Shutao Zheng

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

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.

IL-1β from M2 macrophages promotes migration and invasion of ESCC cells enhancing epithelial-mesenchymal transition and activating NF-κB signaling pathway

Despite the phenotype has been established that M2 macrophages promotes the metastasis of ESCC, question still remains as to how the M2 macrophages facilitated metastasis of ESCC cells. To begin with, immunohistochemistry was performed to detect the expression of CD163, one typical surface marker of M2 macrophages in 90 paired ESCC and its normal controls after meta‐analyzing the relevant studies regarding M2 macrophages in ESCC, confirming that infiltration of M2 macrophages was significantly linked with lymph node metastasis, T classification, and inferior overall survival of ESCC. To explore the mechanism behind, protein factors secreted by M2 macrophages were identified using antibody microarray. Six different significantly differential protein factors were screened out, including IL‐1β, TIMP1, IL‐1α, MDC, TGF‐β1, and TGF‐β2. Among which, IL‐1β was picked up as cytokine as interest based on previous reports and its absolute fold. Functional analysis of IL‐1β showed that IL‐1β was able to promote migration and invasion of ESCC cells, enhancing epithelial‐mesenchymal transition, and activating NF‐κB pathway. Our study supports the promoting role of M2 macrophages in metastasis of ESCC cells, enriching the profile of protein factors released from M2 macrophages.

MCP2 activates NF-κB signaling pathway promoting the migration and invasion of ESCC cells: MCP2 activates NF-κB promoting motility

MCP2, aliased CCL8, has been suggested to be implicated in the metastasis of cancer cells; however, no direct evidence has been established in esophageal squamous cell carcinoma (ESCC). In our present study, to investigate the role MCP2 played in the metastasis of ESCC cells; in vitro cell co‐culture system was established. Wound‐healing and Transwell assays were used to evaluate the migratory and invasive variation of ESCC cells before and after treatment with recombinant human MCP2. It was shown that MCP2 was able to activate the NF‐κB signaling pathway inducing the epithelial‐mesenchymal transition (EMT) and promoting the migration and invasion of ESCC cells in vitro. Our study provides an alternative working mechanism for M2 macrophage mediated the metastasis in tumor microenvironment in ESCC.

MiR-106b promotes migration and invasion through enhancing EMT via downregulation of Smad 7 in Kazakh’s esophageal squamous cell carcinoma

Accumulated evidence suggests that miR-106b played a key role in the promotion of the metastases of cancer; however, little is known about miR-106b in esophageal squamous cell carcinoma (ESCC). To investigate expression level of miR-106b in ESCC tissues, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect miR-106b expression in 35 Kazakh’s ESCC and paired normal adjacent tissues (NATs). To evaluate the role mediated by miR-106b in the proliferation, migration, and invasion, MTT, wound healing, and transwell assays were employed, respectively. Luciferase reporter assay was used to identify the downstream target through miR-106b. To understand the regulation between miR-106b and Smad 7, qRT-PCR and western blot were performed. The present study showed that miR-106b was pronouncedly upregulated in ESCC relative to paired NAT and that upregulated miR-106b was significantly associated with lymph node metastases. MiR-106b was found to be able to promote proliferation, migration, and invasion of ESCC cells in vitro. Smad 7 was confirmed as a downstream target of miR-106b in our experimental setting. Smad 7 was remarkably downregulated in ESCC compared with paired NAT. In addition, upregulation of miR-106b can promote epithelial mesenchymal transition (EMT) in ESCC cell in vitro. Our results indicated that miR-106b can promote migration and invasion of ESCC cells through enhancing EMT process via downregulation of Smad 7, suggesting that miR-106b can be a potential molecular phenotype in ESCC metastases.