Bit1 knockdown contributes to growth suppression as well as the decreases of migration and invasion abilities in esophageal squamous cell carcinoma via suppressing FAK-paxillin pathway

Abstract

BackgroundThere is growing evidence that Bit1 exerts different roles in the development and progression of human cancers. Although Bit1 was highly exhibited in ESCC tissues in our previous study, its roles and molecular mechanisms implicated in development and progression of ESCC remain unknown.MethodsBit1 protein expression in ESCC cell lines and normal esophageal epithelial cell was detected by Western blotting. Bit1 protein expression mediated by Bit1 shRNA was investigated by Western blotting. MTT, migration assay, invasion experiment, ELISA and Flow cytometry were utilized to determine the effects of Bit1 knockdown on cell proliferation, migration, invasion and apoptosis, respectively. A xenograft model was used to examine in vivo tumourigenicity, and immunohistochemistry and TUNEL were utilized to evaluate the related protein expression and apoptosis. Gene microarray was determined by Agilent SurePrint G3 Human GE 8 × 60 K Microarray, the interaction of Bit1 and FAK proteins were detected by Immunoprecipitation and the key protein expressions of FAK-paxillin pathway were detected by Western blotting.ResultsWe found Bit1 expression in all human ESCC cell lines tested was significantly higher than that in normal esophageal epithelial cell Het-1A (P < 0.05), in which EC9706 presented the highest Bit1 level. Bit1 protein level was significantly downregulated at day 1 after transfection with specific shRNA against Bit1 (P < 0.05). At days 2 and 3, Bit1 level reached the lowest value after transfection with Bit1 shRNA. Moreover, Bit1 depletion contributed to growth inhibition in vitro and in vivo, reduced cell migration and invasion abilities, and induced cell apoptosis in EC9706 and TE1 cells. More importantly, Bit1 downregulation significantly lowered Bcl-2 and MMP-2 levels in EC9706 xenografted tumor tissues, meanwhile triggered apoptosis after treatment with different doses of Bit1 shRNA. Further gene microarray revealed that 23 genes in Bit1-RNAi group were markedly downregulated, whereas 16 genes were obviously upregulated. Notably, Bit1 intrinsically interacted with FAK protein in EC9706 cells. Moreover, paxillin was downregulated at mRNA and protein levels in Bit1 shRNA group, coupled with the decreases of FAK mRNA and protein expressions.ConclusionBit1 may be an important regulator in cell growth, apoptosis, migration and invasion of ESCC via targeting FAK-paxillin pathway, and thereby combinative manipulation of Bit1 and FAK-paxillin pathway may be the novel and promising therapeutic targets for the patients with ESCC.

Differential expression of matrix metalloproteinases and miRNAs in the metastasis of oral squamous cell carcinoma

“…[13][14][15][16][17][18][19][20] and seven metastatic OSCC patients (No. [21][22][23][24][25][26][27] were obtained for further experimental validation.…”

“…The qualified total RNA was used to synthetize firststrand cDNA using a cDNA synthesis kit (TIANGEN, China), followed by fluorescent labelling with Agilent's Low Input Quick Amp WT Labeling kit (Agilent Technologies, USA) according to the manufacturer's instructions. The labelled cDNA was purified with an RNeasy Mini kit (Qiagen, Germany) and hybridized onto the Agilent Human SurePrint G3 Human GE 8 × 60 k v16 microarray chip (Agilent Technologies, USA) [21] by Shanghai OE Biotech Company (Shanghai, China). Total RNA was also used for the miRNA microarray experiment.…”

Suppressor of cytokine signaling-1 gene therapy induces potent antitumor effect in patient-derived esophageal squamous cell carcinoma xenograft mice

“…Next, we evaluated the constitutive activation status of STAT3, FAK, and p44/42 MAPK. Similar to STAT3, FAK and p44/42 MAPK signaling pathways are also involved in the proliferation of esophageal cancer . As shown in Figure b, STAT3 was constitutively phosphorylated at high levels in seven ESCC cell lines (TE1, TE4, TE5, TE6, TE8, TE10, and TE15 cells).…”

“…Similar to STAT3, FAK and p44/42 MAPK signaling pathways are also involved in the proliferation of esophageal cancer. 36,37 As shown in Figure 2b, STAT3 was constitutively phosphorylated at high levels in seven ESCC cell lines (TE1, TE4, TE5, TE6, TE8, TE10, and TE15 cells). In contrast, p-STAT3 levels in three ESCC cell lines (TE9, TE11, and TE14) were low.…”

Prdx1 Promotes the Loss of Primary Cilia in Esophageal Squamous Cell Carcinoma

“…We next conducted an animal experiment to test the effect of Prdx1 on tumorigenesis. We used EC9706 cells to induce tumor formation in mice, using immune-deficient nude mice as a transplant carrier to avoid xenogeneic exclusion (35,36). Negative control EC9706 cells in logarithmic growth phase and EC9706 cells with inhibited Prdx1 levels were prepared as cell suspensions and injected subcutaneously into the right axillary of nude mice.…”