| There is a high incidence of gastric cancer in China. Due to the lack of popularity and attention of screening, the majority of gastric cancer has been diagnosed at advanced stages and makes the chemotherapy remaining the primary treatment. But the primary or aquired drug resistance cause decreased sensitivity to chemotherapy and ultimately lead to treatment failure. Although researches about resistant related genes'function have revealed a large number of resistance mechanisms and shown important roles in guiding clinical treatment, these researches still stand alone due to the lack of connection between these genes. In recent years, studies have shown that a class of 21~25 nt single-stranded non-coding microRNAs play important roles in the development of tumors. And bioinformatics studies indicate that microRNAs display important biological effects by regulating different genes, which establish a close correlation between genes that seem independent with each other. Furthermore, one microRNA can modify tumor sensitivity to anticancer drugs through regulating different targets. Cisplatin (cisplatin, CDDP) is still one of basic choices of themotherapy drugs in the treatment of gastric cancer. SGC7901/CDDP cell line is a gastric cancer cisplatin-resistant cell line. Although there have been some reports about durg resistance in this cell, analysis of microRNA expression profile has not been carried out. Meanwhile, the role of microRNA in the process of acquired cisplatin resistance of gastric cancer also has not been studied. In this study, resistance characteristics of SGC7901/CDDP cells were analyzed, and a high-throughput microRNA array analysis of microRNA was used to analyze microRNA expression profiles of cisplatin-resistant SGC7901/CDDP and its parental cisplatin-sensitive SGC7901 cells. A sub-set of differentially expressed microRNAs were then identified when compared with the microRNA expression profile of SGC7901 cells. The application of target prediction softwares were further used to predict targets of these differentially expressed microRNAs, and bioinformatics analysis was also used to analyze tumor biological processes of these microRNAs to find potential targets or regulatory pathway of microRNA to overcome drug resistance of gastric cancer. Based on the establishment of microRNA expression profile in SGC7901/CDDP cells, we further studied the effects of microRNA-200c on the drug resistance and proliferation in the SGC7901/CDDP cells and observed its effects on regulating E-cadherin, PTEN/Akt pathway and apoptotic proteins Bcl-2 and BAX. And changes of drug resistance, proliferation, PTEN/Akt pathway and apoptotic proteins Bcl-2 and BAX after E-cadherin inhibition in SGC7901 cells were further analyzed to explore E-cadherin-mediated biological effects and its mechanisms and identify whether regulation of PTEN/Akt pathway and apoptotic protein by microRNA-200c could be through E-cadherin mediated pathway.Part One microRNA expression profile and its bioinformatics analysis of gastric cancer SGC7901/CDDP cellsObjective: To study drug resistance characteristics of SGC7901/CDDP cells and identify abnormally expressed microRNAs between cisplatin resistant SGC7901/CDDP and its parental SGC7901 cells, and try to find mechanisms of drug resistance and potential targets of reversing drug resistance at microRNA level.Methods: Drug sensitivities of SGC7901/CDDP and SGC7901 cells to cisplatin, doxorubicin, 5-fluorouracil and paclitaxel were analyzed by MTT assay. microRNA microarray was used to analyze microRNA expression profiles of two cells and identify differentially expressed microRNAs. TargetScan (http://www.targetscan.org/) and MicroCosm Targets Version 5 ( http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/ ) softwares were applied to predicted targets of these abnormally expressed microRNAs and the targets which were successfully predicted by both softwares were selected and divided into upregulated microRNA and downregulated microRNA groups according to their corresponding microRNA expression situation. Biological processes notation of targets from each group were further analyzed to predict their biological function using Blast2GO analysis (http://www.blast2go. org/start_blast2go) .Results:1 Drug sensitivities of two cell lines to cisplatin, doxorubicin, 5-fluorouracil and paclitaxel: Cell viability between SGC7901/CDDP and SGC7901 cell lines were different after 48h exposure to different concentrations of cisplatin, doxorubicin, 5-fluorouracil and paclitaxel. IC50 values of cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in SGC7901/CDDP cells were 11.245±0.272, 0.763±0.043, 12.154±2.036 and 3.277±0.426 mg/L respectively; while IC50 values in SGC7901 cells were 2.206±0.256, 0.229±0.020, 4.481±0.544 and 1.528±0.097 mg/L respectively. IC50 values of cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in SGC7901/CDDP cells were significantly higher than in SGC7901 cells (P<0.05), sensitivity of SGC7901/CDDP cells to the four anticancer drugs reduced.2 Results of RNA quality: The concentration of extracted total RNA from SGC7901/CDDP and SGC7901 cells were 1145.77 mg/L and 1853.74 mg/L respectively. And A260/A280 values were between 1.9 to 2.0. From formaldehyde denaturing agarose gel electrophoresis results, the 18S and 28S bands were clear and the brightness ratio was about 1:2, indicating the total RNA purity met to the experiment requirements and had no significant degradation.3 Results of microRNA microarray analysis: After Hy3 fluorescent labeling, purification, the chip hybridization, image acquisition and data normalization, Hy3 fluorescent signal intensity ratios≤0.5 or≥2 were considered as a standard of determining differentially expressed microRNAs. Compared with SGC7901 cells, 14 microRNAs were downregulated more than 2 fold in SGC7901/CDDP cells (microRNA-33a, microRNA-200c, microRNA-302c*, microRNA-488, microRNA-148a, microRNA-141, microRNA-212, microRNA-576-3p, microRNA-660, microRNA-338-3p, microRNA-24-1*, microRNA-589, microRNA-887 and microRNA-22*), while 5 upregulated (microRNA-1246, microRNA-943, microRNA-1290, microRNA-516a-5p and microRNA-34b).4 Bioinformatics analysis of predicted microRNAs'targets: The downregulated microRNA group had a total of 451 predicted targets, and Blast2GO analysis showed that these targets were involved in signal transduction, transcriptional regulation, protein phosphorylation, membrane transport, drug sensitivity, cell cycle, differentiation, apoptosis, proliferation, adhesion, DNA repair and other biological processes. The upregulated microRNA group had a total of 98 predicted targets, Blast2GO analysis showed that these targets were involved in signal transduction, transcription regulation, membrane transport, cell cycle, apoptosis, proliferation, differentiation, tissue and other biological processes.Conclusion:1 SGC7901/CDDP cells were not only resistant to cisplatin, but also resistant to doxorubicin, 5-fluorouracil and paclitaxel, showing a multidrug resistance phenotype.2 A sub-set of microRNAs differentially expressed between gastric cancer SGC7901/CDDP cisplatin-resistant cells and its parental SGC7901 cells were identified using microRNA microarray, among of which 14 microRNAs including microRNA-33a, microRNA-200c, microRNA-302c*, microRNA-488, microRNA-148a, microRNA-141, microRNA-212 and etc were downregulated, and 5 microRNAs including microRNA-1246, microRNA-943, microRNA-1290, microRNA-516a-5p and microRNA-34b were upregulated in SGC7901/CDDP cells.3 GO analysis results of predicted targets of these differentially expressed microRNAs suggested that these microRNAs had complex biological effects, indicating that the process of SGC7901/CDDP cells acquiring cisplatin resistance was the result of combined effects of multiple microRNAs. Part Two Effects of microRNA-200c on drug resistance and proliferation in gastric cancer SGC7901/CDDP cellsObjective: Since microRNA-200c was identified between the SGC7901/CDDP and SGC7901 cells by microRNA array analysis, we aimed to further validate the expression of microRNA-200c from microRNA array analysis and explore effects of microRNA-200c on drug sensitivity and cell proliferation in vitro by changing microRNA-200c expression in SGC7901/CDDP cells.Methods: Real-time fluorescent quantitative PCR was used to test expression levels of microRNA-200c screened by microRNA microarray. And microRNA-200c precursor (pre-200c) or a negative control oligonucleotide was transfected into SGC7901/CDDP cells using siPORT? NeoFX? Transfection Agent. Tewnty-four hours after transfection total cellular RNA was extracted to detect expression level changes of microRNA-200c using real-time fluorescent quantitative PCR, and drug sensitivity changes of transfected cells to cisplatin, doxorubicin, 5-fluorouracil and paclitaxel were also tested by MTT assay. Changes of cell proliferation were further detected by cell counting assay 24h after transfection.Results:1 Difference of microRNA-200c expression in the two cells: Real-time fluorescent quantitative PCR results showed that the△Ct of microRNA-200c in SGC7901/CDDP cells was 15.470±0.036, while△Ct in SGC7901 cells was 14.657±0.229. The relative expression levels of microRNA-200c in SGC7901/CDDP cells was 0.573±0.084 fold lower than in SGC7901 cells, showing a significantly reduction compared with the sensitive cells (P <0.05).2 The expression change of microRNA-200c after 24h transfection in SGC7901/CDDP cells: Real-time fluorescent quantitative PCR results showed that the△Ct of microRNA-200c in SGC7901/CDDP cells transfected with pre-200c was 12.660±0.079, while△Ct in negative control group was 15.493±0.080. The relative expression levels of microRNA-200c in cells 24h after transfected with pre-200c was 7.128±0.159 fold higher than with negative control (P<0.05).3 Effects of microRNA-200c on the drug resistance in SGC7901/CDDP cells: microRNA-200c increased drug sensitivity of SGC7901/CDDP cells to cisplatin, doxorubicin, 5-fluorouracil and paclitaxel. IC50 values of cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in SGC7901/CDDP cells transfected with pre-200c were 7.518±0.186, 0.381±0.053, 7.267±0.090 and 1.705±0.332 mg/L respectively, while IC50 values in SGC7901/CDDP cells transfected with negative control oligonucleotide were 12.180±0.294, 0.721±0.034, 11.534±0.598 and 3.140±0.403 mg/L respectively. IC50 values of cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in SGC7901/CDDP cells transfected with pre-200c were significantly lower than transfected with negative control (P<0.05).4 Effects of microRNA-200c on cell proliferation in SGC7901/CDDP cells: From the third day after inoculation, the cell number of SGC7901/CDDP cells transfected with pre-200c was significantly lower than transfected with negative control oligonucleotide (P<0.05), showing the cell proliferation was significantly reduced.Conclusion:1 Real-time fluorescent quantitative PCR confirmed that microRNA-200c was downregulated in human gastric cancer cisplatin resistant SGC7901/CDDP cells.2 Induction of the microRNA-200c expression can significantly increase the sensitivity of SGC7901/CDDP cells to four anticancer drugs cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in vitro, showing a role of chemotherapy sensitization.3 Induction of the microRNA-200c expression significantly inhibited SGC7901/CDDP cell proliferation in vitro.Part Three The molecular mechanisms of microRNA-200c on reversing drug resistance and inhibiting cell proliferation in SGC7901/CDDP cellsObjective: To clarify mechanisms of microRNA-200c on reversing drug resistance and inhibiting proliferation in SGC7901/CDDP cells, expression changes of E-cadherin, PTEN, p-Akt, Akt, Bcl-2 and BAX protein were tested between SGC7901/CDDP and SGC7901 cells or after forced microRNA-200c expression in SGC7901/CDDP cells. And we further clarify drug resistance related proteins which were regulated by microRNA-200c and involved in the drug resistance formation in SGC7901/CDDP cells.Methods: Western blot was used to detected E-cadherin, PTEN, p-Akt, Akt, Bcl-2 and BAX protein expressions of SGC7901/CDDP and SGC7901 cells. And pre-200c or a negative control oligonucleotide was transfected into SGC7901/CDDP cells using siPORT? NeoFX? Transfection Agent. Seventy-two hours after transfection, total proteins were extracted from the cells and E-cadherin, PTEN, p-Akt, Akt, Bcl-2 and BAX protein expression were tested by Western blot.Results:1 Expressions of E-cadherin, PTEN, p-Akt, Akt, Bcl-2 and BAX protein between the two cells: Relative expressions of E-cadherin, PTEN, p-Akt, Akt, Bcl-2 and BAX protein in SGC7901/CDDP cells were 0.098±0.030, 0.178±0.064, 1.019±0.093, 1.181±0.102, 0.267±0.018 and 0.215±0.026 respectively, while relative expressions in SGC7901 cells were 0.467±0.061, 0.874±0.056, 0.166±0.017, 1.231±0.163, 0.108±0.011 and 0.366±0.017. Compared with SGC7901 cells, p-Akt and Bcl-2 protein expressions in SGC7901/CDDP cells were significantly increased (P<0.05), while E-cadherin, PTEN, and BAX protein expression were decreased (P<0.05). The relative expression of total Akt between the two cells had no significant difference(P>0.05).2 Effects of microRNA-200c on expression changes of E-cadherin, PTEN, p-Akt, Akt, Bcl-2 and BAX protein in SGC7901/CDDP cells: Relative expressions of E-cadherin, PTEN, p-Akt, Akt, Bcl-2 and BAX protein in SGC7901/CDDP cells transfected with pre-200c were 0.471±0.010, 0.589±0.016, 0.220±0.035, 1.255±0.116, 0.074±0.008 and 0.380±0.008 respectively, while relative expressions in SGC7901/CDDP cells transfected with negative control were 0.109±0.002, 0.111±0.002, 0.687±0.086, 1.291±0.117, 0.442±0.020 and 0.111±0.005. Expressions of E-cadherin, PTEN, BAX protein in SGC7901/CDDP cells transfected with pre-200c were significantly higher than transfected with negative control oligonucleotide (P<0.05), while p-Akt and Bcl-2 protein expressions were significantly lower than transfected with negative control (P<0.05). And the relative expression of total Akt between the two groups had no significant differences (P>0.05).Conclusion:1 Resistance phenotype of SGC7901/CDDP cells was associated with decreased expressions of E-cadherin, PTEN and BAX protein, activation of Akt pathway and increased Bcl-2 protein expression.2 The effects of microRNA-200c on reversing drug resistance and inhibiting cell proliferation in SGC7901/CDDP cells was associated with the induction of E-cadherin, PTEN and BAX protein expression, inhibition of Akt pathway and Bcl-2 protein expression. Part Four Effects of E-cadherin siRNA and its molecular mechanisms on drug resistance and cell proliferation in SGC7901 cellsObjective: From last part, we had showed that microRNA-200c could regulate a series of drug resistance related proteins. In order to investigate the relationship between microRNA-200c and these proteins and their signal transduction sequence, we wondered whether signal downstream of microRNA-200c was through E-cadherin regulatory pathway based on previous results. Meanwhile, we also investigated effects of E-cadherin on drug resistance and proliferation in SGC7901 cells.Methods: E-cadherin siRNA or negative control oligonucleotide were transfected into SGC7901 cells using Lipofectamine 2000. Total protein was extracted from cells 24, 48 and 72h after transfection to detect inhibition efficiency of E-cadherin protein by Western blot. MTT assay was used to detect drug sensitivity changes of transfected cells to cisplatin, doxorubicin, 5-fluorouracil and paclitaxel 24h after transfection in SGC7901 cells, and proliferation in vitro was also tested by cell counting after transfection. Total proteins were extracted from SGC7901 cells 72h after transfection, and PTEN, p-Akt, Akt, Bcl-2 and BAX protein expressions were tested by Western blot.Results:1 E-cadherin protein expression changes after transfection in SGC7901 cells: Relative expressions of E-cadherin protein of SGC7901 cells 24, 48 and 72h after transfection with E-cadherin siRNA were 0.244±0.006, 0.155±0.007 and 0.118±0.014 respectively, while the relative expressions was 0.527±0.006 in cells transfected negative control oligonucleotide. E-cadherin protein expression were significantly inhibited after E-cadherin siRNA transfection (P<0.05).2 Drug sensitivity changes of SGC7901 cells transfected with E-cadherin siRNA to chemotherapeutic drugs: E-cadherin siRNA decreased drug sensitivity of SGC7901 to cisplatin, doxorubicin, 5-fluorouracil and paclitaxel. IC50 values of cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in SGC7901 cells transfected with E-cadherin siRNA were 4.375±0.199, 0.368±0.042, 6.855±0.780 and 2.530±0.259 mg/L respectively, while IC50 values in SGC7901 cells transfected with negative control oligonucleotide were 2.882±0.166, 0.228±0.012, 4.058±0.946 and 1.483±0.225mg/L. IC50 values of cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in SGC7901 cells transfected with E-cadherin siRNA were significantly higher than transfected with negative control oligonucleotide (P<0.05), showing less sensitivity to chemotherapy.3 Effects of E-cadherin siRNA on proliferation in SGC7901 cells: From the 4th day after inoculation, the cell number of SGC7901 cells transfected with E-cadherin siRNA was significantly higher than transfected with negative control oligonucleotide (P<0.05), indicating the cell proliferation was significantly enhanced.4 Effects of E-cadherin siRNA on expressions of PTEN, p-Akt, Akt, Bcl-2 and BAX protein in SGC7901 cells: Relative expressions of PTEN, p-Akt, Akt, Bcl-2 and BAX protein in SGC7901 cells 72h after E-cadherin siRNA transfection were 0.148±0.010, 0.455±0.034, 1.045±0.182, 0.328±0.014 and 0.139±0.013 respectively, while relative expressions in SGC7901 cells 72h after negative control oligonucleotide transfection were 0.531±0.014,0.224±0.049,1.158±0.143,0.114±0.012 and 0.369±0.016. The p-Akt and Bcl-2 protein expressions in SGC7901 cells transfected with E-cadherin siRNA was significantly higher than transfected with negative control oligonucleotide, while the relative expressions of PTEN, BAX protein were significantly lower than transfected with negative control (P<0.05). And the relative expression of total Akt between the two groups had no significant differences (P>0.05).Conclusion:1 Inhibition of E-cadherin protein could significantly reduce sensitivity of SGC7901 cells to the four anticancer drugs cisplatin, doxorubicin, 5-fluorouracil and paclitaxel in vitro, promoting the cells resistance to chemotherapeutic drug.2 Inhibition of E-cadherin protein significantly changed the proliferation of SGC7901 cells, leading to increased cell proliferation.3 Drug resistance and proliferation of SGC7901 cells caused by inhibition of E-cadherin expression were associated with downregulation of PTEN and BAX protein expressions, activation of Akt signaling pathway and upregulation of Bcl-2 protein expression.4 microRNA-200c might regulate PTEN/Akt signaling pathway and apoptotic protein Bcl-2 and BAX through E-cadherin mediated pathway, playing roles of reversing drug resistance and inhibiting cell proliferation.
|
PDF Full Text Request