Ilyar Sheyhidin

HMGA2 is down-regulated by microRNA let-7 and associated with epithelial-mesenchymal transition in oesophageal squamous cell carcinomas of Kazakhs.

To investigate the expression of let‐7 and its regulation of high‐mobility group A2 protein (HMGA2), and to verify the relationship between let‐7, HMGA2 and the process of epithelial–mesenchymal transition (EMT), in oesophageal squamous cell carcinomas (OSCC) of Kazakh patients.

MicroRNA-21 promotes the proliferation and inhibits apoptosis in Eca109 via activating ERK1/2/MAPK pathway.

The aim of this study was to investigate how miR-21 promotes proliferation and inhibits apoptosis in esophageal squamous cell carcinoma (ESCC). MTT, wound healing assay and cell cycle showed that proliferation and migration of ESCC cell line Eca109 cells were increased in miR-21 mimics group, and decreased in anti-miR-21 Oligonucleotide (AMO) group after transfection into Eca109 cells with miR-21 mimics, AMO and scramble sequence, respectively. Cell apoptosis assay indicated that cell apoptosis can be obviously inhibited by overexpression of miR-21 and promoted by downregulation of miR-21. Meanwhile, western-blot results showed that p-ERK1/2 expression was elevated in miR-21 mimics group, whereas decreased in AMO group. Furthermore, the ERK1/2, a key component of MAPK signaling pathway, was knocked down, and overexpressed successfully using shRNA-ERK1/2 and overexpressing plasmids containing full length cDNA of ERK1/2, respectively. It was observed that shRNA-ERK1/2 can significantly decreased the level of miR-21 expression, while overexpression of ERK1/2 can up-regulate expression of miR-21. As further confirmation, Eca109 cells were treated with gradient concentration of U0126, a kind of MEK inhibitor, and expression of miR-21 was subsequently examined. It was found that U0126 can significantly decreased endogenous expression of miR-21. In parallel, U0126 decreased cell proliferation, migration and increased the apoptosis in Eca109 cells, with the expression of miR-21 being reduced significantly in U0126 group as compared with control groups. Our findings indicated that miR-21 promoted the proliferation, migration and inhibited apoptosis of Eca109 cells through activating ERK1/2/MAPK pathway, and that targeting miR-21 could be a promising therapeutic strategy in ESCC.

Viral load of HPV 16/18 in esophageal squamous cell carcinoma in three ethnic groups living in Xinjiang Autonomous Region, China

To investigate the viral load of human papillomavirus (HPV) in esophageal squamous cell carcinoma (ESCC) patients from three ethnic groups in Xinjiang. Using Gp5+/Gp6+ consensus primers, the prevalence of HPV DNA was examined in 253 paraffin-embedded ESCC samples. The presence and viral load of HPV 16 and HPV 18 were detected in Kazakhs, Uygurs and Hans using type-specific primers by quantitative real-time PCR (qRT-PCR). Among the 253 ESCC samples, 52 cases were positive for HPV DNA, all the 52 positive cases displayed HPV 16 infection, and six of the 52 cases were co-infected by HPV 16 and 18. HPV 16-positive rate and viral load were higher in lesions, and was inversely correlated with differentiation grades. However, there was no statistic significance among different differentiation grades. Also, there were no significant difference between detection rates of HPV types, viral load and age, gender, ethnic group, and lymph node metastasis. HPV 16 and HPV 18 genotypes could simultaneously be detected in ESCC specimens in three main ethnic groups in Xinjiang. The viral load of HPV 16 is higher in the ESCC lesions, and is inversely correlated with the differentiation grades. These observations reinforce the suggestion that HPV infection may involved in ESCC carcinogenesis; however, high prevalence or viral load of HPV infection does not seem to be related with high incidence of ESCC in Kazakhs, which may be the one element among the multiple risk factors contributing to ESCC.

Roles of phosphorylated JNK in esophageal squamous cell carcinomas of Kazakh ethnic.

The c‐Jun NH2‐terminal kinase (JNK) signal pathway has been implicated in the growth, cellular proliferation, and apoptosis in many kinds of carcinomas. However, the role of JNK in the development of esophageal squamous cell carcinomas (ESCCs) is unknown. To investigate the role of JNK in ESCC, in vitro, esophageal cancer cell line Eca109 was pretreated using SP600125, JNK specific inhibitor, then was subjected to MTT assay to examine cellular proliferation, flow cytometric analysis to detect apoptosis and cell cycle, and wound healing assay to evaluate cell migration. Meanwhile, the mRNA and protein expression of JNK in Eca109 cells pretreated with SP600125 were examined by real‐time quantitative reverse transcription PCR (qRT‐PCR) and Western blotting, respectively. In vivo, 12 paired of fresh ESCC and normal adjacent tissues (NAT) from Kazakh patients were used to validate the expression of JNK by qRT‐PCR and Western blotting. Furthermore, to reconfirm the expression trend of activation JNK (p‐JNK), enlarged 72 paired of Kazakhs ESCC and NAT were subjected to immunohistochemistry. Our results showed that the suppression of p‐JNK could lead to apoptosis and reduce proliferation in Eca109 cells. However, there was an elevated expression of p‐JNK protein in NAT compared with ESCC tissues, and there was significant difference between p‐JNK expression and pathological differentiation (Pu2009<u20090.05) in Kazakh populations. Together, all the data we obtained in the present study indicated that the p‐JNK MAPK pathway was involved in pathogenesis of Kazakhs ESCC, and played a different roles in carcinogenesis and development of Kazakhs ESCC.

Down-regulated miR-26a promotes proliferation, migration, and invasion via negative regulation of MTDH in esophageal squamous cell carcinoma

Numerous studies have reported that the role played by miR‐26a in cancer is controversial, but whether miR26a regulates metadherin (MTDH) expression in esophageal squamous cell carcinoma (ESCC) is unclear. We performed this study to investigate the clinical relevance of miR‐26a expression in ESCC. miR‐26a was detected by using the in situ hybridization method. To functionally analyze the role of miR‐26a in ESCC cell lines in vitro, KYSE‐450 and Eca109 cells were employed, whose endogenous miR‐26a was artificially down‐ or up‐regulated, respectively, by using lentiviral‐based transfection. There was significant association between miR‐26a expression and clinical stage (P = 0.049), lymph node metastasis (P = 0.023), tumor volume (P = 0.003), and poor overall prognosis (P = 0.026). miR‐26a was able to suppress proliferation and migration of ESCC cells in vitro. Moreover, we have confirmed that miR‐26a can negatively regulate MTDH in ESCC cells by using luciferase reporter assay. In addition, to investigate the role miR‐26a plays in cell proliferation, we nude mice were xenografted with ESCC cells whose miR‐26a was stably down‐ and up‐regulated. Together, our results show that miR‐26a is capable of suppressing the proliferation and migration of ESCC cells via negative regulation of MTDH. Moreover, miR‐26a expression was clinically relevant in cancer progression and poor prognosis, which supports the idea that miR‐26a acts as a tumor suppressor in ESCC.—Yang, C., Zheng, S., Liu, T., Liu, Q., Dai, F., Zhou, J., Chen, Y., Sheyhidin, I., Lu, X. Down‐regulated miR‐26a promotes proliferation, migration, and invasion via negative regulation of MTDH in esophageal squamous cell carcinoma. FASEB J. 31, 2114–2122 (2017). www.fasebj.org

M2 isoform of pyruvate kinase (PKM2) is upregulated in Kazakh’s ESCC and promotes proliferation and migration of ESCC cells

The objectives of the present study are to explore role of pyruvate kinase isoenzyme type M2 (PKM2) in progression of Kazakh’s esophageal squamous cell carcinoma (ESCC) in Xinjiang, China, and to clarify mechanism of PKM2 in malignant phenotype. PKM2 expression was examined using immunohistochemistry (IHC) in 101 matched pairs of ESCC and normal adjacent tissues (NATs) and using enzyme-linked immunosorbent assay (ELISA) in 35 serum samples of Kazakh’s ESCC and 8 serum samples of healthy subjects. To investigate mechanism, small interfering RNA (siRNA)-PKM2 was transfected into ESCC cells. Cell migration and invasion were evaluated by wound healing and Transwell assays. Apoptosis and cell cycle were analyzed by flow cytometry (FCM). PKM2 expression was significantly higher in ESCC tissues (77.2xa0%, 78/101) compared with matched NAT (Pu2009=u20090.003) and also higher in serum samples of Kazakh’s ESCC patients (78.84xa0ng/mL) compared with healthy subjects (13.55xa0ng/mL) (Pu2009=u20090.001). Patients with overexpression of PKM2 had a poor prognosis (Pu2009=u20090.032). After knockdown of PKM2, cell proliferation, migration, and invasion were significantly reduced (Pu2009=u20090.001), apoptosis increased (Pu2009=u20090.001), and cell cycle was arrested at G1 phase. PKM2 overexpression was significantly correlated with the worse outcome of Kazakh’s ESCC. Furthermore, PKM2 was involved in progression of ESCC by promoting proliferation and suppressing apoptosis, accelerating invasion, and influencing cell cycle. PKM2 could be a potential biomarker for molecular classification of ESCC.

PIK3CA promotes proliferation and motility but is unassociated with lymph node metastasis or prognosis in esophageal squamous cell carcinoma.

The PIK3CA mutation has been extensively reported in the setting of cancers; however, the clinicopathological significance of PIK3CA expression has rarely been discussed in esophageal squamous cell carcinoma. In the present study, to confirm the significance of PIK3CA expression in association with metastasis and prognosis, which has been somewhat controversial in esophageal squamous cell carcinoma (ESCC), the relationship between clinicopathological features of ESCC and PIK3CA expression was analyzed using immunohistochemistry with a tissue microarray. Meanwhile, as additional verification and an ethnic control, another independent small cohort of Kazakh ESCC were analyzed by immunohistochemistry. To investigate the pilot role of PIK3CA in ESCC cells, ESCC cell lines ECa109 and EC9706 were transiently transfected with specific siRNA against PIK3CA. The silencing effect was detected by Western blot. Cell proliferation was examined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay; apoptosis and the cell cycles were analyzed by flow cytometry. Furthermore, the migratory and invasive ability were evaluated by wound healing and transwell invasion assay, respectively. Expression of PIK3CA was significantly higher in ESCC than in paired normal controls and was ethnicity independent; no statistically significant difference was observed between PIK3CA expression and sex, age, depth of invasion, tumor differentiation, lymph node metastasis, or prognosis. Proliferation, migration, and invasion were all markedly reduced after knockout of PIK3CA. Moreover, the cell cycle was arrested at the S phase, and the apoptosis rate was significantly increased, suggesting that PIK3CA plays a key role in promoting the proliferation and motility of ESCC cells.

Clinicopathological significance of p38β, p38γ, and p38δ and its biological roles in esophageal squamous cell carcinoma

P38β, p38γ, and p38δ have been sporadically and scarcely reported to be involved in the carcinogenesis of cancers, compared with p38α isoform. However, little has been known regarding their clinicopathological significance and biological roles in esophageal squamous cell carcinoma (ESCC). Expression status of p38β, p38γ, and p38δ was assayed using immunohistochemistry with ESCC tissue microarray; ensuing clinicopathological significance was statistically analyzed. To define its biological roles on proliferation, migration and invasion of ESCC cell line Eca109 in vitro, MTT, wound healing, and Transwell assays were employed, respectively. As confirmation, athymic nude mice were taken to verify the effect over proliferation in vivo. It was found that both p38β and p38δ expression, other than p38γ, were significantly higher in ESCC tissues compared with paired normal controls. In terms of prognosis, only p38β expression was observed to be significantly associated with overall prognosis. Clinicopathologically, there was significant association between p38γ expression and clinical stage, lymph nodes metastases, and tumor volume. No significant association was found for p38β and p38δ between its expression and other clinicopathological parameters other than significant difference of expression between ESCC versus normal control. In Eca109, it was observed that p38β, p38γ, and p38δ can promote the cell growth and motility. As verification, over-expression of p38δ can promote, whereas knockdown of p38γ can prevent, the tumorigenesis in nude mice model xenografted with Eca109 cells whose basal level of p38δ was stably over-expressed and p38γ was stably knocked down. Together, our results demonstrate that p38β, p38γ, and p38δ played oncogenic roles in ESCC.

RNF113A promotes the proliferation, migration and invasion, and is associated with a poor prognosis of esophageal squamous cell carcinoma

Ring finger proteinxa0113A (RNF113A) possesses a C3HC4 zinc finger domain and this domain is found in E3xa0ubiquitin ligase and is involved in tumorigenesis. To date, and at least to the best of our knowledge, there are no studies available which have investigated RNF113A in cancer. Thus, this study aimed to explore the role of RNF113A in the development of esophageal squamous cell carcinoma (ESCC). For this purpose, paraffin-embedded samples from 117xa0patients with ESCC were selected, as well as 41xa0pairs of fresh-frozen ESCC and adjacent normal tissue samples. RNF113A expression was examined by immunohistochemistry and reverse transcription-quantitative PCR (RT-qPCR). RNF113A was overexpressed or silenced in the EC9706 and Eca109 cells. The cells were examined for cell cycle progression, apoptosis, invasiveness and migration. Xenograft tumors were also created in mice using the Eca109 cells. Tumor differentiation (P=0.008) and Txa0classification (P<0.001) were found to be significantly associated with RNF113A expression. No statistically significant association was observed between RNF113A expression and sex, age, histological type, tumor location and lymph node metastasis (Nxa0classification). Kaplan-Meier analysis revealed that the patients with ESCC with ahigh expression of RNF113A had a lower survival rate than those with a low expression (P=0.002). Multivariate analysis revealed that RNF113A expression (HR=2.406; 95%xa0CI, 1.301-4.449, P=0.005) was independently associated with overall survival in patients with ESCC. The overexpression of RNF113A promoted proliferation, migration, and invasiveness of ESCC cell lines inxa0vitro, and RNF113A silencing reversed these malignant behaviors. RNF113A knockdown inhibited tumor growth inxa0vivo. Thus, these results indicate that RNF113A promotes the proliferation, migration and invasiveness of ESCC cell lines. RNF113A expression in ESCC is this associated with a poor prognosis of affected patients.

Partition-Defective 3 (PARD3) Regulates Proliferation, Apoptosis, Migration, and Invasion in Esophageal Squamous Cell Carcinoma Cells

Background Altered expression of partition-defective 3 (PARD3), a polarity-related gene associated with oncogenesis, has been identified in some cancers, but the role of PARD3 in esophageal squamous cell carcinoma (ESCC) remains unclear. Material/Methods PARD3 expression in Eca109 cells was silenced using siRNA and overexpressed using an expression vector. We investigated the role of PARD3 in ESCC growth and motility to evaluate its potential role in ESCC. Transwell assay was used to evaluated cell migration and invasion. PARD3 protein expression was assessed by Western blot. Results PARD3 overexpression promoted apoptosis, impaired proliferation, and inhibited cell migration and invasion in Eca109 cells, while PARD3 silencing promoted proliferation and increased migration and invasion. Overexpression of PARD3 exerted its antitumor activity in vitro by impairing cell proliferation, inducing apoptosis, and inhibiting migration and invasion of Eca109 cells, suggesting that PARD3 might play a tumor suppressor role in ESCC. Conclusions Overexpression of PARD3 could be a promising new therapeutic intervention against ESCC.