Candidate oncogene placenta specific 8 affect cell growth and cell migration in non- small cell lung cancers

Candidate oncogene placenta specific 8 affect cell growth and cell migration in non- small cell lung cancers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Candidate oncogene placenta specific 8 affect cell growth and cell migration in non- small cell lung cancers Mei-Lin Zhou, Jin-Ni Ma, Xin Xu, Xin-Yao Gao, Hai-Xia Wang, Jinhua Shen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2533161/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background As a candidate oncogene, PLAC8 participate in genesis and progression of various tumors. However, the role of PLAC8 in lung cancer (LC) especially non-small cell lung cancer (NSCLC) is still limited. Methods We performed Tissue microarray analysis (TMA) and Real-Time PCR (RT-PCR) to detect the expression levels of PLAC8 in LC tissues and cell lines, respectively. Then a series of cellular experiments focusing on cell proliferation, cell cycle, cell motility were conducted to identified the role of PLAC8 in NSCLC-derived cell lines H1299 and A549. Results TMA and RT-PCR showed that PLAC8 played complicated even opposite roles in different LCs. Further cellular experiments confirmed that PLAC8 could promote cell viability, alter cell cycle, and accelerate cell mobility via regulation of cell cyclins or cadherins, respectively. Conclusions Our study indicated that PLAC8 might participate in LC especially NSCLC progression. Our study also shed new light on the potential role of PLAC8 as a therapeutic biomarker in NSCLC. PLAC8 lung cancer non-small cell lung cancer cell proliferation cell migration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction World widely, lung cancer (LC) is one of the leading causes of cancer-related deaths ( 1 – 4 ). In China, LC has become a severe public health problem and an economical burden because of the incorrect diagnosis and high mortality ( 5 , 6 ). Despite advances in our understanding of diverse histological and molecular type of LC ( 1 ), it remains many challenges to identifying more proto-oncogenes and tumor suppressors involved in lung tumorigenesis, which means the exploration of novel molecular targets and biomarkers for early diagnosis and treatment of LC is still necessary. Placenta specific 8 (PLAC8), also known as Onzin , is a 115-amino acid which was first identified from a collection of 15,000 mouse genes with placental and embryonic RNAs ( 7 ). As a small, highly conserved, cysteine-rich protein, PLAC8 was involved in the progression of various cancers including hepatocellular cancer, breast cancer, nasopharyngeal cancer, LC and et al ( 8 – 12 ). For example, down-regulated PLAC8 could promotes cell viability, proliferation and tumor formation via miR-185-5p/PLAC8/b-catenin axis in hepatocellular carcinoma ( 9 ). PLAC8 inhibited cell proliferation, cell invasion and epithelial-mesenchymal transition in oral squamous cell carcinoma ( 10 ). In breast cancer, PLAC8 contributed tamoxifen resistance through MAPK/ERK pathway ( 11 ). Knock out of PLAC8 could increase radiotherapy sensitivity of nasopharyngeal carcinoma cells by promoting apoptosis ( 12 ). Taken together, Placenta-specific 8 (PLAC8) is closely associated with the proliferation, apoptosis, migration, autophagy, chemo-resistance and radio-sensitivity of several tumor cells. Furthermore, the impact of PLAC8 is varied and might have opposite effects in different tumors. Recent studies have also revealed that PLAC8 might play an important role in LC ( 13 , 14 ). The research showed that PLAC8 expression was elevated in lung tumor, and the alteration of PLAC8 expression could affect tumor growth via KLF4/PLAC8 and Wnt/b-Catenin pathway. However, the research about the participation of PLAC8 in tumor cell migration and the underlying mechanism are still limited. Our previous study showed that knockout of PLAC8 could affect proliferation and migration of HEK293T ( 15 ). In current study, we found that PLAC8 expression was decreased in multiple LCs, while the expression of PLAC8 in inflammatory pseudotumor was elevated. Further study revealed that knockout of PLAC8 could inhibit cell growth and decrease cell migration, while overexpression of PLAC8 could promote cell proliferation and enhance cell motility. Taken together, oncogene PLAC8 participated in LCs as a complicated tumor regulator. Our study confirmed certain cell cyclins and epithelial cell adhesion molecules were involved in PLAC8-induced pathological changes in lung tumors. These results might provide a novel molecular target and a novel insight for treatment of LC. Materials And Methods Plasmids, sgRNAs, and transfection The PLAC8 knockout H1299 cell lines were established via CRISPR/Cas9 technology as previously described ( 15 ). Briefly, CRISPR/Cas9 editing plasmid PX458 harboring PLAC8-targeted gRNA (5’-CACCGACTCTCTACAGGACCCGATA-3’) which has been identified the editing efficiency in HEK293T ( 15 ) was transfected into H1299 with lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The monoclonal PLAC8 knockout H1299 cell line was isolated from single GFP + cell which was sorted by fluorescence activated cell sorting (FACS) with a SH800S Cell sorter (Sony, Tokyo, Japan). After the isolated A549 cells reached 90–100% confluent, two independent strains of monoclonal PLAC8 KO cell lines (H2-PLAC8 and H3-PLAC8) were identified and selected for further experiments. The PLAC8-overexpressing A549 cell lines were constructed as previously described with minor modification ( 16 ). Briefly, the coding sequence (CDS) of PLAC8 was synthesized by Bio-Transduction Lab, Wuhan, China. Then the sequence was inserted into a hygromycin resistance mammalian expression vector pCMV-3tag-8 (Agilent Technologies, Palo Alto, LA, USA) and transfected into A549 with lipofectamine 3000 (L3000015, Thermo Fisher Scientific, San Jose, CA, USA), while the original vector was applied as a negative control. To obtain stable PLAC8-overexpressing clones, the transfected A549 cells were treated with 300 µg/mL hygromycin (1366, BioFroxx, Germany) and incubated until normal A549 was killed entirely. A stable PLAC8-overexpressing cell lines (A549-OE8-2) were confirmed by western blotting. Cell culture, Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) The human lung-derived cell lines (MRC5, NCI-H1299, LTEP-A-2, NCI-H490, 95-D, NCI-H1975, SPC-A-1, SK-MES-1, BEAS-2B, H124 and A549) were purchased from ScienCell (Shanghai, China). The purchased cell lines and PLAC8 knockout cell lines (H2-PLAC8 and H3-PLAC8) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a humid atmosphere containing 5% CO 2 , while the PLAC8 overexpressing cell line (A549-OE8-1 and A549-OE8-2) were cultured with the addition of 150 µg/mL hygromycin. To measure the mRNA levels of PLAC8 in human lung-derived cell lines, total RNA was extracted from the cells with an RNA extraction kit (Bioteke, Beijing, China), Then the template cDNA was synthesized with a cDNA synthesis kit (Thermo Fisher Scientific, San Jose, CA, USA). The primers are listed as following: GAPDH-F: 5’-TGACTTCAACAGCGACACCCA-3’; GAPDH-R: 5’-CACCCTGTTGCTGTAGCCAAA-3’; PLAC8-F: 5’-AATTCAGCAGACACCTCTTCAG-3’; PLAC8-R: 5’-GCTAAGTTCAGGGACAACATTCA-3’; The quantitative real-time PCR was performed with SYBR qPCR Mix (Biosharp, Hefei, China) and analysis with a QuantStudio® 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as previously described ( 17 ). The mRNA expression levels of PLAC8 were calculated using the 2 − DDCt method and normalized using the expression levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Immunohistochemical staining The immunohistochemical staining was performed as previously described with minor modification ( 14 ). Briefly, tissue microarrays (LC2083) containing 30 cases of each squamous cell carcinoma and adenocarcinoma, 22 small cell carcinoma, 5 large cell carcinoma, 22 bronchioloalveolar carcinoma, 10 carcinoid, 5 carcinoma sarcomatodes, 30 matched or matched metastatic carcinoma, 10 inflammatory pseudotumor, 20 inflammation, 18 matched or matched adjacent normal tissue and 6 normal tissues were purchased from Biomax, Derwood, MD, USA. Then the TMA slides were stained with PLAC8 antibodies (#13885, Cell Signaling Technology, Beverly, MA, USA) using UltraSensitive™ SP (mouse/rabbit) IHC Kit (KIT-9720, MXB Biotechnologies, Fuzhou, China) according to the manufacturer’s protocol. The stained slides were embedded in neutral balsam (SJ-601, Leica, Heidelberg, Germany), then the bright-field images were photographed under a BX51 microscope (Olympus, Tokyo, Japan). Cell proliferation analysis Cell counting assays were performed to detect the effects of PLAC8 on cell proliferation as previously described with minor modification ( 15 , 17 ). In 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St Louis, MO, USA) assay, the cells were seeded at a density of 2,000 cells/well in 96-well tissue culture plates with 200 µL culture medium. After 4 h incubation of 100 µg MTT reagent at 37℃, 150 µL dimethyl sulfoxide (DMSO, Sigma, St Louis, MO, USA) was added to each well, then the absorbance at 490 nm was recorded at 0, 24, 48, 96, 120 h, respectively. In colony formation assay, approximately 200 cells were plated in 6-well plates and cultured for 10 days. Then the colonies were fixed with 4% paraformaldehyde, and stained with the fast Giemsa Stain kit (40751ES01, Yeasen, Shanghai, China) according to the manufacturer’s protocol. The stained colonies were photographed and counted. The clone formation rate (CFR) = clone counts/seeded cell counts × 100%. Cell cycle analysis The cells were cultured in 60 mm cell culture dishes until achieving 90% confluence. To perform the cell cycle assay, the cells were collected and fixed with 70% ethanol for 2 h on ice. The fixed cells was centrifuged and resuspended with staining buffer (10 mg/mL PI: 1 mg/ml RNase: PBS = 5 : 1 : 94). To perform the cell apoptosis assay, 1 × 10 6 cells were collected and stained with an rh Annexin V/FITC Kit (ANT001, AntGene, Wuhan, China) according to the manufacturer’s protocol. The cell cycle distribution and cell apoptosis were analyzed with a BD FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Cell motility analysis Wound healing and invasion assays were performed as previously described with minor modification ( 17 ). For wound healing assay, the cells were seeded in 60 mm cell culture dishes and allowed to achieved 90% confluence. Then a wound on the cell layer was created with a sterile 10 µL pipette tip. The scraped wounds were photographed and analyzed at 0, 12, 24, 36 h, respectively. The rate of healing = (1-wound width/original width) × 100%. The invasion assay was performed with a 24-well plate Transwell® system with a polycarbonate filter membrane of 8.0 µm pore size (Corning Costar, Corning, NY, USA) as previously mentioned ( 15 ). Briefly, 2 × 10 5 cells resuspended with DMEM including 1% FBS were seeded on the filter, while the bottom plate was filled with DMEM including 10% FBS. The cells were cultured at 37℃ for 48 h. Then the cells on the top side of the filter were wiped out with a sterile cotton swab, while the migrated cells on the bottom side were fixed with 4% polyoxymethylene and stained with the fast Giemsa Stain kit. The numbers of the migrated cells were photographed and counted in five randomly selected per well. Western blot analysis To perform western blot assay, the cells were lysed in protein lysis buffer (P0013J, Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime, Shanghai, China). 20 µg total proteins were separated with 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose (NC) membranes. PLAC8 was blotted with PLAC8 (E1J2Z) Rabbit mAb (#13885, Cell Signaling Technology, Beverly, MA, USA). Cell cyclins were blotted with indicated antibodies in Cell Cycle Regulation Antibody Sampler Kit II (#9870, Cell Signaling Technology, Beverly, MA, USA). Then the bands were visualized by the BeyoECL Plus kit (P0018S, Beyotime, Shanghai, China) according to the manufacturer’s protocol. Quantitatively analysis was performed with the ImageLab® software (Bio-Rad, Hercules, CA, USA). b-actin was applied as an internal control. Statistical analysis All statistical analyses were evaluated using the software origin8.0. All experiments were repeated for at least three independent times. Differences between groups were calculated using a student's t-test. Results were expressed as means ± standard deviation (SD). P < 0.05 was considered to indicate significantly different. Results PLAC8 Expression Is Repressed in LCs PLAC8 is a candidate oncogene associated with multiple diseases especially tumors ( 8 ). Previous study with oncomine database ( 18 , 19 ) has observed that the expression levels of PLAC8 were significantly decreased in LCs compared with the paired adjacent normal tissues ( 14 ). To further identify the role of PLAC8 in LC, the expression pattern of PLAC8 was analyzed in human LC tissue microarrays (TMA). The staining intensity of PLAC8 and the clinicopathological information of TMA was shown in Table 1 . It was found that the staining signals of PLAC8 in inflammatory pseudotumor, chronic pneumonia and adjacent normal lung tissue were much stronger than that in lung carcinomas. Further statistical analysis showed that the expression levels of PLAC8 in 6 types of lung tumors (atypical carcinoid, metastatic squamous cell carcinoma, metastatic adenocarcinoma, squamous cell carcinoma, adenocarcinoma and small cell carcinoma) were much lower compared with adjacent normal lung tissue (Fig. 1 A), while the expression level of PLAC8 in inflammatory pseudotumor was higher than that in adjacent normal lung tissue. Four representative images of PLAC8 expression (negative, weak, moderate and strong) were shown in Fig. 1 B. Taken together, the expression of PLAC8 was decreased in LCs and increased in inflammation, which indicated that PLAC8 might play a crucial role in pathological process. Table 1 The key clinicopathological parameters (pathology diagnosis, organ, cases, sex and age) and the staining intensity of PLAC8 in lung cancer. Pathology diagnosis Organ Cases Sex Age (years) Staining intensity(SI) F M ༜60 ≥60 Squamous cell carcinoma lung 30 28 2 17 13 − (22) +(5) ++(1) +++(2) Adenocarcinoma lung 30 10 20 14 16 − (23) +(5) ++(0) +++(1) Small cell carcinoma lung 22 7 15 13 9 − (18) +(0) ++(1) +++(2) Large cell carcinoma lung 5 1 4 3 2 − (4) +(1) ++(0) +++(0) Invasive adenocarcinoma lung 22 13 9 12 10 − (19) +(2) ++(1) +++(0) Atypical carcinoid lung 10 2 8 5 5 − (8) +(1) ++(1) +++(0) Carcinoma sarcomatodes lung 5 1 4 3 2 − (4) +(1) ++(0) +++(0) Metastatic squamous cell carcinoma from hilum of lung Lymph node 14 0 14 7 7 − (8) +(2) ++(0) +++(2) Metastatic adenocarcinoma from hilum of lung Lymph node 16 5 11 8 8 − (13) +(0) ++(2) +++(1) Inflammatory pseudotumor lung 10 3 7 6 4 − (0) +(1) ++(4) +++(5) Chronic pneumonia lung 20 4 16 10 1 − (0) +(5) ++(14) +++(1) Adjacent normal lung tissue lung 24 4 20 14 10 − (0) +(12) ++(11) +++(1) Knockout of PLAC8 decreased cell proliferation and induced G1 arrest in H1299 cell line Furthermore, we checked the expression levels of PLAC8 in 11 lung-derived cell lines. It was found that PLAC8 was highly expressed in H1299, LTEP-A-2, 95-D, SPC-A-1, BEAS-2B, H125, while expressed at much lower levels in MRC-5, H460, H1975, SK-MES-1 and A549 (Fig. 2 ). The result indicated that PLAC8 might be involved in different signal pathways to affect the pathological process in different cell lines. To further investigate how PLAC8 played a role in tumorigenesis, H1299 was chosen to construct PLAC8-knockout cell line, while A549 was chosen to establish PLAC8-overexpressing cell line for further study. Abnormal cell growth was a typical characteristic of pathological process. Two PLAC8 knockout H1299 cell line were established and the proliferative function of PLAC8 was explored in Fig. 3 . As shown in Fig. 3 A, western blot result showed that PLAC8 was highly expressed in H1299, which is consistent with the mRNA expression result. Meanwhile, the protein expression of PLAC8 could not be detected in two knockout cell lines (H2-PLAC8 and H3-PLAC8), which indicated that the knockout cell lines have been successfully established. The MTT assays revealed that H2-PLAC8 and H3-PLAC8 both exhibited decreased cell viability (Fig. 3 B). Furthermore, colony formation assays showed that knockout of PLAC8 inhibited cell proliferation and growth in H2-PLAC8 and H3-PLAC8 (Fig. 3 C&D). These results indicated that knockout of PLAC8 might play an inhibitory role on H1299 proliferation. Furthermore, the distribution of cell cycle was detected and analyzed with flow cytometry. It turned out that knockout of PLAC8 redistributed the cell cycle of H1299 (Fig. 4 ). In H2-PLAC8 and H3-PLAC8, the cells percentage in G1 phase was increased while that in S phase was reduced (Fig. 4 ). To further explore underlying molecular mechanism, the expression levels of cell cylins were detected via western blot. It was found that the expression levels of P27 kip1 , P21 waf/cip1 , cyclin-dependent kinase 4 (CDK4), cyclin E2 were increased, while the expression levels of CDK2, cyclin D1, cyclin D3 were decreased. These results indicated that PLAC8 might affect cell growth by regulating cell cycle via cell-cyclins induced G1 arrest. Knockout of PLAC8 suppressed cell motility in H1299 cell line The alteration of cell motility is important to cancer metastasis ( 14 ). To determine the effect of PLAC8 on cell motility, we performed wound-healing assay and invasion assay (Fig. 5 ). As shown in Fig. 5 A&B, H2-PLAC8 and H3-PLAC8 exhibited significantly less wound closure than H1299 cells after 24 h, indicating that knockout of PLAC8 inhibit cell migration. Furthermore, the Transwell® chamber was employed to perform the cell invasion assay. It turned out that the number of invading cells in H2-PLAC8 and H3-PLAC8 were statistically lower than that in H1299 cells. Moreover, the expression level of E-cadherin, which is a key negative regulator of cell motility, was increased in H2-PLAC8 and H3-PLAC8. These results indicated that PLAC8 might play a positive role in cell motility via regulating E-cadherin. Overexpressing of PLAC8 promoted cell proliferation and altered cell cycle distribution in A549 cell line The multifaced roles of PLAC8 in LC were further explored in A549. PLAC8-overpressing cell lines was constructed and identified in Fig. 6 A. We successfully constructed a PLAC8-overexpressing cell lines (A549-OE8-2), which could stably express PLAC8 protein. Then MTT assay and colony formation assay were also performed in A549. It turned out that A549-OE8-2 exhibited a statistically higher cell viability than that in A549. Meanwhile, colony formation results also indicated that overexpression of PLAC8 could significantly increase the number of cell colonies in A549. Further flow cytometry analysis revealed that PLAC8 overexpression reduced the percentage of cells in G2/M phase (Fig. 7 A&B), and western blot results showed that up-regulated cell cyclins included cyclin B1, cdc2, P-cdc2, P21 waf/cip1 , P-H3, while down-regulated cell cyclins included cyclin E2, CDK2, P27 kip1 , P-Wee1 (Fig. 7 C). These results demonstrated that PLAC8 might promote cell proliferation of A549 via the altered expression of certain cell cyclins. Overexpressing of PLAC8 enhance cell motility in A549 cell lines Further wound-healing assay result showed that overexpression of PLAC8 could significantly accelerate wound closure in A549 compared with negative control (Fig. 8 A&B). As shown in Fig. 8 C&D, overexpression of PLAC8 also increased the number of invading cells in A549-OE8-2. Moreover, the expression level of N-cadherin was decreased in A549-OE8-2, while vimentin was upregulated. These results indicated that N-cadherin and vimentin might participate in PLAC8-induced cell migration. Discussion As a candidate oncogene, PLAC8 was proven to be involved in solid tumor formation and metastasis, such as liver ( 9 ), prostate ( 20 ), kidney ( 21 ) etc. In current study, we aimed to explore the role of PLAC8 in LC. First, the human LC TMA showed that the expression of PLAC8 was decreased in a series of tumor tissues, while the expression level was up-regulated in inflammatory pseudotumor. These results indicated that PLAC8 might play complicated even opposite roles in different pulmonary pathological process, and also provide a new insight for PLAC8 as a potential biomarker to distinguish inflammatory pseudotumor and tumor. To further investigate the role of PLAC8 in LC tumorigenesis and metastasis, the expression levels of PLAC8 in different lung-derived cell lines were detected. It was revealed that PLAC8 was highly expressed in a NSCLC cell line (H1299), a high metastasis human lung cancer cell line (95-D). three human lung adenocarcinoma cell lines (LTEP-A-2, SPC-A-1 and H125) and a human bronchial epithelial cell line (BEAS-2B), while the expression levels of PLAC8 were much lower in two NSCLC cell lines (A549, H460 and SK-MES-1), a human lung adenocarcinoma cell line (H1975) and MRC-5 derived from normal human embryonic lung. It is hard to reach a conclusion to explain the complicated mRNA expression patterns of PLAC8 in different LC-derived cell lines and normal lung cell lines. However, it is consistent with the above results that PLAC8 plays a complicate even opposite role in different lung-derived cell lines. H1299 and A549 are both NSCLC cell lines. H1299 was derived from a metastatic lymph node and lacked p53 protein expression, while the A549 was established from lung cancer tissue and p53 was normally expressed. Previous study indicated that p53 could suppression tumor growth in NSCLC ( 22 ). The expression levels of PLAC8 in H1299 was much higher than that in A549. A potential molecular mechanism was that PLAC8 might participate in NSCLC metastasis as a part of p53 signaling cascades. Herein two NSCLC cell lines H1299 (p53-null) which highly expresses PLAC8 and A549 (p53-widetype) in which the expression level of PLAC8 is much lower were chosen for further cellular research. To investigate the potential tumor suppressor/activator role PLAC8 in NSCLC, we knockout PLAC8 protein in H1299 while PLAC8 was overexpressed in A549. Primarily, the proliferative capacity of PLAC8 was detected. The cell growth experiments conducted in H1299 and A549 indicated that knockout of PLAC8 could inhibit cell proliferation in H1299, while PLAC8 upregulation could promote cell growth in A549. these results were consistent with other studies about PLAC8 in LC ( 13 , 14 ). These results demonstrated that PLAC8 might be considered as a tumor activator specially in NSCLC. Cell cycle assays showed that knockout of PLAC8 could accumulate the percentage of cells in G1 phase and reduce the proportion of cells in S phase. The potential molecular mechanism is that G1 cyclin-dependent cell cycle inhibitors including P27 kip1 ( 23 , 24 ) and P21 waf/cip1 ( 25 , 26 ) was upregulated and inhibited cell cycle progression at the G1/S interface, resulting in G1 arrest. Meanwhile, the expression of regulators necessary for G1-S transition were also changed. Increase in CDK4 and cyclin E2 and reduction in cyclin D1, cyclin D3 and CDK2 in H1299 were observed with PLAC8 knockout. As well known, cyclinD-CDK4/6 complex and cyclin E/CDK2 complex play important roles in the cell cycle transition from G1 phase to S phase ( 27 – 29 ). As shown in Figure S1, a possible explanation is that the CDK inhibitor P27 kip1 and P21 waf/cip1 bind then inactivate cyclin D/CDK4/6 complex and cyclin E/CDK2 kinase complex to evoke the G1 arrest ( 30 ). On the other hand, alteration of cell cycle was also observed in PLAC8 overexpressed A549. The possible molecular mechanisms were drafted in Figure S1B. The G2/M check point regulators, such as cyclin B1 ( 31 ), P-cdc2 ( 32 ), P21 waf/cip1 ( 33 ), P-H3 ( 34 ) were upregulated, while P-Wee1 ( 33 ), CDK2 ( 35 ), P27 kip1 ( 36 , 37 ) were suppressed. The potential mechanism might be that overexpression of PLAC8 altered the expression of G2/M phase regulators then the cell proportion of G2/M phase was decreased. Taken together, our investigation confirmed that the alteration of PLAC8 could affect cell growth and cell cycle via imbalanced expression of cell cycle regulators. Recent studies reveal that PLAC8 played a crucial role in malignant progressions of various cancers ( 21 , 38 ). The role of PLAC8 in NSCLC metastasis was further explored. It turned out that knockout of PLAC8 inhibits cell mobility in H1299 companying with the up-regulation of E-cadherin, which is a transmembrane protein that mediates cell-cell interaction and acts as a tumor suppressor in tumor metastasis ( 39 , 40 ). On the other hand, PLAC8 overexpression promote cell migration in A549. Meanwhile, the expression level of cell adhesion molecule N-cadherin ( 41 , 42 ) was downregulated, while the intermediate filament protein vimentin ( 43 , 44 ) was increased. As previous studies described, tumor metastasis is often associated with the loss of E-cadherin ( 45 ), while N-cadherin and vimentin could promote cell migration ( 46 , 47 ). Taken together, our investigation assumed that overexpression of PLAC8 could affect cell migration and cell invasion via regulation of cadherins and vimentin. Collectively, these results uncovered that PLAC8 could act as a tumor activator in NSCLC. The underlying molecular mechanism is that PLAC8 promotes cell proliferation via regulation of cell cyclins and accelerates cell migration via alteration of cadherins and vimentin. These findings suggest that abolishment of PLAC8 may be a new strategy for the LC especially NSCLC treatment. Conclusion In current study, we found that PLAC8 could affect cell proliferation and migration in NSCLC-derived cell lines via regulation of cell cyclins and cadherins, respectively. Our study indicated that PLAC8 might play a critical role in the development and progression of LC. Understanding the novel function of PLAC8 might open a door for new chance for the detection and treatment of LC. However, further animal experiments and pre-clinic trial are necessary for us to develop a promising usage of PLAC8 in the therapeutic strategy of LC. Abbreviations CDK, cyclin-dependent kinase; CDS, Coding sequence; CFR, clone formation rate; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NC, nitrocellulose; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethanesulfonyl fluoride; SD, standard deviation. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated and analyzed in this study are available upon reasonable request from the corresponding author. Competing interests The authors declare no competing interests. Funding This project was supported by Fund for Key Laboratory Construction of Hubei Province (Grant No. 2018BFC360), the National Natural Science Foundation of China (Grant No. 31101047 to Lu Xue), “the Fundamental Research Funds for the Central Universities”, South-Central MinZu University (Grant Number：CZQ22013). Authors’ contributions Lu Xue conceived and designed the experiments. Mei-Lin Zhou, Jin-Ni Ma, Xin Xu, Xin-Yao Gao and Hai-Xia Wang performed the experiments. Jinhua Shen and Lu Xue analyzed the data and generated the figures. Lu Xue wrote the manuscript. All authors declared that they have no conflicts of interest in the authorship and publication of this article. Acknowledgements We appreciate all the colleagues who work in institute for medical biology for their scientific and technical support. References Nooreldeen R, Bach H. Current and Future Development in Lung Cancer Diagnosis.International journal of molecular sciences 22 , (2021). Nasim F, Sabath BF, Eapen GA. 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Overexpression of placenta specific 8 is associated with malignant progression and poor prognosis of clear cell renal cell carcinoma. Int Urol Nephrol. 2017;49:1165–76. Zhang T et al. Transcription Factor p53 Suppresses Tumor Growth by Prompting Pyroptosis in Non-Small-Cell Lung Cancer. Oxid Med Cell Longev 2019, 8746895 (2019). Meroni SB, et al. Molecular Mechanisms and Signaling Pathways Involved in Sertoli Cell Proliferation. Front Endocrinol (Lausanne). 2019;10:224. Nakayama KI, Hatakeyama S, Nakayama K. Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1. Biochem Biophys Res Commun. 2001;282:853–60. Kuczyk MA, et al. Prognostic value of p27Kip1 and p21WAF/Cip protein expression in muscle invasive bladder cancer. Oncol Rep. 1999;6:687–93. Kuczyk MA, et al. Predictive value of altered p27Kip1 and p21WAF/Cip1 protein expression for the clinical prognosis of patients with localized prostate cancer. Oncol Rep. 2001;8:1401–7. 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Induction of G2/M Cell Cycle Arrest via p38/p21(Waf1/Cip1)-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells. Molecules 26 , (2021). Xie CL, et al. Antiproliferative Sorbicillinoids From the Deep-Sea-Derived Penicillium allii-sativi. Front Microbiol. 2020;11:636948. Chung JH, Bunz F. Cdk2 is required for p53-independent G2/M checkpoint control. PLoS Genet. 2010;6:e1000863. Font de Mora J, Uren A, Heidaran M, Santos E. Biological activity of p27kip1 and its amino- and carboxy-terminal domains in G2/M transition of Xenopus oocytes. Oncogene. 1997;15:2541–51. Song Z, et al. JS–K induces G2/M phase cell cycle arrest and apoptosis in A549 and H460 cells via the p53/p21WAF1/CIP1 and p27KIP1 pathways. Oncol Rep. 2019;41:3475–87. Huang CC, et al. Gut butyrate-producing organisms correlate to Placenta Specific 8 protein: Importance to colorectal cancer progression. J Adv Res. 2020;22:7–20. Na TY, Schecterson L, Mendonsa AM, Gumbiner BM. The functional activity of E-cadherin controls tumor cell metastasis at multiple steps. Proc Natl Acad Sci U S A. 2020;117:5931–7. Canel M, Serrels A, Frame MC, Brunton VG. E-cadherin-integrin crosstalk in cancer invasion and metastasis. J Cell Sci. 2013;126:393–401. Cao ZQ, Wang Z, Leng P. Aberrant N-cadherin expression in cancer. Biomed pharmacotherapy = Biomedecine pharmacotherapie. 2019;118:109320. Blaschuk OW. N-cadherin antagonists as oncology therapeutics. Philos Trans R Soc Lond B Biol Sci. 2015;370:20140039. Satelli A, Li S. Vimentin in cancer and its potential as a molecular target for cancer therapy. Cell Mol Life Sci. 2011;68:3033–46. Battaglia RA, Delic S, Herrmann H, Snider NT. Vimentin on the move: new developments in cell migration. F1000Res 7 , (2018). Shamir ER, Ewald AJ. Adhesion in mammary development: novel roles for E-cadherin in individual and collective cell migration. Curr Top Dev Biol. 2015;112:353–82. Choi S, Yu J, Kim W, Park KS. N-cadherin mediates the migration of bone marrow-derived mesenchymal stem cells toward breast tumor cells. Theranostics. 2021;11:6786–99. Karoii DH, Azizi H, Amirian M. Signaling Pathways and Protein-Protein Interaction of Vimentin in Invasive and Migration Cells: A Review. Cell Reprogram. 2022;24:165–74. Additional Declarations No competing interests reported. Supplementary Files FigureS1.pdf Figure S1 Possible schematic model of how PLAC8 influenced cell cycle redistribution. A. Possible schematic model of PLAC8 altering cell cycle in H1299. B. Possible schematic model of PLAC8 altering cell cycle in A549. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2533161","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":172324947,"identity":"9287471a-89f6-41a5-b9ef-b9ab5a64c530","order_by":0,"name":"Mei-Lin Zhou","email":"","orcid":"","institution":"South- Central Minzu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mei-Lin","middleName":"","lastName":"Zhou","suffix":""},{"id":172324948,"identity":"2e33dd3f-314a-4d1c-a5c2-a85c5ec3355c","order_by":1,"name":"Jin-Ni Ma","email":"","orcid":"","institution":"South- Central Minzu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jin-Ni","middleName":"","lastName":"Ma","suffix":""},{"id":172324949,"identity":"1713422b-8c8e-4449-9559-a4eae37ac86f","order_by":2,"name":"Xin Xu","email":"","orcid":"","institution":"South- Central Minzu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Xu","suffix":""},{"id":172324950,"identity":"eb4c242e-ad26-4ba5-b686-b13f9b7a2585","order_by":3,"name":"Xin-Yao Gao","email":"","orcid":"","institution":"South- Central Minzu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xin-Yao","middleName":"","lastName":"Gao","suffix":""},{"id":172324951,"identity":"57a37236-6028-46b9-89d4-ab47f6a77e6a","order_by":4,"name":"Hai-Xia Wang","email":"","orcid":"","institution":"South- Central Minzu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai-Xia","middleName":"","lastName":"Wang","suffix":""},{"id":172324953,"identity":"34aa20e8-1ae2-4d37-8e91-56e5c21dce7a","order_by":5,"name":"Jinhua Shen","email":"","orcid":"","institution":"South- Central Minzu University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Shen","suffix":""},{"id":172324954,"identity":"0826f149-1435-481a-bb02-a50b7853497d","order_by":6,"name":"Lu Xue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYHACxgMMFQwMbOwIEQOCeg4wnAFqYSZJC2MbkCRai8GN5AOHeedtk+djZmD+8LPNRp6BvXmbBEPNHZxaJGekJRzm3XbbsI2ZgU2yty3NsIHnWJkEw7FnOLXwS+QYgLQwgrQwM7YdTmCQyDGTYGw4jFMLm0T+h8O8c27bA7Uwf2Zs+5/AIP8GvxagLQyHeRtuJwK1MEgzth0A2sKDX4tkzzODg3OO3U5uA7pKsudcsmEbT1qxRcIx3FoMjic/fPCm5rbt/Pbmwx9+lNnJ87Mf3njjQw1uLUiAsQHiOxCRQIyGUTAKRsEoGAU4AQCgR00uA5+aOgAAAABJRU5ErkJggg==","orcid":"","institution":"South- Central Minzu University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Xue","suffix":""}],"badges":[],"createdAt":"2023-01-31 09:59:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2533161/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2533161/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32406954,"identity":"937a25ca-5969-4182-a299-c3269fdf8e02","added_by":"auto","created_at":"2023-02-02 23:10:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":478932,"visible":true,"origin":"","legend":"\u003cp\u003eThe protein expression patterns of PLAC8 in LC samples.\u003c/p\u003e\n\u003cp\u003eA. PLAC8 protein levels were reduced in 6 LCs (atypical carcinord, metastatic squamous cell carcinoma, metastatic adenocarcinoma, inflammatory pseudotumor, squamous cell carcinoma and adenocarcinoma, respectively) compared with normal lung tissue. B. Four representative images were presented from the IHC analysis of PLAC8 expression (negative, weak, moderate, and strong) obtained from LC samples.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/01245b81723e5fae6c8a9995.jpg"},{"id":32406957,"identity":"4a9102a5-6556-49b2-8a8d-ce7ca1198a60","added_by":"auto","created_at":"2023-02-02 23:10:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":149753,"visible":true,"origin":"","legend":"\u003cp\u003eThe mRNA expression patterns of PLAC8 in 11 human lung cell lines.\u003c/p\u003e\n\u003cp\u003eRelative mRNA expression of PLAC8 in the human lung cell lines H1299, LTEP-A-2, 95-D, SPC-A-1, BEAS-2B, H125, MRC-5, H460, H1975, SK-MES-1 and A549, respectively.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/fd2f75a42f969af47eacfd3c.jpg"},{"id":32407176,"identity":"e0c10fcd-8478-445e-8cf2-eb423789395b","added_by":"auto","created_at":"2023-02-02 23:18:01","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":239006,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of PLAC8 inhibits cellular proliferation and colony formation in H1299.\u003c/p\u003e\n\u003cp\u003eA. Western blot analysis shows knockout of PLAC8 protein in 2 knockout cell lines (H2-PLAC8 and H3-PLAC8) compared with control. B. Knockout of PLAC8 inhibits cell proliferation, valuing at staring point (day 1) set to 1 (***p \u0026lt; 000.1). C\u0026amp;D. Knockout of PLAC8 inhibits colony formation and the number of colonies were quantified.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/8dccf78c7cd0ca77c0eb3bc3.jpg"},{"id":32407177,"identity":"c175c24a-53cf-4e88-a5f1-f0e1b11b64c1","added_by":"auto","created_at":"2023-02-02 23:18:02","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":270999,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of PLAC8 induces G1 arrest in H1299.\u003c/p\u003e\n\u003cp\u003eA\u0026amp;B. Knockout of PLAC8 increases the cell population in G1 phase, while reducing the number of cells in S phase. (*p \u0026lt; 0.05, ***p \u0026lt; 000.1). C. Knockout of PLAC8 alters the expression of cell cycle proteins. Representative blots from three experiments with similar results are shown.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/3acb3b31b994636d189b9b5a.jpg"},{"id":32406958,"identity":"07a875e1-527d-4f98-ad22-c6e847609c2a","added_by":"auto","created_at":"2023-02-02 23:10:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":303741,"visible":true,"origin":"","legend":"\u003cp\u003eKnockout of PLAC8 inhibits cell migration in H1299.\u003c/p\u003e\n\u003cp\u003eA\u0026amp;B. Knockout of PLAC8 inhibits cell motility in a wound-healing assay (*p \u0026lt; 0.05, **p \u0026lt; 0.01). C\u0026amp;D. PLAC8 inhibits cell invasion (***p \u0026lt; 0.001). E. Knockout of PLAC8 increases the expression of E-cadherin.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/23feed0ce62a26ca0c0bf50d.jpg"},{"id":32406955,"identity":"9355d470-6ec5-44b2-93cc-c84b1453d69a","added_by":"auto","created_at":"2023-02-02 23:10:02","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":185687,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of PLAC8 increases cell proliferation in A549.\u003c/p\u003e\n\u003cp\u003eA\u0026amp;B. Western blot analysis shows overexpression of PLAC8 protein in 2 overexpression cell lines (A549-OE8-1 and A549-OE8-2) compared with control. C. Overexpression of PLAC8 increases cell proliferation (**p \u0026lt; 0.01, ***p \u0026lt; 0.001). D\u0026amp;E. Knockout of PLAC8 inhibits colony formation. The number of colonies were quantified (*p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/91cf04b3217e88c5fca9480f.jpg"},{"id":32407178,"identity":"5e8c744f-264a-4f1f-a876-0ff74716ff63","added_by":"auto","created_at":"2023-02-02 23:18:02","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":210832,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of PLAC8 reduces cell proportion in G2/M phase in A549.\u003c/p\u003e\n\u003cp\u003eA\u0026amp;B. Overexpression of PLAC8 reduces the cell population in G2/M phase (*p \u0026lt; 0.05). C. Overexpression of PLAC8 alters the expression of cell cycle proteins. Representative blots from three experiments with similar results are shown.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/a2046327963968ca8b641ece.jpg"},{"id":32406962,"identity":"a30e0e81-accd-45a1-99f8-a304ada60de0","added_by":"auto","created_at":"2023-02-02 23:10:02","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":269110,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of PLAC8 increases cell migration in A549.\u003c/p\u003e\n\u003cp\u003eA\u0026amp;B. Overexpression of PLAC8 increases cell motility in a wound-healing assay (**p \u0026lt; 0.01, ***p \u0026lt; 0.001). C\u0026amp;D. Overexpression of PLAC8 increases cell invasion (**p \u0026lt; 0.01). E. Overexpression of PLAC8 alters the expression of vim and N-cad.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/894a89087e0dae4f8dd28d39.jpg"},{"id":33228008,"identity":"95e2687e-e5dc-4d09-af00-a1e760ee4f07","added_by":"auto","created_at":"2023-02-21 13:44:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":948372,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/897ca6f7-9e5e-421b-b402-a67ca4461b96.pdf"},{"id":32406960,"identity":"5cf0df36-5307-460f-b8ed-826122c6fcd9","added_by":"auto","created_at":"2023-02-02 23:10:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":240786,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1 Possible schematic model of how PLAC8 influenced cell cycle redistribution.\u003c/p\u003e\n\u003cp\u003eA. Possible schematic model of PLAC8 altering cell cycle in H1299. B. Possible schematic model of PLAC8 altering cell cycle in A549.\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2533161/v1/e899de138eaaf2129120b913.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Candidate oncogene placenta specific 8 affect cell growth and cell migration in non- small cell lung cancers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWorld widely, lung cancer (LC) is one of the leading causes of cancer-related deaths (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). In China, LC has become a severe public health problem and an economical burden because of the incorrect diagnosis and high mortality (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Despite advances in our understanding of diverse histological and molecular type of LC (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e), it remains many challenges to identifying more proto-oncogenes and tumor suppressors involved in lung tumorigenesis, which means the exploration of novel molecular targets and biomarkers for early diagnosis and treatment of LC is still necessary.\u003c/p\u003e \u003cp\u003ePlacenta specific 8 (PLAC8), also known as \u003cem\u003eOnzin\u003c/em\u003e, is a 115-amino acid which was first identified from a collection of 15,000 mouse genes with placental and embryonic RNAs (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). As a small, highly conserved, cysteine-rich protein, PLAC8 was involved in the progression of various cancers including hepatocellular cancer, breast cancer, nasopharyngeal cancer, LC and et al (\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). For example, down-regulated PLAC8 could promotes cell viability, proliferation and tumor formation via miR-185-5p/PLAC8/b-catenin axis in hepatocellular carcinoma (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). PLAC8 inhibited cell proliferation, cell invasion and epithelial-mesenchymal transition in oral squamous cell carcinoma (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In breast cancer, PLAC8 contributed tamoxifen resistance through MAPK/ERK pathway (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Knock out of PLAC8 could increase radiotherapy sensitivity of nasopharyngeal carcinoma cells by promoting apoptosis (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Taken together, Placenta-specific 8 (PLAC8) is closely associated with the proliferation, apoptosis, migration, autophagy, chemo-resistance and radio-sensitivity of several tumor cells. Furthermore, the impact of PLAC8 is varied and might have opposite effects in different tumors.\u003c/p\u003e \u003cp\u003eRecent studies have also revealed that PLAC8 might play an important role in LC (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The research showed that PLAC8 expression was elevated in lung tumor, and the alteration of PLAC8 expression could affect tumor growth via KLF4/PLAC8 and Wnt/b-Catenin pathway. However, the research about the participation of PLAC8 in tumor cell migration and the underlying mechanism are still limited.\u003c/p\u003e \u003cp\u003eOur previous study showed that knockout of PLAC8 could affect proliferation and migration of HEK293T (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In current study, we found that PLAC8 expression was decreased in multiple LCs, while the expression of PLAC8 in inflammatory pseudotumor was elevated. Further study revealed that knockout of PLAC8 could inhibit cell growth and decrease cell migration, while overexpression of PLAC8 could promote cell proliferation and enhance cell motility. Taken together, oncogene PLAC8 participated in LCs as a complicated tumor regulator. Our study confirmed certain cell cyclins and epithelial cell adhesion molecules were involved in PLAC8-induced pathological changes in lung tumors. These results might provide a novel molecular target and a novel insight for treatment of LC.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids, sgRNAs, and transfection\u003c/h2\u003e \u003cp\u003eThe PLAC8 knockout H1299 cell lines were established via CRISPR/Cas9 technology as previously described (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Briefly, CRISPR/Cas9 editing plasmid PX458 harboring PLAC8-targeted gRNA (5\u0026rsquo;-CACCGACTCTCTACAGGACCCGATA-3\u0026rsquo;) which has been identified the editing efficiency in HEK293T (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) was transfected into H1299 with lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The monoclonal PLAC8 knockout H1299 cell line was isolated from single GFP\u003csup\u003e+\u003c/sup\u003e cell which was sorted by fluorescence activated cell sorting (FACS) with a SH800S Cell sorter (Sony, Tokyo, Japan). After the isolated A549 cells reached 90\u0026ndash;100% confluent, two independent strains of monoclonal PLAC8 KO cell lines (H2-PLAC8 and H3-PLAC8) were identified and selected for further experiments.\u003c/p\u003e \u003cp\u003eThe PLAC8-overexpressing A549 cell lines were constructed as previously described with minor modification (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Briefly, the coding sequence (CDS) of PLAC8 was synthesized by Bio-Transduction Lab, Wuhan, China. Then the sequence was inserted into a hygromycin resistance mammalian expression vector pCMV-3tag-8 (Agilent Technologies, Palo Alto, LA, USA) and transfected into A549 with lipofectamine 3000 (L3000015, Thermo Fisher Scientific, San Jose, CA, USA), while the original vector was applied as a negative control. To obtain stable PLAC8-overexpressing clones, the transfected A549 cells were treated with 300 \u0026micro;g/mL hygromycin (1366, BioFroxx, Germany) and incubated until normal A549 was killed entirely. A stable PLAC8-overexpressing cell lines (A549-OE8-2) were confirmed by western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture, Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eThe human lung-derived cell lines (MRC5, NCI-H1299, LTEP-A-2, NCI-H490, 95-D, NCI-H1975, SPC-A-1, SK-MES-1, BEAS-2B, H124 and A549) were purchased from ScienCell (Shanghai, China). The purchased cell lines and PLAC8 knockout cell lines (H2-PLAC8 and H3-PLAC8) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37\u0026deg;C in a humid atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e, while the PLAC8 overexpressing cell line (A549-OE8-1 and A549-OE8-2) were cultured with the addition of 150 \u0026micro;g/mL hygromycin.\u003c/p\u003e \u003cp\u003eTo measure the mRNA levels of PLAC8 in human lung-derived cell lines, total RNA was extracted from the cells with an RNA extraction kit (Bioteke, Beijing, China), Then the template cDNA was synthesized with a cDNA synthesis kit (Thermo Fisher Scientific, San Jose, CA, USA). The primers are listed as following:\u003c/p\u003e \u003cp\u003eGAPDH-F: 5\u0026rsquo;-TGACTTCAACAGCGACACCCA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eGAPDH-R: 5\u0026rsquo;-CACCCTGTTGCTGTAGCCAAA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003ePLAC8-F: 5\u0026rsquo;-AATTCAGCAGACACCTCTTCAG-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003ePLAC8-R: 5\u0026rsquo;-GCTAAGTTCAGGGACAACATTCA-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eThe quantitative real-time PCR was performed with SYBR qPCR Mix (Biosharp, Hefei, China) and analysis with a QuantStudio\u0026reg; 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) as previously described (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The mRNA expression levels of PLAC8 were calculated using the 2\u003csup\u003e\u0026minus;\u0026thinsp;DDCt\u003c/sup\u003e method and normalized using the expression levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eThe immunohistochemical staining was performed as previously described with minor modification (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Briefly, tissue microarrays (LC2083) containing 30 cases of each squamous cell carcinoma and adenocarcinoma, 22 small cell carcinoma, 5 large cell carcinoma, 22 bronchioloalveolar carcinoma, 10 carcinoid, 5 carcinoma sarcomatodes, 30 matched or matched metastatic carcinoma, 10 inflammatory pseudotumor, 20 inflammation, 18 matched or matched adjacent normal tissue and 6 normal tissues were purchased from Biomax, Derwood, MD, USA. Then the TMA slides were stained with PLAC8 antibodies (#13885, Cell Signaling Technology, Beverly, MA, USA) using UltraSensitive\u0026trade; SP (mouse/rabbit) IHC Kit (KIT-9720, MXB Biotechnologies, Fuzhou, China) according to the manufacturer\u0026rsquo;s protocol. The stained slides were embedded in neutral balsam (SJ-601, Leica, Heidelberg, Germany), then the bright-field images were photographed under a BX51 microscope (Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell proliferation analysis\u003c/h2\u003e \u003cp\u003eCell counting assays were performed to detect the effects of PLAC8 on cell proliferation as previously described with minor modification (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St Louis, MO, USA) assay, the cells were seeded at a density of 2,000 cells/well in 96-well tissue culture plates with 200 \u0026micro;L culture medium. After 4 h incubation of 100 \u0026micro;g MTT reagent at 37℃, 150 \u0026micro;L dimethyl sulfoxide (DMSO, Sigma, St Louis, MO, USA) was added to each well, then the absorbance at 490 nm was recorded at 0, 24, 48, 96, 120 h, respectively. In colony formation assay, approximately 200 cells were plated in 6-well plates and cultured for 10 days. Then the colonies were fixed with 4% paraformaldehyde, and stained with the fast Giemsa Stain kit (40751ES01, Yeasen, Shanghai, China) according to the manufacturer\u0026rsquo;s protocol. The stained colonies were photographed and counted. The clone formation rate (CFR)\u0026thinsp;=\u0026thinsp;clone counts/seeded cell counts \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eThe cells were cultured in 60 mm cell culture dishes until achieving 90% confluence. To perform the cell cycle assay, the cells were collected and fixed with 70% ethanol for 2 h on ice. The fixed cells was centrifuged and resuspended with staining buffer (10 mg/mL PI: 1 mg/ml RNase: PBS\u0026thinsp;=\u0026thinsp;5 : 1 : 94). To perform the cell apoptosis assay, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were collected and stained with an rh Annexin V/FITC Kit (ANT001, AntGene, Wuhan, China) according to the manufacturer\u0026rsquo;s protocol. The cell cycle distribution and cell apoptosis were analyzed with a BD FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell motility analysis\u003c/h2\u003e \u003cp\u003eWound healing and invasion assays were performed as previously described with minor modification (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). For wound healing assay, the cells were seeded in 60 mm cell culture dishes and allowed to achieved 90% confluence. Then a wound on the cell layer was created with a sterile 10 \u0026micro;L pipette tip. The scraped wounds were photographed and analyzed at 0, 12, 24, 36 h, respectively. The rate of healing = (1-wound width/original width) \u0026times; 100%.\u003c/p\u003e \u003cp\u003eThe invasion assay was performed with a 24-well plate Transwell\u0026reg; system with a polycarbonate filter membrane of 8.0 \u0026micro;m pore size (Corning Costar, Corning, NY, USA) as previously mentioned (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Briefly, 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells resuspended with DMEM including 1% FBS were seeded on the filter, while the bottom plate was filled with DMEM including 10% FBS. The cells were cultured at 37℃ for 48 h. Then the cells on the top side of the filter were wiped out with a sterile cotton swab, while the migrated cells on the bottom side were fixed with 4% polyoxymethylene and stained with the fast Giemsa Stain kit. The numbers of the migrated cells were photographed and counted in five randomly selected per well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eTo perform western blot assay, the cells were lysed in protein lysis buffer (P0013J, Beyotime, Shanghai, China) supplemented with phenylmethanesulfonyl fluoride (PMSF, ST506, Beyotime, Shanghai, China). 20 \u0026micro;g total proteins were separated with 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose (NC) membranes. PLAC8 was blotted with PLAC8 (E1J2Z) Rabbit mAb (#13885, Cell Signaling Technology, Beverly, MA, USA). Cell cyclins were blotted with indicated antibodies in Cell Cycle Regulation Antibody Sampler Kit II (#9870, Cell Signaling Technology, Beverly, MA, USA). Then the bands were visualized by the BeyoECL Plus kit (P0018S, Beyotime, Shanghai, China) according to the manufacturer\u0026rsquo;s protocol. Quantitatively analysis was performed with the ImageLab\u0026reg; software (Bio-Rad, Hercules, CA, USA). b-actin was applied as an internal control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were evaluated using the software origin8.0. All experiments were repeated for at least three independent times. Differences between groups were calculated using a student's t-test. Results were expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate significantly different.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePLAC8 Expression Is Repressed in LCs\u003c/h2\u003e \u003cp\u003ePLAC8 is a candidate oncogene associated with multiple diseases especially tumors (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Previous study with oncomine database (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) has observed that the expression levels of PLAC8 were significantly decreased in LCs compared with the paired adjacent normal tissues (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To further identify the role of PLAC8 in LC, the expression pattern of PLAC8 was analyzed in human LC tissue microarrays (TMA). The staining intensity of PLAC8 and the clinicopathological information of TMA was shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It was found that the staining signals of PLAC8 in inflammatory pseudotumor, chronic pneumonia and adjacent normal lung tissue were much stronger than that in lung carcinomas. Further statistical analysis showed that the expression levels of PLAC8 in 6 types of lung tumors (atypical carcinoid, metastatic squamous cell carcinoma, metastatic adenocarcinoma, squamous cell carcinoma, adenocarcinoma and small cell carcinoma) were much lower compared with adjacent normal lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), while the expression level of PLAC8 in inflammatory pseudotumor was higher than that in adjacent normal lung tissue. Four representative images of PLAC8 expression (negative, weak, moderate and strong) were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Taken together, the expression of PLAC8 was decreased in LCs and increased in inflammation, which indicated that PLAC8 might play a crucial role in pathological process.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe key clinicopathological parameters (pathology diagnosis, organ, cases, sex and age) and the staining intensity of PLAC8 in lung cancer.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePathology diagnosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOrgan\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCases\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStaining intensity(SI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e༜60\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026ge;60\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSquamous cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(22)\u003c/p\u003e \u003cp\u003e+(5)\u003c/p\u003e \u003cp\u003e++(1)\u003c/p\u003e \u003cp\u003e+++(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(23)\u003c/p\u003e \u003cp\u003e+(5)\u003c/p\u003e \u003cp\u003e++(0)\u003c/p\u003e \u003cp\u003e+++(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmall cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(18)\u003c/p\u003e \u003cp\u003e+(0)\u003c/p\u003e \u003cp\u003e++(1)\u003c/p\u003e \u003cp\u003e+++(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarge cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(4)\u003c/p\u003e \u003cp\u003e+(1)\u003c/p\u003e \u003cp\u003e++(0)\u003c/p\u003e \u003cp\u003e+++(0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInvasive adenocarcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(19)\u003c/p\u003e \u003cp\u003e+(2)\u003c/p\u003e \u003cp\u003e++(1)\u003c/p\u003e \u003cp\u003e+++(0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtypical carcinoid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(8)\u003c/p\u003e \u003cp\u003e+(1)\u003c/p\u003e \u003cp\u003e++(1)\u003c/p\u003e \u003cp\u003e+++(0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarcinoma sarcomatodes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(4)\u003c/p\u003e \u003cp\u003e+(1)\u003c/p\u003e \u003cp\u003e++(0)\u003c/p\u003e \u003cp\u003e+++(0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetastatic squamous cell carcinoma from hilum of lung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLymph node\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(8)\u003c/p\u003e \u003cp\u003e+(2)\u003c/p\u003e \u003cp\u003e++(0)\u003c/p\u003e \u003cp\u003e+++(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetastatic adenocarcinoma from hilum of lung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLymph node\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(13)\u003c/p\u003e \u003cp\u003e+(0)\u003c/p\u003e \u003cp\u003e++(2)\u003c/p\u003e \u003cp\u003e+++(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInflammatory pseudotumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(0)\u003c/p\u003e \u003cp\u003e+(1)\u003c/p\u003e \u003cp\u003e++(4)\u003c/p\u003e \u003cp\u003e+++(5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChronic pneumonia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(0)\u003c/p\u003e \u003cp\u003e+(5)\u003c/p\u003e \u003cp\u003e++(14)\u003c/p\u003e \u003cp\u003e+++(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdjacent normal lung tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elung\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;(0)\u003c/p\u003e \u003cp\u003e+(12)\u003c/p\u003e \u003cp\u003e++(11)\u003c/p\u003e \u003cp\u003e+++(1)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eKnockout of PLAC8 decreased cell proliferation and induced G1 arrest in H1299 cell line\u003c/h2\u003e \u003cp\u003eFurthermore, we checked the expression levels of PLAC8 in 11 lung-derived cell lines. It was found that PLAC8 was highly expressed in H1299, LTEP-A-2, 95-D, SPC-A-1, BEAS-2B, H125, while expressed at much lower levels in MRC-5, H460, H1975, SK-MES-1 and A549 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The result indicated that PLAC8 might be involved in different signal pathways to affect the pathological process in different cell lines. To further investigate how PLAC8 played a role in tumorigenesis, H1299 was chosen to construct PLAC8-knockout cell line, while A549 was chosen to establish PLAC8-overexpressing cell line for further study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAbnormal cell growth was a typical characteristic of pathological process. Two PLAC8 knockout H1299 cell line were established and the proliferative function of PLAC8 was explored in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, western blot result showed that PLAC8 was highly expressed in H1299, which is consistent with the mRNA expression result. Meanwhile, the protein expression of PLAC8 could not be detected in two knockout cell lines (H2-PLAC8 and H3-PLAC8), which indicated that the knockout cell lines have been successfully established. The MTT assays revealed that H2-PLAC8 and H3-PLAC8 both exhibited decreased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, colony formation assays showed that knockout of PLAC8 inhibited cell proliferation and growth in H2-PLAC8 and H3-PLAC8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026amp;D). These results indicated that knockout of PLAC8 might play an inhibitory role on H1299 proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the distribution of cell cycle was detected and analyzed with flow cytometry. It turned out that knockout of PLAC8 redistributed the cell cycle of H1299 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In H2-PLAC8 and H3-PLAC8, the cells percentage in G1 phase was increased while that in S phase was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To further explore underlying molecular mechanism, the expression levels of cell cylins were detected via western blot. It was found that the expression levels of P27\u003csup\u003ekip1\u003c/sup\u003e, P21\u003csup\u003ewaf/cip1\u003c/sup\u003e, cyclin-dependent kinase 4 (CDK4), cyclin E2 were increased, while the expression levels of CDK2, cyclin D1, cyclin D3 were decreased. These results indicated that PLAC8 might affect cell growth by regulating cell cycle via cell-cyclins induced G1 arrest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eKnockout of PLAC8 suppressed cell motility in H1299 cell line\u003c/h2\u003e \u003cp\u003eThe alteration of cell motility is important to cancer metastasis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). To determine the effect of PLAC8 on cell motility, we performed wound-healing assay and invasion assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026amp;B, H2-PLAC8 and H3-PLAC8 exhibited significantly less wound closure than H1299 cells after 24 h, indicating that knockout of PLAC8 inhibit cell migration. Furthermore, the Transwell\u0026reg; chamber was employed to perform the cell invasion assay. It turned out that the number of invading cells in H2-PLAC8 and H3-PLAC8 were statistically lower than that in H1299 cells. Moreover, the expression level of E-cadherin, which is a key negative regulator of cell motility, was increased in H2-PLAC8 and H3-PLAC8. These results indicated that PLAC8 might play a positive role in cell motility via regulating E-cadherin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOverexpressing of PLAC8 promoted cell proliferation and altered cell cycle distribution in A549 cell line\u003c/h2\u003e \u003cp\u003eThe multifaced roles of PLAC8 in LC were further explored in A549. PLAC8-overpressing cell lines was constructed and identified in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. We successfully constructed a PLAC8-overexpressing cell lines (A549-OE8-2), which could stably express PLAC8 protein. Then MTT assay and colony formation assay were also performed in A549. It turned out that A549-OE8-2 exhibited a statistically higher cell viability than that in A549. Meanwhile, colony formation results also indicated that overexpression of PLAC8 could significantly increase the number of cell colonies in A549. Further flow cytometry analysis revealed that PLAC8 overexpression reduced the percentage of cells in G2/M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026amp;B), and western blot results showed that up-regulated cell cyclins included cyclin B1, cdc2, P-cdc2, P21\u003csup\u003ewaf/cip1\u003c/sup\u003e, P-H3, while down-regulated cell cyclins included cyclin E2, CDK2, P27\u003csup\u003ekip1\u003c/sup\u003e, P-Wee1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These results demonstrated that PLAC8 might promote cell proliferation of A549 via the altered expression of certain cell cyclins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverexpressing of PLAC8 enhance cell motility in A549 cell lines\u003c/p\u003e \u003cp\u003eFurther wound-healing assay result showed that overexpression of PLAC8 could significantly accelerate wound closure in A549 compared with negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026amp;B). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC\u0026amp;D, overexpression of PLAC8 also increased the number of invading cells in A549-OE8-2. Moreover, the expression level of N-cadherin was decreased in A549-OE8-2, while vimentin was upregulated. These results indicated that N-cadherin and vimentin might participate in PLAC8-induced cell migration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a candidate oncogene, PLAC8 was proven to be involved in solid tumor formation and metastasis, such as liver (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), prostate (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), kidney (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) etc. In current study, we aimed to explore the role of PLAC8 in LC. First, the human LC TMA showed that the expression of PLAC8 was decreased in a series of tumor tissues, while the expression level was up-regulated in inflammatory pseudotumor. These results indicated that PLAC8 might play complicated even opposite roles in different pulmonary pathological process, and also provide a new insight for PLAC8 as a potential biomarker to distinguish inflammatory pseudotumor and tumor.\u003c/p\u003e \u003cp\u003eTo further investigate the role of PLAC8 in LC tumorigenesis and metastasis, the expression levels of PLAC8 in different lung-derived cell lines were detected. It was revealed that PLAC8 was highly expressed in a NSCLC cell line (H1299), a high metastasis human lung cancer cell line (95-D). three human lung adenocarcinoma cell lines (LTEP-A-2, SPC-A-1 and H125) and a human bronchial epithelial cell line (BEAS-2B), while the expression levels of PLAC8 were much lower in two NSCLC cell lines (A549, H460 and SK-MES-1), a human lung adenocarcinoma cell line (H1975) and MRC-5 derived from normal human embryonic lung. It is hard to reach a conclusion to explain the complicated mRNA expression patterns of PLAC8 in different LC-derived cell lines and normal lung cell lines. However, it is consistent with the above results that PLAC8 plays a complicate even opposite role in different lung-derived cell lines.\u003c/p\u003e \u003cp\u003eH1299 and A549 are both NSCLC cell lines. H1299 was derived from a metastatic lymph node and lacked p53 protein expression, while the A549 was established from lung cancer tissue and p53 was normally expressed. Previous study indicated that p53 could suppression tumor growth in NSCLC (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The expression levels of PLAC8 in H1299 was much higher than that in A549. A potential molecular mechanism was that PLAC8 might participate in NSCLC metastasis as a part of p53 signaling cascades. Herein two NSCLC cell lines H1299 (p53-null) which highly expresses PLAC8 and A549 (p53-widetype) in which the expression level of PLAC8 is much lower were chosen for further cellular research.\u003c/p\u003e \u003cp\u003eTo investigate the potential tumor suppressor/activator role PLAC8 in NSCLC, we knockout PLAC8 protein in H1299 while PLAC8 was overexpressed in A549. Primarily, the proliferative capacity of PLAC8 was detected. The cell growth experiments conducted in H1299 and A549 indicated that knockout of PLAC8 could inhibit cell proliferation in H1299, while PLAC8 upregulation could promote cell growth in A549. these results were consistent with other studies about PLAC8 in LC (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). These results demonstrated that PLAC8 might be considered as a tumor activator specially in NSCLC. Cell cycle assays showed that knockout of PLAC8 could accumulate the percentage of cells in G1 phase and reduce the proportion of cells in S phase. The potential molecular mechanism is that G1 cyclin-dependent cell cycle inhibitors including P27\u003csup\u003ekip1\u003c/sup\u003e(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and P21\u003csup\u003ewaf/cip1\u003c/sup\u003e (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) was upregulated and inhibited cell cycle progression at the G1/S interface, resulting in G1 arrest. Meanwhile, the expression of regulators necessary for G1-S transition were also changed. Increase in CDK4 and cyclin E2 and reduction in cyclin D1, cyclin D3 and CDK2 in H1299 were observed with PLAC8 knockout. As well known, cyclinD-CDK4/6 complex and cyclin E/CDK2 complex play important roles in the cell cycle transition from G1 phase to S phase (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). As shown in Figure S1, a possible explanation is that the CDK inhibitor P27\u003csup\u003ekip1\u003c/sup\u003e and P21\u003csup\u003ewaf/cip1\u003c/sup\u003e bind then inactivate cyclin D/CDK4/6 complex and cyclin E/CDK2 kinase complex to evoke the G1 arrest (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, alteration of cell cycle was also observed in PLAC8 overexpressed A549. The possible molecular mechanisms were drafted in Figure S1B. The G2/M check point regulators, such as cyclin B1 (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), P-cdc2 (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), P21 \u003csup\u003ewaf/cip1\u003c/sup\u003e (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), P-H3 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) were upregulated, while P-Wee1 (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), CDK2 (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), P27\u003csup\u003ekip1\u003c/sup\u003e (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) were suppressed. The potential mechanism might be that overexpression of PLAC8 altered the expression of G2/M phase regulators then the cell proportion of G2/M phase was decreased. Taken together, our investigation confirmed that the alteration of PLAC8 could affect cell growth and cell cycle via imbalanced expression of cell cycle regulators.\u003c/p\u003e \u003cp\u003eRecent studies reveal that PLAC8 played a crucial role in malignant progressions of various cancers (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The role of PLAC8 in NSCLC metastasis was further explored. It turned out that knockout of PLAC8 inhibits cell mobility in H1299 companying with the up-regulation of E-cadherin, which is a transmembrane protein that mediates cell-cell interaction and acts as a tumor suppressor in tumor metastasis (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). On the other hand, PLAC8 overexpression promote cell migration in A549. Meanwhile, the expression level of cell adhesion molecule N-cadherin (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) was downregulated, while the intermediate filament protein vimentin (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) was increased. As previous studies described, tumor metastasis is often associated with the loss of E-cadherin (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), while N-cadherin and vimentin could promote cell migration (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Taken together, our investigation assumed that overexpression of PLAC8 could affect cell migration and cell invasion via regulation of cadherins and vimentin. Collectively, these results uncovered that PLAC8 could act as a tumor activator in NSCLC. The underlying molecular mechanism is that PLAC8 promotes cell proliferation via regulation of cell cyclins and accelerates cell migration via alteration of cadherins and vimentin. These findings suggest that abolishment of PLAC8 may be a new strategy for the LC especially NSCLC treatment.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn current study, we found that PLAC8 could affect cell proliferation and migration in NSCLC-derived cell lines via regulation of cell cyclins and cadherins, respectively. Our study indicated that PLAC8 might play a critical role in the development and progression of LC. Understanding the novel function of PLAC8 might open a door for new chance for the detection and treatment of LC. However, further animal experiments and pre-clinic trial are necessary for us to develop a promising usage of PLAC8 in the therapeutic strategy of LC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCDK, cyclin-dependent kinase; CDS, Coding sequence; CFR, clone formation rate; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NC, nitrocellulose; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethanesulfonyl fluoride; SD, standard deviation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll data generated and analyzed in this study are available upon reasonable request from the corresponding author.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis project was supported by Fund for Key Laboratory Construction of Hubei Province (Grant No. 2018BFC360), the National Natural Science Foundation of China (Grant No. 31101047 to Lu Xue), \u0026ldquo;the Fundamental Research Funds for the Central Universities\u0026rdquo;, South-Central MinZu University (Grant Number：CZQ22013).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eLu Xue conceived and designed the experiments. Mei-Lin Zhou, Jin-Ni Ma, Xin Xu, Xin-Yao Gao and Hai-Xia Wang performed the experiments. Jinhua Shen and Lu Xue analyzed the data and generated the figures.\u0026nbsp;Lu Xue wrote the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors declared that they have no conflicts of interest in the authorship and publication of this article.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe appreciate all the colleagues who work in institute for medical biology for their scientific and technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNooreldeen R, Bach H. Current and Future Development in Lung Cancer Diagnosis.International journal of molecular sciences\u003cb\u003e22\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNasim F, Sabath BF, Eapen GA. Lung Cancer. 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Molecular Mechanisms and Signaling Pathways Involved in Sertoli Cell Proliferation. Front Endocrinol (Lausanne). 2019;10:224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakayama KI, Hatakeyama S, Nakayama K. Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1. Biochem Biophys Res Commun. 2001;282:853\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuczyk MA, et al. Prognostic value of p27Kip1 and p21WAF/Cip protein expression in muscle invasive bladder cancer. Oncol Rep. 1999;6:687\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuczyk MA, et al. Predictive value of altered p27Kip1 and p21WAF/Cip1 protein expression for the clinical prognosis of patients with localized prostate cancer. Oncol Rep. 2001;8:1401\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang R, Xu K, Gao F, Huang J, Guan X. 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Vimentin on the move: new developments in cell migration. \u003cem\u003eF1000Res\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShamir ER, Ewald AJ. Adhesion in mammary development: novel roles for E-cadherin in individual and collective cell migration. Curr Top Dev Biol. 2015;112:353\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi S, Yu J, Kim W, Park KS. N-cadherin mediates the migration of bone marrow-derived mesenchymal stem cells toward breast tumor cells. Theranostics. 2021;11:6786\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaroii DH, Azizi H, Amirian M. Signaling Pathways and Protein-Protein Interaction of Vimentin in Invasive and Migration Cells: A Review. Cell Reprogram. 2022;24:165\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PLAC8, lung cancer, non-small cell lung cancer, cell proliferation, cell migration","lastPublishedDoi":"10.21203/rs.3.rs-2533161/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2533161/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAs a candidate oncogene, PLAC8 participate in genesis and progression of various tumors. However, the role of PLAC8 in lung cancer (LC) especially non-small cell lung cancer (NSCLC) is still limited.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe performed Tissue microarray analysis (TMA) and Real-Time PCR (RT-PCR) to detect the expression levels of PLAC8 in LC tissues and cell lines, respectively. Then a series of cellular experiments focusing on cell proliferation, cell cycle, cell motility were conducted to identified the role of PLAC8 in NSCLC-derived cell lines H1299 and A549.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTMA and RT-PCR showed that PLAC8 played complicated even opposite roles in different LCs. Further cellular experiments confirmed that PLAC8 could promote cell viability, alter cell cycle, and accelerate cell mobility via regulation of cell cyclins or cadherins, respectively.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study indicated that PLAC8 might participate in LC especially NSCLC progression. Our study also shed new light on the potential role of PLAC8 as a therapeutic biomarker in NSCLC.\u003c/p\u003e","manuscriptTitle":"Candidate oncogene placenta specific 8 affect cell growth and cell migration in non- small cell lung cancers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-02 23:09:57","doi":"10.21203/rs.3.rs-2533161/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"59711fd1-317d-4bcc-b6f3-6bcb438c1ecb","owner":[],"postedDate":"February 2nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-02-21T13:44:35+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-02 23:09:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2533161","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2533161","identity":"rs-2533161","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}