| Background and Objective Laryngeal carcinoma is one of the tumors with high incidence in the north of China. The tumor incidence is about 1 to 5% of the whole body. Although the diagnosis and treatment of laryngeal carcinoma has made great progress, but the prognosis of some patients is poor, the survival rate is low. For the patients with advanced stage who can not be operated and the patients of postoperative recurrence, chemotherapy is often used, but the effect of chemotherapy is not good. It may be caused by the laryngeal cancer cells’ drug resistance. Multidrug resistance of tumor cells is one of the main reasons for the failure of chemotherapy. The drug resistance of chemotherapy in laryngeal cancer is often expressed as multidrug resistance. To reverse the drug resistance of tumor cells is a key point in tumor research. Progress has been made in the research of mi RNAs reversing tumor drug resistance, but there are few applications of mi RNAs in the treatment of laryngeal cancer and drug resistance which have been studied. We used micro array chip technology to study the difference of mi RNA expression in laryngeal squamous cell carcinoma and adjacent normal mucosa, to screen the specific mi RNAs in laryngeal carcinoma. By comparing the expression difference of mi RNAs between Hep-2 cells and VCR drug resistance cell line Hep-2/v, the specific mi RNAs related to multidrug resistance in laryngeal carcinoma was verified.Materials and Methods miRNA were extracted from 8 cases of laryngeal cancer tissue and its adjacent normal tissue and were labeled with Cy3 for hybridization to the mi RNA microarray. The slides were scanned by Lux Scan 10K/A and images were analyzed by Lux Scan3.0 software. SAM and Clusters were used for different mi RNA Identification and cluster analysis. The difference mi RNA were confirmed by real time quantification RT-PCR with RNA- tailing and primer extension. Hep-2/v cells were treated by 50 u M, 100 u M and 200 u M. The difference between Hep-2 and Hep-2/v on the resistance to cisplatin was determined by cell proliferation assay and apoptosis of Tunel cells. And we confirm the existence of multi drug resistance of Hep-2/v. Detection mi R-133 a level of NP69 of normal pharyngeal epithelial cells, Hep-2 and Hep-2/v cells using Poly tailed PCR. Immunofluorescence staining and Western staining and Blotting PCR were used to detect the changes of two kinds of cells after the addition of ATP7 B. Hep-2 cells and Hep-2/v were transfected with p EZX-mi R-133 a. The effect of different concentrations of cisplatin on cell survival was observed by cck8 test. The transcription and protein expression changes of ATP7 B were detected by immunofluorescence staining and real-time quantitative PCR assay after transfected with p EZX-133 a.Results 1. Totally 47 mi RNAs were found to be differentially expressed in laryngeal cancer through mi RNA expression profile, with 23 of mi RNA expression up-regulated and 24 of mi RNA expression down-regulated. The expression of mir-1,mi R-486-5p, mi R-206, mi R-487 a,mi R-375, mi R-422 a, mi R-144, mi R-384, mi R-378, mi R-133 a were down- regulated by 5 folds and while expression of mi R-93, mi R-31, mi R-20 b were up-regulated by 3 folds too. There are 5 mi RNA clusters with co-expression in laryngeal cancer tissue and located on chromosome 8,13,14,18 and X. Moreover, RT-PCR analysis demonstrated that there was no significantly difference of mi RNA expression between microarray and RT-PCR.2. After treated with 50 uM and 100 u M cisplatin 24 h, the survival of Hep-2 cells were 71% and 73%, while the survival of Hep-2/v cells was not significantly different(p<.0.01). The survival of Hep-2/v cells at 100 u M was 85%, significantly higher than the survival of Hep-2 at the same dose(p<0.05). Treated with 50 u M of cisplatin 24 h, the apoptosis index of Hep-2 cells was 31.12±4.75, and the apoptotic index of Hep-2/v cells was 3.5±1.23, p<0.01. The expression of ATP7 B protein in Hep-2/v cells was significantly higher than that in Hep-2 cells.3. The expression of and ATP7 B in Hep-2 and Hep-2/v treated with cisplatin were increased compared with the control group. Real time quantitative PCR detected ATP7 A and ATP7 B changes at different time after treated with cisplatin, the results showed that 50 u M cisplatin could induce Hep-2 and Hep-2/v expression ATP7 B. The expression of Hep-2 cells was enhanced in 3h, reached the peak in 6h, and sustained expression with no significant change in 24 h. Hep-2 cells and Hep-2/v expression of ATP7 B were significantly higher than that of NP-69 cells.4. The expression level of mi R-133 a in Hep-2 cells was 4.76 ± 0.54 times lower than that of NP-69 cells, and the expression of mi R-133 a in Hep-2/v was decreased by 5.55± 0.14 times compared with that of NP-69. Hep-2 cells and Hep-2/v cells were transfected with recombinant mi R-133 a, using MTT method to detect cell survival after 48 h. The results showed that the survival of the Hep-2/v cells transfected with p EZX-Control were significantly higher than that of Hep-2/v cells transfected with p EZX-mi R-133 a after treated with 50 u M å'Œ 100 u M cisplatin. Mi R-133 a can reduce the transcription and expression of ATP7 B in Hep-2/v cells.Conclusion The expression of mi R-133 a in laryngeal carcinoma tissues was down regulated, and the differentially expression of mi RNA-133 a may be one of the factors involved in the occurrence of laryngeal carcinoma. The expression of ATP7 B protein in laryngeal carcinoma cells was significantly higher than that of Hep-2 cells, and the expression of ATP7 B protein was increased to provide a new way for the study of cisplatin resistance. It was found that mi R-133 a can reincrease the chemotherapeutic sensitivity of Hep-2/v cells to cisplatin, and its mechanism may be related to mi R-133 a the down-regulation of ATP7 B expression. |
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