Efficient detection and post-surgical monitoring of colon cancer with a multi-marker DNA methylation liquid biopsy.

Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

MiR-186-5p upregulation inhibits proliferation, metastasis and epithelial-to-mesenchymal transition of colorectal cancer cell by targeting ZEB1.

MicroRNA-186-5p (miR-186-5p) is upregulated and exhibits as a crucial oncogene in various human tumors. However, the functions and underlying mechanisms of this microRNA on colorectal cancer remain largely unknown. Here, we report that miR-186-5p share a lower expression in colorectal cancer cell lines (HT116, H29, SW620 and LoVo) than in normal colonic epithelial cell line NCM460. MiR-186-5p overexpression inhibits proliferation, metastasis and epithelial-to-mesenchymal transition (EMT) of colorectal cancer cell line LoVo. Zinc Finger E-Box Binding Homeobox 1 (ZEB1), an EMT related marker, is predicted as a target of miR-186-5p. Luciferase reporter assay, qRT-PCR and western blot analysis showed that miR-186-5p directly targeted the 3'-untranslated regions (3'UTR) of ZEB1 messenger RNA. Further functional experiments indicated that overexpression of miR-186-5p suppress the proliferation and metastasis ability of LoVo, which was consistent with the inhibitory effects by knockdown of ZEB1. Additionally, overexpression of ZEB1 could significantly reverse the miR-186-5p mimics initiated suppression impact of proliferation, metastasis and EMT on LoVo. In summary, miRNA-186-5p affects the proliferation, metastasis and EMT process of colorectal cancer cell by inhibition of ZEB1. Hence, it may serve as a promising therapeutic target for colorectal cancer.

Characterization of kiss2 and kissr2 genes and the regulation of kisspeptin on the HPG axis in Cynoglossus semilaevis.

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic-pituitary-gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5'-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.

Identification and characterization of kiss2 and kissr2 homologs in Paralichthys olivaceus.

The role of kisspeptin in puberty onset has been extensively investigated by neuroendocrinologists in the past decade. In the present study, we first cloned and analyzed Pokiss2 and Pokissr2 genes in Paralichthys olivaceus, a Pleuronectiformes fish. By 5'/3' rapid amplification of cDNA ends (RACE), the P. olivaceus kiss2 gene (Pokiss2) and two isoforms of the P. olivaceus kissr2 gene (Pokissr2) transcripts were cloned. During development, Pokissr2 was maternally inherited but Pokiss2 was not, and their expression reached maximum and minimum levels, respectively, when the gonads began to develop. Analysis of tissue distribution revealed that Pokiss2 and Pokissr2 transcripts were predominantly expressed in the brain and gonads, with expression levels in females higher than those in males. Moreover, Pokiss2 and Pokissr2 both showed significantly higher expression in brains and gonads during puberty. In situ hybridization of the ovary at pre-vitellogenesis stage and testis at spermatogonial proliferation stage revealed that both Pokiss2 and Pokissr2 were expressed in spermatocyte, oocytes, and some somatic cells. Our results also showed significantly stronger Pokiss2 expression in the area of the third ventricle of females than males and no Pokissr2 expression in this region in both sexes. These results lay a strong foundation for understanding the role of kisspeptin in neuroendocrine system in teleosts, in particular in Pleuronectiformes.

Functional characterization of the Japanese flounder (Paralichthys olivaceus) Sox2 gene promoter.

Sox2 has essential roles in early embryogenesis and the development of the central nervous system. Sox2 is also necessary in maintaining the identity of progenitor cells. In our study, a 1.8-kb fragment of the 5' flanking region of Paralichthys olivaceus Sox2 (Po-Sox2) gene was cloned and functionally characterized. The activity and specificity of Po-Sox2 promoter were analyzed by comparing various deletion mutants for their ability to direct luciferase and GFP expression in flounder brain cell line. Results indicated that the basal promoter is located between -978 and -442 bp, and the region from -1370 to -978 bp enhances the promoter activity. The regulatory elements in the -1370 to -442 bp fragment were further investigated. Many binding sites of transcription factors closely related to neurogenesis and stem cell properties were found in this region. Mutational analysis indicated that Nanog, Pax6, p53, and POU binding sites play functional roles in the transcription of Po-Sox2 gene, whereas NF-Y binding sites did not affect this gene. In vivo studies using transient transgenic zebrafish embryos showed that the Po-Sox2 promoter region can drive GFP expression in brain, yolk syncytial layer, and notochord. Our results provide valuable information in understanding the molecular regulatory mechanisms of Po-Sox2 gene during neurogenesis and embryonic development.

Characterisation of kisspeptin system genes in an ovoviviparous teleost: Sebastes schlegeli.

Kisspeptins are neuropeptides that play important roles in the reproduction and the onset of puberty in vertebrate by activating their receptor, Kissr. In the present study, we first isolated kiss1 and kissr4 genes from an ovoviviparous fish, the black rockfish (Sebastes schlegeli) by homologue cloning. Phylogenetic analysis indicated that the kiss and kissr of S. schlegeli belonged to kiss1 and kissr4 respectively. Quantitative real-time PCR analysis showed that the kissr4 was expressed mainly in the brain and testis, while the kiss1 was expressed predominantly in the heart of both sexes. As for the different gonadal maturation stages the kiss1 showed different expression patterns in different tissues. During the early development stage, expression levels of the ligand and receptor genes showed similar increasing trends. The promoter region of kissr4 contained several putative transcription factor (TF) binding sites which may have the function of regulating kisspeptin system gene expression, providing potential targets for future in-depth investigation. These results together confirmed that the kisspeptin system in S. schlegeli may be involved in reproduction and other activities. Furthermore, our study laid the groundwork for further learning about the evolution and function of kisspeptin system in fish even vertebrate.

Identification and Characterization of a PRDM14 Homolog in Japanese Flounder (Paralichthys olivaceus).

PRDM14 is a PR (PRDI-BF1-RIZ1 homologous) domain protein with six zinc fingers and essential roles in genome-wide epigenetic reprogramming. This protein is required for the establishment of germ cells and the maintenance of the embryonic stem cell ground state. In this study, we cloned the full-length cDNA and genomic DNA of the Paralichthys olivaceus prdm14 (Po-prdm14) gene and isolated the 5' regulatory region of Po-prdm14 by whole-genome sequencing. Peptide sequence alignment, gene structure analysis, and phylogenetic analysis revealed that Po-PRDM14 was homologous to mammalian PRDM14. Results of real-time quantitative polymerase chain reaction amplification (RT-qPCR) and in situ hybridization (ISH) in embryos demonstrated that Po-prdm14 was highly expressed between the morula and late gastrula stages, with its expression peaking in the early gastrula stage. Relatively low expression of Po-prdm14 was observed in the other developmental stages. ISH of gonadal tissues revealed that the transcripts were located in the nucleus of the oocytes in the ovaries but only in the spermatogonia and not the spermatocytes in the testes. We also presume that the Po-prdm14 transcription factor binding sites and their conserved binding region among vertebrates. The combined results suggest that Po-PRDM14 has a conserved function in teleosts and mammals.

Characterization of the Dmrt1 gene in the black rockfish Sebastes schlegeli revealed a remarkable sex-dimorphic expression.

The Dmrt genes encode a large family of transcription factors with a conserved zinc finger-like DNA-binding DM domain. The function of Dmrt1, one of the family members, in sexual development has been well studied in invertebrates and vertebrates. In the present study, the full-length cDNA of Dmrt1 was isolated from the testis of Sebastes schlegeli. The full-length cDNA of S. schlegeli Dmrt1 (SsDmrt1) was 1,587 bp and contained a 189-bp 5' UTR, a 489-bp 3' UTR and a 909-bp open reading frame, which encoded 302 amino acids with a conserved DM domain and an male-specific motif domain. Phylogenetic analysis showed the evolutionary relationships of SsDmrt1 with other known Dmrt genes in fish and tetrapods. Several transcriptional factor-binding sites in the 5' promoter were identified that might regulate SsDmrt1 expression. Quantitative real-time PCR analysis indicated that SsDmrt1 was expressed in all of the inspected larval developmental stages from 1 to 35 days after birth and that the level of expression gradually decreased. The expression of SsDmrt1 in adult gonads was sexually dimorphic with extremely high expression in the testis, but very low expression in the ovary. No expression was detected in other tissues. Using in situ hybridization, we demonstrated that SsDmrt1 was specifically expressed in the germ cells of both the testis and the ovary. Thus, our results suggest that SsDmrt1 may have an important role in the differentiation of both the testis and the ovary of S. schlegeli.

[A procedure to assist achievement of total mesorectal excision through the extending intersphincteric plane].

OBJECTIVE: To investigate the value of assisted achievement total mesorectal excision (TME) through the extending intersphincteric plane. METHODS: From February 2006 to April 2010, 65 patients with low rectal cancer underwent assisted implementing TME through the extending intersphincteric plane under direct vision and achieved sphincter preservation. The clinical data was summarized and analyzed retrospectively. Follow-up visits were conducted on complications and oncological outcomes. RESULTS: The mean operation time was (245 ± 42) minutes, and the mean intraoperative blood loss was (114 ± 76) ml. There was no postoperative mortality. Postoperative complications included 2 cases of anastomotic leak, 13 cases of anastomotic stenosis, 2 cases of early postoperative inflammatory ileus, 1 case of urinary tract infection, and 1 case of incision infection. Distal margins and circumferential resection margin of all specimens were negative. For pathological stage, there were 26 cases at stage pTNMI, 17 cases at stage pTNMII and 22 cases at stage pTNMIII. The mean follow-up time was (47.9 ± 18.9) months. 10 patients were lost to follow up, 15 cases had distant metastasis or local recurrence in, and 8 cases died of tumor metastasis at the latest follow up. Local recurrence occurred in 3 cases, including recurrence in presacral region, metastasis of lymph node at the left side in pelvis cavity, and metastasis at the sacrum at 35, 36, and 52 months postoperatively. There was no anastomotic recurrence. Log-rank survival analysis showed 5-year cumulative survival rate was 100%, 93.3%, and 63.1% in TNM stage I, II, and III, respectively. The cumulative disease-free survival rate was 96.2%, 83.3%, 44.8% in TNM stage I, II, and III, respectively. CONCLUSION: It has a good oncological effect and was an advantageous procedure to assist achievement total mesorectal excision (TME) through the extending intersphincteric plane as surgeons encountered with difficulties from transabdominal TME.

[Value of protective stoma in intersphincteric resection for ultra-low rectal cancer].

OBJECTIVE: To investigate the value of protective stoma in intersphincteric resection (ISR) for ultra-low rectal cancer. METHODS: Clinical data of 56 ultra-low rectal cancer patients without involvement of external anal sphincter treated during January 1999 to July 2009 with trans-anal ISR plus trans-abdominal total mesorectum excision and coloanal anastomosis were retrospectively analyzed. The patients were divided into two groups based on whether they received protective ostomy: ostomy group (16 cases) and ostomy-free group (40 cases). The postoperative complications as well as anal functional restoration were compared between the two groups. RESULTS: Sixteen cases (32.1%) of the 56 patients received protective stoma. The complication rate of anastomosis and anus complication rate in the ostomy-free group were significantly higher than those in ostomy group [35.0% (14/40) and 40.0% (16/40) vs. 1/16 and 1/16; P < 0.05]. In the ostomy-free group, one patient developed anastomotic dehiscence and tumor recurrence, the patients was given permanent colostomy, and the other three patients with lesions in the anastomosis and anus received ostomy and secondary surgical treatment, with a reoperation rate of 10.0% (4/40). The anal function of patients in the two groups were both decreased after the operation. The rate of patients got Kirwan grade I anal sphincter function in the 3rd, 6th and 12th month after protective stoma operation was 11/16, 13/15 and 11/13 in the ostomy group, respectively; and those were 30.0%, 37.5% and 45.0% in the ostomy-free group, respectively. Anal function was significantly better in the ostomy group than that in the ostomy-free group during the same postoperative period (P < 0.05). CONCLUSION: Protective stoma can avoid anastomotic leakage following ISR for ultra-low rectal cancer, and alleviate the suffering of anal incontinence in the early postoperative period, and is conducive to the restoration of anal function.

[Clinical application of intersphincteric resection in the anal-preserving operation for ultra-low rectal carcinoma].

OBJECTIVE: To investigate the clinical application of intersphincter resection (ISR) combined with total mesorectal excision (TME) and colon-anal anastomosis in the treatment for ultra-low rectal carcinoma. METHODS: To review and analyze retrospectively the data of 34 patients with ultra-low rectal carcinoma (without external anal sphincter involvement) who received treatment of ISR, TME and colon-anal anastomosis. RESULTS: Partial resection of internal sphincter was performed in the patients with a distal edge of the tumor greater than or equal to 2 cm from the dentate line. Subtotal removal of the rectum was performed between 1 cm and 2 cm. Total resection was performed in less than 1 cm or involvement of dentate line. Reconstruction of digestive tract was done by manual colon-anal anastomosis. The average distance from distal excised margin to the tumor was 2.3 (1.8 - 3.2) cm among 34 patients. The pathological types were as follows: 28 cases of adenocarcinoma (11 were well differentiated, 17 moderately differentiated), 1 case of papillary carcinoma and 5 cases of villous adenoma with malignant change. The postoperative pathological stages were: Dukes stage A in 28 cases, stage B in 1 and stage C in 5 cases. The pTNM staging was 28 cases in phase I, 1 in phase IIa, 4 in phase IIIa and 1 in phase IIIb. The T stages of the patients were as following: 16 Tl, 17 T2 and 1 T3. Postoperative anastomotic stenosis occurred in 3 cases, anastomotic dehiscence in 2 cases and rectovaginal fistula in 2 cases. The ability of controlling feces of patients decreased significantly in the early postoperative period, and restored gradually at 6 to 12 months after operation. Anastomotic recurrence occurred in 1 case at 5 months after operation and liver metastasis in 1 case at 40 months. CONCLUSION: With strictly grasping indications, radical resection can be attained and anal sphincter preserved by ISR combined with TME and colon-anal anastomosis. It is an effective sphincter-preserving operation.

[Combination of trans-anal intersphincteric resection and trans-abdominal total mesorectal excision for anus-retained ultra-low rectal tumors].

OBJECTIVE: To study the combination of trans-anal intersphincteric resection and transabdominal total mesorectal excision for anus-retained ultra-low rectal tumors. METHODS: Clinical data of 34 ultra-low rectal tumor patients without external anal sphincter involved, who underwent the combination surgery, were retrospectively analyzed. RESULTS: The distance from the distal incisal margin of the rectum to the inferior margin of the tumor ranged from 1.8 cm to 3.0 cm on an average of 2.1 cm. For pathological types, there were 23 cases of adenocarcinoma (9 well differentiated and 14 moderately differentiated), 1 papillary carcinoma, 2 rectal stromal tumor, 5 rectal villous adenoma with neoplasia and 3 giant villous adenoma. For pathological stages, there were 18 cases at stage pTNM I, 5 at IIA, 1 at IIB, 4 at IIIA, 1 at III and for T grading, there were 15 cases at stage T1, 5 at T2, 8 at T3, 1 at T4. In these 34 patients, there were 3 cases with postoperative anastomotic stenosis, 2 with postoperative anastomotic rupture, 2 with rectovaginal fistula and no operative death. Because of the dysfunction of bowel control, bowel frequency varied from 3 to 12 in the early stage after operation, but with the recovery of anus function, bowel frequency decreased and ranged form 1 to 5 times a day and the time of formed bowel control could be more than 5 min in 6-12 months after operation. However, patients underwent total resection of internal anal sphincter still suffered from incontinence of loose stool after 1 year. After operation, anastomotic recurrence was found in 1 case in 5 months, liver metastasis in 1 case in 10 months and 28 months respectively, cardiac sudden death in 1 case in 26 months. CONCLUSION: The combination of trans-anal ISR and trans-abdominal TME for anus-retained ultra low rectal tumor is not only coincident with radical tumor principle but also retains the function of anus, on the premise of the strict indication.

Tumor-Associated Macrophages Derived TGF-ß‒Induced Epithelial to Mesenchymal Transition in Colorectal Cancer Cells through Smad2,3-4/Snail Signaling Pathway.

PURPOSE: We investigated the role of tumor-associated macrophages (TAMs) on the epithelial to mesenchymal transition (EMT) of colorectal cancer cells and determined the potential mechanism involved in the metastatic process. MATERIALS AND METHODS: In this study, flow cytometry was used to detect the expression of target proteins. We used transwell assay to evaluate the migration of cancer cells under specific conditions. Using real-time polymerase chain reaction, we examined the expressions of cytokines and EMT-related markers in mRNA level. Animal assay was performed for analysis in vivo and hematoxylin and eosin was used to visualize the effect of TAMs on tumor metastasis. We also used immunohistochemistry and Western blotting to detect the expression of target proteins. RESULTS: Here, we observed enrichment of TAMs in colorectal tumor tissues, resulting in high metastasis in clinical therapy. Moreover, those TAMs could facilitate the EMT progression of colorectal cancer cells, which is induced by the transforming growth factor-ß (TGF-ß) derived from TAMs, leading to the invasion and migration of cancer cells. CONCLUSION: Our results demonstrated that TAMs contributed the EMT progression through a TGF-ß/Smad2,3-4/Snail signaling pathway, and disrupting this pathway with TGF-ß receptor inhibitor could suppress metastasis, readjusting our focus to the connection of TAMs and cancer metastasis.

Simple instruments facilitating achievement of transanal total mesorectal excision in male patients.

AIM: To assess the efficacy of a modified approach with transanal total mesorectal excision (taTME) using simple customized instruments in male patients with low rectal cancer. METHODS: A total of 115 male patients with low rectal cancer from December 2006 to August 2015 were retrospectively studied. All patients had a bulky tumor (tumor diameter ≥ 40 mm). Forty-one patients (group A) underwent a classical approach of transabdominal total mesorectal excision (TME) and transanal intersphincteric resection (ISR), and the other 74 patients (group B) underwent a modified approach with transabdominal TME, transanal ISR, and taTME. Some simple instruments including modified retractors and an anal dilator with a papilionaceous fixture were used to perform taTME. The operative time, quality of mesorectal excision, circumferential resection margin, local recurrence, and postoperative survival were evaluated. RESULTS: All 115 patients had successful sphincter preservation. The operative time in group B (240 min, range: 160-330 min) was significantly shorter than that in group A (280 min, range: 200-360 min; P = 0.000). Compared with group A, more complete distal mesorectum and total mesorectum were achieved in group B (100% vs 75.6%, P = 0.000; 90.5% vs 70.7%, P = 0.008, respectively). After 46.1 ± 25.6 mo follow-up, group B had a lower local recurrence rate and higher disease-free survival rate compared with group A, but these differences were not statistically significant (5.4% vs 14.6%, P = 0.093; 79.5% vs 65.1%, P = 0.130). CONCLUSION: Retrograde taTME with simple customized instruments can achieve high-quality TME, and it might be an effective and economical alternative for male patients with bulky tumors.

Native recombinant kisspeptin can induce gnrh1 and kissr2 expression in Paralichthys olivaceus in vitro.

Kisspeptins have been described as one of the most potent activators of the hypothalamic-pituitary-gonadal axis. Kisspeptins control the onset of reproductive functions during puberty by directly stimulating the neuronal activity and release of gonadotropin-releasing hormone (GnRH). The function of kisspeptins has been investigated in vivo and in vitro. In our study, three kinds of recombinant kisspeptin proteins were expressed in Escherichia coli. Kisspeptin fragments Kp54, Kp44, and Kp10 translated from Paralichthys olivaceus kiss2 gene were then obtained. Kp44 contained 44 amide acids (aa) which are the same as the N-terminal of Kp54; Kp10 shares the same 10 aa with the C-terminal of Kp54 but Kp10 also contains some other amide acids. In the dose course of treatments with prokaryotically expressed peptides, Kp54 and Kp10 could induce the expression of kissr2 and gnrh1; by contrast, Kp44 could not induce a similar expression. These results provided direct evidence that the core decapeptide of kisspeptin is necessary to ensure its biological functions. In the time course of the Kp54 treatments on two kinds of cultured brain cells, different patterns of kissr2 and gnrh1 mRNA suggested that the responses of these cells to kisspeptins depends on cell type and treatment duration. Thus, our research provided alternative methods to investigate the functions of kisspeptin in vitro and to detect biological activities; this research also established basis for kisspeptin applications in production processes.

Prognostic Value of Lymph Node Ratio in Locally Advanced Rectal Cancer Patients After Preoperative Chemoradiotherapy Followed by Total Mesorectal Excision.

Although the absolute number of positive lymph nodes (LNs) has been established as 1 of the most important prognostic factors in rectal cancers, many researchers have proposed that the lymph node ratio (LNR) may have better predicted outcomes. We conducted a retrospective study to compare the predictive ability of LNR and ypN category in rectal cancer. A total of 264 locally advanced rectal cancer (LARC) patients who underwent preoperative chemoradiotherapy (CRT) followed by total mesorectal excision (TME) between 2005 and 2012 were reviewed. All patients were categorized into 3 groups or patients with metastatic LNs were categorized into 2 groups according to the LNR. The prognostic effect on overall survival (OS) and disease-free survival (DFS) was evaluated. With a median follow-up of 45 months, the OS and DFS were 68.4% and 59.3% for the entire cohort, respectively. The respective 5-year OS and DFS rates for the 3 groups (LNR = 0, 0 < LNR ≤ 0.20, and 0.20 < LNR ≤ 1.0) were as follows: 83.2%, 72.6%, and 49.4% (P < 0.001) and 79.5%, 57.3%, and 33.5% (P < 0.001), respectively. Multivariate analysis revealed that LNR and differentiation, but not the number of positive LNs, had independent prognostic value for OS (hazard ratio [HR] = 2.328, 95% confidence interval [CI]: 1.850-4.526, P < 0.001) and DFS (HR = 3.004, 95% CI: 1.616-5.980, P < 0.001). As for patients with positive LNs, the respective 5-year OS and DFS rates for the 2 groups (0 < LNR ≤ 0.20, and 0.20 < LNR ≤ 1.0) were 72.6% and 49.4% (P < 0.001) and 57.3% and 33.5% (P < 0.001), respectively. Multivariate analysis revealed that only LNR was an independent factor for OS (HR = 3.214, 95% CI: 1.726-5.986, P < 0.001) and DFS (HR = 4.230, 95% CI: 1.825-6.458, P < 0.001). Subgroups analysis demonstrated that the ypN category had no impact on survival whereas increased LNR was a significantly prognostic indicator for worse survival in the LNs < 12 subgroup. LNR is an independent prognostic factor in LARC patients treated with preoperative CRT followed by TME. It may be a better independent staging method than the number of metastatic LNs when <12 LNs are harvested after preoperative CRT.

Identification, molecular characterization and gene expression analysis of sox1a and sox1b genes in Japanese flounder, Paralichthys olivaceus.

The transcription factor, Sox1 has been implicated in neural determination and differentiation as well as in the maintenance of neural progenitor cell status in mammals. However, the molecular cloning and expression of sox1 gene in marine fish have not been reported yet. In this study, we first cloned and characterized the full-length cDNAs and the partial 5'-flanking regions of Paralichthys olivaceus sox1a (Posox1a) and sox1b (Posox1b). Phylogenetic, gene structure, and chromosome synteny analyses revealed that Posox1a and Posox1b were co-orthologs and homologous to mammalian Sox1. The promoter regions of Posox1a and Posox1b were also analyzed and several potential transcription factor (TF) binding sites were identified which might modulate gene expression. Quantitative real-time RT-PCR (qRT-PCR) results showed that Posox1a and Posox1b were consistently expressed during embryogenesis, with the highest level at the neurula stage. Tissue distribution analyses revealed that Posox1a and Posox1b were abundant in the adult brain. Moreover, Posox1a had a faster evolution rate and much higher expression levels than Posox1b. These results provide a foundation for further surveying the function of PoSox1 genes during Japanese flounder development and neurogenesis.

Comparative Evolution of Duplicated Ddx3 Genes in Teleosts: Insights from Japanese Flounder, Paralichthys olivaceus.

Following the two rounds of whole-genome duplication that occurred during deuterostome evolution, a third genome duplication event occurred in the stem lineage of ray-finned fishes. This teleost-specific genome duplication is thought to be responsible for the biological diversification of ray-finned fishes. DEAD-box polypeptide 3 (DDX3) belongs to the DEAD-box RNA helicase family. Although their functions in humans have been well studied, limited information is available regarding their function in teleosts. In this study, two teleost Ddx3 genes were first identified in the transcriptome of Japanese flounder (Paralichthys olivaceus). We confirmed that the two genes originated from teleost-specific genome duplication through synteny and phylogenetic analysis. Additionally, comparative analysis of genome structure, molecular evolution rate, and expression pattern of the two genes in Japanese flounder revealed evidence of subfunctionalization of the duplicated Ddx3 genes in teleosts. Thus, the results of this study reveal novel insights into the evolution of the teleost Ddx3 genes and constitute important groundwork for further research on this gene family.

Identification and characterization of a Sox2 homolog in the Japanese flounder Paralichthys olivaceus.

Sox2 plays an essential role in maintaining the pluripotency of embryonic and neural stem cells as well as in the neurogenesis. While it has been well studied in mammals, information from lower invertebrate especially marine fish is still limited. In this study, we cloned and sequenced the full-length cDNA and partial 5'-flanking region of the Japanese flounder Sox2. Phylogenetic, gene structure, and protein comparison analyses revealed that Paralichthys olivaceus Sox2 (Po-Sox2) was homologous to mammalian Sox2. Quantitative real-time RT-PCR results showed that Po-Sox2 was not maternal inherited, and the transcripts were present from high blastula-stage onwards, with the highest level at the mid-gastrula stage. Tissue distribution analysis revealed that Po-Sox2 was present not only in neural tissues, but also in gonadal and gill tissues. In addition, we analyzed the Po-Sox2 promoter region for several species-conserved motifs as well as various transcription factor binding sites. The overall hypomethylation status of the identified CpG sites in the 5'-regulatory region revealed that it was not involved in the transcriptional modulation of Po-Sox2. All these results suggest that Po-Sox2 may have a conserved function in neurogenesis and early embryonic development.

Sexually dimorphic expression of vasa isoforms in the tongue sole (Cynoglossus semilaevis).

The vasa gene encodes an ATP-dependent RNA helicase of the DEAD box protein family that functions in a broad range of molecular events involving duplex RNA. In most species, the germline specific expression of vasa becomes a molecular marker widely used in the visualization and labeling of primordial germ cells (PGCs) and a tool in surrogate broodstock production through PGC transplantation. The vasa gene from tongue sole (Cynoglossus semilaevis) was characterized to promote the development of genetic breeding techniques in this species. Three C. semilaevis vasa transcripts were isolated, namely vas-l, vas-m, and vas-s. Quantitative real-time PCR results showed that C. semilaevis vasa transcripts were prevalently expressed in gonads, with very weak expression of vas-s in other tissues. Embryonic development expression profiles revealed the onset of zygotic transcription of vasa mRNAs and the maternal deposit of the three transcripts. The genetic ZW female juvenile fish was discriminated from genetic ZZ males by a pair of female specific primers. Only the expression of vas-s can be observed in both sexes during early gonadal differentiation. Before PGCs started mitosis, there was sexually dimorphic expression of vas-s with the ovary showing higher levels and downward trend. The results demonstrated the benefits of vasa as a germline specific marker for PGCs during embryonic development and gonadal differentiation. This study lays the groundwork for further application of C. semilaevis PGCs in fish breeding.