LncRNA MALAT1 Regulates The Resistance Of Breast Cancer Cells To Paclitaxel Via The MiR-497-5p/SHOC2 Axis

Breast cancer（BC）is a process in which many carcinogenic factors act on the epithelial cells of the breast and produce uncontrolled proliferation of cells.The early symptoms of breast cancer are generally breast lumps,axillary lymph node enlargement and other manifestations.In the late stage,distant metastasis may occur due to the dissemination of malignant tumor cells,which seriously threatens the life and health of patients.Breast cancer,known as"pink killer",is the malignant tumor with the highest incidence rate among women,and has a very high mortality rate.During the treatment of breast cancer,resistance to chemotherapy drugs is often encountered,which seriously affects the prognosis of patients.The resistance to paclitaxel is a common clinical issue,and it is a complex process that may involve multiple factors.In the past few decades,while constantly trying to understand the complexity of drug resistance in breast cancer,researchers have also constantly explored and developed new research ideas.The results showed that the study of paclitaxel resistance in breast cancer based on molecular mechanism would help to fundamentally solve the problem.Metastasis associated lung adenocarcinoma transcript 1（MALAT1）is a highly abundant long chain non-coding RNA,also known as NEAT2,which has been widely studied in the field of cancer.Micro RNA（miRNA）is a kind of non-coding RNA with a length of about 20-25 nucleotides.Among them,miR-497-5p,as a member of the micro RNA family,has low expression in liver cancer,gastric cancer,bladder cancer cancer,colon cancer,breast cancer,leukemia,optic nerve mother cells and other malignant tumors.Some in vitro and in vivo experiments have clearly shown that miR-497-5p is a tumor suppressor factor that can inhibit the proliferation,invasion,and migration of various malignant tumor cells.SHOC2 is a member of the Scaffold protein family,which can positively regulate the Ras-Raf-ERK pathway in a variety of tumor cells and promote the occurrence and development of tumors.This study combined the effects of MALAT1,miR-497-5p and SHOC2 to explore the mechanism of paclitaxel resistance in breast cancer at the molecular level and the interaction between the three,hoping to provide new biological indicators for the study of paclitaxel resistance in breast cancer.Part One The role of lnc RNA MALAT1 in paclitaxel resistance in breast cancerObjective:To analyze the expression of lnc MALAT1 in paclitaxel resistant breast cancer cell lines and explore the role of MALAT1 in paclitaxel resistant breast cancer.Methods:1.Three kinds of breast cancer cells MCF-7,MDA-MB-231 and SKBR3,and human breast epithelial cell MCF-10A were treated with paclitaxel.Select well growing cell lines based on their growth status for further research.By gradually increasing the drug concentration of paclitaxel,we screened paclitaxel resistant cell lines that can stably pass through.We compared the cell viability and half inhibitory concentration(IC₅₀)of the two cell lines before and after paclitaxel treatment,and compared the difference in MALAT1 expression before and after paclitaxel treatment.2.The small interference MALAT1（si-MALAT1）and negative control MALAT1（NC-MALAT1）with the highest transfection rate were transfected into two drug resistant breast cancer cell lines through liposome transfection technology.The expression level of MALAT1 was detected to verify the transfection effect.3.CCK-8 technique was used to detect the cell viability and IC₅₀of drug-resistant breast cancer cell lines after MALAT1 knockdown.Transwell technique was used to detect the cell invasion of drug-resistant breast cancer cell lines after MALAT1 knockdown.Results:1.Among the four cell lines MCF-7 and MDA-MB-231 grew better under the action of paclitaxel and the expression level of MALAT1 was higher;Construct paclitaxel resistant cell lines MCF-7-PR and MDA-MB-231-PR by gradually increasing the concentration of paclitaxel.Compared with the same cell lines before paclitaxel treatment,the cell viability and IC₅₀of both drug resistant cell lines were significantly upregulated,and the expression level of MALAT1 was upregulated.2.Compared with cells transfected with NC-MALAT1,the expression of MALAT1 in cells transfected with si-MALAT1 was significantly downregulated.3.At the same concentration of paclitaxel,the IC₅₀of si-MALAT1transfected cells was lower than that of NC-MALAT1 transfected cells.The cell viability and invasiveness of the si-MALAT1 transfected group were significantly lower than those of NC-MALAT1 transfected group.Conclusions:MALAT1 was up-regulated in drug-resistant breast cancer cell lines.Knockdown of MALAT1 inhibits the cell viability and invasiveness of drug-resistant breast cancer cells.MALAT1 contributes to paclitaxel resistance of breast cancer.Part Two Expression of SHOC2 and miR-497-5p in clinical samples of breast cancerObjective: Analyze the expression of SHOC2 and miR-497-5p in clinical samples.Explore the role of SHOC2 and miR-497-5p in paclitaxel resistance in breast cancer.Methods:1.Collect the breast cancer tissue and adjacent tissue of 40 patients with primary breast cancer confirmed by pathology in our hospital.Before paclitaxel treatment,q RT-PCR was used to detect the expression of SHOC2 and miR-497-5p in breast cancer tissue and adjacent tissue to compare SHOC2 and miR-497-5p in breast cancer tissue and adjacent tissue,and to analyze the correlation between SHOC2 and miR-497-5p and MALAT1.2.After paclitaxel treatment,the treatment response was evaluated according to the evaluation criteria 1.1 for solid tumor response.Patients who achieved complete response,partial response or disease stability were classified as the paclitaxel sensitive group,while patients with disease progression were classified as the paclitaxel resistant group.The difference in the expression of SHOC2 and miR-497-5p between the two groups was compared.3.In order to verify the difference of SHOC2 expression between drug resistant group and sensitive group of breast cancer,GSE22513 data set from GEO was included for analysis.The data set contains the expression profile data of 19 breast cancer puncture biopsy samples from breast cancer patients.Compare the differences in SHOC2 expression between tissues from patients with pathological complete remission（p CR）and non p CR patients.4.In order to verify the effect of miR-497-5p on paclitaxel resistance in breast cancer,miR-497-5p mimic was transfected into MDA-MB-231-PR and MCF-7-PR cells to verify the transfection efficiency of miR-497-5p,and compare the cell viability of miR-497-5p simulant group and miR-497-5p negative control group.Results:1.Analysis of clinical samples of breast cancer before paclitaxel treatment showed that SHOC2 expression in breast cancer tissue was significantly up-regulated,while miR-497-5p expression was significantly down regulated compared with adjacent tissue.The expression of SHOC2 and miR-497-5p in breast cancer tissue was negatively correlated,and SHOC2 was proportional to the expression of MALAT1.2.After paclitaxel treatment,according to the evaluation of treatment response,compared with the paclitaxel sensitive group,the SHOC2 expression in the drug resistant group was significantly upregulated.The expression level of miR-497-5p in the paclitaxel sensitive group was higher than that in the paclitaxel resistant group.3.The analysis of the GSE22513 dataset confirmed that patients who did not achieve p CR had a higher SHOC2 compared to those who achieved p CR.4.When miR-497-5p mimic was transfected into paclitaxel resistant breast cancer cells,the expression of miR-497-5p was significantly up-regulated,and the cell viability was significantly inhibited.Conclusions: SHOC2 was significantly up-regulated and miR-497-5p was significantly down regulated in the paclitaxel resistant breast cancer patients.SHOC2 may contribute to paclitaxel resistance in breast cancer,and miR-497-5p inhibits paclitaxel resistance in breast cancer.Part Three MALAT1/miR-497-5p/SHOC2 axis regulates paclitaxel resistance in breast cancerObjective: MALAT1 and SHOC2 contribute to paclitaxel resistance in breast cancer,and miR-497-5p inhibits paclitaxel resistance in breast cancer.The expression of SHOC2 is negatively correlated with miR-497-5p,while the expression of SHOC2 is directly proportional to MALAT1.Therefore,we speculate that there is a targeted regulatory relationship between the three to regulate paclitaxel resistance in breast cancer.This part aims to explore the role of MALAT1/miR-497-5p/SHOC2 axis in paclitaxel resistance in breast cancer.Methods:1.Bioinformatics online database detects the potential binding sites among MALAT1,miR-497-5p and SHOC2.It was found that MALAT1 does not directly regulate SHOC2.MiR-497-5p shows a potential binding site with MALAT1,and miR-497-5p also shows a potential binding site with SHOC2.2.The targeting relationship between MALAT1 and miR-497-5p was verified by double Luciferase experiment.Detect the difference of miR-497-5p expression in paclitaxel resistant breast cancer cells before and after blocking MALAT1.To analyze the effect of MALAT1 on the expression of miR-497-5p in drug-resistant breast cancer cells.3.The targeting relationship between miR-497-5p and SHOC2 was verified by double Luciferase experiment,and the cell viability was observed after SHOC2 overexpression in breast cancer drug resistant cells with and without miR-497-5p.To analyze the effect of miR-497-5p on SHOC2 expression and paclitaxel resistance in breast cancer resistant cells.4.Build up/down regulation plasmids including [MALAT1 over expression plasmid（MALAT1 OE）,miR-497-5p analog particle（miR-497-5p-mimic）and down regulation SHOC2 plasmid（si SHOC2）] to detect the cell viability of breast cancer paclitaxel resistant cells and the impact of SHOC2 and downstream protein expression.Analyze the impact of MALAT1/miR-497-5p/SHOC2 axis on paclitaxel resistance of breast cancer cells.Results:1.Luciferase reporter gene analysis showed that miR-497-5p could bind to MALAT1.The expression of miR-497-5p in paclitaxel resistant breast cancer cells decreased significantly,but increased after blocking MALAT1.2.Luciferase reporter gene analysis showed that miR-497-5p could target SHOC2,and the overexpression of SHOC2 almost restored the decreased paclitaxel resistance of breast cancer paclitaxel resistant cells due to the upregulation of miR-497-5p.3.The up regulation of MALAT1 in breast cancer cells led to the increase of paclitaxel resistance.The further up regulation of miR-497-5p restored the sensitivity of the two cells to paclitaxel.The knockdown of SHOC2 partially eliminated the increased paclitaxel resistance due to the up regulation of MALAT1 in the two cell lines;The up regulation of MALAT1 increased the protein expression of SHOC2 and its subsequent downstream activating protein p ERK1/2,which was up regulated by miR-497-5p and decreased by SHOC2 knockdown.Conclusions:1.MALAT1 can down regulate the expression of miR-497-5p in breast cancer tissue and increase the purple shirt resistance of breast cancer.2.MiR-497-5p can down regulate SHOC2 to reduce paclitaxel resistance of breast cancer resistant cells.3.MALAT1 increases paclitaxel resistance of breast cancer cells depending on miR-497-5p/SHOC2 axis.