Effect of Qigesan on Proliferation， Apoptosis and miR-133a/Akt/mTOR Signaling Pathway of Esophageal Cancer EC9706 Cells / 中国实验方剂学杂志

Objective:To observe the effect of Qigesan on the proliferation and apoptosis of the human esophageal cancer cell EC9706， and the effect on miR-133a/protein kinase B（Akt）/mammalian target of rapamycin （mTOR） signaling pathway. Method:The effective constituent of Qigesan was extracted by ethyl acetate. Thiazolyl blue tetrazolium bromide（MTT） colorimetric assay was used to determine the dosage of Qigesan on cells and to detect the effect of Qigesan on the proliferation of EC9706 cells. The effect of Qigesan on apoptosis of EC9706 cells was detected by flow cytometry. The effect of Qigesan on miR-133a and insulin-like growth factor 1 receptor（IGF-1R） mRNA expression was detected by Real-time quantitative polymerase chain reaction （Real-time PCR） . The protein expression of Akt and mTOR in EC9706 cells was detected by Western blot. Result:Qigesan can inhibit the proliferation of EC9706 cells in a dose-dependent manner（<italic>P</italic><0.01）. Inhibitory concentrations 30% inhibition concentration（IC₃₀） 40 mg·L^-1 and median inhibition concentration（IC₅₀） 80 mg·L^-1 were selected for follow-up experiments. Compared with the blank group， both the inhibitor group and the combination drug group can inhibit the proliferation of EC9706 cells （<italic>P</italic><0.01）. The inhibitor at 0.25 μmol·L^-1 was selected for subsequent experiments. Compared with the blank group， Qigesan 80 mg·L^-1 dose group could significantly promote the late apoptosis rate and total apoptosis rate of EC9706 cells（<italic>P</italic><0.05）， and the 40 mg·L^-1 dose group could significantly promote the late apoptosis rate of EC9706 cells（<italic>P</italic><0.05）， which shows synergistic effect after concomitant use with Akt/mTOR inhibitor（<italic>P</italic><0.05）. Compared with the blank control group， each group can effectively increase expression of miR-133a（<italic>P</italic><0.05）. The combination of inhibitor and traditional Chinese medicine（TCM） has obvious promotion effect. Compared with blank control group， the expressions of Akt and mTOR were significantly decreased in each group（<italic>P</italic><0.05）. Compared with single medication， the expressions of Akt and mTOR were decreased in combination of inhibitor and TCM group. Conclusion:Qigesan can inhibit the growth of EC9706 cells and promote apoptosis， and its inhibitory mechanism may be related to the Akt/mTOR signaling pathway by regulating the expression of miR-133a.

lncRNANORADpromotestheproliferationandmigrationofesophagealsquamous cell carcinoma EC9706 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects and mechanisms of long non-coding RNA (lncRNA) non-coding RNA-activated by DNA damage (NORAD) on the proliferation and migration of esophageal squamous cell carcinoma (ESCC) EC9706 cells. Methods: RT-PCR was used to detect the mRNA expression level of NORAD in different ESCC cells (EC9706, TE1, YES-2, KYSE150). Small interfering RNA (siRNA) targeting NORAD gene was transfected into EC9706 cells (as si-NORAD group) with RNA interference technique to knockdown NORAD expression; in addition, blank control group (as Ctrl group, without any transfection) as well as negative control group (as NC group, transfected with siRNAnegative control sequence)werealsoestablished. qPCR was used to verify the transfection efficiency. MTT, Colony formation assay and Wound-healing test were used to detect the abilities of proliferation and migration of EC9706 cells before and after NORAD knockdown. Western blotting was used to detect the expressions of E-cadherin, N-cadherin and Snail in EC9706 cells before and after NORAD knockdown. Results: NORAD mRNAwas highly expressed in 4 ESCC cell lines. Comparing with TE1, YES-2 and KYSE150 cells, the expression of NORAD mRNA was significantly higher in EC9706 cells (P<0.01). After transfection of NORAD-siRNA into EC9706 cells, the expression of NORAD was down-regulated significantly as comparing with Ctrl group and NC group (all P<0.01), in the meanwhile, the proliferation and migration abilities of EC9706 cells were also significantly suppressed (P<0.05).After NORAD knockdown, the expression of E-cadherin was up-regulated while the expressions of N-cadherin and Snail were down-regulated in EC9706 cells (all P<0.05). Conclusion: NORAD is highly expressed in EC9706 cells;knockdown of NORAD expression can inhibit the proliferation and migration ability of EC9706 probably through up-regulating E-cadherin and down-regulating N-cadherin and Snail.

Tanshinone IIA inhibits apoptosis, invasion and migration of esophageal cancer EC9706 and KYSE70 cells by regulating epithelial-mesenchymal transition / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of tanshinone IIA on the invasion and migration of esophageal cancer EC9706 and KYSE70 cells, and to explore the underlying mechanism. Methods: Esophageal cancer cells (EC9706 and KYSE70) were divided into 4 groups: control group, tanshinone IIA groups (2, 4, 6 μg/ml). Cell prliferation viability was measured by CCK-8; Apoptosis was detected by flow cytometry; Invasion was tested by Transwell assay; And migration was measured by Scratch assay. The mRNA and protein levels of E-cadherin, Snail-2, Vimentin and N-cadherin were tested by quantitative Real-time reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Results: Tanshinone IIAat concentrations less than 6 μg/ml did not affect the cell viability of esophageal cancer EC9706 and KYSE70 cells. The apoptosis in tanshinone IIA (4, 6 μg/ml) groups was significantly higher than that in control group ( P < 0.01). The number of invasive cells per field and wound-healing rate in tanshinone IIA(2, 4, 6 μg/ml) groups were significantly lower than those in control group (all P <0.01). Moreover, the cell morphology was transformed from a spindle-shaped mesenchymal form into epithelial morphology after tanshinone IIA treatment. Compared with control group, the expression of E-cadherin in tanshinone IIA groups (2, 4, 6 μg/ml) was significantly up-regulated while the expressions of Snail-2, Vimentin and N-cadherin were significantly down-regulated (all P <0.01). Conclusion: Tanshinone IIApromotes apoptosis and attenuates the invasion and migration of esophageal cancer cells by inhibiting the epithelial-mesenchymal transition.