# Reversal of Radiotherapy Resistance of Ovarian Cancer Cell Strain CAOV3/R by Targeting lncRNA CRNDE

Authors: Wencui Yang; Xiaoning Li; Lin Zhao; Fengju Zhao
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

Radiotherapy resistance is one of the key factors of poor prognosis of ovarian cancer clinical treatment. The search for key targets of ovarian cancer radiotherapy resistance has become a high priority. Long noncoding RNA plays an important role in tumor development. However, the key lncRNA in ovarian cancer radiotherapy resistance is not identified. Our finding that lncRNA CRNDE is highly expressed in the radiotherapy resistance cell line CAOV3/R drew our attention. Therefore, in this study, we targeted lncRNA CRNDE to analyze whether it is a key factor of radiotherapy resistance in ovarian cancer. Ultimately, we found that silencing lncRNA CRNDE could reverse CAOV3/R radiotherapy resistance, which would be a boon to clinical treatment.


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## Body

## 1. Introduction

Ovarian malignancy is one of the common malignancies of the female genitalia [1, 2], second only to cervical and corpus uteri cancers in terms of incidence [3]. Therefore, an ideal cancer treatment strategy is to selectively target genes essential for the survival of cancer cells, which are dependent on nonnormally expressed oncogenes for continued growth or maintenance of their malignant phenotype [4, 5]. In recent years, research has identified many genes associated with tumor development by sequencing tumor cells against normal cells or tumor tissue against paracarinoma tissue. These genes are associated with tumor development and can be targeted to achieve therapeutic effects. Long noncoding RNA (lncRNA) is a transcriptional product of noncoding proteins required for tumor development [6, 7]. Therefore, it may inhibit tumors and enhance targeted tumor treatment methods by controlling its expression in cancers [8, 9]. Furthermore, the lncRNA CRNDE was strongly associated with ovarian cancer prognosis and played an essential role in ovarian cancer radiation resistance in our research. As a result, we examined whether the long noncoding RNA CRNDE might be used as a target gene for ovarian cancer radiation resistance by modulating particular signaling pathways.


## 2. Method

### 2.1. Construction of Radiotherapy-Resistant Strain CAOV3/R in Ovarian Cancer

Ovarian cancer cell strain CAOV3 was grown in an iron-walled shape. The cells were cultured using the 1640 (BI) complete medium containing 10% serum (BI) in a 37°C incubator containing 5% CO2 [10]. When the cells grew to 70–80%, 6 MV X-ray irradiation (small animal radiometer) was given at 2 Gy each time, for a total dose of 100 Gy. The medium was changed the day after irradiation. When the cells grow to 85% or more, they are digested by 0.25% trypsin for passaging. The cells were irradiated again when the cell fusion reached 70–80%, and this was repeated until the irradiation dose was completed.


### 2.2. Flat Clone

Cells in the logarithmic growth phase were selected and digested using 0.25% trypsin to make a single cell suspension. After cell counting, the cells were planted in 6-well plates at 1000 cells/well. After the cells were attached to the wall, they were irradiated with 0, 2, 4, 6, 8, and 10 Gy doses, respectively, in three replicate wells per group. After growing until the clonal colonies were visible to the naked eye, they were rinsed twice using PBS, fixed in 4% paraformaldehyde, and stained with crystalline violet. The number of clonal colonies per group was analyzed. The relevant radiobiological parameters were calculated by Graphpad 7.0 software with a single-click multitarget model, and fitted survival fraction curves were plotted.


### 2.3. Analysis of lncRNA CRNDE Expression by qRT-PCR

After successfully constructing the radioresistant strain CAOV3/R, RNA was extracted from the radioresistant strain and the parental strain by using the RNA Fast Extraction Kit. After reverse transcription, lncRNA CRNDE expression differences in the parental and radioresistant strains were analyzed by real-time fluorescence quantitative PCR and calculated as2−ΔΔct.


### 2.4. In Situ Hybridization Experiments (Fish)

CAOV3 and CAOV3/R in the logarithmic growth phase were selected, digested by 0.25% trypsin, and seeded in confocal dishes. After cell apposition, the cells were rinsed twice by PBS and fixed using 4% paraformaldehyde. The prehybridization solution was treated for 30 min, and the hybridization solution was diluted with the probe (Reebok) and incubated overnight at 37°C. The nuclei were eluted with 4x, 2x, and 1x SSC, stained with DAPI, and analyzed for localization by confocal microscopy (Zeiss).


### 2.5. SiRNA Silencing of lncRNA CRNDE Expression

CAOV3/R cells in the logarithmic growth phase were trypsinized, and cells were counted.1∗104 cells/well were selected and seeded in 6-well plates, and the cells adhered to the wall overnight. The siRNA was transfected with 30 nmol by lip 3000, and the medium was changed after 24 h of transfection. Cells were collected 48 h after transfection and analyzed for silencing effect by qRT-PCR. After verifying the silencing effect, the cells were treated with 4 Gy dose irradiation.


### 2.6. Apoptosis Analysis

The cells were washed twice with PBS and digested with 0.25 percent trypsin after treatment. The degree of apoptosis in each group of cells was determined using the ANENXIN-V/PI double-staining technique, and flow cytometry was used to examine apoptosis in each group.


### 2.7. Animal Experiments

The mice were studied according to our hospital’s ethical specifications approved by the Institute of Animal Protection and Research. The nude mice used in this experiment were purchased from the Guangdong Medical Research Animal Center. After one week of acclimatization of nude mice, CAOV3/R was digested into a single cell suspension. Then, 1 : 1 mixed matrix gel, 200μl per nude mouse, containing 2∗106 tumor cells, was injected and planted on the dorsum of nude mice. The treatment was administered when the tumor volume reached 100 mm3. The experimental group received siRNA tail vein injections at 1-day intervals for three consecutive treatments, each with 1 noml siRNA, whereas the control group received NC sequences. During this time, naked mice’s tumor volume and body weight were assessed at 3-day intervals.


## 2.1. Construction of Radiotherapy-Resistant Strain CAOV3/R in Ovarian Cancer

Ovarian cancer cell strain CAOV3 was grown in an iron-walled shape. The cells were cultured using the 1640 (BI) complete medium containing 10% serum (BI) in a 37°C incubator containing 5% CO2 [10]. When the cells grew to 70–80%, 6 MV X-ray irradiation (small animal radiometer) was given at 2 Gy each time, for a total dose of 100 Gy. The medium was changed the day after irradiation. When the cells grow to 85% or more, they are digested by 0.25% trypsin for passaging. The cells were irradiated again when the cell fusion reached 70–80%, and this was repeated until the irradiation dose was completed.


## 2.2. Flat Clone

Cells in the logarithmic growth phase were selected and digested using 0.25% trypsin to make a single cell suspension. After cell counting, the cells were planted in 6-well plates at 1000 cells/well. After the cells were attached to the wall, they were irradiated with 0, 2, 4, 6, 8, and 10 Gy doses, respectively, in three replicate wells per group. After growing until the clonal colonies were visible to the naked eye, they were rinsed twice using PBS, fixed in 4% paraformaldehyde, and stained with crystalline violet. The number of clonal colonies per group was analyzed. The relevant radiobiological parameters were calculated by Graphpad 7.0 software with a single-click multitarget model, and fitted survival fraction curves were plotted.


## 2.3. Analysis of lncRNA CRNDE Expression by qRT-PCR

After successfully constructing the radioresistant strain CAOV3/R, RNA was extracted from the radioresistant strain and the parental strain by using the RNA Fast Extraction Kit. After reverse transcription, lncRNA CRNDE expression differences in the parental and radioresistant strains were analyzed by real-time fluorescence quantitative PCR and calculated as2−ΔΔct.


## 2.4. In Situ Hybridization Experiments (Fish)

CAOV3 and CAOV3/R in the logarithmic growth phase were selected, digested by 0.25% trypsin, and seeded in confocal dishes. After cell apposition, the cells were rinsed twice by PBS and fixed using 4% paraformaldehyde. The prehybridization solution was treated for 30 min, and the hybridization solution was diluted with the probe (Reebok) and incubated overnight at 37°C. The nuclei were eluted with 4x, 2x, and 1x SSC, stained with DAPI, and analyzed for localization by confocal microscopy (Zeiss).


## 2.5. SiRNA Silencing of lncRNA CRNDE Expression

CAOV3/R cells in the logarithmic growth phase were trypsinized, and cells were counted.1∗104 cells/well were selected and seeded in 6-well plates, and the cells adhered to the wall overnight. The siRNA was transfected with 30 nmol by lip 3000, and the medium was changed after 24 h of transfection. Cells were collected 48 h after transfection and analyzed for silencing effect by qRT-PCR. After verifying the silencing effect, the cells were treated with 4 Gy dose irradiation.


## 2.6. Apoptosis Analysis

The cells were washed twice with PBS and digested with 0.25 percent trypsin after treatment. The degree of apoptosis in each group of cells was determined using the ANENXIN-V/PI double-staining technique, and flow cytometry was used to examine apoptosis in each group.


## 2.7. Animal Experiments

The mice were studied according to our hospital’s ethical specifications approved by the Institute of Animal Protection and Research. The nude mice used in this experiment were purchased from the Guangdong Medical Research Animal Center. After one week of acclimatization of nude mice, CAOV3/R was digested into a single cell suspension. Then, 1 : 1 mixed matrix gel, 200μl per nude mouse, containing 2∗106 tumor cells, was injected and planted on the dorsum of nude mice. The treatment was administered when the tumor volume reached 100 mm3. The experimental group received siRNA tail vein injections at 1-day intervals for three consecutive treatments, each with 1 noml siRNA, whereas the control group received NC sequences. During this time, naked mice’s tumor volume and body weight were assessed at 3-day intervals.


## 3. Result

### 3.1. Verification of the Single-Click Multitarget Model

After the cells were irradiated to the end, the parental strain cells after 10, 30, and 50 irradiations were selected as the study targets. After an equal number of cells were walled and irradiated by 2 Gy X-rays, the radiotherapy sensitivity of the irradiated group and the parental strain was analyzed by clone formation assay. The results showed that the cells were most resistant to radiotherapy after a single 2 Gy, irradiated 30 times. The mean lethal dose (OD) was 3.15 Gy, which was 2.35 times higher than that of the parental strain, and the difference was statistically significant. Therefore, it was selected as the radiotherapy-resistant strain CAOV3/R, as shown in Table1 and Figure 1.Table 1
Radiobiological parameters of single-click multitarget models for each group.
GroupODQuasi-threshold measurement (Dq)2 Gy SFCAOV31.3403.15480.658CAOV3/101.8583.45810.764CAOV3/303.150.83650.886CAOV3/501.6853.08950.702Figure 1
Survival curves of CAOV3 with different irradiation times.


### 3.2. Expression Analysis of lncRNA CRNDE in CAOV3/R

RNA was extracted from the parental strain and the resistant strain, and qRT-PCR analysis revealed that lncRNA CRNDE was 23.5-fold higher in CAOV3/R specifically,P<0.001, as shown in Figure 2(a). The lncRNA CRNDE was mainly expressed in the cytoplasm after nuclear plasma isolation, as shown in Figure 2(b). In situ hybridization experiments verified that it was mainly expressed in the cytoplasm, as shown in Figure 2(c).Figure 2
Expression analysis of lncRNA CRNDE in CAOV3/R. (a)P<0.001 compared with the parental strain. (b) qRT-PCR analysis of lncRNA expression in nuclear plasma after nuclear plasma isolation. (c) The localization of lncRNA verified by in situ hybridization.
(a)(b)(c)


### 3.3. lncRNA CRNDE Regulates Radiotherapy Resistance of CAOV3/R

The expression of lncRNA CRNDE in CAOV3/R was knocked down by siRNA. qRT-PCR analysis revealed that si#1 could knock down 85.2% and si#2 could knock down 79.6% compared with the NC group,P<0.05, as shown in Figure 3(a). Therefore, the si#1 and si#2 sequences were selected as valid sequences to analyze the sensitivity of CAOV3/R to radiotherapy after silencing lncRNA CRNDE. The results showed silencing lncRNA CRNDE. This enhanced the sensitivity of CAOV3/R to radiotherapy, as shown in Figure 3(b). In addition, the si#1 and si#2 groups showed a significant decrease in clone formation ability compared with the blank and NC groups, P<0.05, as shown Figures 3(c) and 3(d).Figure 3
lncRNA CRNDE regulates radiotherapy resistance of CAOV3/R. (a) qRT-PCR verifies the knockdown efficiency of siRNA, and∗indicates P<0.001 compared with blank and NC. (b) Knockdown of lncRNA CRNDE significantly reduces the sensitivity of CAOV3/R to radiotherapy. (c), (d) Clone formation assay analyzes the clonogenic ability of cells after knockdown of lncRNA CRNDE at 2 Gy radiation dose, and ∗indicates P<0.001 compared with blank and NC.
(a)(b)(c)(d)


### 3.4. Reversal of Radiotherapy Resistance by Targeted lncRNA CRNDE

Female nude mice at 3-4 weeks were selected, and after one week of acclimatization culture, CAOV3/R was implanted subcutaneously on the back. When the tumor volume was to be 100 mm3, tail vein injection of siRNA targeted to silence the expression of lncRNA CRNDE. Irradiation X-rays were given using a small animal radiometer at 2 Gy each time for 3 consecutive times. Tumor volume in the SiRNA group became significantly smaller over time, as shown Figure 4(c). Tumors were peeled off after 15 days of observation, and tumor weight was recorded, as shown in Figure 4(b). There was no significant difference in the change in body weight between the two groups of nude mice, as shown in Figure 4(d).Figure 4
Reversal of radiotherapy resistance by targeted lncRNA CRNDE. (a) Changes in tumor volume with volume after treatment. (b) Weight of the tumor at the end of observation. (c) The stripped tumors were displayed. (d) Changes in body weight over time in the two groups of nude mice.
(a)(b)(c)(d)


## 3.1. Verification of the Single-Click Multitarget Model

After the cells were irradiated to the end, the parental strain cells after 10, 30, and 50 irradiations were selected as the study targets. After an equal number of cells were walled and irradiated by 2 Gy X-rays, the radiotherapy sensitivity of the irradiated group and the parental strain was analyzed by clone formation assay. The results showed that the cells were most resistant to radiotherapy after a single 2 Gy, irradiated 30 times. The mean lethal dose (OD) was 3.15 Gy, which was 2.35 times higher than that of the parental strain, and the difference was statistically significant. Therefore, it was selected as the radiotherapy-resistant strain CAOV3/R, as shown in Table1 and Figure 1.Table 1
Radiobiological parameters of single-click multitarget models for each group.
GroupODQuasi-threshold measurement (Dq)2 Gy SFCAOV31.3403.15480.658CAOV3/101.8583.45810.764CAOV3/303.150.83650.886CAOV3/501.6853.08950.702Figure 1
Survival curves of CAOV3 with different irradiation times.


## 3.2. Expression Analysis of lncRNA CRNDE in CAOV3/R

RNA was extracted from the parental strain and the resistant strain, and qRT-PCR analysis revealed that lncRNA CRNDE was 23.5-fold higher in CAOV3/R specifically,P<0.001, as shown in Figure 2(a). The lncRNA CRNDE was mainly expressed in the cytoplasm after nuclear plasma isolation, as shown in Figure 2(b). In situ hybridization experiments verified that it was mainly expressed in the cytoplasm, as shown in Figure 2(c).Figure 2
Expression analysis of lncRNA CRNDE in CAOV3/R. (a)P<0.001 compared with the parental strain. (b) qRT-PCR analysis of lncRNA expression in nuclear plasma after nuclear plasma isolation. (c) The localization of lncRNA verified by in situ hybridization.
(a)(b)(c)


## 3.3. lncRNA CRNDE Regulates Radiotherapy Resistance of CAOV3/R

The expression of lncRNA CRNDE in CAOV3/R was knocked down by siRNA. qRT-PCR analysis revealed that si#1 could knock down 85.2% and si#2 could knock down 79.6% compared with the NC group,P<0.05, as shown in Figure 3(a). Therefore, the si#1 and si#2 sequences were selected as valid sequences to analyze the sensitivity of CAOV3/R to radiotherapy after silencing lncRNA CRNDE. The results showed silencing lncRNA CRNDE. This enhanced the sensitivity of CAOV3/R to radiotherapy, as shown in Figure 3(b). In addition, the si#1 and si#2 groups showed a significant decrease in clone formation ability compared with the blank and NC groups, P<0.05, as shown Figures 3(c) and 3(d).Figure 3
lncRNA CRNDE regulates radiotherapy resistance of CAOV3/R. (a) qRT-PCR verifies the knockdown efficiency of siRNA, and∗indicates P<0.001 compared with blank and NC. (b) Knockdown of lncRNA CRNDE significantly reduces the sensitivity of CAOV3/R to radiotherapy. (c), (d) Clone formation assay analyzes the clonogenic ability of cells after knockdown of lncRNA CRNDE at 2 Gy radiation dose, and ∗indicates P<0.001 compared with blank and NC.
(a)(b)(c)(d)


## 3.4. Reversal of Radiotherapy Resistance by Targeted lncRNA CRNDE

Female nude mice at 3-4 weeks were selected, and after one week of acclimatization culture, CAOV3/R was implanted subcutaneously on the back. When the tumor volume was to be 100 mm3, tail vein injection of siRNA targeted to silence the expression of lncRNA CRNDE. Irradiation X-rays were given using a small animal radiometer at 2 Gy each time for 3 consecutive times. Tumor volume in the SiRNA group became significantly smaller over time, as shown Figure 4(c). Tumors were peeled off after 15 days of observation, and tumor weight was recorded, as shown in Figure 4(b). There was no significant difference in the change in body weight between the two groups of nude mice, as shown in Figure 4(d).Figure 4
Reversal of radiotherapy resistance by targeted lncRNA CRNDE. (a) Changes in tumor volume with volume after treatment. (b) Weight of the tumor at the end of observation. (c) The stripped tumors were displayed. (d) Changes in body weight over time in the two groups of nude mice.
(a)(b)(c)(d)


## 4. Discussion

Radiotherapy has significant efficacy in treating many tumors, but resistance to radiotherapy is one of the critical factors affecting the prognosis of tumors [11, 12]. Ovarian cancer is one of the most common gynecologic malignancies, with a high incidence and mortality rate, and is a severe health risk for women worldwide [13, 14]. In response to the limited therapeutic options, radiotherapy has become a standard treatment [15, 16]. Therefore, there is a lack of a critical target or marker for reversing or advancing the assessment of radiotherapy efficacy.Long noncoding RNA has been shown to have a role in developing a variety of tumors [17]. Several lncRNAs resistant to radiation have been identified (for example, AFAP1-AS1, HOTAIR, and lncRNA-P21) [18]. Regulating its expression in tumors may significantly impact tumor cell biology by inducing apoptosis and blocking DNA damage repair [19, 20]. According to human gene sequencing, about 98 percent of genes do not code for proteins, although they have biological roles. As a result, another avenue for targeted tumor treatment will be to investigate how these noncoding RNAs fulfill biological roles.In our study, we found that lncRNA CRNDE was explicitly highly expressed in acquired radiotherapy-resistant strains. However, how it was affected by high expression was not investigated by us. Silencing lncRNA CRNDE by siRNA significantly reduced the sensitivity of CAOV3/R cells to radiotherapy and inhibited clone formation. Furthermore, when treated in animals in vivo, we found that targeted silencing of lncRNA CRNDE reversed CAOV3/R radiotherapy resistance and inhibited tumor growth.


## 5. Conclusions

Our results enrich the view that CRNDE is important in acquired radiotherapy resistance in human ovarian cancer and will be a marker of prognosis for radiotherapy in ovarian cancer. It is also an important indicator to guide clinical chemotherapy treatment. The lncRNA CRNDE was further investigated. lncRNA CRNDE was inhibited from reversing ovarian cancer radiotherapy resistance for patients.



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*Source: 8556965-2021-09-08.xml*