of the Western blots are shown in Figure S5.
Next, we confirmed by luciferase reporter assay that miR-139-3p binds directly to the 3'UTR of the TRIP13 gene.The putative miR-139-3p-binding sequence within TRIP13 is shown in Figure 6C.Luciferase activity was significantly suppressed when miR-139-3p and a vector containing a miR-139-3p-binding sequence were simultaneously transfected into LUAD cells (Figure 6D).In contrast, when a vector lacking the miR-139-3p-binding sequence was used, no reduction in luciferase activity was observed (Figure 6D).
These results revealed that miR-139-3p binds directly to the predicted binding sites in TRIP13 genes and regulates its expression in LUAD cells.

Expression and Clinical Significance of TRIP13 in LUAD
Immunostaining was performed to verify the localization of TRIP13 expression in LUAD clinical specimens.Stronger immunostaining of the TRIP13 protein was observed in cancer than in normal lung tissues (Figure 7A).
Multivariate analysis identified TRIP13 expression as an independent prognostic factor for LUAD after adjusting for the clinical prognostic factors of Stage, T-factor, N-factor, age, and gender (Figure 7B).Specifically, high TRIP13 expression was associated with a poorer 5-year overall survival rate.Furthermore, we performed gene set enrichment analysis using TCGA-LUAD data to investigate which molecular pathways were dysregulated in LUAD patients with high TRIP13 expression.The "cell cycle", "DNA repair", "proteasome", "P53 signaling pathway", homologous recombination", and "mismatch repair" pathways were found to be enriched in patients with high TRIP13 expression (Table 3, Figure 7C).It has been revealed that high expression of TRIP13 affected the prognosis of LUAD patients.An important question is which molecular pathway is affected by the high expression of the TRIP13 gene in lung cancer cells.

Effects of TRIP13 Knockdown by siRNAs and TRIP13 Inhibition by a Specific Inhibitor in LUAD Cells
To investigate the functional significance of TRIP13 in LUAD cells, we performed analysis using siRNA to knock down TRIP13.The two siRNAs used in this study (siTRIP13-1 and siTRIP13-2) were confirmed to significantly suppress TRIP13 expression (both at the mRNA and protein levels) in LUAD cells (Figure 8A,B).Full-size images of the Western blots are shown in Figure S6.Cell proliferation was significantly suppressed after transfection of both siRNAs in the LUAD cells (Figure 8C).Cell cycle assays demonstrated an increased proportion of cells in the G0/G1 phase after transfection of both siRNAs in LUAD cells (Figure 8D).

Combined Effects of a TRIP13 Inhibitor (DCZ0415) and Anticancer Drugs on LUAD Cells
We also investigated the anticancer effects of a TRIP13 inhibitor (DCZ0415) combined with anticancer drugs (cisplatin and carboplatin) on LUAD cells.We found that DCZ0415 inhibited the proliferation of LUAD cells in a concentration-dependent manner (Figure 9).Furthermore, the sensitivity of LUAD cells to anticancer drugs (cisplatin and carboplatin) was increased by DCZ0415.Co-treatment of DCZ0415 together with cisplatin or carboplatin resulted in increased sensitivity of A549 and H1299 cells to cisplatin or carboplatin (Figure 10).The IC50 of cisplatin significantly decreased from 3.55 µM to 2.17 µM in A549 cells and from 2.38 µM to 2.03 µM in H1299 cells.The IC50 of carboplatin significantly decreased from 40.98 µM to 21.22 µM in A549 cells and from 59.42 µM to 28.45 µM in H1299 cells.
Additionally, the Chou-Talalay method was used to determine the synergistic effect of two anticancer drugs (cisplatin and carboplatin) and DCZ0415.The results revealed that simultaneous administration of anticancer drugs and DCZ0415 had synergistic effects (Figure 11).

Discussion
In miRNA biogenesis, miRNAs, which function as single-stranded RNAs, are derived from miRNA precursors.Of the two single strands derived from a miRNA precursor, the functional strand is defined as the guide strand and the non-functional strand as the passenger strand [21,22].Generally, the miRNAs evaluated in cancer research have been guide strands.
From previous reports, it is clear that aberrant expression of miR-139-5p plays important roles in various types of cancer cells.In contrast, there have not been many studies on the role of miR-139-3p, the passenger strand, in cancer cells.
We have generated miRNA expression signatures for various cancer types using RNA sequencing and selected tumor-suppressive miRNA candidates based on those signatures [10,[35][36][37].Of these candidates, we identified downregulation and tumor-suppressive functions of miR-139-3p in various cancers, including bladder cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and colorectal cancer [11][12][13][14].Here, we showed that miR-139-3p functions as a tumor-suppressive miRNA in LUAD in addition to other cancer types.
Previous studies showed that miR-139-3p is downregulated in NSCLC tissues and that its expression suppresses cancer cell aggressiveness in vitro and in vivo [38,39].Furthermore, ELAVL1 is directly regulated by miR-139-3p in NSCLC cells [38].ELAVL1 is a member of the ELAVL family of RNA-binding proteins, which contain several RNA recognition motifs, and ELAVL1 overexpression has been observed in several cancers [40][41][42][43][44].More recently, downregulation of miR-139-3p was detected in lung squamous cell carcinoma, and expression of miR-139-3p restrained cell growth and accelerated the cell cycle in this cancer [45].Moreover, the expression of checkpoint kinase 1 (CHEK1) was regulated directly by miR-139-3p in lung squamous cell carcinoma cells [45].CHEK1 is a serine/threonine protein kinase and a key modulator of DNA damage checkpoints [46,47].Our present analysis and previous reports revealed that miR-139-3p has tumor-suppressive functions in lung cancer.
Next, we aimed to elucidate the oncogenic network that is controlled by tumorsuppressive miR-139-3p in LUAD cells.In this study, 52 genes were identified as putative targets of miR-139-3p in LUAD cells.Among these target genes, 21 were closely involved in the molecular pathogenesis of LUAD.Functional classification of these genes revealed that 11 (HELLS, MELK, CHEK1, CHAF1B, CENPF, KIF18B, MKI67, SPC24, FAM111B, ORC1, and TRIP13) are involved in the cell cycle, DNA replication, mitotic checkpoint, and cell division.A very interesting finding is that miR139-3p, a passenger strand, controls genes essential for cell division and maintenance.
The AAA (ATPases associated with various cellular activities) protein family is widely conserved from bacteria to humans, and these ATPases use the energy from ATP hydrolysis to unfold proteins and dissociate complexes [48,49].TRIP13 is a member of the AAA protein family, and it functions as a spindle assembly checkpoint for accurate chromosome segregation during cell division [50].A recent report showed that TRIP13 plays an important role as a gatekeeper for cell maintenance and management (e.g., DNA break repair, recombination, and chromosome synapsis) [51,52].Aberrant expression of TRIP13 has been reported in a wide range of cancers, including lung cancer [50,53,54].The knockdown of TRIP13 in cancer cells significantly suppressed the malignant phenotypes of the cancer cells (i.e., cell proliferation, invasion, and drug resistance) [55,56].In bladder cancer, overexpression of TRIP13 enhanced the resistance of cancer cells to cisplatin and doxorubicin [56].In head and neck cancer, epidermal growth factor receptor (EGFR)-mediated phosphorylation of TRIP13 (pY56) enhanced non-homologous end-joining repair and induced radiation resistance in cancer cells [57].More recently, overexpression of TRIP13 promoted gefitinib (EGFR inhibitor) resistance in NSCLC cells by controlling autophagy and activating EGFR-mediated oncogenic signaling pathways [58].Since TRIP13 overexpression is involved in drug resistance in various cancer cells, TRIP13 might be a potential target molecule for cancer therapy.TRIP13 overexpression is common in BRCA1-deficient cancers, confers PARP inhibitor resistance, and correlates with poor prognosis [59].
Recent studies have reported that various noncoding RNA molecules are involved in the aberrant expression of TRIP13 in cancer cells.miR-515-5p was found to be downregulated in prostate cancer tissues, and TRIP13 was regulated directly by miR-515-5p [60].In hepatocellular carcinoma (HCC), upregulated TRIP13 induced malignant transformation of HCC in vitro and lung metastasis in vivo [61,62].In addition, miR-192-5p regulated TRIP13 expression in HCC cells [63].In colorectal cancer, aberrant expression of TRIP13 contributed markedly to the aggressive phenotype of cancer cells, and its overexpression induced the downregulation of miR-129-5p and miR-4693-5p [64,65].More recently, miR-30c-1-3p and miR-30c-2-3p were found to be significantly downregulated in breast cancer (BrCa) tissues, and their target gene TRIP13 was overexpressed in BrCa tissues [55].Moreover, overexpression of TRIP13 facilitated the aggressiveness of BrCa cells [55].Based on this study and previous data, TRIP13 overexpression is partially due to the downregulation of several tumor-suppressive miRNAs in cancer cells.

Conclusions
Based on the miRNA expression signature of LUAD, we focused on miR-139-3p, a passenger strand, and clarified its tumor-suppressive function in LUAD cells.We revealed that miR-139-3p target molecules are intricately involved in the molecular pathogenesis of LUAD.Inhibition of TRIP13 using a specific inhibitor (DCZ0415) attenuated the malignant transformation of LUAD cells.Interestingly, DCZ0415 exhibited a synergistic effect on the suppression of cell proliferation when used in combination with anticancer drugs.TRIP13 is a potential therapeutic target for LUAD.

Figure 2 .
Figure 2. Effects of ectopic expression of miR-139-3p in LUAD cells (A549 and H1299).(A) Cell proliferation assessed by XTT assay.At 72 h after transient transfection of miRNAs, cancer cell viability was analyzed.(B) Cell cycle status at 72 h after transfection with miR-139-3p assessed using flow cytometry.(C) Cell invasion assessed using Matrigel invasion assays at 48 h after seeding miR-139-3p-transfected cells into the chambers.(D) Cell migration assessed using a membrane culture system at 48 h after seeding miR-139-3p transfected cells into the chambers.****, p < 0.0001.

Figure 5 .
Figure 5. Gene expression in, and 5-year overall survival rate of, patients with LUAD.Kaplan-Meier curves of the 5-year overall survival rates according to the expression of the 21 target genes (KRT80, CENPM, SPC24, ORC1, MYEOV, TRIP13, GPX8, ARHGEF39, MKI67, KIF18B, CHAF1B, CP, GPRIN1, UCK2, CHEK1, HELLS, CTSV, FAM111B, SLC16A3, MELK, and CENPF) are shown.Low expression of all 21 genes was significantly predictive of poorer overall survival in patients with LUAD.The patients (n = 487) were divided into high-and low-expression groups according to the median gene expression level.The red and blue lines represent the high and low expression groups, respectively.

Figure 6 .
Figure 6.Direct regulation of TRIP13 by miR-139-3p expression in LUAD cells.(A) Significant reduction in the TRIP13 mRNA level by ectopic expression of miR-139-3p in LUAD cells (A549 and H1299).Total RNA was extracted 72 h after miR-139-3p transfection into LUAD cells, and expression levels were analyzed by real-time PCR.For miRNA expression, GAPDH was used as an internal control.(B) Significant reduction in the TRIP13 protein level by ectopic expression of miR-139-3p in LUAD cells (A549 and H1299).Protein level expression was determined by Western blotting.Proteins were collected 72 h after miR-139-3p transfection.GAPDH was used as an internal control.(C) Putative miR-139-3p binding sites in the 3 UTR of the TRIP13 gene detected in the TargetScanHuman database (release 8.0).(D) Direct binding of miR-139-3p to target sequences was analyzed by dual luciferase reporter assays.These data showed that miR-139-3p bound directly to the target sequence.***, p < 0.001; ****, p < 0.0001; N.S., not significant.

Figure 7 .
Figure 7. Clinical significance of TRIP13 expression in LUAD.(A) Immunohistochemical staining of TRIP13.Immunostaining showed that the TRIP13 protein was strongly expressed in cancer lesions and less in non-cancerous tissues.Scale bar: 200 µm (low magnification); 50 µm (high magnification).(B) Forest plot showing the results of multivariate Cox proportional hazards regression analysis of the 5-year overall survival rate.Patients with high TRIP13 expression had a significantly lower overall survival rate.These data were obtained from TCGA-LUAD datasets.(C) Gene set enrichment analysis (GSEA) was applied to explore molecular pathways mediated by TRIP13 in LUAD cells.The top six pathways enriched in LUAD patients with high TRIP13 expression were cell cycle, DNA replication, proteasome, P53 signaling pathway, homologous recombination, and mismatch repair.

Figure 11 .
Figure 11.Synergistic effects between two anticancer drugs (cisplatin and carboplatin) and the TRIP13 inhibitor DCZ0415.The Chou-Talalay method was used to determine the synergistic effects between two anticancer drugs (cisplatin and carboplatin) and DCZ0415 in LUAD cells (A549 and H1299).CDDP, cisplatin; CBDCA, carboplatin.
FDR, false discovery rate.