Pleckstrin homology and RhoGEF domain containing G4 (PLEKHG4) leads to the activation of RhoGTPases promoting the malignant phenotypes of thyroid cancer.

Thyroid cancer (TC) is one of the most common endocrine system cancers, and its incidence is elevating. There is an urgent need to develop a deeper understanding of TC pathogenesis and explore new therapeutic target for its treatment. This study aimed to investigate the effects of pleckstrin homology and RhoGEF domain containing G4 (PLEKHG4) on the progression of TC. Herein, 29 pairs of TC and adjacent tissues were used to assess the expression of PLEKHG4. A xenograft model of mouse was established by subcutaneously injected with TC cells. Lung metastasis model was established through left ventricular injection. The results revealed that PLEKHG4 was up-regulated in human TC tissues. PLEKHG4 level was correlated with clinicopathological parameters of TC patients. In vitro assays revealed that PLEKHG4 promoted TC cell proliferation, migration, invasion, and epithelial-mesenchymal transformation. Knockdown of PLEKHG4 led to the opposite effects, and the loss of PLEKHG4 enhanced the apoptosis ability and inhibited the stemness properties of TC cells. These findings were further confirmed by the in vivo growth and lung metastasis of TC tumor. Mechanistically, PLEKHG4 promoted the activation of RhoGTPases RhoA, Cdc42, and Rac1. The inhibitors of these RhoGTPases reversed the PLEKHG4-induced malignant phenotypes. Additionally, ubiquitin-conjugating enzyme E2O (UBE2O), a large E2 ubiquitin-conjugating enzyme acted as an ubiquitin enzyme of PLEKHG4, facilitated its ubiquitination and degradation. In conclusion, PLEKHG4, regulated by UBE2O, promoted the thyroid cancer progression via activating the RhoGTPases pathway. UBE2O/PLEKHG4/RhoGTPases axis is expected to be a novel a therapeutic target for TC treatment.

FUT8-AS1/miR-944/Fused in Sarcoma/Transcription Factor 4 Feedback Loop Participates in the Development of Oral Squamous Cell Carcinoma through Activation of Wnt/ß-Catenin Signaling Pathway.

As a common type of head and neck squamous cell carcinoma, oral squamous cell carcinoma (OSCC) is a lethal and deforming disease. Long noncoding RNAs have emerged as critical modulators in different malignancies. However, the role of fucosyltransferase 8 antisense RNA 1 (FUT8-AS1) in OSCC still remains elusive. In this study, quantitative RT-PCR and Western blot were used for the measurement of RNAs and proteins. Mechanism assays explored the putative correlation among genes. In vitro assays evaluated the changes in OSCC cell malignant phenotype, whereas in vivo assays highlighted the influence of FUT8-AS1 on tumor growth. FUT8-AS1, aberrantly up-regulated in OSCC tissues and cells, could exacerbate OSCC cell malignant behaviors. The cancerogenic property of FUT8-AS1 in OSCC was further confirmed via animal experiments. Furthermore, FUT8-AS1 enhanced the expression of transcription factor 4 (TCF4) via sponging miR-944 and recruiting fused in sarcoma (FUS), thus affecting OSCC cell biological behaviors via modulation on Wnt/ß-catenin signaling activity. In addition, TCF4 was validated as the transcriptional activator of FUT8-AS1. To conclude, TCF4-mediated FUT8-AS1 could exacerbate OSCC cell malignant behaviors and facilitate tumor growth via modulation on miR-944/FUS/TCF4.

3,3'-Diindolylmethane plus Eflornithine suppress DNA Replication and Cell Cycle in Esophageal Squamous Cell Carcinoma in vivo.

Esophageal squamous cell carcinoma (ESCC) is a malignant cancer that is responsible for a high mortality rate; it accounts for approximately 90% of the 456,000 esophageal cancer (EC) cases diagnosed annually. Effective natural or synthesized compounds to prevent, treat, and/or inhibit ESCC relapse are desperately needed. The natural di-indole compound 3,3'-diindolylmethane (DIM) is abundant in cruciferous vegetables and shows potent anti-tumor effects in multiple cancers. The synthesized Eflornithine (DFMO) is clinically used to treat African sleeping sickness. We demonstrated that the combination of DIM+DFMO could significantly suppress the ESCC growth in the in vivo study of three patient-derived xenograft (PDX) cases. Then, the corresponding underlying anticancer mechanisms were investigated via the isobaric tags for relative and absolute quantification (iTRAQ) on the proteome level. We found that the DNA Replication and Cell Cycle were the top-2 most significantly downregulated signaling pathways following the DIM+DFMO treatment. Correspondingly and interestingly, these two pathways were the top-2 upregulated ones in clinic ESCC tumors. Moreover, the involved differentially expressed genes (DEGs) including MCM2, MCM3, MCM5, MCM6, MCM7, CDK1, and LIG1 were all inversely downregulated by DIM+DFMO treatment. In the limited clinical study in two ESCC cases, the administration of DIM (250mg) +DFMO (500 mg) once daily showed favorable results, including alleviated swallowing difficulties, decreased blood tumor markers (CA19-9, CA15-3 and AFP), and no severe toxicity in at least one month progression free survival period. We concluded that DIM+DFMO is a promising therapeutic combination for ESCC treatment via the suppression of DNA Replication and Cell Cycle activities. However, these therapeutic effects should be verified in large cohort clinical trials with sufficient cases.

IGF2BP2 knockdown suppresses thyroid cancer progression by reducing the expression of long non-coding RNA HAGLR.

BACKGROUND: N6-methyladenosine (m6A), a common internal modification on RNAs, has been found to be closely linked with RNA biosynthesis/metabolism and cancer development. In this text, the roles and molecular mechanisms of m6A-bind protein IGF2BP2 in the development of thyroid cancer (TC) were investigated in vitro. METHODS: IGF2BP2 and lncRNA HAGLR were screened out through multiple public databases such as TCGA, Ualcan, POSTAR2, Starbase, and GEPIA. Cell proliferative, migratory and invasive abilities were assessed by CCK-8, Transwell migration and invasion assays, respectively. Cell cycle distribution and cell apoptotic patterns were measured by flow cytometry. The interaction between HAGLR and IGF2BP2 was examined by RIP, RNA pull-down and luciferase assays and bioinformatics analysis. The effect of IGF2BP2 knockdown on the m6A level of HAGLR was explored by meRIP assay. RESULTS: IGF2BP2 was highly expressed in TC tumor tissues. IGF2BP2 knockdown weakened cell proliferative, migratory, and invasive abilities, and induced cell cycle arrest and cell apoptosis in TC cells. LncRNA HAGLR expression was markedly upregulated and positively associated with IGF2BP2 expression in TC tissues. IGF2BP2 knockdown reduced HAGLR expression and transcript stability in TC cells. IGF2BP2 regulated HAGLR expression in an m6A-dependent manner. HAGLR overexpression weakened the effects of IGF2BP2 loss on cell proliferation, migration, invasion, apoptosis, and cell cycle progression in TC cells. CONCLUSION: IGF2BP2 loss inhibited cell proliferation, migration and invasion, and induced cell apoptosis and cell cycle arrest by down-regulating HAGLR expression in an m6A-dependent manner in TC cells, providing some potential diagnostic and therapeutic targets for TC.

miR-744-5p mediates lncRNA HOTTIP to regulate the proliferation and apoptosis of papillary thyroid carcinoma cells.

Papillary thyroid carcinoma (PTC) is the most common malignancy in thyroid. miR-744-5p plays an efficient role in various cancers, but its role in PTC remains unknown. In this work, we aimed to explore the function of miR-744-5p and the mechanism by which miR-744-5p acted in PTC. We observed that miR-744-5p expression was significantly declined in PTC tissues and cell lines. The high level of miR-744-5p is significantly associated with a better clinical picture of PTC patients. Overexpression of miR-744-5p inhibited the proliferation, arrested the cell cycle, and promoted the apoptosis in PTC cells. Oppositely, down-regulation of miR-744-5p reversed the above tendencies. We also found that miR-744-5p down-regulated its downstream genes c-myc and attenuated cell proliferation induced by c-myc. Long non-coding RNA (lncRNA) HOTTIP was found to be up-regulated and to act as an oncogene in PTC. In this study, miR-744-5p bound to HOTTIP and was negatively regulated by HOTTIP. In conclusion, miR-744-5p acts as a tumor suppressor to inhibit proliferation and promotes the apoptosis of PTC cells via targeting c-myc. Moreover, miR-744-5p expression interferes with lncRNA HOTTIP ability to promote proliferation and downregulate apoptosis in papillary thyroid carcinoma.

Knockout of ASAP1 induces autophagy in papillary thyroid carcinoma by inhibiting the mTOR signaling pathway.

Due to lymph node metastasis and infiltration, surgery for PTC (papillary thyroid carcinoma) is a high-risk treatment strategy. Our work reports for the first time that ASAP1 (ArfGAP with SH3 Domain, Ankyrin Repeat and PH Domain 1) is highly expressed in PTC and that its high expression is related to autophagy. Autophagy and ASAP1 expression in 40 PTC tissues and normal tissues were detected by immunofluorescence. We used the lentiviral CRISPR/Cas9 nickase to generate stable cell lines. The difference in autophagy levels between the ASAP1 KO group and the control group was determined by Western blot and immunofluorescence analyses. We added chloroquine (CQ) to verify that ASAP1 increased the formation of autophagosomes rather than reducing their degradation. The expression of mTOR activity-related proteins (P-P70S6K, P-MTOR) was studied by Western blotting. ASAP1 was upregulated while autophagy was downregulated in PTC tissues compared to normal tissues. Knockout of ASAP1 induced autophagy in both MDA-T32 and MDA-T85 cells. Knockout of ASAP1 attenuated the activation of the mTOR signaling pathway. Our studies demonstrated that ASAP1 is upregulated while autophagy is reduced in PTC tissues. In addition, knockout of ASAP1 induces autophagy in PTC by inhibiting the mTOR signaling pathway.

LncRNA PTCSC3 affects drug resistance of anaplastic thyroid cancer through STAT3/INO80 pathway.

BACKGROUND: LncRNA PTCSC3 is a tumor suppressor in thyroid cancer, and its role in drug resistance of anaplastic thyroid cancer (ATC) to chemotherapy drug doxorubicin was investigated in this study. METHODS: Expression of RNA and protein was analyzed by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze the expression rate of CD133+ cells. The endogenous expression of related genes was modulated by recombinant plasmids and cell transfection. Combination condition and interaction between PTCSC3 and STAT3 were determined by RIP and RNA pull-down assay, respectively. MTT assay was performed to detect cytotoxicity. Chromatin immunoprecipitation was conducted to identify interactions between STAT3 and DNA promoter of INO80. RESULTS: LncRNA PTCSC3 was low-expressed in ATC tissues and cells. Over-expressed PTCSC3 inhibited the drug resistance of ATC to doxorubicin. PTCSC3 negatively regulated STAT3, and STAT3 promoted expression of INO80. PTCSC3 regulated INO80 through STAT3. PTCSC3 suppressed stem cells properties and drug resistance of ATC to doxorubicin. CONCLUSION: LncRNA PTCSC3 inhibits INO80 expression by negatively regulating STAT3, and thereby attenuating drug resistance of ATC to chemotherapy drug doxorubicin.

LncRNA HOTTIP promotes papillary thyroid carcinoma cell proliferation, invasion and migration by regulating miR-637.

BACKGROUND: Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy. Besides, increasing evidence has demonstrated that long non-coding RNA (lncRNA) HOTTIP played a crucial role in cancer pathogenesis. MiR-637-mediated Akt1 was involved in cell growth, invasion and migration in various malignancies. This study was aimed to investigate the potential biological effect and regulatory mechanism of HOTTIP on cell proliferation, invasion and migration in PTC. METHODS: Expression of HOTTIP, miR-637 and Akt1 were determined by quantitative RT-PCR (qRT-PCR) and western blotting in PTC tissues, normal tissues, PTC cells (TPC-1 and HTH83) or non-tumor thyroid cells (Nthy-ori 3-1). Cell proliferation, invasion and migration following HOTTIP knockdown were investigated in PTC cells. The target of HOTTIP was validated by RNA immunoprecipitation (RIP) and pull-down assay. Moreover, a xenograft model was performed. RESULTS: HOTTIP was upregulated in human PTC tissues and PTC cell lines. In addition, HOTTIP knockdown inhibited the proliferation, invasion and migration in vitro together with in vivo tumorigenesis of PTC cells. Additionally, HOTTIP knockdown downregulated Akt1 expression and suppressed cell proliferation, invasion and migration in PTC cells by regulating miR-637. In contrast, miR-637 inhibitor reversed above-mentioned tendencies caused by HOTTIP knockdown. CONCLUSION: HOTTIP is a potential oncogene in PTC and may serve as a therapeutic target for malignancies.

LncRNA PTCSC3/miR-574-5p Governs Cell Proliferation and Migration of Papillary Thyroid Carcinoma via Wnt/ß-Catenin Signaling.

The distance metastases of papillary thyroid carcinoma (PTC) were a major threaten for PTC patients, thus, to study the potential mechanism for the treatment of PTC was essential. Previous studies have shown that PTCSC3 (Thyroid Carcinoma Susceptibility Candidate 3), miR-574-5p and Wnt/ß-catenin were involved in PTC, but the potential pathogenic mechanism among them was still unclear. Real-time PCR and Western blot were used to detect genes expression. Luciferase reporter assay was used to detect the combination of miR-574-5p and suppressor of cancer cell invasion (SCAI), as well as the ratio of TOP/FOP. RNA Pull-down assay verified the bound of PTCSC3 and miR-574-5p. MTT assay, Transwell assay, and wound scratch assay were used to detect cell viability and cell migration. The expression of PTCSC3 and SCAI were decreased, while miR-574-5p and ß-catenin were increased in PTC tissues and cells. Overexpressed PTCSC3 suppressed cell proliferation and migration, promoted the expression of SCAI, but inhibited ß-catenin. PTCSC3 absorbed miR-574-5p, and miR-574-5p targeted to SCAI; SCAI could regulate the activity of Wnt/ß-catenin. PTCSC3/miR-574-5p regulated the activity of Wnt/ß-catenin via SCAI and mediated cell proliferation and migration of PTC-1. In vivo experiments verified the fact that overexpressed PTCSC3 inhibited tumor growth. The signaling PTCSC3-miR-574-5p-SCAI-Wnt/ß-catenin mediated the proliferation and migration of PTC-1 cells, which was vital for the further PTC therapy and prognosis. J. Cell. Biochem. 118: 4745-4752, 2017. © 2017 Wiley Periodicals, Inc.