The Relationship And Mechanism Between Lin28/miR-107and Chemo-drug Sensitivity In Gastric Cancer Cell

BACKGROUNDAs one of common digestive cancers, gastric cancer causes the second leading of cancer mortality worldwide. Gastric cancer is a frequent neoplasm in China, and the prevalence and mortality are both the twice of average level of world. Surgery provide just40%5-year survival rate benefit to gastric patients, only companied with chemotherapy is the best treatment. Although there are multiple of chemotherapy drugs, the combined chemotherapy can increase the efficiency, and continuous development of new drugs, however, the5-year survival rate in advanced gastric cancer is still below30%. To improve the clinical outcome of patients with gastric cancer, it is necessary to study the molecular mechanism of drug resistance in gastric cancer. There are many mechanisms of drug resistance included:the trans-membrane transport of the ABC transporter protein, glutathione S-transferase enzymes, DNA topoisomerase activity, p53mutation and apoptosis related pathway. The tumor stem cells have a high capacity of DNA repair, high expression of the ABC transporters, and the feature of anti-radiotherapy, anti-apoptosis. So the tumor stem cells may have the capacity of drug resistance. Lin28, as a stem cell marker, has been shown to participate in inducing pluripotent stem (iPS) cells and maintenance cancer stem-like cell in cancer cells. Studies have demonstrated that the expression of Lin28was correlated with radio-resistance in lung cancer. However, there is no evidence to support the correlation between Lin28and chemotherapy in gastric cancer. We intend to clarify the relationship between Lin28and the response to chemotherapy in gastric cancer, as well as to find its possible mechanism in vitro.METHODS AND RESULTS1. Correlation analysis between Lin28and the pathological response of gastric cancer following neoadjuvant chemotherapyIn total, there are47gastric cancer patients who had complete data record and have gastroscope tissues before neoadjuvant chemotherapy in our hospital from June2004to June2011. At total of47case,≤50%residual tumor in61.7%(29/47) of patients, and>50%residual tumor in38.3%(18/47) patients. The expression of Lin28in gastric cancer was correlation with pathological response by chi-square statistical analysis (P=0.002), and high Lin28expression levels were more frequently found in nonresponding patients. But the mechanism of how does Lin28can led to chemotherapy resistance is still unclear.2. Construction stable expression Lin28protein gastric cancer cell clone and study upon the relationship and the mechanism between Lin28expression and the drug sensitivity of gastric cancer cells in vitroThe Western blot assay were performed to evaluate gastric cancer cells (MKN28^MKN45,SGC7901,BGC823,AGS,N87) the Lin28expression at the protein level. The all of gastric cancer cells have no expression Lin28at protein level. The plasmids pcDNA3.1+-Lin28and pcDNA3.1+were transfected into MKN45and MKN28cells (MKN45/Lin28,MKN45/Vector and MKN28/Lin28,MKN28/Vector).The MTT assay and flow cytometric assay were performed to evaluate the drug sensitivity of MKN45/Lin28and MKN28/Lin28cells compared with MKN45/Vector and MKN28/Vector. The result showed that the survival rate to Lin28positive gastric cancer cell line MKN45/Lin28and MKN28/Lin28were significantly higher compared with that to Lin28negative gastric cancer cell lines MKN45/Vector and MKN28/Vector in four drug (OXA, PTX, ADM and5-Fu); as well as the apoptosis rate.To examine the primary mechanism of Lin28mediated the change of drug sensitivity in gastric cancer cells by Western Bolt and qPCR assay. We found high expression of C-myc (which is one of the stem cell's molecular makers) and MDRl/P-gp in Lin28positive cell line, and low expression of cyclin Dl. Meanwhile, the proteins of the caspase pathway were detected by Western Bolt. The results indicate that the apoptosis related proteins were decreased in Lin28positive gastric cancer cells. We also knockdown the Lin28expression level of Lin28positive cell lines by siRNA interference technology, found that could increase drug sensitivity by down-regulate the expression of C-myc, MDR1/P-gp and up-regulate the expression of cyclin D1.Therefore we assume that the mechanism of Lin28mediated the change of drug sensitivity in gastric cancer cells may include:up-regulate the expression of C-myc and MDR1/P-gp, and down-regulate the expression of cyclin D1.3. The relationship between miR-107and Lin28, the effect and mechanism of Lin28/miR107in chemo-drug sensitivity change of gastric cancer cell linesWe have detected the miR-107expression in Lin28positive gastric cell line by q-PCR. The suppression of miR-107expression were found in both MKN45/Lin28and MKN28/Lin28; By knockdown the Lin28expression,the miR-107level was increased. After pre-miR-107was transfected into MKN45/Lin28and MKN28/Lin28cells, the Lin28level was suppressed in both RNA and protein level. Thus we proved that miR-107is one of the Lin28target microRNA, which was inhibited by Lin28, and miR-107may in turn work on the regulation of Lin28expression. And the inhibition of miR-107which may participated in Lin28mediated chemo-drug sensitivity change of gastric cancer cell line.So we transfected pre-miR-107into Lin28positive cell line and anti-miR-107into Lin28negative cell line.and the MTT assay and flow cytometric assay were performed. We found that the tumor cells become sensitive to chemotherapeutic drugs by transfection of pre-miR-107,and the cell which transfected by anti-miR-107became drug resistance.As a Lin28downstream target microRNAs,inhibition of miR-107plays an important role of causing Chemo-drug resistance in gastric cancer cells. Also Western-blot and q-PCR slow that miR-107can down-regulate the C-myc,MDR1/P-gP expression and up-regulate the cyclinD1expression.CONCLUSIONLin28can not only predict the efficacy of chemotherapy in the neoadjuvant patients, but also decrease the drug sensitivity in gastric cancer cell lines in vitro. One mechanism may be involved:by suppression of miR-107,up-regulation of C-myc, MDR1/P-gP expression,down-regulation of CyclinD1.Perhaps, the Lin28/miR-107pathway may play an important role in drug resistanee in gastric cancer.