A Novel Oncogenic Target of Lung Adenocarcinoma Regulated by Both Strands of Mir-99A (Mir-99A-5P and Mir-99A-3P)

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Article FAM64A: A Novel Oncogenic Target of Lung Adenocarcinoma Regulated by Both Strands of miR-99a (miR-99a-5p and miR-99a-3p)

1, 1, 2 1 Keiko Mizuno †, Kengo Tanigawa † , Nijiro Nohata , Shunsuke Misono , Reona Okada 3, Shunichi Asai 3, Shogo Moriya 4, Takayuki Suetsugu 1, Hiromasa Inoue 1 and Naohiko Seki 3,*

1 Department of Pulmonary Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan; [email protected] (K.M.); [email protected] (K.T.); [email protected] (S.M.); [email protected] (T.S.); [email protected] (H.I.) 2 MSD K.K., Tokyo 102-8667, Japan; [email protected] 3 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan; [email protected] (R.O.); [email protected] (S.A.) 4 Department of Biochemistry and Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-43-226-2971; Fax: +81-43-227-3442 These authors contributed equally to this paper. † Received: 6 August 2020; Accepted: 10 September 2020; Published: 11 September 2020

Abstract: Lung adenocarcinoma (LUAD) is the most aggressive cancer and the prognosis of these patients is unfavorable. We revealed that the expression levels of both strands of miR-99a (miR-99a-5p and miR-99a-3p) were significantly suppressed in several cancer tissues. Analyses of large The Cancer Genome Atlas (TCGA) datasets showed that reduced miR-99a-5p or miR-99a-3p expression is associated with worse prognoses in LUAD patients (disease-free survival (DFS): p = 0.1264 and 0.0316; overall survival (OS): p = 0.0176 and 0.0756, respectively). Ectopic expression of these miRNAs attenuated LUAD cell proliferation, suggesting their tumor-suppressive roles. Our in silico analysis revealed 23 putative target genes of pre-miR-99a in LUAD cells. Among these targets, high expressions of 19 genes were associated with worse prognoses in LUAD patients (OS: p < 0.05). Notably, FAM64A was regulated by both miR-99a-5p and miR-99a-3p in LUAD cells, and its aberrant expression was significantly associated with poor prognosis in LUAD patients (OS: p = 0.0175; DFS: p = 0.0276). FAM64A knockdown using siRNAs suggested that elevated FAM64A expression contributes to cancer progression. Aberrant FAM64A expression was detected in LUAD tissues by immunostaining. Taken together, our miRNA-based analysis might be effective for identifying prognostic and therapeutic molecules in LUAD.

Keywords: lung adenocarcinoma; microRNA; miR-99a-5p; miR-99a-3p; FAM64A; tumor suppressor

1. Introduction Lung cancer is one of the most common and lethal cancers. In 2018, approximately 2.1 million people were diagnosed with this disease, and 1.8 million patients died from it [1]. Lung cancers are divided into two pathological types: small-cell lung cancer and non-small-cell lung cancer (NSCLC). NSCLC includes squamous cell carcinoma, adenocarcinoma and large-cell carcinoma [2]. Among NSCLCs, lung adenocarcinoma (LUAD) is the most common, and it is often at an advanced

Cells 2020, 9, 2083; doi:10.3390/cells9092083 www.mdpi.com/journal/cells Cells 2020, 9, 2083 2 of 22 stage by the time of diagnosis, and thus the prognosis of the patients is unfavorable (5-year survival rate on average below 20% on average) [3]. Recently, the survival rate of LUAD patients has improved because of the development of molecularly targeted drugs and immune checkpoint inhibitors [4–6]. Various molecular targeted agents have become available, based on driver gene mutations in LUAD [5–7]. However, there is a population of LUAD patients who harbor no driver gene mutations, indicating that several distinct molecular and genetic pathways contribute to LUAD progression. To understand the molecular pathogenesis of LUAD, we applied a microRNA (miRNA)-based approach. miRNAs (19- to 22-nucleotide-long RNA molecules) function as fine-tuners of gene expression regulation in various cells [8,9]. A single miRNA can regulate the expression of a vast number of genes; therefore, aberrant expression of miRNAs disrupts intracellular gene expression networks. A large number of studies have shown that abnormal miRNA expression contributes to several oncogenic pathways [10–14]. We have previously determined miRNA expression signatures in various types of cancers using RNA sequencing [15–19]. Our recent studies have demonstrated that some passenger strands derived from pre-miRNAs contribute to the malignant transformation of cancer cells [15–19]. We have shown that both strands of miRNAs (e.g., pre-miR-144, pre-miR-145 and pre-miR-150) were significantly downregulated in lung cancer tissues, and their ectopic expression attenuated the malignant phenotypes of lung cancer cells (e.g., cancer cell proliferation, migration and invasion) [20–24]. The involvement of the passenger strand of miRNAs in the pathogenesis of cancer is a new theme in cancer research. Based on miRNA signatures by RNA sequencing, we revealed that expression levels of both strands derived from pre-miR-99a (miR-99a-5p: the guide strand; miR-99a-3p: the passenger strand) were suppressed in several types of cancer tissues [15–19]. In the current study, we investigated the tumor-suppressive functions of both strands of pre-miR-99a and identified their oncogenic targets in LUAD cells. Notably, a total of 19 genes (CKS1B, KCMF1, CENPF, CASC5, MKI67, ESCO2, FANCI, SGOL1, MCM4, KIF11, NEK2, MTHFD2, NCAPG, RRM2, FAM136A, ZWINT, CDK1, CDKN3 and FAM64A) were identified as targets of pre-miR-99a regulation, and they significantly predicted the prognosis (5-year overall survival) of the patients with LUAD. Among the targets, we focused on FAM64A, as it was regulated directly by both miR-99a-5p and miR-99a-3p in LUAD cells. Our miRNA analysis strategy will accelerate the understanding of the molecular mechanism of LUAD.

2. Materials and Methods

2.1. Data Mining of miRNA Target Genes and Their Expression in LUAD Clinical Specimens Gene expression data in LUAD obtained from The Cancer Genome Atlas (TCGA) were retrieved on 6 March 2020 from the cBioPortal database (https://www.cbioportal.org/)[25], UCSC Xena platform (https://xena.ucsc.edu/)[26] and Firebrowse (http://ﬁrebrowse.org/). The mRNA expression Z-scores and information on the clinical samples corresponding to LUAD patients were collected from cBioPortal. To categorize genes into molecular pathways based on gene set enrichment analysis (GSEA) [27], we employed the WebGestalt program (http://www.webgestalt.org/)[28]. Putative target genes possessing binding sites for miR-99a-5p and miR-99a-3p were isolated using the TargetScanHuman database ver. 7.2 (http://www.targetscan.org/vert_72/)[29]. Comprehensive correlations between mRNA and miRNA gene expression in LUAD samples from TCGA were analyzed by LinkedOmics (http://www.linkedomics.org/)[30].

2.2. Transfection of miRNAs, siRNAs and Plasmid Vectors into LUAD Cells and Functional Assays The procedures for transfecting miRNAs, siRNAs and plasmid vectors were described in our previous studies [20–24]. Functional assays (cell proliferation and cell cycle) were performed in LUAD cells, as described in our previous studies [20–24]. The reagents used are listed in Supplementary Table S1. Cells 2020, 9, 2083 3 of 22

2.3. Plasmid Construction and Dual-Luciferase Reporter Assays The vectors used for this analysis were constructed as described in our previous studies [20–24]. Supplementary Figure S1 shows the sequences incorporated into the vectors. The analysis was performed according to our previous studies [20–24]. The reagents used are listed in Supplementary Table S1.

2.4. Immunohistochemistry The immunohistochemistry procedure was described in our previous studies [20–24]. The antibodies used in this study are listed in Supplementary Table S1.

2.5. Stastistical Analyses Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) and JMP Pro 14 (SAS Institute Inc., Cary, NC, USA). The Mann–Whitney U test was used to determine the signiﬁcance of diﬀerences between two groups, and one-way analysis of variance and Tukey’s test for post-hoc analysis were used for multiple group comparisons. To evaluate the correlation between two variables, we applied Spearman’s rank test. Overall survival (OS) and disease-free survival (DFS) were assessed using the Kaplan–Meier method and log-rank test. To identify independent factors predicting OS and DFS, we utilized multivariate Cox proportional hazards models.

3. Results

3.1. Downregulation of miR-99a-5p and miR-99a-3p in LUAD Clinical Specimens and Their Clinical Signiﬁcance The expression levels of miR-99a-5p and miR-99a-3p were evaluated using miRNA-seq data of TCGA-LUAD from Firebrowse. The miRNA-seq data showed that expression levels of miR-99a-5p and miR-99a-3p were signiﬁcantly suppressed in LUAD tissues compared with normal lung tissues (Figure1A). According to Spearman’s rank test, a positive correlation was detected between the expression levels of the two miRNA strands (r = 0.7716, p < 0.0001; Figure1B). Cells 2020, 9, 2083 4 of 22 Cells 2020, 9, x 4 of 23

Figure 1. Downregulation of miR-99a-5p and miR-99a-3p in LUAD. (A) Comparison of the expression levelsFigure of miR-99a-5p 1. Downregulationand miR-99a-3p of miR-99a-5pbetween tumorand miR-99a-3p and non-tumor in LUAD. tissues (A in) Comparison paired (left) andof the non-paired expression (right)levels LUAD of miR-99a-5p clinical specimens and miR-99a-3p from TCGAbetween datasets. tumor and (B) Positivenon-tumor correlation tissues in between paired (left) the relative and non- expressionpaired (right) level ofLUADmiR-99a-5p clinicaland specimens that of miR-99a-3p from TCGAin clinicaldatasets. specimens (B) Positive according correlation to Spearman’s between the rankrelative tests. expression level of miR-99a-5p and that of miR-99a-3p in clinical specimens according to Spearman’s rank tests. Kaplan–Meier plot and log-rank test using survival data from TCGA-LUAD revealed that low expression of miR-99a-5p was associated with a worse prognosis compared with high expression (DFS: p = 0.1264; OS: p = 0.00176) (Figure2A). Similarly, low expression of miR-99a-3p was associated with a worse prognosis compared with high expression (DFS: p = 0.0316; OS: p = 0.0756) (Figure2A).

Cells 2020, 9, x 2083 55 of of 23 22

Figure 2. Clinical signiﬁcance of miR-99a-5p and miR-99a-3p expression in LUAD. (A) The patients Figurewere divided 2. Clinical into significance two groups accordingof miR-99a-5p to the and median miR-99a-3p expression expression level ofinmiR-99a-5p LUAD. (A)or ThemiR-99a-3p patients: werehigh (reddivided lines) into and two low groups (blue lines)according expression to the groups.median (expressionB) The patients level were of miR-99a-5p divided into or miR-99a-3p two groups,: hightop 25%(red andlines) low and 25%. low (blu Highe lines) expression expression of miR-99a-5p groups. (Band) ThemiR-99a-3p patients wereis representeddivided into bytwo red groups, lines; toplow 25% expression and low of 25%. these High miRNAs expression is represented of miR-99a-5p by blue and lines. miR-99a-3p is represented by red lines; low expression of these miRNAs is represented by blue lines.

Cells 2020, 9, 2083 6 of 22

Similarly, the patients were divided into two groups according to the expression levels of miR-99a-5p and miR-99a-3p (top 25%: red lines and low 25%: blue lines) and analyzed. Kaplan–Meier plot and log-rank test showed that low expression of miR-99a-5p was associated with a worse prognosis compared with high expression (DFS: p = 0.0035; OS: p = 0.0005) (Figure2B). Low expression of miR-99a-3p was associated with a worse prognosis compared with high expression (DFS: p = 0.0517; OS: p = 0.0139) (Figure2B).

3.2. Tumor-Suppressive Functions of miR-99a-5p and miR-99a-3p Assessed by Ectopic Expression Assays Cells 2020, 9, x 6 of 23 We assessed changes in cell proliferation and cell cycle after ectopic expression of these miRNAs into A5493.2. and Tumor-Suppressive H1299 cells. Cell Functions proliferation of miR-99a-5p (XTT and miR-99a-3p assay) was Assess significantlyed by Ectopic Expression inhibited Assays by miR-99a-5p or miR-99a-3p expressionWe assessed in changes A549 in and cell H1299proliferation LUAD and cellcell cycle lines after (Figure ectopic3 A).expression To investigate of these miRNAs the synergistic effects of intomiR-99a-5p A549 and H1299and miR-99a-3pcells. Cell proliferation, we performed (XTT assay) proliferation was significantly assays inhibited with by miR-99a-5p co-transfection of or miR-99a-3p expression in A549 and H1299 LUAD cell lines (Figure 3A). To investigate the miR-99a-5psynergisticand miR-99a-3p effects ofin miR-99a-5p LUAD cells and (A549miR-99a-3p and, H1299),we performed but theyproliferation did not assays show with synergistic co- effects of these miRNAstransfection transfection of miR-99a-5p (Supplementary and miR-99a-3p in FigureLUAD cells S2). (A549 In the and cell H1299), cycle but analysis they did bynot flowshow cytometry, the numbersynergistic of LUAD effects cells of inthese the miRNAs G0/G1 transfection phase was (Su increasedpplementary after Figure ectopic S2). In expressionthe cell cycle ofanalysis these miRNAs comparedby with flow control cytometry, miRNA the number (Figure of LUAD3B). Our cells data in the suggest G0/G1 phase that ectopicwas increased expression after ectopic of miR-99a-5p expression of these miRNAs compared with control miRNA (Figure 3B). Our data suggest that and miR-99a-3pectopic inducesexpression G1 of miR-99a-5p arrest in and LUAD miR-99a-3p cells. induces G1 arrest in LUAD cells.

Figure 3. Functional assays of cell proliferation and cell cycle arrest following ectopic expression of Figure 3. Functional assays of cell proliferation and cell cycle arrest following ectopic expression of miR-99a-5p or miR-99a-3p in LUAD cell lines (A549 and H1299 cells). (A) Cell proliferation assessed miR-99a-5p or miR-99a-3p in LUAD cell lines (A549 and H1299 cells). (A) Cell proliferation assessed using XTTusing assays XTT at assays 72 h afterat 72 h miRNA after miRNA transfection transfection (* (*p p< <0.0001). 0.0001). (B (B) Flow) Flow cytometric cytometric analysis analysis of the of the cell cycle phasecell distribution cycle phase distribution of control ofcells control and cells cells and transfectedcells transfected with withmiR-99a-5p miR-99a-5p oror miR-99a-3pmiR-99a-3p. Cells. Cells were evaluatedwere at 72 evaluated h after at miRNA 72 h after transfection miRNA transfection (* p < (*0.0001). p < 0.0001).

3.3. Identification of miR-99a-5p and miR-99a-3p Target Genes in LUAD

Cells 2020, 9, 2083 7 of 22

Cells3.3. 2020 Identiﬁcation, 9, x of miR-99a-5p and miR-99a-3p Target Genes in LUAD 7 of 23

ToTo identify identify genes genes regulated byby pre-pre-miR-99amiR-99ain in LUAD LUAD cells, cells, we we applied applied in in silico silico analyses analyses using using the theTargetScanHuman TargetScanHuman (release (release 7.2), 7.2), LinkedOmics LinkedOmics and and cBioportal cBioportal databases databases (Figure (Figure4). A 4). total A oftotal 23 genesof 23 geneswere identifiedwere identified as pre- asmiR-99a pre-miR-99atargets targets in LUAD in LUAD cells (five cellsmiR-99a-5p (five miR-99a-5ptargets andtargets 19 miR-99a-3p and 19 miR-99a-targets; 3pTable targets;1). Notably, Table 1).FAM64A Notably,was FAM64A identified was asidentified a target as of a both targetmiR-99a-5p of both miR-99a-5pand miR-99a-3p and miR-99a-3p. .

Figure 4. Identiﬁcation of putative targets of miR-99a-5p or miR-99a-3p regulation in LUAD cells.

Prediction of miR-99a-5p and miR-99a-3p target genes using the TargetScanHuman, cBioportal and FigureLinkedOmics 4. Identification databases. of Vennputative diagrams targets represent of miR-99a-5p the number or miR-99a-3p of putative regulation target genes in LUAD regulated cells. by PredictionmiR-99a-5p ofor miR-99a-5pmiR-99a-3p and. miR-99a-3p target genes using the TargetScanHuman, cBioportal and LinkedOmics databases. Venn diagrams represent the number of putative target genes regulated by Table 1. Putative targets by miR-99a-5p and miR-99a-3p regulation in LUAD cells. miR-99a-5p or miR-99a-3p. 5-Year OS Entrez Gene ID Gene Symbol Gene Name Location Total Sites Table 1. Putative targets by miR-99a-5p and miR-99a-3p regulation in LUAD cells. p-Value Putative targets by miR-99a-5p regulation in LUAD cells Entrez 1063 CENPF Centromere protein F 1q41 1 0.0059 Gene Total 5-Year OS Gene 1163 CKS1B CDC28Gene protein Name kinase regulatory subunit 1B 1q21.3Location 1 0.0073 Symbol56888 KCMF1 Potassium channel modulatory factor 1 2p11.2 1Sites 0.0125p-Value ID 417685 FAM64A Family with sequence similarity 64 member A 17p13.2 1 0.0176 993 CDC25APutative targets by CellmiR-99a-5p division cycle regulation 25A in LUAD 3p21.31 cells 1 0.1585 miR-99a-3p 1063 CENPF PutativeCentromere targets by protein Fregulation in LUAD cells 1q41 1 0.0059 4751 NEK2 NIMA related kinase 2 1q32.3 1 0.0002 1163 CKS1B57082 CASC5CDC28 protein kinaseCancer susceptibilityregulatory candidatesubunit 5 1B 15q15.1 1q21.3 1 1 0.0003 0.0073 56888 KCMF1983 CDK1Potassium channelCyclin modulatory dependent kinase factor 1 1 10q21.2 2p11.2 1 1 0.0003 0.0125 4288 MKI67 Marker of proliferation Ki-67 10q26.2 1 0.0005 417685 FAM64A1033 FamilyCDKN3 with sequenceCyclin dependentsimilarity kinase 64 member inhibitor 3 A 14q22.2 17p13.2 1 1 0.0017 0.0176 993 CDC25A6241 RRM2 CellRibonucleotide division reductase cycle 25A regulatory subunit M2 2p25.1 3p21.31 1 1 0.002 0.1585 Minichromosome maintenance complex 4173 MCM4 8q11.21 1 0.0032 Putative targets by miR-99a-3pcomponent regulation 4 in LUAD cells 4751 NEK23832 KIF11 NIMA relatedKinesin kinase family member 2 11 10q23.33 1q32.3 1 1 0.0034 0.0002 84908 FAM136A Family with sequence similarity 136 member A 2p13.3 1 0.0087 57082 CASC511130 ZWINT Cancer susceptibilityZW10 interacting candidate kinetochore 5 protein 10q21.1 15q15.1 1 1 0.0094 0.0003 983 CDK155215 FANCI Cyclin dependentFA complementation kinase 1 group I 15q26.1 10q21.2 1 1 0.0108 0.0003 64151 NCAPG Non-SMC condensin I complex subunit G 4p15.31 1 0.0208 4288 MKI67 MarkerEstablishment of proliferation of sister chromatid Ki-67 cohesion 10q26.2 1 0.0005 157570 ESCO2 8p21.1 1 0.0235 1033 CDKN3 Cyclin dependentN-acetyltransferase kinase inhibitor 2 3 14q22.2 1 0.0017 151648 SGOL1 Shugoshin 1 3p24.3 1 0.0235 6241 RRM2 Ribonucleotide Methylenetetrahydrofolatereductase regulatory dehydrogenase subunit M2 2p25.1 1 0.002 4173 MCM410797 MinichromosomeMTHFD2 maintenance(NADP+ complexdependent) component 2, 4 2p13.1 8q11.21 1 1 0.0321 0.0032 methenyltetrahydrofolate cyclohydrolase 3832 KIF1111260 XPOT Kinesin familyExportin member for tRNA 11 12q14.2 10q23.33 1 1 0.0669 0.0034 84908 FAM136A417685 FamilyFAM64A with sequenceFamily with similari sequencety similarity 136 member 64 member A A 17p13.2 2p13.3 1 1 0.0756 0.0087 51290 ERGIC2 ERGIC and golgi 2 12p11.22 1 0.0929 11130 ZWINT55854 ZC3H15ZW10 interactingZinc ﬁnger kinetochore CCCH-type containingprotein 15 2q32.1 10q21.1 1 1 0.0942 0.0094 55215 FANCI FA complementation group I 15q26.1 1 0.0108 64151 NCAPG Non-SMC condensin I complex subunit G 4p15.31 1 0.0208 Establishment of sister chromatid cohesion N- 157570 ESCO2 8p21.1 1 0.0235 acetyltransferase 2 151648 SGOL1 Shugoshin 1 3p24.3 1 0.0235 Methylenetetrahydrofolate dehydrogenase (NADP+ 10797 MTHFD2 dependent) 2, 2p13.1 1 0.0321 methenyltetrahydrofolate cyclohydrolase 11260 XPOT Exportin for tRNA 12q14.2 1 0.0669

Cells 2020, 9, x 8 of 23

417685 FAM64A Family with sequence similarity 64 member A 17p13.2 1 0.0756 Cells512902020 , 9,ERGIC2 2083 ERGIC and golgi 2 12p11.22 1 8 0.0929 of 22 55854 ZC3H15 Zinc finger CCCH-type containing 15 2q32.1 1 0.0942

3.4. Clinical Significance of miR-99a-5p and miR-99a-3p Target Genes in LUAD Pathogenesis 3.4. Clinical Significance of miR-99a-5p and miR-99a-3p Target Genes in LUAD Pathogenesis WeWe evaluated evaluated the the associations associations between between expression expression levels levels of of the the genes genes and and survival survival using using TCGA TCGA FAM64A andand GEO GEO datasets. datasets. Among Among 22 22 of of the the 23 23 target target genes genes (excluding (excluding FAM64A),), high high expression expression of of 18 18 genes genes CKS1B KCMF1 CENPF CASC5 MKI67 ESCO2 FANCI SGOL1 MCM4 KIF11 NEK2 MTHFD2 ( (CKS1B, , KCMF1, , CENPF, , CASC5, , MKI67, , ESCO2,, FANCI, , SGOL1, , MCM4, , KIF11, , NEK2, , MTHFD2,, NCAPG RRM2 FAM136A ZWINT CDK1 CDKN3 NCAPG, , RRM2, , FAM136A, , ZWINT, , CDK1and and CDKN3)) significantly significantly predicted predicted worse worse survival survival (OS: (OS: 5-year5-year survival survival rate) rate) in in patients patients withwith LUADLUAD (Figure(Figure5 ).5). We We also also adjusted adjusted the the multiplicity multiplicity by by using using Benjamini–HochbergBenjamini–Hochberg analysisanalysis andand confirmedconfirmed thatthat 1919 outout ofof these these 23 23 target target genes genes were were significant significant (Supplementary(Supplementary Table Table S3). S3). All genesAll genes were were upregulated upregulated in cancer in canc tissueser tissues compared compared with normal with tissuesnormal (Figuretissues6 ).(Figure We also 6). classified We also classifi these targeted these genes target according genes according to Gene Ontology to Gene (GO:Ontology Biological (GO: Biological Process) criteriaProcess) by criteria using the by GeneCodis using the GeneCodis database. The database. GO classification The GO classification of the genes controlledof the genes by eachcontrolled miRNA by waseach shown miRNA in Supplementarywas shown in Supplem Table S4.entary Each miRNATable S4. has Each regulated miRNA genes has associatedregulated genes with “cellassociated cycle (GO:with 0007049)” “cell cycle and (GO: “cell 0007049)” division and (GO: “cell 0051301)”. division (GO: 0051301)”.

Figure 5. Cont.

Cells 2020, 9, 2083 9 of 22 Cells 2020, 9, x 9 of 23

Figure 5. Clinical signiﬁcance of pre-miR-99a target genes in TCGA database. Kaplan–Meier survival curves and log-rank comparisons of patients with LUAD using TCGA datasets. Patients were divided into two groups according to the median expression of each pre-miR-99a target gene evaluated: high and low expression groups. The red and blue lines represent the high and low expression groups, respectively. High expression mRNA of 18 genes (CKS1B, KCMF1, CENPF, CASC5, MKI67, ESCO2, FANCI, SGOL1, MCM4, KIF11, NEK2, MTHFD2, NCAPG, RRM2, FAM136A, ZWINT, CDK1 and CDKN3) signiﬁcantly predicted worse survival (5-year overall survival rate) in patients with LUAD. The expression data were downloaded from http://www.oncolnc.org. Cells 2020, 9, x 10 of 23

Figure 5. Clinical significance of pre-miR-99a target genes in TCGA database. Kaplan–Meier survival curves and log-rank comparisons of patients with LUAD using TCGA datasets. Patients were divided into two groups according to the median expression of each pre-miR-99a target gene evaluated: high and low expression groups. The red and blue lines represent the high and low expression groups, respectively. High expression mRNA of 18 genes (CKS1B, KCMF1, CENPF, CASC5, MKI67, ESCO2, FANCI, SGOL1, MCM4, KIF11, NEK2, MTHFD2, NCAPG, RRM2, FAM136A, ZWINT, CDK1 and CDKN3) significantly predicted worse survival (5-year overall survival rate) in patients with LUAD. Cells 2020The, 9expression, 2083 data were downloaded from http://www.oncolnc.org. 10 of 22

Figure 6. Cont.

Cells 2020, 9, 2083 11 of 22 Cells 2020, 9, x 11 of 23

Figure 6. Expression levels of pre-miR-99a target genes in LUAD clinical specimens. Using TCGA datasets, theFigure expression 6. Expression levels levels of allof pre- 22miR-99a pre-miR-99a target genestarget in genes LUAD evaluated clinical specimens. were upregulated Using TCGA in LUAD clinical specimensdatasets, the (n expression= 475) comparedlevels of all 22 with pre-miR-99a normal target lung genes tissues evaluated (n = were54). upregulated The expression in LUAD data were downloadedclinical from specimens http:// firebrowse.org(n = 475) compared/. with normal lung tissues (n = 54). The expression data were downloaded from http://firebrowse.org/. 3.5. Clinical Significance of FAM64A in LUAD Pathogenesis 3.5. Clinical Significance of FAM64A in LUAD Pathogenesis OverexpressionOverexpression of FAM64A of FAM64Ain LUAD in LUAD tissues tissues was was confirmed confirmed byby RNA-seqRNA-seq data data from from TCGA- TCGA-LUAD (Figure7A).LUAD Spearman’s (Figure 7A). rank Spearman’s test indicated rank test indicated negative negative correlations correlations of ofFAM64A FAM64A expressionexpression with with both miR-99a-5p and miR-99a-3p expression (Figure7B). We investigated the clinical significance of FAM64A expression in LUAD patients using the TCGA database. High expression of FAM64A was associated with a significantly poor prognosis compared with low expression (DFS: p = 0.0276; OS: p = 0.0175; Figure7C) and was identified as an independent prognostic factor of survival in the multivariate analysis (p < 0.01; Figure7D). FAM64A protein expression was also evaluated in LUAD clinical specimens using immunohistochemistry. Overexpression of FAM64A protein was detected in cancer lesions in LUAD clinical specimens (Figure8). Cells 2020, 9, x 12 of 23

both miR-99a-5p and miR-99a-3p expression (Figure 7B). We investigated the clinical significance of FAM64A expression in LUAD patients using the TCGA database. High expression of FAM64A was associated with a significantly poor prognosis compared with low expression (DFS: p = 0.0276; OS: p = 0.0175; Figure 7C) and was identified as an independent prognostic factor of survival in the multivariate analysis (p < 0.01; Figure 7D). FAM64A protein expression was also evaluated in LUAD Cells 2020, 9clinical, 2083 specimens using immunohistochemistry. Overexpression of FAM64A protein was detected 12 of 22 in cancer lesions in LUAD clinical specimens (Figure 8).

Figure 7. Clinical signiﬁcance of FAM64A expression in LUAD. (A) Comparison of FAM64A expression levels between tumor and non-tumor tissues in paired (left) and non-paired (right) LUAD clinical specimens from TCGA datasets. Upregulation of FAM64A was detected in LUAD tissues. (B) Correlations between the relative expression level of FAM64A and that of miR-99a-5p or miR-99a-3p. Spearman’s rank test showed a negative correlation between FAM64A and miR-99a-5p or miR-99a-3p expression levels in clinical specimens. (C) Kaplan–Meier survival curves and log-rank comparisons of patients with LUAD using TCGA database. Patients were divided into two groups according to the median FAM64A expression level: high and low expression groups. The red and blue lines represent the high and low expression groups, respectively. (D) Forest plot of the multivariate analysis results assessing independent prognostic factors for disease-free and overall survival, including FAM64A expression (high vs. low) (* p < 0.05, ** p < 0.01, *** p < 0.001). Cells 2020, 9, 2083 13 of 22 Cells 2020, 9, x 14 of 23

(x 40) (x 200) B

(x 40) (x 200) C

(x 40) (x 200) D

(x 40) (x 200)

FigureFigure 8. Overexpression 8. Overexpression of FAM64A of FAM64A in LUAD in clinicalLUAD specimens.clinical specimens. (A–C) Immunohistochemical (A–C) Immunohistochemical staining of FAM64Astaining inof LUAD FAM64A tissues. in LUAD Overexpression tissues. Overexpression of FAM64A was of detected FAM64A in the was cytoplasm detected and in /orthe nuclei cytoplasm of cancerand/or cells. nuclei On the of other cancer hand, cells. expression On the other of FAM64A hand, expression was low in of normal FAM64A lung was cells low (D). in normal lung cells (D). 3.6. Direct Regulation of FAM64A by miR-99a-5p and miR-99a-3p in LUAD Cells 3.6.We Direct focused Regulation on FAM64A of FAM64Abecause by miR-99a-5p its expression and was miR-99a-3p found to in beLUAD controlled Cells by both strands of pre-miR-99a (miR-99a-5p and miR-99a-3p). The expression level of FAM64A was signiﬁcantly reduced We focused on FAM64A because its expression was found to be controlled by both strands of after transfection of miR-99a-5p and miR-99a-3p in LUAD cells (Figure9A). pre-miR-99a (miR-99a-5p and miR-99a-3p). The expression level of FAM64A was significantly reduced after transfection of miR-99a-5p and miR-99a-3p in LUAD cells (Figure 9A).

Cells 2020, 9, x 15 of 23

There is one miRNA-binding site for each miRNA strand (miR-99a-5p and miR-99a-3p) in the 3′UTR region of FAM64A (Figure 9B). In dual-luciferase reporter assays, luciferase activity was significantly decreased by co-transfection of miR-99a-5p or miR-99a-3p with the vector containing the wild-type 3′UTR of FAM64A in A549 cells. On the other hand, the transfection of the deletion vector (containing the deletion-type 3′UTR of FAM64A) prevented this decrease in luminescence (Figure Cells 20209B),, suggesting9, 2083 that miR-99a-5p and miR-99a-3p bind directly to the 3′UTR of FAM64A in LUAD cells.14 of 22

FigureFigure 9. Direct 9. Direct regulation regulation of ofFAM64A FAM64A byby miR-99a-5p andand miR-99a-3pmiR-99a-3p in LUADin LUAD cells. cells. (A) Significantly (A) Significantly reducedreduced expression expression of ofFAM64A FAM64AmRNA mRNA by bymiR-99s-5p miR-99s-5p or miR-99a-3pmiR-99a-3p transfectiontransfection in A549 in A549 cells cells (at 72 (at 72 h afterh transfection;after transfection; * p <* p0.0001). < 0.0001). ( B(B)) Predictions Predictions of of miR-99a-miR-99abinding-binding sites sites using using TargetScanHuman TargetScanHuman databasedatabase analyses. analyses. EachEach miRNA miRNA strand strand (miR-99a-5p (miR-99a-5p and miR-99a-3pand miR-99a-3p) had one) hadbinding one site binding in the 3′ siteUTR in the of FAM64A. Dual-luciferase reporter assays showed that luminescence activity was reduced by co- 30UTR of FAM64A. Dual-luciferase reporter assays showed that luminescence activity was reduced transfection of the FAM64A wild-type vector (containing the miR-99a-5p-binding site) with miR-99a- by co-transfection of the FAM64A wild-type vector (containing the miR-99a-5p-binding site) with 5p or of the FAM64A wild-type vector (containing the miR-99a-3p-binding site) with miR-99a-3p in miR-99a-5p or of the FAM64A wild-type vector (containing the miR-99a-3p-binding site) with miR-99a-3p A549 cells. Normalized data were calculated as Renilla/firefly luciferase activity ratios (* p < 0.0001). in A549 cells. Normalized data were calculated as Renilla/firefly luciferase activity ratios (* p < 0.0001). 3.7. Effects of FAM64A Knockdown on Cell Proliferation and Cell Cycle in LUAD Cells There is one miRNA-binding site for each miRNA strand (miR-99a-5p and miR-99a-3p) in the 30UTR region of FAM64A (Figure9B). In dual-luciferase reporter assays, luciferase activity was significantly decreased by co-transfection of miR-99a-5p or miR-99a-3p with the vector containing the wild-type 30UTR of FAM64A in A549 cells. On the other hand, the transfection of the deletion vector (containing the deletion-type 30UTR of FAM64A) prevented this decrease in luminescence (Figure9B), suggesting that miR-99a-5p and miR-99a-3p bind directly to the 30UTR of FAM64A in LUAD cells. Cells 2020, 9, x 16 of 23

To investigate the oncogenic function of FAM64A in LUAD cells, we performed knockdown Cells 2020, 9, 2083 15 of 22 assays using siRNAs. The expression level of FAM64A was successfully reduced by two different siRNAs (siFAM64A-1 and siFAM64A-2; Figure 10A). 3.7. Eﬀects ofThe FAM64A proliferation Knockdown of LUAD on Cell cells Proliferation was attenuated and Cell by the Cycle transfection in LUAD of Cells each siFAM64A (Figure 10B). To investigateCell cycle the assays oncogenic demonstrated function that of FAM64A the numberin LUAD of LUAD cells, cells we in performed the G0/G1 phase knockdown was increased assays using siRNAs.after knockdown The expression of FAM64A level (Figure of FAM64A 10C).was These successfully data indicate reduced that the by expression two diﬀerent of FAM64A siRNAs (siFAM64Aenhances-1 and cell siFAM64A cycle progression.-2; Figure 10A).

Figure 10. Eﬀect of FAM64A knockdown on cell proliferation and cell cycle arrest in LUAD cells. (A) Successful suppression of FAM64A expression by siFAM64A-1 or siFAM64A-2 transfection in A549 and H1299 cells. (B) Cell proliferation assessed by XTT assay at 72 h after miRNA transfection (* p < 0.0001). (C) Flow cytometric analyses of cell cycle phase distributions in control cells and cells transfected with siFAM64A. The cells were assessed at 72 h after miRNA transfection (* p < 0.0001). Cells 2020, 9, x 17 of 23

Figure 10. Effect of FAM64A knockdown on cell proliferation and cell cycle arrest in LUAD cells. (A) Successful suppression of FAM64A expression by siFAM64A-1 or siFAM64A-2 transfection in A549 and H1299 cells. (B) Cell proliferation assessed by XTT assay at 72 h after miRNA transfection (* p < 0.0001). (C) Flow cytometric analyses of cell cycle phase distributions in control cells and cells Cells 2020, 9, 2083 16 of 22 transfected with siFAM64A. The cells were assessed at 72 h after miRNA transfection (* p < 0.0001).

3.8.The FAM64A proliferation Effects ofon LUAD Molecular cells Pathwa was attenuatedys in LUAD by the transfection of each siFAM64A (Figure 10B). CellWe cycle identified assays demonstrateddifferentially expressed that the number genes offrom LUAD TCGA-LUAD cells in the G0RNA-seq/G1 phase between was increased FAM64A afterhigh knockdown expression of groupFAM64A and(Figure low expression 10C). These group. data indicate that the expression of FAM64A enhances cell cycleGSEA progression. showed that the top signaling pathways enriched in the high FAM64A expression group were cell cycle-associated terms, such as E2F targets, G2M checkpoints, MYC targets and mitotic 3.8. FAM64A Effects on Molecular Pathways in LUAD spindle assembly (Figure 11). WeFinally, identified we found differentially that the expressed proportion genes of genome from TCGA-LUAD alterations (percentage RNA-seq between of chromosomeFAM64A regionshigh expressionwith copy group number and alterations low expression relative group. to all regions evaluated) and the mutation count (the number of GSEAmutational showed events that per the case) top signaling were significantly pathways enrichedincreased in in the the high highFAM64A FAM64Aexpression expression group group were(Figure cell cycle-associated12), suggesting terms,that FAM64A such as expression E2F targets, may G2M becheckpoints, associated with MYC genetic targets mutations and mitotic and spindlegenomic assembly instability (Figure in LUAD 11). cells.

Figure 11. Cont.

Cells 2020, 9, 2083 17 of 22 Cells 2020, 9, x 18 of 23

Cells 2020, 9, x 18 of 23

Figure 11. FigureTCGA 11. databaseTCGA database analysis analysis of theof the clinical clinical significancesignificance and and function function of FAM64A of FAM64A in LUADin LUAD clinical specimens.clinical specimens. The bar The graph bar graph shows shows the the results results of of gene gene set set enrichment enrichment analysis analysis (GSEA) (GSEA)of the of the genes differentially expressed between high and low FAM64A expression groups in LUAD patients. genes differentiallyFour representative expressed GSEA between plots are highshown and below low forFAM64A E2F targets,expression G2/M checkpoint, groups MYC in target LUAD 1 patients. Four representativevariant 1 and GSEA mitotic plotsspindle are assembly shown with below q-values for < 0.05. E2F These targets, pathway G2/ Mterms checkpoint, were significantly MYC target 1 variant 1 andenriched mitotic in the spindle high FAM64A assembly expression with group. q-values < 0.05. These pathway terms were significantly enriched in the high FAM64A expression group.

Finally,Figure we 11. found TCGA that database the proportionanalysis of the of clinical genome significance alterations and (percentagefunction of FAM64A of chromosome in LUAD regions with copyclinical number specimens. alterations The bar relative graph shows to all the regions results evaluated)of gene set enrichment and the mutationanalysis (GSEA) count of (thethe number of mutationalgenes differentially events per expressed case) were between signiﬁcantly high and low increased FAM64A expression in the high groupsFAM64A in LUADexpression patients. group (Figure 12Four), suggesting representative that GSEAFAM64A plots expressionare shown below may for be E2F associated targets, G2/M with checkpoint, genetic mutations MYC target and 1 genomic variant 1 and mitotic spindle assembly with q-values < 0.05. These pathway terms were significantly instability in LUAD cells. enriched in the high FAM64A expression group. Figure 12. Associations of genome alterations and mutation counts with FAM64A expression in LUAD clinical specimens. Proportion of genome alterations (percentage of chromosome regions with copy number alterations relative to all regions evaluated; (left)) and the mutation count (number of mutational events per case; (right)) were significantly increased in the high compared with the low FAM64A expression group (*** p < 0.001).

4. Discussion

Figure 12. Associations of genome alterations and mutation counts with FAM64A expression in Figure 12. Associations of genome alterations and mutation counts with FAM64A expression in LUADLUAD clinical clinical specimens. specimens. Proportion Proportion ofof genomegenome al alterationsterations (percentage (percentage of chromosome of chromosome regions regions with with copycopy number number alterations alterations relative relative to to all all regionsregions evaluated; (left (left)) ))and and the the mutation mutation count count (number (number of of mutationalmutational events events per per case; case; (right (right)))) were were signiﬁcantlysignificantly increased increased in inthe the high high compared compared with withthe low the low FAM64AFAM64Aexpression expression group group (*** (***p

4. Discussion

Cells 2020, 9, 2083 18 of 22

4. Discussion Active genomic research has led to the discovery of driver genes/mutations critical to lung cancer [31]. Molecularly targeted drugs were developed based on these driver genes, and the prognosis of advanced LUAD has greatly improved due to the emergence of molecularly targeted therapeutic agents [32]. However, even with these therapeutic agents, it is difficult to eliminate cancer cells from patients. Continued exploration of molecular networks in LUAD cells provides useful information for developing novel therapeutics. To identify novel therapeutic targets and pathways, we have previously identified tumor-suppressive miRNAs and their oncogenic targets in LUAD [22–24]. A feature of our study is that we analyzed both strands of pre-miRNAs: the guide and passenger strands. The general theory regarding miRNA biogenesis so far is that the passenger strand of a miRNA derived from a pre-miRNA is decomposed in the cytoplasm and has no function [9,10]. Contrary to this belief, recent reports have shown that some passenger strands of miRNAs regulate oncogenes in cancer cells and exert tumor-suppressive functions [33]. Our recent studies demonstrated that some passenger strands of miRNAs, e.g., miR-143-5p, miR-145-3p and miR-150-3p, behave as tumor-suppressive miRNAs in LUAD cells by targeting oncogenes, e.g., LMNB2, MCM4 and TNS4, respectively [22–24]. In lung cancer, several studies have shown that miR-99a-5p acts as a tumor-suppressive miRNA by targeting critical oncogenic pathways, including AKT1 and mTOR signaling [34,35]. In contrast, there are few reports on miR-99a-3p function in lung cancer cells. Based on our miRNA signatures, we showed that miR-99a-3p also acts as a tumor-suppressive miRNA in prostate cancer and head and neck squamous cell carcinoma [36,37]. Of particular interest in those papers is that many of the genes identified as targets of miR-99a-3p contribute to malignant phenotypes of cancer cells and significantly predict the worse prognosis of the patients [37]. The search for genes regulated by the passenger strands of miRNAs will provide new information for exploring the molecular mechanisms of LUAD. In this study, a total of 19 genes (CKS1B, KCMF1, CENPF, CASC5, MKI67, ESCO2, FANCI, SGOL1, MCM4, KIF11, NEK2, MTHFD2, NCAPG, RRM2, FAM136A, ZWINT, CDK1, CDKN3 and FAM64A) identified as pre-miR-99a targets appear to be intimately involved in LUAD pathogenesis. Interestingly, many of these genes are involved in the cell cycle, cell division and chromosome segregation. These molecules are essential for cell division and may be potential targets for cancer drug development. For example, KIF11 is a kinesin, a microtubule-based motor protein that mediates diverse intracellular functions, such as its critical roles in cell division and intercellular vesicle and organelle transport [38,39]. Several inhibitors of KIF11 have entered phase I and II clinical trials [39]. Functional analyses of the genes regulated by pre-miR-99a are useful for exploring molecular networks in LUAD. In this study, we focused on FAM64A because its expression is regulated by both strands of pre-miR-99a (miR-99a-5p and miR-99a-3p) in LUAD cells. FAM64A (also known as PIMREG, CAKM, CATS and RCS1) was initially identified as a CALM/PICALM-interacting protein using a yeast two-hybrid system [40]. The fusion protein CALM/AF10, t(10;11)(p13;q14), plays a crucial role in acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma [41,42]. Previous studies demonstrated that FAM64A contributes to cell cycle progression [43–45]. Overexpression of FAM64A was reported in leukemia, lymphoma and several types of solid cancer [46]. In breast cancer, overexpression of FAM64A enhanced the transactivation of NF-κB by disrupting the NF-κB/IκBα negative feedback loop [47]. Another study demonstrated that FAM64A regulates STAT3 activation and is involved in Th17 differentiation, colitis and colorectal cancer development [48]. These findings indicate that FAM64A behaves as a transcriptional regulator contributing to cell cycle progression. FAM64A might be a potential prognostic factor and therapeutic target in LUAD. Cells 2020, 9, 2083 19 of 22

5. Conclusions Both the guide (miR-99a-5p) and passenger (miR-99a-3p) strands of pre-miR-99a showed antitumor functions in LUAD cells. A total of 23 genes were identiﬁed as putative pre-miR-99a targets in LUAD cells. Among these targets, 19 genes (CKS1B, KCMF1, CENPF, CASC5, MKI67, ESCO2, FANCI, SGOL1, MCM4, KIF11, NEK2, MTHFD2, NCAPG, RRM2, FAM136A, ZWINT, CDK1, CDKN3 and FAM64A) were closely associated with the molecular pathogenesis of LUAD. FAM64A was directly regulated by both strands of pre-miR-99a, and its aberrant expression enhanced cancer cell proliferation.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/9/2083/s1, Figure S1: Vectorinserted sequences; Figure S2: Effects of ectopic expression of miR-99a-5p and miR-99a-3p on LUAD cells; Table S1: Reagent used in this study; Table S2: Characteristics of the patients used in immunostaining; Table S3: 23 target genes analyzed by Benjamini-Hochberg method; Table S4: Significantly enriched annotations regulated by miR-99a-5p/miR-99a-3p in LUAD cells and pathways regulated by miR-99a-5p/miR-99a-3p in LUAD cells. Author Contributions: Conceptualization, K.M., N.S.; methodology, N.S.; validation, K.M., H.I. and N.S.; formal analysis, N.N., K.T., S.M. (Shunsuke Misono), R.O., S.A., T.S. and S.M. (Shogo Moriya); investigation, K.T., S.M. (Shunsuke Misono), R.O., S.A., T.S. and S.M. (Shogo Moriya); resources, N.N. and S.A.; data curation, K.T., S.M. (Shunsuke Misono) and R.O.; writing—original draft preparation, K.M., N.S.; writing—review and editing, K.T., S.M. (Shunsuke Misono), R.O. and S.M. (Shogo Moriya); visualization, K.T., S.M. (Shunsuke Misono), R.O. and N.S.; supervision, N.S.; project administration, K.M., H.I. and N.S.; funding acquisition, K.M., H.I. and N.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by JSPS KAKENHI, grant numbers; 18K09338, 19K08605, 19K08627, 19K08656 and 19K17640. Conflicts of Interest: The authors declare no conflict of interest. N.N. is an employee of MSD K.K., a subsidiary of Merck & Co., Inc., and receives personal fees from MSD K.K. outside of this study.

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