| Objective: 1.Doxorubicin is currently one of the most widely used and effective drugs in the treatment of breast cancer as an anthracycline chemotherapeutic drug.Drug resistance of breast cancer is the main factor limiting the ideal therapeutic effect of doxorubicin.Reversing the resistance of breast cancer to doxorubicin is the key to improvethe therapeutic effect,but the underlying mechanism of doxorubicinresistance is still unclear.Recent studies have shown that abnormallyexpressed microRNAs are involved in the formation of drug resistance in tumors.2.MicroRNAs(miRNAs)are highly conserved endogenous,non-coding,single-stranded RNA molecules with a length of 19-25 base pairs.They regulate protein expression by inhibiting gene translationor promoting gene degradation.We searched and downloaded the gene expression profiling data and microRNA expression profiling data related to neoadjuvant chemotherapy for breast cancer(an anthracycline-containing chemotherapy regimen)in the public biomedical database to conduct bioinformatics analysis to construct miRNA-mRNA regulatory network about breast cancer anthracycline sensitivity.Through literature review,miR-520 b and IGF-1R were identified as further research goals.Studies have shown that miRNA is involved in the occurrence ofmany tumors,among which the abnormal expression of miR-520 b is associated with gastric cancer,liver cancer,bladder cancer,breast cancer and so on.In particular,in breast cancer,the expression of miR-520 b was down-regulated in drug-resistant breast cancer cells,and there was a high correlation between miR-520 b and IGF-1R in the sensitivity of doxorubicin in the treatment of breast cancer.In order to elucidate the mechanism of drug resistance of miR-520 b and IGF-1R in drug-resistant breast cancercells and how to reverse the drug resistance of drug-resistant breastcancer cells to anthracycline chemotherapeutics,we investigated the correlation of them and the effect of the dynamic changes of expression of miR-520 b and IGF-1R on drug-resistant breast cancer cells.Methods: Changes of related genes in breast cancer cell lines resistant to anthracycline chemotherapeutics were analyzed by biological software.RT-PCR was used to detect the expression of IGF-1R in breast cancer cell lines and drug-resistant breast cancer cell lines,the expression levels of miR-520 b and IGF-1R in breast cancer tissues before neoadjuvant chemotherapy,and the activity of apoptosis-related protease.The sensitivity of breast cancer cells overexpressing IGF-1R to doxorubicin was detected by MTT,and the expression of pathway protein was detected by WB protein level.The effects of over-expression of miR-520 b on breast cancer cells treated with doxorubicin were investigated by cell proliferation assay.Luciferase assay wasused to verify whether IGF-1R is a direct target gene of miR-520 b.Results:1.Retrieving and downloading gene expression data and microRNA expression data related to neoadjuvant chemotherapy for breast cancer in public biomedical database,analyzing differentially expressed mRNA and differentiallyexpressed miRNA in chemotherapy sensitive group and resistant group in R,and constructing miRNA-mRNA regulatory network associated with drug chemosensitivity.Through literature review,miR-520 b and IGF-1R were identified as further research goals.2.Expression of miR-520 b in tissue samples of breast cancer patients before neoadjuvant chemotherapy(1)The relationship between the expression of miR-520 b and the efficacy of neoadjuvant chemotherapy:The expression of miR-520 b was positively correlated with the efficacy of neoadjuvant chemotherapy in breast cancer patients(P<0.05),indicating that the higher the expression of miR-520 b,the better the efficacy.(2)By PCR detecting the expression of miR-520 b in tumor tissue samples before neoadjuvant chemotherapy.The results showed that the expression of miR-520 b of PCR group was significantly higher than that of non-PCR group.3.Expression of miR-520 b in adriamycin resistant strains MCF-7/ADR and MDA-MB-231/ADRThe results showed that compared with breast cancer cell linesMCF-7 and MDA-MB-231,the expression of miR-520 b in adriamycin-resistant strains MCF-7/ADR and MDA-MB-231/ADR decreased significantly(P<0.01).4.Expression of miR-520 b in co-transfected miR-520 b mimics resistant strainsThe results showed that the expression of miR-520 b in MCF-7/ADR and MDA-MB-231/ADR was significantly higher than that in NC group(P<0.01).5.Overexpression of miR-520 b enhances the sensitivity of breast cancer cells to doxorubicinThe results showed that in breast cancer drug-resistant cells,compared withNC transfection group,the survival rate of breast cancer cells decreased significantly with the increase of drug concentration,and the IC50 value decreased significantly(P<0.01),suggesting that over-expression of miR-520 b increased the sensitivity of breast cancer cells to doxorubicin.6.Effect of over-expression of miR-520 b on proliferation of breast cancer cells treated with doxorubicinThe results showed that there was no significant difference in the number of cells between NC+DOX group and NC+FBS group.The number of cells decreased and cell proliferation was inhibited in the group of miR-520b+DOX and the group of miR-520b+PBS.Compared with NC+DOX group,the number of breast cancer cells decreased significantly and the proliferation of breast cancer cells was significantly inhibited in the miR-520b+DOX group,indicating that overexpression of miR-520 b significantly inhibited the proliferation of breast cancer cells treated with chemotherapy drug doxorubicin.7.Effect of overexpression of miR-520 b on apoptosis of breast cancer cells induced by doxorubicin(1)The effect of over-expression of miR-520 b on apoptosis of drug-resistant breast cancer cells induced by doxorubicin: The results suggest that over-expression of miR-520 b in breast cancer resistant strains MCF-7/ADR and MDA-MB-231/ADR promotes apoptosis of breast cancer resistant strains induced by doxorubicin.(2)Effect of over-expression of miR-520 b on apoptosis-related caspase-3/7 and caspase-9 activity of doxorubicin-induced breast cancer cell lines:The results showed that over-expression of miR-520 b might promote apoptosis of drug-resistant breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR induced by doxorubicin through caspase pathway.(3)Overexpression of apoptosis-related casepase-3 protein in drugresistant breast cancer cell line miR-520b: The results showed that overexpression of miR-520 b could significantly up-regulate the expression of apoptosis-related caspase-3 in drug-resistant breast cancer cells.8.Effect of over-expression of miR-520 b on apoptosis-related genes in breast cancer drug-resistant cellsDetecting the transcription levels of apoptosis-related genes andsurvivalrelated genes by RT-PCR.After transfection of MCF-7/ADRwith miR-520 b mimics,the expression of Bax gene and survivin gene increased significantly(P<0.01).9.Effect of miR-520 b on apoptosis of drug-resistant cells through PI3K/AKT signaling pathway activityThe results showed that PI3 CA and P-AKT protein expression decreased in MCF-7/ADR-miR-520 b group and MDA-MB-231/ADR-miR-520 b group,while t-AKT protein expression showed no significant difference,and the expression of Bcl-2 decreased.The detection of Bax was consistent with that of RT-PCR.10.Expression of IGF-1R in breast cancer cell linesThe expression of IGF-1R in drug-resistant cell lines MCF-7/ADR and MDA-MB-231/ADR was high.The expression of IGF-1R intwo breast cancer drug-resistant cell lines with over-expression of miR-520 b was low.11.The correlation between the expression of miR-520 b and IGF-1R in breast cancer(1)The relationship between the expression of IGF-1R and the efficacy of neoadjuvant chemotherapy: The expression of IGF-1R was negatively correlated with the efficacy of neoadjuvant chemotherapy in breast cancer patients(P<0.05),indicating that the lower the expression of IGF-1R,the better theefficacy of chemotherapy.(2)The expression of IGF-1R was different between chemosensitive patients and drug-resistant patients.(3)Correlation analysis of expression of miR-520 b and IGF-1R:Theexpression of miR-520 b was negatively correlated with that of IGF-1R.The expression of miR-520 b was significantly down-regulated inbreast cancer tissues,and the expression of IGF-1R was increased.12.Effect of IGF-1R on drug resistance of breast cancer cellsOverexpression of IGF-1R plasmid was constructed and transfected into breast cancer cell lines MCF-7 and MDA-MB-231.The sensitivity of IGF-1R plasmid to doxorubicin was decreased.After overexpression of IGF-1R,the apoptotic rates of MCF-7 and MDA-MB-231 breast cancer cells in the doxorubicin drug group decreased significantly,suggesting that IGF-1R can play a role in inhibiting apoptosis of breast cancer cells.MDA-MB-231/ADR drug-resistant cell lineswith si-IGF-1R expression increased the sensitivity to doxorubicin and the IC50 value was lower than that of the control group.13.IGF-1R as a downstream target gene of miR-520 b is directly involved in the effect of breast cancer resistance.(1)These results suggest that IGF-1R and miR-520 b are involved in the effect of breast cancer drug resistance.miR-520 b can effectively regulate the susceptibility of IGF-1R to breast cancer cell resistance.Moreover,miR-520 b can promote the activity of caspase-3/7 andcaspase-9 related to apoptosis through IGF-1R gene,the expression of caspase-3/7 and caspase-9 related to apoptosis is increased in the microRNA-520 b mimimics resistant group.(2)Overexpression of IGF-1R under the regulation of miR-520 b canreduce the level of Bax protein,inhibit the apoptosis of breast cancer drug-resistantcells,increase the level of P13 CA,p-AKT,Bcl-2 protein,and promote the proliferation of breast cancer drug-resistant cells.14.Verification of IGF-1R as a direct target gene of miR-520b(1)Prediction of binding sites between miR-520 b and IGF-1R by target gene prediction software.(2)Using luciferase reporter gene assay,we found that up-regulation of the expression of miR-520 b in breast cancer cell MCF-7 couldsignificantly inhibit the expression of luciferase reporter gene containing IGF-1R 3’UTR.Conclusions: miR-520 b participates in the regulation of resistance of breast cancer cells to doxorubicin,a new adjuvant chemotherapeutic drug,and it is likely that miR-520 b can reverse the resistance of breast cancer cells to doxorubicin by down-regulating the expression of IGF-1R,that is,to enhance the sensitivity of breast cancer cells to doxorubicin. |
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