Coix Seed Oil Inhibits The Expression Of Drug Resistance Genes Through Suppressing LncRNA-PVT1 In Gastric Cancer Cells

BackgroundAt present,the treatment of middle and late stage gastric cancer is mainly applied to alleviate the symptoms of the disease,and is far from achieving radical cure.Treatments include surgery,radiotherapy,and chemotherapy.Chemotherapy of gastric cancer is an important treatment due to the characteristics of convenience,quickness,well effect and so on.However,with the use of a large number of chemicals,multi-drug resistance is also followed.According to the American Cancer Society Statistics,over 90%of the 490 thousand patients who died each year were associated with multidrug resistance(MDR).MDR means that tumor cells after exposure to a single drug develop cross-resistance to a variety of structurally unrelated natural product-derived antitumor agents with different mechanisms.For a long time,the MDR is a difficult problem in cancer chemotherapy,and is also a major obstacle for cancer treatment.There is a great clinical significance for us to understand the mechanism of drug resistance of tumor,and look for high resistance reversal agent.The mechanism of multi-drug resistance of cancer is very complex.Many studies are reported that drug efflux,drug inactivation,DNA damage,apoptosis and survival signaling pathway alteration are the main mechanisms of drug resistance.Many regulatory factors are involved in the drug resistance of gastric cancer,including non-coding RNA,transcription factors,multi-drug resistance related molecules and so on.Although the molecular mechanisms of drug resistance in gastric cancer has been studied in part,it is still far away from meeting the need of clinical application due to the complexity of the drug resistance of gastric cancer and limitations of existing research.Therefore,it is of great significance to further study the molecular mechanism of drug resistance in gastric cancer.According to the traditional Chinese medicine theory,pathological factors of gastric cancer metastasis include Spleen deficiency and damp evil.Spleen deficiency is the internal factor;damp evil is the pathological product.Damp evil is the cause of spleen deficiency.The two factors complement and promote each other.Gastric cancer patients with spleen deficiency and damp evil syndrome patients should be particularly focused on prevention of metastasis after surgery and chemotherapy.Coix seed oil(CSO)is the preferred drug in the treatment of spleen deficiency and damp evil.CSO is a kind of traditional Chinese medicine injection with coix seed as the main raw materials,its effective components is triglyceride playing a role with a variety of anti-tumor mechanism.It plays an important role in immune regulation,the hypoglycemic effect,induced ovulation and anti-tumor.It has been reported that CSO treatments were showed significant therapeutic effect on many kinds of cancer,including lung cancer,pancreatic cancer,liver cancer and gastric cancer.In addition,CSO can inhibit the growth of tumor,also enhance the sensitivity of tumor drug resistance.Studies are reported that chemotherapy in combination with CSO injection is showed better effect on the treatment of gastric cancer.CSO can also inhibit the expression of multidrug resistance related genes in gastric cancer.However,the molecular mechanism of CSO in gastric cancer drug resistance remains to be further studied.Long non-coding RNA(Inc RNA),a kind of non-coding RNA,is widely distributed in mammals.Although IncRNA does not have the function of encoding protein,it is involved in the growth,differentiation,proliferation and apoptosis of the living organisms by regulating the expression of gene.A large number of studies have shown that IncRNA regulated tumor related pathway at transcriptional,post transcriptional and epigenetic level.An increasing number of studies have confirmed that the abnormal expression of IncRNA existed in almost all tumors.Therefore,IncRNA is gradually recognized as an important factor in the pathogenesis of cancer.Because IncRNA is not conservative,it is not only involved in the various processes of life activities,but also in regulating tumor resistance and drug sensitivity.Studies have confirmed that a variety of IncRNA by improving the DNA repair function could modulate drug metabolism and efflux,regulate apoptosis and affect the epithelial-mesenchymal process leading to the drug resistance of cells.LncRNA PVT1 is located at upstream 60kb of promoter for MYC which is an important proto-oncogene.Studies have shown that PVT1 overexpression can promote cancer cells proliferation and invasion,and enhance the drug resistance of tumor cells to chemotherapy.These findings have been confirmed in liver cancer,pancreatic cancer,breast cancer,lung cancer and colon cancer.The expression of PVT1 was also significantly increased in gastric cancer,but its effect on the drug resistance of gastric cancer has not been reported.Therefore,in this study,the expression of PVT1 was detected by real-time quantitative PCR(qRT-PCR)in the drug resistant tissues and cells of gastric cancer.Then,the effect of drug resistance related proteins in gastric cancer drug resistant cells was observed by modulating the expression of PVT1.CSO was used to treat the drug resistant cell lines of gastric cancer,and to observe the expression changes of PVT1.Finally,the expression of gastric cancer resistance related proteins was studied after regulation of PVT1 expression in drug resistant gastric cancer cells and treatment of CSO.This study illustrated the role of PVT1 in the drug resistance of gastric cancer and the molecular mechanism of its function.The results provided a basis for further research on the molecular mechanism of drug resistance in gastric cancer,and a theoretical basis for the treatment of gastric cancer drug resistance.The paper was divided into four sections:1.Effect of IncRNA-PVTl on drug resistance of gastric cancer cellsObjective:To analyze the expression of PVT1 in tumor tissues of drug resistant patients with gastric cancer and drug resistant cell lines,and observe the effect of PVT1 overexpression or downregulation on the drug resistance of gastric cancer cells in vitro.Methods:Tumor tissue was isolated from gastric cancer patients with cisplatin sensitivity or cisplatin resistance,the expression of PVT1 in two groups was detected by qRT-PCR.The expression of PVT1 in gastric cancer cell lines BGC823/DDP and SGC7901/DDP were also detected by qRT-PCR.After BGC823/DDP and SGC7901/DDP cell lines were transfected with si-PVT1,the expression of PVT1 was measured by qRT-PCR.The cell lines were treated with lug/ml cisplatin for 24h,36h and 48h respectively,and then cell survival rate was detected by CCK-8.After BGC823 and SGC7901 were transfected with the expression vector LV-PVT1-GFP,the expression of PVT1 was measured by qRT-PCR.After the cell lines were treated with lug/ml cisplatin for 48h,cell apoptosis was measured by flow cytometry with Annexin V-PI staining.Results:The expression of PVT1 in tumor tissues from drug resistant patients with gastric cancer and drug resistant cell lines(BGC823/DDP and SGC7901/DDP)was significantly increased.The expression of PVT1 in gastric cancer resistant cell line was significantly reduced by PVT1 interference sequence.After cells were treated with si-PVT1 and cisplatin,the survival rate of gastric cancer resistant cells was significantly decreased and the apoptosis rate was increased.After gastric cancer cell line GC823 and SGC7901 were transfected with LV-PVT1-GFP and treated with cisplatin,the expression of PVT1 in cells was significantly increased,and the apoptosis rate was decreased.Conclusion:The expression of PVT1 was increased in tumor tissues in drug resistant patients with gastric cancer and drug resistant cell lines.PVT1 overexpression caused the increased resistance to cisplatin in human gastric cancer cell line.Down-regulation of PVT1 caused the high sensitivity to cisplatin in human gastric cancer cell line.2.Molecular mechanism of PVT1 in increasing drug resistance of gastric cancer cellsObjective:To investigate the expression of multidrug resistance associated molecules MDR1,MRP1,mTOR and HIF-la in the gastric cancer cells treated with cisplatin by up-regulation of PVT 1 in vitro,and explore the molecular mechanism of PVT 1 promoting drug resistance of gastric cancer cells.Methods:We constructed PVT1 expression vector LV-PVT1-GFP and transfected it into gastric cancer cells BGC823 and SGC7901.48 h after treatment with cisplatin,the mRNA expression of MDR-1,MRP,mTOR and HIF-1 a were detected with qRT-PCR.MDR1 and MRP1 protein expression were detected by Western blotting.Results:PVT1 over-expression caused significantly increased expression of MDR1 and MRP1 in gastric cancer cell line treated with cisplatin,while the expression of mTOR and HIF-la was not regulated by PVT1.Conclusion:PVT1 could regulate the expression of MDR1 and MRP1 to control the drug resistance of gastric cancer cells to cisplatin.mTOR and HIF-1? were involved in the mechanism of drug resistance of gastric cancer not regulated by PVT1.3.Effect of CSO on gastric cancer cell resistanceObjective:To investigate the effect of CSO on gastric cancer cell resistance by treating gastric cancer cell lines BGC823/DDP and SGC7901/DDP with different concentrations of CSO.Methods:BGC823/DDP and SGC7901/DDP cell lines were treated with different concentration gradient of CSO(1,2.5,5 ?l/ml)for 24,36,48 h.CCK-8 is used to detect the cell viability.After 48h of CSO treatment,flow cytometry is used to detect cell apoptosis with Annexin V-PI staining.As before,the mRNA expression of MDR1 and MRP1 was detected by qRT-PCR.MDR1 and MRP1 protein expression was detected by western blotting.Results:Under the same time condition,cell viability was decreased significantly with the increased CSO concentration treatment.The apoptosis rate was significantly increased with the increased CSO concentration treatment.Cell viability decreased significantly by the same concentration of CSO treatment,along with the increase of processing time.The apoptosis rate was significantly increased by the same concentration of CSO treatment,along with the increase of processing time.Then we tested the expression of MDR1 and MRP1 of gastric cancer cells BGC823/DDP and SGC7901/DDP under different concentrations of CSO treatment for 24 h and 48 h.The results showed that the expression of MDR1 and MRP1 were significantly decreased with the increase of the CSO concentrations,regardless of the treatment time 24 h or 48 h,which was dose dependent.Conclusion:CSO treatment of resistant gastric cancer cell lines promoted cell apoptosis and inhibited its viability,and decreased the expression of MDR1 and MRP1.CSO treatment significantly decreased the drug resistance of gastric cancer cells to cisplatin.4.Study on the molecular mechanism of CSO suppressed drug resistance in gastric cancer cellsObjective:To further investigate the expression of multidrug resistance associated molecules MDR1,MRP1 in the resistant gastric cancer cells by up-regulation of PVT1 in vitro,and to further explore the molecular mechanism drug resistance of gastric cancer cells.Methods:BGC823/DDP and SGC7901/DDP were treated with different concentration gradient of CSO for 24h.The expression of PVT1 was detected with qRT-PCR.The PVT1 overexpression adenovirus vectors were transfected into BGC823/DDP and SGC7901/DDP with 2.5ul/ml CSO treatment.The apoptosis was detected by flow cytometry.The mRNA expression of MDR1 and MRP1 were detected by qRT-PCR,and the protein expression of MDR1 and MRP1 were detected by western blotting.Results:The expression of PVT1 was significantly decreased with the increase of the concentration of CSO.After BGC823/DDP and SGC7901/DDP were transfected PVT1 overexpression adenovirus vectors with 2.5ul/ml CSO treatment,the apoptosis were significantly inhibited,while expression of MDR1 and MRP1 was significantly up-regulated.Conclusion:CSO inhibited the expression of PVT1 leading to the reduced expression of multidrug resistance related protein MDR1 and MRP1,and then inhibited the drug resistance of gastric cancer cells.To sum up,this study explored the effect of PVT1 on the drug resistance of gastric cancer.We found the significant increase of PVT1 expression in the gastric cancer drug resistant tissues and cells,revealed the mechanism of PVT1 in the drug resistance of gastric cancer.And it was found that the mechanism of PVT1 regulating the drug resistance of gastric cancer cells was achieved by regulating the expression of MDR1 and MRP1.CSO treatment significantly reduced the drug resistance of gastric cancer cells.The specific molecular mechanism was to reduce the expression of multidrug resistance related molecules MDR1 and MRP1 by inhibiting the expression of PVT 1,which can reduce the drug resistance of gastric cancer cells.