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Abstract Background Adoptive transfer of engineered immune cells is a promising strategy for cancer treatment. However, low transduction efficiency particularly when large payload lentiviral vectors are used on primary T cells is a limitation for the development of cell therapy platforms that include multiple constructs bearing long DNA sequences. RB-340-1 is a new CAR T cell that combines two strategies in one product through a CRISPR interference (CRISPRi) circuit. Because multiple regulatory components are included in the circuit, RB-340-1 production needs delivery of two lentiviral vectors into human primary T cells, both containing long DNA sequences. To improve lentiviral transduction efficiency, we looked for inhibitors of receptors involved in antiviral response. BX795 is a pharmacological inhibitor of the TBK1/IKKɛ complex, which has been reported to augment lentiviral transduction of human NK cells and some cell lines, but it has not been tested with human primary T cells. The purpose of this study was to test if BX795 treatment promotes large payload RB-340-1 lentiviral transduction of human primary T cells. Methods To make the detection of gene delivery more convenient, we constructed another set of RB-340-1 constructs containing fluorescent labels named RB-340-1F. We incorporated BX795 treatment into the human primary T cell transduction procedure that was optimized for RB-340-1F. We tested BX795 with T cells collected from multiple donors, and detected the effect of BX795 on T cell transduction, phenotype, cell growth and cell function. Results We found that BX795 promotes RB-340-1F lentiviral transduction of human primary T cells, without dramatic change in cell growth and T cell functions. Meanwhile, BX795 treatment increased CD8+ T cell ratios in transduced T cells. Conclusions These results indicate that BX795 treatment is effective, and might be a safe approach to promote RB-340-1F lentiviral transduction of human primary T cells. This approach might also be helpful for other T cell therapy products that need delivery of complicated platform via large payload lentiviral vectors.

Abstract Background DNA methylation is a heritable epigenetic mark, enabling stable but reversible gene repression. In mammalian cells, DNA methyltransferases (DNMTs) are responsible for modifying cytosine to 5-methylcytosine (5mC), which can be further oxidized by the TET dioxygenases to ultimately cause DNA demethylation. However, the genome-wide cooperation and functions of these two families of proteins, especially at large under-methylated regions, called canyons, remain largely unknown. Results Here we demonstrate that DNMT3A and TET1 function in a complementary and competitive manner in mouse embryonic stem cells to mediate proper epigenetic landscapes and gene expression. The longer isoform of DNMT3A, DNMT3A1, exhibits significant enrichment at distal promoters and canyon edges, but is excluded from proximal promoters and canyons where TET1 shows prominent binding. Deletion of Tet1 increases DNMT3A1 binding capacity at and around genes with wild-type TET1 binding. However, deletion of Dnmt3a has a minor effect on TET1 binding on chromatin, indicating that TET1 may limit DNA methylation partially by protecting its targets from DNMT3A and establishing boundaries for DNA methylation. Local CpG density may determine their complementary binding patterns and therefore that the methylation landscape is encoded in the DNA sequence. Furthermore, DNMT3A and TET1 impact histone modifications which in turn regulate gene expression. In particular, they regulate Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 enrichment to constrain gene expression from bivalent promoters. Conclusions We conclude that DNMT3A and TET1 regulate the epigenome and gene expression at specific targets via their functional interplay.

Purpose: Dendritic cells (DCs) are a heterogeneous cell population which critically regulates the adaptive immune response. Depending on their activation status, DCs can also promote peripheral immune tolerance, thus limiting the activation of the immune system and tissue damage. The N-myc downregulated gene 2 (Ndrg2) is highly expressed in DCs and limits the secretion of vascular endothelial growth factor (VEGF), which is critical for wound healing. Cell based therapy approaches using DCs have been approved by the FDA and clinical trials using DC immunotherapy are being performed against a variety of cancer types. However, the role of DC therapy for wound healing has not yet been investigated. Methods: Hematopoietic progenitor cells were isolated from the bone marrow of mice and differentiated into DCs over a 7-day in vitro culture period. Pharmacologic down-regulation of Ndrg2 was performed by treatment of cultures with 1,25-dihydroxyvitamin D3 (VD3) and the angiogenic potential of the treated cells was evaluated by endothelial cell (EC) tube formation assays. Cytokine secretion of DCs was measured in the conditioned media using Luminex multiplex assays. To permanently knock out Ndrg2 in DCs, a CRISPR/Cas9 gene editing approach was developed, using Cas9/sgRNA-ribonucleoproteins and electroporation. The determine the impact of genetically edited DCs on wound healing, splinted excisional wounds in C57BL6/J (wild-type) mice were treated weekly with pullulan-collagen hydrogels seeded with Ndrg2-knockout (KO) DCs, control DCs which had undergone electroporation only, or blank hydrogels. The transcriptomic impact of Ndrg2 downregulation on DC fate was evaluated by microfluidic single-cell RNA sequencing (scRNA seq) of Ndrg2-KO DCs, VD3-treated DCs and control DCs. Results: Ndrg2 down-regulation lead to a significantly stronger EC tube formation in co-cultures with VD3-treated DCs, and strongly enhanced VEGF secretion compared to untreated DCs in vitro. A CRISPR/Cas9 editing pipeline was developed for KO of Ndrg2 in DCs with a transfection rate and editing efficiency of > 90% shown by Sanger Sequencing. Excisional wounds treated with Ndrg2-KO DCs demonstrated significantly accelerated healing compared to control DCs and blank hydrogels. scRNA seq revealed that Ndrg2 downregulation strongly induced Vegfa expression and anti-oxidant transcriptomic signatures. Conclusion: Our data indicate that KO of Ndrg2 in DCs strongly enhances their secretion of VEGF, thus promoting angiogenesis and accelerating wound healing. Given the ready availability of DCs from the human blood through established leukapheresis protocols and easy multiplication in vitro, CRISPR/Cas9 editing of DCs is a promising new approach to induce wound healing and soft-tissue regeneration.

The presence of extensive infiltrated macrophages with impaired phagocytosis is widely recognised as a significant regulator for the development of endometriosis (EMs). Nevertheless, the metabolic characteristics and the fundamental mechanism of impaired macrophage phagocytosis are yet to be clarified. Here, we observe that there is the decreased expression of haematopoietic cellular kinase (HCK) in macrophage of peritoneal fluid from EMs patients, which might be attributed to high oestrogen and hypoxia condition. Of note, HCK deficiency resulted in impaired macrophage phagocytosis, and increased number and weight of ectopic lesions in vitro and in vivo. Mechanistically, this process was mediated via regulation of glutamine metabolism, and further upregulation of macrophage autophagy in a c-FOS/c-JUN dependent manner. Additionally, macrophages of EMs patients displayed insufficient HCK, excessive autophagy and phagocytosis dysfunction. In therapeutic studies, supplementation with glutamine-pre-treated macrophage or Bafilomycin A1 (an autophagy inhibitor)-pre-treated macrophage leads to the induction of macrophage phagocytosis and suppression of EMs development. This observation reveals that the aberrant HCK-glutamine-autophagy axis results in phagocytosis obstacle of macrophage and further increase the development risk of Ems. Additionally, it offers potential therapeutic approaches to prevent EMs, especially patients with insufficient HCK and macrophage phagocytosis dysfunction., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

Background: Our objective was to assess the viability and oncological security of a gasless, transaxillary single-incision endoscopic procedure for performing total thyroidectomy and bilateral central neck dissection (TT + BCND). This study focused on patients diagnosed with bilateral papillary thyroid microcarcinoma (PTMC)., Method: Between April 2020 and November 2021, 22 patients with bilateral PTMC underwent single-incision, gasless, transaxillary endoscopic TT + BCND. The patients' clinicopathologic characteristics, surgical completeness and complications were analyzed., Result: Single-incision, gasless, transaxillary endoscopic TT + BCND was successful performed in all patients. The median (IQR) total surgical time was 143 (85-160) min. Only two patients experienced transient unilateral RLN palsy or transient hypocalcemia. All these complications resolved within 1 month after surgery. The median duration of hospital stay after surgery was 4 (3-4.5) days. The median hospitalization expense for these patients was 3848 (3781-4145) USD. The median number of lymph node yielded was 10.5 (8-15). The cosmetic outcomes were well-received by all individuals., Conclusion: In certain cases, gasless, transaxillary endoscopic TT + BCND procedure performed through a single incision proved to be a secure alternative for managing bilateral PTMC., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Background: In a previous study, we proposed a novel anatomy-based five-settlement method for transaxillary endoscopic thyroidectomy (fs-TAT) for patients with papillary thyroid carcinoma. The safety of this new method has been reported in a retrospective study of a single cohort. The safety and short-term oncological outcome of this method was confirmed by comparing it with conventional open surgery (COT) in patients with papillary thyroid microcarcinoma., Methods: The medical records of patients who underwent fs-TAT or COT by a single surgeon from February 2019 to December 2021 were reviewed retrospectively. All patients were diagnosed with papillary thyroid microcarcinoma and underwent lobectomy and ipsilateral central compartment neck dissection. Propensity score matching was used to compare the technical safety and short-term oncologic outcomes of fs-TAT and COT for the purpose of reducing potential selection bias. Reporting was consistent with the STROCSS 2021 guidelines., Result: After propensity score matching, 460 (fs-TAT: 230; COT: 230) patients remained in the study population. There were no significant differences in sex, age, tumor size, Hashimoto's thyroiditis, or tumor multifocality between the groups. The operative time was longer [104.5 (90.3, 120.0) vs. 62.0 (52.0, 76.0), P < 0.001] and the total postoperative drainage volume [135(90, 210) vs. 75 (55, 115), P < 0.001] was greater in the fs-TAT group than in the COT group. However, intraoperative bleeding [3.0 (2.0, 5.0) vs. 5.0 (5.0, 7.5), P < 0.001] was greater, and the median number of lymph nodes yielded [5.0 (2.3, 8.0) vs. 7.0 (5.0, 11.0), P < 0.001] was greater in the COT group than in the fs-TAT group. The groups exhibited no significant difference in the rate of complications (fs-TAT: 2.2% vs. COT: 2.6%, P = 0.856), rate of positive lymph nodes (fs-TAT: 32.2% vs. COT: 36.5%, P = 0.377), length of postoperative hospital stay (3 days vs. 3 days, P = 0.305) or total medical costs (26,936 vs. 26,549, P = 0.144)., Conclusion: Compared to conventional open surgery, fs-TAT offered excellent safety and acceptable short-term oncological outcomes in a selected cohort of patients with papillary thyroid microcarcinoma., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: This study aimed to evaluate the relationship between lateral lymph node yield (LLNY) and the ratio of lateral positive lymph nodes to lymph node yield (LPLR) from initial lateral neck dissection (LND) in patients with papillary thyroid carcinoma (PTC), as well as the risk of recurrence in patients undergoing LND reoperations., Methods: This retrospective cohort study enrolled patients with PTC who underwent revision LND between 1 January 2012, and 31 December 2021. The initial and revised clinical data were retrieved. Patient demographics, clinicopathological features, clinical records, and follow-up information were also reviewed. LLNY and LPLR were determined during the initial LND., Results: In total, 156 patients with PTC were included in this study, with a median follow-up of 36.5 months; 107 had recurrent lateral neck disease. The optimal LLNY and LPLR cutoff values for recurrent/persistent disease were 24.5 and 32.74%, respectively. The high-risk group (LLNY<25) had the lowest recurrence-free survival rate compared with to moderate-risk group (LLNY≥25, LPLR≥32.74%) and low-risk group (LLNY≥25, LPLR<32.74%) ( P <0.001). The moderate-risk group had lower recurrence-free survival than the low-risk group. Multivariate analysis revealed that an LLNY less than 25 in the initial LND was an independent risk factor for recurrence/persistence of lateral neck ( P <0.001)., Conclusions: This study identified that LLNY and LPLR were associated with recurrence/persistence in PTC patients at the time of revision surgery was performed., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Endoscopic thyroidectomy (ET) via gasless unilateral axillary (GUA) approach has been widely implemented worldwide. Based on our concept of mesothyroid excision in open surgery, we proposed a novel anatomy-based five-settlement method in ET via the GUA approach. This preliminary report aimed to explore the efficacy and safety of this method in patients with papillary thyroid carcinoma (PTC)., Methods: PTC patients who underwent endoscopic ET and unilateral central compartment neck dissection (CCND) via GUA approach with the five-settlement method at the Department of General Surgery, Nanfang Hospital, Southern Medical University from March 2020 to December 2021 were retrospectively collected. The data included general clinicopathological characteristics, surgical information (including duration, complication, and clinicopathological features), and hospital stay information, and other medical records were documented., Results: In total, 521 patients underwent lobectomy and CCND under the GUA approach with the five-settlement method. The mean number of lymph nodes yielded (LNY) and positive lymph nodes (PLN) was 5.7 ± 4.3 (range, 1-30) and 1.0 ± 1.8 (range, 0-12), respectively. The incidence of transient recurrent laryngeal nerve injury was 1.1%. Chyle leakage and Horner's syndrome respectively occurred in one patient (0.2%). Five (0.9%) patients developed a hematoma. No severe complications or conversion to open surgery have occurred., Conclusion: The five-settlement method could be implemented safely and efficiently in ET+CCND via the GUA approach in selected PTC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ge, Yu, Sun, Wei, Zhang, Chen, Li and Lei.)

Endometriosis (EMs) is characterized as an estrogen-dependent disease. Whereas, the underlying mechanism for activated estrogen biosynthesis in EMs lesions is largely unknown. We analyzed cholesterol metabolism and estrogen biosynthesis condition of EMs lesions by biological information analysis of GEO datasets, and further verified both in vitro and in vivo by constructing EMs models with uterus fragments from donors of PRNP knockout mouse ( Prnp -/- , KO119), Octapeptide repeat region of PRNP knockout mouse (KO120) and PRNP transgenic mouse (Tg20). We found that transcriptome of cholesterol metabolism and estrogen-converting enzymes were disturbed in EMs patients, and cellular cholesterol concentration and local estradiol level were substantially increased in EMs lesions, as well as the high level of prion (PrP C , encoded by PRNP). Notably, 17-β estradiol stimulation significantly up-regulated PrP C expression in endometrial stromal cells (ESC) and PrP C promoted the proliferative, migratory and invasive abilities of ESC, and was further verified to accelerate EMs progression in mouse models. More importantly, PrP C promoted cholesterol accumulation and activated estrogen biosynthesis of ESC in a PPARα pathway-dependent manner. Taken together, this study suggests that PrP C -cholesterol metabolism/estrogen biosynthesis contributes to the progression of EMs by negatively regulating PPARα pathway, and could be potential therapeutic targets for EMs intervention., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Endometriosis (EMS) is a chronic benign inflammatory disease characterized by the growth of endometrial-like tissue in aberrant locations outside of the uterine cavity. Angiogenesis and abnormal immune responses are the fundamental requirements of endometriotic lesion survival in the peritoneal cavity. Follistatin-like I (FSTL1) is a secreted glycoprotein that exhibits varied expression levels in cardiovascular disease, cancer and arthritis. However, the role of FSTL1 in the development of EMS remains to be fully elucidated. Results of the present study demonstrated that the expression of FSTL1 was significantly increased in ectopic endometrial stromal cells (ESCs) and peritoneal fluid from patients with EMS, compared to the control group. Both conditions of hypoxia and estrogen treatment induced human ESCs to produce increased levels of FSTL1 and disco-interacting protein 2 homolog A (DIP2A). Furthermore, the expression levels of DIP2A, IL8 and IL1β were increased in FSTL1 overexpressed HESCs. Additionally, FSTL1 treatment increased the proliferation of HUVECs in a dose-dependent manner in vitro and markedly increased the tube formation of HUVECs. Moreover, treatment with FSTL1 facilitated M1 polarization of macrophages, increased the secretion of proinflammatory factors and inhibited the expression of scavenger receptor CD36. Results of the present study suggested that the elevated expression of FSTL1 may play a key role in accelerating the development of EMS via enhancing the secretion of proinflammatory factors and promoting angiogenesis.

Background: To evaluate the relationship between lymph node yield (LNY) from the initial central neck dissection (CND) and the risk of recurrence in patients undergoing reoperative CND for papillary thyroid cancer (PTC)., Method: We reviewed clinical data from all patients with pathologically proven PTC who underwent central neck and/or lateral neck dissection reoperations at Nanfang Hospital between 2012 and 2020. Patient demographics, tumor characteristics, clinical data and follow-up information were obtained. In the initial CND, the total number of lymph nodes removed (LNY), total positive nodes removed, and the percentage of positive lymph nodes to the number of lymph nodes removed (PLN%) were determined., Results: A total of 162 patients were included in the study, with a median follow-up of 44 months. 62 had central neck disease recurrence. The optimal LNY and PLN% cut-off values for recurrence were 11 and 65%, respectively. Group 2 (LNY ≥ 11, PLN% < 65%) showed a significantly higher RFS rate than group 1 (LNY < 11 and PLN% < 65%; P < 0.001), group 3 (LNY < 11, PLN% ≥ 65%; P < 0.001), and group 4 (LNY ≥ 11, PLN% ≥ 65%; P = 0.038). Furthermore, group 4 had a higher RFS rate than group 1 (P = 0.008) and group 3 (P = 0.001). Multivariate analysis revealed that LNY < 11 in the central neck was an independent risk factor for recurrence/persistence in the initial surgery (P < 0.001)., Conclusion: Higher LNY in central and neck dissections is associated with lower papillary thyroid cancer recurrence rates, which was confirmed by a reoperative CND procedure. To minimize the risk of recurrence and the need for secondary therapy, surgeons should perform compartment-oriented CNDs when indicated., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In our previous study, we have shown that CRLF1 can promote proliferation and metastasis of papillary thyroid carcinoma (PTC); however, the mechanism is unclear. Herein, we investigated whether the interaction of CRLF1 and MYH9 regulates proliferation and metastasis of PTC cells via the ERK/ETV4 axis. Immunohistochemistry (IHC), qPCR, and Western blotting assays were performed on PTC cells and normal thyroid cells to profile specific target genes. In vitro assays and in vivo assays were also conducted to examine the molecular mechanism. Results showed that CRLF1 directly bound MYH9 to enhance the stability of CRLF1 protein. Inhibition of MYH9 in PTC cells overexpressing CRLF1 significantly reversed malignant phenotypes, and CRLF1 overexpression activated ERK pathway, in vitro , and in vivo . RNA-sequencing revealed that ETV4 is a downstream target gene of CRLF1, which was up-regulated following ERK activation. Moreover, it was revealed that ETV4 is highly expressed in PTC tissues and is associated with poor prognosis. Finally, the ChIP assays showed that ETV4 induces the expression of matrix metalloproteinase 1 (MMP1) by binding to its promoter on PTC cells. Altogether, our study demonstrates that CRLF1 interacts with MYH9, promoting cell proliferation and metastasis via the ERK/ETV4 axis in PTC., (Copyright © 2020 Yu, Sun, Ge, Shi, Zhu, Wei, Li, Zhang, Chen and Lei.)

Objectives: Endometriosis (EMS) is a benign disease that is related to estrogen, immune disorders and inflammation. The purpose of this research was to determine the expression of CD200 in EMS and to clarify its role in the pathogenesis of the disease., Methods: The levels of serum CD200 in patients with and without EMS were determined by ELISA. Furthermore, the expression of CD200 in normal eutopic endometrium and ectopic endometrium was detected by immunohistochemistry and western blotting. The CD200 receptor (CD200R) in macrophages in peritoneal fluid (pMØ) obtained from controls and patients with EMS was examined by western blotting. CD200 expression in human endometrial stromal cells (HESCs) stimulated with 17β-estradiol (E 2 ) was measured by western blotting. Furthermore, macrophages were stimulated with different concentrations of CD200 and the effect on phagocytosis was analyzed., Results: The plasma CD200 levels of patients with EMS was significantly increased compared with controls (P = 0.0173, 95%CI [18.75, 159.6]). Compared with normal eutopic endometrium, the expression of CD200 was significantly increased in ectopic endometrial tissues. The CD200R expression in pMØ obtained from patients with EMS was increased compared with the controls (P = 0.0244). CD200 expression in HESCs stimulated with E 2 was up-regulated. As the levels of CD200 increased, macrophage phagocytosis in vitro gradually decreased., Conclusions: CD200 is an estrogen-induced molecule that impairs macrophage phagocytosis and may contribute to the immune escape of ectopic lesions in EMS., Competing Interests: Declaration of Competing Interest The authors declared no conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The objective of the current study was to investigate the clinical significance of the suprasternal space lymph node (SSLN) in pathological node-positive (pN+) papillary thyroid carcinoma (PTC) patients., Method: One hundred and forty patients with pN + PTC who underwent neck dissection were enrolled into this study. SSLN was resected and used as a specimen to investigate the relationship of SSLN with several clinicopathological parameters., Results: The metastasis rate of SSLN was 20.7%. On univariate analysis, we found that SSLN metastasis was significantly associated with primary cancer site (inferior portion), strap muscle invasion, level III metastasis, Level IV metastasis and lymph node metastasis between sternocleidomastoid and sternohyoid muscles. On multivariate analysis, primary cancer site (inferior portion), strap muscle invasion, Level IV metastasis and lymph node metastasis between sternocleidomastoid and sternohyoid muscles were independent risk factors for SSLN metastasis of PTC., Conclusion: For pN + PTC patients, special attention should be paid to the issue of SSLN metastasis., (Copyright © 2019 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.)

Problem: Decidual stromal cells (DSCs) are important origins of cytokines to modulate maternal-fetal immunotolerance and provide a feasible environment for embryo implantation and development. Interleukin (IL)-24 is a multifunctional cancer killing cytokine and a pleiotropic immunoregulator with complex potency according to tissue or cell types. Its role in establishment and maintenance of normal pregnancy is largely unknown. The aim of our study was to investigate the function and significance of IL-24 and its receptor in the coordination between DSCs and natural killer cells (NK) in early pregnancy., Method of Study: The levels of IL-24 in DSC, endometrial stromal cell (ESC), peripheral blood NK cells (pNK), or decidual NK cells (dNK) culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA), and the levels of IL-24 receptors were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry assays. The effect of IL-24 on the functions of decidual NK cells was analyzed by flow cytometry assays in vitro., Results: The concentration of IL-24 in culture supernatant of DSCs was significantly higher than that of ESCs. Both eNK (endometrial NK cells) and dNK highly expressed IL-24 receptors (IL-20R1 and IL-22R1), especially on CD56 dim eNK. However, there were extremely low levels of IL-20R1 and IL-22R1 on pNK. Recombinant human IL-24 or DSCs-secreted IL-24 downregulated the levels of CD16, Granzyme B, perforin, and interferon (IFN)-γ and upregulated the levels of inhibitory receptors killer-cell immunoglobulin-like receptor (KIR)2DL1 and KIR3DL1, or immunotolerant or angiogenic cytokines (eg, transforming growth factor (TGF)-β, IL-10, and IL-8), and elevated the percentage of CD56 bright CD16 - dNK in vitro., Conclusion: These data suggest that DSCs promote the differentiation of CD56 bright CD16 - NK with high levels of inhibitory receptors, immunotolerant, and angiogenic cytokines by secreting IL-24 during decidualization in early pregnancy., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Epstein-Barr virus (EBV) is associated with many epithelial malignancies. A few reports on the association between EBV and thyroid tumorigenesis have been investigated. However, the conclusion is highly contradictory. We aimed to explore the role of EBV in thyroid nodule development and its clinical significance in a cohort from southern China. Method: We conducted a retrospective data abstraction study of patients who underwent thyroidectomy between December 2017 and June 2018. We retrospectively analyzed the clinicopathological parameters and EBV infection status (serological antibodies and in situ hybridization). Result: The cohort comprised 384 patients with newly diagnosed thyroid diseases, including 261 papillary thyroid carcinomas, 87 nodular goiters, 21 follicular adenomas, 12 follicular thyroid carcinomas, and 3 medullary thyroid carcinomas. Forty-two (10.9%) patients were identified as being serological antibody positive. However, there was no association between the clinicopathological parameters and serological antibody positivity. Additionally, none of the patients showed EBER expression in thyroid normal/cancer cell nuclei in in situ hybridization. Conclusion: In this study, no correlation between EBV and thyroid diseases was found in a cohort from southern China.

Background: Tyrosine kinase inhibitors (TKIs) have been administered to advanced or radio-iodine refractory differentiated thyroid carcinoma (RR-DTC) patients for years. We performed a pooled analysis to explore the frequency of severe adverse effects in advanced or RR-DTC patients treated with sorafenib and lenvatinib., Methods: We performed a comprehensive search of computerized databases, including PubMed, Web of Science, Ovid, EMASE, and the Cochrane Library, from the drugs' inception to July 2018 to identify clinical trials. All grade and severe adverse events (AEs; grade ≥3) were analyzed. This meta-analysis was conducted in accordance with PRISMA guidelines., Results: In total, seve studies published from 2012-2018 with 657 patients were eligible for this study. We included two studies (238 patients) that received 200 mg sorafenib twice and five studies (419 patients) that received 24 mg lenvatinib daily. The frequency of AEs was different among the two drugs. Patients in the sorafenib group had a significantly higher frequency of all grade hand-foot syndrome, hypocalcemia, rash, elevated alanine aminotransferase (ALT), and elevated aspartate aminotransferase (AST). Conversely, the lenvatinib group experienced more frequent all grade voice change, hypertension, nausea, and vomiting compared with those with sorafenib. For grade ≥3 adverse effects, hand-foot syndrome, hypocalcemia, and elevated ALT were more frequent in sorafenib-treated patients. Moreover, lenvatinib-treated patients had a significantly higher incidence of severe weight loss, hypertension, and nausea., Conclusion: Significant differences in common adverse effects, such as all-grade and severe AEs, were detected between sorafenib and lenvatinib in the current study. Early intervention and management of treatment-related AEs (TRAEs) can minimize the impact on patients' quality-of-life, and avoid unnecessary dose reductions and treatment-related discontinuations., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Purpose: Currently the extent of lymph node dissection (LND) for papillary thyroid microcarcinoma (PTMC) remains controversial. The present study aims to investigate the clinicopathologic predictors of lymph node metastasis (LNM) and prognosis in PTMC patients from Guangdong to enable appropriate treatment and follow-up., Methods: Data including demographics, tumor size, multifocality, extrathyroidal extension (ETE) and concomitant thyroiditis were collected from 374 untreated PTMC patients from Guangdong, China. Univariate and multivariate analyses were performed to identify clinicopathologic predictors of LNM and prognostic indicators in PTMC patients with LNM., Results: During the follow-up period of 120 months, recurrence was significantly higher in patients with LNM than in patients without LNM (P<0.05). Age <45 years, larger tumor (>5 mm) and multifocality were predictors of LNM; age <45 years, larger tumor size and absence of concomitant thyroiditis were associated with central LNM (CLNM); male sex, ETE and multifocality were correlated with lateral LNM (LLNM) (P<0.05). There was no difference in recurrence between patients with CLNM and LLNM (P>0.05). LNM in PTMC primarily influenced disease-free survival. Age >45 years and male sex were risk factors of recurrence in PTMC patients with LNM. Male patients with CLNM and older patients with LLNM exhibited worse prognosis (P<0.05)., Conclusions: PTMC easily metastasizes to cervical lymph nodes, which significantly influences prognosis. Prophylactic LND is recommended in PTMC patients from Guangdong, China, who have a high risk of CLNM and/or LLNM. More aggressive postoperative treatment and more frequent follow-up could be considered for older and/or male PTMC patients with LNM., Competing Interests: None., (IJCEP Copyright © 2017.)

Accumulating evidence has indicated that aberrantly expressed microRNAs (miRs) are extensively involved in cancer development and progression. MiR-639 has been reported to act as tumor promoter in various types of cancer. However, the biological function and underlying molecular mechanism of miR-639 in thyroid carcinoma (TC) have not been intensively investigated. Herein the present study aimed to investigate the functional role of miR-639 in TC. We found that miR-639 expression was upregulated in TC cells and clinical tissues. Overexpression of miR-639 promoted TC cell proliferation and cell cycle, with increased expression of CyclinE and c-myc, whereas miR-639-in reverses the function. Using prediction software and luciferase reporter assay, we found that CDKN1A was a target of miR-639. CDKN1A small interfering RNA (siRNA) abrogated the role of miR-639-in on cell proliferation of TC. In summary, our data demonstrated that miR-639 upregulation was associated with development of TC, miR-639 promoted cell proliferation and cell cycle by targeting CDKN1A in TC., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Objective: To investigate the regulative effect of microRNA-338-3p on colorectal carcinoma cell invasion and migration., Methods: The microRNA-338-3p expression pattern of colorectal carcinoma tissues and cell lines was detected by real-time reverse transcriptase polymerase chain reaction. The protein level of smoothened was detected by western blot analysis. Furthermore, colorectal carcinoma cells were pretreated with or without anti-smoothened-small interfering ribonucleic acid prior to the addition of pre-microRNA-338-3p or anti-microRNA-338-3p. The status of colorectal carcinoma cell invasion and that of migration were detected by transwell assay and wound healing assay, respectively., Results: The expression of microRNA-338-3p was significantly down-regulated in colorectal carcinoma tissues in comparison with those in the adjacent non-tumorous tissues, and the value was negatively related to advanced tumor, node, metastasis stage and local invasion. The expression of microRNA-338-3p in colorectal carcinoma cells transfected with pre-microRNA-338-3p p was significantly increased. Furthermore, over-expression of microRNA-338-3p inhibited the expression of smoothened protein in colorectal carcinoma cells, which showed obviously suppressed invasion and migration ability. The expression of microRNA-338-3p in colorectal carcinoma cells transfected with anti-microRNA-338-3p was significantly decreased. Moreover, the down-regulated expression of microRNA-338-3p caused the up-regulated expression of smoothened protein in colorectal carcinoma cells, which showed significantly enhanced invasion and migration ability. However, anti-smoothened-small interfering ribonucleic acid largely, but not completely, reversed the effects induced by blockage of microRNA-338-3p, suggesting that the regulative effect of microRNA-338-3p on colorectal carcinoma cell invasion and migration was indeed mediated by smoothened. Additionally, smoothened was identified as a direct target of microRNA-338-3p by luciferase assay., Conclusions: MicroRNA-338-3p could inhibit colorectal carcinoma cell invasion and migration by inhibiting smoothened expression.

Aim: To investigate the regulative effect of miRNA (miR)-221 on colorectal carcinoma (CRC) cell radiosensitivity and the underlying mechanisms., Methods: A human CRC-derived cell line was cultured conventionally and exposed to different doses of X-rays (0, 2, 4, 6 and 8 Gy). The total RNA and protein of the cells were extracted 24 h after irradiation, and the alteration of miR-221 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene mRNA expression was detected by real-time reverse transcriptase polymerase chain reaction (PCR). The protein alteration of PTEN in the cells was detected by Western blotting. Caco2 cells were pretreated with or without anti-PTEN-siRNA prior to the addition of pre-miR-221 or anti-miR-221 using Lipofectamine 2000. Colony formation assay and flow cytometry analysis were used to measure the surviving cell fraction and the sensitizing enhancement ratio after irradiation. Additionally, PTEN 3'-untranslated region fragment was PCR amplified and inserted into a luciferase reporter plasmid. The luciferase reporter plasmid construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was detected., Results: The X-ray radiation dose had a significant effect on the expression of miR-221 and PTEN protein in human Caco2 cells in a dose-dependent manner. The miR-221 expression level improved gradually with the increase in irradiation dose, while the PTEN protein expression level reduced gradually. miR-221 expression was significantly reduced in the anti-miR-221 group compared with the pre-miR-221 and negative control groups (P < 0.01). Anti-miR-221 upregulated expression of PTEN protein and enhanced the radiosensitivity of Caco2 cells (P < 0.01). Moreover, the inhibitory effect was dramatically abolished by pretreatment with anti-PTEN-siRNA, suggesting that the enhancement of radiosensitivity was indeed mediated by PTEN. A significant increase of luciferase activity was detected in CRC cells that were cotransfected with the luciferase reporter plasmid construct and anti-miR-221 (P < 0.01)., Conclusion: Anti-miR-221 can enhance the radiosensitivity of CRC cells by upregulating PTEN.

Aim: To construct a lentivirus-based inhibitor with specific secondary structure that could exert long-term suppression on microRNA-338-3p (miR-338-3p), thus elucidating its molecular function in colorectal carcinoma cells., Methods: The miR-338-3p inhibitor sequence was synthesized and inserted into pLV-THM plasmid. HEK-293T cells were co-transfected with the lentiviral vectors pLV-THM-miR-338-3p-inhibitor, psPAX2, and pMD2.G. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and then used to infect colorectal carcinoma-derived SW-620 cells. eGFP(+) cells were sorted using flow cytometry. The expression of miR-338-3p in SW-620 cells was determined with real-time RT-PCR, and the expression of the smoothened (SMO) protein was detected using Western blot analysis. The migration ability of the transfected SW-620 cells was assessed with transwell assay., Results: Restriction endonuclease analysis and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p-inhibitor was successfully constructed. The expression of miR-338-3p in SW-620 cells was significantly decreased by infection with the lentivirus pLV-THM-miR-338-3p-inhibitor. Moreover, the down-regulated expression of miR-338-3p caused up-regulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced migration in transwell assay., Conclusion: The construction of the lentiviral vector pLV-THM-miR-338-3p-inhibitor with specific secondary structure provides a basis for further studies the molecular function of miR-338-3p in colorectal carcinoma. miR-338-3p may suppress SMO gene expression and thereby inhibit colorectal carcinoma migration.

Aim: To investigate the regulative effect of miRNA-338-3p (miR-338-3p) on cell growth in colorectal carcinoma (CRC)., Methods: The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed. The recombinant viral vector encoding the pre-miR-338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus. The supernatant containing the lentivirus particles was harvested to determine the viral titer, and this supernatant was then used to transduce CRC-derived cell line, SW-620. Flow cytometry was utilized for sorting the green fluorescent protein (GFP)⁺ cells to establish the SW-620 cell line stably expressing pre-miR-338-3p or miR-338-3p-inhibitor. Moreover, the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction, and Western blotting was used to detect the expression of the smoothened (SMO, the possible target of miR-338-3p) protein in SW-620 cells. Furthermore, the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry, respectively., Results: Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully. GFP was expressed after the SW-620 cells were transduced by the lentivirus. Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased (relative expression 3.91 ± 0.51 vs 2.36 ± 0.44, P < 0.01). Furthermore, overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells, which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate (CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%, P < 0.01]. Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased (relative expression 0.92 ± 0.29 vs 2.36 ± 0.44, P < 0.01). Moreover, the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells, which showed significantly enhanced proliferation ability (CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%, P < 0.01). However, anti-SMO-siRNA largely, but not completely, reversed the effects induced by blockage of miR-338-3p, suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO., Conclusion: miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.

Aim: To investigate the regulatory effect of microRNA-221 (miR-221) on CDKN1C/p57 expression in colorectal carcinoma (CRC)., Methods: Thirty four CRC and adjacent non-tumorous tissue samples were collected individually. Total RNA and protein were isolatedand from these samples and four human CRC-derived cell lines (including HT-29, Lovo, SW-480 and Caco2). MiR-221 expression was examined using real-time RT-PCR. CRC cells were treated with or without anti-p57-siRNA prior to the addition of pre-miR-221 or anti-miR-221. The mRNA and protein levels of CDKN1C/p57 were examined using semi-quantitative RT-PCR and Western blot, respectively. CRC cell proliferation and apoptosis were assessed using MTT assay and flow cytometry, respectively. The CDKN1C/p57 3'-UTR fragment was amplified using PCR from the genomic DNA of human colon cells and inserted into a luciferase reporter construct. The reporter construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was examined., Results: MiR-221 expression was significantly up-regulated in 90% of CRC samples compared to that in the adjacent non-tumorous tissue, and the expression level was positively correlated to an advanced TNM stage and local invasion. There was no significant difference in CDKN1C/p57 mRNA expression between CRC and corresponding non-tumorous tissues, whereas CDKN1C/p57 protein expression was markedly decreased in the CRC samples. A significant inverse correlation between miR-221 and CDKN1C/p57 expression was found in CRC cells. Moreover, a miR-221-specific inhibitor significantly increased CDKN1C/p57 protein expression in CRC cells. Anti-miR-221 markedly inhibited CRC cell proliferation and induced apoptosis. This inhibitory effect was abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57. A significant increase of the luciferase activity was observed in CRC cells co-transfected with the luciferase reporter construct and anti-miR-221., Conclusion: MiR-221 binds to the target site in the 3'-UTR of the CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote CRC occurrence and progress, therefore serving as a potential therapeutic target for the prevention and treatment of CRC.