Overexpression Of MiR-203 Sensitizes Paclitaxel (Taxol) Resistant Colorectal Cancer Cells Through Targeting The Saltinducible Kinase 2 (SIK2)

Background and objective:Colorectal cancer(CRC)is a common malignant tumor of the digestive tract.It ranks third in the global incidence rate and poses a serious threat to human health.Chemotherapy is the most important treatment for advanced colorectal cancer.Although Taxol,a powerful antitumor drug,improves the survival of colorectal cancer patients,most people develop drug resistance after initial treatment.Therefore,it has become an important task to study the mechanism of drug resistance and explore new therapeutic targets.Micro RNA(miRNA)is an endogenous non-encoded single stranded small molecule RNA that regulates gene expression after transcription.Through the specific binding of the target m RNA with downstream 3,—UTR of the target gene,the target m RNA translation is inhibited or the target m RNA degradation is induced to play a regulatory role.miRNA is closely related to cell growth and differentiationumor? cell proliferation and invasion?apoptosis and tumor resistance.miR-203 was originally discovered in dermatology-related studies and is located at 14q32,33 sites on the chromosome and belongs to an unstable brittle region.It is known that about 1/8 of human miRNA is encoded by miR-203,and the loss of heterozygous in this area is closely related to a variety of diseases.miR-203 has been reported to have decreased expression in various types of cancer,suggesting that miR-203 may be a tumor suppressor gene.Now,only a small number of target gene,s biological functions have been known,and the specific working mechanism is still unclear,but it has been determined that miR-203 can use different expression patterns to participate in the pathological process of many diseases.In this study,we investigated the sensitive mechanism of taxol regulated by miR-203 in colorectal cancer cells.By assessing the effects of changes in expression levels of colorectal cancer cells miR-203 and SIK2 on drug resistance of colorectal cancer cells,further study whether miR-203 can target SIK2 to affect the resistance of colorectal cancer cells to taxol,in order to provide more effective miRNA-based anti-tumor drugs.Methods:To evaluate the expression level of miR-203 in colorectal cancer cells we use q RT-PCR,then construct the node of taxol resistant colorectal cancer cells,and the lentiviral transfection experiments and MTT method to measure the viability of colorectal cancer cells in order to evaluate the overexpression of miR-203 influence on sensitivity to taxol in colorectal cancer cells.The expression of SIK2,a downstream target gene of micro RNA-203,in colorectal cancer cells transfected with lentivirus was studied by q RT-PCR,double luciferase activity assay and Western blot.The expression of SIK2 in tumor tissues was compared by q RT-PCR and immunohistochemistry.The effects of expression or knockout of SIK2 on the activity of resistant cells in colorectal cancer taxol were detected by Western blot and MTT,and further verified that miR-203 mediated the sensitization of taxol by inhibiting SIK2.Results:1.We compared the expressions of miR-203 in colorectal tumors and their adjacent normal tissues in q RT-PCR.We found the expressions of miR-203 were down-regulated,and the expressions of SIK2 were up-regulated in tumor samples,compared with normal colorectal tissues.2.Overexpression of miR-203 markedly enhanced the Taxol-induced cell cytotoxicity in both cells.The miR-203 was significantly downregulated in Taxol resistant cells,and inhibition of miR-203 in colorectal cancer cells could significantly increase their tolerance toward Taxol.3.The transfection of miR-203 reduced the luciferase activity of wild SIK2 3'-UTR,but did not have a similar effect on SIK2 3'-UTR mutants.Immunohistochemical staining showed that the expression of miR-203 and SIK2 in colorectal cancer was reversed,and the expression of miR-203 and SIK2 in colorectal cancer cells was strongly negatively correlated.4.We observed significantly upregulation of both protein and m RNA levels of SIK2 in Taxol resistant cells compared with parental cells by q RT-PCR.Overexpression of SIK2 could enhance colorectal cancer cell,s resistance to Taxol,which was acquired from si RNA inhibition or knockdown of SIK2 test.5.Transfection with miR-203 decreased the cell viability of Taxol resistant cells,but with the rescue of SIK2,the Taxol resistant cells exhibited increased resistance to Taxol.Conclution:1.miR-203 was downregulated and SIK2 was upregulated in colorectal tumors,indicated a tumor suppressor-like role of miR-203 in colorectal cancer.2.The expressions of miR-203 were down-regulated in Taxol resistance cells.Overexpression miR-203 could sensitize colorectal cancer cells to Taxol?Cisplatin and 5-Fu treatments,suggest overexpression of miR-203 could sensitize colorectal cancer cells to chemotherapeutic agents.3.Knockdown of SIK2 sensitizes colorectal cancer cells to chemotherapeutic agent Taxol,consistent with previous studies that SIK2 exhibits oncogenic potential.4.The findings of the present study showed that overexpression of miR-203 markedly reduced the expression of SIK2.We identified SIK2 as a direct target of miR-203 and first reported the overexpression of miR-203 sensitized colorectal cancer cells to Taxol via the downregulation of SIK2.5.Recovery of SIK2 desensitized the colorectal cancer cells to Taxol,suggesting the miR-203-mediated sensitization to Taxol is through the inhibition of SIK2.In summary,our study proposed mechanisms for the miR-203-mediated Taxol sensitivity of colorectal cancer cells,and will provide experimental basis for the clinical development of anti-chemoresistance drugs.