Function And Mechanism Of MiR-21in Chemoresistance Of Gastric Cancer Cell

Background Gastric cancer is one of the most commonly diagnosed cancer worldwideand ranks second in global cancer mortality. Surgery is the primary treatment for theearly stage of gastric cancer, but most patients are either diagnosed in an advanced stage,or develop a relapse after apparently curative operation. For these patients,chemotherapy is a main treatment option. Unfortunately, many gastric cancer patientsexperience a recurrence of cancer after initial therapy and become refractory tochemotherapy following treatment. Thus, the acquisition of chemo-resistance is a majorclinical obstacle to the successful treatment of gastric cancer. The process ofchemo-resistance appears to be multifactorial and includes changes in drug transportleading to decreasing drug accumulation and increasing drug detoxification, changes inDNA repair and damage and/or alterations in the apoptotic cell death pathways.However, the mechanisms underlying chemo-resistance have not been fullycharacterized.MicroRNAs (miRNAs) are a class of22-nucleotide, non-coding RNA molecules thatnegatively regulate the expression of target genes post-transcriptionally by binding tothe3′untranslated region (3′UTR) of mRNA. It has been demonstrated that miRNAs mediate diverse physiological function, such as cell differentiation, proliferation,apoptosis, and metabolism. Deviations from normal pattern of expression may causehuman diseases. In particular, it has been shown that miRNAs are aberrantly expressedin a variety of human cancers compared with their normal counterparts, suggesting thatmiRNAs contribute to oncogenesis through modulation of key cellular processes byfunctioning as tumor suppressors or oncogenes. Furthermore, miRNAs also playimportant roles in the drug resistance of different tumors. Indeed, recent evidencesindicate that miRNAs can influence tumor cell response to chemotherapy in differentmalignancies.MicroRNA-21(miR-21) has been shown to be implicated in multiple malignancyrelated processes including cell proliferation, apoptosis, invasion, and metastasis. ThismiRNA is frequently over-expressed in a wide variety of cancers. Previous studies havealso shown that miR-21was among the top miRNAs with increased expression ingastric cancer tissue. The oncogenic properties of miR-21are further supported byfunctional studies showing that inhibition of miR-21expression reduced proliferation orgenerated a proapoptotic response of several cancer cells. Recent studies also showed itsmultiple roles in chemoresistance in other cancers.However, whether miR-21can lead to drug resistance in gastric cancer remainsunknown. Considering the critical role of miR-21in a variety of cancers includinggastric cancer and chemo-resistance in other cancers, we hypothesized that theacquisition of chemo-resistance by cancer cells may also be modulated via the changesin miR-21levels. This novel miR-21/PTEN/PI3K/Akt signaling pathway in gastriccancer cells may provide drug targets for the sensitivity of tumor cells and could beapplied to treat chemotherapy resistance in gastric cancer patients. This study containsthree parts. Part l Expression of miR-21and relationship with cisplatin resistance in gastriccancerObjective Investigate the expression of miR-21and relationship with cisplatinresistance in gastric cancer cells. Methods Real-time PCR was used to detectexpression of miR-21in gastric cancer cells. The SGC7901and SGC7901/DDP cellswere transfected with the mimics or inhibitors of miR-21or negative control RNA (NC)by lipofectamine2000. MTT was used to analyze drug sensitivity. Apoptosis analysisand cell cycle were measured by fluorescene activated cell sorter. Results Theexpression of miR-21was an average of (7.65±1.197)-fold higher in SGC7901/DDPcells than in SGC7901cells and there is a significant difference between the two groups(P<0.01). SGC7901cells transfection with mimics of miR-21showed that the IC50(1.085±0.0639μg/mL) was more than the NC (0.487±0.018μg/mL) and veicle group(0.476±0.014μg/mL), apoptosis rate was lower than the NC and veicle group, cell ratioin S phase was higer than the NC and veicle group, and there was a significantdifference between them (P<0.05). Conclusion The expression of miR-21wasupregulated in SGC7901/DDP cells. Transfection with miR-21inhibitors can reversecisplatin resistance in SGC7901/DDP cells. Transfection with miR-21mimics can leadto cisplatin resistance in SGC7901cells.Part2Expression of miR-21and relationship with Adriamycin resistance in gastriccancerObjective Investigate the expression of miR-21and relationship with Adriamycin(ADM) resistance in gastric cancer cells. Methods Real-time PCR was used to detectexpression of miR-21in gastric cancer cells treated with ADM at IC50. The SGC7901and MGC803cells were transfected with the mimics of miR-21or negative controlRNA (NC) by lipofectamine2000. MTT was used to analyze drug sensitivity. Apoptosis analysis measured by fluorescene activated cell sorter. Results The expression ofmiR-21was an average of (2.941±0.225)-fold and (2.164±0.167)-fold higher inSGC7901and MGC803cells treated with ADM at IC50than untreated cells and there isa significant difference between the two groups (P<0.01). SGC7901cells transfectionwith miR-21mimics showed that the IC50(0.942±0.027μg/mL) was more than the NCgroup (0.335±0.029μg/mL), apoptosis rate was lower than the NC group (P<0.05).MGC803cells transfection with miR-21mimics showed that the IC50(1.430±0.124μg/mL) was more than the NC group (0.481±0.034μg/mL)(P<0.05).Conclusion The expression of miR-21was upregulated in SGC7901and MGC803cellstreated with IC50ADM. Transfection with miR-21mimics can lead to ADM resistancein SGC7901and MGC803.Part3the mechanism of miR-21in chemoresistance of gastric cancer cellObjective Investigate the mechanism of miR-21in chemoresistance of gastric cancercell. Methods The expresstion of PTEN mRNA was measured by real-time PCR. Theexpresstion of PTEN, p-Akt, Akt protein was measured by western-blot. The SGC7901and SGC7901/DDP cells were transfected with the mimics or inhibitors of miR-21ornegative control RNA (NC) by lipofectamine2000. MTT was used to analyze drugsensitivity. Results The expression of PTEN mRNA and protein was downreagulated inSGC7901/DDP cells. Overexpression of miR-21decreased the PTEN mRNAexpression and PTEN protein level in SGC7901cells, whereas knockdown of miR-21increased the PTEN mRNA expression and PTEN protein level (P<0.01) inSGC7901/DDP cells. SGC7901cells that were transfected with miR-21increased Aktphosphorylation (P<0.001). Knockdown of miR-21in the SGC7901/DDP cellsdecreased Akt phosphorylation (P<0.01). miR-21transfected SGC7901cells weretreated with serial dilutions of cisplatin and/or PI3K inhibitor LY294002. LY294002abrogated miR-21–activated Akt and significantly inhibited miR-21–induced cell survival and cisplatin resistance. Conclusion The expression of PTEN wasdownregulated in SGC7901/DDP cells. Overexpression of miR-21can activate thePI3K/Akt pathway by decreasing the PTEN protein level.