Effect of miR-181b on the biological characteristics and clinical drug resistance of small cell lung cancer by targeting gene ACE2

Objective To clarify the effect of miR-181b on the biological function of SCLC, to detect and verify its downstream target gene ACE2, and to explore the effect of clinical resistance on SCLC in order to nd new specic diagnostic markers and therapeutic targets. Methods 1. Collect blood samples from 30 SCLC patients and 30 normal persons in our department from 2017 to 2019 to detect the expression level of miR-181b; 2. Detect the expression level of miR-181b in SCLC cells by RT-PCR, and Screening of downstream target genes used by gene chip, verication with luciferase and Western Blot; 3. Collect the general data of 30 SCLC patients and 30 healthy people (control group) (cid:0) the patients were diagnosed by pathology and undergoing EC protocol in the Department of Thoracic Surgery and Oncology of our hospital to detect the expression level of mir-181b in different periods; 4. In the SCLC cell line and the SCLC mouse model constructed, different concentrations of EC chemotherapy and ACEI drugs were administered to detect the sensitivity of drug resistance and non-drug resistance. Results 1. The expression level of miR-181b in SCLC patients was lower than normal people; 2. The expression level of miR-181b in SCLC cell lines was lower than normal cells; ACE2 was veried as a downstream target of miR-181b by gene chip screening; 3. miR-181b is low-expressed in SCLC patients, rst-line chemotherapy can promote its recovery, but cannot repair to normal levels, and miR-181b has a certain effect on SCLC chemotherapy sensitivity; 4. miR-181b can enhance the drug sensitivity of SCLC drug-resistant cells, the application of ACEI should increase the risk of SCLC drug-resistant. Conclusion 1. miR-181b has a low expression level in SCLC, and it can directly target the gene ACE2 to affect the biological characteristics of SCLC; 2. miR-181b is lowly expressed in SCLC patients, and rst-line chemotherapy can promote it recovery, but cannot repair to normal levels, and ACEI drugs can increase the risk of SCLC drug-resistant.


Introduction
Small cell lung cancer (SCLC) is characterized by short tumor cell doubling time, early metastasis, and high recurrence. The incidence rate has been increasing in recent years. Chemotherapy is the main treatment for SCLC. Most patients are very sensitive to chemotherapy drugs, but they are prone to relapse after chemotherapy. At present, it is believed that chemotherapy resistance is the key factor that causes SCLC to relapse. The effectiveness of second-line chemotherapy in patients with relapsed SCLC depends mainly on the length of remission after rst-line chemotherapy. Patients with a remission period of less than 3 months are highly resistant (1,2). miRNA (micro RNA, small RNA) is a type of non-coding RNA about 22 nt long. It can regulate gene expression by binding to speci c mRNA or regulating protein translation process of speci c mRNA, and is widely involved in cell growth and development, differentiation, proliferation and a variety of important biological processes such as apoptosis and tumor formation (3,4). As a member of the mRNA family, miR-181b has been con rmed in recent years that it has important signi cance in the occurrence and development of malignant tumors, but there was little research on the biological characteristics of SCLC and clinical treatment resistance (5). Here, we study the expression level and biological characteristics of miR-181b in SCLC patients and cell lines, screen and clarify their downstream target genes, explore the effect of miR-181b downstream target genes on SCLC resistance, and hope to SCLC treatment helps.
Materials And Methods

General Information
The peripheral blood samples of 30 patients with SCLC con rmed by pathology from the Department of Thoracic Medicine and Oncology Department of Hebei Provincial People's Hospital from December 2017 to December 2019 were collected and tested for miR-181b expression levels. 4. Luciferase report analysis NCI-H446 cells were seeded in 24-well plates at a density of 40-50%. The next day, transfected HEK 293T cells pmirGLO-ACE-2-3′UTR wild type (Wt) or pmirGLO-ACE-2-3′UTR mutant (Mut) (genefarma) with miR-181b mimics or miR-NC. The cells were then incubated in 37% 5% CO 2 for 2 days. Transfected harvested cells and used a dual Lu-ciferase reporter system (Promega Corpo-ration, Madison, WI, USA) for luciferase report analysis. Renin luciferase was used as an internal control to correct differences in transfection and harvest e ciency.

Western blot analysis
Proteins were collected in cold RIPA buffer. Samples were collected and the protein concentration was measured (BCA protein A determination, Byotime, Haimen, China). The proteins were separated by SDS-PAGE and transferred to polyvinylidene uoride membranes (Millipore, Billerica, MA, USA). Seal the cell membrane in TBST-0.1% (0.1% Tween-20, Tris-base buffer) skim milk, and use primary antibodies (including ACE2, ELANE, IL17C, IRF6, MAML1, CYLD, ATF3, BCL2, CCND1, and β-actin (Cell Signaling Technology, Danvers, MA, USA) were overnight at 4°C. The next day, the membrane was washed three times with TBST-0.1% buffer. Then secondary antibodies (Microwell) Incubate the membrane. Use ECL reagent (Thermo Scienti c, Waltham, MA, USA) to display the signal on the membrane. 6. The effect of miR-181b on the biological function of SCLC cell line 1). Multiplication experiment The cloning ability of tumor cells Prepare a cell suspension in the logarithmic growth phase and count. Each experimental group was seeded with 400-1000 cells/well in a 6-well plate culture plate, and three replicate wells were set for each experimental group. Before the experiment was terminated, the cell clones were photographed under a uorescence microscope. After washing and xing, 500 μL of a 1:1 diluted crystal violet aqueous solution was added to each well to stain the cells for 1-5 minutes; ddH2O washed the cells several times and dried them. Digital cameras take pictures and organize them, and clone counts.

Cell cycle detection
Prepare a cell suspension, collect the cells in a 5 mL centrifuge tube, and set up three complex wells for each group; x by centrifugation, washing, re-centrifugation, 75% ethanol xation, centrifugation, washing, centrifugation, and de-xation solution. According to 40 × PI mother liquor (2 mg/mL): 100 × RNase mother liquor (10 mg/mL): 1 × D-Hanks = 25: 10: 1000. Perform cell staining according to the ratio: add a certain volume of cell staining solution (0.6-1 mL) to resuspend according to the amount of cells, so that the cell pass rate is 300~800 Cell/s when it is on the machine. On-board testing and data analysis.

EDU detection
According to the instructions of the EDU kit (C10310 Ruibo), by detecting EDU in ltrating during DNA replication, new proliferating cells can be accurately detected with the help of uorescence detection tools, and the percentage of cells in S phase can be quickly counted to analyze the cell proliferation.
2  as the cell expansion medium, and cultured at 37°C and 5% CO 2 cells Cultivation in the box. The fresh medium was replaced in 48-hour cycles. Select the cells with good growth conditions and quickly ll the bottom of the culture ask. After centrifuging for 5min (1500rpm), slowly discard the supernatant along the direction of precipitation, and then add 1ml of the pre-prepared cell cryopreservation solution (medium: Fetal bovine serum: DMSO=5:4:1), and then repeatedly pipetting with a 1ml pipette to resuspend the pelleted cells. After being placed at 4℃ for 20min, transfer to a -20℃ refrigerator for 2h, then transfer to an ultra-low temperature refrigerator (-80℃), and store it for 48h. If it is to be stored for a long time, it must be transferred to a liquid nitrogen tank. According to the cell survival rate of each drug concentration, make a logarithmic curve to obtain the drug concentration (IC50) at 50% cell survival rate; and calculate the drug resistance index, drug resistance index = IC50 (H446 resistance) / IC50 (H446).

Use of ACEI drugs
Captopril was selected as miR-181b downstream target gene ACE2 inhibitor for sensitivity detection of drug-resistant and non-drug-resistant SCLC cells (same steps as 9).

Statistical analysis
The statistical analysis of this experimental data uses SPSS 21.0 for Windows software, and the results are expressed as mean ± standard deviation. The t test is used for comparison between two groups, and the analysis of variance is used for comparison between multiple groups, p<0.05. Indicates that the difference is statistically signi cant.

Results
1. miR-181b shows low expression abundance in NCI-H446 cell line Using real-time quantitative qPCR method to detect the expression level of miR-181b in NCI-H446 cell line was signi cantly lower than the control group (P <0.01), the veri cation results are shown in Figure 1.

MiR-181b overexpression can promote the proliferation ability of NCI-H446 cell line
The results of cell clone detection showed that overexpression of miR-181b can induce NCI-H446 cells to be subcultured, and the clone generation of NCI-H446 cells increased from before expression (259 ± 5 mean ± standard deviation) to overexpression (334 ± 26 mean ± standard deviation), the difference between the two is statistically signi cant. As can be seen from Figure  The results showed that the overexpression of miR-181b could increase the proportion of two cells in G1 phase, but there was no signi cant difference between the two types. EDU results showed that the proliferation rate of NCI-H446 cells in the miR-181b high expression group was signi cantly higher than that of the control Group (P<0.05) (Figure 2(C)).

miR-181b can inhibit the migration ability of NCI-H446 cell line
The scratch test showed that miR-181b can inhibit the migration ability of NCI-H446 cells (P<0.01). Page 01). (Figure 3(A)) and Transwell show that NCI-H446 cells have weak migration ability before and after miR-181b overexpression, see Figure 3(B), we obtained the same results in the invasion test, see Figure   3(C).

Gene chip targeting of miR-181b downstream regulatory genes
Based on the IPA (Integrated Online Integrated Analysis Software www.ingenuity.com) regulatory effect analysis, we found that the regulators ACE2, ELANE, IL17C, IRF6, MAML1, miR-155-5p (miRNAs w/seed UAAUGCU), etc. passed ATF3, BCL2L11, CCND1, CCNE2, CDKN1A and other genes have an inhibitory effect on Angiogenesis, Binding of tumor cell lines, Migration of tumor cell lines. (Figure 4). The effect correlation of the miR-181b-ACE2-ATF3 pathway can be obtained from this.
Overexpression of ACE2 in turn promotes the expression of miR-181b ( Figure 5 right).

Western Blot veri cation of downstream proteins
We screened for several target protein genes that changed most signi cantly in downstream target regions. We know that the proteins regulated by the target genes include ACE2, ELANE, CYLD, IRF6, MAML1, CCND1, PPARA ( Figure 6). They affect the cell growth cycle, biological characteristics and vascular growth-related factors.

Animal model veri cation
We divided the mice into three groups: blank control group (normal mouse), negative control group (H446 cells) and experimental group (mir-181b overexpressed H446 cells), we named it as negative control in Figure 7 Group "1" and experimental group "2". The tumor growth of the negative control group was signi cantly faster than that of the experimental group ( Figure 7A), the tumor mass of the former was signi cantly higher than that of the control group ( Figure 7A) and the latter (12.3±2.6gVS5.6±1.6gp=0.0023), and the blank control group It is empty by examining the biological characteristics of tumor cells EDU ( Figure 7C) and cell proliferation experiments ( Figure 7D). It can be seen that the negative control group is signi cantly better than the experimental group in terms of cell proliferation and proliferation. Luciferase-labeled ACE2 was overexpressed at different concentrations (1fold, 10-fold, 100-fold) of miR-181b, and then detected by RT-PCR technology ( Figure 7B). The results showed that ACE2 increased signi cantly after overexpression of miR-181b (p=0.021).
8. The clinical resistance of miR-181b and its target genes to SCLC

The expression level of miR-181b in healthy people and SCLC patients after chemotherapy
Comparing the content of miR-181b in the blood samples of each group, miR-181b in the peripheral blood of untreated SCLC patients was signi cantly reduced compared with the normal control group, with a statistically signi cant difference (P<0.01) (as shown in Figure 8A), but after EC treatment, its expression level has increased, but it is still lower than the normal content (P <0.01). (See Table 2) 8.2 miR-181b expression level in SCLC patients with different chemotherapy cycles After 1 cycle of chemotherapy, the expression level of miR-181b increased, but was lower than the normal level (P <0.01) (as shown in Figure 8B); after 6 times of chemotherapy, the content of each group showed a signi cant increase and decrease, including 3 4 There is a peak, and there is a signi cant difference between the groups before and after the comparison, and the content is increased (P <0.01). There is a statistical difference between groups 1 to 4 (P <0.01), indicating that the content is increasing with the progress of chemotherapy, but it has a downward trend since group 5, and there are obvious statistics between 5, 6 and 3, 4 Academic difference (P <0.01) (as shown in Fig8B). As the cycle of chemotherapy increases, its content may indicate that SCLC is resistant. (Table 3)  Table 4).

Sensitivity and resistance of SCLC cells to chemotherapy drugs after miR-181b transfection
The H446 cell line was over-expressed with miR-181b by lentivirus transfection, there was no signi cant difference between the two groups (P = 0.3741>0.05); the drug-resistant group had statistically signi cant difference before and after over-expression of miR-181b (P =0.01047<0.05); In addition, the overexpression of miR-181b in the drug-resistant group was statistically different from that in the sensitive group (P =0.00912<0.01VS P =0.0127<0.05) (Figure 9). After overexpression of miR-181b in the H446 drug-resistant cell line, the IC50 value of chemotherapeutic drugs decreased signi cantly (412.28±12.71 vs 269.55±10.21). (Table 5)

Sensitivity detection of ACEI drugs to resistant and non-resistant SCLC cells
In SCLC cells in drug-resistant and non-drug-resistant groups, different results appeared after the addition of ACEI drugs before and after chemotherapy. For the drug-resistant group, the IC50 value of the drug after the addition of ACEI was higher than that before chemotherapy (p=0.0042), while for the non-drug group, the IC50 value of the drug was higher than that before chemotherapy (p =0.023), there is a signi cant statistical difference between the two groups (see Table 6); and its sensitivity can be seen that both groups showed a decreasing trend after applying ACEI ( Figure 10).  Figure 11). To show the activity of SCLC cells in each group, Ki-67 immunohistochemical staining was performed (see Figure 12). Ki-67 was 80% in the ACEI group and 65% in the non-group.
Discussion miR-181b is down-regulated in human non-small cell lung cancer, glioma, prostate cancer, pancreatic cancer, gastric cancer, and myeloid leukemia tissues, while up-regulated in liver cancer, thyroid and breast cancer, pancreatic cancer, and head and neck tumor tissues, can be used as a biomarker for diagnosis and detection of tumors (6)(7)(8). These results suggest that the function of miR-181b may be unique, depending on the tumor type and cellular environment. Previous studies have shown that miR-181b is an important link between in ammation and malignant tumors. This is due to the transient expression of miR-181b to induce epigenetic switches, which can inhibit miR-181b target CYLD, thereby regulating NF-κB activity. The role. Therefore, miR-181b is part of the positive feedback loop for epigenetic conversion between in ammation and cancer, as shown in Fig. 13. Its target genes are Bcl-2, TIMP3, CYLD and CBX7, etc., which in turn affect the development and invasion of related tumors (9, 10).
The incidence of lung cancer ranks rst among all cancers, and the mortality rate is also the highest. With the advent of targeted drugs, the treatment of NSCLC has made great progress, but the treatment of small cell lung cancer is still at a standstill. At present, the classic treatment plan of platinum plus etoposide combined with radiotherapy, although it has a certain effect on SCLC, is very prone to drug resistance, so it is urgent to study new treatment methods. At present, there are increasing researches on miRNA and tumor, and it is con rmed that there is a clear relationship between tumor and miRNA disorder. In this study, the real-time quantitative PCR method was used to detect the expression level of miR-181b in the peripheral blood of patients with SCLC. The results showed that the low expression level of miR-181b in the occurrence of SCLC may play a role in tumor suppressor genes, which is also the same as Cao et al (12) in the NSCLC study and Cinegaglia NC et al (13) in the study of lung adenocarcinoma on the results of miR-181b on other lung cancer types have similar conclusions.
The miR-181b and ACE genes are not on the same chromosome, and there is a mutual regulation relationship between them. A large number of studies have shown that angiotensin-converting enzyme is involved in the pathogenesis of tumors. For example, Han et al. (14)found that upregulation of ACE in mice increased the risk of laryngeal cancer. Peddiredy, Vidyullatha and others (15) found that in India, the diversity of ACE and eNOS genes increased the risk of non-small cell lung cancer, and the expression of ACE in lung cancer tissues was lower than that of tissue adjacent to cancer tissues. It is just like Blánaid M Hicks and others (16) found that the use of ACEIs is associated with an increased risk of lung cancer, and this correlation is particularly signi cant in people who have used ACEIs for more than 5 years. In short, angiotensin-converting enzyme (ACE) is expressed in a variety of tumors, which can affect tumor proliferation, cell migration, angiogenesis and metastasis. However, after the application of ACE inhibitors, some tumors were suppressed, but the speci c mechanism of action still needs further study.
The miR-181b targeting ACE gene also increases the risk of small cell lung cancer.
The experiment also found that the expression level of miR-181b in plasma of SCLC patients changed with the extension of the treatment period. By detecting the level of miR-181b in peripheral blood after different chemotherapy cycles, it was found that in the 4 cycles before chemotherapy, the miR-181b level continued to rise with the increase in the number of chemotherapy, and reached a peak during 3 and 4 cycles, but its content was still low At the level of healthy people, as the number of chemotherapy increases, the probability of chemotherapy resistance increases, and with the increase in resistance, the level of miR-181b decreases after the fth cycle. The results suggest that there is a concomitant relationship between miR-181b and SCLC resistance, that is, when the expression level of miR-181b decreases again, it may indicate the occurrence of SCLC resistance, and this is also the same as Wang Yuntao et al (17). The expression of miR-181b in lung cancer patients' tissues is low, and the corresponding clinical symptoms are consistent. Voortman J et al. (18) found in research that low expression of a single miRNA predicts poor survival or high recurrence of lung cancer, including miR-181, miR-221, etc. Therefore, changes in the content of miR-181b in peripheral blood of SCLC patients may be Make it an effective indicator to predict the effect of chemotherapy. This is consistent with the results of this experiment.
The previous results showed that miR-181b was under-expressed in H446 cell line, while the expression levels in H446 cell line and corresponding drug-resistant cell line were detected, and the expression level of miR-181b in drug-resistant cell line was signi cantly lower In the H446 cell line, these results have similar results to clinical blood. In the overexpression experiment of miR-181b, we found that the expression level of miR-181b in two cell lines was regulated by overexpression of lentivirus transfection.
After overexpression of its content, the H446 cell line was sensitive to drugs before and after transfection There was no statistically signi cant difference in sex, and the drug sensitivity of drug-resistant strains was statistically different before and after transfection. In addition, the over-expression of miR-181b in the drug-resistant group was statistically different from that in the sensitive group. Moreover, the IC50 value of the H446 drug-resistant cell line after transfection with lentivirus overexpressed miR-181b was signi cantly reduced. It can be inferred that the increased expression of miR-181b in SCLC may directly target the regulation of the activity of downstream substances, thereby affecting the inactivation or enhanced activity of signals such as certain proteins. These changes will lead to increased sensitivity of SCLC to chemotherapy drugs. It is also possible that the increase in miR-181b content will affect upstream genes through a feedback mechanism, resulting in decreased activity of certain proteins. The speci c mechanism is not yet clear, but all tend to think that changes in the expression level of miR-181b will regulate the activity of the target and cause changes in related functions.
MiR-181b also has different mechanisms in other tumor treatments and drug resistance. As Zhu et al. (19) found that in NSCLC-resistant A549/cisplatin (CDDP) cell lines, high expression of miR-181b can be reduced BCL2 protein levels, in turn, make it sensitive to CDDP-induced apoptosis. And to some extent participate in the formation of lung cancer cell line MDR. Studies have also found that increased expression of miR-181b can inactivate the Notch2/Hes1 signaling pathway and enhance the sensitivity to CDDP therapy. In addition, Mir-181b may be involved in the occurrence and development of tumors by regulating various targets such as histone metalloproteinase 3 (TIMP3) inhibitor, insulin-like growth factor 1 receptor (IGF-1R) and cAMP responsive element binding protein 1(CREB1).
ACE2 is the downstream target gene of miR-181b, and after administration of ACEI drugs, it was found that its family increased SCLC tumor resistance. This result is the same as Zosia Kmietowicz et al.(20) in its clinical application of ACEI drugs will increase The risk of lung cancer is similar. But Lever et al. (21) rst proposed that ACEI may have anti-cancer effects. And elaborated that its anti-cancer effect mainly includes inhibiting tumor angiogenesis and extracellular matrix degradation, reducing cancer morbidity and mortality, inhibiting tumor growth, growth and metastasis, etc. (18) Therefore, by regulating the expression of miR-181b, it has certain signi cance for the treatment of patients to increase the sensitivity of SCLC to chemotherapeutic drugs. However, there are many factors affecting SCLC resistance, and the speci c regulatory mechanism still needs further experiments to discover its conduction pathways. Studies have shown that miRNA usually regulates more than 100 target genes to regulate tumor proliferation and metastasis (13). This study provides an experimental basis for further exploring the relationship between miR-181b and SCLC resistance, and the mechanism and pathway through which miR-181b affects SCLC resistance development still needs further research.

Conclusion
There is low expression of miR-181b level in SCLC, and it can directly target the gene ACE2 to affect the biological characteristics of SCLC; 2. miR-181b is low expressed in SCLC patients, and rst-line chemotherapy can promote its recovery, but cannot return to normal levels, and ACEI drugs can increase the risk of SCLC drug-resistant.

Declarations
Ethics approval and consent to participate All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

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Authors' Contributions
HL and JS participated in the design of experiment, the execution of experiment and the writing of the paper; SH was responsible for the collection and analysis of experimental data; QY was responsible for the polishing and modi cation of the paper; YW was responsible for the design of experiment, and guided the execution of experiment, data collection and analysis, writing and revising the paper. All authors read and approved the nal manuscript.
Funding (Not applicable)    Tumor cell migration ability: Figure A shows miR-181b migration distance at different times before and after overexpression and analysis results. The rst two images are visual images of the migration distance at different times under the microscope, the upper image is the migration image before expression, and the lower image is the migration image after expression. The gure below shows the results of data collection and analysis after processing by PS software. Panels B and C show the detection of transwell and migration ability before and after miR-181b overexpression (Giemsa staining).

Figure 3
Tumor cell migration ability: Figure A shows miR-181b migration distance at different times before and after overexpression and analysis results. The rst two images are visual images of the migration distance at different times under the microscope, the upper image is the migration image before expression, and the lower image is the migration image after expression. The gure below shows the results of data collection and analysis after processing by PS software. Panels B and C show the detection of transwell and migration ability before and after miR-181b overexpression (Giemsa staining).

Figure 4
Analysis  Relative expression levels of miR-181b and its target genes The expression levels of the downstream target gene ACE2 after overexpression of different concentrations of miR-181b.  Expression levels of miR-181b before and after chemotherapy (A) and different chemotherapy cycles (B) Figure 8 Expression levels of miR-181b before and after chemotherapy (A) and different chemotherapy cycles (B) Figure 9 Drug sensitivity of each group of cells before and after miR-181b overexpression Figure 9 Drug sensitivity of each group of cells before and after miR-181b overexpression HE staining: the left picture shows the addition of ACEI drugs; the right picture shows the absence of ACEI drugs Figure 11 HE staining: the left picture shows the addition of ACEI drugs; the right picture shows the absence of ACEI drugs Figure 12 Immunohistochemical results: the left picture shows the addition of ACEI drugs; the right picture shows the absence of ACEI drugs Figure 12 Immunohistochemical results: the left picture shows the addition of ACEI drugs; the right picture shows the absence of ACEI drugs Figure 13 MiR-181b regulatory map Figure 13 MiR-181b regulatory map